Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins

Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins Li Su, Laurence Hotel, Cédric Paris, Alexander Brachmann, Jörn Piel, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-222743/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Jan, 2022 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combined multiple, state-of-the-art approaches including modification of docking domains, use of modules of varying domain composition, alternative interdomain fusion sites, and targeted adaptation of key domain-domain interfaces, to reprogram the stambomycin PKS by deleting seven internal modules – the most substantial modification to an intact system reported to date. One such system produced the target 37-membered mini-stambomycin metabolites, a reduction in chain length of 14 carbons relative to the 51-membered parental compounds, but also substantial quantities of shunt metabolites released from the multienzyme subunit upstream of the newly-installed junction. Our data also provide evidence for an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain acting on chains located four modules upstream. The yields of all metabolites were substantially reduced compared to the wild type compounds, likely reflecting the poor tolerance to the non-native substrates of the modules downstream of the introduced interfaces. Taken together, our data demonstrate that even ‘optimized’ PKS engineering strategies remain inadequate for efficient production of target polyketide derivatives, and highlight several areas for future investigation. Chemical Biology polyketide synthases (PKSs) PKS engineering strategies Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction For almost thirty years, efforts have been made to leverage the modular genetic architecture of the type I polyketide synthases (PKSs) to generate novel derivatives, typically by modifying individual catalytic domains. Despite enormous progress in establishing domain structure-function relationships 1 , 2 , such genetic manipulation remains inefficient 3 . Insight into factors potentially contributing to low product yields was provided by cryo-electron microscopy analysis of a model PKS module at multiple stages of its catalytic cycle 4 , 5 . This work revealed that interdomain contacts are critical for establishing the various functional states of the module, and that transitions between such states rely on evolving interfaces between the domains, as well as the intervening ‘linker’ regions. In short, PKS modules appear to be highly integrated units, thus explaining why exchange of catalytic domains for heterologous counterparts is often detrimental 6 . Collectively, these observations motivate future approaches in which modules or multi-modular subunits are employed as the basic building blocks for engineering the assembly lines 7 – 13 . Nevertheless, using modules requires a clear definition of their domain composition. Traditionally, modules encompass the three invariable domains required for monomer selection and chain extension (ketosynthase (KS), acyl transferase (AT) and acyl carrier protein (ACP)), as well as any intervening β-keto processing activities (e.g. ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER)) (Fig. 1 a). However, an alternative definition was recently suggested by the finding that KS domains in certain PKSs co-evolve with the tailoring domains located upstream in the assembly lines 14 , 15 . Accordingly, modules begin with the modifying domains and the associated AT, and terminate with the KS that is classically assigned to the downstream module (Fig. 2 ). Even before a module redefinition was suggested, engineering efforts revealed that maintaining the key ACP n /KS n+1 interface can, in certain cases, be critical for the function of a hybrid PKS 7 , 10 , 16 . Recently, we have carried out module swapping based on both of these definitions, by covalently tethering heterologous modules to a common donor module within a bimodular mini-PKS 17 . Overall, our data demonstrated that both module definitions led to functional hybrid PKSs, and which boundaries worked best depended on the source module 18 . Indeed, regardless of which extremities are employed, module exchange results in non-native interdomain interactions (ACP n /KS n+1 or KS n+1 /ACP n+1 ), and in the case of classical module boundaries, potential incompatibilities in terms of KS substrate specificity (Fig. 2 ) – both of which have been shown to reduce activity via detailed studies in vitro 19 − 21 . In this work, we aimed to investigate the generality of these findings for efforts to create non-native intermodular junctions when the modules are located on distinct subunits 10 . In such cases, the resulting non-covalent interactions are mediated by short sequences at the extreme C- and N-termini of the subunits called docking domains (DDs) 22 (Figs. 1 and 2 ). Matched pairs of DDs form specific complexes at intersubunit interfaces, enforcing a strict subunit ordering within the PKS system. As a test case, we aimed to genetically engineer the biosynthesis of substantially smaller derivatives of the stambomycin family of polyketides in Streptomyces ambofaciens ATCC23877 23 . The stambomycins 1 (Fig. 1 b) are glycosylated macrolides which show promising anti-cancer activity against multiple human cancer cell lines 23 . The six characterized family members (A − F) differ in the alkyl functionality at the C-26 position (Fig. 1 a) which directly impacts their potency, 23 , 24 but have in common modification by trans -acting cytochrome P450 hydroxylases at C-28 and C-50. Notably, at 51-members, the macrolactone ring is among the largest of all known polyketides. Thus, the stambomycins represent an attractive model system for establishing PKS engineering as a means to access structurally-simplified analogues (minimal pharmacophores 25 ) for biological evaluation, as a complement to traditional chemical synthesis. Here we report a comprehensive series of experiments aiming to generate 37-membered ring stambomycin analogues, based on both the classical and revised module definitions. The target mini-stambomycins were detected successfully, albeit in low yields, and the identification of the derivatives allowed us to clarify the relative timing of the two cytochrome P450-catalyzed hydroxylation reactions in the pathway. Attempts to boost titers by ACP/KS interface engineering 19 , 20 , 26 were unsuccessful, but led in certain cases to a surprising increase in the liberation of linear shunt metabolites – only the second report, to our knowledge, of inter-subunit crosstalk resulting in thioesterase (TE)-mediated chain release. Taken together with recent work by others 10 , our data reinforce the idea that in order to boost efficacy, strategies based on modifying PKS intersubunit interfaces must take into account the function of the modules acting downstream from the newly-established junctions. Results Design of engineering experiments based on classical modular boundaries. The stambomycin PKS comprises 25 modules distributed among 9 polypeptides (Pks1 − 9) 23 (Fig. 1 a) (Note: throughout the text, the stambomycin genes have been numbered in accordance with ref. 23 ). To access abridged derivatives using the classical module boundaries, we reasoned that we could engineer novel intersubunit interfaces by suitable manipulation of docking domains. Encouragingly, the extreme C- and N-termini of all subunits (with the exception of the N-terminus of Pks1 and the C-terminus of Pks9) contain sequences with convincing homology to previously-identified DDs 22 , 27 (the C-terminal DDs are referred to hereafter as C DDs and their partner N-terminal DDs as N DDs). By bioinformatics analysis, we were able to confidently assign the DDs acting at 6 of the 8 interfaces to the type ‘1a’ class 22 , and the remaining two sets of DDs as type ‘1b’ 27 ( Supplementary Fig. 1 ). In both cases, docking occurs between an α-helical C DD and a coiled-coil formed by the N DD, with specificity achieved via strategically-placed charge:charge interactions at the complex interface ( Supplementary Fig. 1 ) 22 , 27 . Among the type 1a junctions, there were notably two which appeared compatible in terms of the translocated substrate: PKSs 3/4 + 7/8 and Pks 4/5 + 8/9 ( Supplementary Fig. 2 ). Specifically, the functional groups at the critical α- and β- positions 14 , 28 of the transferred chains are identical at these junctions, and correspondingly, the downstream KSs show similarities across several sequence motifs previously correlated with substrate specificity 14 , 20 , 29 ( Supplementary Fig. 2 ). Targeting such interfaces thus allowed us, at least in principle, to overcome the functional block to the engineered systems represented by poor recognition of the incoming substrate by the directly downstream KS domain 21 . Ultimately, we targeted a new interface between Pks subunits 4 and 9 for two principle reasons. Firstly, Pks4 is at the origin of the structural variation between the stambomycin family members, and thus we anticipated that maintaining the subunit within the hybrid system would give rise to a corresponding series of truncated analogues, providing important evidence for their identity. Secondly, it was genetically more practical to modify the second set of interfaces due to splitting of the PKS subunits between two loci (Fig. 1 ). To establish the novel junction, we initially modified the C DD of Pks4 ( C DD 4 ) to match that of Pks8 (the natural partner of the N DD of Pks9 ( N DD 9 )), either by site-directed mutagenesis of residues previously identified as key mediators of interaction specificity (construct C DD 4 SDM; Supplementary Fig. 3 and Supplementary Table 1 ) 22 , or by exchange of the complete C DD docking α-helix of C DD 4 for that of C DD 8 (construct C DD 4 helix swap; Supplementary Fig. 3 and Supplementary Table 1 ) 30 . Modifying the C DD 4 specificity ‘code’ to match that of C DD 8 required mutation of 3 residues, while for the C DD 4 helix swap, the terminal 16 amino acids of C DD 4 were exchanged for the corresponding 15 residues of C DD 8 ( Supplementary Fig. 3 and Supplementary Table 1 ). The genetic alterations were carried out in two distinct PKS contexts: (i) in the presence of the intervening subunits 5 − 8, which allowed for the possibility of competitive interactions between modified Pks4 and both Pks5 and Pks9; and ii) removing the intervening subunits 5 − 8, thus eliminating competition for binding of Pks4 by Pks5, and of Pks9 by Pks8 ( Supplementary Fig. 3 ). We further generated a mutant in which Pks subunits 5 − 8 were deleted but no modification was made to C DD 4 , in order to judge the intrinsic capacity of Pks4 and Pks9 to interact. Furthermore, genetic engineering was carried out in parallel by both PCR-targeting 31 and CRISPR-Cas9 32 , in order to directly compare the efficacy of these two approaches, as well as evaluate the effect of the short scar sequence remaining in the chromosome following PCR-targeting. Engineering the stambomycin PKS based on the classical module definition. The C DD 4 SDM and C DD 4 helix swap sequences were introduced in parallel into the S. ambofaciens genome using PCR targeting and CRISPR-Cas9 (full experimental details are provided in the Supplementary Methods ). As discussed previously, the modifications were made both in the presence of the intervening subunits Pks5 − 8 and in their absence ( Supplementary Fig. 3 ). As previous work has shown that production from the stambomycin biosynthetic gene cluster requires activation by constitutive overexpression of a pathway-specific LAL (Large ATP-binding regulators of the LuxR family) regulator 23 , we additionally introduced the LAL overexpression plasmid (pOE484) into each of the mutants, using the empty parental plasmid (pIB139 33 ) as a control. In total, this strategy resulted in 20 targeted strains harboring interface mutants (where K7N refers to PCR targeting and CPN to CRISPR-Cas9 engineering): K7N1/pIB139, K7N1/OE484, K7N2/pIB139, K7N2/OE484, K7N3/pIB139, K7N3/OE484, K7N4/pIB139, K7N4/OE484, K7N5/pIB139, K7N5/OE484, K7N6/pIB139, K7N6/OE484, CPN1/pIB139, CPN1/OE484, CPN2/pIB139, CPN2/OE484, CPN4/pIB139, CPN4/OE484, CPN5/pIB139, CPN5/OE484 ( Table 1, Supplementary Tables 2 − 4 ; despite extensive efforts the CPN3 mutant strain was not obtained). The principal difference between the K and CPN series of constructs is the presence of a 33 bp ‘scar’ sequence between the modified pks4 and pks9 genes ( Supplementary Fig. 4 ). Construct K7N6 was assembled specifically to test the effect of this region, without any further modification to C DD 4 and the intervening pks5 − pks8 genes. With the exception of K7N3, CPN4 and CPN5, extracts of the engineered mutant strains harboring pOE484 were analyzed by high performance liquid chromatography heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) on a Dionex UItiMate 3000 HPLC coupled to a Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, and compared to extracts of the control strain containing pIB139 33 as well as the wild type S. ambofaciens containing pOE484, using SIEVE 2.0 screening software. K7N3, CPN4 and CPN5 were analyzed subsequently, and the data inspected manually. Yield quantification was carried out with reference to a calibration curve generated with purified stambomycins A/B 1 (the limit of detection was found to be between 10 and 1 µg L − 1 , and so any yields < 10 µg L − 1 must be considered an estimate). Novel metabolites not present in the control strains, and for which we obtained reliable exact masses, are listed in Table 1 and Supplementary Fig. 5 . The first notable result is that the K7N6/OE484 mutant yielded a similar metabolic profile to S. ambofaciens wt (22 ± 3 mg L − 1 , 73% relative yield ( Supplementary Table 5 )), showing that the scar sequence negatively impacted stambomycin production, but not dramatically (Fig. 3 ). By contrast, no stambomycins were observed, as anticipated, in all constructs in which Pks5 − Pks8 had been removed (K7N1 − 3; CPN1,2) (Fig. 3 ). Stambomycins 1 were present, however, in strains K7N4 and CPN4 harboring C DD 4 site-directed mutations and in the C DD 4 helix swap strain CPN5, all of which still contained Pks5 − Pks8, albeit at reduced amounts relative to the wild type (18%, 23% and 14% of wt, respectively) (Fig. 3 and Supplementary Table 5 ). (Surprisingly, the metabolic profile of K7N5 reproducibly differed from that of CPN5, as no stambomycin-related metabolites were detected (Fig. 3 )). These data suggested that while the mutations introduced into C DD 4 reduced interaction with N DD 5 , they were not sufficient to disrupt natural chain transfer between Pks4 and Pks5, arguing that DD engineering to alter partner choice should be accompanied by removal of competing intersubunit interactions. We did not find any evidence in the DD engineering experiments for any of the target 37-membered metabolites ( Supplementary Figs. 3 and 5 ). However, all strains in which stambomycin production was abolished ( Table 1 ) exhibited four new peaks in common (Fig. 3 b and Supplementary Fig. 5 ) (other peaks corresponding to potentially novel compounds were observed, but none were shared between multiple strains). The determined exact masses and mass spectra (as exemplified by strain CPN2/OE484, Fig. 3 b) correspond to truncated derivatives of stambomycins A/B and C/D respectively, following premature release from modules 13 and 12 of Pks4 (compounds 2 − 5 , Fig. 3 d and Supplementary Fig. 5 ; ca. 8-fold greater yield of the module 13 products ( Supplementary Table 5 )). Further support for the identity of these shunt compounds was obtained by grafting the chain-terminating (type I) thioesterase (TE) domain from the C-terminal end of Pks9 to the C-terminus of Pks4 in order to force chain release at this stage. Indeed, identical compounds were produced, but at 17-fold increased yield relative to CPN2/OE484, consistent with active off-loading of the chains (Fig. 3 c, Supplementary Fig. 6 and Supplementary Table 5 ). Based on their masses, both sets of shunt metabolites were hydroxylated on a single carbon, while none were found to bear the β-mycaminose of the mature stambomycins, consistent with the absence of the tetrahydropyran moiety to which it is normally tethered. To determine the location of the hydroxylation and therefore the hydroxylase responsible, we inactivated in mutant CPN2/OE484 the genes samR0478 and samR0479 encoding respectively, the stambomycin C-28 and C-50 cytochrome P450 hydroxylases 34 . While extracts of CPN2/OE484/Δ478 were unchanged relative to CPN2/OE484 (i.e. the hydroxyl group was still present), the CPN2/OE484/Δ479 mutant exhibited four new peaks with masses corresponding to the dehydroxylated shunt products (Fig. 3 , Supplementary Fig. 7 (compounds 6 − 9 ) and Supplementary Table 5 ). Taken together, these data show that the unusual on-line modification catalyzed by SamR0479 34 which is necessary for macrocyclization, occurs prior to chain extension by Pks5. While SamR0478 has also been speculated to act during chain assembly 34 , hydroxylation evidently occurs downstream of Pks4, at least. The intriguing substrate structural and/or protein-protein recognition features controlling the timing of hydroxylation by these P450 enzymes remain to be elucidated. Role of TE domains in release of the shunt metabolites. We attributed the observed shunt metabolites 2 − 5 to the lack of productive chain translocation between Pks4 and Pks9, causing intermediates to accumulate on ACPs 12 and 13. To evaluate whether these were released by spontaneous hydrolysis or enzymatically, we further investigated the role of Pks9 TEI 34 in chain release, as well as that of SamR0485, a proof-reading type II TE 35 located in the cluster. Both TEs were disabled by site-directed mutagenesis of the active site serines (Ser ◊ Ala) ( Supplementary Fig. 6 ). Interestingly, inactivation of both the type I and type II TEs reduced the yields of shunt products 2 − 5 relative to the parental strain CPN2/OE484 (by 66% and 27%, respectively; average of duplicate experiments) ( Supplementary Fig. 6 and Supplementary Table 5 ). These data clearly show that premature release of the chains is catalyzed, at least in part, by both TEs in the cluster, although spontaneous liberation also occurs. While type II TEs typically interact with acyl-ACPs in trans to release blocked chains 35 , the effect of the Pks9 TEI is less readily explained. One possibility is that the new productive docking interaction between Pks4 and Pks9 allows Pks9 to adopt an alternative conformation from which the TE can off-load intermediates bound to ACPs 12 and 13 of Pks4 ( Supplementary Fig. 6 ). Although this mechanism is reminiscent of that used by the pikromycin PKS to generate both 12- and 14-membered rings 36 , the pikromycin TEI is separated from its alternative ACP target by a single module, while Pks9 TEI is located five or four modules downstream from ACPs 12 and 13 in the engineered system, which would seem to necessitate substantial inter-subunit acrobatics. Understanding the docking domain engineering via studies in vitro with recombinant domains. To better understand the results of the DD engineering, we studied in vitro the wild type DD pairs ( C DD 4 / N DD 5 and C DD 8 / N DD 9 ), as well as binding between the modified versions of C DD 4 and wild type N DD 9 . Design of suitable expression constructs in E. coli ( Supplementary Tables 1 − 3 ) was based on bioinformatics analysis of the C-terminal ends of Pks4 and Pks8, and the N-termini of Pks5 and Pks9, and secondary structure analysis using PSIPRED 37 ( Supplementary Fig. 8 ). Overall, we expressed and purified the following proteins in recombinant form from E. coli : C DD 4 wt, C DD 4 SDM, C DD 4 helix swap, N DD 5 , and C DD 8 ( Supplementary Fig. 8, Supplementary Table 4 ). As N DD 9 proved insoluble when expressed in E. coli , two versions with alternative start sites were obtained as synthetic peptides (Met and Val; Supplementary Fig. 8, Supplementary Table 4 ). Analysis of the individual C DDs by circular dichroism (CD) confirmed their expected high α-helical content ( C DD 4 wt (100 µM): 58%; C DD 8 wt (100 µM): 49%), and showed no evident effect of the introduced mutations on secondary structure ( Supplementary Fig. 8 ). All of the constructs were further confirmed to be homodimeric by size exclusion chromatography-multi-angle light scattering (SEC-MALS) ( Supplementary Fig. 8 ). The two N DDs also exhibited α-helical character, though less pronounced than the C DDs ( N DD 5 (100 µM): 27%; N DD 9 Met (100 µM): 21%; N DD 9 Val (100 µM): 25%), and were monomeric by SEC-MALS ( Supplementary Fig. 8 ). The latter result was surprising, as type 1a N DDs classically form a homodimeric coiled-coil domain (Fig. 1 , Supplementary Fig. 1 ), but we recently identified functional, monomeric type 1 N DDs 38 . Indeed, we detected binding between the native pairs by isothermal titration calorimetry (ITC), with affinities in the range of those determined previously for matched pairs of DDs 27,38−40 ( C DD 4 + N DD 5 , K d = 14.5 ± 0.9 µM; C DD 8 + N DD 9 Met, K d = 33 ± 2 µM; C DD 8 + N DD 9 Val, K d = 22 ± 1 µM) ( Supplementary Fig. 8 ). Thus, while stable homodimerization of the N DDs may depend on the presence of a downstream homodimeric KS domain, their monomeric character did not preclude interaction with their C DD partners. Based on the higher affinity of the interaction, we could identify the N DD 9 Val as the physiologically relevant construct. The observed binding stoichiometry (1 homodimeric C DD:2 monomeric N DDs), is consistent with the known structure of a type 1a complex in which two monomers of each DD are present (Fig. 1 , Supplementary Fig. 1 ) 22 . As expected, no non-specific interaction was detected between native C DD 4 and N DD 9 , explaining the lack of productive interaction between unmodified subunits Pks4 and Pks9 when the intervening multienzymes are deleted (strain K7N3) (Fig. 3 a). Analysis by ITC of binding between C DD 4 SDM or C DD 4 helix swap and N DD 5 revealed the complete absence of interaction ( Supplementary