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

BACKGROUND: Significant 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. RESULTS: Haploid 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. CONCLUSIONS: Understanding 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.

Determining mating type and ploidy in Rhodotorula toruloides and its effect on growth on sugars from lignocellulosic biomass.

Rhodotorula toruloides is being developed for the use in industrial biotechnology processes because of its favorable physiology. This includes its ability to produce and store large amounts of lipids in the form of intracellular lipid bodies. Nineteen strains were characterized for mating type, ploidy, robustness for growth, and accumulation of lipids on inhibitory switchgrass hydrolysate (SGH). Mating type was determined using a novel polymerase chain reaction (PCR)-based assay, which was validated using the classical microscopic test. Three of the strains were heterozygous for mating type (A1/A2). Ploidy analysis revealed a complex pattern. Two strains were triploid, eight haploid, and eight either diploid or aneuploid. Two of the A1/A2 strains were compared to their parents for growth on 75%v/v concentrated SGH. The A1/A2 strains were much more robust than the parental strains, which either did not grow or had extended lag times. The entire set was evaluated in 60%v/v SGH batch cultures for growth kinetics and biomass and lipid production. Lipid titers were 2.33-9.40 g/L with a median of 6.12 g/L, excluding the two strains that did not grow. Lipid yields were 0.032-0.131 (g/g) and lipid contents were 13.5-53.7% (g/g). Four strains had significantly higher lipid yields and contents. One of these strains, which had among the highest lipid yield in this study (0.131 ± 0.007 g/g), has not been previously described in the literature. SUMMARY: The yeast Rhodotorula toruloides was used to produce oil using sugars extracted from a bioenergy grass.

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Increased expression of the fluorescent reporter protein ymNeonGreen in Saccharomyces cerevisiae by reducing RNA secondary structure near the start codon.

Expression of a new fluorescent reporter protein called mNeonGreen, that is not based on the jellyfish green fluorescent protein (GFP) sequence, shows increased brightness and folding speed compared to enhanced GFP. However, in vivo brightness of mNeonGreen and its yeast-optimized variant ymNeonGreen in S. cerevisiae is lower than expected, limiting the use of this high quantum yield, fast-folding reporter in budding yeast. This study shows that secondary RNA structure near the start codon in the ymNeonGreen ORF inhibits expression in S. cerevisiae. Removing secondary structure, without altering the ymNeonGreen protein sequence, led to a 2 and 4-fold increase in fluorescence when expressed in S. cerevisiae and E. coli, respectively. In S. cerevisiae, increased fluorescence was seen with strong and weak promoters and led to higher transcript levels suggesting greater transcript stability and improved expression in the absence of stable secondary RNA structure near the start codon.

Performance of xylose-fermenting yeasts in oat and soybean hulls hydrolysate and improvement of ethanol production using immobilized cell systems.

We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg-1) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersomyces, Sugiymaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidarum UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats® (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g-1, and productivities of 0.39 and 0.29 g (L h)-1, respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEH-CYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g-1 and productivities from 0.14 to 0.23 g (L h)-1. These results warrant further research using Spathaspora yeasts for second-generation ethanol production.

Impact of stress-response related transcription factor overexpression on lignocellulosic inhibitor tolerance of Saccharomyces cerevisiae environmental isolates.

Numerous transcription factor genes associated with stress response are upregulated in Saccharomyces cerevisiae grown in the presence of inhibitors that result from pretreatment processes to unlock simple sugars from biomass. To determine if overexpression of transcription factors could improve inhibitor tolerance in robust S. cerevisiae environmental isolates as has been demonstrated in S. cerevisiae haploid laboratory strains, transcription factors were overexpressed at three different expression levels in three S. cerevisiae environmental isolates. Overexpression of the YAP1 transcription factor in these isolates did not lead to increased growth rate or reduced lag in growth, and in some cases was detrimental, when grown in the presence of either lignocellulosic hydrolysates or furfural and 5-hydroxymethyl furfural individually. The expressed Yap1p localized correctly and the expression construct improved inhibitor tolerance of a laboratory strain as previously reported, indicating that lack of improvement in the environmental isolates was due to factors other than nonfunctional expression constructs or mis-folded protein. Additional stress-related transcription factors, MSN2, MSN4, HSF1, PDR1, and RPN4, were also overexpressed at three different expression levels and all failed to improve inhibitor tolerance. Transcription factor overexpression alone is unlikely to be a viable route toward increased inhibitor tolerance of robust environmental S. cerevisiae strains.

Defining the eco-enzymological role of the fungal strain Coniochaeta sp. 2T2.1 in a tripartite lignocellulolytic microbial consortium.

