北京棒杆菌DAHP合成酶Ⅰ基因的克隆﹑序列分析及表达

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

摘要:

摘要：【目的】从北京棒杆菌(Corynebacterium pekinense)中克隆DAHP合成酶 (EC 2.5.1.54，3-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. pekinen-sePD-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.coli 3257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. pekinense 1.299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。

关键词:关键词：北京棒杆菌;DAHP合成酶;莽草酸合成途径;L-色氨酸

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.

Key words:Keywords: Corynebacterium pekinense; 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; shikimate pathway; L-tryptophan