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 ß-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 ß-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.

Molecular evolutionary insight of structural zinc atom in yeast xylitol dehydrogenases and its application in bioethanol production by lignocellulosic biomass.

Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into D-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates.

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose.

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.

Metabolic and Transcriptional Analysis of Recombinant Saccharomyces Cerevisiae for Xylose Fermentation: A Feasible and Efficient Approach.

Lignocellulose is an abundant xylose-containing biomass found in agricultural wastes, and has arisen as a suitable alternative to fossil fuels for the production of bioethanol. Although Saccharomyces cerevisiae has been thoroughly used for the production of bioethanol, its potential to utilize lignocellulose remains poorly understood. In this work, xylose-metabolic genes of Pichia stipitis and Candida tropicalis, under the control of different promoters, were introduced into S. cerevisiae. RNA-seq analysis was use to examine the response of S. cerevisiae metabolism to the introduction of xylose-metabolic genes. The use of the PGK1 promoter to drive xylitol dehydrogenase (XDH) expression, instead of the TEF1 promoter, improved xylose utilization in "XR-pXDH" strain by overexpressing xylose reductase (XR) and XDH form C. tropicalis, enhancing the production of xylitol (13.66 ± 0.54 g/L after 6 days fermentation). Overexpression of xylulokinase and XR/XDH from P. stipitis remarkably decreased xylitol accumulation (1.13 ± 0.06 g/L and 0.89 ± 0.04 g/L xylitol, respectively) and increased ethanol production (196.14 % and 148.50 % increases during the xylose utilization stage, respectively), in comparison with the results of XR-pXDH. This result may be produced due to the enhanced xylose transport, Embden-Meyerhof and pentose phosphate pathways, as well as alleviated oxidative stress. The low xylose consumption rate in these recombinant as well as alleviated strains comparing with P. stipitis and C. tropicalis may be explained by the insufficient supplementation of NADPH and NAD +. The results obtained in this work provide new insights on the potential utilization of xylose using bioengineered S. cerevisiae strains.

Cloning, expression, and characterization of an arabitol dehydrogenase and coupled with NADH oxidase for effective production of L-xylulose.

A novel arabitol dehydrogenase (ArDH) gene was cloned from a bacterium named Aspergillus nidulans and expressed heterologously in Escherichia coli. The purified ArDH exhibited the maximal activity in pH 9.5 Tris-HCl buffer at 40 °C, showed Km and Vmax of 1.2 mg/mL and 9.1 U/mg, respectively. The ArDH was used to produce the L-xylulose and coupled with the NADH oxidase (Nox) for the regeneration of NAD+. In further optimization, a high conversion of 84.6% in 8 hours was achieved under the optimal conditions: 20 mM of xylitol, 100 µM NAD+ in pH 9.0 Tris-HCl buffer at 30 °C. The results indicated the coupling system with cofactor regeneration provides a promising approach for L-xylulose production from xylitol.

Conditional dependence of enzyme cascade reaction efficiency on the inter-enzyme distance.

A dual-enzyme cascade, xylitol dehydrogenase and xylulose kinase, derived from the xylose metabolic pathway, was constructed on a three-dimensional DNA scaffold which exhibited a dynamic shape transition from an open state to a closed hexagonal prism. Evaluation of the cascade reaction efficiencies in the open and closed states revealed little to no inter-enzyme distance dependence, presumably due to the far larger catalytic constant of the downstream enzyme. The inter-enzyme distance was not the dominant factor for cascade efficiency when the kinetic parameters of the cascade enzymes were imbalanced with the highly efficient downstream enzyme.

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor.

BACKGROUND: Commercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol. RESULTS: The xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L- 1 xylitol in 120 h from 300 g L- 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L- 1 h- 1 and the conversion rate was 92.40 %. CONCLUSIONS: In conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes.

The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of the DNA scaffold, such as the modulation of the local pH near enzymes. In this study, two types of enzymes with optimal activity at pH 6 and 8 equally displayed significant catalytic enhancements on the DNA scaffold surface. By using a ratiometric pH indicator, a lower local pH shift of 0.8 was observed near the DNA scaffold surface. The postulated local pH change near the DNA scaffold surface is unlikely to play a general role in enhancing the activity of the scaffolded enzymes.

Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase and optimization for enhanced L-xylulose biotransformation from xylitol.

