Refactoring the pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli

Refactoring the pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli | 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 Refactoring the pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli Adrian Keatinge-Clay, Takeshi Miyazawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5640596/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract While engineering modular polyketide synthases (PKSs) using the recently updated module boundary has yielded libraries of triketide-pentaketides, this strategy has not yet been applied to the combinatorial biosynthesis of macrolactones or macrolide antibiotics. We developed a 2-plasmid system for the construction and expression of PKSs and employed it to obtain a refactored pikromycin synthase in E. coli that produces 85 mg of narbonolide per liter of culture. The replacement, insertion, deletion, and mutagenesis of modules enabled access to hexaketide, heptaketide, and octaketide derivatives. Supplying enzymes for desosamine biosynthesis and transfer enabled production of narbomycin, pikromycin, YC-17, methymycin, and 6 derivatives thereof. Knocking out pathways competing with desosamine biosynthesis and supplying the editing thioesterase PikAV boosted the titer of narbomycin 55-fold to 37 mgL − 1 . The replacement of the 3rd pikromycin module with its 5th yielded a new macrolide antibiotic and demonstrates how libraries of macrolide antibiotics can be readily accessed. Biological sciences/Biochemistry/Biocatalysis Biological sciences/Biochemistry/Enzymes/Multienzyme complexes Biological sciences/Chemical biology/Biosynthesis/Multienzyme complexes Biological sciences/Drug discovery/Medicinal chemistry/Drug discovery and development Biological sciences/Systems biology/Synthetic biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Antimicrobial resistance is an ever-increasing threat to human health. Consequently, there is a growing need to discover and develop new antibiotics 1 . Unfortunately, the pace of both discovering new antibiotics and developing antibiotics through medicinal chemistry has slowed 2 . Modular synthesis, through which diversity is created from relatively simple building blocks, can accelerate these processes 3 . A modular chemical synthesis recently yielded a library of > 300 macrolide antibiotic derivatives, 2 of which displayed superior potencies compared to those in clinical use 4 . The modular syntheses of macrolide antibiotics, such as erythromycin, pikromycin, and tylosin, were first conducted by polyketide synthase (PKS) assembly lines within actinomyces bacteria 5 – 8 . These assembly lines are themselves modular, where each module (updated definition used 9 , 10 ) adds one ketide monomer to a growing polyketide chain. They minimally contain 3 domains - an acyltransferase (AT), an acyl carrier protein (ACP), and a ketosynthase (KS). During the catalytic cycle of a module, ACP acquires the polyketide chain from the KS of the upstream module through an a-carboxyacyl extender unit obtained from AT, shuttles the extended chain to optional processing enzymes, such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, and to KS, which acquires properly processed chains through transacylation. The most downstream domain of the assembly line is most commonly a thioesterase (TE) that releases the polyketide chain through cyclization or hydrolysis. Most efforts to engineer PKSs have been based on the traditional module boundary and have not been successful 10 . This boundary was defined immediately upstream of KS when the genes encoding the erythromycin PKS were sequenced in 1990, in analogy with the mammalian fatty acid synthase (FAS), an iterative PKS in which KS is the most upstream domain 11 , 12 . In 2017, genome-mining of modular PKSs that produce related aminopolyols helped reveal that the KSs of modular PKSs evolutionarily co-migrate with processing domains upstream of them and that the module boundary is actually immediately downstream of KS 9 , 10 . Indeed, synthases engineered with the updated module boundary consistently outperform synthases engineered with the traditional module boundary 13 – 15 . Our lab recently developed a BioBrick-like platform to rapidly assemble synthases on a single expression plasmid through the ligation of DNA fragments encoding updated modules. We employed it to combinatorially assemble the modules of the pikromycin synthase ( Streptomyces venezuelae ATCC 15439) into 5 triketide, 25 tetraketide, and 125 pentaketide synthases and assess their function 16 . Here, we describe a 2-plasmid system that enables the expression of a refactored pikromycin synthase in E. coli as well as the combinatorial biosynthesis of new macrolactones and macrolide antibiotics (Fig. 1 ) 17 . One plasmid encodes the 1st pikromycin module ( P1 ) and the upstream portion of the 4th module ( N P4 ), while the other plasmid encodes the downstream portion of the 4th module ( C P4 ) and the 7th module ( P7 ). Module-encoding inserts can be sequentially ligated into both plasmids. The incorporation of P2 , P3 , P5 , and P6 into this system yielded a functional, refactored pikromycin synthase in E. coli K207-3, comprised of 6 polypeptides rather than the 4 natural ones (PikAI-PikAIV) 18 . Docking domain optimization and expression of the editing thioesterase PikAV improved narbonolide ( 1 ) production 2.9-fold to 85 mgL − 1 in shake flasks. Replacing modules with others from the pikromycin and spinosyn synthases yielded new narbonolide derivatives. Deleting, inserting, and mutating modules also resulted in new derivatives, including those of the methymycin precursor 10-deoxymethynolide (10-dml, 4 ). The expression of enzymes that biosynthesize desosamine, modify macrolactones with it, hydroxylate the resulting macrolides, and confer macrolide antibiotic resistance to E. coli enabled the generation of narbomycin ( 2 ), pikromycin ( 3 ), YC-17 ( 5 ), and methymycin ( 6 ), as well as 6 derivatives thereof. Knocking out pathways that compete with desosamine biosynthesis with CRISPR/Cas9 increased narbomycin production to 37 mgL − 1 . A new narbomycin derivative ( 17 ), resulting from the replacement of P3 by P5 , was purified, characterized, and observed to possess antibacterial activity. RESULTS Construction and evaluation of the 2-plasmid system Plasmids p P1 - N P4 and p C P4 - P7 were generated through Gibson assembly using pCDF-1b and pET28b, respectively (Fig. 1 and Supplementary Figs. 1–2). Synthetic DNA encoding docking domain motifs ( C DD and N DD) from the 3rd module of the erythromycin synthase, E3 , was inserted to help noncovalently connect N P4 and C P4 19 . A HindIII-N 12 -SpeI insertion site was placed between the regions encoding P1 and N P4 as well as C P4 and P7 to enable the sequential insertion of fragments encoding modules A – D and generate synthases such as P1 - A - B - P4 - C - D - P7 . The C DD/ N DD pairs used for A , B , C , and D are from the S8 , S3 , E5 , and S5 modules of the spinosyn and erythromycin synthases, respectively. The insertion of the DNA encoding a module preserves the HindIII-N 12 -SpeI insertion site and creates a downstream XbaI/SpeI scar that appears on the polypeptide level as 2 serines in a permissive loop at the updated module boundary 20 , 21 . The cloning plasmids that maintain the updated modules have been previously described 22 . Briefly, they were constructed by inserting synthetic DNA between the HindIII and XbaI sites of pUC19 to introduce a T7 promoter, a lac operator, a ribosome binding site, a T7 terminator, restriction sites for importing DNA fragments encoding the upstream and downstream portions of a module (SpeI/BmtI and MfeI/XbaI), and DNA encoding docking domain motifs to aid the noncovalent assembly of the module (Supplementary Fig. 3). To test the 2-plasmid system, p P1 - N P4 and p C P4 - P7 were transformed into E. coli K207-3 18 . This engineered strain harbors the Bacillus subtilis phosphopantetheinyl transferase Sfp and Streptomyces coelicolor propionyl-CoA carboxylase to respectively activate ACP domains and help convert propionate supplied to the media into (2 S )-methylmalonyl-CoA. Transformed cells were cultured in polyketide production media, and the anticipated product of P1 - P4 - P7 , triketide pyrone 7 , was identified by LC/MS (high-resolution used throughout) of the media extract (Fig. 2 a and Supplementary Fig. 4). Plasmids p P1 - P2 - P3 - N P4 and p C P4 - P5 - P6 - P7 were then constructed and respectively co-transformed with p C P4 - P7 and p P1- N P4 . Pentaketides 8 and 9 , the anticipated products of P1 - P2 - P3 - P4 - P7 and P1 - P4 - P5 - P6 - P7 , were identified by LC/MS of the media extracts (Figs. 2 b-c and Supplementary Figs. 5–6). Plasmids p P1 - P2 - P3 - N P4 and p C P4 - P5 - P6 - P7 were then co-transformed. The media extract contained a compound with the mass expected for narbonolide ( 1 ) (Fig. 2 d and Supplementary Fig. 7). It was purified by silica gel chromatography and semi-preparative HPLC, and its identity was confirmed by NMR (Supplementary Figs. 8–10) 23 . Optimizing narbonolide production from the refactored synthase Fermentation conditions were identified (10 d at 16°C with 60 mM sodium propionate) that yield a narbonolide titer of 28.9 mgL − 1 (Supplementary Fig. S11). LC/MS analysis of the media extract revealed that shunt products (the products expected of P1 - P2 - P7 , P1 - P4 - P7 , P1 - P6 - P7 , P1 - P5 - P6 - P7 , P1 - P2 - P3 - P4 - P7 , P1 - P2 - P5 - P6 - P7 , and P1 - P4 - P5 - P6 - P7 ) were also generated at lower levels (Supplementary Fig. 12a) 22 . We previously hypothesized such shunt products can be produced due to ACPs not being sufficiently restrained by their downstream docking domain motifs 22 . In the refactored synthase, the C DD/ N DD pairs of P3 , P5 , and P6 come from S3 , E5 , and S5 , respectively. Thus, replacing these with their natural docking domain motifs could result in more restrained ACPs, fewer shunt products, and an increased narbonolide titer. The DNA fragments encoding the native C DD/ N DD pairs for P3 , P5 , and P6 were inserted into the P3 , P5 , and P6 cloning plasmids and the natural versions of P3 , P5 , and P6 were independently inserted into the refactored pikromycin synthase. This increased narbonolide production 1.1-, 1.3-, and 1.2-fold, respectively, and the combined replacement of natural P3 , P5 , and P6 increased narbonolide production 1.4-fold to 