• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.225-236
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• 2021

The epipyrone (EPN) biosynthetic gene cluster of Epicoccum nigrum is composed of epnC, epnB, and epnA, which encode cytochrome P450 oxidase, glycosyltransferase, and highly reducing polyketide synthase, respectively. Gene inactivation mutants for epnA, epnB, and epnC were previously generated, and it was found that all of them were incapable of producing EPN and any of its related compounds. It was also reported that epnB inactivation abolished epnA transcription, generating ΔepnAB. This study shows that the introduction of native epnC readily restored EPN production in ΔepnC, suggesting that epnC is essential for polyketide release from EpnA and implies that EpnC works during the polyketide chain assembly of EpnA. Introduction of epnC promoter-epnA restored EPN production in ΔepnA. The ΔepnB genotype was prepared by introducing the epnA expression vector into ΔepnAB, and it was found that the resulting recombinant strain did not produce any EPN-related compounds. A canonical epnB inactivation strain was also generated by deleting its 5'-end. At the deletion point, an Aspergllus nidulans gpdA promoter was inserted to ensure the transcription of epnA, which is located downstream of epnB. Examination of the metabolite profile of the resulting ΔepnB mutant via LC-mass spectrometry verified that no EPN-related compound was produced in this strain. This substantiates that C-glycosylation by EpnB is a prerequisite for the release of EpnA-tethered product. In conclusion, it is proposed that cytochrome P450 oxidase and glycosyltransferase work in concert with polyketide synthase to generate EPN without the occurrence of any free intermediates.

• Journal of Applied Biological Chemistry
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• 제65권3호
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• pp.159-166
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• 2022

Epicoccum nigrum produces epipyrone A (orevactaene), a yellow polyketide pigment. Its biosynthetic gene cluster was previously characterized in E. nigrum KACC 40642. The YES liquid culture of this strain revealed high-level production of epicoccamide (EPC), with an identity that was determined using liquid chromatography-mass spectrometry analysis and molecular mass search using the SuperNatural database V2 webserver. The production of EPC was further confirmed by compound isolation and nuclear magnetic resonance spectroscopy. EPC is a highly reduced polyketide with tetramic acid and mannosyl moieties. The EPC structure guided us to localize the hypothetical EPC biosynthetic gene cluster (BGC) in E. nigrum ICMP 19927 genome sequence. The BGC contains genes encoding highly reducing (HR)-fungal polyketide synthase (fPKS)-nonribosomal peptide synthetase (NRPS), glycosyltransferase (GT), enoylreductase, cytochrome P450, and N-methyltrasnferase. Targeted inactivation of the HR-fPKS-NRPS and GT genes abolished EPC production, supporting the successful localization of EPC BGC. This study provides a platform to explore the hidden biological activities of EPC, a bolaamphiphilic compound.