Construction Of Mutated Strain For L-tryptophan Biosynthesis And Study On Its Fermentation Conditions

L-tryptophan that containing indolyl neutral is one of the essential amino acids,which plays an important role in growth and metabolism of human and animal.L-tryptophan referred to as a second essential amino acid is widely used in medicine,food, and feed. With the development of the microbial production of L-tryptophan,microbial fermentation was used to production L-tryptophan and is becoming adominant method. Because of lacking of high active strains in microbial fermentationand the complex biological pathway of L-tryptophan, it is difficult for L-tryptophan toachieve industrial production. The application of modern biotechnology and geneticengineering techniques in breeding, it has become possible to apply more rationalapproaches to strain improvement and promoted the industrial production process ofL-tryptophan.In the present study, we attempted to introduce defined genetic manipulations intoE. coli to develop an L-Trp-producing strain based on known regulatory andmetabolic information. The feedback-resistant3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) is modified in order to achieve the maximum flow ofcarbon into the common pathway. Follow this ideas, the work we have done are:(1) The aroG gene of E. coli was cloned and mutated by site-directed mutagenesisusing splicing overlap extension PCR (SOE-PCR) technique. The feedback-resistantDAHP synthase encoded by aroG was achieved by replacing the residue Leu175ofaroG with Asp and changing Pro150to Leu. The feedback-resistant aroG gene wasnamed aroGM175and aroGM150, respectively. The DNA sequencing result of vectorpGM-T-aroGM showed that the residue Leu175and Pro150of aroG weresuccessfully replaced with Asp and Leu.(2) Mutated genes were successfully assembled on the vectors pET-28b(+)andexpressed in E.coli BL21(DE3).Sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western-blotting were used in the study to test the expression level of protein，the results showed the mutation of residue Leu175andPro150of aroG DNA sequences significantly achieved the over-expression of aroGgene.(3) E.coli BL21(DE3)/pACYCDuet-1-aroGM175was constructed. In order toachieve the co-expression of aroGM175and aroGM150gene in E.coli BL21(DE3),the low copy plasmid pACYCDuet-1that contained two multiple cloning siteswas used to construct the expression vector pACYCDuet-1-aroGM175. SDS-PAGEshowed that the purpose protein quantity of E.coli BL21(DE3)/pACYCDuet-1-aroGM175was significantly lower than E.coli BL21(DE3)/pET-28b-aroGM175after5hours joining the IPTG when the concentration of total protein is same.(4) In the study the effects of different temperature，pH value and loadingvolume on bacterial density，plasmid stability and glucose in fermentation mediumwere researched with The recombined bacterium E.coli BL21(DE3)/pET-28b–aroGM175and E.coli BL21(DE3)/pET-28b-aroGM150as main fermenting microbeunder the conditions of laboratory.