产异丁醇关键基因在大肠杆菌中表达的研究

Coexpression of two essential isobutanol synthesis genes in Escherichia coli

摘要:

【目的】改造大肠杆菌缬氨酸合成途径, 使其能够代谢合成异丁醇。【方法】将乳酸乳球菌(Lactococcus lactis) 1.2829的2-酮异戊酸脱羧酶基因(kivD)和醇脱氢酶基因(adhA)串联克隆到大肠杆菌 DH5α 宿主中表达。【结果】经过改造的宿主菌发酵24 h后异丁醇产量为0.12 g/L。酶活测定实验发现, kivD和adhA基因在宿主菌中均得到表达, 但由于KivD的低表达量导致宿主菌最终的异丁醇合成能力偏低。通过研究温度和pH对KivD和AdhA酶活的影响, 最终选定二者的最适温度为30 °C, 最适pH为6.5。【结论】通过向宿主菌导入外源异丁醇合成基因能够改造其自身代谢途径, 从而合成异丁醇。

关键词:2-酮异戊酸脱羧酶, 醇脱氢酶, 大肠杆菌DH5α, 异丁醇

Abstract:

[Objective] E. coli DH5α modified the valine biosynthesis pathway to biosynthesize isobutanol. [Methods] The 2-ketoisovalerate decarboxylase gene (kivD) and alcohol dehydrogenase gene (adhA) of Lactococcus lactis 1.2829 were tandemly cloned and expressed in E. coli DH5α. [Results] The yield of isobatanol by the engineered E. coli was only 0.12 g/L by 24?h fermentation. Further results revealed that the insufficient KivD activity is the bottleneck for the isobutanol biosynthesis. A series of experiments also showed that the optimal temperature and pH for both KivD and AdhA are 30 °C and pH 6.5, respectively. [Conclusion] Isobatanol fermentation by cloning and expressing essential genes in host is feasible.

Key words:2-Ketoisovalerate decarboxylase, Alcohol dehydrogenase, E. coli DH5α, Isobutanol