Construction Of Recombinant Corynebacterium Glutamicum For 5-aminolevulinic Acid Production And Optimization Of The Fermentation Process

5-aminolevulinic acid(ALA)has been widely used in agricultural and pharmaceutical fields due to its unique physical and chemical properties,and the production of ALA by microbial fermentation has drawn more and more attention.In this study,Corynebacterium glutamicum ATCC13032 was used as the starting strain to carry out metabolic engineering strategies on the C5 pathway of ALA biosynthesis.An engineering strain without IPTG induction was constructed and its fermentation process was optimized.The main research contents and experimental results are as follows:In order to enhance the pathway of glutamate to convert to ALA,the expression patterns of hemA(encoding glutamyl-tRNA reductase)and hemL(encoding glutamate-1-semialdehyde aminotransferase)were investigated.The results indicate that the heterologous key enzyme genes hemAS.arizona.M and hemLE.coll were overexperssed in the form of constitutive expression was better.Thus the production of ALA was 163.87 mg/L,which realized the initial accumulation of ALA.Then the pyc(encoding pyruvate carboxylase)and ppc(encoding phosphoenolpyruvate carboxylase)were overexpressed by means of the Ptuf strong promoter substitution,respectively.The results showed that the ppc enhanced strains was better and the production of ALA reached 416.75 mg/L.The gene gltA(encoding citrate synthase)and gene icd(encoding isocitrate dehydrogenase)were overexpressed by means of strong promoter replacement,which increased the metabolic flow of glutamate synthesis and provided sufficient precursor for ALA synthesis.The production of ALA increased to 795.75 mg/L.In order to reduce the loss of metabolic flow during ALA synthesis,gene knockout and gene attenuate of kgd(encoding ?-ketoglutarate dehydrogenase)were attempted.The results showed that the production of ALA reached 1090.35 mg/L after the original promoter of kgd gene was replaced by PdapA promoter.For purpose of enhance the extracellular transport ability of ALA,plasmids regulated by promoters of different strengths were used to overexpress the gene rhtA(encoding L-threonine/L-homoserine transporter).The results showed that the engineered strain ALA-11 under the regulation of Pacn promoter had the better effect on the production of ALA,which was reached 1596.82 mg/L.Next,the fermentation process of the high yield strain was optimized.By adding 0.5 g/L yeast powder and 1 g/L peptone in the medium,reducing the concentration of Fe2+to 0.01 mg/L,and controlling the shaking speed to maintain at 150 rpm,the production of ALA finally increased to 2384.69 mg/L.This study would supply the reference for the biosynthesis of ALA by C.glutamicum.