摘要
作为8种必需氨基酸之一,L-缬氨酸在生物体内起着重要的生理生化作用。微生物体内丙酮酸是L-缬氨酸生物合成的唯一前体物质,同时也是重要的中间代谢物,因此L-缬氨酸的高效积累和细胞生长是相互影响的。为保证细胞正常生长,同时可以大量积累L-缬氨酸,研究建立了一套应用于谷氨酸棒杆菌的CRISPRi技术,并利用此技术实现了三羧酸循环(tricarboxylic acid cycle,TCA)和磷酸戊糖途径(pentose phosphate pathway,PPP)中多个基因相对转录水平不同程度的调控。通过摇瓶实验,确定了编码丙酮酸脱氢酶的基因aceE和编码6-磷酸葡萄糖脱氢酶的基因Cgl 1576是利于L-缬氨酸积累的有效修饰靶点。对aceE翻译起始区域(DNA序列中起始密码子ATG区域)弱化时,L-缬氨酸可积累34.6 g/L,对Cgl 1576翻译起始区域弱化时,L-缬氨酸可积累32.3 g/L,较对照菌株分别提高了6.1和3.8 g/L。此外,对同时弱化aceE和Cgl 1576两个靶点基因进行了多种尝试并确定同时弱化aceE和Cgl 1576的翻译起始区域效果最佳,最终得到的菌株可积累L-缬氨酸37.1 g/L。该研究为定向改造谷氨酸棒杆菌积累L-缬氨酸提供了重要的修饰靶点及改造思路。
As one of the essential amino acids,L-valine plays an important role in physiology and biochemistry.Pyruvate is not only the precursor needed for L-valine biosynthesis,but also an important intermediate metabolite.Therefore,the efficient accumulation of L-valine and cell growth are mutually affected.In order to keep balance between cell growth and accumulation of L-valine,the CRISPR interference technique for Corynebacterium glutamate was established,and the relative transcription levels of several genes in tricarboxylic acid cycle(TCA)and pentose phosphate pathway(PPP)were regulated in varying degrees.Through shaking flask experiment,the genes aceE encoding pyruvate dehydrogenase and Cgl 1576 encoding glucose-6-phosphate dehydrogenation were confirmed as effective modification targets for L-valine accumulation.When the initiation of aceE and Cgl 1576 were weakened,34.6 and 32.3 g/L L-valine were accumulated,which were 6.1 and 3.8 g/L higher than the control,respectively.In addition,it was confirmed that weakening the initiation of aceE and Cgl 1576 was the best interference strategy,with a final L-valine accumulation of 37.1 g/L.This study not only determined important target genes for modification,but also provided a new direction for accumulation of L-valine in C.glutamate.
作者
杜丽红
熊海波
徐达
徐庆阳
陈宁
DU Lihong;XIONG Haibo;XU Da;XU Qingyang;CHEN Ning(College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;National and Local United Engineering Lab of Metabolic Control Fermentation Technology,Tianjin 300457,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2020年第17期1-8,共8页
Food and Fermentation Industries
基金
国家重点研发计划项目(2018YFA0900300)。
