Metabolic Engineering Of Corynebacterium Glutamicum For The Biosynthesis Of 5-aminolevulinic Acid

5-Aminolevulinic acid（5-ALA）is a kind of non-protein five-carbon amino acid.It is an important precursor for the synthesis of vitamin B₁₂,heme,chlorophyll and other tetrapyrrole compounds,and widely exists in microbial and animal and plant cells.Because of its special physical and chemical properties,it is widely used in medicine,agriculture and other fields.The disadvantages of traditional chemical synthesis methods,such as high cost,low yield and high pollution,restrict the wide application of 5-ALA in various fields.Subsequently,biosynthesis method which is more green and efficient has attracted wide attention of researchers.In this study,the mechanism of Ncgl0580 on 5-ALA synthesis was explored through metabolic engineering strategy,transcriptional level analysis and other methods in Corynebacterium glutamicum.And5-ALA synthase with high 5-ALA yield was screened by rational mutation design,and finally an engineering strain with high 5-ALA yield was obtained.The overexpression and knockout of Ncgl0580 gene increased the yield of 5-ALA by 0.53 folds and 2.49 folds,respectively.After knocking gene Ncgl0580,the transcription levels of several genes in 5-ALA synthesis pathway,downstream heme pathway,glucose transport pathway and TCA cycle were significantly changed.For example,the transcription level of hem A was increased about 29.96 folds.And the transcription level of suc AB were increased about 38.41 and 41.12 folds,which all indicated that Ncgl0580 might be a potential transcriptional regulatory factor in C.glutamicum.As a key enzyme in C4 pathway,5-aminolevulinic acid synthase（ALAS）plays an important role in 5-ALA synthesis.The activity of 5-aminolevulinic acid synthase was affected by sulfhydryl amino acids.Therefore,the 5-aminolevulinic acid synthase mutant with high yield of 5-ALA was obtained by mutating the cysteine into alanine or serine in the amino acid sequence of ALAS from different sources.After overexpressing Hem A_RC^C201A from Rhodobacter capsulatus in CGT0,the titer of 5-ALA increased 21.9%than control.In addition,the overexpression of mutant Hem A_RP^C132A from Rhodopseudomonas palustris improved the titer of 5-ALA by 14.2%than control.After overexpressing Hem A_RP^C132A in strain CGT1 which was knocked Ncgl0580,the titer of 5-ALA increased by 59.1%than strain CGTP,reached to 3.34g/L.This mutation strategy is beneficial for screening ALAS mutants with high 5-ALA yield from other sources of 5-aminolevulinic acid synthase,which provides a basis for realizing high 5-ALA yield.