Abstract
The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism.
Highlights
Biofuels, such as ethanol, produced by microbial fermentation of plant-derived feedstocks offer renewable, carbon-neutral forms of energy
These include evolved mutations in the alkaline phosphatase PHO13, which was implicated in xylose catabolism through transposon library screening [8], the hexose transporter HXT7, which caused an increased xylose uptake rate [9], and GRE3 [10], which encodes an aldose reductase that converts xylose into xylitol [11, 12], an inhibitor of xylose isomerase [13]
We described the generation and characterization of a series of engineered and sequentially evolved S. cerevisiae strains with a range of abilities to consume and metabolize xylose: (i) GLBRCY22-3 (Y22-3), which was generated from a genetically engineered monosporic derivative of the stress-tolerant NRRL YB-210 S. cerevisiae strain [29]; (ii) GLBRCY127 (Y127), which is a single clone isolated from the aerobic directed evolution of Y22-3 on xylose; and (iii) GLBRCY128 (Y128), a single clone isolated from the anaerobic directed evolution of Y127 on xylose (Fig 1A and [10])
Summary
Biofuels, such as ethanol, produced by microbial fermentation of plant-derived feedstocks offer renewable, carbon-neutral forms of energy. These include evolved mutations in the alkaline phosphatase PHO13, which was implicated in xylose catabolism through transposon library screening [8], the hexose transporter HXT7, which caused an increased xylose uptake rate [9], and GRE3 [10], which encodes an aldose reductase that converts xylose into xylitol [11, 12], an inhibitor of xylose isomerase [13] Even with these genetic modifications, S. cerevisiae strains do not achieve industrially acceptable xylose fermentation rates, indicating that additional metabolic and regulatory bottlenecks limit xylose conversion
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