40.2 mgL − 1 (Supplementary Fig. 12b). Shunt product titers from synthases harboring the natural docking domains were slightly lower (Supplementary Fig. 12a). Next, the effect of the editing thioesterase PikAV on the refactored system was investigated. This TEII is thought to hydrolyze acetyl or propionyl groups inappropriately bound to ACPs formed by untimely, KS-mediated decarboxylations of malonyl and methylmalonyl extender units 24 . Within S. venezuelae , the overexpression, but not the inactivation, of PikAIV has been observed to decrease pikromycin production 24 , 25 . However, for other PKS-containing gene clusters the inactivation of TEII has resulted in decreased polyketide production 26 – 29 . Hypothesizing that some level of PikAV would improve the activity of the refactored pikromycin PKS in E. coli 30 – 32 , PikAV expression plasmids were constructed 33 . A single copy of pikAV on a pACYC vector was observed to maximally increase narbonolide production (2.1-fold to 84.6 mgL − 1 )(Supplementary Fig. 13). Module-swapping to yield narbonolide derivatives With the refactored pikromycin synthase in hand, module-swapping experiments were performed (Fig. 3 ). Modules P2 , P3 , P5 , and P6 were replaced with each of the other extension modules of the pikromycin synthase. Of the 16 module-swapped PKSs, only P1 - P2 - P5 - P4 - P5 - P6 - P7 produced its expected macrolactone ( 10 ), as confirmed by LC/MS and NMR (Fig. 3 and Supplementary Figs. 14–15). Interestingly, this synthase produced an equivalent quantity of d-lactone 11 , as determined by LC/MS and its absorbance spectrum (Supplementary Fig. 14). The overall heptaketide titer from P1 - P2 - P5 - P4 - P5 - P6 - P7 ( 10 + 11 ) is 45% that of the narbonolide titer from P1 - P2 - P3 - P4 - P5 - P6 - P7 . To evaluate the compatibility of the refactored pikromycin PKS with modules from other PKSs, P2 and P6 were respectively replaced by E2 and E6 , erythromycin modules that conduct the same chemistry (Fig. 3 ). The hybrid synthases P1 - E2 - P3 - P4 - P5 - P6 - P7 and P1 - P2 - P3 - P4 - P5 - E6 - P7 respectively produce 3% and 74% the narbonolide titer of P1 - P2 - P3 - P4 - P5 - P6 - P7 (Supplementary Figs. 16–17). The hybrid synthase P1 - S2 - P3 - P4 - P5 - P6 - P7 (with S2 from the spinosyn synthase replacing P2 of the refactored pikromycin synthase) was also constructed and observed to produce a compound with the mass expected for narbonolide derivative 12 at 10% the narbonolide titer of P1 - P2 - P3 - P4 - P5 - P6 - P7 and no d-lactone (Fig. 3 and Supplementary Fig. 18). Accessing hexaketide and octaketide derivatives Within S. venezuelae , the heptaketide narbonolide and the hexaketide 10-dml are produced as intermediates in the biosynthesis of pikromycin and methymycin, respectively 17 . Hexaketide production results from an alternate translation start site for PikAIV that inactivates the P6 KS 34 . To produce 10-dml ( 4 ) in E. coli , a mutagenic approach was employed whereby the P6 KS reactive cysteine is replaced with an alanine (Figs. 1 a and 4 a) 35 – 37 . As confirmed by LC/MS and NMR, the mutated synthase P1 - P2 - P3 - P4 - P5 - P6* - P7 selectively produces 10-dml with a titer 1.5-fold greater than the narbonolide titer from P1 - P2 - P3 - P4 - P5 - P6 - P7 (Fig. 4 a and Supplementary Figs. 19–22). P1 - P2 - P5 - P4 - P5 - P6* - P7 and P1 - S2 - P3 - P4 - P5 - P6* - P7 were constructed, and P1 - S2 - P3 - P4 - P5 - P6* - P7 was observed to produce low levels of compounds with the masses anticipated for 10-dml derivatives 13 and 14 (Figs. 4 b-c and Supplementary Figs. 23–24). Hexaketide synthase P1 - P2 - P4 - P5 - P6 - P7 was also constructed and observed to produce d-lactone 15 at a titer 3.2-fold greater than the narbonolide titer from P1 - P2 - P3 - P4 - P5 - P6 - P7 (Fig. 4 a and Supplementary Fig. 25). d-Lactonization of 15 was confirmed by NMR (Supplementary Fig. 26–29). Since octaketide macrolide antibiotics like tylosin and josamycin are naturally-occuring, the octaketide synthase P1 - P2 - P3 - P4 - P5 - P5 - P6 - P7 was also constructed (Fig. 4 e). A product of the expected mass was observed; however, its absorbance spectrum revealed it to be d-lactone 16 (Supplementary Fig. 26). Interestingly, narbonolide is the major product of this synthase, at a titer 2.1-fold that of 16 . Accessing narbomycin, pikromycin, YC-17, methymycin, and derivatives thereof The desosamine moiety is critical to the biological activities of macrolide antibiotics 38 . Plasmid pDes was constructed to express the desosamine biosynthetic pathway (DesI, DesII, DesV, and DesVI), the desosamine transferase (DesVII/DesVIII) 39,40 , and the macrolide antibiotic resistance enzyme ErmE (Fig. 5 a and Supplementary Fig. 30) 41 . The gene encoding the P450 monooxygenase PikC that converts narbomycin into pikromycin and YC-17 into methymycin, was added to create plasmid pDesPikC 42 . pDes and pDesPikC were transformed into E. coli K207-3 harboring PKS expression plasmids. The transformants were cultured in polyketide production media at 16°C for 10 days, and the media extracts were analyzed by LC/MS. E. coli K207-3 with pDes completely converted narbonolide to narbomycin (1.3 mgL-1), as confirmed by LC/MS and NMR (Fig. 5 a and Supplementary Figs. 31–34) 23 . LC/MS indicates that cells with pDesPikC convert 15% of narbomycin to pikromycin, to provide a pikromycin titer of 0.24 mgL-1 (Fig. 5 b and Supplementary Figs. 35–36). Hypothesizing that the low PikC activity is from low protein expression and/or inefficient reduction by E. coli proteins, PikC expression was supplemented with plasmid pPikC, which contains another copy of pikC 43 . This improved the conversion from narbomycin to 20%, and the pikromycin titer reached 0.31 mgL-1 (Supplementary Fig. 36). P1 - P2 - P3 - P4 - P5 - P6* - P7 was also expressed with pDes and pDesPikC to respectively yield compounds with mass spectra consistent with 5 (YC-17) and 6 (methymycin) (Fig. 5 b and Supplementary Figs. 36–38). NMR confirmed the identity of YC-17 (Supplementary Figs. 39–41). Next, cells with P1 - P2 - P5 - P4 - P5 - P6 - P7 , P1 - S2 - P3 - P4 - P5 - P6 - P7 , P1 - P2 - P5 - P4 - P5 - P6* - P7 , and P1 - S2 - P3 - P4 - P5 - P6* - P7 were transformed with pDes. While the desosaminylated macrolide anticipated from P1 - P2 - P5 - P4 - P5 - P6* - P7 was not observed by LC/MS, the desosaminylated macrolides 17 , 19 , and 21 were observed from the other synthases without traces of their respective aglycone precursors 10 , 11 , and 14 (Fig. 5 b and Supplementary Figs. 42–44). Narbomycin derivative 17 was isolated and characterized by NMR (Supplementary Figs. 45–47). The addition of PikC through pDesPikC + pPikC yielded small quantities of compounds that yield LC/MS data consistent with oxidized macrolides 18 , 20 , and 22 (Fig. 5 b and Supplementary Figs. 48–50). Metabolic engineering boosts titers of macrolide antibiotics and their derivatives The low titers of narbomycin, pikromycin, YC-17, methymycin, and their derivatives hampers investigations of their structures and antibacterial activities. Since no aglycones were observed from E. coli K207-3 generating these macrolides, we hypothesized that the overexpression of the relatively small proteins from pDes, pDesPikC, and pDesPikAV outcompeted PKS expression. Thus, the operon from pDes was transferred to the low copy number (1–2) bacterial artificial chromosome (BAC) pMKBAC02 44 . While narbonolide was completely converted to narbomycin, the narbomycin titer was 69% of that cells transformed with pDes (Fig. 6 ). The operon was also integrated into the E. coli K207-3 genome with CRISPR/Cas9 to yield TM1 cells (Supplementary Fig. 51). Although TM1 cells expressing the refactored pikromycin sythase completely convert narbonolide to narbomycin, the productivity is still only 52% that of E. coli K207-3 transformed with pDes. In a previous study, pathways that utilize TDP-4-keto-6-deoxy- d -glucose were knocked out of E. coli K207-3, and the resulting strains, transformed with plasmids encoding desosamine biosynthesis/transfer genes, were observed to more efficiently add desosamine to supplied macrolactones 45 . TDP-4-keto-6-deoxy- d -glucose is not only a precursor in the biosynthesis of the pikromycin monosaccharide, TDP-D-desosamine, but also of the O -specific polysaccharide chain of the lipopolysaccharide and the glycolipid on the cell surface of E. coli (Fig. 6 a) 45 – 47 . Thus, rmlC , wecD , wecE , vioA , and vioB were inactivated by CRISPR/Cas9 in both E. coli K207-3 and TM1 cells to yield TM4 and TM7 cells, respectively (as well as intermediate strains TM2, TM3, TM5, and TM6) (Fig. 6 b and Supplementary Fig. 52). TM4 and TM7 cells, transformed with the plasmids encoding the refactored pikromycin synthase (and pDes for TM4 cells), produce 24- and 12-fold higher narbomycin titers than TM1 cells, respectively (Fig. 6 c). TM7 cells transformed with the plasmids encoding the refactored pikromycin synthase and pDesPikAV produce a 55-fold higher narbomycin titer than TM1 cells (37.1 mgL − 1 ). Co-transforming TM7 cells with PKS expression plasmids and pDesPikAV or pDesPikC was also observed to boost the titers of other desosaminylated macrolides (Supplementary Table 4). Antibacterial activity Following purification and NMR characterization, narbomycin ( 2 ), YC-17 ( 5 ), and 17 were evaluated for antibacterial activity against Bacillus subtilis 168 (Supplementary Fig. 53). Commercial pikromycin ( 3 ) was used as a positive control. The minimal inhibitory concentration (MIC) values for 3 , 2 , 5 , and 17 were measured to be 3, 6, 50, and 200 µM, respectively 48 , 49 . DISCUSSION Since the erythromycin PKS was sequenced in 1990, scientists have been trying to harness the synthetic power of this and other modular PKSs for the production of designer polyketides and new medicines 11 , 12 . As E. coli is more genetically tractable than the natural erythromycin producer Saccharopolyspora erythraea , a major goal was realized in 2001 when the erythromycin skeleton, 6-deoxyerythronolide B (6-dEB), was produced by E. coli 50 . In 2010, production of the macrolide antibiotic erythromycin A by E. coli was reported 51 . Next to the erythromycin synthase, the pikromycin synthase is the most studied modular PKS 17 , 52 , 53 . This is the first report of the reconstitution of the pikromycin synthase in E. coli to yield narbonolide as well as the macrolide antibiotics narbomycin and pikromycin. The pikromycin pathway has several advantages over the erythromycin pathway: 1) Fewer tailoring enzymes are needed. The erythromycin pathway requires 6 plasmids, which are not stably maintained by cells containing them. 2) Macrolide antibiotics such as narbomycin and YC-17 can be accessed without P450 enzymes, which are not very active in E. coli . The erythromycin pathway contains 2 P450 enzymes, with EryF performing a requisite, first tailoring step. 3) Higher yields of narbomycin (37 mgL − 1 ) can be generated compared to erythromycin A (~ 1 mgL − 1 ). 