Directed Evolution Reveals Unexpected Epistatic Interactions That Alter Metabolic Regulation and Enable Anaerobic Xylose Use by <i>Saccharomyces cerevisiae</i>

<div><p>The inability of native <i>Saccharomyces cerevisiae</i> 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 <i>S</i>. <i>cerevisiae</i> 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 <i>S</i>. <i>cerevisiae</i> strains depends upon epistatic interactions among genes encoding a xylose reductase (<i>GRE3</i>), a component of MAP Kinase (MAPK) signaling (<i>HOG1</i>), a regulator of Protein Kinase A (PKA) signaling (<i>IRA2</i>), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (<i>ISU1</i>). Interestingly, the mutation in <i>IRA2</i> only impacted anaerobic xylose consumption and required the loss of <i>ISU1</i> 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 <i>HOG1</i> and <i>ISU1</i> 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.</p></div

Genetic differences between parental and evolved strains.

<p>Genetic differences between parental and evolved strains.</p

Genetic differences between parental and evolved strains.

<p>Genetic differences between parental and evolved strains.</p

Xylose-metabolizing strains have altered pentose phosphate pathways.

<p>Heat maps display intracellular concentrations of proteins and metabolites from xylose metabolism, pentose phosphate pathway and glycolysis (<b>A</b>) from engineered and evolved strains relative to Y22-3^MR. Colors correspond to average Log₂ fold change values from strains cultured under aerobic (<b>B</b> and <b>D</b>) or anaerobic (<b>C</b> and <b>E</b>) conditions in YPX medium from three biological replicates. White boxes indicate strains for which no corresponding peptides were detected. The asterisk (<b>*</b>) in (<b>A</b>) denotes an enzyme activity that is absent in the Y22-3^MR strain. Abbreviations: X-5P, xylulose-5-phosphate; S-7P, sedoheptulose-7-phosphate; F-6P, fructose-6-phosphate; F-1,6-BP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone-phosphate; 3- and 2-PG, 3- and 2-phosphoglycerates; PEP, phosphoenolpyruvate.</p

Proposed model for how the evolved mutations impact biochemical pathways for xylose metabolism.

<p>Text, shapes and arrows in green signify upregulated activities compared to the activities in the parental strain (in black). Under aerobic or anaerobic conditions, the parent strain consumes low amounts of xylose due to insufficient biochemical activities (<b>A</b>). Under aerobic conditions, the evolved <i>hog1</i>^{<i>M282fs</i>} and <i>isu1</i>^<i>H138Y</i> mutations enhance activities (signified in green) of the pentose phosphate and lower glycolytic pathways, as well as respiration, thereby permitting significantly greater growth on and metabolism of xylose (<b>B</b>). Loss of <i>HOG1</i> function caused reduced expression of <i>GRE3</i> and other targets that impair xylose metabolism. Under anaerobic conditions (<b>C</b>), the evolved <i>ira2</i>^{<i>E2928Stop</i>} mutation causes activation of PKA, which in turn activates glycolytic enzymes. This, along with the disabling <i>gre3</i>^<i>A46T</i> mutation, enables the fermentation of xylose into ethanol.</p

Mutations in <i>ISU1</i> enhance respiration of xylose.

<p>Engineered and evolved strains were cultured in aerobic YPX media and analyzed for intracellular protein and metabolite concentrations. Average Log₂ intracellular concentrations of mitochondrial translation and respiration proteins (<b>A</b>) or hexose transporters and glucose-repressed proteins (<b>B</b>) from mutant strains relative to the Y22-3^MR parent are shown. White boxes indicate strains for which no corresponding peptides were detected. Relative protein concentrations were calculated from three independent biological replicates. Y22-3^MR <i>hog1Δ isu1Δ</i> strains were cultured in YP-Ethanol (<b>C</b>), YPD (<b>D</b>) or YPX (<b>E</b>) media and then treated with DMSO control or 0.5 μg/mL Antimycin A. Shaded areas represent the time during which DMSO or Antimycin A were present in the cultures. Average cell density, sugar and ethanol concentration with standard deviations from three independent biological replicates are reported.</p

Proteomic changes across strains implicate physiological responses.

