While the effects of oxygen supply on 5-ALA biosynthesis were investigated in several organisms (Nishikawa et al. 1999; Yu et al. 2015), little effort on this front was made for E. coli. With the implemented the Shemin pathway, 5-ALA biosynthesis in E. coli can critically depend on the availability of the key precursor of succinyl-CoA, whose formation is rather oxygen-sensitive. In E. coli, succinate (and, therefore, succinyl-CoA) can be derived via three oxygen-dependent pathways in the TCA cycle, i.e., (i) reductive TCA branch, (ii) oxidative TCA cycle, and (iii) glyoxylate shunt (Fig. 1) (Cheng et al. 2013). Under anaerobic conditions, succinate accumulates as an end-product of mixed acid fermentation via the reductive TCA branch (Thakker et al. 2012). Although the reductive TCA branch can potentially yield high-level succinate, this pathway is generally unfavorable due to the limited availability of reducing equivalents (Skorokhodova et al. 2015). Under aerobic conditions, succinate is normally used up as a metabolic intermediate of the oxidative TCA cycle without accumulation, except for the conditions of oxidative stress and/or acetate/fatty-acid consumption under which succinate can be aerobically derived via the operational glyoxylate shunt (Thakker et al. 2012). Using the control strain DMH, the effects of oxygenic conditions on 5-ALA biosynthesis were systematically investigated for cell cultivation under microaerobic (AL-I), semiaerobic (AL-II), and aerobic (AL-III) conditions. Note that glycerol utilization and cell growth were severely retarded when DMH was cultivated under a strict anaerobic condition (data not shown). Our results show that biosynthesis of 5-ALA and porphyrins was favored by microaerobic conditions, though the low oxygenic level could trigger high-level acetogenesis and the associated physiological impacts. The results suggest that, in DMH, most of the dissimilated carbon flux was channeled into the Shemin pathway for biosynthesis of 5-ALA and porphyrins via the reductive TCA branch; and the oxidative TCA cycle and glyoxylate shunt contributed minimally toward such carbon flux channeling. Compared to DMH, blocking the oxidative TCA cycle in DMH∆sdhAcould potentially channel more dissimilated carbon flux toward succinyl-CoA via the reductive TCA branch under AL-I and, therefore, improve biosynthesis of 5-ALA and porphyrins with much reduced acetogenesis. With more dissimilated carbon flux channeling into the Shemin pathway, porphyrin biosynthesis was further reduced by repressinghemB expression to enhance 5-ALA accumulation in DMH-L4∆sdhA under AL-I, achieving 5.95 g l-15-ALA and with 36.9% yield while minimizing porphyrin biosynthesis. Note that inactivating the oxidative TCA cycle and/or repressinghemB expression resulted in uncommon accumulation of formate with reduced acetogenesis, compared to the control strain DMH. Such observation suggests that, under these genetic backgrounds and microaerobic conditions, pyruvate formate lyase (PFL; via which formate is coproduced) could be more active than pyruvate dehydrogenase (PDH) for the conversion of pyruvate to acetyl-CoA. In E. coli, PDH and PFL are responsible for decarboxylation of pyruvate to form acetyl-CoA under aerobic and anaerobic conditions, respectively (Wang et al. 2010). It was also reported that acetate and formate could induce opposite proteome responses inE. coli as most proteins induced by one of these two acids are repressed by the other (Kirkpatrick et al. 2001). While the control strain DMH had a low-level biosynthesis of 5-ALA and porphyrins under aerobic conditions such as AL-III, implying a limited carbon flux contribution from the oxidative TCA cycle (and, therefore, limited succinyl-CoA precursor) into the Shemin pathway, repressinghemB expression could also increase 5-ALA accumulation significantly in DMH-L4. Inactivating the TCA oxidative cycle in DMH-L4∆sdhA significantly retarded cell growth with limited glycerol dissimilation, cell growth, and metabolite production under AL-III, suggesting the critical metabolic roles of the TCA oxidative cycle for biomass formation and biosynthesis under aerobic conditions as per previous observations (Guest 1981; Steinsiek et al. 2011). To resolve the apparent succinyl-CoA limitation under AL-III, we explored channeling of the dissimilated carbon flux via a deregulated glyoxylate shunt by mutating iclR in DMH-L4∆iclR and observed a significantly enhanced 5-ALA biosynthesis. Nevertheless, with an active TCA oxidative cycle in DMH-L4∆iclR, the carbon flux arising from the deregulated glyoxylate shunt could divert at the succinate node to either the oxidative or reductive TCA direction. Importantly, the flux diversion could be prevented with the dissimilated carbon being effectively directed into the Shemin pathway by further mutatingsdhA for inactivation of the oxidative TCA cycle in DMH-L4∆sdhA∆iclR, achieving 6.93 g l-15-ALA with 50.9% yield upon its cultivation under AL-III while minimizing porphyrin biosynthesis. Such carbon flux rerouting effects under AL-III could also be observed by the enhanced biosynthesis of 5-ALA and porphyrins (reflected by significant pigmentation of the culture medium in Fig. 6-IV) in DMH∆sdhA∆iclR compared to DMH. Additionally, note that the retarded glycerol utilization and cell growth for DMH-L4ΔsdhA could be complemented by the iclRmutation, suggesting that both the oxidative TCA cycle and glyoxylate shunt contribute to active TCA operation for sustained cell growth and metabolic biosynthesis under aerobic conditions. While this study successfully demonstrated high-level biosynthesis of 5-ALA from structurally unrelated carbons under both microaerobic and aerobic conditions, the overall culture performance was limited by significant acetogenesis, particularly during extended fedbatch cultivation (data not shown).