Lignocellulosic Hydrolysates Research Articles

Biodetoxification of Lignocellulose Hydrolysate for Direct Use in Succinic Acid Production.

The pretreatment of lignocellulosic biomass with acid generates phenolic and furanyl compounds that function as toxins by inhibiting microbial growth and metabolism. Therefore, it is necessary to detoxify acid-pretreated lignocellulosic biomass for better utilization. Among the various detoxification methods that are available, biodetoxification offers advantages that include mild reaction conditions and low energy consumption. In this study, a newly isolated Rhodococcus aetherivorans strain, N1, was found to effectively degrade various lignin-derived aromatic compounds, such as p-coumarate, ferulate, syringaldehyde, furfural, and 5-hydroxymethylfurfural. Furthermore, the metabolic pathway and genes responsible for this degradation were also identified. In addition, the overexpression of a demethylase (DesA) and 3,4-dioxygenase (DesZ) in strain N1 generated a recombinant strain, N1-S, which showed an enhanced ability to degrade syringaldehyde and 80.5% furfural, 50.7% 5-hydroxymethylfurfural, and 71.5% phenolic compounds in corn cob hydrolysate. The resulting detoxified hydrolysate was used directly as a feedstock for succinate production by Escherichia coli suc260. This afforded 35.3 g/l succinate, which was 6.5 times greater than the concentration afforded when nondetoxified hydrolysate was used. Overall, the results of this study demonstrate that strain N1-S is a valuable microbe for the biodetoxification of lignocellulosic biomass.

Improving heterologous expression of laccase by Pichia pastoris via vanillin-induced stress response and its application for removing inhibitors of lignocellulose hydrolysate

Vanillin-sensitive promoters were screened and employed to improve the heterologous expression of laccase in Pichia pastoris. The recombinant yeast could well remove phenolic compounds to improve the fermentability of wheat straw hydrolysate.

Structural and biochemical insights of xylose MFS and SWEET transporters in microbial cell factories: challenges to lignocellulosic hydrolysates fermentation.

The production of bioethanol from lignocellulosic biomass requires the efficient conversion of glucose and xylose to ethanol, a process that depends on the ability of microorganisms to internalize these sugars. Although glucose transporters exist in several species, xylose transporters are less common. Several types of transporters have been identified in diverse microorganisms, including members of the Major Facilitator Superfamily (MFS) and Sugars Will Eventually be Exported Transporter (SWEET) families. Considering that Saccharomyces cerevisiae lacks an effective xylose transport system, engineered yeast strains capable of efficiently consuming this sugar are critical for obtaining high ethanol yields. This article reviews the structure-function relationship of sugar transporters from the MFS and SWEET families. It provides information on several tools and approaches used to identify and characterize them to optimize xylose consumption and, consequently, second-generation ethanol production.

Isolation and Characterization of Enterobacter sp Capable towards Tolerating Degradation Products and Fermenting Pentoses

<p>The depletion of fossil fuel resources, in addition to the growing demand for energy, has prompted the development of renewable energies, including bioethanol from lignocellulosic biomass. The acid pretreatment of hemicellulose releases inhibiting compounds in addition to xylose. With the possibility of exploitation of lignocellulose as a fermentation substrate, we isolated an <em>Enterobacter </em>characterized by its ability to ferment xylose and tolerate high concentrations of inhibitors. The selection was performed in media containing different carbon and energy sources; glucose, cellobiose, CMC, furfural and 5-HMF. Characterization strategies of the selected strain such as, xylose concentration (from 25 g liter-1 to 100 g liter-1), furfural (0 mM to 25 mM), cell immobilization, were used to quantify the maximum yield of ethanol produced. The results obtained show that our strain can ferment up to 100 g liter-1 of xylose in the presence of 20 mM furfural at 37°C to produce ethanol with a maximum yield of 2.22 g liter- 1 for 24 h under 160 rpm magnetic stirring. The results obtained in this study suggest that the isolated <em>Enterobacter </em>sp. is a promising strain for the bioconversion of lignocellulosic biomass pretreatment hydrolysate into bioethanol.</p><p> </p>

