Construction Of Tryptophan Overproducing E.coli Strain

(?)yptophan is an important nutrient essential amino acid, which can only syn (?)sized by microorganisms or plants. L-Tryptophan is one of eight kinds of essential amino acids, humans and animals only intake from food. In order to get a L-tryptophan overproducing strains that can be used in large-scale production, we modify a Escherichia coli strain MD-T109 by means of mutation breeding and gene cloning.Escherichia coli MD-T109 as the starting strain was incubated by nitrosoguanidine (NTG) treatment, and seven tyrosine auxotrophic mutant strains were screened. One of which named E.coli MD-T109-06 showed increasing L-tryptophan production by 11.23% compared with the original strain. E.coli MD-T109-06 exhibited similar growth rate as the starting strain and showed good genetic stability.To further improve the MD-T109-06 mutant, we amplified its TrpEDCBA gene, and cloned into an expression vector pMD-T109 containing a tac promoter, transformed into E.coli MD-T109-06 and got an engineering strain of E.coli MD-trp-06. After flask shake fermentation test, the L-tryptophan production of MD-trp-06 was increased from 0.206 g /dL to 0.238g/dL, increased 15.5%. The anthranilate synthetase activity of the engineering strain increased by 140%, and tryptophan synthetase activity increased 166%.To address dissolved oxygen limitation that is usually present in high-density fermentation, the Vitreoscilla hemoglobin gene (vgb) was cloned into engineering strain MD-trp-06. According to published Genbank vgb gene and its inducible promoter sequence information, and modified according to the E.coli codon usage bias. Synthetic modification vgb fragment was cloned into the expression vector pMD-trp, transformed into E.coli MD-trp-06, and get engineered strain E.coli MD-trp. The vgb gene was hypoxia induced expression in shaking flask fermentation using 250 ml flask with 60ml and 100ml liquid volume conditions. As a result, bacteria concentration of E.coli MD-trp increased by 21% and 16%, compared with E.coli MD-trp-06, while tryptophan production increased by 16.4% and 21%, respectively. VHb expression was detected by SDS-PAGE.