Metabolic Engineering Of Escherichia Coli To Accumulate L-tyrosine And Optimization Of Fermentation Conditions

L-Tyrosine?Tyr?is one of the three major aromatic amino acids,which has been widely used in food,feed,pharmaceutical,and chemical industries.Due to the complexity of the L-tyrosine metabolic pathway and its regulation mechanism,the synthesis efficiency is restricted.This study was based on a L-phenylalanine producing strain constructed in the laboratory,which was able to metabolic engineered to produce L-tyrosine.In addition,the culture conditions and fermentation process of engineering strain were optimized to further improve the ability of strain to synthesize L-tyrosine.The main contents and results of this thesis are summarized as follows:?1?Construction of L-tyrosine recombinant strain and modification of the tyrosine transport system.Escherichia coli HGX strain was obtained by continuous subculture to eliminate the pAP-B03 plasmid of E.coli WSH-Z06?pAP-B03?strain.The heat-inducible plasmid pAP-aroG^fbr-tyrA^fbr was constructed and transformed into E.coli HGX strain to obtain the heat-inducible strain E.coli HGXP.The L-tyrosine yield of E.coli HGXP strain was 3.12 g?L^-1,which was 110.8%higher than that of the E.coli HGX strain.The genes encoding L-tyrosine transport system in E.coli HGXP strain were knocked out to obtained E.coli HGPP?HGXP?aroP?and HGEP?HGXP?aroP?strains.The results of shake flask fermentation indicated that the yields of L-tyrosine reached 3.74 g?L^-1 and 3.45 g?L^-1 by E.coli HGPP and HGEP strains,respectively.The optimum induction temperature was determined to be 38°C.The fed-batch fermentation was carried out on a 3 L fermentor,and the L-tyrosine production of E.coli HGPP and HGEP strains were further increased to 44.5g?L^-1 and 35.1 g?L^-1,respectively.?2?Modification of L-tyrosine competition pathway,repressor regulation and precursor supply.The pheA gene which was related to the L-phenylalanine competition metabolic pathway,trpD gene which was related to the L-tryptophan competition metabolic pathway,tyrR gene which was related to the global regulatory protein encoding,and pykF gene encoding the pyruvate kinase were knocked out on the basis of E.coli HGXP,respectively.The results of shake flask fermentation indicated that pheA single-gene knockout strain E.coli HSP?HGXP?pheA?had the highest L-tyrosine yield of 4.42 g?L^-1 among the four engineering strains.The tyrR gene of the E.coli HSP strain was further knocked out to obtain the pheA/tyrR double-gene knockout strain E.coli HRP?HGXP?pheA/tyrR?.The L-tyrosine production was further increased to 5.84 g?L^-1.The trpD and pykF genes of the E.coli HRP strain were further knocked out to construct multi-gene knockout strains E.coli HRDP?HGXP?pheA/tyrR/trpD?and HKP?HGXP?pheA/tyrR/pykF?,but the growth of HRDP and HKP strains were affected,and the L-tyrosine production was not improved.?3?Optimization of fermentation conditions of L-tyrosine producing strain E.coli HRP on fermentor.The induction time,induction temperature and feeding strategies of E.coli HRP were optimized on a 3 L fermentor.It was found that the optimum induction time was determined to be mid-log phase?OD600 was about 25.60?,and the L-tyrosine yield was 40.23g?L^-1.Induction at 38°C can further increase the L-tyrosine yield to 48.69 g?L^-1.Several feeding strategies were investigated.It was found that L-tyrosine production was further increased to 55.54 g?L^-1 when linearly decreasing glucose feeding strategy was used,the conversion rate was 0.252 mol?mol^-1,and the production intensity was 1.385 g?L^-1?h^-1.The yield of L-tyrosine was 52.33 g?L^-1 when E.coli HRP was fermented on a 15 L fermentor,which was similar to the yield of a 3 L fermentor.