Breeding L-arginine Producing Strain By Metabolic Engineering Modification Of Escherichia Coli

With the deepening of the research on the biochemical effect and physiological function of L-arginine（L-Arg）,the application of L-Arg has been widely expanded.The market for L-Arg is also expanding.It is significant to screen the strain with high titer of L-Arg.Strains of Corynebacterium glutamicum（C.glutamicum）are still the dominant strains producing L-Arg.However,Escherichia coli（E.coli）is a very promising strain of L-Arg.As an industrial strain of microbial fermentation,E.coli has a series of advantages.Therefore,it is necessary to effectively explore the production of L-Arg by E.coli.A mutagenic strain（strain A1）of E.coli K12 MG1655 was preserved in the laboratory with a small amount of L-Arg accumulation capacity.In this thesis,metabolic engineering methods were used to modify the strain to improve the ability of synthesis and accumulation of L-Arg,and further optimize the medium and fermentation conditions to further improve the production of L-Arg.The main results are as follows:（1）Whole genome resequencing was performed for the starting strain A1.A large number of gene mutations such as spe C,spe F,acn A and dcu A were obtained by resequencing analysis.The most important of these are mutations in genes spe C and spe F,which lead to gene inactivation and impair L-Arg degradation pathways,enabling L-Arg to accumulate during metabolism.In addition,the mutations of acn A and dcu A may have important effects on the metabolism of tricarboxylic acid cycle（TCA cycle）.Gene opp A may affect the transport of intracellular substances.Gene fli I may play an important role in the synthesis of intracellular ATP and glt P may play an important role in the efflux of L-glutamate and L-aspartate in cells.The metabolic flow of strain A1 was detected and analyzed.After the mutation of various genes,the carbon flow of strain A1 to each hetero acid pathway was reduced to varying degrees,while the flow of pyruvate to TCA cycle and TCA cycle to L-Arg synthesis pathway were improved.（2）Remove the negative feedback effect of L-Arg synthesis pathway in E.coli,and enhance the metabolic flow of L-Arg synthesis pathway.The arg R gene of strain A1 was knocked out to eliminate the feedback inhibition of the repressing protein Arg R on various Arg synthases during L-Arg synthesis.The allogeneic introduction of the arg J gene of C.glutamicum removed the key rate-limiting effect of L-Arg on the first step in the synthesis pathway.The carbon flow of L-Arg synthesis pathway in E.coli was enhanced by introducing the arg CJBDF gene cluster of C.glutamicum.In the shaking flask fermentation test,the L-Arg titer of strain A7 reached 16.9 g·L^-1,and there was no accumulation of other byproducts except1.8 g·L^-1 acetate.（3）The pox B gene promoter in E.coli was replaced by weak promoter and acs gene was overexpressed,which reduced the accumulation of acetate during fermentation.The expression of acetate synthesizing gene pox B was weakened and the production of acetate was reduced.Acetyl Co A synthetase encoding gene acs was overexpressed to convert acetate produced by fermentation into acetyl Co A,making the produced acetate return to the TCA cycle.The accumulation of acetate in fermentation process was successfully reduced through metabolic modification,which not only avoided the adverse effect of excessive accumulation of acetate on cell growth,but also avoided the loss of carbon flow,and effectively improved the synthesis efficiency of L-Arg.At the same time,by overexpressing the L-Arg transporter gene lys E of C.glutamicum in E.coli,the L-Arg transporter ability of the strain was effectively improved.In the shaking flask fermentation test,the L-Arg titer of strain A14 reached 22.4 g·L^-1,and the acetate accumulation decreased to 0.25 g·L^-1.（4）Improving the supply of L-Arg synthesis precursors and optimizing the supply of NADPH and ATP.Overexpression of the gene gdh A in E.coli redistributes the flow distribution ofα-ketoglutaric acid nodes,promoting the conversion of moreα-ketoglutaric acid to L-glutamate,which then flows into the L-Arg synthesis pathway.Overexpressed genes asp A and car A/B effectively enhanced the supply of L-aspartate and carbamoyl phosphate,which made the supply of nitrogen source more adequate during L-Arg synthesis,and further promoted the synthesis of L-Arg.Overexpression of pnt A/B also optimized the intracellular supply of NADPH.Knockout of amn gene in E.coli significantly increased intracellular ATP content.The L-Arg production of strain A21 was further increased to 27.3 g·L^-1,and the acetate accumulation also reduced to 0.15 g·L^-1.（5）By using single component experiments and response surface design,the fermentation medium composition and fermentation conditions of L-Arg producing strain A21 were optimized.The optimum fermentation medium composition(g·L^-1)was determined:glucose13,peptone 5,Yeast immersion powder 4,K₂HPO₄·3H₂O 5,Mg SO₄·7H₂O 2,Fe SO₄·7H₂O 0.04,Mn SO₄·4H₂O 0.03,defoaming agent 0.05 m L·L^-1.The optimal fermentation culture conditions were（5 L bioreactor）:10%inoculation amount;p H 7.0,Fermentation temperature 35℃,The dissolved oxygen is controlled at about 30%,Residual glucose should be controlled below 2g·L^-1.After the optimization,the titer of L-Arg fermentation reached 89.7 g·L^-1,the conversion rate of glucose to L-Arg reached 0.377 g·g^-1,and the yield was 1.495 g·L^-1·h^-1.