Xylitol Dehydrogenase Activities Research Articles

Metabolic properties of Pachysolen tannophilus mutants producing xylitol and ethanol from D-xylose

Activity of the major enzymes of D-xylose metabolism in the mutants of xylose-utilizing yeasts Pachysolen tannophilus selectively producing xylitol or ethanol was studied. The xylitol-producing strain exhibited low activities of xylitol dehydrogenase, xylose reductase with preferential affinity to NADPH, NAD+-dependent malate dehydrogenase, and cytochrome c oxidase (4.40, 4.80, 1.87, and 0.28 μmol mg−1 min−1, respectively). The cells of the ethanol-producing mutants exhibited elevated activity of NADH/NADPH-xylose reductase, xylitol dehydrogenase, 1-glycerophosphate dehydrogenase, and lactate dehydrogenase to 6.80, 8.60, 4.68, and 16.48 μmol mg−1 min−1, respectively. Effect of the NADPH/NADH imbalance on ethanol production accumulation and xylitol accumulation is discussed.

Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing D-xylose fermentation by recombinant Saccharomyces cerevisiae.

Since uptake of xylose limits its fermentation, we aimed to identify novel sugar transporters from Scheffersomyces stipitis that allow xylose uptake and fermentation by engineered Saccharomyces cerevisiae. An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

Enhanced expression of genes involved in initial xylose metabolism and the oxidative pentose phosphate pathway in the improved xylose-utilizing Saccharomyces cerevisiae through evolutionary engineering

Fermentation of xylose in lignocellulosic hydrolysates by Saccharomycescerevisiae has been achieved through heterologous expression of the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway. However, the fermentation efficiency is far from the requirement for industrial application due to high yield of the byproduct xylitol, low ethanol yield, and low xylose consumption rate. Through evolutionary engineering, an improved xylose-utilizing strain SyBE005 was obtained with 78.3% lower xylitol production and a 2.6-fold higher specific ethanol production rate than those of the parent strain SyBE004, which expressed an engineered NADP(+)-preferring XDH. The transcriptional differences between SyBE005 and SyBE004 were investigated by quantitative RT-PCR. Genes including XYL1, XYL2, and XKS1 in the initial xylose metabolic pathway showed the highest up-regulation in SyBE005. The increased expression of XYL1 and XYL2 correlated with enhanced enzymatic activities of XR and XDH. In addition, the expression level of ZWF1 in the oxidative pentose phosphate pathway increased significantly in SyBE005, indicating an elevated demand for NADPH from XR. Genes involved in the TCA cycle (LAT1, CIT1, CIT2, KGD1, KGD, SDH2) and gluconeogenesis (ICL1, PYC1) were also up-regulated in SyBE005. Genomic analysis revealed that point mutations in transcriptional regulators CYC8 and PHD1 might be responsible for the altered expression. In addition, a mutation (Y89S) in ZWF1 was identified which might improve NADPH production in SyBE005. Our results suggest that increasing the expression of XYL1, XYL2, XKS1, and enhancing NADPH supply are promising strategies to improve xylose fermentation in recombinant S. cerevisiae.

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD(+)/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

Anaerobic xylose fermentation by Spathaspora passalidarum

A cost-effective conversion of lignocellulosic biomass into bioethanol requires that the xylose released from the hemicellulose fraction (20-40% of biomass) can be fermented. Baker's yeast, Saccharomyces cerevisiae, efficiently ferments glucose but it lacks the ability to ferment xylose. Xylose-fermenting yeast such as Pichia stipitis requires accurately controlled microaerophilic conditions during the xylose fermentation, rendering the process technically difficult and expensive. In this study, it is demonstrated that under anaerobic conditions Spathaspora passalidarum showed high ethanol production yield, fast cell growth, and rapid sugar consumption with xylose being consumed after glucose depletion, while P. stipitis was almost unable to utilize xylose under these conditions. It is further demonstrated that for S. passalidarum, the xylose conversion takes place by means of NADH-preferred xylose reductase (XR) and NAD(+)-dependent xylitol dehydrogenase (XDH). Thus, the capacity of S. passalidarum to utilize xylose under anaerobic conditions is possibly due to the balance between the cofactor's supply and demand through this XR-XDH pathway. Only few XRs with NADH preference have been reported so far. 2-Deoxy glucose completely inhibited the conversion of xylose by S. passalidarum under anaerobic conditions, but only partially did that under aerobic conditions. Thus, xylose uptake by S. passalidarum may be carried out by different xylose transport systems under anaerobic and aerobic conditions. The presence of glucose also repressed the enzymatic activity of XR and XDH from S. passalidarum as well as the activities of those enzymes from P. stipitis.

