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

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 wildtype (WT) strain. And we obtained a complemented-type (CT) strain by transforming a modi ed 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.

Analysis of the Mycotoxin Levels and Expression Pattern of SWN Genes at Different Time Points in the Fungus Slafractonia leguminicola

“…This temporal gene expression pattern in S. leguminicola differs from the pattern of M. anisopliae [53,54]. In those studies, the expression levels of swnT and swnK decreased after 3d and 5d.…”

“…In contrast, Huang et al [53] measured the swainsonine concentration in the fermentation broth of M. anisopliae at different timepoints and showed that the content of SW increased at 5d to a maximum of app. 0.3 mg and then decreased.…”

“…The temporal pattern of expression levels of specific SWN genes has been studied in Metarhizium [53,54]. These authors reported that swnT and swnK began high, went down, then went back up.…”

Establishment of RNA Interference Genetic Transformation System and Functional Analysis of FlbA Gene in Leptographium qinlingensis

“…The pSilent-1 vector has been effectively used to investigate the role of genes in a variety of filamentous fungi. In Metarhizium anisopliae , pSilent-1 plasmid was used interfered with the swnk gene of the WT strain [ 42 ]. Using the pSilent-1 vector, the chr-1 gene was suppressed in Neurospora crassa , resulting in transformants that displayed resistance to chromate and reduced accumulation of chromium [ 43 ].…”

Construction of Yeast One-Hybrid Library of Alternaria oxytropis and Screening of Transcription Factors Regulating swnK Gene Expression

“…SW-producing fungi, Alternaria Section Undifilum spp., S. leguminicola , M. robertsii , and Chaetothyriaceae spp., share more than 70% sequence consistency with the swnK gene. The gene has been confirmed to be required for the biosynthesis of SW in M. robertsii [ 14 , 31 ]. Recent research has shown that the expression of the swnK gene in A. oxytropis was closely related to the synthesis of SW [ 32 ].…”