Energetics of Yarrowia lipolytica co-consuming formic acid
In this work, we found that co-feeding formic acid and glucose, up to a molar ratio of ~5:1, linearly increased the biomass yield of Y. lipolytica on glucose (Figure 3). This indicated that under these conditions, consumption of formic acid by this organism has a net positive ATP yield, similar to previous observations for other yeasts (Babel et al., 1983; Bruinenberg et al., 1985; Geertman et al., 2006). At F:G feed ratios ≤5, we observed that 24 ± 2 moles of formic acid were able to displace 1 mole of glucose for dissimilatory requirements in Y. lipolytica (Supplementary Table S9). Since glucose dissimilation provides 4 ATP and 12 NADH equivalents (assuming no energetic costs of glucose transport) compared to 1 NADH from formic acid dissimilation, these results indicate that either: 1) the effective P/O ratio of respiration in Y. lipolytica is low (<1.0), or 2) transport of formic acid comes at a net energetic (ATP) cost, or 3) Y. lipolytica has a different P/O ratio for formic acid-derived electrons compared to glucose-derived electrons. We believe the third scenario is most likely, since electrons derived from glucose dissimilation via glycolysis and the TCA cycle are released in both the cytosol and mitochondria, whereas the electrons released by formic acid dissimilation via FDH are expected to be released exclusively in the cytosol. Y. lipolytica FDH is described as cytosolic in UniProt, accession number Q6C5X6. Therefore, glucose derived electrons can be partially transferred to O2 via proton-pumping complex I in the mitochondria, whereas formic acid derived electrons are likely transferred to O2 via the less efficient external alternative NADH dehydrogenase.
Although no benefit on the biomass yield was observed at higher formic acid to glucose ratios up to 11.5:1, virtually all formic acid was consumed as indicated by the low residual formic acid concentrations in the fermenter (Supplementary Table S9). This is in contrast with observations in other yeasts, as in previous work with aerobic chemostat cultures, formic acid accumulated at F:G ratios higher than 5 inC. utilis cultivations and higher than 2 in S. cerevisiae(Bruinenberg et al., 1985; Overkamp et al., 2002). The ability to consume all formic acid at high ratios demonstrates the potential ofY. lipolytica in formic acid co-fed processes.
In this work we used biomass itself as an ATP-intensive product to investigate the potential of formic acid co-feeding for increasing the product yield. Previous work on antibiotic-producing Penicillium chrysogenum strains (Harris et al. , 2007) demonstrated that in chemostat setups, formic acid co-feeding can also increase the yield of product formation. Since Y. lipolytica is used on an industrial scale for synthesis of other ATP-intensive products, such as citrate, lipids, lipase (Madzak, 2018), a logical next step would be to translate our fed-batch process to an industrial Y. lipolytica strain engineered for synthesis of one of these molecules.
Supplementary materials 4 and 5 present extensions of the formic acid co-feeding concept of this study, illustrating how the co-produced O2 can be valorised and how co-feeding of formic acid can even lead to net-negative CO2 emission processes.