Fig. 8 ), and therefore that the introduced modifications were sufficient to disrupt communication between the native pair. Thus, the continued production of stambomycins 1 by K7N4, CPN4 and K7N5 harboring Pks5 − Pks8 must be due to additional contacts between Pks4 and Pks5 beyond the docking domains, likely including the compatible ACP 13 /KS 14 interface. On the other hand, no interaction was detected between C DD 4 SDM and N DD 9 , showing that this limited number of mutations was inadequate to induce productive contacts. This result is fully in accord with the absence of the expected mini-stambomycin products from these strains (K7N1/CPN1, Fig. 3 a). By contrast, the C DD 4 helix swap exhibited essentially the same binding to N DD 9 Val as C DD 8 (K d = 21.0 ± 0.3 µM), demonstrating that exchange of just this helix is sufficient to redirect docking specificity 30 . Thus, inefficient docking is not at the origin of the failure of the C DD 4 helix swaps to yield chain-extended products in vivo (strains K7N2/CPN2, Fig. 3 a). We could therefore conclude at this stage that the problem arose from the non-native interface generated between ACP 13 and KS 21 , poor acceptance by KS 21 of the incoming substrate during chain transfer and/or chain extension, and/or low activity towards the modified chain of domains/modules acting downstream. Attempted optimization of the stambomycin DD mutants. We aimed next to improve the novel Pks4/Pks9 intersubunit interface in strain CPN2 ( C DD 4 helix swap + deletion of Pks5 − 8) by targeting helix αI of ACP 13 , as the first 10 residues of this helix have been implicated previously in governing the interaction with the downstream KS domain at hybrid junctions 26 . Notably, multiple sequence alignment of all ACPs in the stambomycin PKS located at intersubunit junctions, revealed a unique sequence for each ACP in the helix αI region, consistent with a recognition ‘code’ for the KS partner, and the idea that mismatching these contacts might hamper productive chain transfer ( Supplementary Fig. 9 ). Indeed, as mentioned previously, even when docking is interrupted, contacts between ACP 13 and KS 14 are apparently sufficient to enable chain translocation between Pks4 and Pks5 (Fig. 3 a). In addition, an analogous strategy of optimizing the ACP n /KS n+1 chain transfer interface was shown recently to substantially improve interaction between an ACP (JamC) derived from the jamaicamide B biosynthetic pathway, and the first chain extension module of the lipomycin PKS (LipPKS1) 41 . In our case, the first six residues of ACP 13 helix αI were modified using CRISPR-Cas9 (EADQRR ◊ PSERRQ), so that the full 10-residue recognition sequence matched that of ACP 20 , the natural partner of KS 21 ( Supplementary Fig. 9 ). Analysis of extracts of the resulting strain CPN2/OE484/ACP13 SDM by HPLC-MS revealed at best minute amounts (highest yield of 0.5 µg mL − 1 ) of target cyclic mini-stambomycins A/B ( 11 ), lacking the hydroxyl group introduced by SamR0478 (Fig. 4 and Supplementary Fig. 9 ). Thus, while this experiment finally yielded the first evidence for successful chain transfer between Pks4 and Pks9 followed by subsequent chain extension by Pks9 and TE-catalyzed release, the overall efficiency of the system remained poor. Interestingly, however, the yields of the four shunt metabolites 2 − 5 were as much as 48-fold higher from the ACP 13 helix swap mutant than from CPN2/OE484, showing that improved interactions between ACP 13 and KS 20 facilitated release of the stalled intermediates from ACPs 12 and 13, presumably via remote action by the TEI domain. Engineering mini-stambomycins by maintaining the native ACP 13 /KS 14 junction (alternative module definition). Cumulatively, the results obtained with the docking domain engineering identified KS 21 as one potential bottleneck in the engineered PKS. Our parallel strategy based on the alternative module definition (Fig. 2 ) allowed us to directly test this idea. Specifically, we investigated the effects of preserving the native C DD 4 / N DD 5 pair and either the majority of KS 14 , or a little more than half of the domain, resulting in a KS 14 /KS 21 hybrid. For this, we used two different splice sites in KS 14 : i) at the end of the domain in a highly-conserved region (GTNAHV) exploited recently to efficiently swap downstream AT domains 42 ; and, ii) at a site corresponding to a recombination hot spot identified during induced evolution of the rapamycin (RAPS) PKS 43 , yielding the KS 14 /KS 21 chimera (Fig. 4 and Supplementary Fig. 10 ). Both of these modifications were introduced into S. ambofaciens using CRISPR-Cas9, while simultaneously removing Pks5 − Pks8, yielding respectively after co-transformation with pOE484 and the control plasmid pIB139, strains ATCC/OE484/hy59_S1, ATCC/pIB139/hy59_S1, ATCC/OE484/hy59_S2, and ATCC/pIB139/hy59_S2. Analysis of culture extracts revealed the presence in both ATCC/OE484/hy59_S1 and ATCC/OE484/hy59_S2 relative to the controls, of a novel series of 37-membered metabolites (Fig. 4 ). The measured masses were consistent with the desired mini-stambomycins either as their free acids or in cyclic form (metabolites 10 − 12 , Fig. 4 ). Signals corresponding to the A/B and C/D derivatives of all metabolites were detected, providing important evidence for their identities, as well as both the C-14 hydroxylated 12 and non-hydroxylated 11 forms of the cyclic mini-stambomycins (C-14 corresponds to C-28 in the parental compounds (Fig. 1 )). It is not surprising that the corresponding E and F forms were not detected, as their yields even from the wild type are much lower than the A − D derivatives (Fig. 3 a). The observation of non-hydroxylated 11 shows notably that internal hydroxylation by SamR0478 is not an absolute prerequisite for TE-catalysed macrolactonization, and argues that hydroxylation of the mini-stambomycins only takes place on the macrocyclic compound. Although compounds 11 and 12 incorporate the tetrahydropyran moiety of the parental stambomycins 1 which undergoes glycosylation, derivatives bearing β-mycaminose were not observed, presumably due to poor recognition of the overall modified macrocycle by glycosyl transferase SamR0481 23 . The yields of the target compounds were minor relative to wild type stambomycins (metabolites 10 , 11 and 12 from ATCC/OE484/hy59_S1 were obtained at highest yields of 0.3, 3.2 and 1.0 µg L − 1 (4.5 µg L − 1 total), respectively), but nonetheless approximately 4-fold higher from ATCC/OE484/hy59_S2 incorporating the hybrid KS 14 /KS 21 than from the full KS 14 swap (3.7, 9.7 and 3.6 µg L − 1 (17 µg L − 1 total); 1500-fold lower yields than 1 ) (Fig. 4 and Supplementary Fig. 10 ). Although the low titers of these compounds precluded their structure elucidation by NMR, we obtained additional confirmatory evidence for their identities by inactivation of samR0479 (which introduces the hydroxyl used for macrocyclization), which resulted in exclusive production of linear dehydroxy mini-stambomycins 13 ( Supplementary Fig. 10 ). As observed previously, the strains also produced substantial quantities of the shunt products 2 − 5 (inactivation of samR0479 led correspondingly to the dehydroxy versions of these compounds 6 − 9 ( Supplementary Figs. 7 and 10 )). The yields were ca. 80-fold higher than those of the corresponding mini-stambomycins, with the highest titers observed in the strain incorporating the hybrid KS 14 /KS 21 . The amount of shunt metabolites was also approximately 123-fold higher than from strain CPN2/OE484 (which incorporates an ACP 13 - C DD 4 swap/ N DD 9 -KS 21 interface) (Figs. 3 a and 4 , Supplementary Table 5 ). Thus, contrary to expectation, although using the KS as a fusion site improved communication between Pks4 and Pks9, it also substantially boosted TEI-mediated off-loading of stalled upstream intermediates. In principle, such stalling could result from a slow rate of chain extension in the now hybrid acceptor module (for example, in the full KS swap construct, KS 14 and ACP 21 are completely mismatched for chain extension). To evaluate this idea, we modified ACP 21 within ATCC/OE484/hy59_S1 incorporating the full-length KS 14 , targeting a sequence region previously identified as mediating intramodular communication between the KS and ACP during chain extension ( Supplementary Fig. 11 ) 19 , 26 . Specifically, we exchanged loop 1 and the initial portion of helix αII of ACP 21 for the corresponding sequence of ACP 14 , using CRISPR-Cas9 ( Supplementary Fig. 11 ). As we anticipated that creation of this substantially hybrid ACP might engender structural perturbation, we also engineered a minimal mutant of ACP 21 in which only one of the two most critical residues in the recognition motif was mutated to the corresponding amino acid in ACP 14 (G 1499 of Pks9 ◊ D; the second residue, R, of the motif is already common to the two ACPs) ( Supplementary Fig. 10 ). Analysis of the loop/helix αII swap by HPLC-MS showed that all mini-stambomycin production had been abolished ( Supplementary Fig. 10 ), consistent with the anticipated disruption to ACP 14 structure. Production by the ACP site-directed mutant was not any better than by the full KS swap construct (Fig. 4 and Supplementary Fig. 10 ), as only metabolite 11 remained above the limits of detection. In principle, the hybrid KS 14 /KS 21 domain may have worked better than KS 14 for chain extension due to improved interaction with ACP 21 , with stalling displaced to later modules. If this were the case, we might expect to see accumulation in the medium of shunt metabolites corresponding to the intermediate generated by module 21. Indeed, in the case of strain hy59_S2 (chimeric KS 14 /KS 21 ) but not hy59_S1 (KS 14 ), we detected masses consistent with the A/B and C/D forms of intermediate 14 generated by module 21, at yields comparable to those of the final mini-stambomycins (Fig. 4 , Supplementary Fig. 10 ) (and correspondingly, 15 , the dehydroxylated analogue of 14 , was detected in the SamR0479 mutant ( Supplementary Fig. 10 )). The same metabolite 14 was identified from the ACP 21 G ◊D mutant (Fig. 4 and Supplementary Fig. 11 ), consistent with interrupted chain transfer to KS 22 . Taken together, these data confirm module 22 as a new blockage point in the engineered systems. Relative efficacy of PKS engineering using PCR-targeting and CRISPR-Cas9. As multiple of our core constructs were generated by both PCR-targeting and CRISPR-Cas9, we were able to directly compare the efficiency of the two techniques (Fig. 3 and Supplementary Fig. 4 ). Globally, our results confirm that both approaches can be employed to introduce large-scale modifications to PKS biosynthetic genes (i.e. deletions of single or multi-gene regions) 32,44−46 . We have also demonstrated, for only the second time to our knowledge, that CRISPR-Cas9 can be leveraged to specifically modify modular PKS domains 47 . Of the two methods, CRISPR-Cas9 was the more rapid, as the corresponding constructs were engineered in approximately half of the time. In addition, while CRISPR-Cas9 allowed for direct modification of the host genome, PCR-targeting relied on the availability of suitable cosmids housing the target genes, and resulted in a 33 bp attB -like ‘scar’ sequence in the genome ( Supplementary Fig. 4 ) 48 . In addition to hampering iterative use of the approach, the scar apparently provoked a moderate reduction in stambomycin yields in mutant K7N6 compared to the wild type, an effect also noted upon comparison of several analogous mutant strains (e.g. K7N4 vs. CPN4, Fig. 3 ). Nonetheless, we did encounter certain difficulties with use of CRISPR-Cas9 (i.e. failure to obtain construct CPN3, occasional reversions to wild type, etc.), observations motivating ongoing efforts in other laboratories to further enhance the suitability of CRISPR-Cas9 for editing PKS pathways 47,49−54 . Discussion In this work, we have utilized an approach based on the state-of-the-art in PKS engineering to modify the stambomycin PKS (Fig. 5 ). Specifically, we aimed to remove the four PKS subunits between Pks4 and Pks9 in the assembly line which together house seven chain extension modules, to generate a series of 37-membered ‘mini-stambomycins’. While in principle such a change might have been possible by directly fusing Pks4 and Pks9 via a suitable intermodular linker, this approach would have resulted in a heptamodular subunit whose size is far in excess of the tetramodular multienzymes present in the system. We have also demonstrated recently the low efficacy of this strategy when the module downstream of the linker is N-terminal in its native subunit context (as with module 21 of Pks9) 18 . As an initial approach (Fig. 5 ), we modified C DD 4 to render it compatible with N DD 9 , with the aim of inducing productive communication between Pks4 and Pks9, while leaving all modular units intact. This modified PKS relied for function on both a non-native chain transfer interface (ACP 13 /KS 21 ), and the intrinsic tolerance of the downstream KS/modules to the incoming substrate. We were optimistic this experiment might work given the structural similarities between the native substrates of KS 14 and KS 21 at least directly adjacent to the acyl terminus, as well as the fact that the stambomycin PKS generates a small family of metabolites, and therefore must exhibit some intrinsic tolerance to structural variation. Although we showed in vitro with recombinant DD pairs that a docking helix-swapped mutant of C DD 4 communicated effectively with N DD 9 , chain transfer across the engineered interface did not occur in vivo , as evidenced by the accumulation of multiple shunt products. While our attempt to render the ACP 13 /KS 21 junction more native by site-directed mutagenesis did result in certain target metabolites, the most significant effect was to increase the yields of the truncated chains. Having narrowed down the biosynthetic block to events occurring downstream of the engineered junction, we next carried out interface engineering based on proposed alternative module boundaries, leveraging fusion points within the KS domain (Fig. 5 ). In this case, sites were selected to either maintain essentially the whole of KS 14 , or to create a hybrid KS 14 /KS 21 domain. This strategy at once preserved key elements of the ACP 13 /KS 14 chain transfer junction, and in the case of the almost full-length KS 14 , ensured that the domain had the appropriate substrate specificity for the incoming chain. Interestingly, the construct incorporating the chimeric KS functioned best, producing the desired mini-stambomycins in both linear and macrocyclic forms. These data identify this location in the middle of the KS 43 as a potentially general fusion site, perhaps because it preserves elements of both the chain transfer and chain extension interfaces with the two partner ACP domains (ACP 13 and ACP 21 , in this case). Intriguingly, a site within the analogous condensation (C) domains of modular nonribosomal peptide synthetase (NRPS) systems, has also recently emerged as a useful fusion point for generating engineered hybrids 55 . Our results also showcase the intrinsically high tolerance of the Pks9 TEI domain towards shorter substrates. Indeed, the data also demonstrate that this TEI domain participates in off-loading the shunt metabolites from the upstream subunit, and that this activity interferes with passage of the chain to subsequent modules. Unfortunately, our attempts to boost yields of the mini-stambomycins by engineering the condensation interface between KS 14 and ACP 21 were unsuccessful, both when the full ACP 21 recognition loop/helix αII region was swapped for that of ACP 14 , and when a single site-directed mutation was made at a putatively critical position (Fig. 5 ). This result is surprising in light of the beneficial effects reported in vitro of both of these modifications on chain extension carried out by mis-matched KS and ACP domains sourced from the erythromycin PKS (DEBS) 26 . Apparently, the introduced changes were not sufficient to ensure effective communication between the KS 14 and ACP 21 domains (or were in fact deleterious to function), and/or any benefit was masked by the poor tolerance of the downstream modules to the modified intermediates. To fully judge the efficacy of this work, it is instructive to compare it to the other two examples in the literature in which full biosynthetic systems have been re-engineered to remove multiple internal modules 10 . In the first, recently-reported case, the neoaureothin (Nor) hexamodular PKS was ‘morphed’ into the evolutionarily-related aureothin (Aur) tetramodular PKS by removing the second bimodular subunit, NorA′. As in our work, the authors initially attempted to engineer a new interaction between the monomodular subunits NorA and NorB flanking NorA′ using compatible docking domains, by exchanging the type 1b N DD of NorB for the type 1a N DD of NorA′ (the natural partner of NorA). When the target metabolite was not obtained, they relocated the fusion site to the KS-AT linker downstream of the conserved KS region in NorB, thereby maintaining the native NorA ACP- C DD/ N DD-KS NorA′ junction. Ultimately, several linker variants had to be evaluated before a functional sequence was identified, in part by serendipity (indeed it is 1 residue longer than the native linker). Overall, the yields of the targeted chain-shortened metabolites were reduced approximately 18-fold compared to the parental neoaureothin (to ca. 2.5 mg L − 1 ), a much less significant penalty than engendered by our engineering strategy. Presumably, the superior titers obtained in this experiment reflect the much higher intrinsic amenability of the Nor PKS to conversion into an Aur PKS, as the Nor PKS likely evolved from an Aur PKS by subunit insertion 10 . Nevertheless, the newly-created NorA/NorB interface was also only partially functional, as product corresponding to the intermediate generated by iterative action of the upstream subunit NorA was still obtained. The second relevant investigation concerns the accelerated evolution (AE) of the RAPS PKS, based on spontaneous induced homologous recombination between its component modules 43 . As mentioned earlier, several of the resulting systems incorporated intermodular fusion sites essentially at the mid-point of the respective KS domains, and so can be compared to our best performing construct hy59_S2. Notably, yields from the hybrid RAPS PKSs from which either 3 or 6 modules were removed, were reduced by a maximum of 33% relative to that of the parental compound. We propose two explanations for the higher functionality of these systems relative to hy59_S2. First of all, in every case, the module downstream of the newly-formed junctions in the contracted RAPS PKSs was internal to its respective subunit (unlike module 21 of Pks9), consistent with the idea that such modules boast intrinsically broader substrate specificity 18 . Secondly, the KS domains of the RAPS PKS exhibit unusually high mutual sequence identity (85 − 90%) 56 . This means, in effect, that the same KS domain acts on a large variety of substrates of differing length and functionality, contributing to the tolerance of all modules downstream of the fusion site. In contrast, the KSs in the stambomycin PKS have substantially lower sequence identity (62 − 80%), and thus KSs 22 − 24 in hy59_S2 likely represent a specificity barrier to efficient transfer and extension of the modified intermediates. Taken together, this set of results shows that contracting PKS systems represents a viable approach to accessing truncated polyketide derivatives of variable length, including macrocycles. Whether such systems are generated rationally or using an AE process, the most efficient hybrids will likely result: i) from PKSs whose modules (and in particular KS domains) exhibit a substantial degree of mutual sequence identity and thus intrinsically high substrate tolerance (or which can be adapted by mutagenesis to broaden their specificity 29 ); and, ii) when novel junctions are created with downstream modules which are situated at internal positions within their subunits. The data also reinforce the idea that in cases where communication at modified interfaces occurs via non-covalent protein-protein interactions, at least a portion of the KS downstream from the docking domains should be included to boost efficiency 10 , 11 , 16 . Finally, our work has identified an increase in TEI-mediated proof-reading provoked by such interface engineering. Elucidating the mechanism underlying this unexpected intersubunit release activity, and thus how to effectively suppress it, should be a profitable avenue for further boosting product titers. Methods Bioinformatics analysis. To underpin the interface engineering strategy, the extremities of all the stambomycin PKS subunits were analyzed to identify the boundaries of the most C-terminal and N-terminal function domains (ACP and KS, respectively), and thus the regions potentially containing docking domains (DDs). The resulting sequences were compared by multiple sequence alignment using Clustal Omega ( https://www.ebi.ac.uk/Tools/msa/clustalo ) 57 , to bonafide and putative DD sequences from multiple DD classes, including those present at the DEBS 2/DEBS 3 interface (type 1a, PDB ID:1PZQ, 1PZR 22 ) and the PikAIII/PikAIV junction (type 1b, PDB ID: 3F5H 2 7 ), to allow for type classification. To identify suitable boundaries for DD heterologous expression in E. coli , the secondary structure of the putative DD regions was predicted using PSIPRED 4.0 ( http://bioinf.cs.ucl.ac.uk/psipred/ ) 37 . Analysis for potential specificity-conferring residues in the stambomycin PKS ketosynthase (KS) domains was carried out by multiple sequence alignment against model KS domains 14 , 20 , 29 , using Clustal Omega 57 . General methods. All reagents and chemicals were obtained from Sigma-Aldrich, except the following: BD (tryptone, yeast extract, TSB powder), Thermo Fisher Scientific (Tris), VWR (glycerol, NaCl, NaNO 3 ), ADM, France (NutriSoy flour), and New England Biolabs (T4 DNA ligase, restriction enzymes). Oligonucleotide primers and two additional synthetic DNA fragments for CPN4 and CPN5 constructs were synthesized by Sigma-Aldrich ( Supplementary Table 2 ). The docking domains N DD 9 Val and N DD 9 Met Supplementary Table 1 ) were obtained as synthetic peptides from GeneCust. DNA sequencing of PCR products was performed by Sigma-Aldrich and Eurofins. PCR reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific) or Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) when higher fidelity was required. