Metabolic Engineering Of Escherichia Coli For The High-production Of L-phenylalanine And Optimization Of Its Fermentation Process

L-phenylalanine(L-Phe)is one of the eight essential amino acids for humans,which is widely used in the production of food flavors,pharmaceuticals,chemical industry and other fields.The glucose used in the production of L-Phe by microbial fermentation can be prepared by corn or stale grain.Therefore,research and development of strain and processes for producing L-Phe by microbial fermentation,and inducteialized production,not only meets the market demand for L-Phe,increases the added value of food,and promotes the grain processing industry,but also has important economic and social significance.In this study,the L-Phe producing strain Escherichia coli SQH021 was used as the starting strain.Directed evolution and Metabolic Engineering techniques were used to regulate recombinant strain,which was based on the systematic analysis of the L-Phe metabolic synthesis pathway in Escherichia coli.The aro F and phe A genes for the supplement of PEP and E4 P,the ppa A?tkt A?tal A and aro A genes of shikimic acid metabolic pathway,the trp E gene of tryptophan metabolic pathway,the csr A gene of central metabolic regulator and the pta gene of acetic acid metabolic pathway were modified to improve the production of L-Phe.The yield of L-Phe by strain FS01/p APTA reached 36.85 g/L at the level of 5 L fermentor,which was 269% higher than that by flask fermentation.On this basis,the fermentation process was optimized,including seeding amount,p H value,dissolved oxygen,glucose basic concentration,glucose feeding strategy and temperature control strategy,and the yield of L-Phe reached 53.56 g/L in the5 L fermentor,which increased 45.3% and the conversion rate was 27%.In order to further study the industrial application value of this strain,we verified the recomb inant FS01/p APTA seed medium and the three stage fermentation process,and then the pilot-scale fermentation of recombinant strain in 1 T fermentor according to the principle of dissolved oxygen consistency.The yield of L-Phe was 42.76 g/L,reaching 79.8% of the highest yield of 5 L fermentor,the conversion was 24%,and the yield rate of L-Phe is0.76g/L/h.Based on the phenomenon of insufficient oxygen supply during pilot-scale fermentation,we improved the problem of insufficient oxygen supply during high-density fermentation by introducing Vitreoscilla hemoglobin gene and expressing hemoglobin.The yield of L-Phe was increased to 58.68 g/L in the 5 L fermentor by recombinant strain FS01/p APTA-vgb,and the highest yield of L-Phe was 50.56 g/L in the 1 T fermentor.The conversion rate of glucose was 26.5%,and the yield rate of L-Phe was 0.9g/L/h,which laid the foundation for the industrial application of recombinant strain FS01/p APTA-vgb.