Comprehensive in Vitro Analysis of Acyltransferase Domain

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Comprehensive In Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production Satoshi Yuzawa, Kai Deng, George Wang, Edward E.K. Baidoo, Trent R. Northen, Paul D. Adams, Leonard Katz, and Jay D Keasling ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00176 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 24, 2016

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ACS Synthetic Biology

Comprehensive In Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production Satoshi Yuzawa1,*, Kai Deng2,3, George Wang2, Edward E. K. Baidoo2, Trent R. Northen2,4, Paul D. Adams2,5,6, Leonard Katz1,7, and Jay D. Keasling1,2,5,7-10,* 1

QB3 Institute, University of California, Berkeley, California, 94720, United States Joint BioEnergy Institute, Emeryville, California, 94608, United States 3 Sandia National Laboratories, Livermore, California, 94551, United States 4 Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States 5 Department of Bioengineering, University of California, Berkeley, California, 94720, United States 6 Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States 7 Synthetic Biology Research Center, Emeryville, California, 94608, United States 8 Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, 94720, United States 9 Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, 94720, United States 10 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé, DK2970Hørsholm, Denmark 2

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ABSTRACT

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.

KEYWORDS

Type I modular polyketide synthase, acyltransferase domain, substrate specificity, protein engineering, synthetic biology tool

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INTRODUCTION

Type I modular polyketide synthases (PKSs) are multi-domain enzymes that synthesize polyketides from acyl-CoAs in a fashion similar to fatty acid synthases (FASs).1 However, unlike FASs, each elongation step is carried out by a discrete set of catalytic domains in the PKS defined as a module, which enable them to use a variety of acyl-CoAs. Hence, the modular nature of the PKSs not only accounts for the thousands of diverse product structures currently known, but also offers a wealth of engineering opportunities for the production of novel compounds.2

In most type I modular PKS systems, each module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain that are together responsible for a single round of chain elongation, where the AT domain determines the specific acyl-CoA incorporated into the growing polyketide chain. At least 20 acyl-CoA substrates, all malonyl-CoA analogs, have been found to be incorporated into naturally occurring polyketides, and many of the corresponding AT domains have been identified in sequenced PKSs (Figure S1).3, 4 These acyl-CoAs include long-chain alkylmalonyl-CoAs, halogenated malonylCoAs, and benzylmalonyl-CoA. Although several different PKS engineering strategies have been developed to incorporate different acyl-CoAs,5-7 the most common approach to date is the swapping of an entire AT domain for a homolog with different substrate specificity.8 For example, analogs of the naturally occurring polyketides, 6-deoxyerythronolide B (6-dEB) and geldanamycin, have been produced in vivo by replacing one of the native AT domains with malonyl CoA-specific AT domains in the corresponding respective PKSs, 6-dEB synthase (DEBS) and the geldanamycin PKS.9, 10 Most of the engineered PKSs generated, however, were

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either inactive or showed significant losses of activity, leading to the marked reduction in production levels of the resulting polyketide, except for the cases where the AT domains in terminal PKS modules were replaced. The mechanistic basis for these deleterious effects was previously investigated using a monomodular PKS, DEBS module 6+TE (M6+TE), in vitro, where the native AT domain was replaced with homologs conferring the same substrate specificity.11 Kinetic analyses of the hybrid PKSs revealed 20-fold or more decreases in kcat values compared to the wild-type module, suggesting the use of sub-optimal AT domain boundaries in the construction of the hybrid enzymes.

Until recently, the role of inter-domain linkers in type I modular PKSs had not been rigorously analyzed. Recent biochemical and structural analyses have highlighted the possible functions of the linker sequences. Mutagenesis studies demonstrated that the linker connecting the KS and AT domains (heretofore designated KAL) interacts with the KS and the post AT linker (PAL) segment through the side chain of the conserved arginine residue.12, 13 Furthermore, the four crystal structures of KS-AT didomains all indicate extensive interactions between the highly conserved residues in the last half of PAL (PAL2) and the KS domain (Figures S2 and S4)14-17 although the recent cryo-electron microscopy (EM) study of an intact PKS module has challenged this model.13 It has been argued that these specific interactions appear to be essential to stabilize the correct conformation required for the KS-catalyzed chain elongation reaction,18 which is most likely the rate-determining step in type I modular PKS-catalyzed polyketide biosynthesis.19 Here we report a more thorough investigation of the role of the linkers in AT domain exchanges and its application in the production of industrially important short-chain ketones.

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RESULTS AND DISCUSSION

Role of inter-domain linkers in AT domain replacements

Several different AT domain boundaries have been used previously to produce polyketide analogs in vivo and have shown mixed results. In some cases, the AT-PAL segment in an acceptor PKS was replaced with the corresponding segment in donor PKSs.9, 10, 20, 21 The ATPAL1 (PAL1 is a non-conserved region in PAL, see Figure 2) segment has also been used in AT domain swapping.5, 22-24 When Reeves et al. exchanged only the AT domain, none of the hybrid PKSs generated were active.6 To comprehensively investigate the role of the linkers in AT domain swapping in vitro, we replaced the methylmalonyl-CoA-specific AT domain in DEBS M6+TE with the AT domain from module 4 of the epothilone PKS (EPOS M4), which incorporates either malonyl- or methylmalonyl-CoA to generate the natural products epothilone A and epothilone B, respectively (Figures 1 and 2).25, 26 Using the AT domain from EPOS M4 allowed us to compare the incorporation of two alternative substrates in a single isolated PKS module. The exchanges employed the AT domain along with various combinations of the associated KAL and PAL1 segments, resulting in the construction of the hybrid PKSs D1-D4 (Figure 2 and Table S1). These constructs retained the native sequence of PAL2 to maintain the necessary interaction with the KS domain. We also created a hybrid module where the AT-PAL (= AT-PAL1-PAL2) was replaced (D0).