L-Xylulose is a rare ketopentose which inhibits α-glucosidase and is an indicator of hepatitis or liver cirrhosis. This pentose is also a precursor of other rare sugars such as L-xylose, L-ribose or L-lyxose. Recombinant E. coli expressing xylitol-4-dehydrogenase gene of Pantoea ananatis was constructed. A cost-effective culture media were used for L-xylulose production using the recombinant E. coli strain constructed. Response surface methodology was used to optimize these media components for L-xylulose production. A high conversion rate of 96.5% was achieved under an optimized pH and temperature using 20 g/L xylitol, which is the highest among the reports. The recombinant E. coli cells expressing the xdh gene were immobilized in calcium alginate to improve recycling of cells. Effective immobilization was achieved with 2% (w/v) sodium alginate and 3% (w/v) calcium chloride. The immobilized E. coli cells retained good stability and enzyme activity for 9 batches with conversion between 53 and 92% which would be beneficial for economical production of L-xylulose.

Improving xylitol yield by deletion of endogenous xylitol-assimilating genes: a study of industrial Saccharomyces cerevisiae in fermentation of glucose and xylose.

Engineered Saccharomyces cerevisiae can reduce xylose to xylitol. However, in S.cerevisiae, there are several endogenous enzymes including xylitol dehydrogenase encoded by XYL2, sorbitol dehydrogenases encoded by SOR1/SOR2 and xylulokinase encoded by XKS1 may lead to the assimilation of xylitol. In this study, to increase xylitol accumulation, these genes were separately deleted through CRISPR/Cas9 system. Their effects on xylitol yield of an industrial S. cerevisiae CK17 overexpressing Candida tropicalis XYL1 (encoding xylose reductase) were investigated. Deletion of SOR1/SOR2 or XKS1 increased the xylitol yield in both batch and fed-batch fermentation with different concentrations of glucose and xylose. The analysis of the transcription level of key genes in the mutants during fed-batch fermentation suggests that SOR1/SOR2 are more crucially responsible for xylitol oxidation than XYL2 under the genetic background of S.cerevisiae CK17. The deletion of XKS1 gene could also weaken SOR1/SOR2 expression, thereby increasing the xylitol accumulation. The XKS1-deleted strain CK17ΔXKS1 produced 46.17 g/L of xylitol and reached a xylitol yield of 0.92 g/g during simultaneous saccharification and fermentation (SSF) of pretreated corn stover slurry. Therefore, the deletion of XKS1 gene provides a promising strategy to meet the industrial demands for xylitol production from lignocellulosic biomass.

Different transcriptional responses of haploid and diploid S. cerevisiae strains to changes in cofactor preference of XR.

BACKGROUND: Xylitol accumulation is a major barrier for efficient ethanol production through heterologous xylose reductase-xylitol dehydrogenase (XR-XDH) pathway in recombinant Saccharomyces cerevisiae. Mutated NADH-preferring XR is usually employed to alleviate xylitol accumulation. However, it remains unclear how mutated XR affects the metabolic network for xylose metabolism. In this study, haploid and diploid strains were employed to investigate the transcriptional responses to changes in cofactor preference of XR through RNA-seq analysis during xylose fermentation. RESULTS: For the haploid strains, genes involved in xylose-assimilation (XYL1, XYL2, XKS1), glycolysis, and alcohol fermentation had higher transcript levels in response to mutated XR, which was consistent with the improved xylose consumption rate and ethanol yield. For the diploid strains, genes related to protein biosynthesis were upregulated while genes involved in glyoxylate shunt were downregulated in response to mutated XR, which might contribute to the improved yields of biomass and ethanol. When comparing the diploids with the haploids, genes involved in glycolysis and MAPK signaling pathway were significantly downregulated, while oxidative stress related transcription factors (TFs) were significantly upregulated, irrespective of the cofactor preference of XR. CONCLUSIONS: Our results not only revealed the differences in transcriptional responses of the diploid and haploid strains to mutated XR, but also provided underlying basis for better understanding the differences in xylose metabolism between the diploid and haploid strains.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering.

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins ß actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

Valorization of apple pomace using bio-based technology for the production of xylitol and 2G ethanol.

Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35 g L-1 in 96 h), while K. marxianus produced ethanol as the main product (10.47 g L-1 in 24 h) and xylitol as byproduct (9.10 g L-1 xylitol in 96 h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24 h of fermentation with C. guilliermondii (0.23 and 0.53 U/mgprot, respectively) and with K. marxianus (0.08 e 0.08 U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.

Transcription Analysis of Recombinant Trichoderma reesei HJ-48 to Compare the Molecular Basis for Fermentation of Glucose and Xylose.