4) The methymycin/pikromycin pathway is more versatile, with the PKS having evolved to generate both a hexaketide and a heptaketide and DesVII/DesVIII having evolved to transfer d -desosamine to these 12- and 14-membered macrolactones. 5) Furthermore, the BioBricks-like platform developed here for the refactored pikromycin synthase facilitates the modular synthesis of new macrolide antibacterials through the combinatorial construction of PKSs. Both the recently reported 1-plasmid system and the 2-plasmid system described here for PKS expression in E. coli rely on heterologous docking domain pairs to noncovalently connect the N-terminal and C-terminal portions of modules, which are expressed on separate polypeptides 16 , 54 , 55 . Since the current study employs as many as 6 heterologous docking domain pairs per synthase, we compared the performances of heterologous pairs with their natural counterparts. The combined replacement of the S3 , E5 , and S5 docking domain pairs with the docking domains pairs native to P3 , P5 , and P6 improved narbonolide production by 40% (Supplementary Fig. 12). This difference may reflect some degree of co-evolution between docking domains pairs and the modules which they are associated 9 , 10 . While maximal titers may not be important when first accessing new synthases, native docking domain pairs may be subsequently employed to optimize synthase productivity. The most significant impediment to combinatorially engineering the refactored pikromycin PKS is KS gatekeeping. While the use of the updated modules ensures compatability of KSs with the a- and b-substituents they are presented, it does not ensure compatability with substituents beyond the b-carbon. Comprehensive module swapping experiments with the pikromycin modules showed that only 1 out of the 16 singly-swapped synthases in which P2 , P3 , P4 , P5 , or P6 replaces P2 , P3 , P5 , or P6 in the refactored pikromycin synthase yields its expected product. That P1 - P2 - P5 - P4 - P5 - P6 - P7 is functional is consistent with our studies with the 1-plasmid system in which P5 was observed to function downstream of P1 - P2 in P1 - P2 - P5 - P7 , P1 - P2 - P5 - P5 - P7 , and P1 - P2 - P5 - P6 - P7 16 . In these synthases, the P5 KS accepts a triketide with the same substituents and stereochemistries as in its native substrate at the a-, b-, and g-positions and seems to be tolerant of the differences downstream. However, such differences are not always tolerated by this KS. In the octaketide synthase P1 - P2 - P3 - P4 - P5 - P5 - P6 - P7 , the downstream P5 KS is presented the same substituents and stereochemistries as in its native substrate at the a-, b-, and g-positions, yet two-thirds of the pentaketide intermediates skip the downstream P5 module, likely due to low flux through its KS. Thus, even P5 KS, the most promiscuous of the pikromycin KSs, shows a significant preference for its natural substrate. We are currently experimenting with rapamycin modules, since their KSs have demonstrated greater tolerance to unnatural substrates 16 , 56 , 57 . Our results suggest some level of co-evolution between the upstream modules of synthases. As all of the interactions between acyl-ACPs and their downstream KSs are native in P1 - E2 - P3 - P4 - P5 - P6 - P7 , KS gatekeeping does not account for its poor activity. This hybrid synthase may be suboptimal due to poor intermodular interactions ( i.e. , upstream KSs with downstream ACPs); however, the robust activity of P1 - P2 - P3 - P4 - P5 - E6 - P7 suggests this is not a general phenomenon. The specialization of at least the upstream KS domains of PKSs for receiving short substrates indicates that upstream modules may evolutionarily co-migrate 16 . Disruption of these multimodular collaborations could largely explain why E2 and S2 exhibit low activity between P1 and P3 in P1 - E2 - P3 - P4 - P5 - P6 - P7 and P1 - S2 - P3 - P4 - P5 - P6 - P7 . Some engineered PKSs in this study produce a d-lactone instead of, or in addition to, a macrolactone ( P1 - P2 - P4 - P5 - P6 - P7 produces 15 , P1 - P2 - P5 - P4 - P5 - P6 - P7 produces 11 in addition to 10 , P1 - P2 - P3 - P4 - P5 - P5 - P6 - P7 produces 16 in addition to 1 ) (Figs. 3 and 4 d-e and Supplementary Figs. 14, 25–26). Biochemical and structural studies of the pikromycin TE have revealed that functional groups installed by the first 4 pikromycin modules ( P1 – P4 ) aid in the macrocyclization of the natural hexaketide and heptaketide intermediates 58 – 60 . A crystal structure of a version of the pikromycin TE in which 2,3-diaminopropionate replaces the catalytic serine and is connected through an amide linkage to the heptaketide intermediate shows complementarity between active site pockets and the C8 and C12 methyl groups as well as a hydrogen bond between the side chain hydroxyl group of Thr1125 and the C9 enone oxygen 60 . These interactions help position the l -oriented C13 hydroxyl group for nucleophilic attack on the C1 carbonyl; however, the relative contributions of these interactions and those at the ACP/TE interface to macrocylization remain unclear. In the context of PikAIV, the pikromycin TE can macrocyclize a heptaketide intermediate in which the ketone is replaced by a hydroxyl group 59 . In this study, the production of macrolactones 10 and 13 by P1 - P2 - P5 - P4 - P5 - P6 - P7 and P1 - P2 - P5 - P4 - P5 - P6* - P7 , respectively, demonstrates that the double bond is also not required for macrocyclization. Also in this study, the production of macrolactones 12 and 14 by P1 - S2 - P3 - P4 - P5 - P6 - P7 and P1 - S2 - P3 - P4 - P5 - P6* - P7 , respectively, demonstrates that the nucleophilic hydroxy group does not need to be l -oriented 61 . As combinatorial modular syntheses of macrolide antibiotics are explored, more will be learned about the tolerance of pikromycin TE. If it is a significant bottleneck, promiscuity-enhancing mutations, such as the replacement of the reactive serine by cysteine, will be introduced 62 . The described platform provides a means to study mysterious assembly line phenomena. For example, it has been debated whether the full pikromycin PKS produces the hexaketide 10-dml or not. In this study, no 10-dml was detected from P1 - P2 - P3 - P4 - P5 - P6 - P7 . However, the single cysteine-to-alanine point mutation to the P6 KS renders P1 - P2 - P3 - P4 - P5 - P6* - P7 an efficient producer of 10-dml. Why P6 ACP does not transfer the hexaketide intermediate to TE in P1 - P2 - P3 - P4 - P5 - P6 - P7 is not clear. One hypothesis is that KSs are dimeric when acylated by their polyketide intermediate but monomeric in their absence and that the monomeric state enables the P6 ACP and TE to approach one another. P1 - P2 - P3 - P4 - P5 - P5 - P6 - P7 may predominantly produce narbonolide due to a similar module-skipping phenomenon that can be investigated through the platform. Many opportunities exist to boost the titers of polyketides and macrolide antibiotics from E. coli . Genomic deletion of a putative propionyl CoA:succinate CoA transferase, ygfH , increased the titer of erythromycin A 7-fold 63 , 64 .The use of bioreactors rather than shake flasks increased the titer of 6-dEB 5-fold 65 . E. coli K207-3 has not been optimized for general polyketide production; however, now that this chassis organism can produce macrolide antibiotics, avenues to evolve it for enhanced polyketide production have been opened 18 . For example, the macrolide-sensing transcription factor MphR could be used to help select for E. coli K207-3 variants that produce higher titers of narbomycin 66 . To access higher titers of hydroxylated products such as pikromycin and methymycin, PikC activity could be increased through its fusion to a reductase domain 43 . In summary, we have developed a 2-plasmid BioBrick-like system to assemble PKSs from updated modules and employed it to express a refactored pikromycin PKS in E. coli . Through swapping modules and expressing tailoring enzymes from the pikromycin pathway, we modularly synthesized new derivatives of narbonolide, narbomycin, and pikromycin as well as 10-dml, YC-17, and methymycin. Since many strategies beyond those employed here can further boost the yields of macrolide antibiotics, the described platform may be used beyond advancing our understanding of PKS enzymology to access new macrolide antibiotics at preparative levels. Our E. coli -based PKS engineering platform is also higher throughput than streptomyces-based platforms 67 , 68 . We envision it being employed to generate combinatorial libraries of promising polyketide drug leads that will accelerate the medicinal chemistry of these synthetically-challenging compounds. Declarations Data availability. All data are available through the Supplementary Information document. Biological materials. E. coli K207-3 cells are available from the authors upon request. Acknowledgments. This work was supported by the NIH (GM145992) and the Welch Foundation (F-1712). Author contributions. T.M. and A.T.K. designed the research. T.M. performed experiments. T.M. and A.T.K. wrote the manuscript. Competing interests. The authors declare no competing interests. Inclusion & ethics statement. All participants in this research were properly credited. Supplementary information. Supplementary methods, figures, and tables are available in the Supplementary Information document. References Brown, E. D. & Wright, G. D. Antibacterial drug discovery in the resistance era. 