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Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli
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Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli Satoshi Yuzawa1,3, Woncheol Kim1,3, Leonard Katz1,4 and Jay D Keasling1,2,3,4,5 Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli. Addresses 1 QB3 Institute, University of California, Berkeley, CA 94270, USA 2 Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94270, USA 3 Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA 4 Synthetic Biology Engineering Research Center, 5885 Hollis Street, Emeryville, CA 94608, USA 5 Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94270, USA Corresponding author: Keasling, Jay D ([email protected])
Current Opinion in Biotechnology 2012, 23:727–735 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Hal Alper and Wilfried Weber For a complete overview see the Issue and the Editorial Available online 13th January 2012 0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2011.12.029
Introduction Heterologous natural product biosynthesis has emerged as a strategy to produce clinically important compounds that are too complex to be chemically synthesized. Many polyketides are produced by complex, slow-growing microorganisms such as actinobacteria and myxobacteria that make their production and isolation difficult. To this end, organisms like Escherichia coli are at a great advantage as heterologous hosts because they are well-characterized and easy to culture. Polyketides are linear or cyclic poly-b-ketones with varying degrees of reduction at the b-carbonyl centers (b-C O, b-C–OH, b-C C-a, or b-C–C-a). Polyketides www.sciencedirect.com
are widely used as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. While structurally diverse, all polyketides are assembled by successive rounds of decarboxylative condensation between an acyl thioester and an a-carboxythioester in a fashion similar to fatty acid biosynthesis. The enzymes that catalyze these condensations are referred to as polyketide synthases (PKSs) and they are generally classified into three groups (Type I, Type II, and Type III PKSs). Type I PKSs are further subdivided into two groups, iterative and modular. Modular type I PKSs (mPKSs) are unique among these groups in terms of the chain extension mechanism. Iterative type I PKSs (iPKSs), type II PKSs, and type III PKSs repeatedly use the same catalytic domains for multiple rounds of chain extension whereas mPKSs use different catalytic domains for each round of chain extension as discussed below. Heterologous biosynthesis of polyketides was first demonstrated with Streptomyces parvulus in 1984 [1]. Malpartida, Hopwood, and co-workers introduced Streptomyces coelicolor DNA that carried the biosynthetic gene cluster for the antibiotic actinorhodin into S. parvulus, which does not produce actinorhodin, and showed a similar amount of production as wild-type S. coelicolor. Actinorhodin is a so-called aromatic polyketide; this structural class of polyketides is synthesized by type II PKSs. As exemplified by the first successful heterologous biosynthesis of actinorhodin in S. parvulus, other type II PKS gene clusters have been expressed in Streptomyces strains, mostly S. coelicolor and Streptomyces lividans. By contrast, heterologous polyketide production by type III PKSs, which were initially recognized as a member of the plant chalcone synthase superfamily involved in flavonoid and stillbene biosynthesis, was first demonstrated in E. coli in 1991 [2]. Since then, many type III PKSs, of plant and microbial origin, have been expressed in E. coli. iPKSs were originally thought to be specific to fungi. However, several bacterial iPKSs have been found and characterized since the discovery of avilamycin synthase in Streptomyces viridochromogenes in 1997 [3]. The first example of a heterologously expressed iPKS was 6methylsalicylic acid synthase (6MSAS). The 6MSAS PKS genes from Penicillium patulum were expressed in S. coelicolor, which produced a significant amount of 6methylsalicylic acid (6MSA) [4]. 6MSA production in E. coli was achieved by expressing 6MSAS and a substrate Current Opinion in Biotechnology 2012, 23:727–735
728 Tissue, cell and pathway engineering
Figure 1 DEBS1
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Schematic architecture of 6-deoxyerythronolide B synthase (DEBS). The polyketide product, 6-deoxyerythronolide B (6dEB) is synthesized from propionyl-CoA and six (2S)-methylmalonyl-CoA. Previously reported CoA-linked starters and extension substrates are also shown. Blue arrows indicates the substrate of each AT domain. Domains outlined in gray are inactive. Abbreviations: ACP, acyl carrier protein domain; AT, acyl transferase domain; DH, dehydratase domain; KR, ketoreductase domain; KS, ketosynthase domain; TE, thioesterase domain.
promiscuous surfactin phosphopantetheinyl transferase from Bacillus subtilis, encoded by sfp, that converts the expressed apo 6MSAS to its holo form [5,6]. This posttranslational modification is not required in type III PKS systems. mPKSs are unique not only in the chain extension mechanism but also in their frequent use of (2S)-methylmalonyl-CoA that does not exist naturally in E. coli. Although heterologous polyketide biosynthesis by mPKSs was achieved in 1994 in S. coelicolor, production in E. coli was not reported until 2001 [7,8]. In this work, Pfeifer, Khosla and co-workers developed a new E. coli strain, BAP1, that harbors sfp in the genome and that was engineered to supply the substrates of the mPKS, propionyl-CoA and (2S)-methylmalonyl-CoA, from exogenously added propionate. In this review, we focus on heterologous production of polyketides by mPKSs in E. coli and also speculate on its potential for biosynthetic engineering. For reviews on heterologous expression and biosynthetic mechanisms of other types of PKSs, the reader is directed elsewhere [9–12]. Current Opinion in Biotechnology 2012, 23:727–735
Architecture of modular type I polyketide synthases and assembly line synthesis mPKSs are best exemplified by the PKS that synthesizes 6-deoxyerythronolide B (6dEB), the macrocyclic aglycon of the antibiotic erythromycin, which was the first heterologously expressed mPKS [7,8]. 6dEB synthase (DEBS) from Saccharopolyspora erythraea is composed of three large polypeptides: DEBS1, DEBS2, and DEBS3 (Figure 1). Each subunit harbors two modules, defined as a set of catalytic domains that are together responsible for one round of polyketide chain extension and subsequent processing (reduction) of the newly generated b-ketone. In addition, DEBS1 and DEBS3 contain additional domains capable of selecting the appropriate starter unit and releasing the final product, respectively. Therefore, in mPKSs, the structure of each extension unit is determined by the domain composition of each module, and the number of modules plus the starter unit dictates the chain length. A module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl www.sciencedirect.com
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carrier protein (ACP) domain. The mechanism by which these domains extend the polyketide chain is as follows. First, the KS domain receives the growing polyketide chain from the ACP domain of the preceding module, while the AT domain transfers a coenzyme A-linked acarboxyacyl extender unit to the phosphopantetheine arm of the ACP. Although utilization of extender units other than malonyl-CoA and (2S)-methylmalonyl-CoA is only rarely observed, several other different extender units including ethylmalonyl-CoA and chloroethylmalonyl-CoA have been reported [13]. The KS then catalyzes decarboxylative condensation between the growing chain (n carbon length) and the extender unit, leading to the formation of a (n + 2) b-ketoacyl thioester-ACP intermediate. The b-ketone is then modified by auxiliary enzymes that may be present in the module: the ketoreductase (KR), the dehydratase (DH), and the enoyl reductase (ER) domains. This intermediate is then translocated to the KS domain of the next module for subsequent condensation. Modules that contain a C-methyltransferase domain (cMT), which transfers a methyl group from Sadenosyl-methionine to the a-carbon to generate a rare geminal dimethyl group, are present in yersiniabactin and epothilone synthases. Chemically, the b-ketone of the corresponding nascent polyketide chain draws the required electrons from the a-carbon to allow methylation to take place at that site. Some of the cMT-containing modules contain a functional KR domain; reduction of the b-ketone to the corresponding hydroxyl must occur after methylation of the a-carbon. Further reduction to form an a,bdouble bond is not possible because of the lack of available proton on the a-carbon. mPKSs utilize a wide range of starter units in the assembly of their products. DEBS1, for example, contains a loading didomain, which consists of an AT domain and an ACP domain, before the first two extension modules: The AT domain specifies that propionyl-CoA is preferentially loaded to the cognate ACP domain. Loading AT domains that are predicted to use other CoAs such as isobutyrylCoA, 2-methylbutyryl-CoA, isovaleryl-CoA, cyclohexylCoA, and benzoyl-CoA have also been found in this type I PKS systems [14]. Most other mPKSs, however, have an additional KS domain, termed KSQ, in which the active site cysteine residue has been changed to a glutamine residue. These KSQ-AT-ACP tridomains are known to load malonyl-CoA or methylmalonyl-CoA and catalyze the decarboxylation to generate acetyl or propionyl starter units in situ [15]. On the contrary, in mPKSs such as rifamycin synthase, free carboxylic acids appear to be activated and loaded by a nonribosomal peptide synthase-like adenylation domain at the N-terminus of the PKSs [14]. Thioesterase (TE) domain-mediated macrolactonization is the most-frequent product release mechanism observed in mPKSs [16]. Other minor mechanisms include TEcatalyzed hydrolysis to generate a free acid, and recently www.sciencedirect.com
characterized KR and sulfotranferase domains plus TEmediated dehydrative decarboxylation to generate a terminal alkene [16,17].