The resulting 5 proteins (D0-D4) and DEBS M6+TE were produced in Escherichia coli K207327 and purified using Ni-NTA resins followed by anion exchange column chromatography (Figure S3). D1 was obtained at a level similar to the wild-type protein, indicating stable protein folding (Table 1). In contrast, D0, and D2-D4 showed 10-20-fold lower purified levels although Page 5 of 25



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their expression levels are comparable (within two-fold difference) with D1 right after cell lysis, except for D0 (Figure S3). Interestingly, the model structures of the hybrid modules revealed that D1 contains a single heterologous interface only between the KS domain and the KAL, whereas the other mutants have two or more heterologous interfaces between functional domains and linkers (Figure S4), which would likely render these hybrid enzymes more structurally unstable and could be the cause of the significantly reduced purification yields.

The relative abilities of DEBS M6+TE and the hybrid PKSs to catalyze chain elongation with methylmalonyl-CoA were measured by analyzing the steady-state rates of (4S, 5R)-3-oxo-2,4dimethyl-5-hydroxy-heptanoic acid-δ-lactone (2) formation using (2S, 3R) 3-hydroxy-2methylpentanoyl-S-N-acetylcysteamine thioester (1) as a starter substrate for the reaction (Figures 1B and 1C).28 To simplify the kinetic analysis, we omitted NADPH because addition of NADPH resulted in the production of a mixture of the reduced and the non-reduced forms of the lactone products (data not shown). Having an authentic standard of 2 allowed us to determine and compare the kcat and KM values of the various enzymes employed, as summarized in Table 1 (Figure S5). Consistent with the aforementioned in vitro study, which employed AT-PAL1-PAL2 as an AT source in DEBS M6+TE11, we also found that D0 was inactive, although the in vitro data cannot explain a handful of the very active hybrid PKSs described in vivo where the same domain boundary was employed in the same module. D2 was also inactive, which agrees with previous in vivo observations.6 On the other hand, D1, which carried the AT domain along with its cognate KAL and PAL1 linkers, showed only a slight decrease in kcat, and a ca. 2-fold increase in KM as compared to the wild-type protein. Although D3 showed similar condensation activity as the wild-type protein, which further supports the importance of the native PAL2 sequence, it should be noted that the KM values of D1 and D3 are significantly different.

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To produce the polyketide variant, we incubated D1 and D3 with malonyl-CoA in the presence of the starter substrate, 1 (Figure 1D). As expected, both PKSs produced the corresponding desmethyl version of 2, (4S, 5R)-3-oxo-4-methyl-5-hydroxy-heptanoic acid-δ-lactone (3), whereas the wild-type DEBS M6+TE, as expected, did not produce the desmethylated product (Table 1 and Figure S5). Interestingly, the kcat of producing the desmethyl version was ca. 10-fold slower than that of the reaction with methylmalonyl-CoA, although the KM values for malonylCoA and methylmalonyl-CoA were in the same range, suggesting that the KS domain might prefer the methylmalonyl- over the malonyl- moiety as a substrate for condensation. To our knowledge, this is the first report of the determination of the kinetic parameters of an AT domain that has dual substrate specificity.

Examination of AT domain boundaries in a second PKS system

To investigate the generality of the optimized AT domain boundary (KAL-AT-PAL1), we performed similar experiments using the first PKS module from the antibiotic-producing lipomycin synthase, LIPS M1+TE (Figure 3).29 Although the amino acid sequence is substantially different from DEBS M6, the domain composition of LIPS M1 is exactly the same as that of DEBS M6 except that it contains an upstream loading AT-ACP didomain. When we replaced the methylmalonyl-CoA-specific AT domain with the epothilone KAL-AT4-PAL1 segment (Table S2), we observed increased purification yield of the hybrid PKS (L1), as compared to the wild-type (Table 2).

The relative abilities of LIPS M1+TE and L1 to catalyze chain elongation from butyryl-CoA and methylmalonyl-CoA were measured by analyzing steady-state rates of 2-methyl-3hydroxyhexanoic acid (4) production. Interestingly, L1 showed a comparable kcat of producing 4 Page 7 of 25



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as compared to the wild-type protein (Table 2 and Figure S6), providing further support for the swapping design.

To produce the desmethyl version of 4, 3-hydroxyhexanoic acid (5), we incubated L1 with butyryl-CoA and malonyl-CoA and determined the kinetic parameters. Surprisingly, L1 showed a ca. 10-fold faster kcat as compared to the native reaction with the wild-type PKS (Table 2 and Figure S6). This is the first example, to our knowledge, where an AT-swapped PKS that incorporated a different substrate showed a clearly increased activity over the wild-type enzyme that incorporates the natural substrate. This observation suggests that natural, “wild-type” PKSs may not be fully optimized in catalyzing some reactions, and that AT domain replacements could result in increased amounts of natural polyketide variants.

Validation of AT replacement strategy using several different AT domains

To further validate the methodology, we constructed and analyzed five additional hybrid PKSs, L2-L6, containing heterologous, malonyl-CoA-specific KAL-AT-PAL segments in the LIPS M1+TE system (Table S2). Although L6, containing the AT from module 7 of the curacin PKS, was not solubly produced, all of the other hybrid PKSs were obtained with purification yields comparable to the wild-type LIPS M1+TE (Table 2). L2, L4, and L5 showed similar kcat values of producing 5 as that of L1 (Table 1 and Figure S6). In contrast, L3, containing the AT from module 2 of the rapamycin PKS, had less than 10% of the activity of L1 as shown in Table 1. Although the basis for the decrease is unclear, the size of the swapped region may be critical in AT domain replacements. The lengths of KAL-AT-PAL1 used in the study are summarized in Figure S7. In the wild type and active AT-swapped modules, the lengths of the KAL-AT-PAL1

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segments range from 429-457 residues (WT = 452 residues), but L3 and L6 have the shortest (389 residues) and longest (479 residues) segments, respectively, among the mutants.

