A Phosphopantetheinyl Transferase from Dictyobacter vulcani sp. W12 Expands the Combinatorial Biosynthetic Toolkit

phosphopantetheinyl transferase, acyl carrier protein, polyketide, synthase (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 2

The value of microbial natural product pathways extends beyond the chemicals they produce, as the enzymes they encode can be harnessed as biocatalysts. Microbial type II polyketide synthases (PKSs) are particularly noteworthy , as these enzyme assemblies produce complex polyaromatic pharmacophores. Combinatorial biosynthesis with type II PKSs has been described as a promising route for accessing never-before-seen bioactive molecules, but this potential is stymied in part by the lack of functionally compatible non-cognate proteins across type II PKS systems. Acyl carrier proteins (ACPs) are central to this challenge, as they shuttle reactive intermediates and malonyl building blocks between the other type II PKS domains during biosynthesis. Activating ACPs to their holo state via the phosphopantetheinyl transferase (PPTase) -catalyzed installatio n of a coenzyme A (CoA) -derived phosphopantetheine (Ppant) arm is critical to effectively study and strategically engineer type II PKSs, but not all ACPs can be activated using conventional PPTases. Here, we report the discovery of a previously unexplored non -actinobacterial PPTase from Dictyobacter vulcani sp. nov. W12 (vulcPPT). We explored its compatibility with both native and non-native ACPs, observing that vulcPPT activated all ACPs tested in this study, including a non- cognate, non-actinobacterial ACP which cannot be activated by the prototypical broad substrate PPTases AcpS and Sfp. Strategic optimization of phosphopantetheinylation reaction conditions increased apo to holo conversion. In addition to identifying a promising new promiscuous PPTase, this work establishes a road map for further investigation of PPTase compatibility and increases access to functional synthase components for use in combinatorial biosynthesis. MAIN TEXT (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 3

Microorganisms create a vast array of organic molecules that are not essential to the organism’s survival but confer them with an ecological competitive advantage. Type II polyketides are a particularly exciting class of these secondary metabolites because of their medicinally relevant properties (e.g. the antibiotic tetracycline and anticancer agent doxorubicin).1 These molecules are manufactured by multi-enzyme assemblies known as type II polyketide synthases (PKSs) whic h are spatially encoded within microbial genomes as biosynthetic gene clusters (BGCs ).1 At minimum, a type II PKS comprises an acyl carrier protein (ACP), wh ich must be post - translationally modified via the installation of a phosphopantetheine arm to its active “holo” form, and a ketosynthase -chain length factor (KS -CLF). The holo-ACP and KS -CLF interact to transform malonyl-based building blocks into a nascent beta-keto polyketide chain through a series of Claisen -like decarboxylation reactions.1 Subsequent reactions with a series of accessory enzymes tailor the polyketide intermediate into its final polyaromatic structure. The diversity of type II polyketides originates from variability in both the KS-CLF, which is a primary determinant in the number of carbons in the poly -beta-keto chain, and the members of the “tailoring enzyme roster” which catalyze the late -stage biochemical transformation and functionalization of the polyketide backbone.1 Unlike type I synthases (or synthetases), each protein or enzyme within a type II PKS exists as a discrete domain, making these systems uniquely conducive to mixing-and- matching synthase components in an effort to gain access to non -native chemical diversity. However, impaired ACP-protein interactions prevent such combinatorial biosynthesis efforts,2 so exploring ACPs that are more suitable for mediation of these interactions is crucial. ACPs from non -actinobacterial type II PKS s are historically understudied and represent an interesting set of proteins to explore. While recent studies suggest that non -actinobacterial KS- (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 4 CLFs are uniquely amenable to expression in Escherichia coli , their cognate ACPs can be expressed but not activated to their active holo form using conventional approaches.3–6 For example, the Photorhabdus luminescens TT01 type II PKS ACP could not be activated by the E. coli PPTase, AcpS, requiring the co -expression of