Development of corynebacterium glutamicum expression vectors for the high-level expression of heterologous genes

• Main Authors: Tzu-Ting Pan, 潘姿穎
• Other Authors: 許文輝
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/89518647447728153337

碩士 === 國立中興大學 === 分子生物學研究所 === 90 === Corynebacterium glutamicum is generally recognized as nonhazardous organism, which is safe to handle. Based on its extremely well investigated central metabolism and well-established molecular biology tools, C. glutamicum may be very suitable as an organism for the expression of heterologous gene in high (G+C) bacteria. In this study, We have cloned promoter regions from glutamate dehydrogenase gene (gdh), glutamine synthetase gene (gln A), and 3-phosphoglycerate kinase genes (pgk) into 5’ end of Escherichia coli lacZ gene and then transformed into C. glutamicum. The specific activities of b- galactosidase was 161.2, 97, and 7.97 U/mg under the control of gln A, gdh, and pgk promoters, repectively, using cells growth in CM broth. We have isolated a corynephage P1201 from the culture broth of C. glutamicum in an industrial L-glutamate fermentation. The host range of the virulent bacteriophage P1201 is limited to C. glutamicum CCRC18238, a glutamic acid hyperproducing strain. P1201 is a typtical corynephage of Styloviridae family with a head of 52 nm wide and a long non-contractile and striated tail 348 nm. Its genome consisted of a double-stranded DNA of 70 kb. Sau3AI digested genomic DNA was cloned into pZY2, a promoter probed vector, with lacZ as a reporter gene. We found several efficient promoter clones, (pZY1021-8, —5b, —12, -5c, -11, and -1c) that produced b-galactosidases with specific activities of 13×103、6.6×103、5.2×103、792.86、555 and 332.4U / mg, repectively. Corynephage P1201 genomic DNA was sequenced for realizing the specificity between C. glutamicum strains and its infected phage. Open reading frames and their putative proteins were predicted from about 26.6 kb DNA fragment probably related to major head protein, minor tail subunit, envelop protein, transcription, and DNA replication.