Coniochaeta species are versatile ascomycetes that have great capacity to deconstruct lignocellulose. Here, we explore the transcriptome of Coniochaeta sp. strain 2T2.1 from wheat straw-driven cultures with the fungus growing alone or as a member of a synthetic microbial consortium with Sphingobacterium multivorum w15 and Citrobacter freundii so4. The differential expression profiles of carbohydrate-active enzymes indicated an onset of (hemi)cellulose degradation by 2T2.1 during the initial 24 hours of incubation. Within the tripartite consortium, 63 transcripts of strain 2T2.1 were differentially expressed at this time point. The presence of the two bacteria significantly upregulated the expression of one galactose oxidase, one GH79-like enzyme, one multidrug transporter, one laccase-like protein (AA1 family) and two bilirubin oxidases, suggesting that inter-kingdom interactions (e.g. amensalism) take place within this microbial consortium. Overexpression of multicopper oxidases indicated that strain 2T2.1 may be involved in lignin depolymerization (a trait of enzymatic synergism), while S. multivorum and C. freundii have the metabolic potential to deconstruct arabinoxylan. Under the conditions applied, 2T2.1 appears to be a better degrader of wheat straw when the two bacteria are absent. This conclusion is supported by the observed suppression of its (hemi)cellulolytic arsenal and lower degradation percentages within the microbial consortium.

Genome expansion by allopolyploidization in the fungal strain Coniochaeta 2T2.1 and its exceptional lignocellulolytic machinery.

BACKGROUND: Particular species of the genus Coniochaeta (Sordariomycetes) exhibit great potential for bioabatement of furanic compounds and have been identified as an underexplored source of novel lignocellulolytic enzymes, especially Coniochaeta ligniaria. However, there is a lack of information about their genomic features and metabolic capabilities. Here, we report the first in-depth genome/transcriptome survey of a Coniochaeta species (strain 2T2.1). RESULTS: The genome of Coniochaeta sp. strain 2T2.1 has a size of 74.53 Mbp and contains 24,735 protein-encoding genes. Interestingly, we detected a genome expansion event, resulting ~ 98% of the assembly being duplicated with 91.9% average nucleotide identity between the duplicated regions. The lack of gene loss, as well as the high divergence and strong genome-wide signatures of purifying selection between copies indicates that this is likely a recent duplication, which arose through hybridization between two related Coniochaeta-like species (allopolyploidization). Phylogenomic analysis revealed that 2T2.1 is related Coniochaeta sp. PMI546 and Lecythophora sp. AK0013, which both occur endophytically. Based on carbohydrate-active enzyme (CAZy) annotation, we observed that even after in silico removal of its duplicated content, the 2T2.1 genome contains exceptional lignocellulolytic machinery. Moreover, transcriptomic data reveal the overexpression of proteins affiliated to CAZy families GH11, GH10 (endoxylanases), CE5, CE1 (xylan esterases), GH62, GH51 (α-l-arabinofuranosidases), GH12, GH7 (cellulases), and AA9 (lytic polysaccharide monoxygenases) when the fungus was grown on wheat straw compared with glucose as the sole carbon source. CONCLUSIONS: We provide data that suggest that a recent hybridization between the genomes of related species may have given rise to Coniochaeta sp. 2T2.1. Moreover, our results reveal that the degradation of arabinoxylan, xyloglucan and cellulose are key metabolic processes in strain 2T2.1 growing on wheat straw. Different genes for key lignocellulolytic enzymes were identified, which can be starting points for production, characterization and/or supplementation of enzyme cocktails used in saccharification of agricultural residues. Our findings represent first steps that enable a better understanding of the reticulate evolution and "eco-enzymology" of lignocellulolytic Coniochaeta species.

Development and characterization of vectors for tunable expression of both xylose-regulated and constitutive gene expression in Saccharomyces yeasts.

Synthetic hybrid promoters for xylose-regulated gene expression in the yeast Saccharomyces cerevisiae have recently been developed. However, the narrow range of expression level from these new hybrid promoters limits their utility for pathway optimization in engineered strains. To expand the range of xylose-regulated gene expression, a series of expression vectors was created using a xylose derepressible promoter (PXYL) and varied termination regions from several S. cerevisiae genes. The new set of vectors showed a 26-fold range of gene expression under inducing conditions and a 13-fold average induction due to xylose. In the presence of the XylR repressor, gene expression was very sensitive to xylose concentration and full induction was observed with 0.10 g/L xylose. In the absence of XylR, gene expression from the vector set did not require xylose and was constitutive over a similar 26-fold range of expression. These results show that the vectors are extremely versatile for constitutive expression as well as for fine-tuning both the timing of gene expression and expression level using xylose as an inexpensive inducer.

Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium.

Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

Engineering Candida phangngensis-an oleaginous yeast from the Yarrowia clade-for enhanced detoxification of lignocellulose-derived inhibitors and lipid overproduction.

Candida phangngensis is an ascomycetous yeast and a phylogenetic relative of the industrial workhorse Yarrowia lipolytica. Here, we report that genetic tools already established for use in the latter organism-including promoters, expression vectors, antibiotic resistance genes, a transformation protocol, and the Cre/lox system for marker recycle-can be transferred to the newer member of the Yarrowia clade with little or no need for modifications. Using these tools, we engineered C. phangngensis for improved cellulosic lipid production by introducing two heterologous yeast genes. First, overexpression of Saccharomyces cerevisiae ADH6 enhanced in situ detoxification of aldehyde fermentation inhibitors that are generated during biomass pretreatment (e.g. furfural). Subsequently, Y. lipolytica DGA1 expression boosted lipid accumulation in C. phangngensis by pulling additional carbon flux into the triacylglycerol synthesis pathway. In acid-pretreated switchgrass hydrolysate cultures, the final engineered strain JQCP04 showed a 58% decrease in lag time and a 32% increase in lipid titer as compared to wild-type PT1-17. Furthermore, we expect that this study will generate new interest in the highly oleaginous yeast C. phangngensis, which is closely related to a safe, industrial species, and is shown here to be quite amenable for genetic manipulation.

Association of improved oxidative stress tolerance and alleviation of glucose repression with superior xylose-utilization capability by a natural isolate of Saccharomyces cerevisiae.

BACKGROUND: Saccharomyces cerevisiae wild strains generally have poor xylose-utilization capability, which is a major barrier for efficient bioconversion of lignocellulosic biomass. Laboratory adaption is commonly used to enhance xylose utilization of recombinant S. cerevisiae. Apparently, yeast cells could remodel the metabolic network for xylose metabolism. However, it still remains unclear why natural isolates of S. cerevisiae poorly utilize xylose. Here, we analyzed a unique S. cerevisiae natural isolate YB-2625 which has superior xylose metabolism capability in the presence of mixed-sugar. Comparative transcriptomic analysis was performed using S. cerevisiae YB-2625 grown in a mixture of glucose and xylose, and the model yeast strain S288C served as a control. Global gene transcription was compared at both the early mixed-sugar utilization stage and the latter xylose-utilization stage. RESULTS: Genes involved in endogenous xylose-assimilation (XYL2 and XKS1), gluconeogenesis, and TCA cycle showed higher transcription levels in S. cerevisiae YB-2625 at the xylose-utilization stage, when compared to the reference strain. On the other hand, transcription factor encoding genes involved in regulation of glucose repression (MIG1, MIG2, and MIG3) as well as HXK2 displayed decreased transcriptional levels in YB-2625, suggesting the alleviation of glucose repression of S. cerevisiae YB-2625. Notably, genes encoding antioxidant enzymes (CTT1, CTA1, SOD2, and PRX1) showed higher transcription levels in S. cerevisiae YB-2625 in the xylose-utilization stage than that of the reference strain. Consistently, catalase activity of YB-2625 was 1.9-fold higher than that of S. cerevisiae S288C during the xylose-utilization stage. As a result, intracellular reactive oxygen species levels of S. cerevisiae YB-2625 were 43.3 and 58.6% lower than that of S288C at both sugar utilization stages. Overexpression of CTT1 and PRX1 in the recombinant strain S. cerevisiae YRH396 deriving from S. cerevisiae YB-2625 increased cell growth when xylose was used as the sole carbon source, leading to 13.5 and 18.1%, respectively, more xylose consumption. CONCLUSIONS: Enhanced oxidative stress tolerance and relief of glucose repression are proposed to be two major mechanisms for superior xylose utilization by S. cerevisiae YB-2625. The present study provides insights into the innate regulatory mechanisms underlying xylose utilization in wild-type S. cerevisiae, which benefits the rapid development of robust yeast strains for lignocellulosic biorefineries.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

Draft Genome Sequence of the d-Xylose-Fermenting Yeast Spathaspora xylofermentans UFMG-HMD23.3.

Here, we report the draft genome sequence of the yeast Spathaspora xylofermentans UFMG-HMD23.3 (=CBS 12681), a d-xylose-fermenting yeast isolated from the Amazonian forest. The genome consists of 298 contigs, with a total size of 15.1 Mb, including the mitochondrial genome, and 5,948 predicted genes.

