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.