P122: Identification and characterization of the spiruchostatin biosynthetic gene cluster enables yield improvement by overexpressing a heterologous transcriptional activator

Spiruchostatins A and B are anticancer agents produced by Pseudomonas sp. Q71576. A gene cluster (spi) responsible for spiruchostatin biosynthesis was identified by rapid genome sequencing, and characterized by genetic mutations. This spi gene cluster consists of fourteen genes, among which three NRPS genes, three PKS genes and four tailoring genes collectively encode a hybrid NRPS-PKS biosynthetic pathway similar to the FK228 biosynthetic pathway. Differences between the spi gene cluster and FK228 biosynthetic (dep) gene cluster include a putative aminotransferase gene (depM) found in the dep gene cluster but not in the spi gene cluster, and a malonyl CoA acyltransferase gene (spiP) in the spi gene cluster but not in the dep gene cluster. In an attempt to increase the yield of spiruchostatins, a transcriptional activator gene (depR) from the FK228 biosynthesis pathway was introduced into Pseudomonas sp. Q71576; overexpression of DepR resulted in a significant increase of spiruchostatin production. depR and the newly identified pathway regulatory gene in the spi cluster - spiR - both encode an LysR-type transcriptional regulator. RT-PCR analysis of the spi gene cluster in the presence of depR indicates that DepR upregulates SpiR at the transcriptional level and SpiR in turn activates the rest spi genes. The parallel and convergent properties between the dep and the spi pathways provides opportunities for combinatorial biosynthesis of “FK228-family” of structural analogs; moreover, yield improvement of spiruchostatins demonstrates that the metabolic potential of natural product biosynthetic gene clusters can be tapped into by overexpression of heterologous transcriptional activators.