Profiling the transcriptome changes involved in xylose metabolism by the fungus Trichoderma reesei allows for the identification of potential targets for ethanol production processing. In the present study, the transcriptome of T. reesei HJ-48 grown on xylose versus glucose was analyzed using nextgeneration sequencing technology. During xylose fermentation, numerous genes related to central metabolic pathways, including xylose reductase (XR) and xylitol dehydrogenase (XDH), were expressed at higher levels in T. reesei HJ-48. Notably, growth on xylose did not fully repress the genes encoding enzymes of the tricarboxylic acid and respiratory pathways. In addition, increased expression of several sugar transporters was observed during xylose fermentation. This study provides a valuable dataset for further investigation of xylose fermentation and provides a deeper insight into the various genes involved in this process.

Photosynthetic Reduction of Xylose to Xylitol Using Cyanobacteria.

Photosynthetic generation of reducing power makes cyanobacteria an attractive host for biochemical reduction compared to cell-free and heterotrophic systems, which require burning of additional resources for the supply of reducing equivalent. Here, using xylitol synthesis as an example, efficient uptake and reduction of xylose photoautotrophically in Synechococcus elongatus PCC7942 are demonstrated upon introduction of an effective xylose transporter from Escherichia coli (Ec-XylE) and the NADPH-dependent xylose reductase from Candida boidinii (Cb-XR). Simultaneous activation of xylose uptake and matching of cofactor specificity enabled an average xylitol yield of 0.9 g g-1 xylose and a maximum productivity of about 0.15 g L-1 day-1 OD-1 with increased level of xylose supply. While long-term cellular maintenance still appears challenging, high-density conversion of xylose to xylitol using concentrated resting cell further pushes the titer of xylitol formation to 33 g L-1 in six days with 85% of maximum theoretical yield. While the results show that the unknown dissipation of xylose can be minimized when coupled to a strong reaction outlet, it remains to be the major hurdle hampering the yield despite the reported inability of cyanobacteria to metabolize xylose.

Cloning and functional characterization of xylitol dehydrogenase genes from Issatchenkia orientalis and Torulaspora delbrueckii.

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.

Characterisation of a catalytic triad and reaction selectivity in the dual mechanism of the catalyse hydride transfer in xylitol phosphate dehydrogenase.

Xylitol is a high-value low-calorie sweetener used as sugar substitute in food and pharmaceutical industry. Xylitol phosphate dehydrogenase (XPDH) catalyses the conversion of d-xylulose 5-phosphate (XU5P) and d-ribulose 5-phosphate (RU5P) to xylitol and ribitol respectively in the presence of nicotinamide adenine dinucleotide hydride (NADH). Although these enzymes have been shown to produce xylitol and ribitol, there is an incomplete understanding of the mechanism of the catalytic events of these reactions and the detailed mechanism has yet to be elucidated. The main goal of this work is to analyse the conformational changes of XPDH-bound ligands such as zinc, NADH, XU5P, and RU5P to elucidate the key amino acids involved in the substrate binding. In silico modelling, comparative molecular dynamics simulations, interaction analysis and conformational study were carried out on three XPDH enzymes of the Medium-chain dehydrogenase (MDR) family in order to elucidate the atomistic details of conformational transition, especially on the open and closed state of XPDH. The analysis also revealed the possible mechanism of substrate specificity that are responsible in the catalyse hydride transfer are the residues His58 and Ser39 which would act as the proton donor for reduction of XU5P and RU5P respectively. The structural comparison and MD simulations displayed a significant difference in the conformational dynamics of the catalytic and coenzyme loops between Apo and XPDH-complexes and highlight the contribution of newly found triad residues. This study would assist future mutagenesis study and enzyme modification work to increase the catalysis efficiency of xylitol production in the industry.

Exploring links between antisense RNAs and pathogenesis in Ustilago maydis through transcript and gene characterization.

Biotrophic basidiomycete plant pathogens cause billions of dollars in losses to cereal crops annually. The model for this group of fungi is the corn smut pathogen Ustilago maydis. Annotation of its genome identified antisense RNAs (asRNAs) complementary to over half of the coded mRNAs, some of which are present at high levels in teliospores but detected at very low levels or not at all in other cell types, suggesting they have a function in the teliospore or during teliospore formation. Expression of three such asRNAs (as-UMAG_02150, ncRNA1, and as-UMAG_02151) is controlled by two adjacent genomic regions. Deletion of these regions increased transcript levels of all three asRNAs and attenuated pathogenesis. This study investigated the reason for this marked reduction in pathogenesis by: (1) using deletion analyses to assess the involvement of genes, complementary to the asRNAs, in pathogenesis; (2) determining that one of the linked genes encodes a putative xylitol dehydrogenase; and (3) identifying and functionally characterizing asRNAs that could influence expression of protein-coding genes. The results presented suggest that the influence of the asRNAs on pathogenesis occurs through their action at unlinked loci.

High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains.

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.