大肠杆菌中三酶耦联合成α-酮异戊酸

Three enzyme coupling to synthesizeα-ketoisovalerate in E.coli

α-酮异戊酸是生物合成和化学合成领域中重要的中间体,在医药和化学合成领域中具有广泛的研究和应用价值。将α-酮异戊酸合成途径中的乙酰乳酸合成酶、乙酰乳酸异构还原酶、二羟基酸脱水酶3种关键酶编码基因BsalsS、EcilvC、EcilvD克隆于pETDuet-1载体上,在E.coli BL21(DE3)中加强α-酮异戊酸的合成途径,获得出发菌株SDC。进一步优化3个关键酶基因在质粒上的排列顺序,并分别在基因后添加终止子,通过比较菌株粗酶液催化丙酮酸钠底物合成α-酮异戊酸产物的能力,获得了菌株CTSDT,其α-酮异戊酸合成量比菌株SDC提高了2.28倍。菌株CTSDT以葡萄糖为碳源发酵16 h后,α-酮异戊酸产量达到1.70 g/L,是改造前菌株SDC的3.36倍。研究结果为大肠杆菌中α-酮异戊酸的高效发酵合成奠定了基础。

As an important intermediate product in the fields of biosynthesis and chemical synthesis,α-ketoisovalerate has been widely studied and applied in pharmaceutical and chemical synthesis field.Three key genes,BsalsS,EcilvC and EcilvD(encoding acetolactate synthase,acetolactate isomerase and dihydroxy acid dehydratase respectively),were cloned in this study into plasmid pETDuet-1 in order to enhance the synthesis of α-ketoisovalerate in E.coli BL21(DE3).And the original strain SDC were obtained.Furthermore,the orders of the three genes in plasmid were optimized to balance their expression.After comparing the co-expression result and the ability to catalyze the substrate sodium pyruvate to α-ketoisovalerate using the crude enzyme solution,the optimal strain CSD was obtained.Moreover,terminators of each gene in strain CSD to fine-tuning gene expression were added so as to get the final strain CTSDT.The α-ketoisovalerate conversion level of strain CTSDT was 2.28-fold high than that of strain SDC.Using glucose as the carbon source,strain C TSD T yielded α-ketoisovalerate of 1.70 g/L after 16 h cultivation,which was 3.36-fold higher than the original strain SDC.These results established foundation for efficient production of α-ketoisovalerate in E.coli BL21(DE3).