sucA缺失型大肠杆菌菌株的构建与发酵条件优化  

Construction of sucA-Knockout E. coli Strains and Optimization of Fermentation Conditions

作　　者：林凡[1,2,3] 张震宇 孙付保[1,2,3] 于林[1] LIN Fan;ZHANG Zhenyu;SUN Fubao;YU Lin(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Wuxi 214122,China;Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]工业生物技术教育部重点实验室,江苏无锡214122 [3]糖化学与生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2018年第8期830-837,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(30800018;30970058);国家自然科学基金面上项目(21176106);高等学校博士学科点专项科研基金项目(200802951036);江苏省自然科学基金项目(BK2012554);工业生物技术教育部重点实验室主任基金项目(KLIB-ZR200801)

摘　　要：反式-4-羟脯氨酸在多领域具有重要应用价值。为了进一步提高反式-4-羟脯氨酸的产量,在已构建好的E. coli BL21(DE3)△putA的基础上,敲除基因sucA的同时插入密码子优化后的反式-4-羟化酶基因(hyp),并将构建好的质粒pUC19-ptrp2-hyp-vgb导入该基因敲除菌株中。之后在摇瓶水平上,单因素优化发酵条件与发酵培养基的组分,并通过正交实验进一步确定敲除菌株的培养条件。结果表明:与含有同样质粒的原菌和两种单敲除菌E. coli BL21(DE3)△sucA、E. coli BL21(DE3)△putA相比,E. coli BL21(DE3)△putA△sucA双敲除菌具有最高的全细胞酶活,大约是原菌的2.6倍;在摇瓶水平上,发酵条件优化后,以100 mmol/L的L-脯氨酸为底物进行发酵,12 h后可得到1.08 g/L的羟脯氨酸,是优化前的3.87倍。trans-4-hydroxyproline has important applications in many fields.On the basis of the constructed E.coli BL21(DE3)△putA strain,sucA gene was knocked out and the optimized trans-4-hydroxylase gene(hyp)was introduced.Then,the constructed plasmid pUC19-ptrp2-hyp-vgb which included a tryptophan tandem promoter and Vitreoscilla hemoglobin(VHb)was imported into the recombinant strain and overexpressed.Then in the flask level,fermentation conditions and medium components were confirmed by single factor optimization and orthogonal experiment.The results showed that,compared with the original bacteria and two single knockout strains containing the same plasmid,the double knockout strain had the highest whole-cell activity,which is about 2.6 times as the original bacteria.After optimizing the fermentation conditions,trans-4-hydroxyproline was accumulated to 1.08 g/L,which increased 2.87 fold.

关 键 词：反式-4-羟脯氨酸 sucA 透明颤菌血红蛋白 基因敲除 重组大肠杆菌 全细胞酶活 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]