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.
Jirakkakul
Jiraporn
J
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Punya
Juntira
J
National Center for Genetic Engineering and Biotechnology, Thailand Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Pongpattanakitshote
Somchai
S
National Center for Genetic Engineering and Biotechnology, Thailand Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Paungmoung
Porntip
P
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Vorapreeda
Namol
N
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Tachaleat
Anuwat
A
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Klomnara
Cheeranun
C
School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
Tanticharoen
Morakot
M
National Center for Genetic Engineering and Biotechnology, Thailand Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand.
Cheevadhanarak
Supapon
S
Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi, Bangkok 10150, Thailand.|School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bangkok 10140, Thailand.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D000995:Antitubercular Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016254:Mutagenesis, Insertional; D016366:Open Reading Frames; D010453:Peptide Synthases / Q000235:genetics* / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods; D017434:Protein Structure, Tertiary; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014934:Wood; D013098:Xylariales / Q000201:enzymology* / Q000235:genetics
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D000995:Antitubercular Agents; D017931:DNA Primers; D004271:DNA, Fungal; D010456:Peptides, Cyclic; C035050:bassianolide; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase