High-level Expression Of Bacillus Subtilis HemA Gene In Escherichia Coli And The Effect On The 5-ALA Biosynthesis Pathway

5-Aminolevulinate (5-ALA) is the first common precursor in the biosynthesis oftetrapyrrole compounds such as heme, porphyrins, chlorophyll and vitamin B₁₂, and also is akey metabolic intermediate to regulate the tetrapyrroles biosythesis. 5-ALA is widely existedin cells of microorganisms, plants and animals. Recently, it has found that 5-ALA can be usedas biodegradable, herbicide and insecticides as well as growth-promoting factor in agricultureand is a new generation of photodynamic therapy in medicine, which has potential applicationfor the treatment of various kinds of cancers, for example those developed in skin. oral,esophageal, colon, duodenal, pancreas and bladder, etc. Therefore, it is of great importance toenhance research and development of 5-aminolevulinic acid production process.5-ALA is produced via the Shemin pathway (C₄ pathway) and the C₅ pathway. TheShemin pathway has been found in animals, fungi (including yeasts), andα-proteobacteria,such as the photosynthetic genera Rhodobacter and Bradyrhizobium. In the Shemin pathway,5-ALA is synthesized by the condensation of glycine and succinyl coenzyme A(succinyl-CoA), which is catalyzed by 5-ALA synthase encoded by hemA. The C₅ pathwayhas been found in plants (including algae) and in all other bacteria examined to date. In thispathway, glutamate is coupled with a cognate tRNA in a reaction catalyzed by glutamylt-RNA synthase and then it is reduced to glutamate-1-semialdehyde (GSA) in a reactioncatalyzed by glutamyl-tRNA reductase. Finally, the transamination of GSA, catalyzed by GSAaminomutase, yields 5-ALA. The glutamyl-tRNA reductase is the rate-limitation enzyme inbiosynthesis of 5-ALA in the C₅ pathway. Porphobilinogen (PBG) is formed by thecondensation of two molecules of 5-ALA, in a reaction catalyzed by 5-aminolevulinic aciddehydratase (HemB), and the immediate precursor of the tetrapyrrole uroporphyrinogen(urogen)â…¢. Urogenâ…¢is the common precursor to all tetrapyrroles, and thus it represents themajor branching point in pathways to protoheme, vitamin B12, hemed1, and siroheme. InBacillus subtilis and Escherichia coli, 5-ALA is produced both via the C₅ pathway. In this work, The hemA gene was polymerase chain reaction amplified from B. subtilisgenome DNA and cloned into the expression vector pET28a. The hemA gene was High-levelexpressed in E. coli BL21 (DE3) when induced by IPTG, in which the recombinant proteinmakes up 20ï¼…of total soluble protein through SDS-PAGE analysis. We also obtained thepurified protein of HemA through the Ni²⁺-NAT affinity chromatography resin. Biosynthesislevel of 5-ALA was enhanced responding to expression of the recombinant protein. Theproduction of 5-ALA reached 36.6 mg/L. The catabolite metabolism of 5-ALA porphyrinswas also accelerated.The cultivation condition of strain E. coli BL21(DE3)(pET28a-hemA) was evaluated atshake-flask scale. The effects of dissolved oxygen, the kind of culture mediem were examined,and the cultivation as well as induction conditions were optimized. The hightest 5-ALAproductivity reached 52.1 mg/L.