Identification of the nonribosomal peptide synthetase gene responsible for bassianolide synthesis in wood-decaying fungus Xylaria sp. BCC1067.

Intensive study of gene diversity of bioactive compounds in a wood-rot fungus, Xylaria sp. BCC1067, has made it possible to identify polyketides and nonribosomal peptides (NRPs) unaccounted for by conventional chemical screening methods. Here we report the complete nonribosomal peptide synthetase (NRPS) gene responsible for the biosynthesis of an NRP, bassianolide, using a genetic approach. Isolation of the bassianolide biosynthetic gene, nrpsxy, was achieved using degenerate primers specific to the adenylation domain of NRPS. The complete ORF of nrpsxy is 10.6 kb in length. Based on comparisons with other known NRPSs, the domain arrangement of NRPSXY is most likely to be C-A-T-C-A-M-T-T-C-R. The other ORF found upstream of nrpsxy, designated efxy, is 1.8 kb in length and shows high similarity to members of the major facilitator superfamily of transporters. Functional analysis of the nrpsxy gene was conducted by gene disruption, and the missing metabolite in the mutant was identified. Chemical analysis revealed the structure of the metabolite to be a cyclooctadepsipeptide, bassianolide, which has been found in other fungi. A bioassay of bassianolide revealed a wide range of biological activities other than insecticidal uses, which have been previously reported, thus making bassianolide an interesting candidate for future structural modification. This study is the first evidence for a gene involved in the biosynthesis of bassianolide.

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA.

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.

Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067.

Fungal type I polyketide (PK) compounds are highly valuable for medical treatment and extremely diverse in structure, partly because of the enzymatic activities of reducing domains in polyketide synthases (PKSs). We have cloned several PKS genes from the fungus Xylaria sp. BCC 1067, which produces two polyketides: depudecin (reduced PK) and 19,20-epoxycytochalasin Q (PK-nonribosomal peptide (NRP) hybrid). Two new degenerate primer sets, KA-series and XKS, were designed to amplify reducing PKS and PKS-NRP synthetase hybrid genes, respectively. Five putative PKS genes were amplified in Xylaria using KA-series primers and two more with the XKS primers. All seven are predicted to encode proteins homologous to highly reduced (HR)-type PKSs. Previously designed primers in LC-, KS-, and MT-series identified four additional PKS gene fragments. Selected PKS fragments were used as probes to identify PKS genes from the genomic library of this fungus. Full-length sequences for five PKS genes were obtained: pks12, pks3, pksKA1, pksMT, and pksX1. They are structurally diverse with 1-9 putative introns and products ranging from 2162 to 3654 amino acids in length. The finding of 11 distinct PKS genes solely by means of PCR cloning supports that PKS genes are highly diverse in fungi. It also indicates that our KA-series primers can serve as powerful tools to reveal the genetic potential of fungi in production of multiple types of HR PKs, which the conventional compound screening could underestimate.