Molecular mechanism of tolerance evolution in Candida tropicalis to inhibitory compounds derived from corn stover hydrolysis

Candida tropicalis is distinguished from Saccharomyces cerevisiae by its great xylose utilization ability, maximizing its capability to ferment both hexoses and xylose to produce high-value products, and its ability to overcome the problem of end-product inhibition derived from lignocellulosic hydrolysis. However, little or no information about the molecular evolution mechanism of C. tropicalis to inhibitory compounds (acetic acid, phenol, and furfural) derived from lignocellulosic hydrolysis is known. In this study, the adaptation of a C. tropicalis strain SHC-03 to corn stover hydrolysate after mutagenesis through stepwise evolution resulted in multi-generation evolved strains after 60 rounds of evolution using the C. tropicalis strain SHC-03 as the parental strain. The evolved strain, generation B-60, (GB-60) showed the highest tolerance (15.81 g/L) and resistance as well as the lowest subcellular damage to inhibitory compounds derived from corn stover hydrolysis. Comparative transcriptome and genomic analyses between the evolved strain GB-60 and the control strain SHC-03 revealed enhanced functional and mutated genes including CTRG_03726, CTRG_01923, CTRG_04945, CTRG_05115, CTRG_03102, CTRG_05127, CTRG_00952 and CTRG_03034 involved in increased cell wall biosynthesis and remodeling, pentose phosphate pathway, metabolic and carbon pathways, detoxification of inhibitory compounds, and the multidrug/multixenobiotic resistance (MDR/MXR) transporters. These genes also played active roles in oxidative stress response, mitochondrial stress response, ER stress response, vacuole stress response and autophagy, DNA repair, and chromatin protection resulting in less ROS accumulation, as well as lower damage to some subcellular organelles in the evolved strain. The information from this study will provide guidelines for the development of engineering strains with high tolerance towards inhibitors derived from corn stover hydrolysis.

Effect of various aromatic compounds with different functional groups on enzymatic hydrolysis of microcrystalline cellulose and alkaline pretreated wheat straw

Low molecular aromatic compounds are detrimental to the enzymatic hydrolysis of lignocellulose. However, the specific role of their functional groups remains unclear. Here, a series of nine aromatic compounds as additives were tested to understand their effect on the hydrolysis yield of microcrystalline cellulose (MCC) and alkaline pretreated wheat straw. Based on the results, the inhibition of aldehyde groups on MCC was greater than that of carboxyl groups, whereas for the alkaline pretreated wheat straw case, the inhibitory effect of aldehyde groups was lower than that of carboxyl groups. Increased methoxyl groups of aromatic compounds reduced the inhibitory effect on enzymatic hydrolysis of both substrates. Stronger inhibition of aromatic compounds on MCC hydrolysis was detected in comparison with the alkaline pretreated wheat straw, indicating that the substrate lignin can offset the inhibition to a certain extent. Among all aromatic compounds, syringaldehyde with one aldehyde group and two methoxyl groups improved the glucan conversion of the alkaline pretreated wheat straw.

A closed-loop strategy for on-site production of saccharolytic enzymes for lignocellulose biorefinery using internal lignocellulosic hydrolysates