Precursor supply in Escherichia coli Malonyl-CoA and (2S)-methylmalonyl-CoA are the most commonly used substrates for mPKSs. Native E. coli metabolism, however, only produces malonyl-CoA at level sufficient to promote polyketide biosynthesis. Thus, E. coli has to be engineered to supply (2S)-methylmalonyl-CoA. As shown in Figure 2, there are currently three recognized reactions to generate (2S)-methylmalonylCoA: (1) carboxylation of propionyl-CoA, (2) isomerization of TCA cycle-derived succinyl-CoA, and (3) oxidation of (2S)-methylmalonyl-CoA semialdehyde, which is produced from valine [13]. Besides decarboxylation of (2S)methylmalonyl-CoA, propionyl-CoA can be produced from at least five routes: b-oxidation of odd-chain and branched-chain fatty acids; catabolism of threonine; from acetyl-CoA via the 3-hydroxypropionate pathway [18–23]; conversion of exogenously supplied propionate; and via the ethylmalonyl-CoA pathway [24,25,26–28,29]. Of these, the exogenous propionate-dependent pathway and succinyl-CoA isomerization pathway have been reconstituted in E. coli (Figure 2) [8,30]. Although mPKSs commonly use malonyl-CoA and methylmalonyl-CoA as starter and extender units, a wide variety of other starter units can be employed in polyketide biosynthesis, as described earlier. For example, isobutyryl-CoA and 2-methylbutyryl-CoA are used as starters for avermectin synthase, and cyclohexyl-CoA, benzoyl-CoA, and 3-amino-5-hydroxybenzoic acid (AHBA) start phoslactomycin, soraphen, and rifamycin biosynthesis, respectively [14]. Isobutyryl-CoA, 2methylbutyryl-CoA, and isovaleryl-CoA are well-known intermediates in valine, isoleucine, and leucine catabolism. E. coli metabolism, however, insufficiently supplies these acyl-CoAs, as well as propionyl-CoA. CyclohexylCoA and benzoyl-CoA are also not produced at appreciable levels in E. coli. Seven genes are believed to be necessary for the biosynthesis of AHBA and were heterologously expressed in E. coli. The production of AHBA was confirmed and a tetraketide intermediate of rifamycin has been synthesized in the engineered strain [31].
Protein expression of modular type I polyketide synthases in Escherichia coli Functional expression of mPKSs in E. coli has proven to be challenging owing to two key issues, phosphopantetheinylation of ACP domains and folding. The attempts to express functional mPKSs in E. coli began with DEBS in 1993 [32]. In this work, Roberts, Staunton, and Leadlay observed that purified DEBS3 was not post-translationally modified with the phosphopantetheinyl group, although this 332-kDa protein was properly folded. To address this issue, sfp was co-expressed or integrated into Current Opinion in Biotechnology 2012, 23:727–735
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Figure 2
Even-chain fatty acids 2x Acetyl-CoA etyl-CoA
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Figure 3
Module substitution [44]
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Biosynthetic engineering of 6-deoxyerythronolide B synthase (DEBS). Red color indicates manipulation of DEBS by domain or module engineering. Nacetyl cysteamine (NAC) substrates used in unnatural feeding experiments are also shown in red. Blue arrows indicates the substrate of the redcolored AT domain or of the KS domain in module 2, respectively. Domains outlined in gray are inactive. [35,39,40–44] indicate references cited. Abbreviations: ACP, acyl carrier protein domain; AT, acyl transferase domain; DH, dehydratase domain; KR, ketoreductase domain; KS, ketosynthase domain; TE, thioesterase domain; TKL, triketide lactone.
the genome to allow successful in vivo phosphopantetheinylation of ACP domains of mPKSs [8,33]. The other key challenge to functional mPKS expression is protein folding, which is greatly impacted by the postinduction temperature. In the first production runs of 6dEB in E. coli, active DEBS1, DEBS2, and DEBS3 were only observed by lowering the expression temperature from 30 8C to 22 8C, probably owing to misfolding of the proteins at the elevated temperatures [8]. More recently, it has been shown that co-expression with E. coli or S. coelicolor chaperones can improve the solubility and purification yield of DEBS in E. coli [34].
