Metabolic engineering to enhance 5-ALA biosynthesis under microaerobic conditions
To alleviate growth limitation and acetogenesis in DMH under AL-I, we derived a single-knockout mutant DMH∆sdhA , in which the oxidative TCA cycle was inactivated (Fig. 5-I). In addition to reduced acetogenesis, DMH∆sdhA produced 0.94 g l-15-ALA with 6.61% yield. With an increased carbon flux toward porphyrin biosynthesis, pigmentation of the DMH∆sdhA culture medium was significantly enhanced. On the other hand, while glycerol consumption and cell growth were significantly retarded upon cultivation of thehemB -repressed strain DMH-L4 under AL-I, 5-ALA biosynthesis was drastically improved with much reduced acetogenesis, achieving 4.73 g l-1 5-ALA with 32.0% yield (Fig. 5-II). The improved 5-ALA biosynthesis was also evidenced by significant reduction in pigmentation of the culture medium compared to DMH. Notably, compared to DMH∆sdhA or DMH-L4, 5-ALA biosynthesis was further improved upon cultivation of DMH-L4∆sdhA , in which the sdhA mutation andhemB -repression was simultaneously introduced, under AL-I, achieving 5.95 g l-1 5-ALA with 36.9% yield (Fig. 5-III). These results suggest that the dissimilated carbon flux was directed toward the succinyl-CoA node for 5-ALA biosynthesis primarily via the reductive TCA branch under microaerobic conditions, and such carbon flux direction was rather effective upon simultaneous disruption of the oxidative TCA cycle and hemB repression. Finally, compared to DMH, higher levels of succinate, formate, and ethanol were observed upon cultivation of all engineered strains, i.e. DMH∆sdhA , DMH-L4, and DMH-L4∆sdhA , under microaerobic conditions for enhanced 5-ALA biosynthesis.