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∆iclRsdhA 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∆iclRsdhA 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∆sdhAiclR 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∆iclRsdhA and DMH∆iclRsdhA 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∆iclRsdhA 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.