Eastern gamagrass as an alternative cellulosic feedstock for bioethanol production

Eastern gamagrass (Trypsacum dactyloides) is a C₄ perennial grass, native to the USA with desirable characteristics that warrants further investigation as a new lignocellulosic crop for bioethanol production. Chemical composition assays showed that eastern gamagrass had comparable cellulose, hemicellulose and lignin compositions to those of switchgrass (Panicum virgatum). With the cellulose solvent-based lignocellulose fractionation (CSLF) pretreatment and subsequent enzymatic saccharification, 80.5–99.8% of cellulosic glucose was released from the gamagrass biomass, which was 10–17% greater than the glucose release efficiency from switchgrass (73.5–87.1%). Furthermore, the hydrolysate of gamagrass supported greater ethanol fermentation yield (up to 0.496 g/g glucose) than the hydrolysates of switchgrass. As such, in the whole process of biomass-to-ethanol conversion, gamagrass could yield 13–35% more ethanol per gram of biomass than switchgrass, indicating that gamagrass has high potential as an alternative energy feedstock for lignocellulosic ethanol production.     

Removal of enzymatic and fermentation inhibitory compounds from biomass slurries for enhanced biorefinery process efficiencies

Within the biorefinery paradigm, many non-monomeric sugar compounds have been shown to be inhibitory to enzymes and microbial organisms that are used for hydrolysis and fermentation. Here, two novel separation technologies, polyelectrolyte polymer adsorption and resin-wafer electrodeionization (RW-EDI), have been evaluated to detoxify a dilute acid pretreated biomass slurry. Results showed that detoxification of a dilute acid pretreated ponderosa pine slurry by sequential polyelectrolyte and RW-EDI treatments was very promising, with significant removal of acetic acid, 5-hydroxymethyl furfural, and furfural (up to 77%, 60%, and 74% removed, respectively) along with >97% removal of sulfuric acid. Removal of these compounds increased the cellulose conversion to 94% and elevated the hydrolysis rate to 0.69 g glucose/L/h. When using Saccharomyces cerevisiae D₅A for fermentation of detoxified slurry, the process achieved 99% of the maximum theoretical ethanol yield and an ethanol production rate nearly five-times faster than untreated slurry.     

Ethanol production from lignocellulosic biomass of energy cane

Ethanol produced from lignocellulosic biomass is a renewable alternative to diminishing petroleum based liquid fuels. The release of many new sugarcane varieties by the United States Department of Agriculture to be used as energy crops is a promising feedstock alternative. Energy cane produces large amounts of biomass that can be easily transported, and production does not compete with food supply and prices because energy cane can be grown on marginal land instead of land for food crops. The purpose of this study was to evaluate energy cane for lignocellulosic ethanol production. Energy cane variety L 79-1002 was pretreated with weak sulfuric acid to remove lignin. In this study, 1.4 M sulfuric acid pretreated type II energy cane had a higher ethanol yield after fermentation by Klebsiella oxytoca without enzymatic saccharification than 0.8 M and 1.6 M sulfuric acid pretreated type II energy cane. Pretreated biomass was inoculated with K. oxytoca for cellulose fermentation and Pichia stipitis for hemicellulose fermentation under simultaneous saccahrification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) conditions. For enzymatic saccharification of cellulose, the cellulase and ??-glucanase cocktail significantly increased ethanol production compared to the ethanol production of fermented acid pretreated energy cane without enzymatic saccharification. The results revealed that energy cane variety L 79-1002 produced maximum cellulosic ethanol under SHF (6995 mg/L) and produced 3624 mg/L ethanol from fermentation of hemicellulosic sugars.     