Biosynthetic engineering of modular type I polyketide synthases The modular nature of mPKSs and the colinearity between the module activities and the structure of the polyketide products offers a wealth of engineering opportunities. In fact, a variety of ‘unnatural’ polyketides (>200) have been produced by engineered DEBSs in a programmed manner
since Donadio, Katz, and co-workers first demonstrated the potential of mPKS engineering in 1991 [35–38]. Manipulation of DEBS has included (1) domain deletion, inactivation, insertion, and substitution to modify the structure of each extension unit, (2) module deletion and substitution to alter the chain length of the resulting polyketides or to construct hybrid mPKSs, and (3) unnatural substrate feeding (Figure 3) [39–44]. Beyond simple domain and module swapping approaches, a major challenge for biosynthetic engineering will be to overcome a severe loss in overall catalytic efficiency of the engineered mPKSs compared with natural mPKSs. Recently, detailed structural studies of catalytic domains have revealed functional domain boundaries that can now be exploited for more accurate swapping experiments, even though module structures remain unresolved [45– 48,49,50,51]. In addition, dedicated analyses of intermodule and intra-module interactions have begun to reveal the determinants of the observed binding specificities at amino acid residue level, which would greatly aid the
(Figure 2 Legend) Metabolic pathways leading to the formation of (2S)-methylmalonyl-CoA from central metabolites, such as acetyl-CoA, amino acids, and fatty acids shown in red. Three reactions to generate (2S)-methylmalonyl-CoA, carboxylation of propionyl-CoA, isomerization of succinylCoA, and oxidation of (2S)-methylmalonyl-CoA semialdehyde are numbered as (1), (2), and (3), respectively. The pathways that were reconstituted in E. coli are shown in blue. Solid arrows indicate one-step reactions catalyzed by enzymes. Dashed arrows indicate multiple enzymatic processes. www.sciencedirect.com
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Figure 4
L1: KSQ-mAT-ACP
L2: KSQ-mmAT-ACP
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E12: KSR-mmAT-KRA-ACP
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Current Opinion in Biotechnology
Biological parts that constitute modular type I polyketide synthases. L1-L8 indicate loading domains. E1-E19 indicate extension modules. R1-R3 indicate release domains. Abbreviations: ACP, acyl carrier protein domain; mAT, methylmalonyl-CoA specific acyl transferase domain; mAT, malonylCoA specific acyl transferase domain; mmAT, methylmalonyl-CoA specific acyl transferase domain; pAT, propionyl-CoA specific acyl transferase domain; ibAT, isobutyryl-CoA specific acyl transferase domain; ivAT, isovaleryl-CoA specific acyl transferase domain; mbAT, 2-methylbutyryl-CoA specific acyl transferase domain; chAT, cyclohexyl-CoA specific acyl transferase domain; bzAT, benzoyl-CoA specific acyl transferase domain; emAT, ethylmalonyl-CoA specific acyl transferase domain; cemAT, chloroethylmalonyl-CoA specific acyl transferase domain; cMT, C-methyltransferase domain; DH, dehydratase domain; KR, ketoreductase domain; KRA, A-type ketoreductase domain; KRB, B-type ketoreductase domain; KS, ketosynthase domain; KSQ, ketosynthase domain with decarboxylation activity; KSR, ketosynthase domain with racemization activity; ST, sulfotransferase domain; TE, thioesterase domain. Current Opinion in Biotechnology 2012, 23:727–735
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design that allows noncognate protein-protein interaction [52,53,54,55,56,57,58]. Another major challenge in mPKS engineering is how to manipulate the substrate specificity of domains to allow the efficient transfer of the growing, unnatural, polyketide chain from one domain to the next and maintain the enzyme kinetics. Although this challenge remains to be solved, a product-specific selection strategy should play an important role in biosynthetic engineering. For example, Lee and Khosla reported a bactericidal activity-based selection strategy to identify erythromycin-overproducing E. coli strains [59]. However, clearly, other strategies have to be developed if target polyketide compounds are not antibiotics.
Modular type I polyketide synthases, a platform of synthetic biology One definition of synthetic biology is ‘‘the deliberate design and fabrication of novel biologically-based components as well as the redesign of existing biological systems’’. Although many challenges still exist as described earlier, mPKSs have the potential as a platform to synthesize thousands of chemicals. As shown in Figure 4, at least 19 types of extension modules are available in nature, as well as 8 types of loading domains and 3 types of release domains, which account for >2000 naturally occurring polyketides. Recently, the cost of DNA synthesis and gene assembly has declined exponentially [60], which should accelerate design and fabrication of mPKSs with DNA parts where codons are optimized for designated heterologous hosts and domains and modules are flanked by unique restriction enzyme sites to facilitate domain and module swapping [37]. An example is the re-construction of the DEBS system using the parts L3 + E13 + E7 + E11 + E14 + E7 + E7 + R1 shown in Figure 4. Currently, approximately 50 genome sequences of Streptomyces strains are available (http://www.ncbi.nlm.nih.gov/ genomes/MICROBES/microbial_taxtree.html). Further, genome sequencing and genome mining with appropriate search algorithms would expand the scope of starter units, extension modules and release mechanisms. From this point of view, bioinformatics and chemoinformatics to connect between chemical products and the gene clusters should play an important role in future mPKS engineering. Efforts relating to isolation and structural characterization of products and related biocatalytic mechanisms also continue to be important for development of reliable computational algorithms.
knowledge of fermentation strategies. Preliminary studies have highlighted both the promise and limitations of polyketide biosynthesis in E. coli. Insufficient supply of polyketide precursors is one challenge in using E. coli as a convenient host for heterologous polyketide production. Functional folding of mPKSs at 37 8C is another issue to be addressed. Rational exploitation of the modular nature of mPKSs is only just beginning to emerge, even for a prototypical mPKSs such as DEBS. Given the accumulating knowledge of the functional boundaries of catalytic domains, we need to re-evaluate past engineering efforts to develop more accurate predictive algorithms that exploit modularity, which are currently exemplified by MapsiDB and SBSPKS [61,62]. Understanding the fundamentals of the molecular enzymology and structural biology of not only DEBS but also other mPKSs is important to design kinetically competent chimeric mPKSs that will rapidly and in high yield produce natural and unnatural polyketides.
Acknowledgements This work was funded by the Advanced Research Projects Agency – Energy (ARPA-E), U.S. Department of Energy, under award number DEAR0000091, by the National Science Foundation, award No. EEC-0540879 to the Synthetic Biology 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 number DE-AC02-05CH11231.
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Conclusions E. coli offers unquestioned advantages to support heterologous production of polyketides including fast growth in simple culture conditions, well understood metabolism, available recombinant DNA tools, and extensive www.sciencedirect.com
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8.