The cost-effective production of lignocellulose-saccharolytic enzymes is crucial for the bioconversion of plant biomass into fuels and chemicals. The integrated mode for producing these enzymes using lignocellulosic feedstocks as natural inducers has a cost advantage; however, enzyme production level is limited because of the solid-state and complex composition of the raw materials. This study aimed to establish an innovative process for the integrated production of lignocellulolytic enzymes using lignocellulosic hydrolysate as the carbon source. The cellulase-producing workhorse Trichoderma reesei was reprogrammed by combinatorial engineering of transcription factors and the introduction of heterologous β-glucosidases. This enabled inducer-independent production of a complete lignocellulose-saccharolytic enzyme cocktail using lignocellulose-derived monosaccharides as sole carbon source. Via continuous feeding of the enzymatic hydrolysate of pretreated corn stover, crude enzymes with filter paper enzyme activity of 60.4 U/ml and β-glucosidase activity of 503 U/ml were produced. Crude fermentation broth can be used directly to prepare hydrolysates, enabling nex-round enzyme production by recycling 3.1% of the hydrolysate. These results demonstrate a closed-loop strategy for integrated enzyme production using internally produced lignocellulosic sugars from biorefinery plants.

Continuous dark-photo fermentative H2 production from synthetic lignocellulose hydrolysate with different photoheterotrophic cultures: Sequential vs. co-culture processes

Integration of dark and photo-fermentation is a promising strategy to efficiently produce renewable hydrogen from different types of waste biomass. In this study, sequential and co-culture dark-photo fermentations were evaluated and compared during continuous operation using synthetic lignocellulose hydrolysate as a substrate. Mixed dark-fermentative bacterial consortium was combined with two different photo-fermentative bacterial cultures: pure culture of Rhodobacter sphaeroides (RSC) and mixed photoheterotrophic consortium (MPC). The sequential process showed higher total H2 yields compared to co-culture fermentation for both RSC and MPC. The highest yield of 4.15 mol H2/molsugar was obtained for the sequential process with RSC at an HRT of 3.5 days and pH 7.5. For the co-culture process, the yield reached 2.88 mol H2/molsugar at pH 7.0 and an HRT of 3.5 days. Complete sugar utilization was observed for dark fermentation. RSC preferred acetate as a carbon source, while MPC preferred butyrate. Photoheterotrophic bacteria, Rhodopseudomonas pseudopalustris and Rhodoplanes piscinae, present in the MPC inoculum were outcompeted by undesirable microorganisms during the continuous process, resulting in low HPR. On the other hand, in both sequential and co-culture processes, a high relative abundance of R. sphaeroides (>40%) was observed in the RSC, indicating effective cooperation between dark and photo bacteria.

Four ways of implementing robustness quantification in strain characterisation

BackgroundIn industrial bioprocesses, microorganisms are generally selected based on performance, whereas robustness, i.e., the ability of a system to maintain a stable performance, has been overlooked due to the challenges in its quantification and implementation into routine experimental procedures. This work presents four ways of implementing robustness quantification during strain characterisation. One Saccharomyces cerevisiae laboratory strain (CEN.PK113-7D) and two industrial strains (Ethanol Red and PE2) grown in seven different lignocellulosic hydrolysates were assessed for growth-related functions (specific growth rate, product yields, etc.) and eight intracellular parameters (using fluorescent biosensors).ResultsUsing flasks and high-throughput experimental setups, robustness was quantified in relation to: (i) stability of growth functions in response to the seven hydrolysates; (ii) stability of growth functions across different strains to establish the impact of perturbations on yeast metabolism; (iii) stability of intracellular parameters over time; (iv) stability of intracellular parameters within a cell population to indirectly quantify population heterogeneity. Ethanol Red was the best-performing strain under all tested conditions, achieving the highest growth function robustness. PE2 displayed the highest population heterogeneity. Moreover, the intracellular environment varied in response to non-woody or woody lignocellulosic hydrolysates, manifesting increased oxidative stress and unfolded protein response, respectively.ConclusionsRobustness quantification is a powerful tool for strain characterisation as it offers novel information on physiological and biochemical parameters. Owing to the flexibility of the robustness quantification method, its implementation was successfully validated at single-cell as well as high-throughput levels, showcasing its versatility and potential for several applications.Graphical

Novel insights into the effects of 5-hydroxymethfurural on genomic instability and phenotypic evolution using a yeast model.

