CloninCloning, expression and sequence analysis of DSⅠgene in Corynebacterium pekinense AS1.299 and PD-67 g, expression and sequence analysis of DSⅠgene in Corynebacterium pekinense AS1.299 and PD-67

Abstract:

Abstract: [Objective]3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54;DS) is the key enzyme in tryptophan synthesis pathway. Cloning DSⅠgene from Corynebacterium pekinense and expression of DSⅠgene might facilitate testing the existence and function of DSⅠin Corynebacterium pekinense. [Methods] According to the homology between Corynebacterium glutamicum ATCC13032 and Corynebacterium pekinense, we designed a pair of PCR primers to clone the DSⅠgene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant DSⅠgene was ex-pressed in C. pekinense PD-67 by subcloning the the PCR fragment into plasmid pAK6. [Results]Analysis of PCR frag-ments revealed that they contained the whole DSⅠgene. There was no base change all over the structure genes and regu-latory sequences between C. pekinense AS1.299 and PD-67. An internal promoter was found in the upstream of the DSⅠgene from C. pekinense and it functioned in E. coli 3257. The DSⅠgene from C. pekinense PD-67 was expressed ho-mogenously, and the specific enzyme activity of DSⅠin C. pekinense PD-67(pAD1) was much higher than that of the control strain C. pekinense PD-67(pAK6). [Conclusion] This is the first report that DSⅠgene existed in Corynebaterium Pekinense, The amplification of the specific activity of DSⅠis expected to increase L-tryptophan accumulation of C. pekinense PD-67.