Engineering the acyltransferase domain of epothilone polyketide synthase to alter the substrate specificity (original) (raw)
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ACS synthetic biology, 2016
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 a 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 optimi...
Modular Construction of a Functional Artificial Epothilone Polyketide Pathway
ACS Synthetic Biology, 2012
Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.
New Start and Finish for Complex Polyketide Biosynthesis
Chemistry & Biology, 2004
as well as a loading module for transferring the starter acyl group onto the first KS domain. This modular orga-Biosynthesis nization allows programmed assembly of a defined sequence of starter and extender units, together with controlled processing of each -ketone group. The final The polyketide vicenistatin has significant anticancer product may be cyclized by a thioesterase (TE) to give activity. In the January issue of Chemistry & Biology, a macrolactone.
A Model of Structure and Catalysis for Ketoreductase Domains in Modular Polyketide Synthases
Biochemistry, 2003
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr f Phe, Ser f Ala, and Lys f Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces liVidans for in vivo analysis. In this case, the Tyr f Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser f Ala and Lys f Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immoblized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.
An artificial pathway for polyketide biosynthesis
Nature Catalysis, 2020
Polyketide synthases are multi-domain enzymes that catalyse the construction of many bioactive natural products. Now, some of the inefficiencies and limitations of these systems have been solved by designing an artificial pathway for carbon-carbon bond formation via iterative rounds of non-decarboxylative thio-Claisen reactions. Anuran k. Gayen, lindsay nichols and Gavin J. Williams P olyketides are a class of natural products with diverse and potent biological activities, owing to their structural complexity and chemical diversity. Notable examples of polyketides include lovastatin (anticholesterol), doxorubicin (anticancer), ivermectin (antiparasitic), and erythromycin A (macrolide antibiotic). All polyketides are assembled via the repeated decarboxylative thio-Claisen condensation between malonyl-coenzyme A (malonyl-CoA) derivatives catalysed by enzyme machinery called polyketide synthases (PKSs). Type I PKSs are multi-modular mega-enzyme complexes, type II PKSs are a collection of cooperative enzymes, and type III PKSs iteratively catalyse chain elongation via ketosynthase domains. Though they differ in complexity and molecular organization, the three PKS types share similar substrate scopes, which largely consist of malonyl-and methylmalonyl-CoA extender units and simple acyl-CoA starter units, such as acetyl-, propionyl-, and isobutyryl-CoA. Site-selective modular control of oxidation levels by PKSs facilitates structural diversity in the polyketide scaffold that contributes to their biological activity 1. In spite of the proven ability to leverage PKSs for the production of blockbuster polyketide drugs, polyketide biosynthesis suffers from slow reaction kinetics, low energy efficiency, and poor carbon economy-only two out of three extender unit carbons incorporate into the polyketide backbone with loss of CO 2. In addition, the formation of the commonly utilized malonyl-CoA extender unit from acetyl-CoA, ATP and bicarbonate by acetyl-CoA carboxylase (ACC) is highly regulated and a likely rate-limiting step 2. While type II and III PKSs can be less complicated than their type I counterparts, they often do not express well in tractable heterologous hosts such as Escherichia coli (E. coli) and access to substrates is susceptible to competition with primary metabolism. Now, writing in Nature Catalysis, Gonzalez and colleagues report the
Applied and Environmental Microbiology, 2011
ABSTRACTPolyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT0in the nystatin PKS loading module NysA with the propionate-specific AT1from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT0domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT0and an active site residue in the ketosynthase (KS)-like domain of...
Proceedings of the National Academy of Sciences, 2008
The enediynes, unified by their unique molecular architecture and mode of action, represent some of the most potent anticancer drugs ever discovered. The biosynthesis of the enediyne core has been predicted to be initiated by a polyketide synthase (PKS) that is distinct from all known PKSs. Characterization of the enediyne PKS involved in C-1027 (SgcE) and neocarzinostatin (NcsE) biosynthesis has now revealed that (i) the PKSs contain a central acyl carrier protein domain and C-terminal phosphopantetheinyl transferase domain; (ii) the PKSs are functional in heterologous hosts, and coexpression with an enediyne thioesterase gene produces the first isolable compound, 1,3,5,7,9,11,13-pentadecaheptaene, in enediyne core biosynthesis; and (iii) the findings for SgcE and NcsE are likely shared among all nine-membered enediynes, thereby supporting a common mechanism to initiate enediyne biosynthesis.