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.

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose.

BACKGROUND: Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts. RESULTS: In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media. CONCLUSION: Our results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.

Engineering and two-stage evolution of a lignocellulosic hydrolysate-tolerant Saccharomyces cerevisiae strain for anaerobic fermentation of xylose from AFEX pretreated corn stover.

The inability of the yeast Saccharomyces cerevisiae to ferment xylose effectively under anaerobic conditions is a major barrier to economical production of lignocellulosic biofuels. Although genetic approaches have enabled engineering of S. cerevisiae 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 S. cerevisiae 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 S. cerevisiae 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 GRE3, 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 S. cerevisiae strain with the ability to ferment xylose anaerobically from ACSH.

Phenotypic selection of a wild Saccharomyces cerevisiae strain for simultaneous saccharification and co-fermentation of AFEX™ pretreated corn stover.

BACKGROUND: Simultaneous saccharification and co-fermentation (SSCF) process involves enzymatic hydrolysis of pretreated lignocellulosic biomass and fermentation of glucose and xylose in one bioreactor. The optimal temperatures for enzymatic hydrolysis are higher than the standard fermentation temperature of ethanologenic Saccharomyces cerevisiae. Moreover, degradation products resulting from biomass pretreatment impair fermentation of sugars, especially xylose, and can synergize with high temperature stress. One approach to resolve both concerns is to utilize a strain background with innate tolerance to both elevated temperatures and degradation products. RESULTS: In this study, we screened a panel of 108 wild and domesticated Saccharomyces cerevisiae strains isolated from a wide range of environmental niches. One wild strain was selected based on its growth tolerance to simultaneous elevated temperature and AFEX™ (Ammonia Fiber Expansion) degradation products. After engineering the strain with two copies of the Scheffersomyces stipitis xylose reductase, xylitol dehydrogenase and xylulokinase genes, we compared the ability of this engineered strain to the benchmark 424A(LNH-ST) strain in ethanol production and xylose fermentation in standard lab medium and AFEX pretreated corn stover (ACS) hydrolysates, as well as in SSCF of ACS at different temperatures. In SSCF of 9% (w/w) glucan loading ACS at 35°C, the engineered strain showed higher cell viabilities and produced a similar amount of ethanol (51.3 g/L) compared to the benchmark 424A(LNH-ST) strain. CONCLUSION: These results validate our approach in the selection of wild Saccharomyces cerevisiae strains with thermo-tolerance and degradation products tolerance properties for lignocellulosic biofuel production. The wild and domesticated yeast strains phenotyped in this work are publically available for others to use as genetic backgrounds for fermentation of their pretreated biomass at elevated temperatures.