Perennial Grass Bioenergy Cropping on Wet Marginal Land: Impacts on Soil Properties,Soil Organic Carbon,and Biomass During Initial Establishment

The control of soil moisture, vegetation type, and prior land use on soil health parameters of perennial grass cropping systems on marginal lands is not well known. A fallow wetness-prone marginal site in New York (USA) was converted to perennial grass bioenergy feedstock production. Quadruplicate treatments were fallow control, reed canarygrass (Phalaris arundinaceae L. Bellevue) with nitrogen (N) fertilizer (75 kg N ha^?1), switchgrass (Panicum virgatum L. Shawnee), and switchgrass with N fertilizer (75 kg N ha^?1). Based on periodic soil water measurements, permanent sampling locations were assigned to various wetness groups. Surface (0–15 cm) soil organic carbon (SOC), active carbon, wet aggregate stability, pH, total nitrogen (TN), root biomass, and harvested aboveground biomass were measured annually (2011–2014). Multi-year decreases in SOC, wet aggregate stability, and pH followed plowing in 2011. For all years, wettest soils had the greatest SOC and active carbon, while driest soils had the greatest wet aggregate stability and lowest pH. In 2014, wettest soils had significantly (p?<?0.0001) greater SOC and TN than drier soils, and fallow soils had 14 to 20% greater SOC than soils of reed canarygrass + N, switchgrass, and switchgrass + N. Crop type and N fertilization did not result in significant differences in SOC, active carbon, or wet aggregate stability. Cumulative 3-year aboveground biomass yields of driest switchgrass + N soils (18.8 Mg ha^?1) were 121% greater than the three wettest switchgrass (no N) treatments. Overall, soil moisture status must be accounted for when assessing soil dynamics during feedstock establishment.     

Fermentation of soybean hulls to ethanol while preserving protein value

Soybean hulls were evaluated as a resource for production of ethanol by the simultaneous saccharification and fermentation (SSF) process, and no pretreatment of the hulls was found to be needed to realize high ethanol yields with Saccharomyces cerevisiae D₅A. The impact of cellulase, β-glucosidase and pectinase dosages were determined at a 15% biomass loading, and ethanol concentrations of 25–30 g/L were routinely obtained, while under these conditions corn stover, wheat straw, and switchgrass produced 3–4 times lower ethanol yields. Removal of carbohydrates also concentrated the hull protein to over 25% w/w from the original roughly 10%. Analysis of the soybean hulls before and after fermentation showed similar amino acid profiles including an increase in the essential amino acids lysine and threonine in the residues. Thus, eliminating pretreatment should assure that the protein in the hulls is preserved, and conversion of the carbohydrates to ethanol with high yields produces a more concentrated and valuable co-product in addition to ethanol. The resulting upgraded feed product from soybean hulls would likely to be acceptable to monogastric as well as bovine livestock.     

Quantifying Actual and Theoretical Ethanol Yields for Switchgrass Strains Using NIRS Analyses

Quantifying actual and theoretical ethanol yields from biomass conversion processes such as simultanteous saccharification and fermentation (SSF) requires expensive, complex fermentation assays, and extensive compositional analyses of the biomass sample. Near-infrared reflectance spectroscopy (NIRS) is a non-destructive technology that can be used to obtain rapid, low-cost, high-throughput, and accurate estimates of agricultural product composition. In this study, broad-based NIRS calibrations were developed for switchgrass biomass that can be used to estimate over 20 components including cell wall and soluble sugars and also ethanol production and pentose sugars released as measured using a laboratory SSF procedure. With this information, an additional 13 complex feedstock traits can be determined including theoretical and actual ethanol yields from hexose fermentation. The NIRS calibrations were used to estimate feedstock composition and conversion information for biomass samples from a multi-year switchgrass (Panicum virgatum L.) biomass cultivar evaluation trial. There were significant differences among switchgrass strains for all biomass conversion and composition traits including actual ethanol yields, ETOHL (L Mg^?1) and theoretical ethanol yields, ETOHTL (L Mg^?1), based on cell wall and non-cell wall composition NIRS analyses. ETOHL means ranged from 98 to 115 L Mg^?1 while ETOHTL means ranged from 203 to 222 L Mg^?1. Because of differences in both biomass yields and conversion efficiency, there were significant differences among strains for both actual (2,534?C3,720 L ha^?1) and theoretical (4,878?C7,888 L ha^?1) ethanol production per hectare. It should be feasible to improve ethanol yields per hectare by improving both biomass yield and conversion efficiency by using NIRS analyses to quantify differences among cultivars and management practices.     