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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-5640596","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":398788470,"identity":"bed530f3-1c14-4842-9c2a-df99fff5d3e3","order_by":0,"name":"Adrian Keatinge-Clay","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYFCCBIYDIIIfWYQ4LZINzCRoARMGB4jVwt+e+/DAzx1p8sY38g8+5qmpY+BnzzHAq0XizHODg71ncgy33UhmNpxx7DCDZM8b/FoYbqQxHOBtq0gwu5HMJvGB7QCDwQ0CtsgDtRz8C9RiPCOZ/UfCvzoGe0JaDIBaDvO25SQYSCSzMXxsY2YwkCCgxfDMM4bDsm1phjPOPDaWnNl3mEfizLMCvFrkjqcxf3zblizP35748DPPtzo5/vbkDXi1YAAe0pSPglEwCkbBKMAKAA8TSQMRjKDfAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4358-7628","institution":"UT Austin","correspondingAuthor":true,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Keatinge-Clay","suffix":""},{"id":398788471,"identity":"f9b44496-4b8a-42c8-b06c-320116f0024f","order_by":1,"name":"Takeshi Miyazawa","email":"","orcid":"","institution":"UT Austin","correspondingAuthor":false,"prefix":"","firstName":"Takeshi","middleName":"","lastName":"Miyazawa","suffix":""}],"badges":[],"createdAt":"2024-12-13 21:35:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5640596/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5640596/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73303002,"identity":"3164d406-60d9-45f7-9a8a-1ff6bb2e4338","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":418773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePikromycin PKS and 2-plasmid platform for constructing engineered PKSs. a\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003ePikromycin/methymycin biosynthetic pathway (PKS colored by updated modules).\u003cstrong\u003e b\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eConstruction of the BioBrick-like units encoding pikromycin modules. T7 promoters and terminators are shown. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eThe 2-plasmid system enables the BioBrick-like assembly of PKSs such as \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eA\u003c/strong\u003e-\u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e. Docking domains (DDs) are specific for each position in the constructed synthase. The ligation of HindIII-XbaI fragments into the HindIII-N\u003csub\u003e12\u003c/sub\u003e-SpeI insertion sites yields XbaI/SpeI scars (*) encoding 2 serines at module boundaries.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/95387b667ad725496b7f3864.png"},{"id":73303724,"identity":"52a49e22-e499-401f-96c8-c73a2687aa56","added_by":"auto","created_at":"2025-01-08 16:33:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":284247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of the 2-plasmid system and narbonolide biosynthesis by refactored pikromycin PKS. \u003c/strong\u003eExpected products are observed from\u003cstrong\u003e P1\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e,\u003cstrong\u003e P1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, and\u003cstrong\u003e P1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/80db6414134e23f91dafd9c0.png"},{"id":73303001,"identity":"69c46207-9e4a-4dd0-bd7d-2b904f578a40","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":139984,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModule swapping of the refactored pikromycin PKS. \u003c/strong\u003eWhile 16 swaps with pikromycin modules were attempted, only the replacement of \u003cstrong\u003eP3\u003c/strong\u003e with \u003cstrong\u003eP5\u003c/strong\u003eyielded an active PKS. This synthase generates anticipated macrolactone \u003cstrong\u003e10\u003c/strong\u003eas well as d-lactone \u003cstrong\u003e11\u003c/strong\u003e. Swapping \u003cstrong\u003eP2\u003c/strong\u003e and \u003cstrong\u003eP6\u003c/strong\u003e for \u003cstrong\u003eE2\u003c/strong\u003eand \u003cstrong\u003eE6\u003c/strong\u003e (from the erythromycin PKS), respectively, yielded hybrid synthases that generate narbonolide (\u003cstrong\u003e1\u003c/strong\u003e). Swapping \u003cstrong\u003eP2\u003c/strong\u003e for \u003cstrong\u003eS2\u003c/strong\u003e(from the spinosyn PKS) yielded a hybrid synthase that generates macrolactone \u003cstrong\u003e12\u003c/strong\u003e. Titers are reported in comparison to that of narbonolide by the refactored pikromycin PKS.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/e19bd1cac2f73e5d620225e2.png"},{"id":73303004,"identity":"6457a17a-a2f1-4c2e-b250-3898d8e1acdd","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":347962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConstruction of hexaketide and octaketide synthases. \u003c/strong\u003eHexaketide synthases \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6*\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6*\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eS2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6*\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, and \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, respectively produce \u003cstrong\u003e4\u003c/strong\u003e (10-dml),\u003cstrong\u003e 13\u003c/strong\u003e, \u003cstrong\u003e14\u003c/strong\u003e, and \u003cstrong\u003e15\u003c/strong\u003e. The asterisk (*) next to the \u003cstrong\u003eP6\u003c/strong\u003e KS indicates the C-\u0026gt;A mutation. The octaketide synthase \u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7 \u003c/strong\u003edoes not produce the anticipated octaketide macrolactone but does produce octaketide d-lactone \u003cstrong\u003e16\u003c/strong\u003e and twice as much heptaketide macrolactone \u003cstrong\u003e1\u003c/strong\u003e (narbonolide). Docking domain (DD) motifs from \u003cstrong\u003eA1\u003c/strong\u003e of the amphotericin synthase were used for the upstream \u003cstrong\u003eP5\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/61a488fd5cb7fd02765e5a76.png"},{"id":73303010,"identity":"42adf2b7-c536-439f-b722-a311ebbeeb29","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":814264,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProduction of macrolide antibiotics in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e E. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003ePlasmids pDes, pDesPikC, and pDesPikAV encode desosamine biosynthesis/transfer and macrolide resistance enzymes. Plasmids pDesPikC and pDesPikAV additionally encode the P450 monooxygenase PikC and the editing thioesterase PikAV, respectively. PikAV is controlled by a second T7 promoter in pDesPikAV. \u003cstrong\u003eb\u003c/strong\u003e, With the exception of \u003cstrong\u003e13\u003c/strong\u003e, each of the macrolactones in this study is glycosylated by DesVII/DesVIII and hydroxylated by PikC to yield known macrolide antibiotics or derivatives thereof.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/cc061f0be6ba9a383653f6ed.png"},{"id":73303011,"identity":"ff171d8e-884f-4c52-8ed7-dea529cc6268","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":271432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRISPR/Cas9 engineering helps boost titers of macrolide antibiotics. a\u003c/strong\u003e, Biosynthetic pathways in \u003cem\u003eE. coli\u003c/em\u003e compete with desosamine biosynthesis for TDP-4-keto-6-deoxy-d-glucose. \u003cstrong\u003eb\u003c/strong\u003e, The TM1 strain was engineered by inserting the desosamine biosynthesis/transfer genes into the \u003cem\u003eE. coli\u003c/em\u003e K207-3 genome. The TM2-TM4 and TM5-TM7 strains were engineered through sequentially inactivating \u003cem\u003ermlC\u003c/em\u003e, \u003cem\u003ewecD\u003c/em\u003e/\u003cem\u003eE\u003c/em\u003e, \u003cem\u003evioA\u003c/em\u003e/\u003cem\u003eB\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e K207-3 and TM1, respectively (Supplementary Table 1). \u003cstrong\u003ec\u003c/strong\u003e, Narbomycin production is reported for engineered \u003cem\u003eE. coli\u003c/em\u003e strains transformed with p\u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003csup\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP4\u003c/strong\u003e \u0026amp; p\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, encoding the refactored pikromycin synthase, as well as an additional plasmid or BAC that encodes enzymes from the pikromycin biosynthetic pathway (Fig. 5a). TM7 cells with p\u003cstrong\u003eP1\u003c/strong\u003e-\u003cstrong\u003eP2\u003c/strong\u003e-\u003cstrong\u003eP3\u003c/strong\u003e-\u003csup\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP4\u003c/strong\u003e, p\u003csup\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eP4\u003c/strong\u003e-\u003cstrong\u003eP5\u003c/strong\u003e-\u003cstrong\u003eP6\u003c/strong\u003e-\u003cstrong\u003eP7\u003c/strong\u003e, and pDesPikAIV yield a narbomycin (\u003cstrong\u003e2\u003c/strong\u003e) titer of 37.1 mgL\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/084243e117b5eac30ecc9c26.png"},{"id":73304733,"identity":"2fc378b2-0b12-4614-a012-7d7b3e628c37","added_by":"auto","created_at":"2025-01-08 16:49:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3102738,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/3b68d52f-d9dc-4adb-aa58-f142927c6a40.pdf"},{"id":73303008,"identity":"f298d2a5-a6aa-419e-a099-b44bd767eed9","added_by":"auto","created_at":"2025-01-08 16:25:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10780047,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SI.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5640596/v1/71d83ad579cb03bed4e142dc.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Refactoring the pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAntimicrobial resistance is an ever-increasing threat to human health. Consequently, there is a growing need to discover and develop new antibiotics\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Unfortunately, the pace of both discovering new antibiotics and developing antibiotics through medicinal chemistry has slowed\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Modular synthesis, through which diversity is created from relatively simple building blocks, can accelerate these processes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. A modular chemical synthesis recently yielded a library of \u0026gt;\u0026thinsp;300 macrolide antibiotic derivatives, 2 of which displayed superior potencies compared to those in clinical use\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe modular syntheses of macrolide antibiotics, such as erythromycin, pikromycin, and tylosin, were first conducted by polyketide synthase (PKS) assembly lines within actinomyces bacteria\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These assembly lines are themselves modular, where each module (updated definition used\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e) adds one ketide monomer to a growing polyketide chain. They minimally contain 3 domains - an acyltransferase (AT), an acyl carrier protein (ACP), and a ketosynthase (KS). During the catalytic cycle of a module, ACP acquires the polyketide chain from the KS of the upstream module through an a-carboxyacyl extender unit obtained from AT, shuttles the extended chain to optional processing enzymes, such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, and to KS, which acquires properly processed chains through transacylation. The most downstream domain of the assembly line is most commonly a thioesterase (TE) that releases the polyketide chain through cyclization or hydrolysis.