Dataset Information

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

ABSTRACT: Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C-terminal domain with significant homology to carnitine O-acyltransferase (cAT). Characterization of one such HRPKS (Tv6-931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, as confirmed through the holo ACP-dependent transesterification of the released product. The methyltransferase (MT) domain of Tv6-931 can perform two consecutive ?-methylation steps on the last ?-keto intermediate to yield an ?,?-gem-dimethyl product, a new programing feature among HRPKSs. Recapturing of the released product by cAT domain is suggested to facilitate complete gem-dimethylation by the MT.

SUBMITTER: Hang L

PROVIDER: S-EPMC5580239 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Publications

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

Hang Leibniz L Tang Man-Cheng MC Harvey Colin J B CJB Page Claire G CG Li Jian J Hung Yiu-Sun YS Liu Nicholas N Hillenmeyer Maureen E ME Tang Yi Y

Angewandte Chemie (International ed. in English) 20170705 32

Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C-terminal domain with significant homology to carnitine O-acyltransferase (cAT). Characterization of one such HRPKS (Tv6-931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, ...[more]

PMID: 28679030

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Project description:BackgroundPolyketide synthases (PKSs) include ketone synthase (KS), acyltransferase (AT) and acyl carrier protein (ACP) domains to catalyse the elongation of polyketide chains. Some PKSs also contain ketoreductase (KR), dehydratase (DH) and enoylreductase (ER) domains as modification domains. Insertion, deletion or substitution of the catalytic domains may lead to the production of novel polyketide derivatives or to the accumulation of desired products. Epothilones are 16-membered macrolides that have been used as anticancer drugs. The substrate promiscuity of the module 4 AT domain of the epothilone PKS (EPOAT4) results in production of epothilone mixtures; substitution of this domain may change the ratios of epothilones. In addition, there are two dormant domains in module 9 of the epothilone PKS. Removing these redundant domains to generate a simpler and more efficient assembly line is a desirable goal.ResultsThe substitution of module 4 drastically diminished the activity of epothilone PKS. However, with careful design of the KS-AT linker and the post-AT linker, replacing EPOAT4 with EPOAT2, EPOAT6, EPOAT7 or EPOAT8 (specifically incorporating methylmalonyl-CoA (MMCoA)) significantly increased the ratio of epothilone D (4) to epothilone C (3) (the highest ratio of 4:3 = 4.6:1), whereas the ratio of 4:3 in the parental strain Schlegelella brevitalea 104-1 was 1.4:1. We also obtained three strains by swapping EPOAT4 with EPOAT3, EPOAT5, or EPOAT9, which specifically incorporate malonyl-CoA (MCoA). These strains produced only epothilone C, and the yield was increased by a factor of 1.8 compared to that of parental strain 104-1. Furthermore, mutations of five residues in the AT domain identified Ser310 as the critical factor for MMCoA recognition in EPOAT4. Then, the mutation of His308 to valine or tyrosine combined with the mutation of Phe310 to serine further altered the product ratios. At the same time, we successfully deleted the inactive module 9 DH and ER domains and fused the ΨKR domain with the KR domain through an ~ 25-residue linker to generate a productive and simplified epothilone PKS.ConclusionsThese results suggested that the substitution and deletion of catalytic domains effectively produces desirable compounds and that selection of the linkers between domains is crucial for maintaining intact PKS catalytic activity.

Dataset Information

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

Publications

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

Similar Datasets

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