植物乳杆菌asd2基因的表达及其对多种氨基酸合成的促进作用

Expression of the asd2 Gene and Its Enhancement Effect on Biosynthesis of Various Amino Acids in Lactobacillus plantarum

从植物乳杆菌WQ0815中克隆并表达天冬氨酸半醛脱氢酶基因asd2,评估asd2表达对WQ0815中多种氨基酸合成的促进作用。结果表明,asd2大小为1 062 bp,编码354个氨基酸的蛋白质,具有保守的N端NAD结合域和C端二聚化结构域。利用乳酸菌表达载体p BEmp M4a构建了asd2表达工程菌株WQ0815-asd2;q RT-PCR结果表明,发酵4,7 h和10 h,重组菌株中asd2的转录水平分别是对照菌株的16,23倍和32倍。氨基酸含量检测结果表明,重组菌株中赖氨酸、苏氨酸、异亮氨酸和蛋氨酸的消耗速率明显小于对照菌株;发酵10h,重组菌株中赖氨酸、苏氨酸、异亮氨酸和蛋氨酸的含量约为对照菌株的1.5~2倍。研究表明,asd2的表达对植物乳杆菌WQ0815中赖氨酸、苏氨酸、异亮氨酸和蛋氨酸的合成具有显著促进作用。

The aspartate semialdehyde dehydrogenase gene asd2 was cloned from and overexpressed in Lactobacillus plantarum WQ0815, and the effect of asd2 expression on various amino acids biosynthesis was further investigated. Sequence analysis showed that the cloned asd2 gene was 1 062 bp in size and was predicted to encode a putative protein containing 354 amino acid residues. Two conserved domains of dehydrogenase, one NAD binding domain and one dimerization domain were detected in the N-terminal and C-terminal of ASD2, respectively. The asd2 gene was overexpressed in L. plantarum WQ0815 via the expression vector p BEmp M4 a, generating the recombinant strain L. plantarum WQ0815-asd2. q RT-PCR assay showed that the relative transcript level of asd2 in the recombinant strain were about 16-fold,23-fold and 32-fold higher than that in the wild type strain after 4 h, 7 h and 10 h of fermentation. Amino acids detection by HPLC showed that the consumption rates of lysine, threonine, isoleucine and methionine in the recombinant strain were obviously slower than that in the wild type strain. The concentrations of lysine, threonine, isoleucine and methionine in the recombinant strain were around 1.5～2-fold higher than that in the wild type strain after 10 h of fermentation. In conclusion, over-expression of asd2 exerted a significant enhancement effect on biosynthesis of lysine,threonine, isoleucine and methionine in L. plantarum WQ0815.