Abstract: 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 CTSDT 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).
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LI Yating,ZHOU Li,ZHOU Zhemin. Three enzyme coupling to synthesize α-ketoisovalerate in E. coli[J]. Food and Fermentation Industries, 2020, 46(13): 18-23.
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