Correction: Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

[This corrects the article DOI: 10.1371/journal.pgen.1006372.].

Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by Saccharomyces cerevisiae.

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.

Comparative functional genomics identifies an iron-limited bottleneck in a Saccharomyces cerevisiae strain with a cytosolic-localized isobutanol pathway.

Metabolic engineering strategies have been successfully implemented to improve the production of isobutanol, a next-generation biofuel, in Saccharomyces cerevisiae. Here, we explore how two of these strategies, pathway re-localization and redox cofactor-balancing, affect the performance and physiology of isobutanol producing strains. We equipped yeast with isobutanol cassettes which had either a mitochondrial or cytosolic localized isobutanol pathway and used either a redox-imbalanced (NADPH-dependent) or redox-balanced (NADH-dependent) ketol-acid reductoisomerase enzyme. We then conducted transcriptomic, proteomic and metabolomic analyses to elucidate molecular differences between the engineered strains. Pathway localization had a large effect on isobutanol production with the strain expressing the mitochondrial-localized enzymes producing 3.8-fold more isobutanol than strains expressing the cytosolic enzymes. Cofactor-balancing did not improve isobutanol titers and instead the strain with the redox-imbalanced pathway produced 1.5-fold more isobutanol than the balanced version, albeit at low overall pathway flux. Functional genomic analyses suggested that the poor performances of the cytosolic pathway strains were in part due to a shortage in cytosolic Fe-S clusters, which are required cofactors for the dihydroxyacid dehydratase enzyme. We then demonstrated that this cofactor limitation may be partially recovered by disrupting iron homeostasis with a fra2 mutation, thereby increasing cellular iron levels. The resulting isobutanol titer of the fra2 null strain harboring a cytosolic-localized isobutanol pathway outperformed the strain with the mitochondrial-localized pathway by 1.3-fold, demonstrating that both localizations can support flux to isobutanol.

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

Invited for this month's cover is the research team from the D.O.E. Great Lake Bioenergy Research Center (GLBRC) at the University of Wisconsin-Madison. The cover image shows how a diverse team with expertise in many different fields works together in an integrated fashion to address complex problems. Only when the whole system, from field to the liquid fuels and co-products, is assessed, can we identify the key parameters needed to design an economically viable biorefinery-based economy. Cover art by Chelsea Mamott. The Full Paper itself is available at 10.1002/cssc.201903345.

Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams.

The hydroxycinnamic acids p-coumaric acid (pCA) and ferulic acid (FA) add diversity to the portfolio of products produced by using grass-fed lignocellulosic biorefineries. The level of lignin-bound pCA in Zea mays was modified by the alteration of p-coumaroyl-CoA monolignol transferase expression. The biomass was processed in a lab-scale alkaline-pretreatment biorefinery process and the data were used for a baseline technoeconomic analysis to determine where to direct future research efforts to couple plant design to biomass utilization processes. It is concluded that future plant engineering efforts should focus on strategies that ramp up accumulation of one type of hydroxycinnamate (pCA or FA) predominantly and suppress that of the other. Technoeconomic analysis indicates that target extraction titers of one hydroxycinnamic acid need to be >50 g kg-1 biomass, at least five times higher than observed titers for the impure pCA/FA product mixture from wild-type maize. The technical challenge for process engineers is to develop a viable process that requires more than 80 % reduction of the isolation costs.

Exploiting natural variation in Saccharomyces cerevisiae to identify genes for increased ethanol resistance.

Ethanol production from lignocellulosic biomass holds promise as an alternative fuel. However, industrial stresses, including ethanol stress, limit microbial fermentation and thus prevent cost competitiveness with fossil fuels. To identify novel engineering targets for increased ethanol tolerance, we took advantage of natural diversity in wild Saccharomyces cerevisiae strains. We previously showed that an S288c-derived lab strain cannot acquire higher ethanol tolerance after a mild ethanol pretreatment, which is distinct from other stresses. Here, we measured acquired ethanol tolerance in a large panel of wild strains and show that most strains can acquire higher tolerance after pretreatment. We exploited this major phenotypic difference to address the mechanism of acquired ethanol tolerance, by comparing the global gene expression response to 5% ethanol in S288c and two wild strains. Hundreds of genes showed variation in ethanol-dependent gene expression across strains. Computational analysis identified several transcription factor modules and known coregulated genes as differentially expressed, implicating genetic variation in the ethanol signaling pathway. We used this information to identify genes required for acquisition of ethanol tolerance in wild strains, including new genes and processes not previously linked to ethanol tolerance, and four genes that increase ethanol tolerance when overexpressed. Our approach shows that comparative genomics across natural isolates can quickly identify genes for industrial engineering while expanding our understanding of natural diversity.