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Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

ABSTRACT: Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, and phosphoproteomics with network analysis to understand the physiology of improved anaerobic xylose fermentation. Our results show that upstream regulatory changes produce a suite of physiological effects that collectively impact the phenotype. Evolved strains show an unusual co-activation of Protein Kinase A (PKA) and Snf1, thus combining responses seen during feast on glucose and famine on non-preferred sugars. Surprisingly, these regulatory changes were required to mount the hypoxic response when cells were grown on xylose, revealing a previously unknown connection between sugar source and anaerobic response. Network analysis identified several downstream transcription factors that play a significant, but on their own minor, role in anaerobic xylose fermentation, consistent with the combinatorial effects of small-impact changes. We also discovered that different routes of PKA activation produce distinct phenotypes: deletion of the RAS/PKA inhibitor IRA2 promotes xylose growth and metabolism, whereas deletion of PKA inhibitor BCY1 decouples growth from metabolism to enable robust fermentation without division. Comparing phosphoproteomic changes across ira2? and bcy1? strains implicated regulatory changes linked to xylose-dependent growth versus metabolism. Together, our results present a picture of the metabolic logic behind anaerobic xylose flux and suggest that widespread cellular remodeling, rather than individual metabolic changes, is an important goal for metabolic engineering.

SUBMITTER: Myers KS

PROVIDER: S-EPMC6428351 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

Myers Kevin S KS Riley Nicholas M NM MacGilvray Matthew E ME Sato Trey K TK McGee Mick M Heilberger Justin J Coon Joshua J JJ Gasch Audrey P AP

PLoS genetics 20190311 3

Microbes can be metabolically engineered to produce biofuels and biochemicals, but rerouting metabolic flux toward products is a major hurdle without a systems-level understanding of how cellular flux is controlled. To understand flux rerouting, we investigated a panel of Saccharomyces cerevisiae strains with progressive improvements in anaerobic fermentation of xylose, a sugar abundant in sustainable plant biomass used for biofuel production. We combined comparative transcriptomics, proteomics, ...[more]

PMID: 30856163

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Project description:BackgroundThe production of ethanol and other fuels and chemicals from lignocellulosic materials is dependent of efficient xylose conversion. Xylose fermentation capacity in yeasts is usually linked to xylose reductase (XR) accepting NADH as cofactor. The XR from Scheffersomyces stipitis, which is able to use NADH as cofactor but still prefers NADPH, has been used to generate recombinant xylose-fermenting Saccharomyces cerevisiae. Novel xylose-fermenting yeasts species, as those from the Spathaspora clade, have been described and are potential sources of novel genes to improve xylose fermentation in S. cerevisiae.ResultsXylose fermentation by six strains from different Spathaspora species isolated in Brazil, plus the Sp. passalidarum type strain (CBS 10155(T)), was characterized under two oxygen-limited conditions. The best xylose-fermenting strains belong to the Sp. passalidarum species, and their highest ethanol titers, yields, and productivities were correlated to higher XR activity with NADH than with NADPH. Among the different Spathaspora species, Sp. passalidarum appears to be the sole harboring two XYL1 genes: XYL1.1, similar to the XYL1 found in other Spathaspora and yeast species and XYL1.2, with relatively higher expression level. XYL1.1p and XYL1.2p from Sp. passalidarum were expressed in S. cerevisiae TMB 3044 and XYL1.1p was confirmed to be strictly NADPH-dependent, while XYL1.2p to use both NADPH and NADH, with higher activity with the later. Recombinant S. cerevisiae strains expressing XYL1.1p did not show anaerobic growth in xylose medium. Under anaerobic xylose fermentation, S. cerevisiae TMB 3504, which expresses XYL1.2p from Sp. passalidarum, revealed significant higher ethanol yield and productivity than S. cerevisiae TMB 3422, which harbors XYL1p N272D from Sc. stipitis in the same isogenic background (0.40 vs 0.34 g gCDW (-1) and 0.33 vs 0.18 g gCDW (-1) h(-1), respectively).ConclusionThis work explored a new clade of xylose-fermenting yeasts (Spathaspora species) towards the engineering of S. cerevisiae for improved xylose fermentation. The new S. cerevisiae TMB 3504 displays higher XR activity with NADH than with NADPH, with consequent improved ethanol yield and productivity and low xylitol production. This meaningful advance in anaerobic xylose fermentation by recombinant S. cerevisiae (using the XR/XDH pathway) paves the way for the development of novel industrial pentose-fermenting strains.

Project description:BackgroundCost-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.ResultsWe 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.ConclusionCollectively, 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.

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Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

Publications

Rewired cellular signaling coordinates sugar and hypoxic responses for anaerobic xylose fermentation in yeast.

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