5-Hydroxymethfurural (5-HMF) is naturally found in a variety of foods and beverages and represents a main inhibitor in the lignocellulosic hydrolysates used for fermentation. This study investigated the impact of 5-HMF on the genomic stability and phenotypic plasticity of the yeast Saccharomyces cerevisiae. Using next-generation sequencing technology, we examined the genomic alterations of diploid S. cerevisiae isolates that were subcultured on a medium containing 1.2 g/L 5-HMF. We found that in 5-HMF-treated cells, the rates of chromosome aneuploidy, large deletions/duplications, and loss of heterozygosity were elevated compared with that in untreated cells. 5-HMF exposure had a mild impact on the rate of point mutations but altered the mutation spectrum. Contrary to what was observed in untreated cells, more monosomy than trisomy occurred in 5-HMF-treated cells. The aneuploidy mutant with monosomic chromosome IX was more resistant to 5-HMF than the diploid parent strain because of the enhanced activity of alcohol dehydrogenase. Finally, we found that overexpression of ADH6 and ZWF1 effectively stabilized the yeast genome under 5-HMF stress. Our findings not only elucidated the global effect of 5-HMF on the genomic integrity of yeast but also provided novel insights into how chromosomal instability drives the environmental adaptability of eukaryotic cells.IMPORTANCESingle-cell microorganisms are exposed to a range of stressors in both natural and industrial settings. This study investigated the effects of 5-hydroxymethfurural (5-HMF), a major inhibitor found in baked foods and lignocellulosic hydrolysates, on the chromosomal instability of yeast. We examined the mechanisms leading to the distinct patterns of 5-HMF-induced genomic alterations and discovered that chromosomal loss, typically viewed as detrimental to cell growth under most conditions, can contribute to yeast tolerance to 5-HMF. Our results increased the understanding of how specific stressors stimulate genomic plasticity and environmental adaptation in yeast.

Simultaneous enhancement of lignin-derived inhibitor tolerance and lipid accumulation of oleaginous yeast Trichosporon cutaneum by adaptive evolution

Inhibitory compounds should be removed from the pretreated lignocellulose feedstock before fermentative production of microbial lipid by oleaginous yeast. Biodetoxification effectively degrades furan aldehydes and weak organic acids, but is less efficient against complex and various lignin-derived phenolic aldehydes. Residual phenolics in lignocellulose hydrolysates require a tolerant fermentation strain. This study adopted the long-term adaptive evolution combined with centrifugal enrichment in corn stover hydrolysate to simultaneously improve the phenolic inhibitors tolerance and lipid synthesis of Trichosporon cutaneum. The obtained mutant T. cutaneum MS28 showed significant improved performances of phenolic aldehydes tolerance and lipid accumulation. The final cellulosic lipid production of T. cutaneum MS28 utilizing corn stover reached 33.8 ± 0.1 g/L, approximately 6-fold higher than that of the starting strain. Transcriptional analysis suggested that T. cutaneum MS28 had more active lignocellulose-derived sugars metabolism and lipid synthesis shunts. The intracellular contents of lipid synthesis precursors including NADPH and acetyl-CoA increased 1.7–3.0 folds. This adaptive evolution in lignocellulose hydrolysate combined with centrifugal enrichment is an efficient tool to targeted enhancement of inhibitors tolerance and cellulosic lipid production of oleaginous yeast.

Metabolic engineering of a stable haploid strain derived from lignocellulosic inhibitor tolerant Saccharomyces cerevisiae natural isolate YB-2625

