swnK plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae

Abstract

Abstract Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by fungi including Metarhizium anisopliae, Slafractonia leguminicola, and Alternaria oxytropis. A key enzyme in the SW synthesis pathway is a hybrid SWNK/NRPS. The downstream biosynthetic pathways of SW have been clearly analyzed by a knockout and complement of swnK in Metarhizium robertsii, but the upstream steps and the associated regulatory proteins have not been thoroughly investigated. To verify the role of swnK in the SW biosynthesis pathway of M. anisopliae, the concentration of SW and the swnK expression in M. anisopliae fermentation from 1st to 7th d were determined using LC-MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnK expression on the 5th day of fermentation; We obtained a mutant strain (MT) by PEG-mediated homologous recombination (HR) to knock out swnK in the wild-type (WT) strain. And we obtained a complemented-type (CT) strain by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; We also used a Psilent-1 plasmid with Benomyl (ben)-resistant that interfered with swnK of wild-type strain (WT). The level of SW in the RNAi strain was markedly diminished. RNAi of swnK affected the formation of the cell wall in M. anisopliae. These results indicate that swnK plays a crucial role in the SW biosynthesis of M. anisopliae.