Corynebacterium pekinense transketolase: gene cloning, sequence analysis and expression

Abstract:

Abstract: [Objective] Transketolase (EC 2.2.1.1；TKT) 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. [Methods] 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. [Results] Analysis of PCR fragments reveals that, besides the regulatory sequence, they contain the whole structure of tkt gene. There is no basic 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 TKT in C. pekinense PD-67(pTK3) is twice as much as 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. [Conclusion] The moderate amplification of TKT activity resulted in increase of L-tryptophan production without affecting the cell growth.