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin® Gel and PCR Cleanup or NucleoSpin® Plasmid DNA kits (Macherey Nagel, Hoerdt, France). Strains and media. E. coli BL21 strains were obtained from Novagen. Unless otherwise specified, all E. coli strains were cultured in LB medium (yeast extract 10 g, tryptone 5 g, NaCl 10 g, distilled water up to 1 L, pH 7.0) 58 or on LB agar plates (LB medium supplemented with 20 g L − 1 agar) at 37°C. Streptomyces ambofaciens ATCC23877 and the derived mutants were grown in TSB (TSB powder 30 g (tryptone 17 g, soy 3 g, NaCl 5 g, K 2 HPO 4 2.5 g, glucose 2.5 g), distilled water up to 1 L, pH 7.3) or on TSA plates (TSB medium supplemented with 20 g L − 1 agar), and sporulated on SFM 59 agar plates (NutriSoy flour 20 g, D-mannitol 20 g, agar 20 g, tap water up to 1 L) at 30°C. All strains were maintained in 20% ( v/v) glycerol in 2 mL Eppendorf tubes and stored at −80°C. For fermentation of S. ambofaciens ATCC23877 and its mutants, spores were streaked on TSA with appropriate antibiotics and after incubation 48 h at 30°C, a loop of mycelium was used to inoculate 7 ml of MP5 medium (yeast extract 7 g, NaCl 5 g, NaNO 3 1 g, glycerol 36 mL, MOPS 20.9 g, distilled water up to 1 L, pH 7.4) supplemented with selective antibiotics and sterile glass beads, followed by incubation at 30°C and 200 rpm for 24 − 48 h. Finally, the seed culture was centrifuged and resuspended into 2 mL fresh MP5 before being inoculated into 50 mL MP5 medium in a 250 mL Erlenmeyer flask, and cultivated at 200 rpm and 30°C for 4 days. PCR-targeting-based genetic engineering (exemplified by mutant ATCC/OE484/K7N1, Supplementary Fig. 4 ). To render the BAC BAA9ZA8 proficient for selection following conjugation, its chloramphenicol resistance gene was replaced using a PCR-targeting approach, by a kanamycin resistance gene cassette sourced from pIJ776 31 , resulting in BAC1 ( Supplementary Table 3 ). The cassette attL + aac(3)IV + oriT + attR was amplified from the plasmid pSPM88T 60 ( Supplementary Fig. 4 ) using primers 9996 and 9997 ( Supplementary Table 2 ), affording PCR amplicon PCR-K7N1. The PCR fragment was then electro-transformed into E. coli BW25113/pKD20/BAC1 61 , giving rise to mutant BAC1_K7N1 (in which the C-terminus of pks4 was modified and the genes pks5 − pks8 , were deleted) 31 . The BAC1_K7N1 was then introduced into E. coli ET12567/pUZ8002 62 and then transferred to S. ambofaciens wild type via intergeneric conjugation. The resulting exconjugants (ATCC/K7N1_ aac(3)IV + oriT ) were selected for their apramycin resistance and kanamycin sensitivity (i.e. a phenotype consistent with successful double cross-over). The correct mutations were confirmed by PCR and sequencing. Subsequently, the disruption cassette was excised using the excisionase and integrase of pSAM2 encoded by pOSK111 as described 60 , leaving a 33 bp ‘scar’ sequence (mutant ATCC/K7N1). Successful removal of the cassette was verified by PCR and DNA sequencing Finally, the LAL regulator overexpression plasmid pOE484 23 or the parental vector pIB139 33 was introduced into the strain giving rise to mutants K7N1/OE484 and K7N1/pIB139, respectively. The same overall procedure was applied to construct mutants K7N2/OE483, K7N2/pIB139, K7N3/OE483, K7N3/pIB139, K7N4/OE483, K7N4/pIB139, K7N5/OE483, K7N5/pIB139, K7N6/OE483 and K7N6/pIB139. An analogous PCR-targeting approach ( Supplementary Fig. 4 ) was also employed to inactivate samR0478 and samR0479 using appropriate BACS 34 ( Supplementary Table 3 ). CRISPR-Cas9-mediated genetic engineering. Plasmids pCRISPomyces-2 (and associated cloning and screen protocols) 32 (used for construction of all mutants except ATCC/hy59_S1 and ATCC/hy59_S2) and pCRISPR-Cas9 45 , respectively, were used for CRISPR-Cas9-based genome editing, as described previously. The two systems differ in the way in which Cas9 is expressed; in the case of pCRISPomyces-2, the nuclease is expressed constitutively, while in the pCRISPR-Cas9 system, its expression is under inductive control by thiostrepton (Tsr). The crRNA sequence was selected to match the DNA segment which contains NGG on its 3’ end (N is any nucleotide, and the NGG corresponds to the protospacer-adjacent motif (PAM). The annealed crRNA fragment and two homologous arms (HAL and HAR, flanking the target region) were sequentially inserted into the delivery plasmid pCRISPomyces-2 using the respective restriction sites Bbs I and Xba I, to afford the specific recombinant plasmid for each mutant ( Supplementary Fig. 4 ). Correspondingly, an sgRNA cassette (tracrRNA + sgRNA) and two homologous arms were inserted into the plasmid pCRIPR-Cas9 using sites Nco I, SnaB I and Stu I, respectively ( Supplementary Fig. 10 ). In addition, the crRNA was designed to be located within the region to be deleted ( Supplementary Fig. 4 ) to avoid Cas9-catalyzed cleavage occurring in the genome of the resulting mutant. In the case of site-directed mutants, additional DNA fragments containing the targeted mutations were inserted between the two homologous arms. In addition, the DNA sequence with the fragments identical to the crRNA was modified, so as to avoid subsequent Cas9-catalyzed cleavage of the obtained mutants ( Supplementary Figs. 9 and 11 ). Overexpression and purification of docking domains. The wild-type docking domains ( C DD 4 , N DD 5 , C DD 8 , N DD 9 Val and N DD 9 Met) and mutant docking domains ( C DD 4 SDM, C DD 4 helix swap) were amplified from genomic DNA of S. ambofaciens wild type and the relevant mutants, using forward and reverse primers incorporating Bam HI and Hin dIII restriction sites, respectively ( Supplementary Table 2 ). The PCR amplicons were digested using FD Bam HI and FD Hin dIII, and then ligated into the equivalent sites of vector pBG-102 (Center for Structural Biology, Vanderbilt University). In the case of all C DDs which lacked aromatic residues, a tyrosine residue (codon TAT incorporated in the forward primer, Supplementary Table 1 ) was added at the N-terminal ends (so as not to interfere with docking with the N DD partner) to allow efficient monitoring by UV-Vis during the purification, as well as reliable measurement of protein concentration necessary for binding studies by ITC. The resulting constructs pBG102- N DD 5 , pBG102- C DD 8 and pBG102- N DD 9 were used to transform E. coli BL21 (DE3). For C DD 4 and its mutants, these were transformed into Rosetta™ 2(DE3), as these constructs contain 8 codons rarely used in E. coli . Positive transformants were selected on LB agar supplemented with kanamycin (50 µg mL − 1 ) (25 µg mL − 1 chloramphenicol was also added for expression in Rosetta™ 2(DE3)). A single colony was transferred to LB (10 mL) supplemented with antibiotics, and the culture grown at 37°C and 200 rpm for overnight. The 1 mL overnight culture was used to inoculate LB media (1 L) supplemented with appropriate antibiotics, and then incubated at 37°C and 200 rpm to an optical density of 0.8, at which point protein synthesis was induced by the addition of IPTG (final concentration 0.1 mM). After incubation at 18°C and 200 rpm for 18 h, cells were collected by centrifugation at 8000 g for 30 min, resuspended in 40 mL protein purification buffer A (50 mM Tris-HCl, 400 mM NaCl, 10 mM imidazole, pH 8.0), and lysed by sonication. Following centrifugation at 20000 g and filtration using a 0.45 µm membrane, the soluble cell lysates were loaded onto 2 ⋅ 5 mL HisTrap HP (GE Healthcare) columns (two 5 mL columns in series) equilibrated in buffer A, and purified by preparative protein purification chromatography using an ÄKTA Avant system. The following program was applied: sample loading, 1 mL min − 1 ; washing, 2 mL min − 1 , 10 column volumes of buffer A; elution, 2 mL min − 1 , 5 column volumes of buffer B (50 mM Tris-HCl, 400 mM NaCl, 250 mM imidazole, pH 8.0); elution, 2 mL min − 1 , 2 column volumes of buffer C (50 mM Tris-HCl, 400 mM NaCl, 500 mM imidazole, pH 8.0). All His 6 -SUMO-tagged proteins were collected (fractions containing the protein of interest were selected based on the UV chromatography and SDS-PAGE gel), and transferred into dialysis bag containing His 6 -tagged human rhinovirus 3C protease (H3C) (1 − 2 µM). The dialysis bag was then placed into a container filled with buffer D (50 mM Tris-HCl, 400 mM NaCl, pH 8.0), and the cleavage allowed to proceed at 4°C overnight. The resulting proteins, which incorporated a non-native N-terminal GPGS sequence, were then separated from the remaining His 6 -tagged SUMO and His 6 -tagged human rhinovirus 3C protease by re-loading onto the 2 ⋅ 5 mL HisTrap HP columns pre-equilibrated in buffer A. Purification was then carried out with the following program: sample loading, 1 mL min − 1 ; washing, 2 mL min − 1 , 4 column volumes of buffer A; elution, 2 mL min − 1 , 2 column volumes of buffer B; elution, 2 mL min − 1 , 2 column volumes of buffer C. The untagged docking domains passed through the column during the washing step, and were collected and concentrated to 5 − 7 mL using an Amicon Ultra 3000 MWCO centrifuge filter (Millipore Corp). Subsequently, the concentrated docking domains were loaded onto a size exclusion chromatography column (Superdex 75 26/60 column, GE Healthcare) equilibrated in buffer GF (20 mM HEPES, 100 mM NaCl, 0.5 mM TCEP, pH 7.5). Following a concentration step, the purity of the purified proteins was verified by SDS-PAGE, and their concentrations were determined by NanoDrop (Thermo Scientific) with extinction coefficients calculated using the ExPASy ProtParam tool 63 . Isothermal Titration Calorimetry measurements. ITC measurements were performed at 20°C in buffer GF using a MicroCal ITC200 (Malvern Instruments) (A2F Plateforme ASIA: Approches fonctionnelles et Structurales des InterActions cellulaires). A 300 µL aliquot of N DD 5 at 70 µM was placed in the calorimeter cell and titrated with 700 µM of the C DD 4 s ( C DD 4 wild type, C DD 4 SDM and C DD 4 helix swap) in the syringe. In the case of the binding experiments between N DD 9 Met and C DD 8 , the C DD 8 (700 µM) was added to the N DD 9 Met (80 µM in the cell), while for the binding between N DD 9 Val and the C DDs ( C DD 8 , C DD 4 wild type, C DD 4 SDM and C DD 4 helix swap), the C DDs (700 µM) were added to N DD 9 Val in the cell (120 µM). The ITC experiments were then carried out as followed: initial waiting time 120 s, initial injection of 0.5 µL over 1 s followed by 19 serial injections of 2 µL over 4 s, separated by an interval of 120 s. For each experiment, the reference power was set to 5 µcal − 1 , stirring speed to 750 rpm, and the high feedback mode was selected. Two independent titrations were performed for each combination of DDs. The heat of reaction per injection (µcal s − 1 ) was determined by integration of the peak areas using the Origin 7.0 (OriginLab) software, assuming a one-site binding model (consistent with the solved structures of the types of DDs 22 , 27 ), yielding the best-fit values for the heat of binding (ΔH), the stoichiometry of binding (N) and the dissociation constant (K d ). The heats of dilution of the DDs were determined by injecting them into the cell containing buffer only, and these were subtracted from the corresponding binding data prior to curve fitting. In some cases, when a plateau (binding saturation) was not reached at the final titration step, and the problem could not be solved by increasing the concentration of DD in syringe, we initially placed C DD/ N DD complex in the ITC cell (at the concentration of the two partners reached in the previous titration), filled the syringe with additional DD, and performed a second titration experiment. This procedure was then repeated until binding saturation was reached. To fit the data, the MicroCal ITC concatenation software was used to combine two ITC data files together. Most importantly, the critical parameter dimensionless constant (C-value) was calculated as follows: C = NK a [M] T where K a is the binding constant, [M] T is the total macromolecular concentration in the cell, and N is the stoichiometry of interaction. A reliable ITC binding isotherm is evidenced by ITC data with C-values > 1 (the optimal range is 5 < C < 500) 64 , as was the case for all of our measurements. Circular Dichroism measurements. CD spectra were recorded on a Chirascan CD (Applied Photophysics, United Kingdom) (IBS-Lor UMS 2008 Plateforme de Biophysique et Biologie Structurale) at 0.5 nm intervals in the wavelength range of 180 − 260 nm at 20°C, using a temperature-controlled chamber. A 0.01 cm quartz cuvette containing 30 µL of docking domain at 100 µM, a 0.1 cm cuvette with 200 µL of sample at 10 µM, and a 1 cm cuvette containing 1.5 mL of sample at 1 µM, were used for all the measurements. All measurements were performed at least in triplicate, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone. The CD spectra were deconvoluted using the deconvolution software CDNN2.1 65 to estimate the secondary structure present in the docking domains. SEC-MALS analysis of docking domains. The oligomeric state of all the docking domains was determined by size-exclusion chromatography multi-angle light scattering (SEC-MALS) on the A2F Plateforme ASIA. For this, SEC was first carried out on a Superdex75 10/300 column (GE Healthcare) at 20°C using a flow rate of 0.5 mL min − 1 in HEPES buffer (20 mM HEPES, 100 mM NaCl, 0.5mM TCEP, pH 7.5) using a ÄKTA-Purifier FPLC (GE Healthcare). Multi-angle light scattering (MALS) was measured using a MiniDAWN TREOS II (Wyatt Technology), while refractometry was monitored using an Optilab T-rEX (Wyatt Technology). Data processing was carried out with the manufacturer supplied software (ASTRA 6.1, Wyatt Technology) to determine the protein oligomerization state. HPLC-MS analysis of fermentation metabolites and purified docking domains. The fermentation broth of Streptomyces was centrifuged at 4000 g for 10 min. As described previously 23 , the stambomycins and their derivatives were then extracted from the mycelia, by first resuspending the cells in 40 mL distilled water, followed by centrifugation (4000 g , 10 min, repeated 3×) to remove water-soluble components. After decanting the water, the cell pellets were weighed and extracted with methanol by shaking at 150 rpm for 2 h at room temperature. Thereafter, the methanol extracts were filtered to remove the cell debris, followed by rotary evaporation to dryness. The obtained extracts were then dissolved in methanol, whose volume was determined according to the initial weight of the mycelia (70 µL methanol to 1 g of initial cell pellet). The resulting mycelial crude extracts were then passed through a 0.4 µm syringe filter and analyzed in positive electrospray mode (ESI + ) by HPLC-HRMS (Thermo Scientific Orbitrap LTQ XL or an Orbitrap ID-X Tribrid Mass Spectrometer) (Plateau d’Analyse Structurale et Métabolomique (PASM)-SF4242 EFABA) using an Alltima™ C18 column (2.1 ⋅ 150mm, 5 µm particle size). Separation was carried out with Milli-Q water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) using the following elution profile: 0 − 48 min, linear gradient 5 − 95% solvent B; 48 − 54 min, constant 95% solvent B; 54 − 60 min, constant 5% solvent B. Mass spectrometry operating parameters were: spray voltage, 5 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 30, 10 and 10 arbitrary units min − 1 ; capillary temperature, 275°C; capillary voltage, 4 V; tube lens, split lens and front lens voltages 155 V, −28 V and −6 V, respectively. Due to the much lower sensitivity of the Orbitrap LTQ XL relative to the Orbitrap ID-X Tribrid as evidenced by comparative analysis of identical samples on the two instruments, we introduced a 10× correction factor to the the yields determined using the Orbitrap LTQ XL ( Supplementary Table 5 ). The purified docking domains in buffer GF were diluted with Milli-Q water to a concentration of 50 µM and injected onto an Alltima™ C18 column (2.1 ⋅ 150mm, 5 µm particle size). Analysis was carried out with Milli-Q water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B), using the elution profile: 0 − 15 min, constant 10% solvent B; 15 − 20 min, linear gradient of 10% solvent B to 95%; 20 − 25 min, constant 10% solvent B. Mass spectrometry operating parameters were set as above. Metabolite profiling of engineered strains. Comparative analysis of fermentation extracts (of all strains containing pOE484 except K7N3, CPN4 and CPN5, relative to control mutant containing empty plasmid pIB139) was conducted on a Dionex Ultimate 3000 HPLC system coupled to a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer. MS-settings: spray voltage 3.5 kV; capillary temperature 320°C; sheath gas (52.50), auxiliary gas (13.75), sweep gas (2.75); probe heater 437.50°C; S-Lens RF (50), positive mode, resolution 70.000; AGC target 1e6, microscans 1, maximum IT 75 ms, scan range 200 − 1800 m / z . Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 µm XB-C18 150 × 4.6 mm column with solvents (A, H 2 O + 0.1% formic acid) and (B, MeCN + 0.1% formic acid) and the following gradient: flow rate 0.7 mL min − 1 , 20% B for 2 min, 20–98% B over 18 min, 98% B for 5 min, 98–20% B in 0.5 min and 20% B for 4 min. Metabolic differences within the obtained data ( Supplementary Table 6 ) were identified using SIEVE 2.0 screening software, applying the default settings for component extraction of small molecules, except that of the base peak minimum intensity, which was set to 5000000. Quantification of metabolites. To quantify the yields of native stambomycins and the newly-generated derivatives by HPLC-MS, we generated a calibration curve with previously-purified stambomycin A/B 1 as the standard (using a concentration range between 1 µg L − 1 and 50 mg L − 1 ). This approach yielded a linear correlation between the quantity of 1 and the respective peak area in the extracted ion chromatogram (EIC) (the areas of all peaks corresponding to the parental ions were used) ( Supplementary Fig. 12, Supplementary Table 5 ). The titers of all stambomycin derivatives were then determined using this calibration curve, based on the peak areas corresponding to the parental ions in their respective EIC chromatograms ( Supplementary Table 5 ). As the limit of detection in these experiments lay between 1 and 10 µg L − 1 , yields in this range must be viewed as estimates. Declarations Acknowledgements We acknowledge financial support from the Agence Nationale de la Recherche (grant number ANR-16-CE92-0006-01, PKS STRUCTURE to K.J.W.), the Université de Lorraine, the Centre National de la Recherche Scientifique (CNRS), and the IMPACT Biomolecules project of the Lorraine Université d’Excellence (Investissements d’avenir - ANR 15-004). J.P. acknowledges funding by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 742739). S. Collin and B. Chagot are thanked for help with the analysis of the DDs in vitro , A. Kriznik for assistance with the biophysical experiments, J. Grosjean for help with the HPLC-MS analysis and S. Rousselot for contributing to developing the genetic engineering strategy. Author contributions L.S. constructed plasmids, generated and fermented recombinant strains, analyzed and interpreted HPLC-MS data, expressed and purified recombinant docking domains, carried out biophysical analysis of DD interactions, and generated all of the manuscript figures. L.H. and C.J. constructed certain plasmids, mutant BACs and/or mutant strains. C.P. performed HPLC analyses. A.B. and J.P. carried out HPLC-MS analyses and metabolic profiling. B.A., C.J. and K.J.W. designed the research and supervised the project. K.J.W. performed in silico analyses and wrote the manuscript, with contributions from all authors. Additional information Supplementary Information accompanies this paper at https://doi.org/ Competing interests: The authors declare no competing interests. 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Biol. 14 , 898–908 (2007). Table 1 Ta ble 1 Summary of various strains generated, as well as the novel metabolites detected. Strain Modifications introduced Stambomycins 1 Novel metabolites ATCC/OE484 Wild type ü n.d. K7N6/OE484 a 33 bp scar c ü n.d. K7N5/OE484 C DD 4 helix swap, 33 bp scar û 2 , 3 , 4 , 5 CPN5/OE484 b C DD 4 helix swap ü n.d. K7N4/OE484 C DD 4 site-directed mutagenesis (SDM), 33 bp scar ü n.d. CPN4/OE484 C DD 4 SDM ü n.d. K7N3/OE484 D pks5−8, 33 bp scar û 2 , 3 , 4 , 5 K7N2/OE484 C DD 4 helix swap, D pks5−8, 33 bp scar û 2 , 3 , 4 , 5 CPN2/OE484 C DD 4 helix swap, D pks5−8 û 2 , 3 , 4 , 5 K7N1/OE484 C DD 4 SDM, D pks5−8, 33 bp scar û 2 , 3 , 4 , 5 CPN1/OE484 C DD 4 SDM, D pks5−8 û 2 , 3 , 4 , 5 ATCC/OE484/Pks4+TEI TEI fused to Pks4 û 2 , 3 , 4 , 5 CPN2/OE484/TEI SDM TEI inactivation (Ser à Ala), C DD 4 helix swap, D pks5−8 û 2 , 3 , 4 , 5 CPN2/OE484/TEII SDM TEII inactivation (Ser à Ala), C DD 4 helix swap, D pks5−8 û 2 , 3 , 4 , 5 CPN2/OE484/ D 478 D samR0478, C DD 4 helix swap, D pks5−8 û 2 , 3 , 4 , 5 CPN2/OE484/ D 479 D samR0479, C DD 4 helix swap, D pks5−8 û 6 , 7 , 8 , 9 CPN2/OE484/ACP 13 SDM ACP 13 H1 modified d , C DD 4 helix swap, D pks5−8 û 2 , 3 , 4 , 5 , 11 ATCC/OE484/hy59_S1 N DD 9 +KS 21 replaced by N DD 5 +KS 14 , D pks5−8 û 2 , 3 , 4 , 5 , 10 , 11 , 12 ATCC/OE484/hy59_S2 N DD 9 +KS 21 replaced by N DD 5 +KS 14/21 , D pks5−8 û 2 , 3 , 4 , 5 , 10 , 11 , 12 , 14 ATCC/OE484/hy59_S1/ D 479 D samR0479 , N DD 9 +KS 21 replaced by N DD 5 +KS 14 , D pks5−8 û 6 , 7 , 8 , 9 , 13 ATCC/OE484/hy59_S2/ D 479 D samR0479 , N DD 9 +KS 21 replaced by N DD 5 +KS 14/21 , D pks5−8 û 6 , 7 , 8 , 9 , 13 , 15 ATCC/OE484/hy59_S1/ACP 21 region swap ACP 21 L1+H2 modified e , N DD 9 +KS 21 replaced by N DD 5 +KS 14 , D pks5−8 û 2 , 3 , 4 , 5 ATCC/OE484/hy59_S1/ACP 21 GtoD ACP 21 L1 modified f , N DD 9 +KS 21 replaced by N DD 5 +KS 14 , D pks5−8 û 2 , 3 , 4 , 5 , 11 , 14 a K7N (pronounced “cassette number”) refers to mutants generated by PCR-targeting. b CPN refers to mutants generated using CRISPR-Cas9. c Use of PCR-targeting technique introduced a ‘scar’ sequence between pks4 and pks9 ; for details, see Supplementary Fig. 4 . d H1 modified refers to mutation of six residues within the helix α1 region of ACP 13 (EADQRR à PSERRQ); for details, see Supplementary Fig. 9 . e L1+H2 modified refers to exchange of the loop 1+helix α2 region of ACP 21 ; for details, see Supplementary Fig. 11 . f L1 modified indicates that one residue within the loop 1 region of ACP 21 was mutated (G 1499 à D); for details, see Supplementary Fig. 11 . n.d. indicates no novel metabolites were detected. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-222743","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":12391487,"identity":"f47379ae-2e94-43fc-8da7-55f94cd21971","order_by":0,"name":"Li Su","email":"","orcid":"https://orcid.org/0000-0002-7787-684X","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Su","suffix":""},{"id":12391488,"identity":"91552c93-c5ea-4262-a79d-98978d49101a","order_by":1,"name":"Laurence Hotel","email":"","orcid":"","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laurence","middleName":"","lastName":"Hotel","suffix":""},{"id":12391489,"identity":"e1353706-932d-409a-b3f9-ed2e55acac91","order_by":2,"name":"Cédric Paris","email":"","orcid":"","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cédric","middleName":"","lastName":"Paris","suffix":""},{"id":12391490,"identity":"a522066b-05f5-49dc-9921-3c3bb32a62f0","order_by":3,"name":"Alexander Brachmann","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Brachmann","suffix":""},{"id":12391491,"identity":"c6e2f35d-4c26-45bd-9268-9f6492a2197b","order_by":4,"name":"Jörn Piel","email":"","orcid":"","institution":"ETH Zurich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jörn","middleName":"","lastName":"Piel","suffix":""},{"id":12391492,"identity":"75af98bc-a06d-46cd-8a46-92fdff655ef7","order_by":5,"name":"Christophe Jacob","email":"","orcid":"","institution":"Lorraine University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Jacob","suffix":""},{"id":12391493,"identity":"6aa3398a-13eb-404d-b11d-f7a9de419668","order_by":6,"name":"Bertrand Aigle","email":"","orcid":"https://orcid.org/0000-0001-5266-5926","institution":"University of Lorraine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bertrand","middleName":"","lastName":"Aigle","suffix":""},{"id":12391494,"identity":"c1c6fd17-09bd-42ee-97da-b0aa2e6ba04b","order_by":7,"name":"Kira Weissman","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-3012-2960","institution":"University of Lorraine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kira","middleName":"","lastName":"Weissman","suffix":""}],"badges":[],"createdAt":"2021-02-08 12:17:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-222743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-222743/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-022-27955-z","type":"published","date":"2022-01-26T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":6120079,"identity":"1517c866-1158-487c-bbec-eb3ca75a53cc","added_by":"auto","created_at":"2021-02-19 00:56:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":140726,"visible":true,"origin":"","legend":"Stambomycin polyketide synthase (PKS) and structures of stambomycin derivatives produced by S. ambofaciens ATCC23877. a Organization of the stambomycin biosynthetic gene cluster, and schematic of the encoded PKS subunits (Pks1−Pks9) showing the component modules and domains, as well as the intersubunit docking domains. The DDs belong to two distinct structural classes (type 1a and type 1b), for which representative NMR structures of complexes are shown22,27. The AT domain of PKS module 12 (green box) is responsible for recruiting six alternative extender units, resulting in a small family of stambomycins. The last KR domain of module 24 (grey) is inactive. b Structure of stambomycins 1 (A-F), which differ from each other in the alkyl functionality (R group) at position C-26 (the indicated stereochemistries23 have been predicted based on analysis of known domain stereochemical determinants66, but have not been directly confirmed). The sites of glycosylation and hydroxylation are highlighted with their responsible enzymes indicated. Abbreviations: AT, acyl transferase; KS, ketosynthase (KSQ refers to replacement of the active site cysteine residue by glutamine); ACP, acyl carrier protein; DH, dehydratase; ER, enoyl reductase; KR, ketoreductase; TE, thioesterase; CDD, C-terminal docking domain; NDD, N-terminal docking domain. ","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/aaa527d81a678b9c3b1a3599.jpg"},{"id":6120082,"identity":"9c2ebd7f-c5b9-4ce6-b7af-1e7a61659eec","added_by":"auto","created_at":"2021-02-19 00:56:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91034,"visible":true,"origin":"","legend":"A schematic representation of classical and alternative module definitions. A classical module (black) is defined as the catalytical unit responsible for incorporation of one building block into the growing polyketide chain, and associated functional group modifications. CDD/NDD pairs (shown) mediate communication between such traditionally-defined modules. According to this definition, KS/ACP chain extension interactions (left-pointing arrows) occur within the modules, whereas ACP/KS chain transfer interactions (right-pointing arrows) occur between modules, and the incoming substrate for the KS domain is generated by the upstream module. The alternative module boundaries (shown in green) were inspired by the evolutionary co-migration in certain systems of the KSs and the upstream processing domains14,15. Under this revised definition, the KS/ACP chain extension interaction (left-pointing arrows) is inter-modular, while the ACP/KS chain transfer contacts (right-pointing arrows) are intra-modular.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/a000f86358a173eb346d1f91.jpg"},{"id":6120523,"identity":"56759259-1461-492e-99cb-bf378baa48c1","added_by":"auto","created_at":"2021-02-19 01:02:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147399,"visible":true,"origin":"","legend":"Analysis of metabolites derived from PKS engineering based on the classical module definition. a HPLC-PDA analysis at lmax 238 nm of stambomycins 1 from the wild type strain and various mutants. Quantification of all derivatives (Supplementary Table 5) was based on comparison to the yields of the wild type stambomycins 1 as determined using a calibration curve (Supplementary Fig. 13) (30 mg L-1 total yield of stambomycins A/B and C/D, set to 100%). b LC-ESI-HRMS analysis of mutants in which 1 was absent revealed a serious of shunt products (2-5) (the average yields (two measurements) relative to 1 in the wt are indicated). Shown are the extracted ion chromatograms (EICs) of 2-5, using the calculated m/z values shown in Supplementary Fig.5. c LC-ESI-HRMS analysis of several CPN2-derived mutants (the yields of shunt products 2-5 are shown relative to 1 in the wt (average of four measurements)). Notably, the combined yield of 2-5 in mutant ATCC/OE484/Pks4+TEI was 17-fold higher than that from CPN2/OE484. A series of new compounds 6-9 was generated in strain CPN2/OE484 in which the gene samR0479 was deleted. d Chemical structures of shunts 2-9. The structural differences among them are highlighted (green = R group; red = hydroxyl). Shunt products 2, 4, 6 and 8 correspond to stambomycin C/D derivatives, and 3, 5, 7 and 9 to stambomycin A/B derivatives. M12 and M13 indicate shunt compounds released from modules 12 and 13, respectively.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/e667131b18a8ad4a5a2e117f.jpg"},{"id":6120264,"identity":"1247fe9b-1995-4a67-96cb-3849caaa93af","added_by":"auto","created_at":"2021-02-19 00:59:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135839,"visible":true,"origin":"","legend":"Engineering of functional mini-stambomycin PKSs. The various strategies used in each case are represented schematically, along with the obtained products and their maximum yields (estimated for \u003c10 mg L-1) (full analysis of all constructs is provided in Supplementary Table 5). The engineering starting point, CPN2 PKS, contains a functional CDD4 helix swap/NDD9 docking interaction (swapped docking a-helix shown in dark yellow), but a mis-matched ACP13/KS21 interdomain interaction. This PKS yielded only shunt products 2-5. The CPN2/ACP13 SDM PKS, in which the ACP13 helix aI has been modified to match that of ACP20 (dark yellow ball), generates mini-stambomycin derivatives (11, a cyclic form lacking the internal hydroxy, Supplementary Fig. 9). The ATCC/hy59_S1 and S2 constructs were based on the alternative module definition, as the engineering point was selected downstream of the CDD4/NDD5 interface within the KS21 domain. Of the two junctions, that in which the fusion was located essentially at the midpoint of the domain (ATCC/hy59_S2) functioned better than that which included the majority of KS14 (ATCC/hy59_S1), although both PKSs led to successful generation of three novel mini-stambomycins, both as their free acids (10) and in macrolide form (11 and 12) (Supplementary Fig. 10). In an attempt to boost yields from ATCC/hy59_S1, two further constructs were created by modification of ACP21 – either by swapping a region implicated in KS/ACP communication during extension (ATCC/hy59_S1/ACP21 region swap), or by mutating a single key residue within the motif (ATCC/hy59_S1/ACP21 GtoD). The ATCC/hy59_S1/ACP21 region swap yielded only the shunt metabolites 2−5, while solely 11 was detected from ATCC/hy59_S1/ACP21 GtoD, showing that the introduced changes did not work as intended. We observed in addition from ATCC/hy59_S2 PKS and ATCC/hy59_S1/ACP21 GtoD, shunt product 14 corresponding to the chain released from module 21 (Supplementary Figs. 9 and 10), identifying the downstream module 22 as a blockage point.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/b235dc5ad88f45fbf01f1b57.jpg"},{"id":6120262,"identity":"5fc5f0ff-e59e-491b-8f57-c38065fe39c6","added_by":"auto","created_at":"2021-02-19 00:59:20","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179587,"visible":true,"origin":"","legend":"Summary of the engineering strategies applied in this work to the stambomycin PKS. Inset are the six distinct approaches used, and the structures of the resulting metabolites are shown. The strategies giving rise to the target mini-stambomycins 10−12 are indicated in red. The hydroxyl group shown in pink is introduced by the P450-hydroxylase SamR0478, and that in red, by SamR0479.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/fa0b5b64d62dd9434a0fa831.jpg"},{"id":19033667,"identity":"b640ce6c-6d26-4257-83e4-16e7a09a1baa","added_by":"auto","created_at":"2022-03-09 18:06:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1473058,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/99162f36-6354-4e51-ad20-83a67219e5b3.pdf"},{"id":6120084,"identity":"f9bfbc90-3fdd-4450-8483-91bf70c6f9fd","added_by":"auto","created_at":"2021-02-19 00:56:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":25849482,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationSuetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-222743/v1/7c3778b1a6c9efe4f4d0f646.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFor almost thirty years, efforts have been made to leverage the modular genetic architecture of the type I polyketide synthases (PKSs) to generate novel derivatives, typically by modifying individual catalytic domains. Despite enormous progress in establishing domain structure-function relationships\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, such genetic manipulation remains inefficient\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Insight into factors potentially contributing to low product yields was provided by cryo-electron microscopy analysis of a model PKS module at multiple stages of its catalytic cycle\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. This work revealed that interdomain contacts are critical for establishing the various functional states of the module, and that transitions between such states rely on evolving interfaces between the domains, as well as the intervening \u0026lsquo;linker\u0026rsquo; regions. In short, PKS modules appear to be highly integrated units, thus explaining why exchange of catalytic domains for heterologous counterparts is often detrimental\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Collectively, these observations motivate future approaches in which modules or multi-modular subunits are employed as the basic building blocks for engineering the assembly lines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNevertheless, using modules requires a clear definition of their domain composition. Traditionally, modules encompass the three invariable domains required for monomer selection and chain extension (ketosynthase (KS), acyl transferase (AT) and acyl carrier protein (ACP)), as well as any intervening \u0026beta;-keto processing activities (e.g. ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER)) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). However, an alternative definition was recently suggested by the finding that KS domains in certain PKSs co-evolve with the tailoring domains located upstream in the assembly lines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Accordingly, modules begin with the modifying domains and the associated AT, and terminate with the KS that is classically assigned to the downstream module (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Even before a module redefinition was suggested, engineering efforts revealed that maintaining the key ACP\u003csub\u003en\u003c/sub\u003e/KS\u003csub\u003en+1\u003c/sub\u003e interface can, in certain cases, be critical for the function of a hybrid PKS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recently, we have carried out module swapping based on both of these definitions, by covalently tethering heterologous modules to a common donor module within a bimodular mini-PKS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Overall, our data demonstrated that both module definitions led to functional hybrid PKSs, and which boundaries worked best depended on the source module\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Indeed, regardless of which extremities are employed, module exchange results in non-native interdomain interactions (ACP\u003csub\u003en\u003c/sub\u003e/KS\u003csub\u003en+1\u003c/sub\u003e or KS\u003csub\u003en+1\u003c/sub\u003e/ACP\u003csub\u003en+1\u003c/sub\u003e), and in the case of classical module boundaries, potential incompatibilities in terms of KS substrate specificity (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) \u0026ndash; both of which have been shown to reduce activity via detailed studies \u003cem\u003ein vitro\u003c/em\u003e\u003csup\u003e19\u0026thinsp;\u0026minus;\u0026thinsp;21\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this work, we aimed to investigate the generality of these findings for efforts to create non-native intermodular junctions when the modules are located on distinct subunits\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In such cases, the resulting non-covalent interactions are mediated by short sequences at the extreme C- and N-termini of the subunits called docking domains (DDs)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Matched pairs of DDs form specific complexes at intersubunit interfaces, enforcing a strict subunit ordering within the PKS system. As a test case, we aimed to genetically engineer the biosynthesis of substantially smaller derivatives of the stambomycin family of polyketides in \u003cem\u003eStreptomyces ambofaciens\u003c/em\u003e ATCC23877\u003csup\u003e23\u003c/sup\u003e. The stambomycins \u003cstrong\u003e1\u003c/strong\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb) are glycosylated macrolides which show promising anti-cancer activity against multiple human cancer cell lines\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The six characterized family members (A\u0026thinsp;\u0026minus;\u0026thinsp;F) differ in the alkyl functionality at the C-26 position (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) which directly impacts their potency,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e but have in common modification by \u003cem\u003etrans\u003c/em\u003e-acting cytochrome P450 hydroxylases at C-28 and C-50. Notably, at 51-members, the macrolactone ring is among the largest of all known polyketides. Thus, the stambomycins represent an attractive model system for establishing PKS engineering as a means to access structurally-simplified analogues (minimal pharmacophores\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e) for biological evaluation, as a complement to traditional chemical synthesis.\u003c/p\u003e\n\u003cp\u003eHere we report a comprehensive series of experiments aiming to generate 37-membered ring stambomycin analogues, based on both the classical and revised module definitions. The target mini-stambomycins were detected successfully, albeit in low yields, and the identification of the derivatives allowed us to clarify the relative timing of the two cytochrome P450-catalyzed hydroxylation reactions in the pathway. Attempts to boost titers by ACP/KS interface engineering\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e were unsuccessful, but led in certain cases to a surprising increase in the liberation of linear shunt metabolites \u0026ndash; only the second report, to our knowledge, of inter-subunit crosstalk resulting in thioesterase (TE)-mediated chain release. Taken together with recent work by others\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, our data reinforce the idea that in order to boost efficacy, strategies based on modifying PKS intersubunit interfaces must take into account the function of the modules acting downstream from the newly-established junctions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDesign of engineering experiments based on classical modular boundaries.\u003c/strong\u003e The stambomycin PKS comprises 25 modules distributed among 9 polypeptides (Pks1\u0026thinsp;\u0026minus;\u0026thinsp;9)\u003csup\u003e23\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea) (Note: throughout the text, the stambomycin genes have been numbered in accordance with ref. \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e). To access abridged derivatives using the classical module boundaries, we reasoned that we could engineer novel intersubunit interfaces by suitable manipulation of docking domains. Encouragingly, the extreme C- and N-termini of all subunits (with the exception of the N-terminus of Pks1 and the C-terminus of Pks9) contain sequences with convincing homology to previously-identified DDs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (the C-terminal DDs are referred to hereafter as \u003csup\u003eC\u003c/sup\u003eDDs and their partner N-terminal DDs as \u003csup\u003eN\u003c/sup\u003eDDs). By bioinformatics analysis, we were able to confidently assign the DDs acting at 6 of the 8 interfaces to the type \u0026lsquo;1a\u0026rsquo; class\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and the remaining two sets of DDs as type \u0026lsquo;1b\u0026rsquo;\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1\u003c/strong\u003e). In both cases, docking occurs between an \u0026alpha;-helical \u003csup\u003eC\u003c/sup\u003eDD and a coiled-coil formed by the \u003csup\u003eN\u003c/sup\u003eDD, with specificity achieved via strategically-placed charge:charge interactions at the complex interface (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAmong the type 1a junctions, there were notably two which appeared compatible in terms of the translocated substrate: PKSs 3/4\u0026thinsp;+\u0026thinsp;7/8 and Pks 4/5\u0026thinsp;+\u0026thinsp;8/9 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2\u003c/strong\u003e). Specifically, the functional groups at the critical \u0026alpha;- and \u0026beta;- positions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e of the transferred chains are identical at these junctions, and correspondingly, the downstream KSs show similarities across several sequence motifs previously correlated with substrate specificity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;2\u003c/strong\u003e). Targeting such interfaces thus allowed us, at least in principle, to overcome the functional block to the engineered systems represented by poor recognition of the incoming substrate by the directly downstream KS domain\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Ultimately, we targeted a new interface between Pks subunits 4 and 9 for two principle reasons. Firstly, Pks4 is at the origin of the structural variation between the stambomycin family members, and thus we anticipated that maintaining the subunit within the hybrid system would give rise to a corresponding series of truncated analogues, providing important evidence for their identity. Secondly, it was genetically more practical to modify the second set of interfaces due to splitting of the PKS subunits between two loci (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eTo establish the novel junction, we initially modified the \u003csup\u003eC\u003c/sup\u003eDD of Pks4 (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e) to match that of Pks8 (the natural partner of the \u003csup\u003eN\u003c/sup\u003eDD of Pks9 (\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e)), either by site-directed mutagenesis of residues previously identified as key mediators of interaction specificity (construct \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM; \u003cstrong\u003eSupplementary Fig.\u0026nbsp;3\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, or by exchange of the complete \u003csup\u003eC\u003c/sup\u003eDD docking \u0026alpha;-helix of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e for that of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e (construct \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap; \u003cstrong\u003eSupplementary Fig.