<p>The heat map shows the average relative protein abundance for 3,660 proteins (rows) in each denoted strain (columns) compared to Y22-3^MR grown aerobically (left) or anaerobically (right) on xylose as the sole carbon source. Genotypes indicated in green or blue denote strains that can grow on xylose aerobically or contain mutations in <i>IRA2</i>, respectively. Yellow indicates higher abundance and blue indicates lower abundance according to the key. Data were pooled and organized by hierarchical clustering [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006372#pgen.1006372.ref095" target="_blank">95</a>]. Functional groups enriched in denoted clusters at p < 1e-5 (hypergeometric test [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006372#pgen.1006372.ref094" target="_blank">94</a>]) are annotated to the right of each cluster. Proteins encoded by mRNAs whose salt-dependent expression is dependent on Hog1 or the phosphodiesterase Pde2 (which indirectly represses PKA activity through cAMP degradation, [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006372#pgen.1006372.ref021" target="_blank">21</a>]) as defined in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006372#pgen.1006372.ref048" target="_blank">48</a>] are shown as orange or magenta boxes, respectively.</p

Deletions of <i>ISU1</i>, <i>HOG1</i>, <i>GRE3</i> and <i>IRA2</i> are sufficient to increase cell growth and xylose consumption rates.

<p>Indicated strains were cultured in YPX media under aerobic (<b>A-B</b>) or anaerobic (<b>C-F</b>) conditions. Average growth and specific xylose consumption rates with standard deviations are reported in g of dry cell mass•hr^-1 (<b>A, C</b>) or OD600•hr^-1 (<b>E</b>), and g of xylose consumed•g of dry cell mass^-1•h^-1 (<b>B, D</b>) or g of xylose consumed•OD₆₀₀^-1•h^-1 (<b>F</b>), respectively, from the indicated strains cultured in YPX media from three independent biological replicates. Asterisks denote statistically significant differences (*; <i>P</i> < 0.05, **; <i>P</i> < 0.061) between specified strains by paired Student’s t-test. Xylose consumption rates for all strains in (<b>D</b>) were significantly faster (<i>P</i> < 0.05) than Y22-3^MR.</p

Deletions of <i>HOG1</i> and <i>ISU1</i> have different effects on the metabolism of other carbon substrates.

<p>Graphs display growth and consumption rates for Y22-3^MR strains containing the indicated genotypes cultured aerobically in YP media containing glucose (<b>A-B</b>), glycerol (<b>C-D</b>), acetate (<b>E-F</b>), or anaerobically with glucose (<b>G-H</b>). Reported values are averages and standard deviations from biological triplicate experiments, and in g substrate consumed or ethanol produced•L^-1•h^-1•cell mass (in OD₆₀₀)^-1. Asterisks denote statistically significant differences (<i>*</i>, <i>P</i> < 0.05; **, <i>P</i> < 0.068) by Student’s t-test.</p

Engineering and Two-Stage Evolution of a Lignocellulosic Hydrolysate-Tolerant <i>Saccharomyces cerevisiae</i> Strain for Anaerobic Fermentation of Xylose from AFEX Pretreated Corn Stover

<div><p>The inability of the yeast <i>Saccharomyces cerevisiae</i> to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of <i>S. cerevisiae</i> to convert xylose efficiently into ethanol in defined lab medium, few strains are able to ferment xylose from lignocellulosic hydrolysates in the absence of oxygen. This limited xylose conversion is believed to result from small molecules generated during biomass pretreatment and hydrolysis, which induce cellular stress and impair metabolism. Here, we describe the development of a xylose-fermenting <i>S. cerevisiae</i> strain with tolerance to a range of pretreated and hydrolyzed lignocellulose, including Ammonia Fiber Expansion (AFEX)-pretreated corn stover hydrolysate (ACSH). We genetically engineered a hydrolysate-resistant yeast strain with bacterial xylose isomerase and then applied two separate stages of aerobic and anaerobic directed evolution. The emergent <i>S. cerevisiae</i> strain rapidly converted xylose from lab medium and ACSH to ethanol under strict anaerobic conditions. Metabolomic, genetic and biochemical analyses suggested that a missense mutation in <i>GRE3</i>, which was acquired during the anaerobic evolution, contributed toward improved xylose conversion by reducing intracellular production of xylitol, an inhibitor of xylose isomerase. These results validate our combinatorial approach, which utilized phenotypic strain selection, rational engineering and directed evolution for the generation of a robust <i>S. cerevisiae</i> strain with the ability to ferment xylose anaerobically from ACSH.</p></div