Repression of xylose‐specific enzymes by ethanol inScheffersomyces(Pichia)stipitisand utility of repitching xylose‐grown populations to eliminate diauxic lag

During the fermentation of lignocellulosic hydrolyzates to ethanol by native pentose-fermenting yeasts such as Scheffersomyces (Pichia) stipitis NRRL Y-7124 (CBS 5773) and Pachysolen tannophilus NRRL Y-2460, the switch from glucose to xylose uptake results in a diauxic lag unless process strategies to prevent this are applied. When yeast were grown on glucose and resuspended in mixed sugars, the length of this lag was observed to be a function of the glucose concentration consumed (and consequently, the ethanol concentration accumulated) prior to the switch from glucose to xylose fermentation. At glucose concentrations of 95 g/L, the switch to xylose utilization was severely stalled such that efficient xylose fermentation could not occur. Further investigation focused on the impact of ethanol on cellular xylose transport and the induction and maintenance of xylose reductase and xylitol dehydrogenase activities when large cell populations of S. stipitis NRRL Y-7124 were pre-grown on glucose or xylose and then presented mixtures of glucose and xylose for fermentation. Ethanol concentrations around 50 g/L fully repressed enzyme induction although xylose transport into the cells was observed to be occurring. Increasing degrees of repression were documented between 15 and 45 g/L ethanol. Repitched cell populations grown on xylose resulted in faster fermentation rates, particularly on xylose but also on glucose, and eliminated diauxic lag and stalling during mixed sugar conversion by P. tannophilus or S. stipitis, despite ethanol accumulations in the 60 or 70 g/L range, respectively. The process strategy of priming cells on xylose was key to the successful utilization of high mixed sugar concentrations because specific enzymes for xylose utilization could be induced before ethanol concentration accumulated to an inhibitory level.

Characterization of Bioethanol Production from Hexoses and Xylose by the White Rot Fungus Trametes versicolor

Bioethanol production by white rot fungus (Trametes versicolor), identified from fungal mixture in naturally decomposing wood samples, from hexoses and xylose was characterized. Results showed that T. versicolor can grow in culture, under hypoxic conditions, with various mixtures of hexoses and xylose and only xylose. Xylose was efficiently fermented to ethanol in media containing mixtures of hexoses and xylose, such as MBMC and G11XY11 media (Table 1), yielding ethanol concentrations of 20.0 and 9.02 g/l, respectively, after 354 h of hypoxic culture. Very strong correlations were found between ethanolic fermentation (alcohol dehydrogenase activity and ethanol production), sugar consumption and xylose catabolism (xylose reductase, xylitol dehydrogenase and xylulokinase activities) after 354 h in culture in MBMC medium. In a medium (G11XY11) containing a 1:1 glucose/xylose ratio, fermentation efficiency of total sugars into ethanol was 80% after 354 h.

Enhancement of xylitol production by attenuation of intracellular xylitol dehydrogenase activity in Candida tropicalis

To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62gl(-1)h(-1), respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.

Metabolism of glucose and xylose as single and mixed feed in Debaryomyces nepalensis NCYC 3413: production of industrially important metabolites

Efficient conversion of hexose and pentose (glucose and xylose) by a single strain is a very important factor for the production of industrially important metabolites using lignocellulose as the substrate. The kinetics of growth and polyol production by Debaryomyces nepalensis NCYC 3413 was studied under single and mixed substrate conditions. In the presence of glucose, the strain produced ethanol (35.8 ± 2.3g/l), glycerol (9.0 ± 0.2g/l), and arabitol (6.3 ± 0.2g/l). In the presence of xylose, the strain produced xylitol (38 ± 1.8g/l) and glycerol (18 ± 1.0g/l) as major metabolites. Diauxic growth was observed when the strain was grown with different combinations of glucose/xylose, and glucose was the preferred substrate. The presence of glucose enhanced the conversion of xylose to xylitol. By feeding a mixture of glucose at 100g/l and xylose at 100g/l, it was found that the strain produced a maximum of 72 ± 3g/l of xylitol. A study of important enzymes involved in the synthesis of xylitol (xylose reductase (XR) and xylitol dehydrogenase (XDH)), glycerol (glycerol-3-phosphate dehydrogenase (G3PDH)) and ethanol (alcohol dehydrogenase (ADH)) in cells grown in the presence of glucose and xylose revealed high specific activity of G3PDH and ADH in cells grown in the presence of glucose, whereas high specific activity of XR, XDH, and G3PDH was observed in cells grown in the presence of xylose. To our knowledge, this is the first study to elaborate the glucose and xylose metabolic pathway in this yeast strain.