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10. Hertweck C, Luzhetskyy A, Rebets Y, Bechthold A: Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork. Natural Product Reports 2007, 24:162-190. 11. Fujii I: Heterologous expression systems for polyketide synthases. Natural Product Reports 2009, 26:155-169. 12. Fujii I: Functional analysis of fungal polyketide biosynthesis genes. The Journal of Antibiotics 2010, 63:207-218. 13. Chan YA, Podevels AM, Kevany BM, Thomas MG: Biosynthesis of polyketide synthase extender units. Natural Product Reports 2009, 26:90-114. 14. Moore BS, Hertweck C: Biosynthesis and attachment of novel bacterial polyketide synthase starter units. Natural Product Reports 2002, 19:70-99. 15. Bisang C, Long PF, Cortes J, Westcott J, Crosby J, Matharu AL, Cox RJ, Simpson TJ, Staunton J, Leadlay PF: A chain initiation factor common to both modular and aromatic polyketide synthases. Nature 1999, 401:502-505. 16. Du L, Lou L: PKS and NRPS release mechanisms. Natural Product Reports 2010, 27:255-278. 17. Gu L, Wang B, Kulkarni A, Gehret JJ, Lloyd KR, Gerwick L, Gerwick WH, Wipf P, Hakansson K, Smith JL et al.: Polyketide decarboxylative chain termination preceded by o-sulfonation in curacin a biosynthesis. Journal of the American Chemical Society 2009, 131:16033-16035. An precedented decarboxylative chain termination mechanism was described for the polyketide synthase of curacin A. This enzymatic process results in the formation of a rare terminal olefin in the final polyketide product. 18. Hugler M, Menendez C, Schagger H, Fuchs G: Malonylcoenzyme A reductase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO(2) fixation. Journal of Bacteriology 2002, 184:2404-2410. 19. Alber BE, Fuchs G: Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3hydroxypropionate cycle for autotrophic CO2 fixation. The Journal of Biological Chemistry 2002, 277:12137-12143. 20. Alber B, Olinger M, Rieder A, Kockelkorn D, Jobst B, Hugler M, Fuchs G: Malonyl-coenzyme A reductase in the modified 3hydroxypropionate cycle for autotrophic carbon fixation in archaeal Metallosphaera and Sulfolobus spp. Journal of Bacteriology 2006, 188:8551-8559. 21. Alber BE, Kung JW, Fuchs G: 3-Hydroxypropionyl-coenzyme A synthetase from Metallosphaera sedula, an enzyme involved in autotrophic CO2 fixation. Journal of Bacteriology 2008, 190:1383-1389. 22. Kockelkorn D, Fuchs G: Malonic semialdehyde reductase, succinic semialdehyde reductase, and succinyl-coenzyme A reductase from Metallosphaera sedula: enzymes of the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle in Sulfolobales. Journal of Bacteriology 2009, 191:6352-6362. 23. Teufel R, Kung JW, Kockelkorn D, Alber BE, Fuchs G: 3hydroxypropionyl-coenzyme A dehydratase and acryloyl-coenzyme A reductase, enzymes of the autotrophic 3hydroxypropionate/4-hydroxybutyrate cycle in the Sulfolobales. Journal of Bacteriology 2009, 191:4572-4581. 24. Alber BE, Spanheimer R, Ebenau-Jehle C, Fuchs G: Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides. Molecular Microbiology 2006, 61:297-309. Current Opinion in Biotechnology 2012, 23:727–735
25. Erb TJ, Berg IA, Brecht V, Muller M, Fuchs G, Alber BE: Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. In Proceedings of the National Academy of Sciences of the United States of America 2007, 104:10631-10636. A pathway termed the ethylmalonyl-CoA pathway is described. This pathway starts from conversion of crotonyl-CoA into ethylmalonyl-CoA by crotonyl-CoA carboxylase/reductase in the presence of CO2 and NADPH. The enzyme from Rhodobacter sphaeroides was heterologously produced in E. coli and characterized. Ethylmalonyl-CoA is a precursor in the production of numerous polyketide-based macrolide antibiotics. 26. Zarzycki J, Schlichting A, Strychalsky N, Muller M, Alber BE, Fuchs G: Mesaconyl-coenzyme A hydratase, a new enzyme of two central carbon metabolic pathways in bacteria. Journal of Bacteriology 2008, 190:1366-1374. 27. Erb TJ, Retey J, Fuchs G, Alber BE: Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases. The Journal of Biological Chemistry 2008, 283:32283-32293. 28. Erb TJ, Fuchs G, Alber BE: (2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. Molecular Microbiology 2009, 73:992-1008. 29. Erb TJ, Brecht V, Fuchs G, Muller M, Alber BE: Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase. In Proceedings of the National Academy of Sciences of the United States of America 2009, 106:8871-8876. Crotonyl-CoA carboxylase/reductase catalyzes both reduction of crotonyl-CoA to butyryl-CoA and carboxylation of crotonyl-CoA to ethylmalonyl-CoA. In this report, the complete stereochemical courses of both reactions are described. 30. Dayem LC, Carney JR, Santi DV, Pfeifer BA, Khosla C, Kealey JT: Metabolic engineering of a methylmalonyl-CoA mutaseepimerase pathway for complex polyketide biosynthesis in Escherichia coli. Biochemistry 2002, 41:5193-5201. 31. Watanabe K, Rude MA, Walsh CT, Khosla C: Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli. In Proceedings of the National Academy of Sciences of the United States of America 2003, 100:9774-9778. 32. Roberts GA, Staunton J, Leadlay PF: Heterologous expression in Escherichia coli of an intact multienzyme component of the erythromycin-producing polyketide synthase. European Journal of Biochemistry/FEBS 1993, 214:305-311. 33. Gokhale RS, Tsuji SY, Cane DE, Khosla C: Dissecting and exploiting intermodular communication in polyketide synthases. Science 1999, 284:482-485. 34. Betancor L, Fernandez MJ, Weissman KJ, Leadlay PF: Improved catalytic activity of a purified multienzyme from a modular polyketide synthase after coexpression with Streptomyces chaperonins in Escherichia coli. Chembiochem: A European Journal of Chemical Biology 2008, 9:2962-2966. 35. Donadio S, Staver MJ, McAlpine JB, Swanson SJ, Katz L: Modular organization of genes required for complex polyketide biosynthesis. Science 1991, 252:675-679. 36. McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Betlach M, Ashley G: Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel ‘‘unnatural’’ natural products. In Proceedings of the National Academy of Sciences of the United States of America 1999, 96:1846-1851. 37. Menzella HG, Reid R, Carney JR, Chandran SS, Reisinger SJ, Patel KG, Hopwood DA, Santi DV: Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes. Nature Biotechnology 2005, 23:1171-1176. 38. Kellenberger L, Galloway IS, Sauter G, Bohm G, Hanefeld U, Cortes J, Staunton J, Leadlay PF: A polylinker approach to reductive loop swaps in modular polyketide synthases. Chembiochem: A European Journal of Chemical Biology 2008, 9:2740-2749. 39. Donadio S, McAlpine JB, Sheldon PJ, Jackson M, Katz L: An erythromycin analog produced by reprogramming of www.sciencedirect.com
