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

The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recently-published proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm.

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Directed biosynthesis of fluorinated polyketides

10.1101/2021.07.30.454469 ◽

2021 ◽

Author(s):

Alexander Rittner ◽

Mirko Joppe ◽

Jennifer J. Schmidt ◽

Lara Maria Mayer ◽

Elia Heid ◽

...

Keyword(s):

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Coenzyme A ◽

Chain Extension ◽

Type I ◽

Complex Molecules ◽

Site Specific ◽

Directed Biosynthesis ◽

Polyketide Chain ◽

Selection Of

Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-specific incorporation of fluorine in complex molecules have improved in recent years, approaches for the direct biosynthetic fluorination of natural compounds are still rare. Herein, we present a broadly applicable approach for site-specific, biocatalytic derivatization of polyketides with fluorine. Specifically, we exchanged the native acyltransferase domain (AT) of a polyketide synthase (PKS), which acts as the gatekeeper for selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase (FAS). The resulting PKS/FAS hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl disubstitutions in polyketide scaffolds. Addition of a fluorine atom is often a decisive factor toward developing superior properties in next-generation antibiotics, including the macrolide solithromycin. We demonstrate the feasibility of our approach in the semisynthesis of a fluorinated derivative of the macrolide antibiotic YC-17.

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Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide β-methyl branch incorporation

Scientific Reports ◽

10.1038/s41598-020-71850-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Asha V. Nair ◽

Alice Robson ◽

Thomas D. Ackrill ◽

Marisa Till ◽

Matthew J. Byrne ◽

...

Keyword(s):

Polyketide Synthase ◽

Acyl Carrier Protein ◽

Type Systems ◽

Site Directed Mutagenesis ◽

Methyl Branch ◽

New Paradigm ◽

X Ray Crystallography ◽

Polyketide Chain ◽

Modular Polyketide Synthase

Abstract Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions β to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in β-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.

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Faculty Opinions recommendation of Structural and stereochemical analysis of a modular polyketide synthase ketoreductase domain required for the generation of a cis-alkene.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.720372137.793511631 ◽

2015 ◽

Author(s):

Zixin Deng ◽

Tiangang Liu

Keyword(s):

Polyketide Synthase ◽

Stereochemical Analysis ◽

Modular Polyketide Synthase

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Promiscuity of a modular polyketide synthase towards natural and non-natural extender units

Organic & Biomolecular Chemistry ◽

10.1039/c3ob40633d ◽

2013 ◽

Vol 11 (27) ◽

pp. 4449 ◽

Cited By ~ 37

Author(s):

Irina Koryakina ◽

John B. McArthur ◽

Matthew M. Draelos ◽

Gavin J. Williams

Keyword(s):

Polyketide Synthase ◽

Modular Polyketide Synthase

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Type II thioesterase ScoT is required for coelimycin production by the modular polyketide synthase Cpk of Streptomyces coelicolor A3(2).

Acta Biochimica Polonica ◽

10.18388/abp.2014_1936 ◽

2014 ◽

Vol 61 (1) ◽

Cited By ~ 6

Author(s):

Magdalena Kotowska ◽

Jarosław Ciekot ◽

Krzysztof Pawlik

Keyword(s):

Culture Medium ◽

Polyketide Synthase ◽

Streptomyces Coelicolor ◽

Acyl Chain ◽

No Production ◽

Type I ◽

Type Ii ◽

Peptide Synthetases ◽

Modular Polyketide Synthase ◽

Editing Enzyme

Type II thioesterases were shown to maintain efficiency of modular type I polyketide synthases and nonribosomal peptide synthetases by removing acyl residues blocking extension modules. We found that thioesterase ScoT from Streptomyces coelicolor A3(2) is required for the production of the yellow-pigmented coelimycin by the modular polyketide synthase Cpk. No production of coelimycin was observed in cultures of scoT disruption mutant. Polyketide production was restored upon complementation with an intact copy of the scoT gene. An enzymatic assay showed that ScoT thioesterase can hydrolyse a 12-carbon acyl chain but the activity is too low to play a role in product release from the polyketide synthase. We conclude that ScoT is an editing enzyme necessary to maintain the activity of polyketide synthase Cpk. We provide a HPLC based method to measure the amount of coelimycin P2 in a culture medium.

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