Co-expression of xylose reductase gene from Candida shehatae and endogenous xylitol dehydrogenase gene in Saccharomyces cerevisiae and the effect of metabolizing xylose to ethanol

The inability of Saccharomyces cerevisiae to utilize xylose is attributed to its inability to convert xylose to xylulose. Low xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in S. cerevisiae are regarded as the reason of blocking the pathway from xylose to xylulose. We had found that Candida shehatae could also be another source for XR gene except Pichia stipitis in the previous study. In this study, we tried to investigate if the expressed XR from C. shehatae could work with the over-expressed endogenous XDH together to achieve the same goal of converting xylose to ethanol in S. cerevisiae. The XR gene (XYL1) from C. shehatae and endogenous XDH gene (XYL2) were both cloned and over-expressed in host S. cerevisiae cell. The specific enzyme activities of XR and XDH were measured and the result of fermentation revealed that the new combination of two enzymes from different sources other than P. stipitis could also coordinate and work with each other and confer xylose utilization ability to S. cerevisiae.

Cosubstrate effect on xylose reductase and xylitol dehydrogenase activity levels, and its consequence on xylitol production by Candida tropicalis

Due to its anticariogenic properties, the five-carbon sugar alcohol xylitol is finding increasing use in food industry as a functional sweetener. Although xylitol is currently manufactured by the chemical reduction of xylose, its biological production would be attractive because of lower costs and better organoleptic features. In xylose-fermenting yeasts, the substrate is directly converted to xylitol by an NAD(P)H-dependent reductase (XR). Xylitol is oxidized in turn by an NAD +-dependent dehydrogenase (XDH) yielding xylulose, which is channelled into the pentose phosphate pathway. In the absence of other carbon sources, a significant fraction of xylitol is used to fuel cell growth. To avoid excess yield loss and maximize xylitol production, a series of mono- and disaccharides were tested as cosubstrates for a hyper-acidophilic strain of Candida tropicalis. Their effect upon XR and XDH specific activity levels was evaluated, and the resulting expression of the inducible pathway and the XR:XDH ratio were related to either non-optimized or optimized yields. Glucose strongly inhibited xylose reduction, whereas galactose stimulated xylitol utilization. On the contrary, the addition of low concentrations of maltose significantly increased biomass growth and xylitol accumulation.

A New Strategy to Improve the Efficiency and Sustainability of Candida parapsilosis Catalyzing Deracemization of (R,S)-1-Phenyl-1,2-Ethanediol Under Non-Growing Conditions: Increase of NADPH Availability

Microbial oxidoreductive systems have been widely used in asymmetric syntheses of optically active alcohols. However, when reused in multi-batch reaction, the catalytic efficiency and sustainability of non-growing cells usually decreased because of continuous consumption of required cofactors during the reaction process. A novel method for NADPH regeneration in cells was proposed by using pentose metabolism in microorganisms. Addition of D-xylose, L-arabinose, or D-ribose to the reaction significantly improved the conversion efficiency of deracemization of racemic 1-phenyl-1,2-ethanediol to (S)-isomer by Candida parapsilosis cells already used once, which afforded the product with high optical purity over 97%e.e. in high yield over 85% under an increased substrate concentration of 15 g/l. Compared with reactions without xylose, xylose added to multi-batch reactions had no influence on the activity of the enzyme catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the non-growing cells with its consumption in each batch. The detection of activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis made xylose metabolism feasible in cells, and the depression of the pentose phosphate pathway inhibitor to this reaction further indicated that xylose facilitated the NADPH-required deracemization through the pentose phosphate pathway in C. parapsilosis. moreover, by investigating the cofactor pool, the xylose addition in reaction batches giving more NADPH, compared with those without xylose, suggested that the higher catalytic efficiency and sustainability of C. parapsilosis non-growing cells had resulted from xylose metabolism recycling NADPH for the deracemization.

Tyr-51 is the proton donor–acceptor for NAD(H)-dependent interconversion of xylose and xylitol by Candida tenuis xylose reductase (AKR2B5)

Substitution of active-site Tyr-51 by Ala (Y51A) disrupted the activity of Candida tenuis xylose reductase by six orders of magnitude. External bromide brought about unidirectional rate enhancement (≈2 × 10 3-fold at 300 mM) for NAD +-dependent xylitol oxidation by Y51A. Activity of the wild-type reductase was dependent on a single ionizable protein group exhibiting a p K of 9.2 ± 0.1 and 7.3 ± 0.3 in the holo-enzyme bound with NADH and NAD +, respectively. This group which had to be protonated for xylose reduction and unprotonated for xylitol oxidation was eliminated in Y51A, consistent with a catalytic acid–base function of Tyr-51. Bromide may complement the xylitol dehydrogenase activity of Y51A by partly restoring the original hydrogen bond between the reactive alcohol and the phenolate of Tyr-51.