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polyketide synthesis. In Proceedings of the National Academy of Sciences of the United States of America 1993, 90:7119-7123. 40. Kao CM, Luo G, Katz L, Cane DE, Khosla C: Engineered biosynthesis of a triketide lactone from an incomplete modular polyketide synthase. Journal of the American Chemical Society 1994, 116:11612-11613. 41. Oliynyk M, Brown MJ, Cortes J, Staunton J, Leadlay PF: A hybrid modular polyketide synthase obtained by domain swapping. Chemistry & Biology 1996, 3:833-839. 42. Jacobsen JR, Hutchinson CR, Cane DE, Khosla C: Precursordirected biosynthesis of erythromycin analogs by an engineered polyketide synthase. Science 1997, 277:367-369. 43. McDaniel R, Kao CM, Fu H, Hevez P, Gustafsson C, Betlach M, Ashley G, Cane DE, Khosla C: Gain-of-function mutagenesis of a modular polyketide synthase. Journal of the American Chemical Society 1997, 119:4309-4310. 44. Ranganathan A, Timoney M, Bycroft M, Cortes J, Thomas IP, Wilkinson B, Kellenberger L, Hanefeld U, Galloway IS, Staunton J et al.: Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues. Chemistry & Biology 1999, 6:731-741. 45. Cane DE: Programming of erythromycin biosynthesis by a modular polyketide synthase. The Journal of Biological Chemistry 2010, 285:27517-27523. 46. Scaglione JB, Akey DL, Sullivan R, Kittendorf JD, Rath CM, Kim ES, Smith JL, Sherman DH: Biochemical and structural characterization of the tautomycetin thioesterase: analysis of a stereoselective polyketide hydrolase. Angewandte Chemie 2010, 49:5726-5730. 47. Akey DL, Razelun JR, Tehranisa J, Sherman DH, Gerwick WH, Smith JL: Crystal structures of dehydratase domains from the curacin polyketide biosynthetic pathway. Structure 2010, 18:94-105. 48. Zheng J, Taylor CA, Piasecki SK, Keatinge-Clay AT: Structural and functional analysis of A-type ketoreductases from the amphotericin modular polyketide synthase. Structure 2010, 18:913-922. 49. Gehret JJ, Gu L, Gerwick WH, Wipf P, Sherman DH, Smith JL: Terminal alkene formation by the thioesterase of curacin A biosynthesis: structure of a decarboxylating thioesterase. The Journal of Biological Chemistry 2011, 286:14445-14454. The 1.7-A˚ curacin thioesterase (TE) crystal structure was reported. In this study, the authors revealed how the active site is adapted to specificity for b-sulfonated substrates. Interestingly, the essential features of curacin TE appears to be conserved in five other putative ORFs encoding acyl-carrier protein–sulforansferase–TE tridomains. 50. Zheng J, Keatinge-Clay AT: Structural and functional analysis of C2-type ketoreductases from modular polyketide synthases. Journal of Molecular Biology 2011, 410:105-117. 51. Wong FT, Jin X, Mathews II, Cane DE, Khosla C: Structure and mechanism of the trans-acting acyltransferase from the disorazole synthase. Biochemistry 2011, 50:6539-6548. 52. Wu N, Tsuji SY, Cane DE, Khosla C: Assessing the balance between protein-protein interactions and enzyme-substrate interactions in the channeling of intermediates between polyketide synthase modules. Journal of the American Chemical Society 2001, 123:6465-6474.
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53. Weissman KJ, Muller R: Protein-protein interactions in multienzyme megasynthetases. Chembiochem: A European Journal of Chemical Biology 2008, 9:826-848. 54. Buchholz TJ, Geders TW, Bartley FE 3rd, Reynolds KA, Smith JL, Sherman DH: Structural basis for binding specificity between subclasses of modular polyketide synthase docking domains. ACS Chemical Biology 2009, 4:41-52. Using surface plasmon resonance, a comprehensive analysis of intermodule interaction in 10-deoxymethynolide/narbonolide and 6-deoxyerythronolide B synthases was reported. The stractural basis for the observed binding specificity was obtained by X-ray crystallographic analysis of the PikAIII/PikAIV docking domain interface. 55. Wong FT, Chen AY, Cane DE, Khosla C: Protein-protein recognition between acyltransferases and acyl carrier proteins in multimodular polyketide synthases. Biochemistry 2010, 49:95-102. 56. Tran L, Broadhurst RW, Tosin M, Cavalli A, Weissman KJ: Insights into protein-protein and enzyme-substrate interactions in modular polyketide synthases. Chemistry & Biology 2010, 17:705-716. 57. Kapur S, Chen AY, Cane DE, Khosla C: Molecular recognition between ketosynthase and acyl carrier protein domains of the 6-deoxyerythronolide B synthase. In Proceedings of the National Academy of Sciences of the United States of America 2010, 107:22066-22071. Using both experimental and computational approaches, the authors decoded the molecular basis for ketosynthase (KS)-acyl carrier protein (ACP) interactions in the 6-deoxyerythronolide B synthase. This is the first study that revealed that KS-ACP recognition is controlled at different interfaces during chain elongation versus chain transfer. 58. Charkoudian LK, Liu CW, Capone S, Kapur S, Cane DE, Togni A, Seebach D, Khosla C: Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase. Protein Science: A Publication of the Protein Society 2011, 20:1244-1255. 59. Lee HY, Khosla C: Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli. PLoS Biology 2007, 5:e45. Bactericidal activity-based selection strategy was used to evolve genes in the plasmids encoding the erythromycin biosynthetic pathway. The resulting plasmids showed more balanced expression of enzymes. 60. Mitchell W: Natural products from synthetic biology. Current Opinion in Chemical Biology 2011, 15:505-515. 61. Tae H, Sohng JK, Park K: MapsiDB: an integrated web database for type I polyketide synthases. Bioprocess and Biosystems Engineering 2009, 32:723-727. MapsiDB (http://gate.smallsoft.co.kr:8080/pks/) is an integrated web database formulated to contain data on type I polyketide synthases (PKSs), including domain and module compoition and related genome information. MapsiDB contains data entries on 45 modular and 21 iterative type I PKSs. 62. Anand S, Prasad MV, Yadav G, Kumar N, Shehara J, Ansari MZ, Mohanty D: SBSPKS: structure based sequence analysis of polyketide synthases. Nucleic Acids Research 2010, 38:W487-W496. SBSPKS (http://www.nii.ac.in/pksdb/sbspks/master.html) consists of Model_3D_PKS, Dock_Dom_Anal, and NRPS-PKS. NRPS-PKS comprises of 167 experimentally characterized polyketide synthase and nonribosomal peptide synthase gene clusters consisting of 4400 catalytic domains.
