Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension

Böhm, Ines; Holzbaur, Inès E.; Hanefeld, Ulf; Cortési, Jesus; Staunton, Jim; Leadlay, Peter F.

doi:10.1016/s1074-5521(98)90157-0

Cited by 52 publications

(26 citation statements)

References 32 publications

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“…[30] In the present case, bringing KR 5 into a chimaeric module with KS 2 did not lead to any loss of stereospecificity or stereoselectivity; this supports a model for catalysis [26,27,30] in which DEBS KS 2 differs from KS 1 in that it cannot catalyse the epimerisation of the (2R)-2-methyl-3-ketopentanoyl-ACP intermediate, or cannot do so fast enough to compete with ketoreduction. This experiment also shows that the ACP-tethered diketide is reduced with full fidelity by KR 5 , even though previous in vitro studies have shown that recombinant purified KR 5 reduces the (untethered) racemic substrate (2RS)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester to give three stereoisomeric products.…”

supporting

confidence: 80%

“…Lactones 4 a and 5 a are the hypothetical products from hybrid DEBS1-TE containing either DEBS KR 1 or RIFS KR 7 in the reductive loop of module 2 based on the activity of these reductive enzymes in their native context, but are not expected to form owing to the absence of catalysed epimerisation activity in DEBS module 2. [26,27,42] has shown the importance from a synthetic viewpoint of trying more than one example of a desired hybrid. The results found here illustrate clearly that a different choice of splice sites, and/or use of a different donor reductive loop, can very significantly affect the yield of the desired novel polyketide.…”

Section: Discussionmentioning

confidence: 99%

“…[25] There is an additional obligatory epimerisation step in module 1, after condensation, to produce the substrate for ketoreduction. [26][27][28][29] The stereochemical outcome therefore essentially depends on the interplay between the properties of individual KS and KR domains. In any chimaeric extension module that generates a methyl centre at C-2 and a hydroxy centre at C-3, if the product of condensation is exactly that stereoisomer of the 2-methyl-3-ketoacyl thioester intermediate which matches the normal preference of the incoming KR, then the heterologous KR often imposes the stereochemical course of its normal reaction on the new substrate.…”

Section: Introductionmentioning

confidence: 99%

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A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

et al. 2008

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Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.

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confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

et al. 2008

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“…30 Since each of these modules is known to act completely stereospecifically when processing both natural and unnatural enzyme-bound substrates, 5,22b it is likely that the loss of stereochemical control in reduction of the ketodiketide-SNAC analog 13 may reflect the requirement that the substrate be covalently anchored to the ACP domain for proper positioning and orientation in the KR active site. Indeed, the results reported here confirm that recombinant DEBS KR2 and KR6 both catalyze completely stereospecific reductions of ACPbound 2-methyl-3-ketotriketides that have been generated in situ by the action of a 32 No conclusions could be drawn as to whether KR2 exercises any selection against the diastereomeric L-2-methyl-3-ketopentanoyl-ACP, which may or may not have been transiently formed by the hybrid DKS1-2 enzyme.…”

Section: Analysis Of Kr Stereospecificitymentioning

confidence: 63%

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Castonguay

Chen

et al. 2007

J. Am. Chem. Soc.

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Abstract6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS) -acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base-or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-mass spectrometry technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-δ-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACPbound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized D-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.Modular polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of polyketides metabolites that exhibit a wide range of important biological properties, including antibacterial, antifungal, anticancer, and immunosuppressive activity. 1 They catalyze repetitive Claisen-like condensations between methylmalonyl or malonyl thioester building blocks and the growing polyketide acyl thioesters. Using an assembly line of active sites, each module is responsible for one cycle of polyketide chain elongation and *To whom correspondence should be addressed. E-mail: David_Cane@brown.edu. (TE) domain, located at the C-terminus of the furthest downstream module, catalyzes release and concomitant cyclization of the mature, full-length macrocyclic polyketide. NIH Public AccessThe 6-deoxyerythronolide synthase (DEBS) from Saccharopolyspora erythraea, which is by far the most thoroughly studied modular PKS, is responsib...

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“…1B). Progress has recently been made in this direction by studying the 6-deoxyerythronolide B synthase in vivo or in vitro, leading to the finding that the stereochemistry of the alkyl-branched center or the oxygenated center is controlled by the ketoacyl synthase (KS) (10,31,52) or the ketoreductase (KR) (10,25), respectively, in the specific cases examined. However, these studies fall short of providing a satisfactory explanation at the molecular level of how a given KS or KR exhibits its stereospecificity; sequence comparison among various KS or KR domains has also failed to reveal any conserved motifs that could potentially be ascribed to their opposite stereospecificities.…”

Section: Discussionmentioning

confidence: 99%

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

Smith

Xiang

Shen

2000

Antimicrob Agents Chemother

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The macrotetrolides are a family of cyclic polyethers derived from tetramerization, in a stereospecific fashion, of the enantiomeric nonactic acid (NA) and its homologs. Isotope labeling experiments established that NA is of polyketide origin, and biochemical investigations demonstrated that 2-methyl-6,8-dihydroxynon-2E-enoic acid can be converted into NA by a cell-free preparation from Streptomyces lividans that expresses nonS. These results lead to the hypothesis that macrotetrolide biosynthesis involves a pair of enantiospecific polyketide pathways. In this work, a 55-kb contiguous DNA region was cloned from Streptomyces griseus DSM40695, a 6.3-kb fragment of which was sequenced to reveal five open reading frames, including the previously reported nonR and nonS genes. Inactivation of nonS in vivo completely abolished macrotetrolide production. Complementation of the nonS mutant by the expression of nonS in trans fully restored its macrotetrolide production ability, with a distribution of individual macrotetrolides similar to that for the wild-type producer. In contrast, fermentation of the nonS mutant in the presence of exogenous (؎)-NA resulted in the production of nonactin, monactin, and dinactin but not in the production of trinactin and tetranactin. These results prove the direct involvement of nonS in macrotetrolide biosynthesis. The difference in macrotetrolide production between in vivo complementation of the nonS mutant by the plasmid-borne nonS gene and fermentation of the nonS mutant in the presence of exogenously added (؎)-NA suggests that NonS catalyzes the formation of (؊)-NA and its homologs, supporting the existence of a pair of enantiospecific polyketide pathways for macrotetrolide biosynthesis in S. griseus. The latter should provide a model that can be used to study the mechanism by which polyketide synthase controls stereochemistry during polyketide biosynthesis.

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Engineering of a minimal modular polyketide synthase, and targeted alteration of the stereospecificity of polyketide chain extension

Cited by 52 publications

References 32 publications

A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

A Polylinker Approach to Reductive Loop Swaps in Modular Polyketide Synthases

Stereospecificity of Ketoreductase Domains of the 6-Deoxyerythronolide B Synthase

Genetic Localization and Molecular Characterization of the nonS Gene Required for Macrotetrolide Biosynthesis in Streptomyces griseus DSM40695

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