The methoxymalonyl-acyl carrier protein biosynthesis locus and the nearby gene with the β-ketoacyl synthase domain are involved in the biosynthesis of galbonolides in Streptomyces galbus, but these loci are separate from the modular polyketide synthase ge

Abstract

Galbonolides A and B are antifungal compounds, which are produced by Streptomyces galbus. A multimodular polyketide synthase (PKS) was predicted to catalyze their biosynthesis, and a methoxymalonyl-acyl carrier protein (methoxymalonyl-ACP) was expected to be involved in the biosynthesis of galbonolide A. Cloning of a methoxymalonyl-ACP biosynthesis locus (galGHIJK) and the flanking regions has revealed that the locus is colocalized with beta-ketoacyl synthase (KAS)-related genes (orf3, 4, and 5), but separated from any multimodular PKS gene cluster in S. galbus. A galI-disruption mutant (SK-galI-5) is unable to produce galbonolide A, but can synthesize galbonolide B, indicating that galGHIJK is involved in the biosynthesis of galbonolide A. A disruption mutant of orf4 is severely impaired in the production of both galbonolides A and B. These results indicate that galGHIJK and the KAS genes are involved in the biosynthesis of galbonolides, although they are not colocalized with a multimodular PKS gene cluster. We further propose that a single galbonolide PKS generates two discrete structures, galbonolides A and B, by alternatively incorporating methoxymalonate and methylmalonate, respectively.

A Single Module Type I Polyketide Synthase Directs de Novo Macrolactone Biogenesis during Galbonolide Biosynthesis in Streptomyces galbus

“…It was previously shown that an inactivation of galI, one of the methoxymalonyl-ACP biosynthetic genes, selectively abolished GAL-A biosynthesis, whereas a vector-integrating gene disruption of galB dramatically reduced the level of GAL-A and -B (12). This galB disruption study implied that galA-E are involved in GAL biosynthesis; however, their biochemical roles remained veiled.…”

“…The PCR-targeting mutagenesis method was used in the preparation of gene inactivation constructs (15). Intergeneric conjugation was used to introduce plasmids into S. galbus (12). Specifically, the conjugation experiment was started with the introduction of a gene inactivation construct into E. coli ET12567/pUZ8002.…”

“…The cosmid library of S. galbus total DNA in SuperCos1 (Stratagene), the gal cosmid clone of pHJK1011, and its nucleotide sequence (GenBank TM accession number GU300145) were previously described (12). The isolation of the cosmid clones harboring the vicenistatin biosynthetic genes (vin) (17) was achieved by colony hybridization with a type I PKS-KS fragment, which was obtained through degenerate PCR.…”

“…The fourth extension units were thus proposed to be methoxymalonyl-ACP and methylmalonyl-CoA for GAL-A and -B, respectively. The cloning of the methoxymalonyl-ACP biosynthesis locus (galG to K) in S. galbus revealed that this locus was neighbored by a gene set (galA to E) that encodes a single module PKS, a cytochrome P450 protein, and a Rieske N-terminal domain protein (12). GalA contains an AT domain and a thiolation domain, which may act as an ACP (Fig.…”

“…Mutant-It was previously shown that a vector-integrating knock-out of galB, generated with the temperature-sensitive replicon vector pKC1139, severely impaired GAL production (12). To assure that galA-E encode GAL biosynthetic enzymes, gene-replacement mutants of galB, galC, and galD (⌬galB::aacIV, ⌬galC::aacIV, and ⌬galD::aacIV, respectively) were prepared (data not shown).…”