Modification Of L-tryptophan-producing E.Coli Recombinant Strains And Control Of Fermentation Conditions

L-tryptophan,as one of the 20 amino acids essential for humans and animals,plays a key role in the growth and development,life activities and metabolism of humans and animals.At present,with the continuous development and progress of synthetic biology,systems biology and metabolic engineering,L-tryptophan is widely used in medicine,food and feed industries.The production of L-tryptophan by microbial fermentation has attracted widespread.In this study,the E.coli strain FJN010 was used as the starting strain,and the high-yield L-tryptophan-producing strain was constructed using metabolic engineering techniques.(1)Using L-Trp production strain E.coli FJN010 as the original strain,the key enzymes genes tktA and pckA were used as research objects.Using high-efficiency expression of foreign plasmids and seamless cloning technology to express key enzyme genes,to examine the effect of strains containing different foreign genes on the production of L-tryptophan.The shake flask fermentation results showed that the strain FJN010/TE-3produced the highest level of L-tryptophan,reaching 3.71 g/L,which was 39.0%higher than the original strain FJN010,and the enzyme activities of PckA and TktA were increased by 3.7 times and 3.4 times respectively.(2)Based on the plasmid pBV-pckA-pL-tktA,the anthranilate synthase gene trp ED was continuously introduced to the original strain.Using error-prone PCR technology to direct evolution of anthranilate synthase,successfully constructed a strain FJN010/TE-5(pBV-pckA-pL-tktA-pL-trp ED^fbr)that can resist L-tryptophan feedback inhibition.The results showed that the cysteine residue(C465)in the amino acid sequence of anthranilate synthase was mutated to tyrosine(Y465).L-tryptophan production and enzyme activity of strain FJN010/TE-5 were greatly improved,and the final L-tryptophan production could reach 4.80 g/L compared to strain FJN010/TE-3 increased by 29.4%and the enzyme activity increased by 4 times.(3)Effects on E.coli metabolism and L-tryptophan synthesis by transaminase of L-alanine synthesis.Threee genes encoding L-alanine aminotransferase(alaA,alaC and avtA)were disrupted by Red recombination technique.The accumulation of L-tryptophan,L-alanine metabolism and cell growth were investigated by fermentation experiments.The shake flask results showed that the strain FJN010/TE-9 that simultaneously knocked out the alanine aminotransferase genes alaA and alaC had the highest L-tryptophan production capacity,up to 6.08 g/L,which was 26.7%higher than the strain FJN010/TE-5.And the content of alanine was only 0.16 g/L,a decrease of 91%.(4)On the basis of the original medium,the residue after the extraction of L-phenylalanine was used to replace the L-phenylalanine powder in the initial fermentation medium.The effects of L-phenylalanine residues on the growth and L-tryptophan production of strain FJN010/TE-9 were investigated in shake flasks and 50 L tank.The shake flask results showed that the addition of 0.5 g/L of L-phenylalanine residue in the fermentation broth can effectively increase the production of L-tryptophan up to 7.40 g/L,and the bacterial cell concentration also increased.While adding a certain amount of L-phenylalanine residue to the 50 L tank,the final L-tryptophan accumulation of strain FJN010/TE-9 was only 20.01 g/L.It may be that the residual liquid contains too many harmful substances,which inhibits the growth of the bacterial body and the level of L-tryptophan production.(5)On the basis of the original culture medium,fermentation conditions of strain FJN010/TE-9 and optimization experiment of shake flask fermentation medium.On the basis of the optimization of the culture medium formula,by feeding batch fermentation to verification on a 50 L tank,through the existing fermentation technology,the concentration of L-tryptophan in the fermentation broth of the modified strain can reach up to 49.3 g/L,it was 17.7%more than the original formula.