Ji Weike, Zhao Zhi, Zhang Yingzi, Wang Yu, Ding Jiuyuan
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Wei Sheng Wu Xue Bao. 2010 Nov;50(11):1474-80.
Transketolase (EC 2. 2. 1. 1; TK) is the key enzyme in non-oxidative phosphate pentose pathway. We cloned tkt gene from Corynebacterium pekinense AS 1.299 and its mutant PD-67 in order to investigate the effect of gene expression on physiological characteristics of C. pekinense. PD-67.
According to the homology between Corynebacterium glutamicum ATCC13032 and C. pekinense, we designed a pair of PCR primers to clone the tkt gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant tkt gene was expressed in C. pekinense PD-67 by subcloning the PCR fragment into plasmid pAK6. The physiological characteristics of the recombinant C. pekinense PD-67 was investigated by fermentation.
Analysis of PCR fragments reveals that, besides the regulatory sequence, they contain the whole structure of tkt gene. There is no base change all over the structure genes and regulatory sequences between C. pekinense AS1. 299 and PD-67. Comparing with Corynebacterium glutamicum ATCC 13032, there exist 5 amino acids change in amino acid sequence. Four of them were located in the motifs involved in thiamine pyrophosphate binding sites. The tkt gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of TK in C. pekinense PD-67 (pTK3) is two times over that of the control strain C. pekinense PD-67 (pAK6). The recombinant C. pekinense PD-67 exhibits higher cell mass and accumulation of more tryptophan.
The moderate amplification of TK activity resulted in increase of L-tryptophan production without affecting the cell growth.
转酮醇酶（EC 2.2.1.1；TK）是非氧化磷酸戊糖途径中的关键酶。我们从北京棒杆菌AS 1.299及其突变体PD - 67中克隆了tkt基因，以研究基因表达对北京棒杆菌生理特性的影响。
根据谷氨酸棒杆菌ATCC13032与北京棒杆菌的同源性，设计一对PCR引物从野生型北京棒杆菌AS1.299及其突变体PD - 67中克隆tkt基因，然后通过将PCR片段亚克隆到质粒pAK6中，使突变的tkt基因在北京棒杆菌PD - 67中表达。通过发酵研究重组北京棒杆菌PD - 67的生理特性。
PCR片段分析表明，除调控序列外，它们包含tkt基因的完整结构。北京棒杆菌AS1.299和PD - 67的结构基因和调控序列在整个区域没有碱基变化。与谷氨酸棒杆菌ATCC 13032相比，氨基酸序列中有5个氨基酸变化。其中4个位于硫胺素焦磷酸结合位点相关的基序中。北京棒杆菌PD - 67的tkt基因均匀表达，北京棒杆菌PD - 67（pTK3）中TK的比酶活性是对照菌株北京棒杆菌PD - 67（pAK6）的两倍。重组北京棒杆菌PD - 67表现出更高的细胞量和更多色氨酸的积累。
TK活性的适度增强导致L - 色氨酸产量增加而不影响细胞生长。