We have also constructed additional AT-swapped mutants in the LIPS M1+TE system using ATPAL1-PAL2, AT, or AT-PAL1 segments from module 1 of the borrelidin PKS in attempts to compare their activities to the L2 construct shown in Table 2. Although soluble protein bands were observed for these hybrid constructs right after the cell lysis, we were not able to purify these proteins due to protein degradation during the purification (data not shown). Hence, as in the case with the DEBS M6+TE mutants (D0, D2, and D3), protein stability appears to be highly dependent upon AT domain boundaries used.

In vitro short-chain ketone production by AT-swapped PKSs

Short-chain alkyl ketones such as acetone, 2-butanone (6) and 4-methyl-2-pentanone (10) are industrially important solvents that are used in a variety of applications, including paints, coatings, adhesives, magnetic tapes, inks, as well as for cleaning and extraction. Although some microbes (mainly Clostridium strains) naturally produce acetone, no microbe or metabolic pathway, to our knowledge, is known to produce detectable levels of 6 and 10; all of these ketones are currently derived from petroleum sources. Engineering microbes to synthesize various short-chain ketones from available biomass could be a promising approach for producing them renewably.

3-Ketocarboxylic acids are chemically unstable and known to decompose to ketones spontaneously under mild conditions.30-33 To produce various 3-ketocarboxylic acids by exploiting the substrate promiscuity of the loading AT domain of LIPS M129, we incubated L2

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with various starter acyl-CoAs and malonyl-CoA in the absence of NADPH in vitro (Figure 4). As expected, L2 was capable of producing 6, 2-pentanone (7), 3-methyl-2-butanone (8), 3methyl-2-pentanone (9), and 10, which were verified against the authentic standards. Importantly, to our knowledge, this work represents the first time that these ketones have been produced biologically.

Microbial short-chain ketone production

Toward in vivo short-chain ketone production, we sought to engineer LIPS M1+TE and L2 to produce ketones in the presence of NADPH. Specifically, we changed the predicted active site serine residue of the KR domain (LFSSIAG) to alanine to inactivate the catalytic function34, 35. These engineered PKSs were designated as LIPS M1+TE (KR null) and L2 (KR null), respectively. These proteins were able to produce ketones in vitro in the presence of NAPDH, employed at a concentration equivalent to its intracellular level in E. coli (Figure 5).36

E. coli K207-3 is an engineered strain whose genome encodes the substrate promiscuous phosphopantetheinyl transferase Sfp from Bacillus subtilis that converts the expressed apo-PKSs to their corresponding holo forms.37 This strain can also convert exogenously fed propionate into propionyl-CoA efficiently via overexpression of the native propionyl-CoA ligase.38 In addition, the host contains the genes accA and pccB from Streptomyces coelicolor that encode propionylCoA carboxylase, which converts propionyl-CoA to methylmalonyl-CoA.39

To demonstrate microbial short-chain ketone production, we transformed E. coli K207-3 with a plasmid encoding LIPS M1+TE (KR null) or L2 (KR null) and cultured the two strains at 18°C for five days in the presence of propionate. As shown in Figure S8, LC/MS analysis of the E. coli

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cultures expressing LIPS M1+TE (KR null) or L2 (KR null) revealed the presence of the expected ketone, respectively (3.2 mg/L of 3-pentanone (11) or 4.1 mg/L of 6).

In summary, AT domain swapping has been used extensively to change the nature of the side chain on the α-carbon in polyketide biosynthesis. Previous approaches, however, often resulted in PKSs that had significantly lower activity compared to their wild-type PKS counterparts. We used two monomodular PKS systems and several AT domains to identify the optimal domain boundary to replace the original AT domains. Our comprehensive in vitro analysis revealed that including both the KAL and the PAL1 segments with the AT domain is essential to maintain both protein stability and enzyme activity. Although the KAL-AT-PAL1 boundary has been rarely used in AT domain swapping experiments, Sugimoto et al. recently demonstrated the utility of a similar domain boundary in the production of an aureothin analog in vivo.40 Our results also indicate that retaining native KS-PAL2 interaction(s) is important, which is consistent with the previous biochemical observations in DEBS and all KS-AT didomain crystal structures currently available.12, 14-18 The recent cryo-EM study of an intact PKS module from the pikromycin PKS conflicts with this model, however.13 The reason is unclear but, as demonstrated in the cryo-EM study, the movements of type I PKS modules during the catalytic cycle are dynamic41 and the required KS-PAL2 interaction could be occurring transiently during the chain elongation reaction. Multiple mechanisms for evolution of type I modular PKSs were previously proposed and two homologous recombination hot spots were suggested for AT domain swapping, which are conserved residues in KAL and PAL2.42, 43 Although our observations are partially consistent with this proposal, our in vitro analysis of hybrid PKS modules suggests that the N-terminal hot spot is a GTNAH motif in the KS domain, which is also highly conserved in type I modular PKSs (Figure S2).

