• Korean Journal of Microbiology
• /
• v.24 no.4
• /
• pp.357-363
• /
• 1986

Synthesis of threonine and methionine in yeast, Saccharomyces cerevisiae shares a common pathway from aspartate via homoserine. HOM6 gene encodes homoserine dehydrogenase (HSDH) which catalyzes the inter-conversion of beta-aspartate semialdehyde and homoserine. The level of HSDH is under methionine specific control. A recombinant plasmid (pEK1: 13.3kb), containing HOM6 gene, has been isolated and cloned into E. coli by complenemtary transformation of a homoserine auxotrophic yeast strain M-20-20D (hom6, trp1, ura3) to a prototrophic M20-20D/pEK1, using a library of yeast genomic DNA fragments in a yeast centromeric plasmid, YCp50(8.0kb). Isolation of HOM6has been primarily confirmed by retransformation of the original yeast strain M20-20D, using the recombinant plasmid DNA which was extracted from M20-20D/pEK1 and subsequently amplified in E. coli. Eleven cleavage sites in the insery (5.3kb) have been localized through fragment analysis for 8 restriction endonucleases; Bgl II(2 site), Bgl II(1), Cla I(3), Eco RI(1), Hind III(2), Kpn I (1), Pvu II(1) and Xho I(1).

• Microbiology and Biotechnology Letters
• /
• v.19 no.1
• /
• pp.31-36
• /
• 1991

A 7.5 kilobases hybrid plasmid, designated as pCE1301, was constructed by combining Eschurichia cwli plasmid pBELl which carries the kanamycin resistance gene of Tn5 with a cryptic plasmid, pSRl of Corynebacterium glutamicum. pCE1301 was transformed C. glutaicum by PEG-mediated protoplast method and its transformation efficiency was about $3.0\times 10^3$ transformants per $\mu g$ of the hybrid plasmid DNA. The physical map reveals that pCE1301 has single restriction sites for SalI and EcoRl, respectively. 'The kanamycin resistance of pCE1301 was stably maintained in C. glutamicum up to 25 generations and any segregation was not detected. pCI31301 was also introduced into Brevibacterium flavum and E coil, and replicated in those strains. pCE1301 was proved to be useiul in cloning the homoscrine dehydrogenase gene from C. glutamicum.

• Korean Journal of Microbiology
• /
• v.29 no.3
• /
• pp.149-154
• /
• 1991

Escherichia coli/Corynebacterium glutamicum shuttle vectors, pECCG1 and pECCG2 were constructed by joining a 3.00 kb cryptic plasmid pCB 1 from C. glutamicum and a 3.94 kb plasmid pACYC 177 from E. coli. By trimming unessential parts and introducing mulitiple cloning site into the plasmid pECCG 1, a plasmid pECCG122(5.1kb) was constructed. All the shuttle vectors were stably maintained in C. glutamicum up to about 40 generations irrespective of kanamycin addition in the medium. Threonine operon (homoserine dehydrogenase/homoserine kinase) and dapA gene (dihydrodipicolinate synthetase) of C. glutamicum were cloned into the plasmid pECCG122, and the resultant plasmids were designated pTN31 and pDHDP19, respectively. They were used to study the effect of cloned foreign gene on the stability of the plasmid pECCG122. Plasmids pTN31 and pDHDP19 were segregated rapidly from C. glutamicum when cultured in the medium without kanamycin. In medium with $50\mu${\g/ml} of kanamycin, their segregation rates were much slower than those in medium without kanamycin, but the danamycin addition didn't guarantee the complete maintenance of the plasmids in C. glutamicum.