Draft Genome Sequence of Coniochaeta ligniaria NRRL 30616, a Lignocellulolytic Fungus for Bioabatement of Inhibitors in Plant Biomass Hydrolysates.

Here, we report the first draft genome sequence (42.38 Mb containing 13,657 genes) of Coniochaeta ligniaria NRRL 30616, an ascomycete with biotechnological relevance in the bioenergy field given its high potential for bioabatement of toxic furanic compounds in plant biomass hydrolysates and its capacity to degrade lignocellulosic material.

A Synthetic Hybrid Promoter for Xylose-Regulated Control of Gene Expression in Saccharomyces Yeasts.

Metabolism of non-glucose carbon sources is often highly regulated at the transcriptional and post-translational levels. This level of regulation is lacking in Saccharomyces cerevisiae strains engineered to metabolize xylose. To better control transcription in S. cerevisiae, the xylose-dependent, DNA-binding repressor (XylR) from Caulobacter crescentus was used to block transcription from synthetic promoters based on the constitutive Ashbya gossypii TEF promoter. The new hybrid promoters were repressed in the absence of xylose and showed up to a 25-fold increase in the presence of xylose. Activation of the promoter was highly sensitive to xylose with activity seen at concentrations below 2 µM xylose. These new xylose-inducible promoters allow improved control of gene expression for engineered strains of Saccharomyces yeasts.

Triacetic acid lactone production in industrial Saccharomyces yeast strains.

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into 13 industrial yeast strains of varied genetic background. TAL production varied 63-fold between strains when compared in batch culture with glucose. Ethanol, acetate, and glycerol were also tested as potential carbon sources. Batch cultures with ethanol medium produced the highest titers. Therefore, fed-batch cultivation with ethanol feed was assayed for TAL production in bioreactors, producing our highest TAL titer, 5.2 g/L. Higher feed rates resulted in a loss of TAL and subsequent production of additional TAL side products. Finally, TAL efflux was measured and TAL is actively exported from S. cerevisiae cells. Percent yield for all strains was low, indicating that further metabolic engineering of the strains is required.

Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL-pretreated lodgepole pine.

The performances of five yeast strains under three levels of toxicity were evaluated using hydrolysates from lodgepole pine pretreated by Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL). The highest level of toxicity was represented by the whole pretreated biomass slurry, while intermediate toxicity was represented by the hydrolysate with partial loading of pretreatment spent liquor. The zero toxicity was represented using the enzymatic hydrolysate produced from thoroughly washed SPORL lodgepole pine solids. The results indicate that strains D5A and YRH400 can tolerate the whole pretreated biomass slurry to produce 90.1 and 73.5% theoretical ethanol yield. Strains Y1528, YRH403, and FPL450 did not grow in whole hydrolysate cultures and were observed to have lower ethanol productivities than D5A and YRH400 on the hydrolysate with intermediate toxicity. Both YRH400 and YRH403 were genetically engineered for xylose fermentation but were not able to consume xylose efficiently in hydrolysate.

Conversion of switchgrass to ethanol using dilute ammonium hydroxide pretreatment: influence of ecotype and harvest maturity.

Switchgrass (Panicum virgatum L.) is a perennial C4 grass that is being developed as a bioenergy crop because it has high production yields and suitable agronomic traits. Five switchgrass biomass samples from upland and lowland switchgrass ecotypes harvested at different stages or maturity were used in this study. Switchgrass samples contained 317.0-385.0 g glucans/kg switchgrass dry basis (db) and 579.3-660.2 g total structural carbohydrates/kg switchgrass, db. Carbohydrate contents were greater for the upland ecotype versus lowland ecotype and increased with harvest maturity. Pretreatment of switchgrass with dilute ammonium hydroxide (8% w/w ammonium loading) at 170 degrees C for 20 min was determined to be effective for preparing switchgrass for enzymatic conversion to monosaccharides; glucose recoveries were 66.9-90.5% and xylose recoveries 60.1-84.2% of maximum and decreased with increased maturity at harvest. Subsequently, pretreated switchgrass samples were converted to ethanol by simultaneous saccharification and fermentation using engineered xylose-fermenting Saccharomyces cerevisiae strain YRH400. Ethanol yields were 176.2-202.01/Mg of switchgrass (db) and followed a similar trend as observed for enzymatic sugar yields.

Growth and fermentation of D-xylose by Saccharomyces cerevisiae expressing a novel D-xylose isomerase originating from the bacterium Prevotella ruminicola TC2-24.

BACKGROUND: Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation. RESULTS: Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain's ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 µmole/min/mg protein and a Km of 34 mM. CONCLUSION: This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.