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Use of truncated, engineered type I modular PKSs provides a promising route for production of many small molecules that cannot be produced by other enzymes. Efficient swapping of AT domains allowed us to construct a hybrid PKS to produce the industrially important short-chain ketones 6 and 10 that heretofore have not been produced biologically. Although PKS reactions in E. coli are currently limited by available substrates, all of the CoA substrates used in this study could be generated from renewable carbon sources by engineered E. coli.44-47 These engineered PKSs could also be moved into other bacterial or fungal hosts in order to produce the desired ketones in an economically favorable manner. Recent advances in genome sequencing and the study of crotonyl-CoA carboxylase/reductase homologues identified many extender substrates and their corresponding AT domains in nature.3, 4 In the future, more exotic ketones and other chemicals (e.g. lactones and 3-hydroxycarboxylic acids) could be produced by replacing the original AT domain with AT domains that naturally incorporate rare malonyl-CoA analogs and expressing these engineered PKSs in hosts capable of producing these extender substrates.

METHODS

Chemicals. All chemicals were purchased from Sigma-Aldrich unless otherwise described. (2S,3R) 3-hydroxy-2-methylpentanoyl-S-N-acetylcysteamine thioester (1), (4S, 5R)-3-oxo-2,4dimethyl-5-hydroxy-heptanoic acid-δ-lactone (2), (4S, 5R)-3-oxo-4-methyl-5-hydroxy-heptanoic acid-δ-lactone (3) were synthesized as previously described48-50. 2-4-dimethylpentanoic acid (4) was previously synthesized in our laboratory29.

Plasmids. Plasmids used in this study are listed in Table S3. Briefly (see Supporting Information for details), PKS genes were codon-optimized for E. coli and synthesized. These genes were subcloned into pET vectors. Page 12 of 25


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Protein expression and purification. PKS modules were produced in E. coli K207-327 and purified as described previously.29 Briefly (see Supporting Information for details), PKS genes were expressed in E. coli K207-3 at 18°C in the presence of IPTG and the corresponding proteins purified using Ni-NTA resins followed by anion exchange column chromatography at 4°C. The fractions containing target PKSs were concentrated and stored at -80°C.

In vitro polyketide biosynthesis. TKL production was carried out in 200 mM phosphate buffer, pH 7.2, containing 2 mM DTT, 1 mM EDTA, 0.8% DMSO, and 20% glycerol at 23°C in the presence of 1 µM DEBS M6+TE (or the AT-swapped mutants), 5 mM 1, and various concentrations of methylmalonyl-CoA or malonyl-CoA. The reactions were quenched at different time points by adding methanol and analyzed by LC/MS (see Supporting Information for details). 3-HA production was carried out in 100 mM phosphate buffer, pH 7.2, containing 2.5 mM TCEP at 23°C in the presence of 0.5 µM LipPks1+TE (or the AT-swapped mutants), 200 µM butyryl-CoA, various concentrations of methylmalonyl-CoA (or malonyl-CoA) and 500 µM NADPH. The reactions were quenched at different time points by adding methanol and analyzed by LC/MS (see Supporting Information for details). Ketone production was carried out in 100 mM phosphate buffer, pH 7.2, containing 2.5 mM TCEP at 23°C in the presence of 0.5 µM LipPks1+TE (or the AT-swapped mutants), 200 µM starter acyl-CoA (propionyl-CoA, nbutyryl-CoA, isobutyryl-CoA, 2-methylbutyryl-CoA, or isovaleryl-CoA), and 200 µM methylmalonyl-CoA (or malonyl-CoA). The reactions were quenched at different time points by adding methanol and incubated at 50°C for overnight to accelerate the decarboxylation reactions (the reaction tubes were covered with parafilm to prevent evaporation of ketones). The resulting

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solutions were cooled down at 4°C and centrifuged. The supernatants were then analyzed by LC/MS (see Supporting Information for details).

Microbial ketone production. E. coli K207-3 harboring a plasmid encoding LIPS M1+TE (KR null) or L2 (KR null) were grown in 50 mL of TB medium supplemented with appropriate antibiotics at 37°C until the OD600 reached 0.4. The cultures were then transferred into nonbaffled flasks and incubated at 4°C for 1 h. Protein production was induced with 250 µM IPTG in the presence of 5 mM sodium propionate. The E. coli were cultured for five days at 18°C. For each analysis, 100 µL of the cultures were quenched with an equal amount of methanol and incubated at 50°C for overnight (the reaction tubes were covered with parafilm). The resulting solutions were cooled down at 4°C, centrifuged, and filtered using Amicon Ultra Centrifugal filters, 3 kDa Ultracel, 0.5 mL device (Millipore). The resulting solutions were then analyzed by LC/MS (see Supporting Information for details).

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: Supplemental figures, tables, and methods.

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected] or [email protected]

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*Email: [email protected]

Author Contributions

S.Y. constructed plasmids, purified proteins, and determined kinetic parameters. K.D. synthesized authentic chemical standards. G.W. and E. B established ketone detection conditions in LC/MS analysis and analyzed ketone production in vitro and in vivo. S.Y., L.K., and J.D.K. were responsible for experimental design. All authors contributed to the preparation of the manuscript.

Note

J.D.K. has a financial interest in Amyris and Lygos. L.K. has a financial interest in Lygos.

ACKNOWLEDGEMENTS

We thank Ryan Phelan and Samuel Deutsch for providing synthetic DNA. This work was funded by the Defense Advanced Research Projects Agency (DARPA), U.S. Department of Defense, via Award HR001148071, by the National Science Foundation, via Awards MCB-1341894 and EEC-0540879 to the Synthetic Biology Engineering Research Center, and by the Joint BioEnergy Institute, which is funded by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under Contract DEAC02-05CH11231.

REFERENCES

1. Smith, S., and Tsai, S. C. (2007) The type I fatty acid and polyketide synthases: a tale of two megasynthases, Nat Prod Rep 24, 1041-1072.