two additional auxiliary enzymes for the successful in vivo production of the type II polyketide in E. coli.4 A similar barrier was encountered in the in vitro reconstitution of the Gloeocapsa sp. PC7428 type II PKS system in which neither Sfp nor AcpS could convert the E. coli heterologously expressed apo-ACP to its holo form, requiring strategic mutation of the ACP to enable type II PKS reconstitution in vitro.3 This activation, called phosphopantetheinylation, is catalyzed by a n enzyme known as a phosphopantetheinyl transferase (PPTase) that installs a coenzyme A ( CoA)-derived 18-Å long, 4’-phosphopantetheine prosthetic group (Ppant arm, Figure 1A) on a conserved serine located on the N-terminus of helix II of the ACP .7 The Ppant arm enables the ACP to tether and transport molecular building blocks and the growing polyketide chain between the other discrete type II PKS domains. It is well -documented that ACPs and peptidyl carrier proteins (PCPs) can be compatible with PPT ases encoded by different BGCs within an organism ;8–10 similarly, carrier proteins (CPs) from one organism can be activated by PPTases from different organisms altogether11. The PPTase from the E. coli fatty acid synthase ( AcpS)12 and the PPTase from the Bacillus subtilis surfactin non-ribosomal peptide synthetase (Sfp) 13 and its commonly used R4 -4 variant14 (referred to as ‘Sfp’ from this point forward), are well -known for their promiscuity in converting non-cognate CPs to their holo form and are therefore widely used as biosynthetic tools. However, as noted above, these broad substrate PPTases have limited success with activating non- actinobacterial ACPs. Whilst the s trategic mutation of non -actinobacterial ACPs can confer Sfp compatibility,3 identifying new PPTases which can readily activate wild type non -actinobacterial (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 5 ACPs will improve access to type II PKS components. Herein, we report the E. coli heterologous expression and subsequent characterization of a previously unexplored PPTase encoded in the Dictyobacter vulcani sp. W12 genome (vulcPPT). vulcPPT demonstrates the ability to activate ACPs that are incompatible with Sfp and AcpS, and therefore represents an important new contribution to the biosynthetic toolkit. RESULTS/DISCUSSION To identify novel promiscuous PPTases that could readily activate non -actinobacterial ACPs, we turned to previously uncharacterized putative non-actinobacterial type II PKSs BGCs and their associated PPTases15,16 for E. coli heterologous expression and subsequent characterization. One such type II PKS BGC was identified in the Dictyobacter vulcani sp. W12 genome. Isolated from the soil of the Mt. Zao volcano in Japan, D. vulcani sp. W12 is a member of the Ktedonobacteria class, which is well known for actinomycete -like morphology and capability for secondary metabolite production .17 antiSMASH18 analysis of the D. vulcani sp. W12 genome revealed a putative type II PKS BGC with a unique, triad-like condensation (KS-CLF) domain architecture16 and an ACP with a non-canonical PPTase recognition motif (IDSI instead of LDSL). We selected the sole PPTase (vulcPPT) from the annotated protein list provided in the NCBI whole genome shotgun (WGS) Sequence Set Browser for D. vulcani sp. W12 for our heterologous expression efforts, but three additional proteins with homology to Bacillus subtilis Sfp and Escherichia coli AcpS were identified through BLASTp similarity searches. Interestingly, the genome is predicted to harbor >70 CPs across several putativ e non-ribosomal peptide synthetase (NRPS) and PKS BGCs, suggesting to us that vulcPPT might display unique and/or broad substrate activity. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 6 The vulcPPT gene (see Methods and Table S1 in Supporting Information) was cloned into a pET28a-derived construct for E. coli heterologous expression with an N-terminal His 6 tag and subsequently purified via affinity column purification to a yield of 60 mg/L. The protein was characterized via SDS-PAGE and liquid chromatography mass spectrometry (LC-MS; Supporting Figures S1 and S2, respectively). The AlphaFold 319-predicted vulcPPT structure depicts vulcPPT as a pseudo -dimer consisting of two structurally similar subdomains attached by a polypeptide loop with high confidence (Figure S3). Together, the sequence, predicted structure, and size (249 aa) of vulcPPT suggest that vulcPPT is an Sfp-type PPTase.7 Circular dichroism (CD) wavelength experiments demonstrate that vulcPPT contains 26.8% ɑ-helical, 8.4% antiparallel ꞵ -sheets, and 6.1% parallel ꞵ -sheets content. 