Studies of the dapA and the lysC genes of lysine biosynthetic pathway of Escherichia coli

• Main Authors: Liao, Hui-Hua, 廖惠華
• Other Authors: T. H. Hseu
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/40715350765752011997

博士 === 國立清華大學 === 生命科學系 === 86 === The dapA gene of Escherichia coli encoding dihydrodipicolinate synthase (DHDPS), the first enzyme of the biosynthetic branch leading to diaminopimelate and lysine, was cloned in this study. We amplified the dapA gene by PCR (polymerase chain reaction) from a minilibrary of E. coli DH1 chromosome, inserted the PCR product into vector pUC18, and confirmed the insert first by restriction patterns and finally by DNA sequence analysis. Plasmid pCR13, the multicopy plasmid carrying the dapA gene, wasintroduced into both E. coli AT998 and DH1. Expression of dihydrodipicolinate synthase was detected from SDS-PAGE analysis and theenzyme activity of the crude cell extract. SDS-PAGE analysis revealed aspecific band of about 35 kDa present on E. coli strains harboring pCR13.SDS-PAGE analysis and enzyme assay showed that expression of the dapA geneon pCR13 varied with the host strains. The expression level was higher in DH1 than in AT998. SDS-PAGE analysis and enzyme assay also indicated thatincrease of the copy number of the dapA gene led to increase of the DHDPS activity. Moreover, DHDPS activity was inhibited 80 to 84 % by 20 mM lysinein vitro. A lysC-lac￠Z fusion plasmid was constructed to study the regulatoryregion of the lysC gene. Analysis by deletion mutations confirmed theexistence of an alternative promoter, P2, located upstream of the previouslyidentified promoter, P1. The transcription start site of promoter P2 was located 85 base pairs upstream the transcription start site of promoter P1.Both promoters are regulated by lysine. The untranslated region of the lysCgene was analyzed by Bal 31 nested deletion and specific-site insertion mutations. It seemed that the regulation mechanism might not be conventionalrepressor-operator binding.