Metabolic engineering to enhance 5-ALA biosynthesis under
aerobic conditions
We also explore 5-ALA biosynthesis under aerobic conditions, which often
facilitate carbon utilization and cell growth. While the DMH-L4 culture
under AL-III showed effective glycerol dissimilation and cell growth,
biosynthesis of 5-ALA and porphyrins was much lower than that of the
DMH-L4 culture under AL-I (Fig. 6-I vs. Fig. 5-II), suggesting a
potential limitation in succinyl-CoA precursor under aerobic conditions.
Nevertheless, the significant enhancing effects ofhemB -repression on 5-ALA biosynthesis were still observable under
aerobic conditions by comparing the two cultures of DMH-L4 and DMH under
AL-III (Fig. 6-I vs. Fig. 4-III). To overcome the limitation in
succinyl-CoA under AL-III, we derived another mutant of
DMH-L4∆iclR with a deregulated glyoxylate shunt. Compared to the
parental strain DMH-L4, DMH-L4∆iclR had a much higher 5-ALA
biosynthesis with effective glycerol dissimilation and cell growth under
AL-III (Fig. 6-III vs. Fig 6-I), suggesting successful direction of the
dissimilated carbon flux toward succinyl-CoA for 5-ALA biosynthesis via
the glyoxylate shunt under aerobic conditions. For more effective carbon
flux direction, we derived another double mutant of
DMH-L4∆iclR ∆sdhA with a disruptive oxidative TCA cycle
such that the directed carbon flux at the succinate node via the
glyoxylate shunt could be further directed toward succinyl-CoA via the
reductive TCA branch. Compared to the parental strain
DMH-L4∆iclR , DMH-L4∆iclR ∆sdhA had even higher 5-ALA
biosynthesis under AL-III (Fig. 6-V vs. Fig. 6-III), achieving 6.93 g
l-1 5-ALA with 50.9% yield, though the single
mutation of ∆sdhA appeared to be rather harmful to cell
physiology and, therefore, culture performance (Fig. 6-II vs. Fig. 6-I).
Note that the 5-ALA yield for DMH-L4∆sdhA ∆iclR was
3.4-fold that for DMH-L4 and 1.3-fold that for DMH-L4∆iclR . Also,
note that the significant enhancing effects of hemB -repression on
5-ALA biosynthesis were further confirmed by comparing the two cultures
of DMH-L4∆iclR ∆sdhA and DMH∆iclR ∆sdhA under
AL-III (Fig. 6-V vs. Fig. 6-IV). On the other hand, the successful
carbon flux direction toward the Shemin pathway via the glyoxylate shunt
and reductive TCA branch for biosynthesis of both 5-ALA and porphyrin
pigments can be also observed by comparing the two cultures of
DMH∆iclR ∆sdhA and DMH under AL-III (Fig. 6-IV vs. Fig.
4-III). These results successfully demonstrated the consolidated
strategy based on carbon flux redirection in the TCA cycle toward the
Shemin pathway with repressed hemB expression to enhance 5-ALA
biosynthesis.