\u0026nbsp;3\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Modifying the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e specificity \u0026lsquo;code\u0026rsquo; to match that of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e required mutation of 3 residues, while for the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap, the terminal 16 amino acids of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e were exchanged for the corresponding 15 residues of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e). The genetic alterations were carried out in two distinct PKS contexts: (i) in the presence of the intervening subunits 5\u0026thinsp;\u0026minus;\u0026thinsp;8, which allowed for the possibility of competitive interactions between modified Pks4 and both Pks5 and Pks9; and ii) removing the intervening subunits 5\u0026thinsp;\u0026minus;\u0026thinsp;8, thus eliminating competition for binding of Pks4 by Pks5, and of Pks9 by Pks8 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3\u003c/strong\u003e). We further generated a mutant in which Pks subunits 5\u0026thinsp;\u0026minus;\u0026thinsp;8 were deleted but no modification was made to \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e, in order to judge the intrinsic capacity of Pks4 and Pks9 to interact. Furthermore, genetic engineering was carried out in parallel by both PCR-targeting\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and CRISPR-Cas9\u003csup\u003e32\u003c/sup\u003e, in order to directly compare the efficacy of these two approaches, as well as evaluate the effect of the short scar sequence remaining in the chromosome following PCR-targeting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEngineering the stambomycin PKS based on the classical module definition.\u003c/strong\u003e The \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap sequences were introduced in parallel into the \u003cem\u003eS. ambofaciens\u003c/em\u003e genome using PCR targeting and CRISPR-Cas9 (full experimental details are provided in the \u003cstrong\u003eSupplementary Methods\u003c/strong\u003e). As discussed previously, the modifications were made both in the presence of the intervening subunits Pks5\u0026thinsp;\u0026minus;\u0026thinsp;8 and in their absence (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;3\u003c/strong\u003e). As previous work has shown that production from the stambomycin biosynthetic gene cluster requires activation by constitutive overexpression of a pathway-specific LAL (Large ATP-binding regulators of the LuxR family) regulator\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, we additionally introduced the LAL overexpression plasmid (pOE484) into each of the mutants, using the empty parental plasmid (pIB139\u003csup\u003e33\u003c/sup\u003e) as a control. In total, this strategy resulted in 20 targeted strains harboring interface mutants (where K7N refers to PCR targeting and CPN to CRISPR-Cas9 engineering): K7N1/pIB139, K7N1/OE484, K7N2/pIB139, K7N2/OE484, K7N3/pIB139, K7N3/OE484, K7N4/pIB139, K7N4/OE484, K7N5/pIB139, K7N5/OE484, K7N6/pIB139, K7N6/OE484, CPN1/pIB139, CPN1/OE484, CPN2/pIB139, CPN2/OE484, CPN4/pIB139, CPN4/OE484, CPN5/pIB139, CPN5/OE484 (\u003cstrong\u003eTable\u0026nbsp;1, Supplementary Tables\u0026nbsp;2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e4\u003c/strong\u003e; despite extensive efforts the CPN3 mutant strain was not obtained). The principal difference between the K and CPN series of constructs is the presence of a 33 bp \u0026lsquo;scar\u0026rsquo; sequence between the modified \u003cem\u003epks4\u003c/em\u003e and \u003cem\u003epks9\u003c/em\u003e genes (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e). Construct K7N6 was assembled specifically to test the effect of this region, without any further modification to \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e and the intervening \u003cem\u003epks5\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003epks8\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003eWith the exception of K7N3, CPN4 and CPN5, extracts of the engineered mutant strains harboring pOE484 were analyzed by high performance liquid chromatography heated electrospray ionization high-resolution mass spectrometry (HPLC-HESI-HRMS) on a Dionex UItiMate 3000 HPLC coupled to a Q Exactive\u0026trade; Hybrid Quadrupole-Orbitrap\u0026trade; Mass Spectrometer, and compared to extracts of the control strain containing pIB139\u003csup\u003e33\u003c/sup\u003e as well as the wild type \u003cem\u003eS. ambofaciens\u003c/em\u003e containing pOE484, using SIEVE 2.0 screening software. K7N3, CPN4 and CPN5 were analyzed subsequently, and the data inspected manually. Yield quantification was carried out with reference to a calibration curve generated with purified stambomycins A/B \u003cstrong\u003e1\u003c/strong\u003e (the limit of detection was found to be between 10 and 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and so any yields\u0026thinsp;\u0026lt;\u0026thinsp;10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e must be considered an estimate). Novel metabolites not present in the control strains, and for which we obtained reliable exact masses, are listed in \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;5\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eThe first notable result is that the K7N6/OE484 mutant yielded a similar metabolic profile to \u003cem\u003eS. ambofaciens\u003c/em\u003e wt (22\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 73% relative yield (\u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e)), showing that the scar sequence negatively impacted stambomycin production, but not dramatically (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). By contrast, no stambomycins were observed, as anticipated, in all constructs in which Pks5\u0026thinsp;\u0026minus;\u0026thinsp;Pks8 had been removed (K7N1\u0026thinsp;\u0026minus;\u0026thinsp;3; CPN1,2) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Stambomycins \u003cstrong\u003e1\u003c/strong\u003e were present, however, in strains K7N4 and CPN4 harboring \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e site-directed mutations and in the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap strain CPN5, all of which still contained Pks5\u0026thinsp;\u0026minus;\u0026thinsp;Pks8, albeit at reduced amounts relative to the wild type (18%, 23% and 14% of wt, respectively) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e). (Surprisingly, the metabolic profile of K7N5 reproducibly differed from that of CPN5, as no stambomycin-related metabolites were detected (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e)). These data suggested that while the mutations introduced into \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e reduced interaction with \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e, they were not sufficient to disrupt natural chain transfer between Pks4 and Pks5, arguing that DD engineering to alter partner choice should be accompanied by removal of competing intersubunit interactions.\u003c/p\u003e\n\u003cp\u003eWe did not find any evidence in the DD engineering experiments for any of the target 37-membered metabolites (\u003cstrong\u003eSupplementary Figs.\u0026nbsp;3 and 5\u003c/strong\u003e). However, all strains in which stambomycin production was abolished (\u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e) exhibited four new peaks in common (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;5\u003c/strong\u003e) (other peaks corresponding to potentially novel compounds were observed, but none were shared between multiple strains). The determined exact masses and mass spectra (as exemplified by strain CPN2/OE484, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb) correspond to truncated derivatives of stambomycins A/B and C/D respectively, following premature release from modules 13 and 12 of Pks4 (compounds \u003cstrong\u003e2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e5\u003c/strong\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;5\u003c/strong\u003e; ca. 8-fold greater yield of the module 13 products (\u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e)). Further support for the identity of these shunt compounds was obtained by grafting the chain-terminating (type I) thioesterase (TE) domain from the C-terminal end of Pks9 to the C-terminus of Pks4 in order to force chain release at this stage. Indeed, identical compounds were produced, but at 17-fold increased yield relative to CPN2/OE484, consistent with active off-loading of the chains (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;6\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eBased on their masses, both sets of shunt metabolites were hydroxylated on a single carbon, while none were found to bear the \u0026beta;-mycaminose of the mature stambomycins, consistent with the absence of the tetrahydropyran moiety to which it is normally tethered. To determine the location of the hydroxylation and therefore the hydroxylase responsible, we inactivated in mutant CPN2/OE484 the genes \u003cem\u003esamR0478\u003c/em\u003e and \u003cem\u003esamR0479\u003c/em\u003e encoding respectively, the stambomycin C-28 and C-50 cytochrome P450 hydroxylases\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. While extracts of CPN2/OE484/\u0026Delta;478 were unchanged relative to CPN2/OE484 (i.e. the hydroxyl group was still present), the CPN2/OE484/\u0026Delta;479 mutant exhibited four new peaks with masses corresponding to the dehydroxylated shunt products (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;7\u003c/strong\u003e (compounds \u003cstrong\u003e6\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e9\u003c/strong\u003e) and \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e). Taken together, these data show that the unusual on-line modification catalyzed by SamR0479\u003csup\u003e34\u003c/sup\u003e which is necessary for macrocyclization, occurs prior to chain extension by Pks5. While SamR0478 has also been speculated to act during chain assembly\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, hydroxylation evidently occurs downstream of Pks4, at least. The intriguing substrate structural and/or protein-protein recognition features controlling the timing of hydroxylation by these P450 enzymes remain to be elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRole of TE domains in release of the shunt metabolites.\u003c/strong\u003e We attributed the observed shunt metabolites \u003cstrong\u003e2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e5\u003c/strong\u003e to the lack of productive chain translocation between Pks4 and Pks9, causing intermediates to accumulate on ACPs 12 and 13. To evaluate whether these were released by spontaneous hydrolysis or enzymatically, we further investigated the role of Pks9 TEI\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e in chain release, as well as that of SamR0485, a proof-reading type II TE\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e located in the cluster. Both TEs were disabled by site-directed mutagenesis of the active site serines (Ser \u0026loz; Ala) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;6\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eInterestingly, inactivation of both the type I and type II TEs reduced the yields of shunt products \u003cstrong\u003e2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e5\u003c/strong\u003e relative to the parental strain CPN2/OE484 (by 66% and 27%, respectively; average of duplicate experiments) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;6\u003c/strong\u003e and \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e). These data clearly show that premature release of the chains is catalyzed, at least in part, by both TEs in the cluster, although spontaneous liberation also occurs. While type II TEs typically interact with acyl-ACPs \u003cem\u003ein trans\u003c/em\u003e to release blocked chains\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, the effect of the Pks9 TEI is less readily explained. One possibility is that the new productive docking interaction between Pks4 and Pks9 allows Pks9 to adopt an alternative conformation from which the TE can off-load intermediates bound to ACPs 12 and 13 of Pks4 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;6\u003c/strong\u003e). Although this mechanism is reminiscent of that used by the pikromycin PKS to generate both 12- and 14-membered rings\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, the pikromycin TEI is separated from its alternative ACP target by a single module, while Pks9 TEI is located five or four modules downstream from ACPs 12 and 13 in the engineered system, which would seem to necessitate substantial inter-subunit acrobatics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnderstanding the docking domain engineering via studies\u003c/strong\u003e \u003cspan class=\"BoldItalic\"\u003ein vitro\u003c/span\u003e \u003cstrong\u003ewith recombinant domains.\u003c/strong\u003e To better understand the results of the DD engineering, we studied \u003cem\u003ein vitro\u003c/em\u003e the wild type DD pairs (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e/\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e/\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e), as well as binding between the modified versions of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e and wild type \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e. Design of suitable expression constructs in \u003cem\u003eE. coli\u003c/em\u003e (\u003cstrong\u003eSupplementary Tables\u0026nbsp;1\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e3\u003c/strong\u003e) was based on bioinformatics analysis of the C-terminal ends of Pks4 and Pks8, and the N-termini of Pks5 and Pks9, and secondary structure analysis using PSIPRED\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). Overall, we expressed and purified the following proteins in recombinant form from \u003cem\u003eE. coli\u003c/em\u003e: \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e wt, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e, and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8, Supplementary Table\u0026nbsp;4\u003c/strong\u003e). As \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e proved insoluble when expressed in \u003cem\u003eE. coli\u003c/em\u003e, two versions with alternative start sites were obtained as synthetic peptides (Met and Val; \u003cstrong\u003eSupplementary Fig.\u0026nbsp;8, Supplementary Table\u0026nbsp;4\u003c/strong\u003e). Analysis of the individual \u003csup\u003eC\u003c/sup\u003eDDs by circular dichroism (CD) confirmed their expected high \u0026alpha;-helical content (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e wt (100 \u0026micro;M): 58%; \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e wt (100 \u0026micro;M): 49%), and showed no evident effect of the introduced mutations on secondary structure (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). All of the constructs were further confirmed to be homodimeric by size exclusion chromatography-multi-angle light scattering (SEC-MALS) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe two \u003csup\u003eN\u003c/sup\u003eDDs also exhibited \u0026alpha;-helical character, though less pronounced than the \u003csup\u003eC\u003c/sup\u003eDDs (\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e (100 \u0026micro;M): 27%; \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met (100 \u0026micro;M): 21%; \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val (100 \u0026micro;M): 25%), and were monomeric by SEC-MALS (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). The latter result was surprising, as type 1a \u003csup\u003eN\u003c/sup\u003eDDs classically form a homodimeric coiled-coil domain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;1\u003c/strong\u003e), but we recently identified functional, monomeric type 1 \u003csup\u003eN\u003c/sup\u003eDDs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Indeed, we detected binding between the native pairs by isothermal titration calorimetry (ITC), with affinities in the range of those determined previously for matched pairs of DDs\u003csup\u003e27,38\u0026minus;40\u003c/sup\u003e (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e + \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e, K\u003csub\u003ed\u003c/sub\u003e = 14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 \u0026micro;M; \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e + \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met, K\u003csub\u003ed\u003c/sub\u003e = 33\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026micro;M; \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e + \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val, K\u003csub\u003ed\u003c/sub\u003e = 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026micro;M) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e). Thus, while stable homodimerization of the \u003csup\u003eN\u003c/sup\u003eDDs may depend on the presence of a downstream homodimeric KS domain, their monomeric character did not preclude interaction with their \u003csup\u003eC\u003c/sup\u003eDD partners. Based on the higher affinity of the interaction, we could identify the \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val as the physiologically relevant construct. The observed binding stoichiometry (1 homodimeric \u003csup\u003eC\u003c/sup\u003eDD:2 monomeric \u003csup\u003eN\u003c/sup\u003eDDs), is consistent with the known structure of a type 1a complex in which two monomers of each DD are present (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;1\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. As expected, no non-specific interaction was detected between native \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e and \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e, explaining the lack of productive interaction between unmodified subunits Pks4 and Pks9 when the intervening multienzymes are deleted (strain K7N3) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eAnalysis by ITC of binding between \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM or \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap and \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e revealed the complete absence of interaction (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;8\u003c/strong\u003e), and therefore that the introduced modifications were sufficient to disrupt communication between the native pair. Thus, the continued production of stambomycins \u003cstrong\u003e1\u003c/strong\u003e by K7N4, CPN4 and K7N5 harboring Pks5\u0026thinsp;\u0026minus;\u0026thinsp;Pks8 must be due to additional contacts between Pks4 and Pks5 beyond the docking domains, likely including the compatible ACP\u003csub\u003e13\u003c/sub\u003e/KS\u003csub\u003e14\u003c/sub\u003e interface. On the other hand, no interaction was detected between \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM and \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e, showing that this limited number of mutations was inadequate to induce productive contacts. This result is fully in accord with the absence of the expected mini-stambomycin products from these strains (K7N1/CPN1, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). By contrast, the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap exhibited essentially the same binding to \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val as \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e (K\u003csub\u003ed\u003c/sub\u003e = 21.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 \u0026micro;M), demonstrating that exchange of just this helix is sufficient to redirect docking specificity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Thus, inefficient docking is not at the origin of the failure of the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swaps to yield chain-extended products \u003cem\u003ein vivo\u003c/em\u003e (strains K7N2/CPN2, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). We could therefore conclude at this stage that the problem arose from the non-native interface generated between ACP\u003csub\u003e13\u003c/sub\u003e and KS\u003csub\u003e21\u003c/sub\u003e, poor acceptance by KS\u003csub\u003e21\u003c/sub\u003e of the incoming substrate during chain transfer and/or chain extension, and/or low activity towards the modified chain of domains/modules acting downstream.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAttempted optimization of the stambomycin DD mutants.