Metabolic Engineering Of Escherichia Coli For Increased Synthesis Of L-histidine

L-histidine is one of the 20 amino acids that constitute human protein,which is an essential amino acid for infants and young children.It has many physiological functions,such as antioxidant,immune regulation and anti-inflammatory.It is widely used in food and medicine industries.The traditional methods of protein hydrolysis and chemical synthesis have some problems,such as raw materials are not easy to obtain,equipment loss rate is high and racemic mixture is produced.Therefore,microbial fermentation has become the mainstream method of L-histidine production.However,most of the existing researches screen strains through irrational design methods such as mutation,which has great randomness and blindness.Therefore,a rational design method for the production of L-histidine by Escherichia coli was established in this research.Firstly,in order to remove the L-histidine regulation of the His LGDBHAFI operon,an artificial open reading frame（hisl’-latt B’-trpe）was cloned and integrated into the genome of E.coli MG1655 and E.coli BL21（DE3）by CRISPR/Cas9 genome editing technology.In the same time,the precursor region of L-histidine operon in E.coli was replaced and recombinant bacteria M1 and B1 were obtained.Then,the recombinant strains M4 and B4 were obtained by expressing his G^E271K in M1 and B1,which is a mutant gene encoding ATP ribosyltransferase from E.coli.The L-histidine titer of strain B4 was highest.It was screened by shake flask fermentation,and the titer of L-histidine was increased to 0.87 g/L.The E.coli BL21（DE3）was identified as the starting strain of this study,the strain B1 was selected as the main research objects.Secondly,the enzyme activities were compared under different concentrations of inhibitors,and L-histidine titer was analyzed when 11 kinds of his G from Corynebacterium glutamicum ATCC 130132,Serratia marcescens ZJZ626 and E.coli BL21（DE3）were expressed in B1.Then,the optimal his G was selected and L-histidine titer was increased to 1.48g/L.Then,CRISPR/cas9 technology was used to integrate the optimal his G into the genome.Finally,after comparing the effects of two different precursor synthesis gene Prs from E.coli BL21（DE3）and S.marcescens ZJZ626 on L-histidine production,the better one was selected and integrated in the genome,resulting in the L-histidine titer of 3.90 g/L in 72 h and5.57 g/L in 96 h.