Wang Yulong, Liu Shihong, Yin Xuebing, Yu Deshui, Xie Xiangyun, Huang Bo
Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei 230036, China.
Engineering Research Center of Fungal Biotechnology, Ministry of Education, Anhui Agricultural University, Hefei 230036, China.
J Fungi (Basel). 2021 Sep 8;7(9):737. doi: 10.3390/jof7090737.
Ketol-acid reductoisomerase (ILVC) is the second enzyme in the branched-chain amino acid (BCAA) biosynthesis, which regulates many physiological activities in a variety of organisms from bacteria to fungi and plants. In this work, function mechanisms of ILVC in Metchnikoff (Hypocreales: Clavicipitaceae) were explored with site-directed mutagenesis, reductase activity assays and transcriptomics analysis. The reductase activity assays showed that ILVC from phytopathogenic fungi exhibited significantly higher activities than those from entomopathogenic fungi but lower than those from yeast. Site-directed mutagenesis and enzymatic activities of MrILVC with different active-site mutants (Arg-113, Ser-118, Asp-152, Asp-260, and Glu-264) confirmed that active sites of MrILVC are conserved with plant and bacterial ILVCs. Deleting causes the complete failures of vegetative growth and conidial germination, feeding with branched-chain amino acids (BCAAs) recovers the fungal growth but not conidial germination, while both characteristics are restored when supplemented with yeast extract. Compared to Δ cultured in czapek agar (CZA), plenty of genes involved in the biosynthesis of antibiotics and amino acids were up- or down-regulated in the wild type or Δ feeding with either BCAAs or yeast extract. Further analysis showed some genes, such as , participate in mycelial growth and conidial germination was down-regulated in Δ from CZA, revealing that MrILVC might control the fungal development by gene regulation and BCAAs or yeast extract could play partial roles of MrILVC. This study will advance our understanding of ILVC function mechanisms in fungi.
酮醇酸还原异构酶（ILVC）是支链氨基酸（BCAA）生物合成中的第二种酶，它调节从细菌到真菌和植物等多种生物体中的许多生理活动。在这项工作中，通过定点诱变、还原酶活性测定和转录组学分析，探索了ILVC在肉座菌（Hypocreales: Clavicipitaceae）中的功能机制。还原酶活性测定表明，植物病原真菌中的ILVC活性显著高于昆虫病原真菌，但低于酵母中的ILVC活性。对具有不同活性位点突变体（Arg-113、Ser-118、Asp-152、Asp-260和Glu-264）的MrILVC进行定点诱变和酶活性测定，证实MrILVC的活性位点与植物和细菌的ILVC保守。缺失导致营养生长和分生孢子萌发完全失败，用支链氨基酸（BCAAs）喂养可恢复真菌生长，但不能恢复分生孢子萌发，而补充酵母提取物时，这两个特征均得以恢复。与在察氏琼脂（CZA）中培养的Δ相比，在野生型或用BCAAs或酵母提取物喂养的Δ中，许多参与抗生素和氨基酸生物合成的基因上调或下调。进一步分析表明，一些基因，如，参与菌丝体生长和分生孢子萌发在来自CZA的Δ中下调，表明MrILVC可能通过基因调控控制真菌发育，BCAAs或酵母提取物可能发挥MrILVC的部分作用。这项研究将增进我们对真菌中ILVC功能机制的理解。