\u003c/strong\u003e We aimed next to improve the novel Pks4/Pks9 intersubunit interface in strain CPN2 (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap\u0026thinsp;+\u0026thinsp;deletion of Pks5\u0026thinsp;\u0026minus;\u0026thinsp;8) by targeting helix \u0026alpha;I of ACP\u003csub\u003e13\u003c/sub\u003e, as the first 10 residues of this helix have been implicated previously in governing the interaction with the downstream KS domain at hybrid junctions\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Notably, multiple sequence alignment of all ACPs in the stambomycin PKS located at intersubunit junctions, revealed a unique sequence for each ACP in the helix \u0026alpha;I region, consistent with a recognition \u0026lsquo;code\u0026rsquo; for the KS partner, and the idea that mismatching these contacts might hamper productive chain transfer (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;9\u003c/strong\u003e). Indeed, as mentioned previously, even when docking is interrupted, contacts between ACP\u003csub\u003e13\u003c/sub\u003e and KS\u003csub\u003e14\u003c/sub\u003e are apparently sufficient to enable chain translocation between Pks4 and Pks5 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). In addition, an analogous strategy of optimizing the ACP\u003csub\u003en\u003c/sub\u003e/KS\u003csub\u003en+1\u003c/sub\u003e chain transfer interface was shown recently to substantially improve interaction between an ACP (JamC) derived from the jamaicamide B biosynthetic pathway, and the first chain extension module of the lipomycin PKS (LipPKS1)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn our case, the first six residues of ACP\u003csub\u003e13\u003c/sub\u003e helix \u0026alpha;I were modified using CRISPR-Cas9 (EADQRR \u0026loz; PSERRQ), so that the full 10-residue recognition sequence matched that of ACP\u003csub\u003e20\u003c/sub\u003e, the natural partner of KS\u003csub\u003e21\u003c/sub\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;9\u003c/strong\u003e). Analysis of extracts of the resulting strain CPN2/OE484/ACP13 SDM by HPLC-MS revealed at best minute amounts (highest yield of 0.5 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of target cyclic mini-stambomycins A/B (\u003cstrong\u003e11\u003c/strong\u003e), lacking the hydroxyl group introduced by SamR0478 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;9\u003c/strong\u003e). Thus, while this experiment finally yielded the first evidence for successful chain transfer between Pks4 and Pks9 followed by subsequent chain extension by Pks9 and TE-catalyzed release, the overall efficiency of the system remained poor. Interestingly, however, the yields of the four shunt metabolites \u003cstrong\u003e2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e5\u003c/strong\u003e were as much as 48-fold higher from the ACP\u003csub\u003e13\u003c/sub\u003e helix swap mutant than from CPN2/OE484, showing that improved interactions between ACP\u003csub\u003e13\u003c/sub\u003e and KS\u003csub\u003e20\u003c/sub\u003e facilitated release of the stalled intermediates from ACPs 12 and 13, presumably via remote action by the TEI domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEngineering mini-stambomycins by maintaining the native ACP\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e13\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003e/KS\u003c/strong\u003e \u003csub\u003e \u003cstrong\u003e14\u003c/strong\u003e \u003c/sub\u003e \u003cstrong\u003ejunction (alternative module definition).\u003c/strong\u003e Cumulatively, the results obtained with the docking domain engineering identified KS\u003csub\u003e21\u003c/sub\u003e as one potential bottleneck in the engineered PKS. Our parallel strategy based on the alternative module definition (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) allowed us to directly test this idea. Specifically, we investigated the effects of preserving the native \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e/\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e pair and either the majority of KS\u003csub\u003e14\u003c/sub\u003e, or a little more than half of the domain, resulting in a KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e hybrid. For this, we used two different splice sites in KS\u003csub\u003e14\u003c/sub\u003e: i) at the end of the domain in a highly-conserved region (GTNAHV) exploited recently to efficiently swap downstream AT domains\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e; and, ii) at a site corresponding to a recombination hot spot identified during induced evolution of the rapamycin (RAPS) PKS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, yielding the KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e chimera (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e). Both of these modifications were introduced into \u003cem\u003eS. ambofaciens\u003c/em\u003e using CRISPR-Cas9, while simultaneously removing Pks5\u0026thinsp;\u0026minus;\u0026thinsp;Pks8, yielding respectively after co-transformation with pOE484 and the control plasmid pIB139, strains ATCC/OE484/hy59_S1, ATCC/pIB139/hy59_S1, ATCC/OE484/hy59_S2, and ATCC/pIB139/hy59_S2.\u003c/p\u003e\n\u003cp\u003eAnalysis of culture extracts revealed the presence in both ATCC/OE484/hy59_S1 and ATCC/OE484/hy59_S2 relative to the controls, of a novel series of 37-membered metabolites (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). The measured masses were consistent with the desired mini-stambomycins either as their free acids or in cyclic form (metabolites \u003cstrong\u003e10\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e12\u003c/strong\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Signals corresponding to the A/B and C/D derivatives of all metabolites were detected, providing important evidence for their identities, as well as both the C-14 hydroxylated \u003cstrong\u003e12\u003c/strong\u003e and non-hydroxylated \u003cstrong\u003e11\u003c/strong\u003e forms of the cyclic mini-stambomycins (C-14 corresponds to C-28 in the parental compounds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e)). It is not surprising that the corresponding E and F forms were not detected, as their yields even from the wild type are much lower than the A\u0026thinsp;\u0026minus;\u0026thinsp;D derivatives (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). The observation of non-hydroxylated \u003cstrong\u003e11\u003c/strong\u003e shows notably that internal hydroxylation by SamR0478 is not an absolute prerequisite for TE-catalysed macrolactonization, and argues that hydroxylation of the mini-stambomycins only takes place on the macrocyclic compound. Although compounds \u003cstrong\u003e11\u003c/strong\u003e and \u003cstrong\u003e12\u003c/strong\u003e incorporate the tetrahydropyran moiety of the parental stambomycins \u003cstrong\u003e1\u003c/strong\u003e which undergoes glycosylation, derivatives bearing \u0026beta;-mycaminose were not observed, presumably due to poor recognition of the overall modified macrocycle by glycosyl transferase SamR0481\u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe yields of the target compounds were minor relative to wild type stambomycins (metabolites \u003cstrong\u003e10\u003c/strong\u003e, \u003cstrong\u003e11\u003c/strong\u003e and \u003cstrong\u003e12\u003c/strong\u003e from ATCC/OE484/hy59_S1 were obtained at highest yields of 0.3, 3.2 and 1.0 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (4.5 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e total), respectively), but nonetheless approximately 4-fold higher from ATCC/OE484/hy59_S2 incorporating the hybrid KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e than from the full KS\u003csub\u003e14\u003c/sub\u003e swap (3.7, 9.7 and 3.6 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (17 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e total); 1500-fold lower yields than \u003cstrong\u003e1\u003c/strong\u003e) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e). Although the low titers of these compounds precluded their structure elucidation by NMR, we obtained additional confirmatory evidence for their identities by inactivation of \u003cem\u003esamR0479\u003c/em\u003e (which introduces the hydroxyl used for macrocyclization), which resulted in exclusive production of linear dehydroxy mini-stambomycins \u003cstrong\u003e13\u003c/strong\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eAs observed previously, the strains also produced substantial quantities of the shunt products \u003cstrong\u003e2\u003c/strong\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cstrong\u003e5\u003c/strong\u003e (inactivation of \u003cem\u003esamR0479\u003c/em\u003e led correspondingly to the dehydroxy versions of these compounds \u003cstrong\u003e6\u0026thinsp;\u0026minus;\u0026thinsp;9\u003c/strong\u003e (\u003cstrong\u003eSupplementary Figs.\u0026nbsp;7 and 10\u003c/strong\u003e)). The yields were ca. 80-fold higher than those of the corresponding mini-stambomycins, with the highest titers observed in the strain incorporating the hybrid KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e. The amount of shunt metabolites was also approximately 123-fold higher than from strain CPN2/OE484 (which incorporates an ACP\u003csub\u003e13\u003c/sub\u003e-\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e swap/\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e-KS\u003csub\u003e21\u003c/sub\u003e interface) (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e). Thus, contrary to expectation, although using the KS as a fusion site improved communication between Pks4 and Pks9, it also substantially boosted TEI-mediated off-loading of stalled upstream intermediates.\u003c/p\u003e\n\u003cp\u003eIn principle, such stalling could result from a slow rate of chain extension in the now hybrid acceptor module (for example, in the full KS swap construct, KS\u003csub\u003e14\u003c/sub\u003e and ACP\u003csub\u003e21\u003c/sub\u003e are completely mismatched for chain extension). To evaluate this idea, we modified ACP\u003csub\u003e21\u003c/sub\u003e within ATCC/OE484/hy59_S1 incorporating the full-length KS\u003csub\u003e14\u003c/sub\u003e, targeting a sequence region previously identified as mediating intramodular communication between the KS and ACP during chain extension (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;11\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Specifically, we exchanged loop 1 and the initial portion of helix \u0026alpha;II of ACP\u003csub\u003e21\u003c/sub\u003e for the corresponding sequence of ACP\u003csub\u003e14\u003c/sub\u003e, using CRISPR-Cas9 (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;11\u003c/strong\u003e). As we anticipated that creation of this substantially hybrid ACP might engender structural perturbation, we also engineered a minimal mutant of ACP\u003csub\u003e21\u003c/sub\u003e in which only one of the two most critical residues in the recognition motif was mutated to the corresponding amino acid in ACP\u003csub\u003e14\u003c/sub\u003e (G\u003csub\u003e1499\u003c/sub\u003e of Pks9 \u0026loz; D; the second residue, R, of the motif is already common to the two ACPs) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e). Analysis of the loop/helix \u0026alpha;II swap by HPLC-MS showed that all mini-stambomycin production had been abolished (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e), consistent with the anticipated disruption to ACP\u003csub\u003e14\u003c/sub\u003e structure. Production by the ACP site-directed mutant was not any better than by the full KS swap construct (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e), as only metabolite \u003cstrong\u003e11\u003c/strong\u003e remained above the limits of detection.\u003c/p\u003e\n\u003cp\u003eIn principle, the hybrid KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e domain may have worked better than KS\u003csub\u003e14\u003c/sub\u003e for chain extension due to improved interaction with ACP\u003csub\u003e21\u003c/sub\u003e, with stalling displaced to later modules. If this were the case, we might expect to see accumulation in the medium of shunt metabolites corresponding to the intermediate generated by module 21. Indeed, in the case of strain hy59_S2 (chimeric KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e) but not hy59_S1 (KS\u003csub\u003e14\u003c/sub\u003e), we detected masses consistent with the A/B and C/D forms of intermediate \u003cstrong\u003e14\u003c/strong\u003e generated by module 21, at yields comparable to those of the final mini-stambomycins (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e) (and correspondingly, \u003cstrong\u003e15\u003c/strong\u003e, the dehydroxylated analogue of \u003cstrong\u003e14\u003c/strong\u003e, was detected in the SamR0479 mutant (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e)). The same metabolite \u003cstrong\u003e14\u003c/strong\u003e was identified from the ACP\u003csub\u003e21\u003c/sub\u003e G \u0026loz;D mutant (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;11\u003c/strong\u003e), consistent with interrupted chain transfer to KS\u003csub\u003e22\u003c/sub\u003e. Taken together, these data confirm module 22 as a new blockage point in the engineered systems.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative efficacy of PKS engineering using PCR-targeting and CRISPR-Cas9.\u003c/strong\u003e As multiple of our core constructs were generated by both PCR-targeting and CRISPR-Cas9, we were able to directly compare the efficiency of the two techniques (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e). Globally, our results confirm that both approaches can be employed to introduce large-scale modifications to PKS biosynthetic genes (i.e. deletions of single or multi-gene regions)\u003csup\u003e32,44\u0026minus;46\u003c/sup\u003e. We have also demonstrated, for only the second time to our knowledge, that CRISPR-Cas9 can be leveraged to specifically modify modular PKS domains\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Of the two methods, CRISPR-Cas9 was the more rapid, as the corresponding constructs were engineered in approximately half of the time. In addition, while CRISPR-Cas9 allowed for direct modification of the host genome, PCR-targeting relied on the availability of suitable cosmids housing the target genes, and resulted in a 33 bp \u003cem\u003eattB\u003c/em\u003e-like \u0026lsquo;scar\u0026rsquo; sequence in the genome (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In addition to hampering iterative use of the approach, the scar apparently provoked a moderate reduction in stambomycin yields in mutant K7N6 compared to the wild type, an effect also noted upon comparison of several analogous mutant strains (e.g. K7N4 vs. CPN4, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Nonetheless, we did encounter certain difficulties with use of CRISPR-Cas9 (i.e. failure to obtain construct CPN3, occasional reversions to wild type, etc.), observations motivating ongoing efforts in other laboratories to further enhance the suitability of CRISPR-Cas9 for editing PKS pathways\u003csup\u003e47,49\u0026minus;54\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this work, we have utilized an approach based on the state-of-the-art in PKS engineering to modify the stambomycin PKS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, we aimed to remove the four PKS subunits between Pks4 and Pks9 in the assembly line which together house seven chain extension modules, to generate a series of 37-membered \u0026lsquo;mini-stambomycins\u0026rsquo;. While in principle such a change might have been possible by directly fusing Pks4 and Pks9 via a suitable intermodular linker, this approach would have resulted in a heptamodular subunit whose size is far in excess of the tetramodular multienzymes present in the system. We have also demonstrated recently the low efficacy of this strategy when the module downstream of the linker is N-terminal in its native subunit context (as with module 21 of Pks9)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs an initial approach (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), we modified \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e to render it compatible with \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e, with the aim of inducing productive communication between Pks4 and Pks9, while leaving all modular units intact. This modified PKS relied for function on both a non-native chain transfer interface (ACP\u003csub\u003e13\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e), and the intrinsic tolerance of the downstream KS/modules to the incoming substrate. We were optimistic this experiment might work given the structural similarities between the native substrates of KS\u003csub\u003e14\u003c/sub\u003e and KS\u003csub\u003e21\u003c/sub\u003e at least directly adjacent to the acyl terminus, as well as the fact that the stambomycin PKS generates a small family of metabolites, and therefore must exhibit some intrinsic tolerance to structural variation. Although we showed \u003cem\u003ein vitro\u003c/em\u003e with recombinant DD pairs that a docking helix-swapped mutant of \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e communicated effectively with \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e, chain transfer across the engineered interface did not occur \u003cem\u003ein vivo\u003c/em\u003e, as evidenced by the accumulation of multiple shunt products. While our attempt to render the ACP\u003csub\u003e13\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e junction more native by site-directed mutagenesis did result in certain target metabolites, the most significant effect was to increase the yields of the truncated chains.\u003c/p\u003e\n\u003cp\u003eHaving narrowed down the biosynthetic block to events occurring downstream of the engineered junction, we next carried out interface engineering based on proposed alternative module boundaries, leveraging fusion points within the KS domain (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). In this case, sites were selected to either maintain essentially the whole of KS\u003csub\u003e14\u003c/sub\u003e, or to create a hybrid KS\u003csub\u003e14\u003c/sub\u003e/KS\u003csub\u003e21\u003c/sub\u003e domain. This strategy at once preserved key elements of the ACP\u003csub\u003e13\u003c/sub\u003e/KS\u003csub\u003e14\u003c/sub\u003e chain transfer junction, and in the case of the almost full-length KS\u003csub\u003e14\u003c/sub\u003e, ensured that the domain had the appropriate substrate specificity for the incoming chain. Interestingly, the construct incorporating the chimeric KS functioned best, producing the desired mini-stambomycins in both linear and macrocyclic forms. These data identify this location in the middle of the KS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e as a potentially general fusion site, perhaps because it preserves elements of both the chain transfer and chain extension interfaces with the two partner ACP domains (ACP\u003csub\u003e13\u003c/sub\u003e and ACP\u003csub\u003e21\u003c/sub\u003e, in this case). Intriguingly, a site within the analogous condensation (C) domains of modular nonribosomal peptide synthetase (NRPS) systems, has also recently emerged as a useful fusion point for generating engineered hybrids\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOur results also showcase the intrinsically high tolerance of the Pks9 TEI domain towards shorter substrates. Indeed, the data also demonstrate that this TEI domain participates in off-loading the shunt metabolites from the upstream subunit, and that this activity interferes with passage of the chain to subsequent modules. Unfortunately, our attempts to boost yields of the mini-stambomycins by engineering the condensation interface between KS\u003csub\u003e14\u003c/sub\u003e and ACP\u003csub\u003e21\u003c/sub\u003e were unsuccessful, both when the full ACP\u003csub\u003e21\u003c/sub\u003e recognition loop/helix \u0026alpha;II region was swapped for that of ACP\u003csub\u003e14\u003c/sub\u003e, and when a single site-directed mutation was made at a putatively critical position (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). This result is surprising in light of the beneficial effects reported \u003cem\u003ein vitro\u003c/em\u003e of both of these modifications on chain extension carried out by mis-matched KS and ACP domains sourced from the erythromycin PKS (DEBS)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Apparently, the introduced changes were not sufficient to ensure effective communication between the KS\u003csub\u003e14\u003c/sub\u003e and ACP\u003csub\u003e21\u003c/sub\u003e domains (or were in fact deleterious to function), and/or any benefit was masked by the poor tolerance of the downstream modules to the modified intermediates.\u003c/p\u003e\n\u003cp\u003eTo fully judge the efficacy of this work, it is instructive to compare it to the other two examples in the literature in which full biosynthetic systems have been re-engineered to remove multiple internal modules\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In the first, recently-reported case, the neoaureothin (Nor) hexamodular PKS was \u0026lsquo;morphed\u0026rsquo; into the evolutionarily-related aureothin (Aur) tetramodular PKS by removing the second bimodular subunit, NorA\u0026prime;. As in our work, the authors initially attempted to engineer a new interaction between the monomodular subunits NorA and NorB flanking NorA\u0026prime; using compatible docking domains, by exchanging the type 1b \u003csup\u003eN\u003c/sup\u003eDD of NorB for the type 1a \u003csup\u003eN\u003c/sup\u003eDD of NorA\u0026prime; (the natural partner of NorA). When the target metabolite was not obtained, they relocated the fusion site to the KS-AT linker downstream of the conserved KS region in NorB, thereby maintaining the native NorA ACP-\u003csup\u003eC\u003c/sup\u003eDD/\u003csup\u003eN\u003c/sup\u003eDD-KS NorA\u0026prime; junction. Ultimately, several linker variants had to be evaluated before a functional sequence was identified, in part by serendipity (indeed it is 1 residue longer than the native linker). Overall, the yields of the targeted chain-shortened metabolites were reduced approximately 18-fold compared to the parental neoaureothin (to ca. 2.5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), a much less significant penalty than engendered by our engineering strategy. Presumably, the superior titers obtained in this experiment reflect the much higher intrinsic amenability of the Nor PKS to conversion into an Aur PKS, as the Nor PKS likely evolved from an Aur PKS by subunit insertion\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the newly-created NorA/NorB interface was also only partially functional, as product corresponding to the intermediate generated by iterative action of the upstream subunit NorA was still obtained.