过量表达甲酸脱氢酶提高大肠杆菌合成L-2-氨基丁酸的效率  

Increase of L-2-aminobutyric acid yield via formate dehydrogenase overexpression in Escherichia coli

作　　者：李莹[1] 史红玲[1,2] 王喆 冉璐妮 薛闯 唐存多[1] LI Ying;SHI Hongling;WANG Zhe;RAN Luni;XUE Chuang;TANG Cunduo(Henan Provincial Key Laboratory of Funiu Mountain Insect Biology,Nanyang Normal University,Nanyang 473061,Henan,China;School of Bioengineering,Dalian University of Technology,Dalian 116024,Liaoning,China)

机构地区：[1]南阳师范学院河南省伏牛山昆虫生物学重点实验室,河南南阳473061 [2]大连理工大学生物工程学院,辽宁大连116024

出　　处：《精细化工》2023年第5期1048-1054,1072,共8页Fine Chemicals

基　　金：国家自然科学基金(31900916);河南省青年人才托举工程项目(2021HYTP036);河南省高校科技创新人才(21HASTIT041)。

摘　　要：为了实现L-2-氨基丁酸(L-ABA)的高效生物合成,借助pACYCDuet-1和pET28a共表达系统,构建了携带L-苏氨酸脱氨酶(L-TD)、L-亮氨酸脱氢酶(L-L-DH)和不同活性甲酸脱氢酶(FDH)编码基因的重组大肠杆菌,分别命名为:E.coli BL21(DE3)/pACYCDuet-1-Ec TD-Es LeuDH:pET28a-Cb FDH和E.coli BL21(DE3)/p ACYCDuet-1-Ec TD-Es Leu DH:p ET28a-Cb FDH^(M)。经诱导表达后,L-苏氨酸脱氨酶和L-亮氨酸脱氢酶在两个重组大肠杆菌中的表达水平基本一致,而后者的甲酸脱氢酶酶活表达水平为(342.00±9.40)IU/mL,显著高于前者的(196.00±6.20)IU/mL。在50 mL反应体系中,200 mmol/L L-苏氨酸经220 r/min、30℃下反应6 h时,前者L-ABA得率为71%,后者为85%。结果表明,提高甲酸脱氢酶的表达水平可以显著提高L-ABA的合成效率。此外,优化反应温度后,在35℃下反应6 h时,L-ABA的得率可达90%。In order to achieve the efficient biosynthesis of L-2-aminobutyric acid(L-ABA),two recombinant Escherichia coli strains carrying threonine deaminase(TD),leucine dehydrogenase(LeuDH)and different formate dehydrogenases(FDH)encoding genes were established by using a dual plasmid co-expression system of pACYCDuet-1 and pET28a,and named E.coli BL21(DE3)/pACYCDuet-1-EsLeuDH-EcTD:pET28a-CbFDH and E.coli BL21(DE3)/pACYCDuet-1-EsLeuDH-EcTD:pET28a-CbFDH^(M),respectively.After induction,the expression levels of L-threonine deaminase and L-leucine dehydrogenase in the two recombinant E.coli strains were basically the same,while that of formate dehydrogenase in the latter was 342 IU/mL,significantly higher than 196 IU/mL expressed in the former strain.In a 50 mL reaction system with 200 mmol/L L-threonine reacted at 220 r/min and 30℃for 8 h,the yield of L-ABA was 71%in the former and 85%in the latter.The results showed that formate dehydrogenase overexpression could significantly improve the synthesis efficiency of L-ABA.Moreover,the yield of L-ABA reached 90%when catalyzed at 220 r/min and the optimized temperature of 35℃for 8 h.

关 键 词：L-2-氨基丁酸 L-苏氨酸 共表达 微生物细胞工厂 全细胞催化 生物工程 

分 类 号：TQ922[轻工技术与工程—发酵工程]