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Cgl1576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Cgl1576 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Cgl1576两个靶点基因进行了多种尝试并确定同时弱化aceE和Cgl1576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 Cgl1576 encoding glucose-6-phosphate dehydrogenation were confirmed as effective modification targets for L-valine accumulation. When the initiation of aceE and Cgl1576 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 Cgl1576 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.
[1] 占米林,阚宝军,张辉,等.谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较[J].微生物学通报,2019,46(2):278-291.
[2] 王书平,李玉玺.L-谷氨酰胺高产菌株的诱变选育[J].食品与发酵工业,2010,36(8):64-67.
[3] 路志强,龚建华,丁久元,等.L-精氨酸产生菌诱变育种的研究[J].微生物学报,1988,28(2):131-135.
[4] 杨宁,王惠娟,郭利健.工业微生物育种综述[J].湖北农机化,2008(3):28-29.
[5] 程明,崔承彬,李长伟,等.化学诱变技术在微生物育种研究中的应用[J].国际药学研究杂志,2009,36(6):412-417.
[6] SUZUKI N,OKAI N,NONAKA H,et al.High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library[J].Appl Environ Microbiol,2006,72(5):3 750-3 755.
[7] NOBUAKI S,MASAYUKI I,HIDEAKI Y.High-throughput transposon mutagenesis of Corynebacterium glutamicum[J].Strain Engineering,2011,765:409-417.
[8] FOLLETTIE M T,SINSKEY A J.Recombinant DNA technology for Corynebacterium glutamicum[J].Food Technol,1986,40(10):88.
[9] TAN Yanzhen,XU Daqing,LI Ye,et al.Construction of a novel sacB-based system for marker-free gene deletion in Corynebacterium glutamicum[J].Plasmid,2012,67(1):44-52.
[10] XU Rongfang,QIN Ruiying,LI Hao,et al.Generation of targeted mutant rice using a crispr-cpf1 system[J].Plant Biotechnology Journal,2017,15(6):713-717.
[11] BERND Z,MATTHIAS H,PRARTHANA M,et al.Erratum:multiplex gene editing by CRISPR-Cpf1 using a single crRNA array[J].Nat Biotechnol,2017,35(2):178.
[12] 张海灵,李颜颜,王小元.代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸[J].生物工程学报,2018,34(10):1 606-1 619.
[13] 严婉荣,戴良英.3种基因突变方法在微生物中的应用研究进展[J].中国农学通报,2012,28(18):179-184.
[14] 刘焕民,张伟国.L-缬氨酸生物合成的基础研究和菌种选育的进展[J].食品工业科技,2010,31(10):408-412.
[15] 张伟国,钱和.L-缬氨酸高产菌诱变育种的研究[J].食品与生物技术学报,2011,30(3):394-397.
[16] BASTIAN B,MARK S,TOBIAS B,et al.Corynebacterium glutamicum tailored for high-yield L-valine production[J].Appl Microbiol Biotechnol,2008,79(3):471-479.
[17] LI Y,CHEN J,LUN S Y.Biotechnological production of pyruvic acid[J].Applied Microbiology and Biotechnology,2002,57(4):451-459.
[18] JIN H P,SANG Y L.Fermentative production of branched chain amino acids:a focus on metabolic engineering[J].Appl Microbiol Biotechnol,2010,85(3):491-506.
[19] HASEGAWA S,SUDA M,UEMATSU K,et al.Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions[J].Appl Environ Microbiol,2013,79(4):1 250-1 257.
[20] 赵丽丽,陈宁,熊明勇,等.L-缬氨酸发酵生产的育种思路及发酵条件优化策略[J].中国生物工程杂志,2003(1):17-21.
[21] HASEGAWA S,UEMATSU K,SUDA M,et al.Improvement of the redox balance increases L-valine production by Corynebacterium glutamicum under oxygen deprivation conditions[J].Appl Environ Microbiol,2012,78(3):865-875.
[22] MA Yuechao,CUI Yi,DU Lihong,et al.Identification and application of a growth-regulated promoter for improving L-valine production in Corynebacterium glutamicum[J].Microbial Cell Factories,2018,17(1):185.
