Assessing the effect of coupling glycerol to xylose metabolism in native xylose-consuming yeasts

BOLZICO, BRUNA C.; COMELLI, RAÚL N.

Congreso; 36th International Specialised Symposium on Yeasts (ISSY36); 2022

In recent years, the use of microbial fermentation to produce chemicals and biofuels from biomass resources has attracted great attention. Conversion of all sugars present at lignocellulosic biomass would increase production and reduce cost of second-generation ethanol. Saccharomyces cerevisiae is the main yeast used for alcohol production worldwide, but it cannot produce ethanol from xylose, the second most abundant sugar in nature. In general, xylose-consuming yeasts are capable of fermenting xylose only when the oxygen flow is tightly regulated. The cofactor balances play critical roles in maintaining intracellular redox homeostasis, which has been recognized to be a prerequisite for robust and cost-efficient bioprocesses. An interesting case occurs in native xylose-consuming yeasts: the first two reactions in xylose assimilation pathway, i.e., xylose reductase (XR) and xylitol dehydrogenase (XDH), prefer different cofactors. XR prefers NADPH while XDH strictly depends on NAD+, which leads to redox imbalance. Saccharomyces engineered-strains containing these genes still lack the capability of produce ethanol efficiently. It has been suggested that a major cause for the limited growth performance and ethanol production with xylose as substrate is the redox bottleneck, rather than enzyme activity deficiency. Our work is focused on understanding on how different metabolic pathways involving production/consumption of cofactors shift in a coherent fashion in response to reduced oxygen supply to produce ethanol. So, we present an initial approach of the impact on fermentation performance when glycerol and xylose metabolism are coupled in native xylose-consuming yeasts. Scheffersomyces stipitis, Spathaspora passalidarum, Pachysolen tannophilus, Ogataea siamensis, Kluyveromyces marxianus, K. lactis, Meyerozyma guilliermondii, Yamadazyma sp., Candida sp., Naganishia sp. and Rhodotorula sp. were tested. Fermentation assays were performed under non-strictly anaerobic cultivation and monitored over time analyzing biomass growth, substrates consumption and products formation. Among the yeasts evaluated, S. passalidarum, P. tannophilus, and both Kluyveromyces consumed considerable amounts of glycerol as sole carbon source under non-strictly anaerobic cultivation, yet only P tannophilus could yield ethanol from glycerol under the experimental conditions. When evaluating glycerol as co-substrate, xylose utilization was not enhanced, and the present of the pentose sugar altered glycerol consumption by the majority of the yeasts. Furthermore, glycerol had no impact on xylose fermentation in S. passalidarum and S. stipitis strains since an increase in ethanol production was not observed. These results suggest that glycerol is not a spread metabolic partner of xylose. Because we not performed enzymatic activities yet, low activities for glycerol metabolism in anaerobic conditions, like glycerol dehydrogenase (Gcy) and dihydroxyacetone kinase (Dak) not to be discarded. Future works will be focused in transfer P. tannophilus glycerol-pathway genes to engineered xylose-fermenting Saccharomyces in attempt to develop more ethanologenic strains from xylose.