Simultaneous saccharification and fermentation of lime-treated biomass

Simultaneous saccharification and fermentation (SSF) was performed on lime-treated switchgrass and corn stover, and oxidatively lime-treated poplar wood to determine their compatibility with Saccharomyces cerevisiae. Cellulose-derived glucose was extensively utilized by the yeast during SSF. The ethanol yields from pretreated switchgrass, pretreated corn stover, and pretreated-and-washed poplar wood were 72%, 62% and 73% of theoretical, respectively, whereas those from -cellulose were 67 to 91% of theoretical. The lower ethanol yields from treated biomass resulted from lower cellulose digestibilities rather than inhibitors produced by the pretreatment. Oxidative lime pretreatment of poplar wood increased the ethanol yield by a factor of 5.6, from 13% (untreated) to 73% (pretreated-and-washed).     

Improved Sugar Conversion and Ethanol Yield for Forage Sorghum (Sorghum bicolor L. Moench) Lines with Reduced Lignin Contents

Lignin is known to impede conversion of lignocellulose into ethanol. In this study, forage sorghum plants carrying brown midrib (bmr) mutations, which reduce lignin contents, were evaluated as bioenergy feedstocks. The near-isogenic lines evaluated were: wild type, bmr-6, bmr-12, and bmr-6 bmr-12 double mutant. The bmr-6 and bmr-12 mutations were equally efficient at reducing lignin contents (by 13% and 15%, respectively), and the effects were additive (27%) for the double mutant. Reducing lignin content was highly beneficial for improving biomass conversion yields. Sorghum biomass samples were pretreated with dilute acid and recovered solids washed and hydrolyzed with cellulase to liberate glucose. Glucose yields for the sorghum biomass were improved by 27%, 23%, and 34% for bmr-6, bmr-12, and the double mutant, respectively, compared to wild type. Sorghum biomass was also pretreated with dilute acid followed by co-treatment with cellulases and Saccharomyces cerevisiae for simultaneous saccharification and fermentation (SSF) into ethanol. Conversion of cellulose to ethanol for dilute-acid pretreated sorghum biomass was improved by 22%, 21%, and 43% for bmr-6, bmr-12, and the double mutant compared to wild type, respectively. Electron microscopy of dilute-acid treated samples showed an increased number of lignin globules in double-mutant tissues as compared to the wild-type, suggesting the lignin had become more pliable. The mutations were also effective for improving ethanol yields when the (degrained) sorghum was pretreated with dilute alkali instead of dilute acid. Following pretreatment with dilute ammonium hydroxide and SSF, ethanol conversion yields were 116 and 130 mg ethanol/g dry biomass for the double-mutant samples and 98 and 113 mg/g for the wild-type samples.     

Comparison of solid-state and submerged-state fermentation for the bioprocessing of switchgrass to ethanol and acetate by Clostridium phytofermentans

The conversion of sustainable energy crops using microbiological fermentation to biofuels and bioproducts typically uses submerged-state processes. Alternatively, solid-state fermentation processes have several advantages when compared to the typical submerged-state processes. This study compares the use of solid-state versus submerged-state fermentation using the mesophilic anaerobic bacterium Clostridium phytofermentans in the conversion of switchgrass to the end products of ethanol, acetate, and hydrogen. A shift in the ratio of metabolic products towards more acetate and hydrogen production than ethanol production was observed when C. phytofermentans was grown under solid-state conditions as compared to submerged-state conditions. Results indicated that the end product concentrations (in millimolar) obtained using solid-state fermentation were higher than using submerged-state fermentation. In contrast, the total fermentation products (in weight of product per weight of carbohydrates consumed) and switchgrass conversion were higher for submerged-state fermentation. The conversion of xylan was greater than glucan conversion under both fermentation conditions. An initial pH of 7 and moisture content of 80 % resulted in maximum end products formation. Scanning electron microscopy study showed the presence of biofilm formed by C. phytofermentans growing on switchgrass under submerged-state fermentation whereas bacterial cells attached to surface and no apparent biofilm was observed when grown under solid-state fermentation. To our knowledge, this is the first study reporting consolidated bioprocessing of a lignocellulosic substrate by a mesophilic anaerobic bacterium under solid-state fermentation conditions.     

