To date, metabolic engineering of E. coli for 1-propanol biosynthesis has been conducted through two major pathways, i.e. (1) the keto-acid biosynthetic pathway [6–8] and (2) the extended 1,2-propanediol pathway . Unlike these approaches, our strategy focused on activation of the endogenous but often silent Sbm operon for extended conversion of succinate into 1-propanol. The 1-propanol-producing capacity was implemented by transforming a wild-type E. coli strain, BW25141, with three plasmids respectively harboring the Sbm operon genes (with the exception of ygfG), sucCD, and adhE2 for expression of these key genes. Using the metabolically engineered strains for anaerobic fermentation, we obtained 1-propanol titers up to 150 mg/L which is comparable to those of other studies [5, 9]. In addition, we identified several potential factors limiting 1-propanol production, in particular the abundance of precursors and the conversion step catalyzed by a bi-functional alcohol/aldehyde dehydrogenase. While it is possible to perform this biotransformation aerobically, anaerobic cultivation was chosen for two reasons. Firstly, the two TCA intermediates of succinate and succinyl-CoA are the precursors for 1-propanol biosynthesis and their abundance can potentially limit 1-propanol production. Under anaerobic, but not aerobic, conditions, E. coli generates both succinate and succinyl-CoA as fermentation end products via a reductive reverse TCA pathway (Figure 1). Secondly, potential oxygen-sensitivity of AdhE2 and other ADHs is another limitation for oxygenic production of 1-propanol.
While the expression of enzymes encoded by the Sbm operon is potentially detectable, their levels are far too low to form a functional pathway [13, 14, 23]. Moreover, due to E. coli’s inability to produce coenzyme B12, the expressed Sbm remains as an inactive apo-enzyme, but nano-molar supplementation of cyanocobalamin can result in the formation of active Sbm [24, 25]. Our observations of no detectable titers of propionate and 1-propanol for wild-type BW25141 as well as the production of 1-propanol upon heterologous expression of the Sbm operon genes with proper supplementation of cyanocobalamin was associated with the activation of the Sbm-pathway. While the activated Sbm-pathway can result in 1-propanol production, the expression of SucCD was deemed crucial to increase the succinyl-CoA pool and consequently the 1-propanol titer. In addition, 1-propanol production was enhanced by exogenous supplementation of succinate. These results suggest that 1-propanol production can be limited by the availability of various precursors and key enzymes along this 1-propanol-producing pathway.
While the metabolic context for the three enzymes encoded by the four-gene Sbm operon, i.e. Sbm, YgfG, and YgfH, has been unraveled, the biological role of the other member, i.e. YgfD/ArgK, remains ambiguous. Earlier studies determined that YgfD/ArgK is a putative arginine kinase interacting with Sbm in vivo and in vitro and involved in the phosphorylation of periplasmic binding proteins for amino acid translocation . The activity of YgfD/ArgK was shown to be potentially essential for 1-propanol biosynthesis since the 1-propanol titer was significantly reduced by the ygfD/argK deletion. Interestingly, propionate production was hardly affected by the ygfD/argK deletion, and this result is consistent with a previous report , where propionate was derived from fatty acids by expressing the Sbm-operon genes excluding ygfD/argK in an engineered E. coli strain.
A selection of native and non-native ADHs were heterologously expressed for evaluation of their effects on 1-propanol-producing capacity of various metabolically engineered E. coli strains, with AdhE2 and BdhB being identified as the most prominent ones for 1-propanol production. Nevertheless, our consistent observation that ethanol titers were significantly higher than 1-propanol implies that propionyl-CoA or propionaldehyde might have less affinity towards ADHs than acetyl-CoA or acetaldehyde. Several native E. coli ADHs (e.g. YqhD, AdhP, and AdhEMUT) were also active in driving 1-propanol production, but in a much lower titer. In particular, the generation of the aerotolerent AdhE mutant (AdhEMUT) opens an avenue for aerobic production of 1-propanol. Under anaerobic conditions, the maximum theoretical yield (on the molar basis) of 1-propanol from glucose is less than one due to limited NADH availability. Thus, developing an oxygenic production system would be beneficial as it increases the carbon throughout whilst improving cell growth and physiology.
Under anoxic conditions for anaerobic fermentation in E. coli, the carbon flux at the PEP node favors reduction into pyruvate rather than carboxylation into oxaloacetate (OAA), with lactate, acetate, and ethanol as major metabolites (Figure 1). Note that there are four NADH-consuming steps along the 1-propanol-producing pathway downstream of phosphoenolpyruvate (PEP), whereas only one or two NADH-consuming steps for the other pathways associated with the major metabolites. The anaplerotic reactions within the metabolic network are optimized in order to balance the cell’s energy budget and electrons. Consequently, only ~10% of glucose consumed is channeled towards succinate and cell mass . Our results suggest that the production of 1-propanol was potentially hampered by the inherent limitation in succinate production and a metabolic deficiency in NADH generation. Interestingly, propionate was also concomitantly produced with 1-propanol in our metabolically engineered strains (Tables 1 and 2). Additional studies are needed to elucidate the dichotomy between 1-propanol and propionate accumulation.
There is an apparent need to reduce the amounts of major metabolites, i.e. ethanol, acetate, and lactate. This could be achieved by knocking out relevant native genes in the hope to redirect the carbon flux into the 1-propanol-producing pathway. While deletions of both adhE and pta were previously found to improve succinate titers , these mutations abolished 1-propanol production in our study (data not shown). Deletion of pta resulted in the channeling of the carbon flux towards lactate accumulation. In addition, heterologous expression of E. coli AdhE or other ADH homologs failed to complement the adhE genomic knockout in terms of restoring 1-propanol production, potentially due to unknown perturbations in the metabolite pool or gene regulation. While the lactate level was significantly reduced for the ldhA null mutants, they produced considerable levels of both acetate and ethanol, thus reducing the carbon flux towards 1-propanol production (Table 3). Nonetheless, the ldhA mutation was deemed beneficial since it offers an additional NADH source and greatly reduces the acidification of the medium, thus improving cell growth.
Another critical factor limiting the production of 1-propanol (and other desired metabolites, such as succinate  and malate ) is the energetically favored diversion of carbon flux at the node of PEP towards pyruvate, resulting in the production of the major metabolites ethanol, lactate, and acetate. Blocking the production of one of these major metabolites (i.e. lactate, acetate, or ethanol) causes the accumulation of the others without improving the overall production of 1-propanol since these major metabolites all share the same precursor of pyruvate. Therefore, the implementation of a “driving force” diverting the carbon flux from pyruvate to OAA appears to be inevitable. Several metabolic engineering strategies to improve this are currently under our investigation Since a considerable amount of succinate accumulated in the extracellular medium potentially due to the poor affinity of succinate to SucCD (K
of ~0.25 mM with succinyl-CoA as the substrate in comparison to K
of ~4 mM with succinate as the substrate ), we are also identifying novel succinyl-CoA synthethases with a higher affinity for succinate to alleviate this limitation in 1-propanol production.