[Cloning, expression and sequence analysis of DS I gene in Corynebacterium pekinense AS1.299 and PD-67].
OBJECTIVE
3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (EC 2.5.1.54;DS) is the key enzyme in tryptophan synthesis pathway. Cloning DS I gene from Corynebacterium pekinense and expression of DS I gene might facilitate testing the existence and function of DS I in Corynebacterium pekinense.


METHODS
According to the homology between Corynebacterium glutamicum ATCC13032 and Corynebacterium pekinense, we designed a pair of PCR primers to clone the DS I gene from wild-type C. pekinense AS1.299 and its mutant PD-67, then the mutant DS I gene was expressed in C. pekinense PD-67 by subcloning the the PCR fragment into plasmid pAK6.


RESULTS
Analysis of PCR fragments revealed that they contained the whole DS I gene. There was no base change all over the structure genes and regulatory sequences between C. pekinense AS1.299 and PD-67. An internal promoter was found in the upstream of the DS I gene from C. pekinense and it functioned in E. coli 3257. The DS I gene from C. pekinense PD-67 was expressed homogenously, and the specific enzyme activity of DS I in C. pekinense PD-67 (pAD1) was much higher than that of the control strain C. pekinense PD-67(pAK6).


CONCLUSIONS
This is the first report that DS I gene existed in Corynebaterium Pekinense, The amplification of the specific activity of DS I is expected to increase L-tryptophan accumulation of C. pekinense PD-67.
Zhang
Chunhua
C
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Zhao
Zhi
Z
Zhang
Yingzi
Y
Wang
Yu
Y
Ding
Jiuyuan
J
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D019976:Cloning, Organism; D003352:Corynebacterium / Q000145:classification / Q000235:genetics*; D030561:Databases, Nucleic Acid; D008969:Molecular Sequence Data; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis
D011993:Recombinant Fusion Proteins; D010723:3-Deoxy-7-Phosphoheptulonate Synthase