\u003c/p\u003e \u003cp\u003eMost efforts to engineer PKSs have been based on the traditional module boundary and have not been successful\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. This boundary was defined immediately upstream of KS when the genes encoding the erythromycin PKS were sequenced in 1990, in analogy with the mammalian fatty acid synthase (FAS), an iterative PKS in which KS is the most upstream domain\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In 2017, genome-mining of modular PKSs that produce related aminopolyols helped reveal that the KSs of modular PKSs evolutionarily co-migrate with processing domains upstream of them and that the module boundary is actually immediately downstream of KS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Indeed, synthases engineered with the updated module boundary consistently outperform synthases engineered with the traditional module boundary\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Our lab recently developed a BioBrick-like platform to rapidly assemble synthases on a single expression plasmid through the ligation of DNA fragments encoding updated modules. We employed it to combinatorially assemble the modules of the pikromycin synthase (\u003cem\u003eStreptomyces venezuelae\u003c/em\u003e ATCC 15439) into 5 triketide, 25 tetraketide, and 125 pentaketide synthases and assess their function\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we describe a 2-plasmid system that enables the expression of a refactored pikromycin synthase in \u003cem\u003eE. coli\u003c/em\u003e as well as the combinatorial biosynthesis of new macrolactones and macrolide antibiotics (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. One plasmid encodes the 1st pikromycin module (\u003cb\u003eP1\u003c/b\u003e) and the upstream portion of the 4th module (\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e), while the other plasmid encodes the downstream portion of the 4th module (\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e) and the 7th module (\u003cb\u003eP7\u003c/b\u003e). Module-encoding inserts can be sequentially ligated into both plasmids. The incorporation of \u003cb\u003eP2\u003c/b\u003e, \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e into this system yielded a functional, refactored pikromycin synthase in \u003cem\u003eE. coli\u003c/em\u003e K207-3, comprised of 6 polypeptides rather than the 4 natural ones (PikAI-PikAIV)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Docking domain optimization and expression of the editing thioesterase PikAV improved narbonolide (\u003cb\u003e1\u003c/b\u003e) production 2.9-fold to 85 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in shake flasks. Replacing modules with others from the pikromycin and spinosyn synthases yielded new narbonolide derivatives. Deleting, inserting, and mutating modules also resulted in new derivatives, including those of the methymycin precursor 10-deoxymethynolide (10-dml, \u003cb\u003e4\u003c/b\u003e). The expression of enzymes that biosynthesize desosamine, modify macrolactones with it, hydroxylate the resulting macrolides, and confer macrolide antibiotic resistance to \u003cem\u003eE. coli\u003c/em\u003e enabled the generation of narbomycin (\u003cb\u003e2\u003c/b\u003e), pikromycin (\u003cb\u003e3\u003c/b\u003e), YC-17 (\u003cb\u003e5\u003c/b\u003e), and methymycin (\u003cb\u003e6\u003c/b\u003e), as well as 6 derivatives thereof. Knocking out pathways that compete with desosamine biosynthesis with CRISPR/Cas9 increased narbomycin production to 37 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. A new narbomycin derivative (\u003cb\u003e17\u003c/b\u003e), resulting from the replacement of \u003cb\u003eP3\u003c/b\u003e by \u003cb\u003eP5\u003c/b\u003e, was purified, characterized, and observed to possess antibacterial activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and evaluation of the 2-plasmid system\u003c/h2\u003e \u003cp\u003ePlasmids p\u003cb\u003eP1\u003c/b\u003e-\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and p\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were generated through Gibson assembly using pCDF-1b and pET28b, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary Figs.\u0026nbsp;1\u0026ndash;2). Synthetic DNA encoding docking domain motifs (\u003csup\u003eC\u003c/sup\u003eDD and \u003csup\u003eN\u003c/sup\u003eDD) from the 3rd module of the erythromycin synthase, \u003cb\u003eE3\u003c/b\u003e, was inserted to help noncovalently connect \u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and \u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e\u003csup\u003e19\u003c/sup\u003e. A HindIII-N\u003csub\u003e12\u003c/sub\u003e-SpeI insertion site was placed between the regions encoding \u003cb\u003eP1\u003c/b\u003e and \u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e as well as \u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and \u003cb\u003eP7\u003c/b\u003e to enable the sequential insertion of fragments encoding modules \u003cb\u003eA\u003c/b\u003e\u0026ndash;\u003cb\u003eD\u003c/b\u003e and generate synthases such as \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eA\u003c/b\u003e-\u003cb\u003eB\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eC\u003c/b\u003e-\u003cb\u003eD\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e. The \u003csup\u003eC\u003c/sup\u003eDD/\u003csup\u003eN\u003c/sup\u003eDD pairs used for \u003cb\u003eA\u003c/b\u003e, \u003cb\u003eB\u003c/b\u003e, \u003cb\u003eC\u003c/b\u003e, and \u003cb\u003eD\u003c/b\u003e are from the \u003cb\u003eS8\u003c/b\u003e, \u003cb\u003eS3\u003c/b\u003e, \u003cb\u003eE5\u003c/b\u003e, and \u003cb\u003eS5\u003c/b\u003e modules of the spinosyn and erythromycin synthases, respectively. The insertion of the DNA encoding a module preserves the HindIII-N\u003csub\u003e12\u003c/sub\u003e-SpeI insertion site and creates a downstream XbaI/SpeI scar that appears on the polypeptide level as 2 serines in a permissive loop at the updated module boundary\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The cloning plasmids that maintain the updated modules have been previously described\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Briefly, they were constructed by inserting synthetic DNA between the HindIII and XbaI sites of pUC19 to introduce a T7 promoter, a \u003cem\u003elac\u003c/em\u003e operator, a ribosome binding site, a T7 terminator, restriction sites for importing DNA fragments encoding the upstream and downstream portions of a module (SpeI/BmtI and MfeI/XbaI), and DNA encoding docking domain motifs to aid the noncovalent assembly of the module (Supplementary Fig.\u0026nbsp;3).\u003c/p\u003e \u003cp\u003eTo test the 2-plasmid system, p\u003cb\u003eP1\u003c/b\u003e-\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and p\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were transformed into \u003cem\u003eE. coli\u003c/em\u003e K207-3\u003csup\u003e18\u003c/sup\u003e. This engineered strain harbors the \u003cem\u003eBacillus subtilis\u003c/em\u003e phosphopantetheinyl transferase Sfp and \u003cem\u003eStreptomyces coelicolor\u003c/em\u003e propionyl-CoA carboxylase to respectively activate ACP domains and help convert propionate supplied to the media into (2\u003cem\u003eS\u003c/em\u003e)-methylmalonyl-CoA. Transformed cells were cultured in polyketide production media, and the anticipated product of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, triketide pyrone \u003cb\u003e7\u003c/b\u003e, was identified by LC/MS (high-resolution used throughout) of the media extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;4). Plasmids p\u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and p\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were then constructed and respectively co-transformed with p\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and p\u003cb\u003eP1-\u003c/b\u003e\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e. Pentaketides \u003cb\u003e8\u003c/b\u003e and \u003cb\u003e9\u003c/b\u003e, the anticipated products of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, were identified by LC/MS of the media extracts (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c and Supplementary Figs.\u0026nbsp;5\u0026ndash;6). Plasmids p\u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003csup\u003e\u003cb\u003eN\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e and p\u003csup\u003e\u003cb\u003eC\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were then co-transformed. The media extract contained a compound with the mass expected for narbonolide (\u003cb\u003e1\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed and Supplementary Fig.\u0026nbsp;7). It was purified by silica gel chromatography and semi-preparative HPLC, and its identity was confirmed by NMR (Supplementary Figs.\u0026nbsp;8\u0026ndash;10)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOptimizing narbonolide production from the refactored synthase\u003c/h3\u003e\n\u003cp\u003eFermentation conditions were identified (10 d at 16\u0026deg;C with 60 mM sodium propionate) that yield a narbonolide titer of 28.9 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig. S11). LC/MS analysis of the media extract revealed that shunt products (the products expected of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e) were also generated at lower levels (Supplementary Fig.