Profiles of xylose reductase, xylitol dehydrogenase and xylitol production under different oxygen transfer volumetric coefficient values

AbstractBACKGROUND: Xylitol is a sugar alcohol (polyalcohol) with many interesting properties for pharmaceutical and food products. It is currently produced by a chemical process, which has some disadvantages such as high energy requirement. Therefore microbiological production of xylitol has been studied as an alternative, but its viability is dependent on optimisation of the fermentation variables. Among these, aeration is fundamental, because xylitol is produced only under adequate oxygen availability. In most experiments with xylitol‐producing yeasts, low oxygen transfer volumetric coefficient (KLa) values are used to maintain microaerobic conditions. However, in the present study the use of relatively high KLa values resulted in high xylitol production. The effect of aeration was also evaluated via the profiles of xylose reductase (XR) and xylitol dehydrogenase (XD) activities during the experiments.RESULTS: The highest XR specific activity (1.45 ± 0.21 U mgprotein−1) was achieved during the experiment with the lowest KLa value (12 h−1), while the highest XD specific activity (0.19 ± 0.03 U mgprotein−1) was observed with a KLa value of 25 h−1. Xylitol production was enhanced when KLa was increased from 12 to 50 h−1, which resulted in the best condition observed, corresponding to a xylitol volumetric productivity of 1.50 ± 0.08 gxylitol L−1 h−1 and an efficiency of 71 ± 6.0%.CONCLUSION: The results showed that the enzyme activities during xylitol bioproduction depend greatly on the initial KLa value (oxygen availability). This finding supplies important information for further studies in molecular biology and genetic engineering aimed at improving xylitol bioproduction. Copyright © 2008 Society of Chemical Industry

Efficient bioethanol production from xylose by recombinant saccharomyces cerevisiae requires high activity of xylose reductase and moderate xylulokinase activity

We varied the promoter strength of xylose reductase (XR) gene and the copy number of xylulokinase (XK) gene to determine how XR and XK activities affect the xylose-fermenting abilities of recombinant Saccharomyces cerevisiae expressing xylitol dehydrogenase (XDH). The most enhanced ethanol yield and lowered xylitol yield occurred in strain I-PGK/AUR, which has high activity of both XR and XDH and moderate XK activity.

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae

BackgroundMetabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274→Arg Asn276→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.ResultsAn isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 ± 0.02 h-1) in both strains.ConclusionIntegration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.

Effects of Oxygen Limitation on Xylose Fermentation, Intracellular Metabolites, and Key Enzymes of Neurospora crassa AS3.1602

The effects of oxygen limitation on xylose fermentation of Neurospora crassa AS3.1602 were studied using batch cultures. The maximum yield of ethanol was 0.34 g/g at oxygen transfer rate (OTR) of 8.4 mmol/L.h. The maximum yield of xylitol was 0.33 g/g at OTR of 5.1 mmol/L.h. Oxygen limitation greatly affected mycelia growth and xylitol and ethanol productions. The specific growth rate (micro) decreased 82% from 0.045 to 0.008 h(-1) when OTR changed from 12.6 to 8.4 mmol/L.h. Intracellular metabolites of the pentose phosphate pathway, glycolysis, and tricarboxylic acid cycle were determined at various OTRs. Concentrations of most intracellular metabolites decreased with the increase in oxygen limitation. Intracellular enzyme activities of xylose reductase, xylitol dehydrogenase, and xylulokinase, the first three enzymes in xylose metabolic pathway, decreased with the increase in oxygen limitation, resulting in the decreased xylose uptake rate. Under all tested conditions, transaldolase and transketolase activities always maintained at low levels, indicating a great control on xylose metabolism. The enzyme of glucose-6-phosphate dehydrogenase played a major role in NADPH regeneration, and its activity decreased remarkably with the increase in oxygen limitation.

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomyces cerevisiae

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.

Effect of glucose:xylose ratio on xylose reductase and xylitol dehydrogenase activities from Candida guilliermondii in sugarcane bagasse hydrolysate

AbstractThe influence of glucose on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzyme activity was evaluated from sugarcane bagasse hydrolysate fermentations with different glucose:xylose ratios (1:25, 1:12, 1:5 and 1:2.5) by employing an inoculum of Candida guilliermondii grown in media containing glucose, a mixture of glucose and xylose, or only xylose as carbon sources. According to the results, the glucose:xylose ratio affected positively this bioconversion and a correlation was not observed between the favourable conditions for xylitol production and the XR and XDH activities. Also, the results were influenced not only by the glucose:xylose ratio in the fermentation medium, but also by the carbon source employed in the growth medium of the inoculum. The optimum condition for xylitol production by C. guilliermondii in sugarcane bagasse hemicellulosic hydrolysate should use hydrolysate with a 1:5 glucose:xylose ratio and inoculum grown in medium containing xylose as the only carbon source. Copyright © 2006 Society of Chemical Industry