Chen Xiao-fang, Zhao Zhi, Zhang Ying-zi, Wang Yu, Ding Jiu-yuan
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Wei Sheng Wu Xue Bao. 2007 Feb;47(1):48-53.
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.
对野生型北京棒杆菌AS1.299及其突变体PD-67的邻氨基苯甲酸合成酶（EC4.1.3.27；AS）基因进行了克隆和测序。对PCR片段的分析表明存在三个开放阅读框（ORF），分别对应trpL、trpE和trpG基因。在北京棒杆菌PD-67的trpE结构基因中发现了六个碱基变化，导致五个氨基酸发生改变，在北京棒杆菌PD-67的trpG结构基因中发现了一个导致氨基酸替换的单碱基变化。同源性比较表明，北京棒杆菌AS1.299与谷氨酸棒杆菌ATCC 13032和乳糖发酵短杆菌密切相关。在北京棒杆菌trpL基因的上游发现了一个内部启动子，它在大肠杆菌中起作用，但在PD-67的-35框中存在一个单碱基交换（A到G）。野生型菌株及其突变体的trpEG基因在北京棒杆菌AS1.299和PD-67中均有表达，转化后的北京棒杆菌的比酶活远高于亲本菌株。AS活性的提高使L-色氨酸产量增加了22.39%，但细胞生长比PD-67慢。