BackgroundSignificant genetic diversity exists across Saccharomyces strains. Natural isolates and domesticated brewery and industrial strains are typically more robust than laboratory strains when challenged with inhibitory lignocellulosic hydrolysates. These strains also contain genes that are not present in lab strains and likely contribute to their superior inhibitor tolerance. However, many of these strains have poor sporulation efficiencies and low spore viability making subsequent gene analysis, further metabolic engineering, and genomic analyses of the strains challenging. This work aimed to develop an inhibitor tolerant haploid with stable mating type from S. cerevisiae YB-2625, which was originally isolated from bagasse.ResultsHaploid spores isolated from four tetrads from strain YB-2625 were tested for tolerance to furfural and HMF. Due to natural mutations present in the HO-endonuclease, all haploid strains maintained a stable mating type. One of the haploids, YRH1946, did not flocculate and showed enhanced tolerance to furfural and HMF. The tolerant haploid strain was further engineered for xylose fermentation by integration of the genes for xylose metabolism at two separate genomic locations (ho∆ and pho13∆). In fermentations supplemented with inhibitors from acid hydrolyzed corn stover, the engineered haploid strain derived from YB-2625 was able to ferment all of the glucose and 19% of the xylose, whereas the engineered lab strains performed poorly in fermentations.ConclusionsUnderstanding the molecular mechanisms of inhibitor tolerance will aid in developing strains with improved growth and fermentation performance using biomass-derived sugars. The inhibitor tolerant, xylose fermenting, haploid strain described in this work has potential to serve as a platform strain for identifying pathways required for inhibitor tolerance, and for metabolic engineering to produce fuels and chemicals from undiluted lignocellulosic hydrolysates.

Recycling enzymatic hydrolysis lignin residues saved cellulase in enzymatic hydrolysis of lignocellulose: An insight from cellulase adsorption mechanism

Enzymatic hydrolysis lignin residues (EHLRs) of lignocellulose usually adsorbs cellulase, which can be recycled and used to replace parts of cellulase in the hydrolysis process. To understand this phenomenon during enzymatic hydrolysis (EH) of sugarcane bagasse (SCB) treated with sulfite (SPORL) and dilute acid (DA), the adsorption characteristics between lignin and cellulase in EHLRs were investigated, focusing on interaction force at molecular level and enzymatic activity. The results revealed that SPORL-EHLR adsorbed more β-glucosidases (β-GLs) through the stronger electrostatic attraction and hydrogen bond force, causing higher cellulase adsorption amount compared to DA-EHLR. Further exploration demonstrated that the cellobiose’s catalytic and binding sites on β-GLs were separated from the binding site of SPORL-EHLR on β-GLs, resulting in minimal inhibition of β-GLs activity when bound to SPORL-EHLR. Furthermore, adding SPORL-EHLR in SCB hydrolysis saved 40% cellulase. This study deepens the understanding of the adsorption behavior between lignin and cellulase.

Installing xylose assimilation and cellodextrin phosphorolysis pathways in obese Yarrowia lipolytica facilitates cost-effective lipid production from lignocellulosic hydrolysates

BackgroundYarrowia lipolytica, one of the most charming chassis cells in synthetic biology, is unable to use xylose and cellodextrins.ResultsHerein, we present work to tackle for the first time the engineering of Y. lipolytica to produce lipids from cellodextrins and xylose by employing rational and combinatorial strategies. This includes constructing a cellodextrin-phosphorolytic Y. lipolytica by overexpressing Neurospora crassa cellodextrin transporter, Clostridium thermocellum cellobiose/cellodextrin phosphorylase and Saccharomyces cerevisiae phosphoglucomutase. The effect of glucose repression on xylose consumption was relieved by installing a xylose uptake facilitator combined with enhanced PPP pathway and increased cytoplasmic NADPH supply. Further enhancing lipid production and interrupting its consumption conferred the obese phenotype to the engineered yeast. The strain is able to co-ferment glucose, xylose and cellodextrins efficiently, achieving a similar μmax of 0.19 h−1, a qs of 0.34 g-s/g-DCW/h and a YX/S of 0.54 DCW-g/g-s on these substrates, and an accumulation of up to 40% of lipids on the sugar mixture and on wheat straw hydrolysate.ConclusionsTherefore, engineering Y. lipolytica capable of assimilating xylose and cellodextrins is a vital step towards a simultaneous saccharification and fermentation (SSF) process of LC biomass, allowing improved substrate conversion rate and reduced production cost due to low demand of external glucosidase.