\u0026nbsp;12a)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We previously hypothesized such shunt products can be produced due to ACPs not being sufficiently restrained by their downstream docking domain motifs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In the refactored synthase, the \u003csup\u003eC\u003c/sup\u003eDD/\u003csup\u003eN\u003c/sup\u003eDD pairs of \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e come from \u003cb\u003eS3\u003c/b\u003e, \u003cb\u003eE5\u003c/b\u003e, and \u003cb\u003eS5\u003c/b\u003e, respectively. Thus, replacing these with their natural docking domain motifs could result in more restrained ACPs, fewer shunt products, and an increased narbonolide titer. The DNA fragments encoding the native \u003csup\u003eC\u003c/sup\u003eDD/\u003csup\u003eN\u003c/sup\u003eDD pairs for \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e were inserted into the \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e cloning plasmids and the natural versions of \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e were independently inserted into the refactored pikromycin synthase. This increased narbonolide production 1.1-, 1.3-, and 1.2-fold, respectively, and the combined replacement of natural \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e increased narbonolide production 1.4-fold to 40.2 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;12b). Shunt product titers from synthases harboring the natural docking domains were slightly lower (Supplementary Fig.\u0026nbsp;12a).\u003c/p\u003e \u003cp\u003eNext, the effect of the editing thioesterase PikAV on the refactored system was investigated. This TEII is thought to hydrolyze acetyl or propionyl groups inappropriately bound to ACPs formed by untimely, KS-mediated decarboxylations of malonyl and methylmalonyl extender units\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Within \u003cem\u003eS. venezuelae\u003c/em\u003e, the overexpression, but not the inactivation, of PikAIV has been observed to decrease pikromycin production\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, for other PKS-containing gene clusters the inactivation of TEII has resulted in decreased polyketide production\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Hypothesizing that some level of PikAV would improve the activity of the refactored pikromycin PKS in \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, PikAV expression plasmids were constructed\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. A single copy of \u003cem\u003epikAV\u003c/em\u003e on a pACYC vector was observed to maximally increase narbonolide production (2.1-fold to 84.6 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)(Supplementary Fig.\u0026nbsp;13).\u003c/p\u003e\n\u003ch3\u003eModule-swapping to yield narbonolide derivatives\u003c/h3\u003e\n\u003cp\u003eWith the refactored pikromycin synthase in hand, module-swapping experiments were performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Modules \u003cb\u003eP2\u003c/b\u003e, \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e were replaced with each of the other extension modules of the pikromycin synthase. Of the 16 module-swapped PKSs, only \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e produced its expected macrolactone (\u003cb\u003e10\u003c/b\u003e), as confirmed by LC/MS and NMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Figs.\u0026nbsp;14\u0026ndash;15). Interestingly, this synthase produced an equivalent quantity of d-lactone \u003cb\u003e11\u003c/b\u003e, as determined by LC/MS and its absorbance spectrum (Supplementary Fig.\u0026nbsp;14). The overall heptaketide titer from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e (\u003cb\u003e10\u003c/b\u003e\u0026thinsp;+\u0026thinsp;\u003cb\u003e11\u003c/b\u003e) is 45% that of the narbonolide titer from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo evaluate the compatibility of the refactored pikromycin PKS with modules from other PKSs, \u003cb\u003eP2\u003c/b\u003e and \u003cb\u003eP6\u003c/b\u003e were respectively replaced by \u003cb\u003eE2\u003c/b\u003e and \u003cb\u003eE6\u003c/b\u003e, erythromycin modules that conduct the same chemistry (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The hybrid synthases \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eE2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eE6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e respectively produce 3% and 74% the narbonolide titer of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e (Supplementary Figs.\u0026nbsp;16\u0026ndash;17). The hybrid synthase \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e (with \u003cb\u003eS2\u003c/b\u003e from the spinosyn synthase replacing \u003cb\u003eP2\u003c/b\u003e of the refactored pikromycin synthase) was also constructed and observed to produce a compound with the mass expected for narbonolide derivative \u003cb\u003e12\u003c/b\u003e at 10% the narbonolide titer of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and no d-lactone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Fig.\u0026nbsp;18).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAccessing hexaketide and octaketide derivatives\u003c/h3\u003e\n\u003cp\u003eWithin \u003cem\u003eS. venezuelae\u003c/em\u003e, the heptaketide narbonolide and the hexaketide 10-dml are produced as intermediates in the biosynthesis of pikromycin and methymycin, respectively\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Hexaketide production results from an alternate translation start site for PikAIV that inactivates the \u003cb\u003eP6\u003c/b\u003e KS\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. To produce 10-dml (\u003cb\u003e4\u003c/b\u003e) in \u003cem\u003eE. coli\u003c/em\u003e, a mutagenic approach was employed whereby the \u003cb\u003eP6\u003c/b\u003e KS reactive cysteine is replaced with an alanine (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. As confirmed by LC/MS and NMR, the mutated synthase \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e selectively produces 10-dml with a titer 1.5-fold greater than the narbonolide titer from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Supplementary Figs.\u0026nbsp;19\u0026ndash;22). \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were constructed, and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e was observed to produce low levels of compounds with the masses anticipated for 10-dml derivatives \u003cb\u003e13\u003c/b\u003e and \u003cb\u003e14\u003c/b\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c and Supplementary Figs.\u0026nbsp;23\u0026ndash;24). Hexaketide synthase \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e was also constructed and observed to produce d-lactone \u003cb\u003e15\u003c/b\u003e at a titer 3.2-fold greater than the narbonolide titer from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;25). d-Lactonization of \u003cb\u003e15\u003c/b\u003e was confirmed by NMR (Supplementary Fig.\u0026nbsp;26\u0026ndash;29). Since octaketide macrolide antibiotics like tylosin and josamycin are naturally-occuring, the octaketide synthase \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e was also constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). A product of the expected mass was observed; however, its absorbance spectrum revealed it to be d-lactone \u003cb\u003e16\u003c/b\u003e (Supplementary Fig.\u0026nbsp;26). Interestingly, narbonolide is the major product of this synthase, at a titer 2.1-fold that of \u003cb\u003e16\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAccessing narbomycin, pikromycin, YC-17, methymycin, and derivatives thereof\u003c/h3\u003e\n\u003cp\u003eThe desosamine moiety is critical to the biological activities of macrolide antibiotics\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Plasmid pDes was constructed to express the desosamine biosynthetic pathway (DesI, DesII, DesV, and DesVI), the desosamine transferase (DesVII/DesVIII)\u003csup\u003e39,40\u003c/sup\u003e, and the macrolide antibiotic resistance enzyme ErmE (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;30)\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The gene encoding the P450 monooxygenase PikC that converts narbomycin into pikromycin and YC-17 into methymycin, was added to create plasmid pDesPikC\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. pDes and pDesPikC were transformed into \u003cem\u003eE. coli\u003c/em\u003e K207-3 harboring PKS expression plasmids. The transformants were cultured in polyketide production media at 16\u0026deg;C for 10 days, and the media extracts were analyzed by LC/MS. \u003cem\u003eE. coli\u003c/em\u003e K207-3 with pDes completely converted narbonolide to narbomycin (1.3 mgL-1), as confirmed by LC/MS and NMR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Supplementary Figs.\u0026nbsp;31\u0026ndash;34)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. LC/MS indicates that cells with pDesPikC convert 15% of narbomycin to pikromycin, to provide a pikromycin titer of 0.24 mgL-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Figs.\u0026nbsp;35\u0026ndash;36). Hypothesizing that the low PikC activity is from low protein expression and/or inefficient reduction by \u003cem\u003eE. coli\u003c/em\u003e proteins, PikC expression was supplemented with plasmid pPikC, which contains another copy of \u003cem\u003epikC\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. This improved the conversion from narbomycin to 20%, and the pikromycin titer reached 0.31 mgL-1 (Supplementary Fig.\u0026nbsp;36). \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e was also expressed with pDes and pDesPikC to respectively yield compounds with mass spectra consistent with \u003cb\u003e5\u003c/b\u003e (YC-17) and \u003cb\u003e6\u003c/b\u003e (methymycin) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Figs.\u0026nbsp;36\u0026ndash;38). NMR confirmed the identity of YC-17 (Supplementary Figs.\u0026nbsp;39\u0026ndash;41).\u003c/p\u003e \u003cp\u003eNext, cells with \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e were transformed with pDes. While the desosaminylated macrolide anticipated from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e was not observed by LC/MS, the desosaminylated macrolides \u003cb\u003e17\u003c/b\u003e, \u003cb\u003e19\u003c/b\u003e, and \u003cb\u003e21\u003c/b\u003e were observed from the other synthases without traces of their respective aglycone precursors \u003cb\u003e10\u003c/b\u003e, \u003cb\u003e11\u003c/b\u003e, and \u003cb\u003e14\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Figs.\u0026nbsp;42\u0026ndash;44). Narbomycin derivative \u003cb\u003e17\u003c/b\u003e was isolated and characterized by NMR (Supplementary Figs.\u0026nbsp;45\u0026ndash;47). The addition of PikC through pDesPikC\u0026thinsp;+\u0026thinsp;pPikC yielded small quantities of compounds that yield LC/MS data consistent with oxidized macrolides \u003cb\u003e18\u003c/b\u003e, \u003cb\u003e20\u003c/b\u003e, and \u003cb\u003e22\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Supplementary Figs.\u0026nbsp;48\u0026ndash;50).