lysC定点突变及lysC、asdA串联表达对谷氨酸棒杆菌L-苏氨酸积累的影响  

Effects of Site-directed Mutation of Gene lysC and Co-expression of lysC-asdA Cluster on L-threonine Accumulation in Corynebacterium glutamate

作　　者：黄勤勤 王慧梅 梁玲 黄钦耿 吴松刚[1] 黄建忠[1] HUANG Qin-qin;WANG Hui-mei;LIANG Ling;HUANG Qin-geng;WU Song-gang;HUANG Jian-zhong(Engineering Research Center of Industrial Microbiology of the Ministry of Education,College of Life Science,Fujian Normal University,Fuzhou 350117)

机构地区：[1]福建师范大学生命科学学院福建师范大学工业微生物教育部工程研究中心,福州350117

出　　处：《生物技术通报》2019年第2期93-100,共8页Biotechnology Bulletin

基　　金："863"国家高技术研究开发计划项目(2015AA021005)

摘　　要：lysC、asdA基因分别编码的天冬氨酸激酶(Aspartate kinase,AK)和天冬氨酸半醛脱氢酶(Aspartate semi-aldehyde dehydrogenase,ASD)是L-苏氨酸合成途径中两个关键限速酶基因,其中AK受到代谢产物赖氨酸与苏氨酸的协同抑制。以选育获得的一株谷氨酸棒状杆菌T11(Corynebacterium glutamicum T11)为出发菌株,通过构建lysC-asdA串联表达盒,并对其关键限速酶基因lysC进行定点突变,突变位点为Ala279Thr,获得抗反馈抑制突变型编码基因lysCr-asdA,将其插入含强启动子tac的穿梭表达载体pZ8-1中成功构建串联表达质粒pZ8-1-lysCr-asdA转化出发菌株,筛选获得工程菌株T11/pZ8-1-lysCr-asdA。摇瓶发酵其L-苏氨酸产量达到7.18 g/L,较出发菌株提高27.8%。进一步的30 L发酵罐补料分批发酵结果显示,发酵60 h L-苏氨酸产量达65.5 g/L,糖酸转化率达到39.5%,较出发菌株分别提高29.5%和33.9%,为后续的进一步构建高产L-苏氨酸的谷氨酸棒杆菌工程菌株提供强有力的基础。Aspartate kinase(AK)and aspartate semi-aldehyde dehydrogenase(ASD)encoded by gene lysC and gene asdA are two key rate-limiting enzymes playing vital roles in L-threonine synthesis pathway,and AK was co-inhibited by lysine and threonine.Selecting Corynebacterium glutamicum T11 as the original strain,the lysC-asdA gene co-expression cassette were constructed,the key rate-limiting enzyme gene lysC was mutated by site-directed mutation at Ala279Th,and the anti-feedback inhibition mutant gene,named lysC^r-asdA,was obtained,and was inserted into the shuttle expression vector pZ8-1 containing the strong promoter tac,thus the tandem expression plasmid pZ8-1-lysC^r-asdA was constructed successfully and transformed into the starting strain,then,the engineering strain T11/pZ8-1-lysC^r-asdA was obtained.The production of L-threonine by shaking flask was 7.18 g/L,which was 27.8% higher than that by the original strain.The results of further batch fermentation in 30 L fermenter showed that the yield of L-threonine reached 65.5 g/L for 60 h and the conversion rate of sugar and acid reached 39.5 g/L,increasing by 29.5% and 33.9% higher than by the original strain.It provides a strong basis for further constructing Corynebacterium glutamicum engineering strain highly yielding threonine.

关 键 词：L-苏氨酸 谷氨酸棒状杆菌 天冬氨酸激酶 天冬氨酸半醛脱氢酶 共表达 

分 类 号：Q78[生物学—分子生物学] Q936