Yasuyo Seshime

Biosynthesis of Steroidal Antibiotic Fusidanes: Functional Analysis of Oxidosqualene Cyclase and Subsequent Tailoring Enzymes from Aspergillus fumigatus

Three putative oxidosqualene cyclase (OSC) genes exist in the genome of the fungus Aspergillus fumigatus that produces a steroidal antibiotic, helvolic acid. One of these genes, Afu4g14770, designated AfuOSC3, is clustered with genes of cytochrome P450 monooxygenases (P450s), a short-chain dehydrogenase/reductase (SDR), and acyltransferases, which presumably function in triterpene tailoring steps, suggesting that this gene cluster codes for helvolic acid biosynthesis. AfuOSC3 was PCR amplified from A. fumigatus IFO8866 genomic DNA and expressed in yeast. The yeast transformant accumulated protosta-17(20)Z,24-dien-3beta-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3beta-ol, was also isolated from the transformed yeast. To further identify the function of triterpene tailoring enzymes, four P450 genes (CYP5081A1-D1) and a SDR gene (AfuSDR1) in the cluster were each coexpressed with AfuOSC3 in yeast. As a result, coexpression of AfuSDR1 gave a 3-keto derivative of protostadienol. On the other hand, coexpression with CYP5081A1 gave protosta-17(20)Z,24-diene-3beta,29-diol and protosta-17(20)Z,24-dien-3beta-ol-29-oic acid. These metabolites are in well accord with the oxidative modification involved in helvolic acid biosynthesis. AfuSDR1 and CYP5081A1 presumably function together to catalyze demethylation of C-29 methyl group. These results provided a firm ground for identification of the present gene cluster to be involved in helvolic acid biosynthesis.

Functional expression of the Aspergillus flavus PKS-NRPS hybrid CpaA involved in the biosynthesis of cyclopiazonic acid.

alpha-Cyclopiazonic acid (CPA) is an indole tetramic acid mycotoxin. Based on our identification of the polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) hybrid gene cpaA involved in cyclopiazonic acid biosynthesis in Aspergillus fungi, we carried out heterologous expression of Aspergillus flavuscpaA under alpha-amylase promoter in Aspergillus oryzae and identified its sole product to be the CPA biosynthetic intermediate cyclo-acetoacetyl-l-tryptophan (cAATrp). This result rationalized that the PKS-NRPS hybrid enzyme CpaA catalyzes condensation of the diketide acetoacetyl-ACP formed by the PKS module and l-Trp activated by the NRPS module. This CpaA expression system provides us an ideal platform for PKS-NRPS functional analysis, such as adenylation domain selectivity and product releasing mechanism.

Identification of csypyrone B1 as the novel product of Aspergillus oryzae type III polyketide synthase CsyB.

As a novel superfamily of type III polyketide synthases (PKSs) in microbes, four genes, csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. Although orthologs of csyA, csyC, and csyD genes are present in a closely related species, Aspergillus flavus, csyB gene is unique to A. oryzae. To identify its function, we carried out overexpression of csyB gene under the control of alpha-amylase promoter in A. oryzae. 3-(3-Acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)propanoic acid, named csypyrone B1, was identified as a CsyB product. Feeding experiments of (13)C-labeled acetate indicated that five acetate units were incorporated into csypyrone B1. Two possible mechanisms are proposed for the biosynthesis of cycpyrone B1: (1) condensation of succinyl-CoA with three acetyl/malonyl-CoAs, and the following pyrone ring cyclization; (2) condensation of butyryl-CoA with three acetyl/malonyl-CoAs, and the following pyrone ring cyclization and side-chain oxidation.

Genetic safeguard against mycotoxin cyclopiazonic acid production in Aspergillus oryzae.

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Its inability to produce mycotoxins, due to mutation or transcriptional repression of the genes responsible for their biosynthesis, is consistent with the hypothesis that A. oryzae is a domesticated species derived from A. flavus, a wild species that is a well‐known producer of aflatoxin. In contrast, the cyclopiazonic acid (CPA) biosynthetic gene (cpa) cluster in A. oryzae contains genes that have been lost in A. flavus. Through targeted gene inactivation, isolation of the corresponding metabolite, and evaluation of biological activity of the metabolite, we demonstrated that an A. oryzae‐specific gene—cpaH—mediates the conversion of CPA into the less toxic 2‐oxocyclopiazonic acid, a new analogue of CPA. The detoxifying properties of cpaH, which have been lost in the A. flavus pathway, reflect the relationship of the two species.

Identification of csypyrone B2 and B3 as the minor products of Aspergillus oryzae type III polyketide synthase CsyB

Since our first report on the identification of the fungal type III polyketide synthase (PKS) genes csyA~D in Aspergillus oryzae RIB40, type III PKS homologues have also been found in other fungal species. We previously reported the isolation and structural determination of csypyrone B1 as the main product of CsyB when inductively expressed in Aspergillus oryzae. Herein we report the isolation and identification of the two minor products of the csyB transformant in addition to csypyrone B1 as 4-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)butyric acid and 5-(3-acetyl-4-hydroxy-2-oxo-2H-pyran-6-yl)pentanoic acid. These compounds were named csypyrone B2 and B3, respectively, and both are homologues of main product csypyrone B1 with different side chain lengths. This result suggests that the carbon skeleton of the csypyrone B precursor is constructed by the condensation of fatty acyl-CoA and acetylmalonyl-CoA followed by pyrone formation. The alkyl side chain of the precursor may be oxidatively cleaved by enzyme(s) in the host fungus to give variations of csypyrone B with propanoic acid, butyric acid, or pentanoic acid side chains.

Aspergillus oryzae type III polyketide synthase CsyB uses a fatty acyl starter for the biosynthesis of csypyrone B compounds.

Csypyrones B1, B2 and B3 are α-pyrones that can be obtained from Aspergillus oryzae expressing CsyB, which is a type III polyketide synthase. We investigated the biosynthesis of the csypyrone B compounds using [1-(13)C] and [2-(13)C] acetate feeding experiments. (13)C NMR analyses of the methyl esters of the csypyrone B compounds fed with the (13)C-labeled acetates showed that the carboxyl carbons of the csypyrone B side-chains were derived from the C-2 methyl carbon of the acetate. These results indicated that fatty acyl starters are involved in the CsyB reaction and that the csypyrone B compounds are formed by the oxidation of side-chains by the host fungus.