20,21 Further protein melting experiments reveal a melting temperature (Tmelt) of 45.53 ℃ (± 0.15 ℃) for vulcPPT (Figure S4 and T able S2). Preliminary examination of its in vitro activity demonstrates that vulcPPT can fully convert its putative cognate ACP (hereafter referred to as vulcACP) from the inactive apo form to active holo form within an hour (Figure 1B and Supporting Information). Figure 1. The PPTase from Dictyobacter vulcani sp. W12 (vulcPPT) converts its putative cognate ACP from the inactive apo form to active holo form. (A) ACP activation from apo to holo form is facilitated by a PPTase that installs the 18 Å CoA-derived phosphopantetheine (Ppant) arm on the (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 7 conserved serine at the N-terminus of helix II. (B) The reaction of vulcACP:vulcPPT at over 0 to 60 min (see Supporting Information for reaction conditions) , monitored by LC -MS, shows that vulcPPT efficiently activates its native ACP partner to nearly 100% within 60 min. To evaluate vulcPPT’s substrate scope relative to PPTases routinely used in the field, we assessed the ability of vulcPPT, AcpS, and Sfp to phosphopantetheinylate four ACPs: (i) vulcACP, (ii) the ACP from the E. coli fatty acid synthase (FAS) system (AcpP), (iii) the ACP from the Streptomyces coelicolor actinorhodin type II PKS (actACP), and (iv) a previously unexplored ACP from the Zooshikella sp. WH53 putative type II PKS BGC (zooACP). These four ACPs were selected to represent diverse substrates , including the putative cognate ACP for vulcPPT (i), the prototypical FAS ACP and native substrate for AcpS (ii), the prototypical type II PKS ACP (iii), and a never-before-studied non-cognate non-actinobacterial ACP (iv). Plasmids encoding for the four ACPs were transformed into E. coli BL21 (DE3) cells for expression in their majority (~90– 95%) inactive apo form. Phosphopantetheinylation reactions were performed by reacting an apo- ACP with a PPTase in the presence of CoA, dithiothreitol (DTT), and MgCl 2 before be ing quenched at 18 hours with formic acid and analyzed via liquid chromatography mass spectrometry (LC-MS). Under the LC conditions used (see Supporting Information for details) the two forms of the ACP ( apo and holo) elute at distinct retention times, allowing for percent conversion to be quantified. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 8 Figure 2. The PPTase from Dictyobacter vulcani sp. W12 (vulcPPT) shows expanded substrate scope compared to Sfp and AcpS for the ACPs studied. (A) The structures of Sfp (PDB 1QR0), AcpS (PDB 5SUV), and vulcPPT (AlphaFold3 prediction) suggest that vulcPPT belongs to the Sfp-family of PPTases. (B) VulcP PT can activate a range of ACPs, including ACPs that are not readily activated using AcpS and Sfp. When apo-vulcACP (blue), apo-actACP (orange), apo- AcpP (purple), and apo-zooACP (green) were reacted with Sfp, AcpS, and vulcPPT (see Supporting Information for reaction conditions) for 18 hrs, vulcACP, actACP, AcpP were converted to their holo forms with efficiencies ranging from 5 8% (± 4.5%) to 100% (± 0.0%) by (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 9 all three PPTases. In contrast, zooACP was only activated by vulcPPT, achieving a conversion rate of 11% (± 1.5%). See Figures S5-S8 for corresponding LCMS data. VulcPPT fully converts apo-actACP and apo-AcpP to their active holo states, matching the activity of both AcpS and Sfp (Figure 2B). However, vulcPPT was significantly more efficient in converting its cognate apo-vulcACP to its holo form than AcpS (100% (± 0.0%) versus 58% (± 4.5%), respectively). Interestingly, of the three PPTases studied, only vulcPPT was capable of converting zooACP to its holo state, albeit at low efficiency (~11% (± 1.5%) holo, Figure 2B) under non -optimized conditions. Together these data highlight the broad substrate scope of vulcPPT and its utility in obtaining holo-ACPs that cannot be readily obtained using field-standard PPTases. These results are particularly significant in the context of recent work on cyanobacterial PPTases and ACPs, in which diverse PPTases could not outperform Sfp in activating cyanobacterial CPs.22 Given promising preliminary results, we next sought to determine whether the apo to holo conversion efficiency observed for the zooACP -vulcPPT reaction could be improved by altering the phosphopantetheinylation conditions. The four reaction variables-- (i) temperature, (ii) pH, (iii) vulcPPT concentration, and (iv) CoA concentration-- were assessed independently with 25 °C, pH 7.6, 1.0 µM vulcPPT, and 10x CoA as the standard conditions. First, a 35 °C incubation temperature yielded the highest conversion to holo-zooACP (85% (± 2.2%)) within the range 20– 37 °C (Figure 3A). Second, vulcPPT produced the highest % holo conversion (27% (± 1.9%)) at pH 7.5, with a broad pH range of activity (Figure 3B). Interestingly, optimal conversion from apo- zooACP to holo-zooACP was observed at 3.0 µM vulcPPT and 750 µM CoA (Figures S11 and S12, respectively). Taken together, these experiments reveal that the vulcPPT phosphopantetheinylation reaction conditions can be tuned to improve conversion efficiency. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 10 Figure 3. By optimizing reaction conditions, the phosphopantetheinylation of zooACP by vulcPPT could be improved from 11% (± 1.5%) to 87% (± 6.4%). To optimize the activation of apo-zooACP by vulcPPT, different variables were tested: temperature at a constant pH of 7.6 (A, more detailed version in Figure S9), pH at a constant temperature of 25 °C (B, more detailed version in Figure S10), vulcPPT concentration (Figure S11), and CoA concentration (Figure S12). The condition with the highest percent conversion for each condition explored is marked with a triangle. (C) Under the optimized conditions for vulcPPT [35 °C, 7 50 µM ( 5 molar equivalents relative to apo-zooACP = 5x) CoA, 3.0 µM PPT, pH 7.5], apo-zooACP was converted to holo- zooACP at 87% (± 6.4%) , which is 8 -fold higher than the initial conditions explored. (D) Phosphopantetheinylation of apo-zooACP by Sfp, AcpS, and vulcPPT under their respective optimal conditions—pH 6.0 at 37 °C for Sfp, pH 8.8 at 37 °C for AcpS, and pH 7.5 at 35 °C for (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 11 vulcPPT—followed by analysis after 18 hours. The results confirm that only vulcPPT effectively activates zooACP into its holo form under these conditions. Next, vulcPPT phosphopantetheinylation reactions were performed on apo-zooACP using the conditions determined to be optimal for each variable explored (3.0 µM vulcPPT, 35 °C, pH 7.5, and 750 µM (5x) CoA , Figure 3C ). Under these conditions, we observed over 87% (± 6.4%) conversion of apo-zooACP to its holo form. The nearly 8 -fold improvement in efficiency compared to the result from unoptimized conditions (11% (± 1.5%)) seems to be primarily influenced by temperature (Figure 3A). The zooACP phosphopantetheinylation react ion was performed under identical conditions using Sfp and AcpS in place of vulcPPT which yielded only 0.5% (± 0.2%) and 3% (± 0.3%) holo-zooACP, respectively (Figure 3C). Finally, to more accurately compare the efficiency of all three PPTases, phosphopantetheinylation reactions were conducted on apo-zooACP using the literature-reported optimal conditions for Sfp (pH 6.0, 37 °C, Figure 3D) and AcpS (pH 8.8, 37 °C, Figure 3D). 23,24 These reactions yielded 1% (± 0.4%) and 6% (± 1.0%) holo-zooACP, respectively–an improvement over the initial reactions using vulcPPT optimal conditions (pH 7.6, 35 °C), yet still much lower than the >87% holo-zooACP achieved by vulcPPT. These data support the conclusion that among the three PPTases studied, vulcPPT is the most effective at activating zooACP. Compared to the optimal pH and temperature of AcpS and Sfp, vulcPPT behaves optimally at less harsh pH conditions (pH 7.6) and cooler temperatures (35 °C). VulcPPT is therefore useful for reactions where substrates require more amenable conditions. We hypothesized that the broad substrate compatibility of vulcPPT is derived from its ability to recognize a broader spectrum of ACP motifs , as compared to Sfp and AcpS .25 To gain further insight, the D. vulcani sp. W12 genome was analyzed using antiSMASH18 to collate a set of putative CPs, including ACPs from type I/II PKSs and fatty acid synthases as well as PCPs from (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 12 non-ribosomal peptide synthetases. 