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A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxyacids

ABSTRACT: Modular polyketide synthases (PKSs) have been proposed as promising megasynthases for retrobiosynthesis because of their fitness for rational carbon skeleton design. However, over the last three decades, all engineered PKSs produce carboxylic acids via terminal thioesterase (TE), which significantly narrows available chemical scope. We proposed the possibility of expanding PKS chemical diversity via terminal thioreductase (TR) engineering, and comprehensively characterised PKS TRs as NADPH-dependent enzymes to terminate polyketides with aldehyde. These aldehyde products can be readily converted to industrially valuable alcohols and amines, demonstrated by an engineered rimocidin PKS-TR producing 1,3-butanediol, 1,3-pentanediol, and 1,3-hexanediol with a total titer of 1008 mg/L in Streptomyces albus, or producing 554 mg/L 1-amino-3-alcohols via post-PKS transamination, both in shake flasks. Furthermore, efficient control of the product profile was achieved by coenzyme A (CoA) substrate regulation, as elevating butyryl-CoA level resulted in 1,3-hexanediol product ratio increased from 11% to 77%. We also demonstrated that PKS-TR can produce biosynthetically challenging branched-chain diols, culminating in production of 166 mg/L branched-chain 2-methyl-1,3-diols via acyltransferase exchange.

ORGANISM(S): Streptomyces

SUBMITTER: Chris Petzold

PROVIDER: PXD047663 | panorama | Tue Dec 17 00:00:00 GMT 2024

REPOSITORIES: PanoramaPublic

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A polyketide-based biosynthetic platform for diols, amino alcohols and hydroxyacids

Project description:Medium- and branched-chain diols and amino alcohols are important industrial solvents, polymer building blocks, cosmetics and pharmaceutical ingredients, yet biosynthetically challenging to produce. Here, we present a novel approach utilising a modular polyketide synthase (PKS) platform for the efficient production of these compounds. This platform takes advantage of a versatile loading module from the rimocidin PKS and NADPH-dependent terminal thioreductases (TRs), previously untapped in engineered PKSs. Reduction of the terminal aldehyde with specific alcohol dehydrogenases enables production of diols, oxidation enables production of hydroxy acids, and transamination with specific transaminases enables production of various amino alcohols. Furthermore, replacement of the malonyl-coenzyme A (CoA)–specific acyltransferase (AT) in the extension module with methyl- or ethylmalonyl-CoA–specific ATs enables production of branched-chain diols and amino alcohols. In total, we demonstrated production of nine 1,3-diols (including the difficult-to-produce insect repellent and cosmetic ingredient 2-ethyl-1,3-hexanediol), six amino alcohols, and two carboxylic acids using our PKS platform in Streptomyces albus. Finally, tuning production of the PKS acyl-CoA substrates enabled production of high titers of specific diols and amino alcohols (1 g/L diol titer in shake flasks), demonstrating high tunability and efficiency of the platform.

2024-12-02 | PXD055149 | Pride

Stabilized biological production by product addiction

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Identification of ACLA, an adipoyl Co enzyme A ligase in Penicillium chrysogenum, a key enzyme of cephem antibiotics

Project description:Production of cephalosporin precursors with recombinant strains of Penicillium chrysogenum has improved the economics and reduced the environmental impact of industrial cephalosporin production. The engineered P. chrysogenum strains used in these processes express heterologous enzymes that convert the intermediate acyl-6-aminopenicillanic acid into different tailor-made compounds. Activation of the cephalosporin side-chain precursor to its corresponding CoA thioester is an essential step for its incorporation into the β-lactam backbone. To identify the acyl-CoA ligase involved in activation of adipic acid, a frequently used cephalosporin side-chain precursor, we searched the genome of P.chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was then used to identify the one presenting the highest expression level when cells were grown in the presence of adipic acid. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipic acid consumption and a three-fold reduction of adipoyl-6-aminopenicillanic acid levels in chemostat cultures of P. chrysogenum, but did not affect penicillin production. After cloning the gene and overexpressing it in Escherichia coli, its purified protein product was shown to have adipoyl-CoA ligase, but no phenylacetyl-CoA ligtase activity. Finally, by fusing the gene to a sequence encoding cyan fluorescent protein, the resulting fusion protein localized to microbodies, which indicates that activation of the side-chain precursor adipic acid takes place in this compartment, where also the subsequent acyltransferase step takes place. Identification and functional characterization of this adipoyl-CoA ligtase gene may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins.

2009-08-01 | GSE12617 | GEO

Engineering acetyl-CoA supply: Functional expression of a bacterial pyruvate-dehydrogenase complex in the cytosol of Saccharomyces cerevisiae

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2020-02-26 | GSE134767 | GEO

Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor

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2009-01-05 | GSE12612 | GEO

Systems-level engineering and characterization of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB)

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Transcriptome- and fluxome-guided metabolic engineering of 3-hydroxypropionic acid production in Saccharomyces cerevisiae

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2022-08-30 | E-MTAB-7819 | biostudies-arrayexpress

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