Metabolic Engineering Of Corynebacterium Glutamicum For 5-aminolevulinic Acid Production

In this study,central metabolism,heme biosynthesis pathway,cell wall biosynthesis pathway and glycine biosynthesis pathway were modified to study the metabolic capacity of Corynebacterium glutamicum for 5-aminolevulinic acid(5-ALA)production.HemA encoding 5-aminolevulinic acid synthase from Rhodobacter sphaeroides was firstly expressed in C.glutamicum,resulting in accumulation of 5-ALA;Secondly,5-ALA was enhanced through deletion of all known genes responsible for the formation of acetate and lactate;Thirdly,modification of anaplerotic reaction resulted in an accumulation of 5-ALA up to 2.06 g/L;The strain with the disruption of succinyl-Co A competition pathway did not show any increase in 5-ALA production.Biosynthesis of 5-ALA is the rate-limiting step for heme biosynthesis.Hem C,hemD,hemE and hemN were individually overexpressed in C.glutamicum.The results showed that 5-ALA production increased by 29.2% through up-regulation of hemD with a low-copy number plasmid pEP2.Additionally,5-ALA production increased by 14.0% and 5.3%,respectively,through down-regulation of hemH and hemY.Penicillin-binding proteins play an important role in biosynthesis of cell wall,which is barrier for 5-ALA secretion.In order to improve the export of 5-ALA,highmolecular-weight penicillin-binding proteins(HMW-PBPs)genes including pbp1 a,pbp1b,pbp2 a and pbp2 b were deleted.The results indicated that deletion of HMWPBPs genes pbp1 a,pbp1b and pbp2 b led to an increase in 5-ALA production of 13.5%,29.5% and 22.2%,respectively.To further improve 5-ALA secretion,the genes encoding threonine transporter including rhtA and rhtC from Escherichia coli and thrE from C.glutamicum were overexpressed using a low copy plasmid pEP2.Extracellular 5-ALA increased by 18.5% after expressing rhtA gene from E.coli.After optimizing the composition of medium and fermentation condition,the strain could produce 3.40 g/L 5-ALA.C.glutamicum R1 produced 10.43 g/L 5-ALA with penicillin supplement in a 5 L fermentor.The glycine biosynthesis pathway was further engineered to improve the availability of glycine.The strain overexpressing serine operon serA?197CB from C.glutamicum accumulated 69.2% more 5-ALA than the control strain without the addition of glycine;5-ALA production decreased drasticly with the deletion of sdaA gene;5-ALA increased by 1.48 times when glyA gene was overexpressed along with serA?197CB in C.glutamicum AG3.Metabolic engineering of glycine synthesis pathway could enhance 5-ALA production when the medium was supplemented with glycine,C.glutamicum AG3 produced 2.72 g/L 5-ALA,39.5% higher compared to reference strain in the present of 7.5 g/L glycine.