Oxygenation influences xylose fermentation and gene expression in the yeast genera Spathaspora and Scheffersomyces

Abstract

Background: Cost-effective production of biofuels from lignocellulose requires the fermentation of D-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD⁺ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments.

Results: We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis.

Conclusion: Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.

Keywords: Aeration; Cofactors; Ethanol; Gene expression; Serinales (CUG-Ser1 clade); Xylitol; Xylitol dehydrogenase; Xylose fermentation; Xylose reductase.

Fig. 1

Yeast species from the order Serinales generate different metabolic end-products based on oxygen levels. Production of ethanol, xylitol, and/or glycerol by species of Scheffersomyces and Spathaspora genera under moderate (shake flask—SF) and high (baffled flask—BF) aeration conditions. Colored squares: metabolite(s) mainly produced by each species based on yields. Gray squares: the species produced low yields or did not produce the metabolite. Complete data are in Additional file 1

Fig. 2

Scheffersomyces xylosifermentans and Spathaspora passalidarum ferment xylose into ethanol at high yield and titer. Consumption of xylose and production of biomass, ethanol, xylitol, and glycerol by Sc. xylosifermentans, Sp. passalidarum, Scheffersomyces amazonensis, and Scheffersomyces coipomoensis under moderate (shake flask—SF) and high (baffled flask—BF) aeration conditions. Error bars indicate the standard deviation from the three biological replicates

Fig. 3

Xylose reductase from Scheffersomyces xylosifermentans lacks a cofactor preference. A XR and XDH activities under moderate aeration and high aeration conditions expressed in units (U) per mg protein [U (mg protein)⁻¹]. B Production of ethanol, xylitol, and/or glycerol by the species tested for XR and XDH activities (reproduced from Fig. 1). Colored squares: metabolite(s) mainly produced by each species based on yields. Gray squares: the species produced low yields or did not produce the metabolite. Spa—Spathaspora passalidarum, Sxy—Scheffersomyces xylosifermentans, Scoip—Scheffersomyces coipomoensis, Sama—Scheffersomyces amazonensis, and Scer—Saccharomyces cerevisiae (negative control). Error bars indicate the standard deviation from the three biological replicates. Asterisks denote significant differences between the activities on NADH and NADPH for each species (P < 0.05)

Fig. 4

Several genes related to xylose fermentation were upregulated only for ethanol producers at lower oxygenation. Xylose metabolism and related pathways. Dashed lines correspond to the < -1 and > 1 confidence intervals for the log₂-fold change from RNA-Seq analysis (moderate aeration/high aeration). Asterisks indicate genes that did not exhibit differential expression. Positive and negative values indicate upregulated and downregulated genes, respectively, and the colors are associated with the species as shown in the right side of the figure. Protein products encoded by each gene: HXK—hexokinase; PGI1—phosphoglucose isomerase; PFK—phosphofructokinase; FBA1—fructose 1,6-bisphosphate aldolase; TPI1—triose phosphate isomerase; TDH—glyceraldehyde-3-phosphate dehydrogenase; PGK1—3-phosphoglycerate kinase; GPM1—phosphoglycerate mutase; ENO—enolase; PYK1—pyruvate kinase; PDC1—pyruvate decarboxylase, ADH1/2—alcohol dehydrogenase; ALD6—aldehyde dehydrogenase; GPD1—glycerol-3-phosphate dehydrogenase; GPP1—glycerol-3-phosphate phosphatase; ZWF1—glucose-6-phosphate dehydrogenase; SOL—6-phosphogluconolactonase; GND1—6-phosphogluconate dehydrogenase; RKI1—ribose-5-phosphate ketol-isomerase; RPE1—d-ribulose-5-phosphate 3-epimerase; TKL1—transketolase; TAL1—transaldolase; XYL1—xylose reductase; XYL2—xylitol dehydrogenase, and XYL3—xylulokinase. Created with BioRender.com [75] with license number EN2651FNZW

Fig. 5

Metabolic and carbohydrate metabolic processes are highly affected by oxygenation. Enrichment analysis for Sc. xylosifermentans, Sc. coipomoensis, Sc. amazonensis, and Sp. passalidarum. On the right are the biological processes enriched in the upregulated gene list, while those the left side are enriched in the downregulated gene list (moderate aeration/high aeration)

Fig. 6

Saccharomyces cerevisiae with XYL genes from Scheffersomyces xylosifermentans ferments xylose anaerobically. Growth curves of Sc. xylosifermentans, Sc. coipomoensis, and Sc. cerevisiae strains carrying different XYL genes under anoxic conditions A OD, B xylose consumption, C ethanol production, and D xylitol accumulation. Error bars indicate the standard deviation from the three biological replicates. Asterisks denote significant differences related to S. cerevisiae + SstipitisXYL1/XYL2/XYL3). SxylosiXYL1 and SxylosiXYL1/XYL2—XYL genes from Sc. xylosifermentans; ScoipXYL1 and ScoipXYL1/XYL2—XYL genes from Sc. coipomoensis; and SstipitisXYL1/XYL2/XYL3—XYL genes from Sc. stipitis