北京棒杆菌邻氨基苯甲酸合成酶基因的克隆、序列分析及表达

Cloning, sequence analysis and expression of anthranilate synthetase gene in Corynebacterium pekinense

摘要:

以北京棒杆菌（Corynebacterium pekinense）野生株AS1.299和突变株PD-67的基因组为模板，用PCR方法扩增了邻氨基苯甲酸合成酶（AS）基因（trpEG）片段和前端控制序列。核酸序列分析结果表明，该片段全长3374bp，包含3个ORF，推测分别为前导肽基因trpL、AS componentⅠ基因trpE和AS componentⅡ基因trpG。C. pekinense野生株AS1.299与突变株PD-67相比较，trpL基因完全一样；trpE基因有6个碱基的突变，导致了5个氨基酸残基的改变；trpG基因有1个碱基的突变，导致了1个氨基酸残基的改变；同时它们在-35序列处还有一个A→G的突变。通过同源性比较发现，C. pekinense AS1.299与Corynebacterium glutamicum ATCC 13032和Brevibacterium lactofermentum的亲缘关系是很近的。trpL基因上游存在启动子区域，并能被Escherichia coli的RNA聚合酶所识别，实现异源互补。野生型和突变型AS基因在C. pekinense AS1.299和PD-67中都得到表达，并且重组菌相对于宿主菌的酶活都有了很大提高。摇瓶发酵实验结果表明，带有突变型AS基因的PD-67重组菌生长比较慢，稳定期比PD-67推迟24h，但产生的L-色氨酸比PD-67高22.39%。

Abstract:

Anthranilate synthetase (EC4.1.3.27;AS) genes from wild-type Corynebacterium pekinense AS1.299 and its mutant PD-67 were cloned and sequenced. Analysis of PCR fragments revealed that three ORFs existed, which corresponded to trpL, trpE and trpG gene, respectively. Six bases changes that resulted in the changes of five amino acids were found in the trpE structural gene of C. pekinense PD-67 and a single-base change that resulted in an amino acid substitution was found in the trpG structural gene of C. pekinense PD-67.A homology comparison revealed that C. pekinense AS1.299 was closely related to Corynebacterim glutamicum ATCC 13032 and Brevibacterium lactofermentum. An internal promoter was found in the upstream of the trpL gene from C. pekinense and it functioned in E. coli, but a single-base exchange (A to G) existed in the-35 box of PD-67.The trpEG genes from the wild-type strain and its mutant were expressed both in C. pekinense AS1.299 and PD-67, and the specific enzyme activities of transformed C. pekinense were much higher than that of the parental strains. The amplification of the activity of AS yielded 22.39% increase of L-tryptophan production, but the cell growth became slower than PD-67.