As a non-proteinogenic amino acid and precursor for synthesis of various tetrapyrrole compounds, 5-aminolevulinic acid has been widely used in medicine, agriculture and husbandry. In this study, Corynebacterium glutamicum ATCC13032 was used as a chassis for efficient 5-aminolevulinic acid biosynthesis. The MscCG protein is the exporter of L-glutamate. To prevent L-glutamate secretion, NCgl1221 was deleted, resulting in a production of 0.62 g/L 5-aminolevulinic acid. The reactions in microorganisms are mediated by simple diffusion and random collisions of metabolites and enzymes, which lowers the local metabolite concentration around the enzymes and results in the inefficiency of metabolic reactions. Furthermore, the cell growth will be inhibited by the accumulation of toxic intermediates. The DNA scaffold system can organize enzymes spatially and temporally and increases the local concentration of intermediates. So, this system was used to assemble glutamyl-tRNA reductase and glutamate-1-semialdehyde aminotransferase. The ratio of the two enzymes was set as 1∶1, 1∶2 and 2∶1, respectively. The highest production of 5-aminolevulinic acid (0.84 g/L) was achieved when the ratio of the two enzymes is 2∶1. To enhance TCA cycle for α-ketoglutarate supply, the anaplerotic pathwaywas strengthened by overexpressing pyc, ppc and pckA through individually integrating the genes together with the strong promoter Ptuf to genome DNA. The production of 5-aminolevulinic acid by the resulted strains was increased by 14.2%, 27.4% and 33.3%, indicating the feasibility of enhancing anaplerotic pathway. Meanwhile, gltA was co-overexpressed with pckA, resulting in 12.5% increase in 5-aminolevulinic acid production (1.26 g/L). As a bypass of TCA cycle, the glyoxylate cycle competes isocitrate with α-ketoglutarate synthesis. Meanwhile, the biosynthesis of 5-aminolevulinic acid requires NADPH. Therefore, a copy transhydrogenase encoding gene pntAB driven Ptuf was integrated at aceA locus, aiming at increasing blocking the glyoxylate cycle to improve α-ketoglutarate synthesis and promoting NADHP supply. The production of 5-aminolevulinic acid by the final strain ALA-10 reached 1.47 g/L. This study could provide reference for improving synthesis of 5-aminolevulinic acid.
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