79 CP genes were identified and further analyzed via multiple sequence alignment (Figure S13). Notably, only a small percentage (6.3%) of these CPs contained the traditionally Sfp-favored amino acid motif of DSL (where the S is the conserved serine at the N-terminus of helix II that is the attachment point for the Ppant arm). Instead, this DSL motif was frequently replaced by other motifs, such as DSI (44.3%, 35/79) and HSL (34.1%, 27/79) among others. These data suggest t hat vulcPPT, which is the sole PPTase identified from the annotated protein list in the NCBI whole genome shotgun (WGS) Sequence Set Browser for D. vulcani sp. W12, may be capable of recognizing a broader range of interaction motifs. This sort of “crosstalk”, where a PPTase activates more than one ACP in an organism (as opposed to having each PPTase be specific to a single CP ), has been observed in recent years and can be leveraged in strategic engineering work.8–11,26 Additional evidence to support this hypothesis lies in the fact that a DST motif has been identified as a key residue that makes an ACP incompatible with Sfp. 3 Moreover, basal expression of the E. coli AcpS could not convert the Rhizobia CP SMb20651, which contains a DST motif, to its holo form. Conversion was only achieved by co-overexpression of the E. coli or S. meliloti AcpS, suggesting that the presence of a DST motif instead of a DSL motif presents barriers to accessing CPs in their holo form.7 ZooACP features a DST motif and cannot be converted by Sfp to its holo form in any meaningful quantity. In comparison, vulcPPT has demonstrated the ability to convert zooACP to its holo form in significant quantities, suggesting that vulcPPT is compatible not only with the more accessible DSL, DSI, and HSL motifs but also with the previously difficult to access DST motif. The impact of discovering and characterizing new PPTases is highlighted by the ability of vulcPPT to convert apo-zooACP, a non -cognate CP which was previously inaccessible using conventional PPTases, to its active holo form. It is critical to expand the biosynthetic toolkit such (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 13 that diverse holo-ACPs can be accessed given that 1) holo-ACPs are an essential component to any PKS, whether native or created via combinatorial biosynthesis, and 2) impaired ACP -protein interactions lead to failure of a PKS to produce a polyketide product. PPTases like vulcPPT allow us to obtain ACPs that are not accessible in their WT form and can currently only be activated by Sfp if the ACP is strategically engineered .3,26 We hope that the introduction of vulcPPT to the combinatorial biosynthetic toolkit will improve access to functional ACPs while also providing additional clues that can be used to uncover the molecular underpinnings of PPTase -ACP compatibility. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge at [doi link will be inserted here later]. Detailed descriptions of materials and methods; plasmid, primer, and amino acid sequence information; SDS -PAGE of the purified ACPs and phosphopantetheinyl transferases; LC -MS spectra of the apo- and holo-ACPs upon phosphopantetheinylation; CD spectrum and T melt of vulcPPT; temperature, pH, vulcPPT concentration, and coenzyme A concentration optimization of the phosphopantetheinylation of apo-zooACP by vulcPPT; multiple sequence alignment of 79 CPs encoded by the Dictyobacter vulcani sp. W12 genome. AUTHOR INFORMATION Corresponding Authors (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 14 Yae In Cho ([email protected]) and Louise K. Charkoudian ([email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. *Co-corresponding authors. C.M.M., C.M.F., K.K.H., L.K.C., N.B.M., K.N.M., R.F., Y.I.C. designed the research. C.M.M., C.M.F., K.K.H., N.B.M., K.N.M., Y.I.C. collected and analyzed data. C.M.M., C.M.F., K.K.H., L.K.C., Y.I.C. wrote the manuscript. Funding Sources We are grateful for generous support from the National Science Foundation (CHE2201984 to L.K.C.), a 2021 Arnold and Mabel Beckman Foundation Scholarship and 2022 Goldwater Scholarship (C.M.M) and Haverford College. ACKNOWLEDGMENTS We thank Professor Joris Beld (Drexel University) for helpful discussions as well as Gabriel Rocco Sotero for technical support. We are also grateful to the 2023 Haverford College Laboratory in Biochemical Research class for their support and guidance. ABBREVIATIONS PKS, polyketide synthase; BGC, biosynthetic gene cluster; ACP, acyl carrier protein; PPTase, phosphopantetheinyl transferase; CoA, coenzyme A; AcpS, holo-(acyl carrier protein) synthase; Sfp, 4' -phosphopantetheinyl transferase protein from Bacillus subtilis ; NRPS, nonribosomal (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 26, 2025. ; https://doi.org/10.1101/2025.03.24.645039doi: bioRxiv preprint 15 peptide synthetase; KS-CLF, ketosynthase chain length factor; Ppant arm, 4’-phosphopantetheine prosthetic group; SDS -PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC - MS, liquid chromatography mass spectrometry; CD, circular dichroism.

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