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMetabolic engineering boosts titers of macrolide antibiotics and their derivatives\u003c/h2\u003e \u003cp\u003eThe low titers of narbomycin, pikromycin, YC-17, methymycin, and their derivatives hampers investigations of their structures and antibacterial activities. Since no aglycones were observed from \u003cem\u003eE. coli\u003c/em\u003e K207-3 generating these macrolides, we hypothesized that the overexpression of the relatively small proteins from pDes, pDesPikC, and pDesPikAV outcompeted PKS expression. Thus, the operon from pDes was transferred to the low copy number (1\u0026ndash;2) bacterial artificial chromosome (BAC) pMKBAC02\u003csup\u003e44\u003c/sup\u003e. While narbonolide was completely converted to narbomycin, the narbomycin titer was 69% of that cells transformed with pDes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The operon was also integrated into the \u003cem\u003eE. coli\u003c/em\u003e K207-3 genome with CRISPR/Cas9 to yield TM1 cells (Supplementary Fig.\u0026nbsp;51). Although TM1 cells expressing the refactored pikromycin sythase completely convert narbonolide to narbomycin, the productivity is still only 52% that of \u003cem\u003eE. coli\u003c/em\u003e K207-3 transformed with pDes.\u003c/p\u003e \u003cp\u003eIn a previous study, pathways that utilize TDP-4-keto-6-deoxy-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-glucose were knocked out of \u003cem\u003eE. coli\u003c/em\u003e K207-3, and the resulting strains, transformed with plasmids encoding desosamine biosynthesis/transfer genes, were observed to more efficiently add desosamine to supplied macrolactones\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. TDP-4-keto-6-deoxy-\u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-glucose is not only a precursor in the biosynthesis of the pikromycin monosaccharide, TDP-D-desosamine, but also of the \u003cem\u003eO\u003c/em\u003e-specific polysaccharide chain of the lipopolysaccharide and the glycolipid on the cell surface of \u003cem\u003eE. coli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea)\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Thus, \u003cem\u003ermlC\u003c/em\u003e, \u003cem\u003ewecD\u003c/em\u003e, \u003cem\u003ewecE\u003c/em\u003e, \u003cem\u003evioA\u003c/em\u003e, and \u003cem\u003evioB\u003c/em\u003e were inactivated by CRISPR/Cas9 in both \u003cem\u003eE. coli\u003c/em\u003e K207-3 and TM1 cells to yield TM4 and TM7 cells, respectively (as well as intermediate strains TM2, TM3, TM5, and TM6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;52). TM4 and TM7 cells, transformed with the plasmids encoding the refactored pikromycin synthase (and pDes for TM4 cells), produce 24- and 12-fold higher narbomycin titers than TM1 cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). TM7 cells transformed with the plasmids encoding the refactored pikromycin synthase and pDesPikAV produce a 55-fold higher narbomycin titer than TM1 cells (37.1 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Co-transforming TM7 cells with PKS expression plasmids and pDesPikAV or pDesPikC was also observed to boost the titers of other desosaminylated macrolides (Supplementary Table\u0026nbsp;4).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibacterial activity\u003c/h3\u003e\n\u003cp\u003eFollowing purification and NMR characterization, narbomycin (\u003cb\u003e2\u003c/b\u003e), YC-17 (\u003cb\u003e5\u003c/b\u003e), and \u003cb\u003e17\u003c/b\u003e were evaluated for antibacterial activity against \u003cem\u003eBacillus subtilis 168\u003c/em\u003e (Supplementary Fig.\u0026nbsp;53). Commercial pikromycin (\u003cb\u003e3\u003c/b\u003e) was used as a positive control. The minimal inhibitory concentration (MIC) values for \u003cb\u003e3\u003c/b\u003e, \u003cb\u003e2\u003c/b\u003e, \u003cb\u003e5\u003c/b\u003e, and \u003cb\u003e17\u003c/b\u003e were measured to be 3, 6, 50, and 200 \u0026micro;M, respectively\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSince the erythromycin PKS was sequenced in 1990, scientists have been trying to harness the synthetic power of this and other modular PKSs for the production of designer polyketides and new medicines\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. As \u003cem\u003eE. coli\u003c/em\u003e is more genetically tractable than the natural erythromycin producer \u003cem\u003eSaccharopolyspora erythraea\u003c/em\u003e, a major goal was realized in 2001 when the erythromycin skeleton, 6-deoxyerythronolide B (6-dEB), was produced by \u003cem\u003eE. coli\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In 2010, production of the macrolide antibiotic erythromycin A by \u003cem\u003eE. coli\u003c/em\u003e was reported\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNext to the erythromycin synthase, the pikromycin synthase is the most studied modular PKS\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. This is the first report of the reconstitution of the pikromycin synthase in \u003cem\u003eE. coli\u003c/em\u003e to yield narbonolide as well as the macrolide antibiotics narbomycin and pikromycin. The pikromycin pathway has several advantages over the erythromycin pathway: 1) Fewer tailoring enzymes are needed. The erythromycin pathway requires 6 plasmids, which are not stably maintained by cells containing them. 2) Macrolide antibiotics such as narbomycin and YC-17 can be accessed without P450 enzymes, which are not very active in \u003cem\u003eE. coli\u003c/em\u003e. The erythromycin pathway contains 2 P450 enzymes, with EryF performing a requisite, first tailoring step. 3) Higher yields of narbomycin (37 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) can be generated compared to erythromycin A (~\u0026thinsp;1 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). 4) The methymycin/pikromycin pathway is more versatile, with the PKS having evolved to generate both a hexaketide and a heptaketide and DesVII/DesVIII having evolved to transfer \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003ed\u003c/span\u003e-desosamine to these 12- and 14-membered macrolactones. 5) Furthermore, the BioBricks-like platform developed here for the refactored pikromycin synthase facilitates the modular synthesis of new macrolide antibacterials through the combinatorial construction of PKSs.\u003c/p\u003e \u003cp\u003eBoth the recently reported 1-plasmid system and the 2-plasmid system described here for PKS expression in \u003cem\u003eE. coli\u003c/em\u003e rely on heterologous docking domain pairs to noncovalently connect the N-terminal and C-terminal portions of modules, which are expressed on separate polypeptides\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Since the current study employs as many as 6 heterologous docking domain pairs per synthase, we compared the performances of heterologous pairs with their natural counterparts. The combined replacement of the \u003cb\u003eS3\u003c/b\u003e, \u003cb\u003eE5\u003c/b\u003e, and \u003cb\u003eS5\u003c/b\u003e docking domain pairs with the docking domains pairs native to \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, and \u003cb\u003eP6\u003c/b\u003e improved narbonolide production by 40% (Supplementary Fig.\u0026nbsp;12). This difference may reflect some degree of co-evolution between docking domains pairs and the modules which they are associated\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While maximal titers may not be important when first accessing new synthases, native docking domain pairs may be subsequently employed to optimize synthase productivity.\u003c/p\u003e \u003cp\u003eThe most significant impediment to combinatorially engineering the refactored pikromycin PKS is KS gatekeeping. While the use of the updated modules ensures compatability of KSs with the a- and b-substituents they are presented, it does not ensure compatability with substituents beyond the b-carbon. Comprehensive module swapping experiments with the pikromycin modules showed that only 1 out of the 16 singly-swapped synthases in which \u003cb\u003eP2\u003c/b\u003e, \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP4\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, or \u003cb\u003eP6\u003c/b\u003e replaces \u003cb\u003eP2\u003c/b\u003e, \u003cb\u003eP3\u003c/b\u003e, \u003cb\u003eP5\u003c/b\u003e, or \u003cb\u003eP6\u003c/b\u003e in the refactored pikromycin synthase yields its expected product. That \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e is functional is consistent with our studies with the 1-plasmid system in which \u003cb\u003eP5\u003c/b\u003e was observed to function downstream of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e in \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e\u003csup\u003e16\u003c/sup\u003e. In these synthases, the \u003cb\u003eP5\u003c/b\u003e KS accepts a triketide with the same substituents and stereochemistries as in its native substrate at the a-, b-, and g-positions and seems to be tolerant of the differences downstream. However, such differences are not always tolerated by this KS. In the octaketide synthase \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, the downstream \u003cb\u003eP5\u003c/b\u003e KS is presented the same substituents and stereochemistries as in its native substrate at the a-, b-, and g-positions, yet two-thirds of the pentaketide intermediates skip the downstream \u003cb\u003eP5\u003c/b\u003e module, likely due to low flux through its KS. Thus, even \u003cb\u003eP5\u003c/b\u003e KS, the most promiscuous of the pikromycin KSs, shows a significant preference for its natural substrate. We are currently experimenting with rapamycin modules, since their KSs have demonstrated greater tolerance to unnatural substrates\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results suggest some level of co-evolution between the upstream modules of synthases. As all of the interactions between acyl-ACPs and their downstream KSs are native in \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eE2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, KS gatekeeping does not account for its poor activity. This hybrid synthase may be suboptimal due to poor intermodular interactions (\u003cem\u003ei.e.