北京棒杆菌DAHP合成酶Ⅰ基因的克隆﹑序列分析及表达  

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

作　　者：张春花[1,2] 赵智[1] 张英姿[1] 王宇[1] 丁久元[1] 

机构地区：[1]中国科学院微生物研究所,北京100101 [2]中国科学院研究生院,北京100049

出　　处：《微生物学报》2008年第11期1466-1472,共7页Acta Microbiologica Sinica

摘　　要：【目的】从北京棒杆菌(Corynebacterium pekinense)中克隆DAHP合成酶(EC2.5.1.54,3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,DS)Ⅰ基因,对其进行功能验证;并将DAHP合成酶Ⅰ基因在C.pekinensePD-67进行同源表达,研究该酶的比活力与生长的相关性。【方法】分别以C.pekinense野生株AS1.299和突变株PD-67的基因组为模板,用PCR方法扩增了DAHP合成酶Ⅰ的全基因序列aroⅠ和前端控制序列;通过pAK6载体提高DAHP合成酶Ⅰ基因在C.pekinensePD-67中的拷贝数实现其同源表达。【结果】核苷酸序列分析结果表明,C.pekinense野生株AS1.299与突变株PD-67相比较,DAHP合成酶Ⅰ基因序列完全一样;通过PCR方法得到的DAHP合成酶Ⅰ基因结构功能完整,能与DAHP合成酶完全缺陷的E.coli3257实现异源互补。突变株PD-67来源的DAHP合成酶Ⅰ基因在重组菌PD-67(pAD1)中进行了表达,在稳定期初期重组菌PD-67(pAD1)的DAHP合成酶Ⅰ的酶比活力比同期的对照菌株PD-67(pAK6)中的该酶酶比活力提高了约5倍。【结论】本工作首次证实了C.pekinense1.299和PD-67中存在DAHP合成酶Ⅰ基因,异源互补试验证明扩增得到的DNA片段编码DAHP合成酶Ⅰ,酶学性质研究表明DAHP合成酶Ⅰ基因在C.pekinensePD-67中的同源表达将有助于提高该菌的色氨酸积累。[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 expressed 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 regulatory 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 homogenously, 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.

关 键 词：北京棒杆菌 DAHP合成酶 莽草酸合成途径 L-色氨酸 

分 类 号：Q93[生物学—微生物学] Q78