| 5-aminolevulinic acid(5-ALA)plays important roles in medicine and agriculture,and synthesis of 5-ALA by microbes shows application prospect.In this study,heme biosynthesis pathway,glycine biosynthesis pathway and transporter proteins of5-ALA of C.glutamicum were researched based on C4 pathway.Co-overexpression of codon-optimized hemA of Salmonella typhimurium and hemL of Escherichia coli were studied for their effects on 5-ALA production.In this study,we researched the effects of regulations of heme pathway on 5-ALA production.Firstly,we down-regulated hemH and hemY genes of heme pathway with their initial codon ATG changed by GTG,the results showed 5-ALA production increased by 14.0% and 5.3%,respectively.Finally,overexpression of genes of heme pathway with IPTG induced,5-ALA accumulation decreased when IPTG was added,5-ALA production was higher than the control by 295% and 12.9% respectively when hemC and hemN genes were leakily expressed..According to amino acid sequence homology,potential genes Ncgl2065 and Ncgl0580 encoding 5-ALA transporter were found.And 5-ALA production of C.glutamicum R1 and R2,in which Ncgl2065 and Ncgl0580 was overexpressed respectively,increased by 129.5% and 100.4% than the control,and the production were 1.90±0.1 g/L and 1.84±0.2 g/L in CGXIIM,respectively。The effects of the serine and glycine biosynthesis pathway on 5-ALA production were studied.We investigated the 5-ALA production by C.glutamicum AG3 with different concentrations of glycine,and the result showed production of 5-ALA increased with the glycine addition.AG3 produced 2.72 g/L 5-ALA,39.5% higher compared to control strain in the present of 7.5 g/L glycine in CGXIIM.According to the effect of different expression of glyA and addition of serine on 5-ALA production,the glyA reaction rather than synthesis of serine maybe the limiting step in glycine synthesizing pathway for 5-ALA production.However,the enzyme activity of SHMT should be controlled in an appropriate rang,not the more the better.In C5 pathway,hemA of S.typhimurium was synthesized and codon-optimized,and the gene was co-overexpressed with codon-optimized hemL of S.typhimurium and hemL of E.coli,and the strain with hemL of E.coli overexpressed,produced 3.3times that of S.Typhimurium. |