Environmental assessment of Rhodosporidium toruloides-1588 based oil production using wood hydrolysate and crude glycerol

Rhodosporidium toruloides, an oleaginous yeast, is a potential feedstock for biodiesel production due to its ability to utilize lignocellulosic biomass-derived hydrolysate with a considerably high lipid titer of 50–70 % w/w. Hence, for the first-time environmental assessment of large-scale R. toruloides-based biodiesel production from wood hydrolysate and crude glycerol was conducted. The global warming potential was observed to be 0.67 kg CO2 eq./MJ along with terrestrial ecotoxicity of 1.37 kg 1,4-DCB eq./MJ and fossil depletion of 0.13 kg oil eq./MJ. The highest impacts for global warming (∼45 %) and fossil depletion (∼37 %) are attributed to the use of chloroform for lipid extraction while fuel consumption for transportation contributed more than 50 % to terrestrial ecotoxicity. Further, sensitivity analysis revealed that maximizing biodiesel yield by increasing lipid yield and solid loading could contribute to reduced environmental impacts. In nutshell, this investigation reveals that environmental impact varies with the type of chemical utilized.

Efficient Production of Triacetic Acid Lactone from Lignocellulose Hydrolysate by Metabolically Engineered Yarrowia lipolytica.

Lignocellulose is a promising renewable feedstock for the bioproduction of high-value biochemicals. The poorly expressed xylose catabolic pathway was the bottleneck in the efficient utilization of the lignocellulose feedstock in yeast. Herein, multiple genetic and process engineering strategies were explored to debottleneck the conversion of xylose to the platform chemical triacetic acid lactone (TAL) in Yarrowia lipolytica. We identified that xylose assimilation generating more cofactor NADPH was favorable for the TAL synthesis. pH control improved the expression of acetyl-CoA carboxylase and generated more precursor malonyl-CoA. Combined with the suppression of the lipid synthesis pathway, 5.03 and 4.18 g/L TAL were produced from pure xylose and xylose-rich wheat straw hydrolysate, respectively. Our work removed the bottleneck of the xylose assimilation pathway and effectively upgraded wheat straw hydrolysate to TAL, which enabled us to build a sustainable oleaginous yeast cell factory to cost-efficiently produce green chemicals from low-cost lignocellulose by Y. lipolytica.

Genomic dissection of Niallia sp. for potential application in lignocellulose hydrolysis and bioremediation.

Genus Niallia has recently been separated taxonomic group from the Bacillus based on conserved signature indels in the genome. Unlike bioremediation, its role in plant biomass hydrolysis has not garnered considerable attention. The present study investigates the genomic potential of a novel Niallia sp. CRN 25 for applications in lignocellulose hydrolysis, significant enzyme production, and bioremediation. The CRN 25 strain exhibits xylosidase, cellobiosidase, α-arabinosidase, and α-D-galactosidase activity as 0.03U/ml whereas β-D-glucosidase and glucuronidase as 0.06 U/ml and 0.01U/ml, respectively. Further genome sequencing reveals nine copies of GH43 gene coding for hemicellulose-specific xylanase enzyme attached to the CBM 6 domain for increased processivity. The presence of β-glucosidase and β-galactosidase indicates the possible application of CRN 25 in facilitating the valorization of plant biomass into value-added products. Apart from this, genes of FMN-dependent NADH-azoreductase, cytochrome P450, and nitrate reductase, playing a crucial role in bioremediation processes, were annotated. Biosynthetic gene clusters (BGCs), responsible for synthesizing specialized metabolites of terpenes and lasso peptides, were also found in the genome. Conclusively genomic sketch of Niallia sp. CRN 25 reveals versatile metabolic potential for diverse environmental applications.

Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals.

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.

Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two β-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1Δ gre3Δ), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.