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2. Poust, S., Hagen, A., Katz, L., and Keasling, J. D. (2014) Narrowing the gap between the promise and reality of polyketide synthases as a synthetic biology platform, Curr Opin Biotechnol 30, 32-39. 3. Wilson, M. C., and Moore, B. S. (2012) Beyond ethylmalonyl-CoA: the functional role of crotonyl-CoA carboxylase/reductase homologs in expanding polyketide diversity, Nat Prod Rep 29, 72-86. 4. Chang, C., Huang, R., Yan, Y., Ma, H., Dai, Z., Zhang, B., Deng, Z., Liu, W., and Qu, X. (2015) Uncovering the formation and selection of benzylmalonyl-CoA from the biosynthesis of splenocin and enterocin reveals a versatile way to introduce amino acids into polyketide carbon scaffolds, J Am Chem Soc 137, 4183-4190. 5. Oliynyk, M., Brown, M. J. B., Cortes, J., Staunton, J., and Leadlay, P. F. (1996) A hybrid modular polyketide synthase obtained by domain swapping, Chem Biol 3, 833-839. 6. Reeves, C. D., Murli, S., Ashley, G. W., Piagentini, M., Hutchinson, C. R., and McDaniel, R. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations, Biochemistry 40, 1546415470. 7. Kumar, P., Li, Q., Cane, D. E., and Khosla, C. (2003) Intermodular communication in modular polyketide synthases: structural and mutational analysis of linker mediated protein-protein recognition, J Am Chem Soc 125, 4097-4102. 8. Dunn, B. J., and Khosla, C. (2013) Engineering the acyltransferase substrate specificity of assembly line polyketide synthases, J R Soc Interface 10, 20130297. 9. Patel, K., Piagentini, M., Rascher, A., Tian, Z. Q., Buchanan, G. O., Regentin, R., Hu, Z., Hutchinson, C. R., and McDaniel, R. (2004) Engineered biosynthesis of geldanamycin analogs for Hsp90 inhibition, Chem Biol 11, 1625-1633. 10. McDaniel, R., Thamchaipenet, A., Gustafsson, C., Fu, H., Betlach, M., Betlach, M., and Ashley, G. (1999) Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel ‘‘unnatural’’ natural products, Proc Natl Acad Sci U S A 96, 1846-1851. 11. Hans, M., Hornung, A., Dziarnowski, A., Cane, D. E., and Khosla, C. (2003) Mechanistic analysis of acyl transferase domain exchange in polyketide synthase modules, J Am Chem Soc 125, 5366-5374. 12. Yuzawa, S., Kapur, S., Cane, D. E., and Khosla, C. (2012) Role of a conserved arginine residue in linkers between the ketosynthase and acyltransferase domains of multimodular polyketide synthases, Biochemistry 51, 3708-3710. 13. Dutta, S., Whicher, J. R., Hansen, D. A., Hale, W. A., Chemler, J. A., Congdon, G. R., Narayan, A. R., Hakansson, K., Sherman, D. H., Smith, J. L., and Skiniotis, G. (2014) Structure of a modular polyketide synthase, Nature 510, 512-517. 14. Tang, Y., Kim, C. Y., Mathews, I. I., Cane, D. E., and Khosla, C. (2006) The 2.7-Angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase, Proc Natl Acad Sci U S A 103, 11124-11129. 15. Tang, Y., Chen, A. Y., Kim, C. Y., Cane, D. E., and Khosla, C. (2007) Structural and mechanistic analysis of protein interactions in module 3 of the 6deoxyerythronolide B synthase, Chem Biol 14, 931-943. 16. Whicher, J. R., Smaga, S. S., Hansen, D. A., Brown, W. C., Gerwick, W. H., Sherman, D. H., and Smith, J. L. (2013) Cyanobacterial polyketide synthase docking domains: a tool for engineering natural product biosynthesis, Chem Biol 20, 1340-1351. Page 16 of 25


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17. Herbst, D. A., Jakob, R. P., Zahringer, F., and Maier, T. (2016) Mycocerosic acid synthase exemplifies the architecture of reducing polyketide synthases, Nature 531, 533-537. 18. Chen, A. Y., Cane, D. E., and Khosla, C. (2007) Structure-based dissociation of a type I polyketide synthase module, Chem Biol 14, 784-792. 19. Khosla, C., Tang, Y., Chen, A. Y., Schnarr, N. A., and Cane, D. E. (2007) Structure and mechanism of the 6-deoxyerythronolide B synthase, Annu Rev Biochem 76, 195-221. 20. Lau, J., Fu, H., Cane, D. E., and Khosla, C. (1999) Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units, Biochemistry 38, 1643-1651. 21. Ruan, X., Pereda, A., Stassi, D. L., Zeidner, D., Summers, R. G., Jackson, M., Shivakumar, A., Kakavas, S., Staver, M. J., Donadio, S., and Katz, L. (1997) Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives, J Bacteriol 179, 6416-6425. 22. Petkovic, H., Sandmann, A., Challis, I. R., Hecht, H. J., Silakowski, B., Low, L., Beeston, N., Kuscer, E., Garcia-Bernardo, J., Leadlay, P. F., Kendrew, S. G., Wilkinson, B., and Muller, R. (2008) Substrate specificity of the acyl transferase domains of EpoC from the epothilone polyketide synthase, Org Biomol Chem 6, 500-506. 23. Stassi, D. L., Kakavas S.J., Reynolds, K. A., Gunawardana, G., Swanson, S., Zeidner, D., Jackson, M., Liu, H., Buko, A., and Katz, L. (1998) Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering, Proc Natl Acad Sci U S A 95, 7305-7309. 24. Liu, L., Thamchaipenet, A., Fu, H., Betlach, M., and Ashley, G. (1997) Biosynthesis of 2nor-6-deoxyerythronolide B by rationally designed domain substitution, J Am Chem Soc 119, 10553-10554. 25. Gerth, K., Bedorf, N., Hofle, G., Irschik, H., and Reichenbach, H. (1996) Epothilons A and B: antifungal and cytotoxic compounds from Sorangium cellulosum (Myxobacteria). Production, physico-chemical and biological properties, J Antibiot (Tokyo) 49, 560563. 26. Tang, L. (2000) Cloning and heterologous expression of the epothilone gene cluster, Science 287, 640-642. 27. Murli, S., Kennedy, J., Dayem, L. C., Carney, J. R., and Kealey, J. T. (2003) Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production, J Ind Microbiol Biotechnol 30, 500-509. 28. Wu, F., Kudo, F., Cane, D. E., and Khosla, C. (2000) Analysis of the molecular recognition features of individual modules derived from the erythromycin polyketide synthase, J Am Chem Soc 122, 4847-4852. 29. Yuzawa, S., Eng, C. H., Katz, L., and Keasling, J. D. (2013) Broad substrate specificity of the loading didomain of the lipomycin polyketide synthase, Biochemistry 52, 37913793. 30. Goh, E. B., Baidoo, E. E., Keasling, J. D., and Beller, H. R. (2012) Engineering of bacterial methyl ketone synthesis for biofuels, Appl Environ Microbiol 78, 70-80. 31. Yuzawa, S., Katz, L., and Keasling, J. D. (2015) Producing 3-hydroxycarboxylic acid and ketone using polyketide synthase, US Patent Application, 20150307855. 32. Goh, E. B., Baidoo, E. E., Burd, H., Lee, T. S., Keasling, J. D., and Beller, H. R. (2014) Substantial improvements in methyl ketone production in E. coli and insights on the pathway from in vitro studies, Metab Eng 26, 67-76. Page 17 of 25