The Presence of Pretreated Lignocellulosic Solids from Birch during Saccharomyces cerevisiae Fermentations Leads to Increased Tolerance to Inhibitors – A Proteomic Study of the Effects

The fermentation performance of Saccharomyces cerevisiae in the cellulose to ethanol conversion process is largely influenced by the components of pretreated biomass. The insoluble solids in pretreated biomass predominantly constitute cellulose, lignin, and -to a lesser extent- hemicellulose. It is important to understand the effects of water-insoluble solids (WIS) on yeast cell physiology and metabolism for the overall process optimization. In the presence of synthetic lignocellulosic inhibitors, we observed a reduced lag phase and enhanced volumetric ethanol productivity by S. cerevisiae CEN.PK 113-7D when the minimal medium was supplemented with WIS of pretreated birch or spruce and glucose as the carbon source. To investigate the underlying molecular reasons for the effects of WIS, we studied the response of WIS at the proteome level in yeast cells in the presence of acetic acid as an inhibitor. Comparisons were made with cells grown in the presence of acetic acid but without WIS in the medium. Altogether, 729 proteins were detected and quantified, of which 246 proteins were significantly up-regulated and 274 proteins were significantly down-regulated with a fold change≥1.2 in the presence of WIS compared to absence of WIS. The cells in the presence of WIS up-regulated several proteins related to cell wall, glycolysis, electron transport chain, oxidative stress response, oxygen and radical detoxification and unfolded protein response; and down-regulated most proteins related to biosynthetic pathways including amino acid, purine, isoprenoid biosynthesis, aminoacyl-tRNA synthetases and pentose phosphate pathway. Overall, the identified differentially regulated proteins may indicate that the likelihood of increased ATP generation in the presence of WIS was used to defend against acetic acid stress at the expense of reduced biomass formation. Although, comparative proteomics of cells with and without WIS in the acetic acid containing medium revealed numerous changes, a direct effect of WIS on cellular physiology remains to be investigated.     

Effect of dilute acid pretreatment on the saccharification and fermentation of rye straw

This research shows the effect of dilute acid pretreatment with various sulfuric acid concentrations (0.5–2.0% [wt/vol]) on enzymatic saccharification and fermentation yield of rye straw. After pretreatment, solids of rye straw were suspended in Na citrate buffer or post-pretreatment liquids (prehydrolysates) containing sugars liberated after hemicellulose hydrolysis. Saccharification was conducted using enzymes dosage of 15 or 25 FPU/g cellulose. Cellulose saccharification rate after rye straw pretreatment was enhanced by performing enzymatic hydrolysis in sodium citrate buffer in comparison with hemicellulose prehydrolysate. The maximum cellulose saccharification rate (69%) was reached in sodium citrate buffer (biomass pretreated with 2.0% [wt/vol] H₂SO₄). Lignocellulosic complex of rye straw after pretreatment was subjected to separate hydrolysis and fermentation (SHF) or separate hydrolysis and co-fermentation (SHCF). The SHF processes conducted in the sodium citrate buffer using monoculture of Saccharomyces cerevisiae (Ethanol Red) were more efficient compared to hemicellulose prehydrolysate in respect with ethanol yields. Maximum fermentation efficiency of SHF processes obtained after rye straw pretreatment at 1.5% [wt/vol] H₂SO₄ and saccharification using enzymes dosage of 25 FPU/g in sodium citrate buffer, achieving 40.6% of theoretical yield. However, SHCF process using cocultures of pentose-fermenting yeast, after pretreatment of raw material at 1.5% [wt/vol] H₂SO₄ and hydrolysis using enzymes dosage of 25 FPU/g, resulted in the highest ethanol yield among studied methods, achieving 9.4 g/L of ethanol, corresponding to 55% of theoretical yield.     

Production of cellulosic ethanol in Saccharomyces cerevisiae heterologous expressing Clostridium thermocellum endoglucanase and Saccharomycopsis fibuligera β-glucosidase genes

Heterologous secretory expression of endoglucanase E (Clostridium thermocellum) and β-glucosidase 1 (Saccharomycopsis fibuligera) was achieved in Saccharomyces cerevisiae fermentation cultures as an α-mating factor signal peptide fusion, based on the native enzyme coding sequence. Ethanol production depends on simultaneous saccharification of cellulose to glucose and fermentation of glucose to ethanol by a recombinant yeast strain as a microbial biocatalyst. Recombinant yeast strain expressing endoglucanase and β-glucosidase was able to produce ethanol from β-glucan, CMC and acid swollen cellulose. This indicates that the resultant yeast strain of this study acts efficiently as a whole cell biocatalyst.     