Current Opinion in Biotechnology 2012, 23:727–735
Heterologous production of polyketides by modular type I polyketide synthases in Escherichia coli Satoshi Yuzawa1,3, Woncheol Kim1,3, Leonard Katz1,4 and Jay D Keasling1,2,3,4,5 Heterologous production of polyketide compounds, an important class of natural products with complex chemical structures, was first demonstrated with Streptomyces parvulus in 1984. Although Streptomyces strains are good first options for heterologous polyketide biosynthesis, their slow growth kinetics prompt other hosts to also be considered. Escherichia coli provides key elements of an ideal host in terms of the growth rate, culture conditions, and available recombinant DNA tools. Here we review the current status and potential for metabolic engineering of polyketides in E. coli. Addresses 1 QB3 Institute, University of California, Berkeley, CA 94270, USA 2 Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94270, USA 3 Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA 94608, USA 4 Synthetic Biology Engineering Research Center, 5885 Hollis Street, Emeryville, CA 94608, USA 5 Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94270, USA Corresponding author: Keasling, Jay D ([email protected])
Current Opinion in Biotechnology 2012, 23:727–735 This review comes from a themed issue on Tissue, cell and pathway engineering Edited by Hal Alper and Wilfried Weber For a complete overview see the Issue and the Editorial Available online 13th January 2012 0958-1669/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.copbio.2011.12.029
Introduction Heterologous natural product biosynthesis has emerged as a strategy to produce clinically important compounds that are too complex to be chemically synthesized. Many polyketides are produced by complex, slow-growing microorganisms such as actinobacteria and myxobacteria that make their production and isolation difficult. To this end, organisms like Escherichia coli are at a great advantage as heterologous hosts because they are well-characterized and easy to culture. Polyketides are linear or cyclic poly-b-ketones with varying degrees of reduction at the b-carbonyl centers (b-C O, b-C–OH, b-C C-a, or b-C–C-a). Polyketides www.sciencedirect.com
are widely used as antibiotic, immunosuppressant, antitumor, antifungal, and antiparasitic agents. While structurally diverse, all polyketides are assembled by successive rounds of decarboxylative condensation between an acyl thioester and an a-carboxythioester in a fashion similar to fatty acid biosynthesis. The enzymes that catalyze these condensations are referred to as polyketide synthases (PKSs) and they are generally classified into three groups (Type I, Type II, and Type III PKSs). Type I PKSs are further subdivided into two groups, iterative and modular. Modular type I PKSs (mPKSs) are unique among these groups in terms of the chain extension mechanism. Iterative type I PKSs (iPKSs), type II PKSs, and type III PKSs repeatedly use the same catalytic domains for multiple rounds of chain extension whereas mPKSs use different catalytic domains for each round of chain extension as discussed below. Heterologous biosynthesis of polyketides was first demonstrated with Streptomyces parvulus in 1984 [1]. Malpartida, Hopwood, and co-workers introduced Streptomyces coelicolor DNA that carried the biosynthetic gene cluster for the antibiotic actinorhodin into S. parvulus, which does not produce actinorhodin, and showed a similar amount of production as wild-type S. coelicolor. Actinorhodin is a so-called aromatic polyketide; this structural class of polyketides is synthesized by type II PKSs. As exemplified by the first successful heterologous biosynthesis of actinorhodin in S. parvulus, other type II PKS gene clusters have been expressed in Streptomyces strains, mostly S. coelicolor and Streptomyces lividans. By contrast, heterologous polyketide production by type III PKSs, which were initially recognized as a member of the plant chalcone synthase superfamily involved in flavonoid and stillbene biosynthesis, was first demonstrated in E. coli in 1991 [2]. Since then, many type III PKSs, of plant and microbial origin, have been expressed in E. coli. iPKSs were originally thought to be specific to fungi. However, several bacterial iPKSs have been found and characterized since the discovery of avilamycin synthase in Streptomyces viridochromogenes in 1997 [3]. The first example of a heterologously expressed iPKS was 6methylsalicylic acid synthase (6MSAS). The 6MSAS PKS genes from Penicillium patulum were expressed in S. coelicolor, which produced a significant amount of 6methylsalicylic acid (6MSA) [4]. 6MSA production in E. coli was achieved by expressing 6MSAS and a substrate Current Opinion in Biotechnology 2012, 23:727–735
728 Tissue, cell and pathway engineering
Figure 1 DEBS1
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Schematic architecture of 6-deoxyerythronolide B synthase (DEBS). The polyketide product, 6-deoxyerythronolide B (6dEB) is synthesized from propionyl-CoA and six (2S)-methylmalonyl-CoA. Previously reported CoA-linked starters and extension substrates are also shown. Blue arrows indicates the substrate of each AT domain. Domains outlined in gray are inactive. Abbreviations: ACP, acyl carrier protein domain; AT, acyl transferase domain; DH, dehydratase domain; KR, ketoreductase domain; KS, ketosynthase domain; TE, thioesterase domain.
promiscuous surfactin phosphopantetheinyl transferase from Bacillus subtilis, encoded by sfp, that converts the expressed apo 6MSAS to its holo form [5,6]. This posttranslational modification is not required in type III PKS systems. mPKSs are unique not only in the chain extension mechanism but also in their frequent use of (2S)-methylmalonyl-CoA that does not exist naturally in E. coli. Although heterologous polyketide biosynthesis by mPKSs was achieved in 1994 in S. coelicolor, production in E. coli was not reported until 2001 [7,8]. In this work, Pfeifer, Khosla and co-workers developed a new E. coli strain, BAP1, that harbors sfp in the genome and that was engineered to supply the substrates of the mPKS, propionyl-CoA and (2S)-methylmalonyl-CoA, from exogenously added propionate. In this review, we focus on heterologous production of polyketides by mPKSs in E. coli and also speculate on its potential for biosynthetic engineering. For reviews on heterologous expression and biosynthetic mechanisms of other types of PKSs, the reader is directed elsewhere [9–12]. Current Opinion in Biotechnology 2012, 23:727–735
Architecture of modular type I polyketide synthases and assembly line synthesis mPKSs are best exemplified by the PKS that synthesizes 6-deoxyerythronolide B (6dEB), the macrocyclic aglycon of the antibiotic erythromycin, which was the first heterologously expressed mPKS [7,8]. 6dEB synthase (DEBS) from Saccharopolyspora erythraea is composed of three large polypeptides: DEBS1, DEBS2, and DEBS3 (Figure 1). Each subunit harbors two modules, defined as a set of catalytic domains that are together responsible for one round of polyketide chain extension and subsequent processing (reduction) of the newly generated b-ketone. In addition, DEBS1 and DEBS3 contain additional domains capable of selecting the appropriate starter unit and releasing the final product, respectively. Therefore, in mPKSs, the structure of each extension unit is determined by the domain composition of each module, and the number of modules plus the starter unit dictates the chain length. A module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl www.sciencedirect.com
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carrier protein (ACP) domain. The mechanism by which these domains extend the polyketide chain is as follows. First, the KS domain receives the growing polyketide chain from the ACP domain of the preceding module, while the AT domain transfers a coenzyme A-linked acarboxyacyl extender unit to the phosphopantetheine arm of the ACP. Although utilization of extender units other than malonyl-CoA and (2S)-methylmalonyl-CoA is only rarely observed, several other different extender units including ethylmalonyl-CoA and chloroethylmalonyl-CoA have been reported [13]. The KS then catalyzes decarboxylative condensation between the growing chain (n carbon length) and the extender unit, leading to the formation of a (n + 2) b-ketoacyl thioester-ACP intermediate. The b-ketone is then modified by auxiliary enzymes that may be present in the module: the ketoreductase (KR), the dehydratase (DH), and the enoyl reductase (ER) domains. This intermediate is then translocated to the KS domain of the next module for subsequent condensation. Modules that contain a C-methyltransferase domain (cMT), which transfers a methyl group from Sadenosyl-methionine to the a-carbon to generate a rare geminal dimethyl group, are present in yersiniabactin and epothilone synthases. Chemically, the b-ketone of the corresponding nascent polyketide chain draws the required electrons from the a-carbon to allow methylation to take place at that site. Some of the cMT-containing modules contain a functional KR domain; reduction of the b-ketone to the corresponding hydroxyl must occur after methylation of the a-carbon. Further reduction to form an a,bdouble bond is not possible because of the lack of available proton on the a-carbon. mPKSs utilize a wide range of starter units in the assembly of their products. DEBS1, for example, contains a loading didomain, which consists of an AT domain and an ACP domain, before the first two extension modules: The AT domain specifies that propionyl-CoA is preferentially loaded to the cognate ACP domain. Loading AT domains that are predicted to use other CoAs such as isobutyrylCoA, 2-methylbutyryl-CoA, isovaleryl-CoA, cyclohexylCoA, and benzoyl-CoA have also been found in this type I PKS systems [14]. Most other mPKSs, however, have an additional KS domain, termed KSQ, in which the active site cysteine residue has been changed to a glutamine residue. These KSQ-AT-ACP tridomains are known to load malonyl-CoA or methylmalonyl-CoA and catalyze the decarboxylation to generate acetyl or propionyl starter units in situ [15]. On the contrary, in mPKSs such as rifamycin synthase, free carboxylic acids appear to be activated and loaded by a nonribosomal peptide synthase-like adenylation domain at the N-terminus of the PKSs [14]. Thioesterase (TE) domain-mediated macrolactonization is the most-frequent product release mechanism observed in mPKSs [16]. Other minor mechanisms include TEcatalyzed hydrolysis to generate a free acid, and recently www.sciencedirect.com
characterized KR and sulfotranferase domains plus TEmediated dehydrative decarboxylation to generate a terminal alkene [16,17].