\u003c/p\u003e\n\u003cp\u003eThe second relevant investigation concerns the accelerated evolution (AE) of the RAPS PKS, based on spontaneous induced homologous recombination between its component modules\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. As mentioned earlier, several of the resulting systems incorporated intermodular fusion sites essentially at the mid-point of the respective KS domains, and so can be compared to our best performing construct hy59_S2. Notably, yields from the hybrid RAPS PKSs from which either 3 or 6 modules were removed, were reduced by a maximum of 33% relative to that of the parental compound. We propose two explanations for the higher functionality of these systems relative to hy59_S2. First of all, in every case, the module downstream of the newly-formed junctions in the contracted RAPS PKSs was internal to its respective subunit (unlike module 21 of Pks9), consistent with the idea that such modules boast intrinsically broader substrate specificity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Secondly, the KS domains of the RAPS PKS exhibit unusually high mutual sequence identity (85\u0026thinsp;\u0026minus;\u0026thinsp;90%)\u003csup\u003e56\u003c/sup\u003e. This means, in effect, that the same KS domain acts on a large variety of substrates of differing length and functionality, contributing to the tolerance of all modules downstream of the fusion site. In contrast, the KSs in the stambomycin PKS have substantially lower sequence identity (62\u0026thinsp;\u0026minus;\u0026thinsp;80%), and thus KSs 22\u0026thinsp;\u0026minus;\u0026thinsp;24 in hy59_S2 likely represent a specificity barrier to efficient transfer and extension of the modified intermediates.\u003c/p\u003e\n\u003cp\u003eTaken together, this set of results shows that contracting PKS systems represents a viable approach to accessing truncated polyketide derivatives of variable length, including macrocycles. Whether such systems are generated rationally or using an AE process, the most efficient hybrids will likely result: i) from PKSs whose modules (and in particular KS domains) exhibit a substantial degree of mutual sequence identity and thus intrinsically high substrate tolerance (or which can be adapted by mutagenesis to broaden their specificity\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e); and, ii) when novel junctions are created with downstream modules which are situated at internal positions within their subunits. The data also reinforce the idea that in cases where communication at modified interfaces occurs via non-covalent protein-protein interactions, at least a portion of the KS downstream from the docking domains should be included to boost efficiency\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Finally, our work has identified an increase in TEI-mediated proof-reading provoked by such interface engineering. Elucidating the mechanism underlying this unexpected intersubunit release activity, and thus how to effectively suppress it, should be a profitable avenue for further boosting product titers.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eBioinformatics analysis.\u003c/strong\u003e To underpin the interface engineering strategy, the extremities of all the stambomycin PKS subunits were analyzed to identify the boundaries of the most C-terminal and N-terminal function domains (ACP and KS, respectively), and thus the regions potentially containing docking domains (DDs). The resulting sequences were compared by multiple sequence alignment using Clustal Omega (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/msa/clustalo\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e57\u003c/sup\u003e, to bonafide and putative DD sequences from multiple DD classes, including those present at the DEBS 2/DEBS 3 interface (type 1a, PDB ID:1PZQ, 1PZR\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e) and the PikAIII/PikAIV junction (type 1b, PDB ID: 3F5H\u003csup\u003e2\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e), to allow for type classification. To identify suitable boundaries for DD heterologous expression in \u003cem\u003eE. coli\u003c/em\u003e, the secondary structure of the putative DD regions was predicted using PSIPRED 4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e37\u003c/sup\u003e. Analysis for potential specificity-conferring residues in the stambomycin PKS ketosynthase (KS) domains was carried out by multiple sequence alignment against model KS domains\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, using Clustal Omega\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral methods.\u003c/strong\u003e All reagents and chemicals were obtained from Sigma-Aldrich, except the following: BD (tryptone, yeast extract, TSB powder), Thermo Fisher Scientific (Tris), VWR (glycerol, NaCl, NaNO\u003csub\u003e3\u003c/sub\u003e), ADM, France (NutriSoy flour), and New England Biolabs (T4 DNA ligase, restriction enzymes). Oligonucleotide primers and two additional synthetic DNA fragments for CPN4 and CPN5 constructs were synthesized by Sigma-Aldrich (\u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e). The docking domains \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val and \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e) were obtained as synthetic peptides from GeneCust. DNA sequencing of PCR products was performed by Sigma-Aldrich and Eurofins.\u003c/p\u003e\n\u003cp\u003ePCR reactions were performed with Taq DNA polymerase (Thermo Fisher Scientific) or Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) when higher fidelity was required. Isolation of DNA fragments from agarose gel, purification of PCR products and extraction of plasmids were carried out using the NucleoSpin\u0026reg; Gel and PCR Cleanup or NucleoSpin\u0026reg; Plasmid DNA kits (Macherey Nagel, Hoerdt, France).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrains and media.\u003c/strong\u003e \u003cem\u003eE. coli\u003c/em\u003e BL21 strains were obtained from Novagen. Unless otherwise specified, all \u003cem\u003eE. coli\u003c/em\u003e strains were cultured in LB medium (yeast extract 10 g, tryptone 5 g, NaCl 10 g, distilled water up to 1 L, pH 7.0)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e or on LB agar plates (LB medium supplemented with 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar) at 37\u0026deg;C. \u003cem\u003eStreptomyces ambofaciens\u003c/em\u003e ATCC23877 and the derived mutants were grown in TSB (TSB powder 30 g (tryptone 17 g, soy 3 g, NaCl 5 g, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 2.5 g, glucose 2.5 g), distilled water up to 1 L, pH 7.3) or on TSA plates (TSB medium supplemented with 20 g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e agar), and sporulated on SFM\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e agar plates (NutriSoy flour 20 g, D-mannitol 20 g, agar 20 g, tap water up to 1 L) at 30\u0026deg;C. All strains were maintained in 20% (\u003cem\u003ev/v)\u003c/em\u003e glycerol in 2 mL Eppendorf tubes and stored at \u0026minus;80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003eFor fermentation of \u003cem\u003eS. ambofaciens\u003c/em\u003e ATCC23877 and its mutants, spores were streaked on TSA with appropriate antibiotics and after incubation 48 h at 30\u0026deg;C, a loop of mycelium was used to inoculate 7 ml of MP5 medium (yeast extract 7 g, NaCl 5 g, NaNO\u003csub\u003e3\u003c/sub\u003e 1 g, glycerol 36 mL, MOPS 20.9 g, distilled water up to 1 L, pH 7.4) supplemented with selective antibiotics and sterile glass beads, followed by incubation at 30\u0026deg;C and 200 rpm for 24\u0026thinsp;\u0026minus;\u0026thinsp;48 h. Finally, the seed culture was centrifuged and resuspended into 2 mL fresh MP5 before being inoculated into 50 mL MP5 medium in a 250 mL Erlenmeyer flask, and cultivated at 200 rpm and 30\u0026deg;C for 4 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR-targeting-based genetic engineering\u003c/strong\u003e (exemplified by mutant ATCC/OE484/K7N1, \u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e). To render the BAC BAA9ZA8 proficient for selection following conjugation, its chloramphenicol resistance gene was replaced using a PCR-targeting approach, by a kanamycin resistance gene cassette sourced from pIJ776\u003csup\u003e31\u003c/sup\u003e, resulting in BAC1 (\u003cstrong\u003eSupplementary Table\u0026nbsp;3\u003c/strong\u003e). The cassette \u003cem\u003eattL\u0026thinsp;+\u0026thinsp;aac(3)IV\u0026thinsp;+\u0026thinsp;oriT\u0026thinsp;+\u0026thinsp;attR\u003c/em\u003e was amplified from the plasmid pSPM88T\u003csup\u003e60\u003c/sup\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e) using primers 9996 and 9997 (\u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e), affording PCR amplicon \u003cem\u003ePCR-K7N1.\u003c/em\u003e The PCR fragment was then electro-transformed into \u003cem\u003eE. coli\u003c/em\u003e BW25113/pKD20/BAC1\u003csup\u003e61\u003c/sup\u003e, giving rise to mutant BAC1_K7N1 (in which the C-terminus of \u003cem\u003epks4\u003c/em\u003e was modified and the genes \u003cem\u003epks5\u0026thinsp;\u0026minus;\u0026thinsp;pks8\u003c/em\u003e, were deleted)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. The BAC1_K7N1 was then introduced into \u003cem\u003eE. coli\u003c/em\u003e ET12567/pUZ8002\u003csup\u003e62\u003c/sup\u003e and then transferred to \u003cem\u003eS. ambofaciens\u003c/em\u003e wild type \u003cem\u003evia\u003c/em\u003e intergeneric conjugation. The resulting exconjugants (ATCC/K7N1_\u003cem\u003eaac(3)IV\u0026thinsp;+\u0026thinsp;oriT\u003c/em\u003e) were selected for their apramycin resistance and kanamycin sensitivity (i.e. a phenotype consistent with successful double cross-over). The correct mutations were confirmed by PCR and sequencing. Subsequently, the disruption cassette was excised using the excisionase and integrase of pSAM2 encoded by pOSK111 as described\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, leaving a 33 bp \u0026lsquo;scar\u0026rsquo; sequence (mutant ATCC/K7N1). Successful removal of the cassette was verified by PCR and DNA sequencing Finally, the LAL regulator overexpression plasmid pOE484\u003csup\u003e23\u003c/sup\u003e or the parental vector pIB139\u003csup\u003e33\u003c/sup\u003e was introduced into the strain giving rise to mutants K7N1/OE484 and K7N1/pIB139, respectively. The same overall procedure was applied to construct mutants K7N2/OE483, K7N2/pIB139, K7N3/OE483, K7N3/pIB139, K7N4/OE483, K7N4/pIB139, K7N5/OE483, K7N5/pIB139, K7N6/OE483 and K7N6/pIB139. An analogous PCR-targeting approach (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e) was also employed to inactivate \u003cem\u003esamR0478\u003c/em\u003e and \u003cem\u003esamR0479\u003c/em\u003e using appropriate BACS\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (\u003cstrong\u003eSupplementary Table\u0026nbsp;3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRISPR-Cas9-mediated genetic engineering.\u003c/strong\u003e Plasmids pCRISPomyces-2 (and associated cloning and screen protocols)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (used for construction of all mutants except ATCC/hy59_S1 and ATCC/hy59_S2) and pCRISPR-Cas9\u003csup\u003e45\u003c/sup\u003e, respectively, were used for CRISPR-Cas9-based genome editing, as described previously. The two systems differ in the way in which Cas9 is expressed; in the case of pCRISPomyces-2, the nuclease is expressed constitutively, while in the pCRISPR-Cas9 system, its expression is under inductive control by thiostrepton (Tsr). The crRNA sequence was selected to match the DNA segment which contains NGG on its 3\u0026rsquo; end (N is any nucleotide, and the NGG corresponds to the protospacer-adjacent motif (PAM). The annealed crRNA fragment and two homologous arms (HAL and HAR, flanking the target region) were sequentially inserted into the delivery plasmid pCRISPomyces-2 using the respective restriction sites \u003cem\u003eBbs\u003c/em\u003eI and \u003cem\u003eXba\u003c/em\u003eI, to afford the specific recombinant plasmid for each mutant (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e). Correspondingly, an sgRNA cassette (tracrRNA\u0026thinsp;+\u0026thinsp;sgRNA) and two homologous arms were inserted into the plasmid pCRIPR-Cas9 using sites \u003cem\u003eNco\u003c/em\u003eI, \u003cem\u003eSnaB\u003c/em\u003eI and \u003cem\u003eStu\u003c/em\u003eI, respectively (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;10\u003c/strong\u003e). In addition, the crRNA was designed to be located within the region to be deleted (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;4\u003c/strong\u003e) to avoid Cas9-catalyzed cleavage occurring in the genome of the resulting mutant. In the case of site-directed mutants, additional DNA fragments containing the targeted mutations were inserted between the two homologous arms. In addition, the DNA sequence with the fragments identical to the crRNA was modified, so as to avoid subsequent Cas9-catalyzed cleavage of the obtained mutants (\u003cstrong\u003eSupplementary Figs.\u0026nbsp;9 and 11\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression and purification of docking domains.\u003c/strong\u003e The wild-type docking domains (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val and \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met) and mutant docking domains (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap) were amplified from genomic DNA of \u003cem\u003eS. ambofaciens\u003c/em\u003e wild type and the relevant mutants, using forward and reverse primers incorporating \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eHin\u003c/em\u003edIII restriction sites, respectively (\u003cstrong\u003eSupplementary Table\u0026nbsp;2\u003c/strong\u003e). The PCR amplicons were digested using FD \u003cem\u003eBam\u003c/em\u003eHI and FD \u003cem\u003eHin\u003c/em\u003edIII, and then ligated into the equivalent sites of vector pBG-102 (Center for Structural Biology, Vanderbilt University). In the case of all \u003csup\u003eC\u003c/sup\u003eDDs which lacked aromatic residues, a tyrosine residue (codon TAT incorporated in the forward primer, \u003cstrong\u003eSupplementary Table\u0026nbsp;1\u003c/strong\u003e) was added at the N-terminal ends (so as not to interfere with docking with the \u003csup\u003eN\u003c/sup\u003eDD partner) to allow efficient monitoring by UV-Vis during the purification, as well as reliable measurement of protein concentration necessary for binding studies by ITC.\u003c/p\u003e\n\u003cp\u003eThe resulting constructs pBG102-\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e, pBG102-\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e and pBG102-\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e were used to transform \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). For \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e and its mutants, these were transformed into Rosetta\u0026trade; 2(DE3), as these constructs contain 8 codons rarely used in \u003cem\u003eE. coli\u003c/em\u003e. Positive transformants were selected on LB agar supplemented with kanamycin (50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (25 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e chloramphenicol was also added for expression in Rosetta\u0026trade; 2(DE3)). A single colony was transferred to LB (10 mL) supplemented with antibiotics, and the culture grown at 37\u0026deg;C and 200 rpm for overnight. The 1 mL overnight culture was used to inoculate LB media (1 L) supplemented with appropriate antibiotics, and then incubated at 37\u0026deg;C and 200 rpm to an optical density of 0.8, at which point protein synthesis was induced by the addition of IPTG (final concentration 0.1 mM). After incubation at 18\u0026deg;C and 200 rpm for 18 h, cells were collected by centrifugation at 8000\u003cem\u003eg\u003c/em\u003e for 30 min, resuspended in 40 mL protein purification buffer A (50 mM Tris-HCl, 400 mM NaCl, 10 mM imidazole, pH 8.0), and lysed by sonication. Following centrifugation at 20000\u003cem\u003eg\u003c/em\u003e and filtration using a 0.45 \u0026micro;m membrane, the soluble cell lysates were loaded onto 2 \u0026sdot; 5 mL HisTrap HP (GE Healthcare) columns (two 5 mL columns in series) equilibrated in buffer A, and purified by preparative protein purification chromatography using an \u0026Auml;KTA Avant system. The following program was applied: sample loading, 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; washing, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10 column volumes of buffer A; elution, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 5 column volumes of buffer B (50 mM Tris-HCl, 400 mM NaCl, 250 mM imidazole, pH 8.0); elution, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2 column volumes of buffer C (50 mM Tris-HCl, 400 mM NaCl, 500 mM imidazole, pH 8.0).\u003c/p\u003e\n\u003cp\u003eAll His\u003csub\u003e6\u003c/sub\u003e-SUMO-tagged proteins were collected (fractions containing the protein of interest were selected based on the UV chromatography and SDS-PAGE gel), and transferred into dialysis bag containing His\u003csub\u003e6\u003c/sub\u003e-tagged human rhinovirus 3C protease (H3C) (1\u0026thinsp;\u0026minus;\u0026thinsp;2 \u0026micro;M). The dialysis bag was then placed into a container filled with buffer D (50 mM Tris-HCl, 400 mM NaCl, pH 8.0), and the cleavage allowed to proceed at 4\u0026deg;C overnight. The resulting proteins, which incorporated a non-native N-terminal GPGS sequence, were then separated from the remaining His\u003csub\u003e6\u003c/sub\u003e-tagged SUMO and His\u003csub\u003e6\u003c/sub\u003e-tagged human rhinovirus 3C protease by re-loading onto the 2 \u0026sdot; 5 mL HisTrap HP columns pre-equilibrated in buffer A. Purification was then carried out with the following program: sample loading, 1 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; washing, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 4 column volumes of buffer A; elution, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2 column volumes of buffer B; elution, 2 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 2 column volumes of buffer C. The untagged docking domains passed through the column during the washing step, and were collected and concentrated to 5\u0026thinsp;\u0026minus;\u0026thinsp;7 mL using an Amicon Ultra 3000 MWCO centrifuge filter (Millipore Corp).\u003c/p\u003e\n\u003cp\u003eSubsequently, the concentrated docking domains were loaded onto a size exclusion chromatography column (Superdex 75 26/60 column, GE Healthcare) equilibrated in buffer GF (20 mM HEPES, 100 mM NaCl, 0.5 mM TCEP, pH 7.5). Following a concentration step, the purity of the purified proteins was verified by SDS-PAGE, and their concentrations were determined by NanoDrop (Thermo Scientific) with extinction coefficients calculated using the ExPASy ProtParam tool\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsothermal Titration Calorimetry measurements.\u003c/strong\u003e ITC measurements were performed at 20\u0026deg;C in buffer GF using a MicroCal ITC200 (Malvern Instruments) (A2F Plateforme ASIA: Approches fonctionnelles et Structurales des InterActions cellulaires). A 300 \u0026micro;L aliquot of \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e at 70 \u0026micro;M was placed in the calorimeter cell and titrated with 700 \u0026micro;M of the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003es (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e wild type, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap) in the syringe. In the case of the binding experiments between \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e, the \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e (700 \u0026micro;M) was added to the \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Met (80 \u0026micro;M in the cell), while for the binding between \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val and the \u003csup\u003eC\u003c/sup\u003eDDs (\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e8\u003c/sub\u003e, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e wild type, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM and \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap), the \u003csup\u003eC\u003c/sup\u003eDDs (700 \u0026micro;M) were added to \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e Val in the cell (120 \u0026micro;M). The ITC experiments were then carried out as followed: initial waiting time 120 s, initial injection of 0.5 \u0026micro;L over 1 s followed by 19 serial injections of 2 \u0026micro;L over 4 s, separated by an interval of 120 s. For each experiment, the reference power was set to 5 \u0026micro;cal\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, stirring speed to 750 rpm, and the high feedback mode was selected. Two independent titrations were performed for each combination of DDs. The heat of reaction per injection (\u0026micro;cal s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was determined by integration of the peak areas using the Origin 7.0 (OriginLab) software, assuming a one-site binding model (consistent with the solved structures of the types of DDs\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e), yielding the best-fit values for the heat of binding (\u0026Delta;H), the stoichiometry of binding (N) and the dissociation constant (K\u003csub\u003ed\u003c/sub\u003e). The heats of dilution of the DDs were determined by injecting them into the cell containing buffer only, and these were subtracted from the corresponding binding data prior to curve fitting.\u003c/p\u003e\n\u003cp\u003eIn some cases, when a plateau (binding saturation) was not reached at the final titration step, and the problem could not be solved by increasing the concentration of DD in syringe, we initially placed \u003csup\u003eC\u003c/sup\u003eDD/\u003csup\u003eN\u003c/sup\u003eDD complex in the ITC cell (at the concentration of the two partners reached in the previous titration), filled the syringe with additional DD, and performed a second titration experiment. This procedure was then repeated until binding saturation was reached. To fit the data, the MicroCal ITC concatenation software was used to combine two ITC data files together. Most importantly, the critical parameter dimensionless constant (C-value) was calculated as follows:\u003c/p\u003e\n\u003cp\u003eC\u0026thinsp;=\u0026thinsp;NK\u003csub\u003ea\u003c/sub\u003e[M]\u003csub\u003eT\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ewhere K\u003csub\u003ea\u003c/sub\u003e is the binding constant, [M]\u003csub\u003eT\u003c/sub\u003e is the total macromolecular concentration in the cell, and N is the stoichiometry of interaction. A reliable ITC binding isotherm is evidenced by ITC data with C-values\u0026thinsp;\u0026gt;\u0026thinsp;1 (the optimal range is 5\u0026thinsp;\u0026lt;\u0026thinsp;C\u0026thinsp;\u0026lt;\u0026thinsp;500)\u003csup\u003e64\u003c/sup\u003e, as was the case for all of our measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCircular Dichroism measurements.