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33. Eng, C. H., Yuzawa, S., Wang, G., Baidoo, E. E., Katz, L., and Keasling, J. D. (2016) Alteration of polyketide stereochemistry from anti to syn by a ketoreductase domain exchange in a type I modular polyketide synthase subunit, Biochemistry 55, 1677-1680. 34. Keatinge-Clay, A. T. (2012) The structures of type I polyketide synthases, Nat Prod Rep 29, 1050-1073. 35. Keatinge-Clay, A. T., and Stroud, R. M. (2006) The structure of a ketoreductase determines the organization of the beta-carbon processing enzymes of modular polyketide synthases, Structure 14, 737-748. 36. Bennett, B. D., Kimball, E. H., Gao, M., Osterhout, R., Van Dien, S. J., and Rabinowitz, J. D. (2009) Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli, Nat Chem Biol 5, 593-599. 37. Lambalot, R. H., Gehring, A. M., Flugel, R. S., Zuber, P., LaCelle, M., Marahiel, M. A., Reid, R., Khosla, C., and Walsh, C. T. (1996) A new enzyme superfamily - the phosphopantetheinyl transferases, Chemistry & biology 3, 923-936. 38. Pfeifer, B. A., Admiraal, S. J., Gramajo, H., Cane, D. E., and Khosla, C. (2001) Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli, Science 291, 1790-1792. 39. Murli, S., Kennedy, J., Dayem, L. C., Carney, J. R., and Kealey, J. T. (2003) Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production, Journal of industrial microbiology & biotechnology 30, 500-509. 40. Sugimoto, Y., Ding, L., Ishida, K., and Hertweck, C. (2014) Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line, Angew Chem Int Ed Engl 53, 1560-1564. 41. Whicher, J. R., Dutta, S., Hansen, D. A., Hale, W. A., Chemler, J. A., Dosey, A. M., Narayan, A. R., Hakansson, K., Sherman, D. H., Smith, J. L., and Skiniotis, G. (2014) Structural rearrangements of a polyketide synthase module during its catalytic cycle, Nature 510, 560-564. 42. Jenke-Kodama, H., Borner, T., and Dittmann, E. (2006) Natural biocombinatorics in the polyketide synthase genes of the actinobacterium Streptomyces avermitilis, PLoS Comput Biol 2, e132. 43. Ridley, C. P., Lee, H. Y., and Khosla, C. (2008) Evolution of polyketide synthases in bacteria, Proc Natl Acad Sci U S A 105, 4595-4600. 44. Yuzawa, S., Kim, W., Katz, L., and Keasling, J. D. (2012) Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli, Curr Opin Biotechnol 23, 727-735. 45. Yuzawa, S., Kim, W., Katz, L., and Keasling, J. D. (2012) Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli, Current opinion in biotechnology 23, 727-735. 46. Haushalter, R. W., Kim, W., Chavkin, T. A., The, L., Garber, M. E., Nhan, M., Petzold, C. J., Katz, L., and Keasling, J. D. (2014) Production of anteiso-branched fatty acids in Escherichia coli; next generation biofuels with improved cold-flow properties, Metab Eng. 47. Jiang, M., and Pfeifer, B. A. (2013) Metabolic and pathway engineering to influence native and altered erythromycin production through E. coli, Metab Eng 19, 42-49.

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48. Sharma, K. K., and Boddy, C. N. (2007) The thioesterase domain from the pimaricin and erythromycin biosynthetic pathways can catalyze hydrolysis of simple thioester substrates, Bioorg Med Chem Lett 17, 3034-3037. 49. Castonguay, R., He, W., Chen, A. Y., Khosla, C., and Cane, D. E. (2007) Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase, J Am Chem Soc 129, 13758-13769. 50. Hinterding, K., Singhanat, S., and Oberer, L. (2001) Stereoselective synthesis of polyketide fragments using a novel intramolecular Claisen-like condensation/reduction sequence, Tetrahedron Lett 42, 8463-8465.

