一株黄色短杆菌基因工程菌株的构建及其L- 缬氨酸积累

从谷氨酸棒杆菌模式菌株C. glutamicum ATCC13032 中克隆出L- 缬氨酸合成途径上的限速酶--乙酰羟酸合酶编码基因ilvBN。对ilvBN 进行定点突变，获得其抗反馈抑制突变型ilvBNr。以大肠杆菌- 黄色短杆菌穿梭表达载体pDXW-10 为基础，构建重组质粒pDXW-10-ilvBNr，并转化野生型黄色短杆菌B. flavum ATCC14067，获得工程菌株ATCC14067/pDXW-10-ilvBNr。3L 罐发酵实验结果显示：在野生型菌株发酵液中检测不到L- 缬氨酸积累，而工程菌株发酵液中L- 缬氨酸积累达5.0g/L。

Construction and L-valine Accumulation of a Genetic Engineering Brevibacterium flavum Strain

As a rate-limiting enzyme for L-valine biosynthesis, the ilvBN gene encoding acetyl-carboxylic acid synthase (AHAS) from Corynebacterium glutamicum ATCC13032 was amplified by PCR, followed by site-direct mutagenesis to obtain an ilvBNr gene, the anti-feedback inhibition gene of ilvBN. The ilvBNr gene inserted into E. coli-Brevibacterium flavum shuttle expression vector pDXW-10 to construct a recombinant plasmid pDXW-10-ilvBNr, which was subsequently transformed into B. flavum ATCC14067, producing a genetic engineering strain ATCC14067/pDXW-10-ilvBNr. The fermentation experiments conducted in a 3 L fermentor showed that no L-valine accumulation was detected in the fermentation broth of the original strain, while an L-valine accumulation of 5.0 g/L was observed in the fermentation broth of the constructed engineering strain.