\u003c/strong\u003e CD spectra were recorded on a Chirascan CD (Applied Photophysics, United Kingdom) (IBS-Lor UMS 2008 Plateforme de Biophysique et Biologie Structurale) at 0.5 nm intervals in the wavelength range of 180\u0026thinsp;\u0026minus;\u0026thinsp;260 nm at 20\u0026deg;C, using a temperature-controlled chamber. A 0.01 cm quartz cuvette containing 30 \u0026micro;L of docking domain at 100 \u0026micro;M, a 0.1 cm cuvette with 200 \u0026micro;L of sample at 10 \u0026micro;M, and a 1 cm cuvette containing 1.5 mL of sample at 1 \u0026micro;M, were used for all the measurements. All measurements were performed at least in triplicate, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone. The CD spectra were deconvoluted using the deconvolution software CDNN2.1\u003csup\u003e65\u003c/sup\u003e to estimate the secondary structure present in the docking domains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEC-MALS analysis of docking domains.\u003c/strong\u003e The oligomeric state of all the docking domains was determined by size-exclusion chromatography multi-angle light scattering (SEC-MALS) on the A2F Plateforme ASIA. For this, SEC was first carried out on a Superdex75 10/300 column (GE Healthcare) at 20\u0026deg;C using a flow rate of 0.5 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in HEPES buffer (20 mM HEPES, 100 mM NaCl, 0.5mM TCEP, pH 7.5) using a \u0026Auml;KTA-Purifier FPLC (GE Healthcare). Multi-angle light scattering (MALS) was measured using a MiniDAWN TREOS II (Wyatt Technology), while refractometry was monitored using an Optilab T-rEX (Wyatt Technology). Data processing was carried out with the manufacturer supplied software (ASTRA 6.1, Wyatt Technology) to determine the protein oligomerization state.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHPLC-MS analysis of fermentation metabolites and purified docking domains.\u003c/strong\u003e The fermentation broth of \u003cem\u003eStreptomyces\u003c/em\u003e was centrifuged at 4000\u003cem\u003eg\u003c/em\u003e for 10 min. As described previously\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, the stambomycins and their derivatives were then extracted from the mycelia, by first resuspending the cells in 40 mL distilled water, followed by centrifugation (4000\u003cem\u003eg\u003c/em\u003e, 10 min, repeated 3\u0026times;) to remove water-soluble components. After decanting the water, the cell pellets were weighed and extracted with methanol by shaking at 150 rpm for 2 h at room temperature. Thereafter, the methanol extracts were filtered to remove the cell debris, followed by rotary evaporation to dryness. The obtained extracts were then dissolved in methanol, whose volume was determined according to the initial weight of the mycelia (70 \u0026micro;L methanol to 1 g of initial cell pellet). The resulting mycelial crude extracts were then passed through a 0.4 \u0026micro;m syringe filter and analyzed in positive electrospray mode (ESI\u003csup\u003e+\u003c/sup\u003e) by HPLC-HRMS (Thermo Scientific Orbitrap LTQ XL or an Orbitrap ID-X Tribrid Mass Spectrometer) (Plateau d\u0026rsquo;Analyse Structurale et M\u0026eacute;tabolomique (PASM)-SF4242 EFABA) using an Alltima\u0026trade; C18 column (2.1 \u0026sdot; 150mm, 5 \u0026micro;m particle size). Separation was carried out with Milli-Q water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) using the following elution profile: 0\u0026thinsp;\u0026minus;\u0026thinsp;48 min, linear gradient 5\u0026thinsp;\u0026minus;\u0026thinsp;95% solvent B; 48\u0026thinsp;\u0026minus;\u0026thinsp;54 min, constant 95% solvent B; 54\u0026thinsp;\u0026minus;\u0026thinsp;60 min, constant 5% solvent B. Mass spectrometry operating parameters were: spray voltage, 5 kV; source gases were set respectively for sheath gas, auxiliary gas and sweep gas at 30, 10 and 10 arbitrary units min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; capillary temperature, 275\u0026deg;C; capillary voltage, 4 V; tube lens, split lens and front lens voltages 155 V, \u0026minus;28 V and \u0026minus;6 V, respectively. Due to the much lower sensitivity of the Orbitrap LTQ XL relative to the Orbitrap ID-X Tribrid as evidenced by comparative analysis of identical samples on the two instruments, we introduced a 10\u0026times; correction factor to the the yields determined using the Orbitrap LTQ XL (\u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe purified docking domains in buffer GF were diluted with Milli-Q water to a concentration of 50 \u0026micro;M and injected onto an Alltima\u0026trade; C18 column (2.1 \u0026sdot; 150mm, 5 \u0026micro;m particle size). Analysis was carried out with Milli-Q water containing 0.1% TFA (A) and acetonitrile containing 0.1% TFA (B), using the elution profile: 0\u0026thinsp;\u0026minus;\u0026thinsp;15 min, constant 10% solvent B; 15\u0026thinsp;\u0026minus;\u0026thinsp;20 min, linear gradient of 10% solvent B to 95%; 20\u0026thinsp;\u0026minus;\u0026thinsp;25 min, constant 10% solvent B. Mass spectrometry operating parameters were set as above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolite profiling of engineered strains.\u003c/strong\u003e Comparative analysis of fermentation extracts (of all strains containing pOE484 except K7N3, CPN4 and CPN5, relative to control mutant containing empty plasmid pIB139) was conducted on a Dionex Ultimate 3000 HPLC system coupled to a Thermo Scientific\u0026trade; Q Exactive\u0026trade; Hybrid Quadrupole-Orbitrap mass spectrometer. MS-settings: spray voltage 3.5 kV; capillary temperature 320\u0026deg;C; sheath gas (52.50), auxiliary gas (13.75), sweep gas (2.75); probe heater 437.50\u0026deg;C; S-Lens RF (50), positive mode, resolution 70.000; AGC target 1e6, microscans 1, maximum IT 75 ms, scan range 200\u0026thinsp;\u0026minus;\u0026thinsp;1800 \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 \u0026micro;m XB-C18 150 \u0026times; 4.6 mm column with solvents (A, H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;0.1% formic acid) and (B, MeCN\u0026thinsp;+\u0026thinsp;0.1% formic acid) and the following gradient: flow rate 0.7 mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 20% B for 2 min, 20\u0026ndash;98% B over 18 min, 98% B for 5 min, 98\u0026ndash;20% B in 0.5 min and 20% B for 4 min. Metabolic differences within the obtained data (\u003cstrong\u003eSupplementary Table\u0026nbsp;6\u003c/strong\u003e) were identified using SIEVE 2.0 screening software, applying the default settings for component extraction of small molecules, except that of the base peak minimum intensity, which was set to 5000000.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of metabolites.\u003c/strong\u003e To quantify the yields of native stambomycins and the newly-generated derivatives by HPLC-MS, we generated a calibration curve with previously-purified stambomycin A/B \u003cstrong\u003e1\u003c/strong\u003e as the standard (using a concentration range between 1 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). This approach yielded a linear correlation between the quantity of \u003cstrong\u003e1\u003c/strong\u003e and the respective peak area in the extracted ion chromatogram (EIC) (the areas of all peaks corresponding to the parental ions were used) (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;12, Supplementary Table\u0026nbsp;5\u003c/strong\u003e). The titers of all stambomycin derivatives were then determined using this calibration curve, based on the peak areas corresponding to the parental ions in their respective EIC chromatograms (\u003cstrong\u003eSupplementary Table\u0026nbsp;5\u003c/strong\u003e). As the limit of detection in these experiments lay between 1 and 10 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, yields in this range must be viewed as estimates.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge financial support from the Agence Nationale de la Recherche (grant number ANR-16-CE92-0006-01, PKS STRUCTURE to K.J.W.), the Universit\u0026eacute; de Lorraine, the Centre National de la Recherche Scientifique (CNRS), and the IMPACT Biomolecules project of the Lorraine Universit\u0026eacute; d\u0026rsquo;Excellence (Investissements d\u0026rsquo;avenir - ANR 15-004). J.P. acknowledges funding by the European Research Council (ERC) under the European Union\u0026rsquo;s Horizon 2020 research and innovation programme (grant agreement No. 742739). S. Collin and B. Chagot are thanked for help with the analysis of the DDs \u003cem\u003ein vitro\u003c/em\u003e, A. Kriznik for assistance with the biophysical experiments, J. Grosjean for help with the HPLC-MS analysis and S. Rousselot for contributing to developing the genetic engineering strategy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.S. constructed plasmids, generated and fermented recombinant strains, analyzed and interpreted HPLC-MS data, expressed and purified recombinant docking domains, carried out biophysical analysis of DD interactions, and generated all of the manuscript figures. L.H. and C.J. constructed certain plasmids, mutant BACs and/or mutant strains. C.P. performed HPLC analyses. A.B. and J.P. carried out HPLC-MS analyses and metabolic profiling. B.A., C.J. and K.J.W. designed the research and supervised the project. K.J.W. performed \u003cem\u003ein silico\u003c/em\u003e analyses and wrote the manuscript, with contributions from all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e accompanies this paper at https://doi.org/\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests: \u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permission\u003c/strong\u003e information is available online at \u003ca href=\"http://npg.nature.com/reprintsandpermissions/\"\u003ehttp://npg.nature.com/reprintsandpermissions/\u003c/a\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWeissman, K. J. The structural biology of biosynthetic megaenzymes. \u003cem\u003eNat. Chem. 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Biol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 898\u0026ndash;908 (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 469.8pt;border-top: 1pt solid windowtext;border-right: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-bottom: none;background: rgb(251, 228, 213);padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:150%;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003eTa\u003cspan style=\"color:black;\"\u003eble 1 Summary of various strains generated, as well as the novel metabolites detected.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 469.8pt;border-top: none;border-left: 1pt solid windowtext;border-bottom: none;border-right: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003ctable style=\"width:453.0pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003eStrain\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003eModifications introduced\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-4.9pt;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003eStambomycins\u0026nbsp;\u003c/strong\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003eNovel metabolites\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eWild type\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026uuml;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eK7N6/OE484\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e33 bp scar\u003csup\u003ec\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026uuml;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eK7N5/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap, 33 bp scar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN5/OE484\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026uuml;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eK7N4/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e site-directed mutagenesis (SDM), 33 bp scar\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026uuml;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN4/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026uuml;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003en.d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eK7N3/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8,\u0026nbsp;\u003c/em\u003e33 bp scar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eK7N2/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8,\u0026nbsp;\u003c/em\u003e33 bp scar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN1/OE484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e SDM,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/Pks4+TEI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eTEI fused to Pks4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:-5.55pt;margin-bottom: 0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484/TEI SDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eTEI inactivation (Ser\u0026nbsp;\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026agrave;\u003c/span\u003e Ala), \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484/TEII SDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eTEII inactivation (Ser\u0026nbsp;\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026agrave;\u003c/span\u003e Ala), \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484/\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003esamR0478,\u0026nbsp;\u003c/em\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484/\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003esamR0479,\u0026nbsp;\u003c/em\u003e\u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e6\u003c/strong\u003e, \u003cstrong\u003e7\u003c/strong\u003e, \u003cstrong\u003e8\u003c/strong\u003e, \u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eCPN2/OE484/ACP\u003csub\u003e13\u003c/sub\u003e SDM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eACP\u003csub\u003e13\u0026nbsp;\u003c/sub\u003eH1 modified\u003csup\u003ed\u003c/sup\u003e, \u003csup\u003eC\u003c/sup\u003eDD\u003csub\u003e4\u003c/sub\u003e helix swap,\u0026nbsp;\u003cspan style=\"font-family: Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;10\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;11\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;12\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14/21\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;10\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;11\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;12\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S1/\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003esamR0479\u003c/em\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e6\u003c/strong\u003e, \u003cstrong\u003e7\u003c/strong\u003e, \u003cstrong\u003e8\u003c/strong\u003e, \u003cstrong\u003e9\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;13\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S2/\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e479\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003esamR0479\u003c/em\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14/21\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e6\u003c/strong\u003e, \u003cstrong\u003e7\u003c/strong\u003e, \u003cstrong\u003e8\u003c/strong\u003e, \u003cstrong\u003e9\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;13\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;15\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S1/ACP\u003csub\u003e21\u003c/sub\u003e region swap\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eACP\u003csub\u003e21\u003c/sub\u003e L1+H2 modified\u003csup\u003ee\u003c/sup\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 132.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eATCC/OE484/hy59_S1/ACP\u003csub\u003e21\u003c/sub\u003e GtoD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 185.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003eACP\u003csub\u003e21\u003c/sub\u003e L1 modified\u003csup\u003ef\u003c/sup\u003e, \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e9\u003c/sub\u003e+KS\u003csub\u003e21\u003c/sub\u003e replaced by \u003csup\u003eN\u003c/sup\u003eDD\u003csub\u003e5\u003c/sub\u003e+KS\u003csub\u003e14\u003c/sub\u003e,\u0026nbsp;\u003cspan style=\"font-family:Symbol;\"\u003eD\u003c/span\u003e\u003cem\u003epks5\u0026minus;8\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.6pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026ucirc;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71.4pt;border-top: none;border-right: none;border-left: none;border-image: initial;border-bottom: 1pt solid windowtext;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e2\u003c/strong\u003e, \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, \u003cstrong\u003e5\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;11\u003c/strong\u003e,\u003cstrong\u003e\u0026nbsp;14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 469.8pt;border-right: 1pt solid windowtext;border-bottom: 1pt solid windowtext;border-left: 1pt solid windowtext;border-image: initial;border-top: none;padding: 0in 5.4pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003ea\u003c/sup\u003eK7N (pronounced \u0026ldquo;cassette number\u0026rdquo;) refers to mutants generated by PCR-targeting.\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003eb\u003c/sup\u003eCPN refers to mutants generated using CRISPR-Cas9.\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003ec\u003c/sup\u003eUse of PCR-targeting technique introduced a \u0026lsquo;scar\u0026rsquo; sequence between \u003cem\u003epks4\u003c/em\u003e and \u003cem\u003epks9\u003c/em\u003e; for details, see \u003cstrong\u003eSupplementary Fig. 4\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003ed\u003c/sup\u003eH1 modified refers to mutation of six residues within the helix\u0026nbsp;\u0026alpha;1 region of ACP\u003csub\u003e13\u003c/sub\u003e (EADQRR\u0026nbsp;\u003cspan style=\"font-family: Wingdings;\"\u003e\u0026agrave;\u003c/span\u003e PSERRQ); for details, see \u003cstrong\u003eSupplementary Fig. 9\u003c/strong\u003e.\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003ee\u003c/sup\u003eL1+H2 modified refers to exchange of the loop 1+helix\u0026nbsp;\u0026alpha;2 region of ACP\u003csub\u003e21\u003c/sub\u003e;\u003csub\u003e\u0026nbsp;\u003c/sub\u003efor details, see \u003cstrong\u003eSupplementary Fig. 11\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003e\u003csup\u003ef\u003c/sup\u003eL1 modified indicates that one residue within the loop 1 region of ACP\u003csub\u003e21\u003c/sub\u003e was mutated (G\u003csub\u003e1499\u0026nbsp;\u003c/sub\u003e\u003cspan style=\"font-family:Wingdings;\"\u003e\u0026agrave;\u003c/span\u003e D); for details, see \u003cstrong\u003eSupplementary Fig. 11\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;text-align:justify;'\u003en.d. indicates no novel metabolites were detected.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;line-height:normal;font-size:15px;font-family:\"Calibri\",sans-serif;'\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"polyketide synthases (PKSs), PKS engineering strategies","lastPublishedDoi":"10.21203/rs.3.rs-222743/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-222743/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe modular organization of the type I polyketide synthases (PKSs) would seem propitious for rational engineering of desirable analogous. However, despite decades of efforts, such experiments remain largely inefficient. Here, we combined multiple, state-of-the-art approaches including modification of docking domains, use of modules of varying domain composition, alternative interdomain fusion sites, and targeted adaptation of key domain-domain interfaces, to reprogram the stambomycin PKS by deleting seven internal modules \u0026ndash; the most substantial modification to an intact system reported to date. One such system produced the target 37-membered mini-stambomycin metabolites, a reduction in chain length of 14 carbons relative to the 51-membered parental compounds, but also substantial quantities of shunt metabolites released from the multienzyme subunit upstream of the newly-installed junction. Our data also provide evidence for an unprecedented off-loading mechanism of such stalled intermediates involving the C-terminal thioesterase domain acting on chains located four modules upstream. The yields of all metabolites were substantially reduced compared to the wild type compounds, likely reflecting the poor tolerance to the non-native substrates of the modules downstream of the introduced interfaces. Taken together, our data demonstrate that even \u0026lsquo;optimized\u0026rsquo; PKS engineering strategies remain inadequate for efficient production of target polyketide derivatives, and highlight several areas for future investigation.\u003c/p\u003e","manuscriptTitle":"Successes, surprises and pitfalls in modular polyketide synthase engineering: generation of ring-contracted stambomycins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-19 00:56:18","doi":"10.21203/rs.3.rs-222743/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"213d4124-60b5-4794-ac60-9913d37b2185","owner":[],"postedDate":"February 19th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":2501140,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2022-03-09T18:06:13+00:00","versionOfRecord":{"articleIdentity":"rs-222743","link":"https://doi.org/10.1038/s41467-022-27955-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2022-01-26 05:00:00","publishedOnDateReadable":"January 26th, 2022"},"versionCreatedAt":"2021-02-19 00:56:18","video":"","vorDoi":"10.1038/s41467-022-27955-z","vorDoiUrl":"https://doi.org/10.1038/s41467-022-27955-z","workflowStages":[]},"version":"v1","identity":"rs-222743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-222743","identity":"rs-222743","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}