Metabolic Engineering Of Escherichia Coli For L-Homoserine Production

L-homoserine is a non-essential amino acid and an important precursor for the synthesis of threonine,methionine and isoleucine.L-homoserine is an important functional amino acid although it is not involved in protein synthesis.As a pharmaceutical intermediate,L-homoserine and its derivatives have a wide range of applications in the food,pharmaceutical and agricultural sectors.To address the problems of high cost of chemical synthesis and low production efficiency of biosynthesis in the current research on L-homoserine synthesis,this study was conducted to construct L-homoserine production strains,and the specific research contents and results are as follows.（1）Attenuation of L-homoserine degradation pathway to achieve initial accumulation of L-homoserine.A gene encoding the key enzyme of the homoserine synthesis pathway,thr A^C1034T,was overexpressed in E.coli,and the promoter of the thr ABC manipulator itself was replaced by the weak promoters P₁,P₂and P₃to weaken the degradation pathway of L-homoserine.（2）To enhance the L-homoserine synthesis pathway,the gene encoding the key enzyme of the L-homoserine synthesis pathway,thr A^C1034T,was overexpressed,and the fermentation results showed that double-copy H-5 accumulated 2.6 g/L of homoserine,a73.3%increase in yield,while triple-copy strain H-6 only produced 30.7%more.（3）To increase the supply of oxaloacetate,a precursor of homoserine synthesis,ppc encoding phosphoenolpyruvate carboxylase was overexpressed,and fermentation results showed that strain H-7 accumulated 5.8 g/L of homoserine.（4）To balance the supply of coenzymes in the L-homoserine synthesis pathway,an exogenous glutamate dehydrogenase-encoding gene roc G from Bacillus subtilis dependent on NADH was introduced to regenerate NAD⁺and glutamate using a cofactor self-sustaining system to provide sufficient glutamate and NADPH supply for the aspartate synthesis pathway,and the L-homoserine yield of strain H-8 shake flask fermentation reached The fermentation L-homoserine yield reached 7.1 g/L.（5）To continue to increase the supply of cofactor NADPH,the strong promoter P_trcand its own P_{pnt AB}promoter were used to increase a copy of pnt AB to promote the conversion of NADH to NADPH and enhance the intracellular reducing power supply of the strain,respectively.The yield of strain H-9 overexpressing pnt AB with its own promoter reached 8.8 g/L,but the strain biomass was slightly reduced.（6）To promote intracellular homoserine efflux,the gene rht A,encoding a transporter protein with homoserine efflux function,was overexpressed with the high-copy inducible plasmid p Trc99a(P_trc),the high-copy constitutive plasmid p UC19(P_lac)and the low-copy constitutive plasmid p STV28(P_lac),respectively.strain H-9 carrying the p STV28-rht A plasmid was overexpressed with the high-copy inducible plasmid p UC19(P_lac)and the low-copy constitutive plasmid p STV28(P_lac).13 shake flask fermentation reached a high serine accumulation of 12.5 g/L and the biomass returned to normal levels.（7）To investigate the fermentation performance of the strain,the recombinant strain H-13 was subjected to batch replenishment fermentation experiments using a 5 L fermenter.After the strain was incubated for 48 h in the fermenter,the L-homoserine yield was 52.1g/L and the sugar-acid conversion rate was 0.43 g L-homoserine/g glucose.In this study,a stable and high yielding,non-inducible,non-defective E.coli L-homoserine producing strain was constructed,and the results of the study can provide reference for the research of biological synthesis of L-homoserine.