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Page 20 of 26

FIGURES O

(A)

DEBS3 (M5, M6)

DEBS2 (M3, M4

DEBS1 (M1, M2) S-CoA

AT ACP KS

Propionyl-CoA

HO

S

O

O

HO

HO

O

O

HO

HO

O

HO

HO

O

HO

HO

O

HO

HO

O

HO

HO

S-CoA

Methylmalonyl-CoA +

AT KR ACP TE

S

S

S

O

O

O

KS AT KR ACP KS

KS AT ACP KS AT DH ER KR ACP

S

S

S

+ O

AT KR ACP KS AT KR ACP

O

O

HO

NADPH OH

HO O

OH

O

OH

6-dEB

(B)

OH O

H N

S

DEBS M6+TE KS

O

1

O

O

HO

O

S

S O

O

+

O

AT KR ACP TE O

2

O

HO S-CoA

HO

Methylmalonyl-CoA

(C)

OH O

AT-swapped DEBS M6+TE

H N

S

KS

O

1

O HO

O

S

O

HO

O

AT-swapped DEBS M6+TE

H N

KS

O O

S

O

S-CoA

OH O

AT KR ACP TE

S

+

(D)

O

1

O

2 O HO

HO

S

S

+ O S-CoA

O

AT KR ACP TE

O

O

HO

O

O

O

3

HO

Malonyl-CoA

Methylmalonyl-CoA

Figure 1. A model PKS system used in this study. (A) Proposed model for 6-dEB biosynthesis by DEBS. (B) Proposed model for biosynthesis of 2 by DEBS M6+TE. (C) Proposed model for biosynthesis of 2 by AT-swapped DEBS M6+TE. (D) Proposed model for biosynthesis of 3 by AT-swapped

DEBS

M6+TE.

Abbreviations:

6-dEB,

6-deoxyerythronolide

B;

AT,

acyltransferase; ACP, acyl carrier protein; CoA, coenzyme A; DH, dehydratase; DEBS, 6deoxyerythronolide B synthase; ER, enoyl reductase; KR, ketoreductase; KS, ketosynthetase; M, module; TE, thioesterase; TKL, triketide lactone.

Page 20 of 25


Page 21 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

DEBS M6: VFVFPGQG EPOS M4: AFLFTGQG

KAL

DEBS M6: IGSLHRDTAE EPOS M4: LLPACLPEAE

PAL1

KS

PAL2 KR

AT

DEBS M6: GTNAHVIIA EPOS M4: GTNAHVVLE

ACP

DEBS M6: LPNYPFEPQRYWL EPOS M4: LPTYPWQRQRYWL

(B) PAL1

KAL

DEBS M6+TE

KS

D0

KS

D1

KS

D2

KS

D3

KS

D

KS

PAL2

AT

PAL1

KAL

PAL1

PAL1

PAL1

PAL1 AT

ACP

TE

KR

ACP

TE

KR

ACP

TE

KR

ACP

TE

KR

ACP

TE

PAL2

AT

KAL

KR

PAL2

AT

KAL

TE

PAL2

AT

KAL

ACP

PAL2

AT

KAL

KR

PAL2

Figure 2. AT domain boundaries used in this study. (A) Sequence alignment of DEBS M6 and EPOS M4. The conserved sequences between domain and linker boundaries are shown. (B) Schematic representation of AT-swapped mutants of DEBS M6+TE, D0-D4. The boxes and lines highlighted in red are derived from their counterparts in EPOS M4. Abbreviations: EPOS, epothilone polyketide synthase; KAL, KS to AT linker; PAL, post AT linker; all others as in Figure 1.

Page 21 of 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

(A)

Page 22 of 26

lipPks3 (M4, M5

lipPks2 (M2, M3

lipPks1 (M1 S-CoA

AT ACP KS

Isobutyryl-CoA

S

S

KS AT DH KR ACP KS

S O

O

O

HO S-CoA

HO HO

Methylmalonyl-CoA + O

AT DH KR ACP

S

S

O

O

O

HO

KS AT DH KR ACP KS AT DH KR ACP

S

O

+ O

AT KR ACP

HO

O

HO

HO

OH

S-CoA

O

OH O

Malonyl-CoA +

O

N

OH

β-lipomycin

NADPH

O

(B)

S-CoA

AT ACP KS

n-Butyryl-CoA

HO

S O

+ O

OH O

AT KR ACP TE S O

S-CoA

OH

AT ACP KS

n-Butyryl-CoA

4

S-CoA

HO

O

OH

AT KR ACP TE

S O

+ O

O

O

AT-swapped LIPS M1+TE

O

(C)

LIPS M1+TE

S O

O OH

5

HO S-CoA

Methylmalonyl-CoA +

Malonyl-CoA +

NADPH

NADPH

Figure 3. A second model PKS system used in this study. (A) Proposed model for β-lipomycin biosynthesis by LIPS. (B) Proposed model for biosynthesis of 4 by LIPS M1+TE. (C) Proposed model for biosynthesis of 5 by AT-swapped LIPS M1+TE. Abbreviations: HA, hydroxycaboxylic acid; LIPS, lipomycin polyketide synthase; all others as in Figure 1.

Page 22 of 25


Page 23 of 26

O

(A) R

L2

S-CoA

AT ACP KS

acyl-CoA

O HO

O

AT KR ACP TE

S

R

- CO2

O

O

O

O

OH

7

6

S

O

+

8

O

O

R

O

O

O R

S-CoA

9

10

Malonyl-CoA

~~

~~

~~

~~

R=

~~

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(B) Propionyl-CoA + Malonyl-CoA

n-Butyryl-CoA + Malonyl-CoA

O

O

6

Isobutyryl-CoA + Malonyl-CoA

O

7 8

2-Methylbutyryl-CoA + Malonyl-CoA

Isovaleryl-CoA + Malonyl-CoA

O

O

10 9

Figure 4. In vitro short-chain ketone production. (A) Proposed model for biosynthesis of 6-10 by L2. (B) LC/MS analysis of each enzyme reaction in the absence of NADPH (top chromatograms) and 25 µM of authentic standards 6-10 (bottom chromatograms). Abbreviations as in Figure 1.

