Optimization Of Escherichia Coli Engineered Strain For L-phenylalanine Production

L-phenylalanine（L-phe）has been widely used and of great market value in food,medicine and other fields.Therefore,the research strategies to improve the industrial production of L-phe have also received wide attention and been deeply developed.As the mainstream method of industrial production of L-phe,microbial fermentation has the advantages of low cost and high efficiency.Traditional microbial fermentation mainly relies on mutagenesis screening orharboring plasmids to obtain producing strains with excellent traits.However,the above methods not only suffers from low breeding efficiency,but also difficult to obtain producing strains with stable genetic characteristics.Therefore,this study used CRISPR/Cas9 system to modify the metabolic pathway of a L-phenylalanine producing strain PHE13 at the genome level,and finally obtained a genetically stable L-phe engineered strain（PHE28）with higher yield.The specific work is as follows.First,the shikimate kinase encoding genes,aro K and aro L and the shikimate dehydrogenase encoding genes,ydi B and aro E were overexpressed with the strong promoter P_trc,respectively,so as to evaluate their effects on L-phe production.The shake flask results showed that the strain PHE16(harboring P_trc-aro K and P_trc-ydi B)produced23.9 g/L L-phe,which increased by 12.7%compared to the starting strain PHE13.It shows that further improving the flux of the shikimate pathway promotes the synthesis of L-phe.Subsequently,in order to improve the supply of precursor PEP,the Enzyme IIBC expression gene pts G of Phosphotransferase（PTS）system was knocked out,and the promoter of different strengths was overexpressed for galactoside permease and glucose uptake pathway in the non-PEP system.The shake flask results showed that the strain replacing the PTS system did not exceed the control strain PHE16 in either yield or conversion rate,indicating that modifying the PTS system alone failed to improve L-phenylalanine accumulation.To improve the supply of the precursor E4P,the phosphotransketase gene xfp K from bifidobacterium virgens was introduced in this study and was overexpressed with the strong promoter P_trc.The shake flask results showed that the titer and glucose conversion rate of strain PHE23 were 26.2 g/L and 24%,respectively increased by 4.8%and 1.1%.Meanwhile,however,its acetic acid accumulation reached 4.9g/L,which was 53.1%higher than PHE16.This results showed that although the introduction of xfp K gene promoted the synthesis of L-phenylalanine,the enzyme produced acetyl phosphate as well,which increases the accumulation of acetate.Considering the negative effects of high concentrations of acetate on strain growth and metabolism,the key genes ack A,tdc D,pox B for acetate synthesis were knocked out in strain PHE23 to explore their effects on acetate accumulation and L-phenylalanine production.Shake flask showed that deletion of ack A,tdc D and pox B had the least acetate accumulation and the highest yield,1.6 g/L and 28.1 g/L,respectively.On this basis,to further reduce acetate synthesis,the acetylphosphotransferase encoding gene pta was knocked out.The results showed that the acetic acid accumulation of strain PHE27 was reduced to 1.2 g/L and L-phenylalanine production and glucose conversion rate increased to28.5 g/L and 25.6%,respectively.This result indicates that block of acetate synthesis pathway has significant effects on reducing acetate accumulation while contributing to the synthesis of L-phenylalanine.In order to direct the carbon flow from PEP nodes to the shikimate pathway while reducing the metabolic overflow of the central metabolic pathway,a mutanted aro G（F146R）gene without feedback inhibition was introduced in PHE27.Shake flasks showed that the yield and conversion rate of the constructed strain PHE28 were 29.3 g/L and 26.9%,respectively,indicating that the introduction of this gene contributes to L-phenylalanine production.Finally,the L-phenylalanine production of strain PHE28 using 5 L bioreactor was 82g/L for 48 h,with an average productivity of 1.7 g/L/h and a conversion rate of 28%,which was 4%higher compared to the starting strain.It can be shown that this strain has a good potential for industrialization.In addition,the metabolic engineering modification strategy used in this study can be extended to the construction of strains for producingother aromatic amino acids and their derivatives.