Huang Enxia, Zhang Yu, Sun Lu, Zhu Yiru, Tang Shiyu, Mo Chonghui, Zhao Baoyu, Lu Hao
College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, China.
College of Agriculture and Animal Husbandry, Qinghai University, Xining, 810016, Qinghai, China.
Biotechnol Lett. 2023 Apr;45(4):509-519. doi: 10.1007/s10529-023-03356-0. Epub 2023 Jan 28.
Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae.
The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day 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; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained 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; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects 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.
苦马豆素（SW）是疯草的主要毒性成分，由多种真菌产生。SW合成途径中的关键酶是一种杂合的swnk/nrps。分析swnk在绿僵菌SW生物合成途径中的作用。
分别采用液相色谱-质谱联用（LC-MS）和实时荧光定量聚合酶链反应（RT-qPCR）测定绿僵菌发酵第1天至第7天SW的浓度和swnk的表达。绿僵菌在发酵第5天SW含量和swnk表达最高；通过聚乙二醇（PEG）介导的同源重组（HR）获得突变株（MT），该突变株敲除了野生型（WT）菌株中的swnk。通过转化含有遗传霉素（G418）抗性基因和swnK的改良PUC19互补载体获得互补型（CT）菌株。在MT菌株中未检测到SW，而在CT菌株中恢复到原始水平；使用具有苯菌灵（ben）抗性的Psilent-1质粒干扰WT菌株的swnk。RNAi菌株中SW水平显著降低。swnk的RNA干扰影响绿僵菌细胞壁的形成。
这些结果表明swnk在绿僵菌SW生物合成中起关键作用。
