Genetic Modification Of Escherichia Coli And Optimization Of Fermentation Conditions For L-tiyptophan Production

Tryptophan is one of the eight essential amino acids for human and animal life activities,and it is widely used in the pharmaceutical, food and animal feed industries. At present, theworld market demands for tryptophan are more than ten thousand tons per year, soL-tryptophan has a huge market. Microbial fermentation is a dominant method for theproduction of L-tryptophan. E. coli is extensively used in industry as a host for producingL-tryptophan due to its clear genetic background, the easeness of genetic manipulation and itsfast growth rate.Two geneticly engineered strains of L-tryptophan biosynthesis were constructed from E.coli JLTRP to investigate the effects of pyruvate dehydrogenase gene (poxB), Phosphatetransacetylase gene (pta) knockout and pyruvate dehydrogenase gene, phosphate transacetylasegene, lactate dehydrogenase (ldh) gene knockout of E.coli on the fermentation of L-tryptophan.The main research contents and results are as follows:（1）The concentration of L-tryptophan was measured using the method of anti-dimethylbenzaidehyde. The linear equation of L-tryptophan standard curve was y=0.3893+0.0061, andthe correlation index R2was0.9972. The quantitative analysis of L-tryptophan was carriedout using high-performance liquid chromatography (HPLC). The linear equation of L-tryptophan standard curve was y=34.102x+0.214and the correlation index was R2=0.9998.（2）Geneticly engineered bacteria E.coli BZ007was constructed by knocking-out pyruvatedehydrogenase gene, phosphate transacetylase gene of Escherichia coli JLTRP using P1phage.Geneticly engineered bacteria E.coli BZ010was constructed by knocking-out pyruvatedehydrogenase gene, phosphate transacetylase gene, Lactate dehydrogenase of Escherichiacoli JLTRP using P1phage. Then，the strains which could produce more tryptophan wereselected by shaking flask fermentation. （3）E.coli BZ007and E.coli BZ010cultures were performed in a5L fermenter. The initialconcentration of glucose was40g/L. The concentration of glucose in the process of cultivationwas close to zero. The seed was cultured untill the OD600of seed medium reached12-14.（4）The2L culture grown in the seed fermenter was inoculated aseptically into14Lproduction medium in a30L fermenter. Under the best culture conditions, The maxL-tryptophan production of E.coli BZ007and E.coli BZ010were up to33.5g/L and18.5g/L，respectively. The results proved that the knockout of poxB, pta genes were beneficial to theaccumulation of tryptophan, and the knockout of poxB, pta, ldh genes affected the normalgrowth of the bacteria, which may be related to other factors, and should be studied further.