Precursor supply in Escherichia coli Malonyl-CoA and (2S)-methylmalonyl-CoA are the most commonly used substrates for mPKSs. Native E. coli metabolism, however, only produces malonyl-CoA at level sufficient to promote polyketide biosynthesis. Thus, E. coli has to be engineered to supply (2S)-methylmalonyl-CoA. As shown in Figure 2, there are currently three recognized reactions to generate (2S)-methylmalonylCoA: (1) carboxylation of propionyl-CoA, (2) isomerization of TCA cycle-derived succinyl-CoA, and (3) oxidation of (2S)-methylmalonyl-CoA semialdehyde, which is produced from valine [13]. Besides decarboxylation of (2S)methylmalonyl-CoA, propionyl-CoA can be produced from at least five routes: b-oxidation of odd-chain and branched-chain fatty acids; catabolism of threonine; from acetyl-CoA via the 3-hydroxypropionate pathway [18–23]; conversion of exogenously supplied propionate; and via the ethylmalonyl-CoA pathway [24,25,26–28,29]. Of these, the exogenous propionate-dependent pathway and succinyl-CoA isomerization pathway have been reconstituted in E. coli (Figure 2) [8,30]. Although mPKSs commonly use malonyl-CoA and methylmalonyl-CoA as starter and extender units, a wide variety of other starter units can be employed in polyketide biosynthesis, as described earlier. For example, isobutyryl-CoA and 2-methylbutyryl-CoA are used as starters for avermectin synthase, and cyclohexyl-CoA, benzoyl-CoA, and 3-amino-5-hydroxybenzoic acid (AHBA) start phoslactomycin, soraphen, and rifamycin biosynthesis, respectively [14]. Isobutyryl-CoA, 2methylbutyryl-CoA, and isovaleryl-CoA are well-known intermediates in valine, isoleucine, and leucine catabolism. E. coli metabolism, however, insufficiently supplies these acyl-CoAs, as well as propionyl-CoA. CyclohexylCoA and benzoyl-CoA are also not produced at appreciable levels in E. coli. Seven genes are believed to be necessary for the biosynthesis of AHBA and were heterologously expressed in E. coli. The production of AHBA was confirmed and a tetraketide intermediate of rifamycin has been synthesized in the engineered strain [31].
Protein expression of modular type I polyketide synthases in Escherichia coli Functional expression of mPKSs in E. coli has proven to be challenging owing to two key issues, phosphopantetheinylation of ACP domains and folding. The attempts to express functional mPKSs in E. coli began with DEBS in 1993 [32]. In this work, Roberts, Staunton, and Leadlay observed that purified DEBS3 was not post-translationally modified with the phosphopantetheinyl group, although this 332-kDa protein was properly folded. To address this issue, sfp was co-expressed or integrated into Current Opinion in Biotechnology 2012, 23:727–735
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Figure 2
Even-chain fatty acids 2x Acetyl-CoA etyl-CoA
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Heterologous production of polyketides in Escherichia coli Yuzawa et al. 731
Figure 3
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Malonyl-CoA Domain substitution [41]
Current Opinion in Biotechnology
Biosynthetic engineering of 6-deoxyerythronolide B synthase (DEBS). Red color indicates manipulation of DEBS by domain or module engineering. Nacetyl cysteamine (NAC) substrates used in unnatural feeding experiments are also shown in red. Blue arrows indicates the substrate of the redcolored AT domain or of the KS domain in module 2, respectively. Domains outlined in gray are inactive. [35,39,40–44] indicate references cited. Abbreviations: ACP, acyl carrier protein domain; AT, acyl transferase domain; DH, dehydratase domain; KR, ketoreductase domain; KS, ketosynthase domain; TE, thioesterase domain; TKL, triketide lactone.
the genome to allow successful in vivo phosphopantetheinylation of ACP domains of mPKSs [8,33]. The other key challenge to functional mPKS expression is protein folding, which is greatly impacted by the postinduction temperature. In the first production runs of 6dEB in E. coli, active DEBS1, DEBS2, and DEBS3 were only observed by lowering the expression temperature from 30 8C to 22 8C, probably owing to misfolding of the proteins at the elevated temperatures [8]. More recently, it has been shown that co-expression with E. coli or S. coelicolor chaperones can improve the solubility and purification yield of DEBS in E. coli [34].
