Studies On Construction Of DapA Gene Deletion Mutant And Its Fermentation Conditions

Based on the principle of pathway engineering and the theory of metabolic control fermentation, the dissertation focuses on the construction of dapA gene deletion mutant by Red recombination and its fermentation conditions.The main research contents and result are as follows:1. In the research, PCR products(dapAkan) were generated by using primers with 50-nt extensions that are homologous to regions adjacent to the dapA gene to be inactivated and template plasmids carrying kanamycin resistance genes that are flanked by FRT (FLP recognition target) sites. The linear fragments is electroporated into the E.coli K12/ pKD46. Mutants of the dapA genes were replaced by homologous recombination as kanamycin-resistant colonies after the introduction into bacteria carrying a Red expression plasmid pKD46. After selection and PCR identification reaction of recombinants, the resistance gene located in the K12â–³dapA::kan mutant was eliminated in the second recombination, by using the FLP recombinase, which acts on the directly repeated FRT sites flanking the kanamycin resistance gene. The Mutants were named K12â–³dapA which were screened and identified.2. The L-threonine in the samples of Escherichia coli K12 and K12â–³dapA respectively were compared quantitatively after fermentation under the fermentation condition without being optimized.The results indicate that the L-thr of K12â–³dapA was as 3 times as that of wild type Escherichia coli K12.The strain with dapA gene deletion were obtained and was characterized as L-lys auxotrophy and L-thr high-yield. This strain could produce L-threonine 3.7 g/L.3. The batch fermentation conditions of strain K12â–³dapA in shake flask were studied in this dissertation. The proportion of seed medium and fermentation medium components were optimized by orthogonal design,uniform design experiment and SPSS and MDAS software.The seed culture conditions and fermentation conditions were also studied.Under the optimum conditions, strain K12â–³dapA could produce L-threonine 8.2 g/L after fermentation for 72 hours by flask-shaking batch fermentation.