\u003c/em\u003e, upstream KSs with downstream ACPs); however, the robust activity of \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eE6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e suggests this is not a general phenomenon. The specialization of at least the upstream KS domains of PKSs for receiving short substrates indicates that upstream modules may evolutionarily co-migrate\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Disruption of these multimodular collaborations could largely explain why \u003cb\u003eE2\u003c/b\u003e and \u003cb\u003eS2\u003c/b\u003e exhibit low activity between \u003cb\u003eP1\u003c/b\u003e and \u003cb\u003eP3\u003c/b\u003e in \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eE2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eSome engineered PKSs in this study produce a d-lactone instead of, or in addition to, a macrolactone (\u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e produces \u003cb\u003e15\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e produces \u003cb\u003e11\u003c/b\u003e in addition to \u003cb\u003e10\u003c/b\u003e, \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e produces \u003cb\u003e16\u003c/b\u003e in addition to \u003cb\u003e1\u003c/b\u003e) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e and Supplementary Figs.\u0026nbsp;14, 25\u0026ndash;26). Biochemical and structural studies of the pikromycin TE have revealed that functional groups installed by the first 4 pikromycin modules (\u003cb\u003eP1\u003c/b\u003e\u0026ndash;\u003cb\u003eP4\u003c/b\u003e) aid in the macrocyclization of the natural hexaketide and heptaketide intermediates\u003csup\u003e\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. A crystal structure of a version of the pikromycin TE in which 2,3-diaminopropionate replaces the catalytic serine and is connected through an amide linkage to the heptaketide intermediate shows complementarity between active site pockets and the C8 and C12 methyl groups as well as a hydrogen bond between the side chain hydroxyl group of Thr1125 and the C9 enone oxygen\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. These interactions help position the \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-oriented C13 hydroxyl group for nucleophilic attack on the C1 carbonyl; however, the relative contributions of these interactions and those at the ACP/TE interface to macrocylization remain unclear. In the context of PikAIV, the pikromycin TE can macrocyclize a heptaketide intermediate in which the ketone is replaced by a hydroxyl group\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In this study, the production of macrolactones \u003cb\u003e10\u003c/b\u003e and \u003cb\u003e13\u003c/b\u003e by \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, respectively, demonstrates that the double bond is also not required for macrocyclization. Also in this study, the production of macrolactones \u003cb\u003e12\u003c/b\u003e and \u003cb\u003e14\u003c/b\u003e by \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e and \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eS2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e, respectively, demonstrates that the nucleophilic hydroxy group does not need to be \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003el\u003c/span\u003e-oriented\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. As combinatorial modular syntheses of macrolide antibiotics are explored, more will be learned about the tolerance of pikromycin TE. If it is a significant bottleneck, promiscuity-enhancing mutations, such as the replacement of the reactive serine by cysteine, will be introduced\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe described platform provides a means to study mysterious assembly line phenomena. For example, it has been debated whether the full pikromycin PKS produces the hexaketide 10-dml or not. In this study, no 10-dml was detected from \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e. However, the single cysteine-to-alanine point mutation to the \u003cb\u003eP6\u003c/b\u003e KS renders \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6*\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e an efficient producer of 10-dml. Why \u003cb\u003eP6\u003c/b\u003e ACP does not transfer the hexaketide intermediate to TE in \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e is not clear. One hypothesis is that KSs are dimeric when acylated by their polyketide intermediate but monomeric in their absence and that the monomeric state enables the \u003cb\u003eP6\u003c/b\u003e ACP and TE to approach one another. \u003cb\u003eP1\u003c/b\u003e-\u003cb\u003eP2\u003c/b\u003e-\u003cb\u003eP3\u003c/b\u003e-\u003cb\u003eP4\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP5\u003c/b\u003e-\u003cb\u003eP6\u003c/b\u003e-\u003cb\u003eP7\u003c/b\u003e may predominantly produce narbonolide due to a similar module-skipping phenomenon that can be investigated through the platform.\u003c/p\u003e \u003cp\u003eMany opportunities exist to boost the titers of polyketides and macrolide antibiotics from \u003cem\u003eE. coli\u003c/em\u003e. Genomic deletion of a putative propionyl CoA:succinate CoA transferase, \u003cem\u003eygfH\u003c/em\u003e, increased the titer of erythromycin A 7-fold\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.The use of bioreactors rather than shake flasks increased the titer of 6-dEB 5-fold\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eE. coli\u003c/em\u003e K207-3 has not been optimized for general polyketide production; however, now that this chassis organism can produce macrolide antibiotics, avenues to evolve it for enhanced polyketide production have been opened\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. For example, the macrolide-sensing transcription factor MphR could be used to help select for \u003cem\u003eE. coli\u003c/em\u003e K207-3 variants that produce higher titers of narbomycin\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. To access higher titers of hydroxylated products such as pikromycin and methymycin, PikC activity could be increased through its fusion to a reductase domain\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, we have developed a 2-plasmid BioBrick-like system to assemble PKSs from updated modules and employed it to express a refactored pikromycin PKS in \u003cem\u003eE. coli\u003c/em\u003e. Through swapping modules and expressing tailoring enzymes from the pikromycin pathway, we modularly synthesized new derivatives of narbonolide, narbomycin, and pikromycin as well as 10-dml, YC-17, and methymycin. Since many strategies beyond those employed here can further boost the yields of macrolide antibiotics, the described platform may be used beyond advancing our understanding of PKS enzymology to access new macrolide antibiotics at preparative levels. Our \u003cem\u003eE. coli\u003c/em\u003e-based PKS engineering platform is also higher throughput than streptomyces-based platforms\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. We envision it being employed to generate combinatorial libraries of promising polyketide drug leads that will accelerate the medicinal chemistry of these synthetically-challenging compounds.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability.\u003c/strong\u003e All data are available through the Supplementary Information document.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiological materials.\u003c/strong\u003e \u003cem\u003eE. coli\u003c/em\u003e K207-3 cells are available from the authors upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eThis work was supported by the NIH (GM145992) and the Welch Foundation (F-1712).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions.\u0026nbsp;\u003c/strong\u003eT.M. and A.T.K. designed the research. T.M. performed experiments. T.M. and A.T.K.\u0026nbsp;wrote the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInclusion \u0026amp; ethics statement.\u0026nbsp;\u003c/strong\u003eAll participants in this research were properly credited.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information.\u0026nbsp;\u003c/strong\u003eSupplementary methods, figures, and tables are available in the Supplementary Information document.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrown, E. D. \u0026amp; Wright, G. D. Antibacterial drug discovery in the resistance era. 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We developed a 2-plasmid system for the construction and expression of PKSs and employed it to obtain a refactored pikromycin synthase in \u003cem\u003eE. coli\u003c/em\u003e that produces 85 mg of narbonolide per liter of culture. The replacement, insertion, deletion, and mutagenesis of modules enabled access to hexaketide, heptaketide, and octaketide derivatives. Supplying enzymes for desosamine biosynthesis and transfer enabled production of narbomycin, pikromycin, YC-17, methymycin, and 6 derivatives thereof. Knocking out pathways competing with desosamine biosynthesis and supplying the editing thioesterase PikAV boosted the titer of narbomycin 55-fold to 37 mgL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The replacement of the 3rd pikromycin module with its 5th yielded a new macrolide antibiotic and demonstrates how libraries of macrolide antibiotics can be readily accessed.\u003c/p\u003e","manuscriptTitle":"Refactoring the pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-08 16:25:05","doi":"10.21203/rs.3.rs-5640596/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-synthesis","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"natsynth","sideBox":"Learn more about [Nature Synthesis](https://www.nature.com/natsynth/)","snPcode":"","submissionUrl":"https://mts-natsynth.nature.com/cgi-bin/main.plex","title":"Nature Synthesis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"46217300-6131-40cc-8193-43fe031abda8","owner":[],"postedDate":"January 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":42484864,"name":"Biological sciences/Biochemistry/Biocatalysis"},{"id":42484865,"name":"Biological sciences/Biochemistry/Enzymes/Multienzyme complexes"},{"id":42484866,"name":"Biological sciences/Chemical biology/Biosynthesis/Multienzyme complexes"},{"id":42484867,"name":"Biological sciences/Drug discovery/Medicinal chemistry/Drug discovery and development"},{"id":42484868,"name":"Biological sciences/Systems biology/Synthetic biology"}],"tags":[],"updatedAt":"2026-04-20T11:03:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-08 16:25:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5640596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5640596","identity":"rs-5640596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}