A kinetic model for quantitative evaluation of the effect of hydrogen and osmolarity on hydrogen production by Caldicellulosiruptor saccharolyticus

Background

The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.

Results

We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel?) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.

Conclusions

Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

A Rapid Simultaneous Saccharification and Fermentation (SSF) Technique to Determine Ethanol Yields

We have developed a relatively simple simultaneous saccharification and fermentation (SSF) technique to determine the ethanol production potential for large sets of biomass samples. The technique is based on soaking approximately 0.5 grams of a biomass sample in aqueous ammonia at room temperature and at atmospheric pressure for 24 h, then fermenting with Saccharomyces cerevisiae D₅A for 24 h using Spezyme CP, for enzymatic hydrolysis of structural polysaccharides. We have tested the technique on a set of corn stover samples representing much of the genetic variability in the commercial corn hybrid population. The samples were weighed into modified Ankom filter bags (F57) before soaking to avoid biomass loss during the process. Fermentation samples were analyzed for ethanol after 24 h by HPLC. Percentages of theoretical maximum ethanol yields of the samples ranged between 44.9 and 73%. We observed that percentages of theoretical maximum ethanol yields were highly correlated (r ²?=?0.90) with acid detergent lignin concentration while a low correlation was observed between cellulose concentration and ethanol yield. Near infrared spectra of corn stover samples were also examined. The coefficient of determination (r ²) from regression of predicted versus measured percent theoretical maximum ethanol yield was 0.96. This result suggests that using NIRS is a promising method for predicting ethanol yield, but larger calibration sets are necessary for obtaining improved accuracy for larger sample populations. We conclude that the developed SSF technique could be applied to large numbers of biomass samples to rapidly estimate ethanol yields and to compare different biomass samples in terms of ethanol yields.     

Impact of Dilute Sulfuric Acid,Ammonium Hydroxide,and Ionic Liquid Pretreatments on the Fractionation and Characterization of Engineered Switchgrass

Improving plant characteristics for better environmental resilience and more cost-effective transformation to fuels and chemicals is one of the focus areas in biomass feedstock development. In order to bridge lignin engineering and conversion technologies, this study aimed to fractionate and characterize lignin streams from wild-type and engineered switchgrass using three different pretreatment methods, i.e., dilute sulfuric acid (DA), ammonium hydroxide (AH), and aqueous ionic liquid (IL). Results demonstrate the low lignin content and high S/G ratio switchgrass mutant (4CL) was more susceptible to pretreatment and subsequently more digestible by enzymes as compared to wild-type switchgrass and AtLOV1 mutant. In addition, when compared to DA and AH pretreatment, aqueous IL (cholinium lysinate) was demostrated to be an efficient lignin solvent, as indicated by the high (> 80%) lignin solubility and reduced lignin molecular weight. FTIR and differential scanning calorimetry measurements suggest that pretreatment chemistry greatly influenced the structural and compositional changes and thermal properties of the pretreated switchgrass and recovered lignin-rich streams. The comparative data obtained from this work deepen our understanding of how lignin modification impacts the fractionation and properties of biomass feedstocks.     

Industrial yeast strain engineered to ferment ethanol from lignocellulosic biomass

In this study an industrial Saccharomyces cerevisiae yeast strain capable of fermenting ethanol from pretreated lignocellulosic material was engineered. Genes encoding cellulases (endoglucanase, exoglucanase and β-glucosidase) were integrated into the chromosomal ribosomal DNA and delta regions of a derivative of the K1-V1116 wine yeast strain. The engineered cellulolytic yeast produces ethanol in one step through simultaneous saccharification and fermentation of pretreated biomass without the addition of exogenously produced enzymes. When ethanol fermentation was performed with 10% dry weight of pretreated corn stover, the recombinant strain fermented 63% of the cellulose in 96 h and the ethanol titer reached 2.6% v/v. These results demonstrate that cellulolytic S. cerevisiae strains can be used as a platform for developing an economical advanced biofuel process.     

Pectin-rich biomass as feedstock for fuel ethanol production

The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae

Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.