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 by overexpression formate dehydrogenase in Escherichia coli

摘要:

L-2-氨基丁酸 (L-2-aminobutyric acid, L-ABA) 是重要的医药中间体和食品添加剂。为了实现L-ABA的高效生物合成，本研究借助pACYCDuet-1和pET28a共表达系统，构建携带L-苏氨酸脱氨酶 (Threonine deaminase, EcTD)、L-亮氨酸脱氢酶 (Leucine dehydrogenase, EsLeuDH) 和不同活性甲酸脱氢酶 (Formate dehydrogenase, CbFDH) 编码基因的重组大肠杆菌，分别命名为：为E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-EcTD:pET28a- CbFDH和E. coli BL21(DE3)/pACYCDuet-1-EsLeuDH-EcTD:pET28a- CbFDHM。经诱导表达后，两个重组大肠杆菌均成功表达了3个目标酶。结果表明，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表达水平为342 IU/mL，显著高于前者的196 IU/mL。在50 mL反应体系中，200 mM L-苏氨酸，经220 rpm、30℃反应8 h后，前者L-ABA的得率为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。优化反应温度后，在35℃下反应8 h，L-ABA的得率可达90%。本研究为未来更大规模的L-苏氨酸转产增值及L-ABA的生物合成奠定了坚实的理论基础。

Abstract:

L-2-aminobutyric acid (L-ABA) is an important pharmaceutical intermediate and food additive. In order to achieve the efficient biosynthesis of L-ABA, two recombinant Escherichia coli strains carrying threonine deaminase, leucine dehydrogenase and different formate dehydrogenases encoding genes were established by using a dual plasmid co-expression system of pACYCDuet-1 and pET28a, which were named as E. coli BL21(DE3)/ pACYCDuet-1-EsLeuDH-EcTD: pET28a- CbFDH and E. coli BL21(DE3)/ pACYCDuet-1-EsLeuDH-EcTD: pET28a- CbFDHM, respectively. After induction, the two recombinant E. coli strains successfully expressed three target enzymes. The results showed that the expression levels of L-threonine deaminase and L-leucine dehydrogenase in the two recombinant E. coli strains were basically the same. The expression level of formate dehydrogenase in the latter was 342 IU/mL, which was significantly higher than 196 IU/mL in the former. In the 50 mL reaction system, 200 mM L-threonine was reacted at 220 rpm and 30 ℃ for 8 hours, the yield of L-ABA was 71% in the former and 85% in the latter. The results showed that increasing the expression level of formate dehydrogenase could significantly improve the synthesis efficiency of L-ABA. After the reaction temperature was optimized, the yield of L-ABA reached 90% catalyzed at 220 rpm and 30 ℃ for 8 hours. This study laid a solid theoretical foundation for the large-scale conversion and value-added of L-threonine and the biosynthesis of L-ABA in the future.