Biosynthetic engineering of modular type I polyketide synthases The modular nature of mPKSs and the colinearity between the module activities and the structure of the polyketide products offers a wealth of engineering opportunities. In fact, a variety of ‘unnatural’ polyketides (>200) have been produced by engineered DEBSs in a programmed manner
since Donadio, Katz, and co-workers first demonstrated the potential of mPKS engineering in 1991 [35–38]. Manipulation of DEBS has included (1) domain deletion, inactivation, insertion, and substitution to modify the structure of each extension unit, (2) module deletion and substitution to alter the chain length of the resulting polyketides or to construct hybrid mPKSs, and (3) unnatural substrate feeding (Figure 3) [39–44]. Beyond simple domain and module swapping approaches, a major challenge for biosynthetic engineering will be to overcome a severe loss in overall catalytic efficiency of the engineered mPKSs compared with natural mPKSs. Recently, detailed structural studies of catalytic domains have revealed functional domain boundaries that can now be exploited for more accurate swapping experiments, even though module structures remain unresolved [45– 48,49,50,51]. In addition, dedicated analyses of intermodule and intra-module interactions have begun to reveal the determinants of the observed binding specificities at amino acid residue level, which would greatly aid the
(Figure 2 Legend) Metabolic pathways leading to the formation of (2S)-methylmalonyl-CoA from central metabolites, such as acetyl-CoA, amino acids, and fatty acids shown in red. Three reactions to generate (2S)-methylmalonyl-CoA, carboxylation of propionyl-CoA, isomerization of succinylCoA, and oxidation of (2S)-methylmalonyl-CoA semialdehyde are numbered as (1), (2), and (3), respectively. The pathways that were reconstituted in E. coli are shown in blue. Solid arrows indicate one-step reactions catalyzed by enzymes. Dashed arrows indicate multiple enzymatic processes. www.sciencedirect.com
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Figure 4
L1: KSQ-mAT-ACP
L2: KSQ-mmAT-ACP
L3: pAT-ACP
L6: mbAT-ACP
L7: chAT-ACP
L8: bzAT-ACP
E1: KS-mAT-ACP
E2: KS-mAT-KRA-ACP
E6: KS-mmAT-ACP
E11: KSR-mmAT-ACP
L4: ibAT-ACP
L5: ivAT-ACP
E3: KS-mAT-KRB-ACP
E4: KS-mAT-KR-DH-ACP
E5: KS-mAT-KR-DH-ER-ACP
E7: KS-mmAT-KRA-ACP
E8: KS-mmAT-KRB-ACP
E9: KS-mAT-KR-DH-ACP
E10: KS-mAT-KR-DH-ER-ACP
E12: KSR-mmAT-KRA-ACP
E13: KSR-mmAT-KRB-ACP
E14: KSR-mAT-KR-DH-ER-ACP
E15: KS-mAT-cMT-ACP E16 :KS-mAT-KRB-cMT-ACP
E17: KSR-emAT-ACP
E18: KSR-emAT-KR-DH-ER-ACP
R1: TE
R2: TE
E19: KS-cemAT-ACP
R3: KR-ST-TE
Current Opinion in Biotechnology
Biological parts that constitute modular type I polyketide synthases. L1-L8 indicate loading domains. E1-E19 indicate extension modules. R1-R3 indicate release domains. Abbreviations: ACP, acyl carrier protein domain; mAT, methylmalonyl-CoA specific acyl transferase domain; mAT, malonylCoA specific acyl transferase domain; mmAT, methylmalonyl-CoA specific acyl transferase domain; pAT, propionyl-CoA specific acyl transferase domain; ibAT, isobutyryl-CoA specific acyl transferase domain; ivAT, isovaleryl-CoA specific acyl transferase domain; mbAT, 2-methylbutyryl-CoA specific acyl transferase domain; chAT, cyclohexyl-CoA specific acyl transferase domain; bzAT, benzoyl-CoA specific acyl transferase domain; emAT, ethylmalonyl-CoA specific acyl transferase domain; cemAT, chloroethylmalonyl-CoA specific acyl transferase domain; cMT, C-methyltransferase domain; DH, dehydratase domain; KR, ketoreductase domain; KRA, A-type ketoreductase domain; KRB, B-type ketoreductase domain; KS, ketosynthase domain; KSQ, ketosynthase domain with decarboxylation activity; KSR, ketosynthase domain with racemization activity; ST, sulfotransferase domain; TE, thioesterase domain. Current Opinion in Biotechnology 2012, 23:727–735
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design that allows noncognate protein-protein interaction [52,53,54,55,56,57,58]. Another major challenge in mPKS engineering is how to manipulate the substrate specificity of domains to allow the efficient transfer of the growing, unnatural, polyketide chain from one domain to the next and maintain the enzyme kinetics. Although this challenge remains to be solved, a product-specific selection strategy should play an important role in biosynthetic engineering. For example, Lee and Khosla reported a bactericidal activity-based selection strategy to identify erythromycin-overproducing E. coli strains [59]. However, clearly, other strategies have to be developed if target polyketide compounds are not antibiotics.
Modular type I polyketide synthases, a platform of synthetic biology One definition of synthetic biology is ‘‘the deliberate design and fabrication of novel biologically-based components as well as the redesign of existing biological systems’’. Although many challenges still exist as described earlier, mPKSs have the potential as a platform to synthesize thousands of chemicals. As shown in Figure 4, at least 19 types of extension modules are available in nature, as well as 8 types of loading domains and 3 types of release domains, which account for >2000 naturally occurring polyketides. Recently, the cost of DNA synthesis and gene assembly has declined exponentially [60], which should accelerate design and fabrication of mPKSs with DNA parts where codons are optimized for designated heterologous hosts and domains and modules are flanked by unique restriction enzyme sites to facilitate domain and module swapping [37]. An example is the re-construction of the DEBS system using the parts L3 + E13 + E7 + E11 + E14 + E7 + E7 + R1 shown in Figure 4. Currently, approximately 50 genome sequences of Streptomyces strains are available (http://www.ncbi.nlm.nih.gov/ genomes/MICROBES/microbial_taxtree.html). Further, genome sequencing and genome mining with appropriate search algorithms would expand the scope of starter units, extension modules and release mechanisms. From this point of view, bioinformatics and chemoinformatics to connect between chemical products and the gene clusters should play an important role in future mPKS engineering. Efforts relating to isolation and structural characterization of products and related biocatalytic mechanisms also continue to be important for development of reliable computational algorithms.
knowledge of fermentation strategies. Preliminary studies have highlighted both the promise and limitations of polyketide biosynthesis in E. coli. Insufficient supply of polyketide precursors is one challenge in using E. coli as a convenient host for heterologous polyketide production. Functional folding of mPKSs at 37 8C is another issue to be addressed. Rational exploitation of the modular nature of mPKSs is only just beginning to emerge, even for a prototypical mPKSs such as DEBS. Given the accumulating knowledge of the functional boundaries of catalytic domains, we need to re-evaluate past engineering efforts to develop more accurate predictive algorithms that exploit modularity, which are currently exemplified by MapsiDB and SBSPKS [61,62]. Understanding the fundamentals of the molecular enzymology and structural biology of not only DEBS but also other mPKSs is important to design kinetically competent chimeric mPKSs that will rapidly and in high yield produce natural and unnatural polyketides.
Acknowledgements This work was funded by the Advanced Research Projects Agency – Energy (ARPA-E), U.S. Department of Energy, under award number DEAR0000091, by the National Science Foundation, award No. EEC-0540879 to the Synthetic Biology 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 number DE-AC02-05CH11231.
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Current Opinion in Biotechnology 2012, 23:727–735