Page 23 of 25



O

(A) R

AT ACP KS

Propionyl-CoA or Isovaleryl-CoA + O

O

R

L2 (KR null)

S-CoA

AT KR X ACP TE

AT ACP KS

11

S

S O

O

O

(B)

LIPS M1+TE (KR null)

S-CoA

R

Propionyl-CoA or Isovaleryl-CoA

R

O

O

AT KR X ACP TE

S O

R

or

O

O

R

O HO

S-CoA

O HO

12

Methylmalonyl-CoA + NADPH

6

S

O

+

or

O

O

S-CoA

10

Malonyl-CoA + NADPH

(C) 40

25

35 20

25

6 (µM)

11 (µM)

30

20 15

15

10

10 5 5 0

LIPS M1+TE LIPS M1+TE (KR null) NADPH

0

+ -

+ +

+ -

+ +

L2 L2 (KR null) NADPH

+ -

+ +

+ -

+ +

30

45 40

25 35 20

10 (µM)

30

12 (µM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 26

25 20

15

10

15 10

5 5 0

0

LIPS M1+TE LIPS M1+TE (KR null) NADPH

+ -

+ +

+ -

+ +

L2 L2 (KR null) NADPH

+ -

+ +

+ -

+ +

Figure 5. In vitro short-chain ketone production in the presence of NADPH. (A) Proposed model for biosynthesis of 11 and 12 by LIPS M1+TE (KR null) in the presence of NADPH. (B) Proposed model for biosynthesis of 6 and 10 by L2 (KR null) in the presence of NADPH. (B) LC/MS analysis of 11 (top left), 12 (bottom left), 6 (top right), and 10 (bottom right) production in the presence of 500 µM NADPH. Abbreviations as in Figure 1.

Page 24 of 25


Page 25 of 26

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TABLES

TABLE 1 kcat and KM values of wild type and hybrid PKS modules for triketide lactone (2, 3) production

PKSs

Swapped regions (donor segments)

Source AT

Purification yields (relative to wild-type)

kcat (methylmalonylCoA) (min-1)

KM (methylmalonylCoA) (µ µM)

kcat (malonylCoA) (min-1)

KM (malonylCoA) (µ µM)

DEBS M6+TE

-

-

1.00

2.74 ± 0.02

6.9 ± 0.4

n.d.a

n.d.a

D0

AT-PAL1PAL2

EPOS M4

0.05

n.d.a

n.d.a

n.d.a

n.d.a

D1

KAL-ATPAL1

EPOS M4

0.72

2.34 ± 0.06

12 ± 2

0.21

9±3

D2

AT

EPOS M4

0.16

n.d.a

n.d.a

n.d.a

n.d.a

D3

AT-PAL1

EPOS M4

0.12

1.9 ± 0.3

47 ± 12

0.23

21 ± 3

D4

KAL-AT

EPOS M4

0.03

n.d.b

n.d.b

n.d.a

n.d.a

of

Abbreviations: DEBS, 6-deoxyerythronolide B synthase; EPOS, epothilone polyketide synthase. n.d., not determined because no product was detected (kcat < 0.0005 min-1) (a) or product formation was too slow to determine kinetic parameters (kcat < 0.017 min-1) (b).

TABLE 2 kcat and KM values of wild type and hybrid PKS modules for 3-hydroxycarboxylic acid (4, 5) production

PKSs

Swapped regions (donor segments)

Source AT

Purification yields (relative to wild-type)

kcat (methylmalonylCoA) (min-1)

KM (methylmalonylCoA) (µ µM)

kcat (malonylCoA) (min-1)

KM (malonylCoA) (µ µM)

LIPS M1+TE

-

-

1.00

0.04 ± 0.02

2 ± 1.5

n.d.b

n.d.b

L1

KAL-ATPAL1

EPOS M4

2.00

0.061 ± 0.004

13 ± 3

0.45 ± 0.01

4±1

L2

KAL-ATPAL1

BORS M1

1.71

n.d.a

n.d.a

0.34 ± 0.01

2±1

L3

KAL-ATPAL1

RAPS M2

1.13

n.d.a

n.d.a

0.033 ± 0.005

11 ± 7

L4

KAL-ATPAL1

INDS M9

2.20

n.d.a

n.d.a

0.46 ± 0.01

0.93 ± 0.14

L5

KAL-ATPAL1

SPIS M2

0.76

n.d.a

n.d.a

0.35 ± 0.02

1.5 ± 1

of

Abbreviations: LIPS, lipomycin polyketide synthase; EPOS, epothilone polyketide synthase; BORS, borrelidin polyketide synthase; RAPS, rapamycin polyketide synthase; INDS, indanomycin polyketide synthase; and SPIS, spinosad polyketide synthase. n.d., not determined because no product was detected (kcat < 0.001 min-1) (a) or product formation was too slow to determine kinetic parameters (kcat < 0.002 min-1) (b).

Page 25 of 25



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Page 26 of 26

Table of Contents PAL1

KAL

Acceptor PKS

KS

AT

PAL1

KAL

AT-swapped mutant library

KS

PAL1

KS

AT


Reduction loop

ACP

Reduction loop

ACP

Reduction loop

ACP

PAL2

AT

KAL

Donor PKS

PAL2

PAL2

Comprehensive in Vitro Analysis of Acyltransferase Domain

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