l-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to l-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and l-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance l-leucine production and cell growth. Subsequently, the genes involved in l-leucine biosynthesis were overexpressed by replacing the native promoters P_leuA and P_ilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter P_tuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36 g/L l-leucine in shake flask fermentation.

1-亮氨酸是一种必需氨基酸，在工业上具有广泛的应用。当前快速增长的市场需求为进一步提高其生物转化生产率和生产稳定性提供了强大的动力。在这项研究中，我们合理地设计了谷氨酸棒杆菌中从丙酮酸到1-亮氨酸合成的代谢通量，以提高丙酮酸的利用率和1-亮氨酸的合成。首先，pyc（编码丙酮酸羧化酶）和avtA（编码丙氨酸-缬氨酸转氨酶）基因被删除，以削弱三羧酸循环的代谢通量，并减少丙酮酸的竞争性消费。接下来，通过插入终止子以平衡1-亮氨酸产生和细胞生长来下调alaT基因（编码丙氨酸转氨酶）的转录水平。随后，参与基因升-亮氨酸的生物合成是由代替天然启动子过表达P_的leuA和P _ilvBNC所述的的leuA基因和ilvBNC分别操纵子，与助催化剂P _TUF的EFTU（编码延伸因子Tu）并使用穿梭表达载体。所得菌株WL-14在摇瓶发酵中产生28.47±0.36g / L的1-亮氨酸。