Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.     

Optimized extraction by cetyl trimethyl ammonium bromide reversed micelles of xylose reductase and xylitol dehydrogenase from Candida guilliermondii homogenate

The intracellular enzymes xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugar cane bagasse hydrolysate, were separated by reversed micelles of cetyl trimethyl ammonium bromide (CTAB) cationic surfactant. An experimental design was employed to optimize the extraction conditions of both enzymes. Under these conditions (temperature = 5 degree C, hexanol: isooctane proportion = 5% (v/v), 22 %, surfactant concentration = 0.15M, pH = 7.0 and electrical conductivity = 14 mScm(-1)) recovery values of about 100 and 80% were achieved for the enzymes XR and XD, respectively. The purity of XR and XD increased 5.6- and 1.8-fold, respectively. The extraction process caused some structural modifications in the enzymes molecules, as evidenced by the alteration of K(M) values determined before and after extraction, either in regard to the substrate (up 35% for XR and down 48% for XD) or cofactor (down 29% for XR and up 11% for XD). However, the average variation of V(max) values for both enzymes was not higher than 7%, indicating that the modified affinity of enzymes for their respective substrates and cofactors, as consequence of structural modifications suffered by them during the extraction, are compensated in some extension. This study demonstrated that liquid-liquid extraction by CTAB reversed micelles is an efficient process to separate the enzymes XR and XD present in the cell extract, and simultaneously increase the enzymatic activity and the purity of both enzymes produced by C. guilliermondii.     

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP⁺, which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP⁺ and NAD⁺. One of the XDH variants utilized NADP⁺ about 4 times more efficiently than NAD⁺. This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.     

The activity of xylose reductase and xylitol dehydrogenase in yeasts

The activity and the cofactor specificity of xylose reductase and xylitol dehydrogenase were studied in extracts of yeasts from the genera Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis grown under microaerobic conditions. It was found that xylitol dehydrogenase in all of the yeast species studied is specific for NAD+; xylose reductase in the xylitol-producing species C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, Kl. fragilis, Kl. marxianus, P. guillermondii, and T. molishiama is specific for NADPH; and xylose reductase in the ethanol-producing species P. stipitis, C. shehatae, and Pa. tannophilus is specific for both NADPH and NADH.     

Conversion of D-xylose into xylitol by xylose reductase from Candida pelliculosa coupled with the oxidoreductase system of methanogen strain HU

Summary A preliminary test for the enzymatic conversion of D-xylose into xylitol by the intact cells of Candida pelliculosa (xylose reductase) coupled with the intact cells of a formate-utilizing methanogen strain HU (hydrogenase and F₄₂₀-NADP oxidoreductase) was conducted by using H₂ as an electron donor of NADP⁺. In the system, NADP(H) was well regenerated via the methanogen cells and about 90% conversion of xylose to xylitol (ca. 8 g/l) could be achieved at 35°C and pH 7.5 after 24 h incubation.     

Response surface methodology as an approach to determine the optimal activities of xylose reductase and xylitol dehydrogenase enzymes from Candida Mogii

A central composite experimental design leading to a set of 16 experiments with different combinations of pH and temperature was performed to attain the optimal activities of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes from Candida mogii cell extract. Under optimized conditions (pH 6.5 and 38°C), the XR and XDH activities were found to be 0.48 U/ml and 0.22 U/ml, respectively, resulting in an XR to XDH ratio of 2.2. Stability, cofactor specificity and kinetic parameters of the enzyme XR were also evaluated. XR activity remained stable for 3 h under 4 and 38°C and for 4 months of storage at −18°C. Studies on cofactor specificity showed that only NADPH-dependent XR was obtained under the cultivation conditions employed. The XR present in C. mogii extracts showed a superior K _m value for xylose when compared with other yeast strains. Besides, this parameter was not modified after enzyme extraction by aqueous two-phase system.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Production of xylitol from D-xylose by a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis

Xylitol dehydrogenase (XDH) is one of the key enzymes in d-xylose metabolism, catalyzing the oxidation of xylitol to d-xylulose. Two copies of the XYL2 gene encoding XDH in the diploid yeast Candida tropicalis were sequentially disrupted using the Ura-blasting method. The XYL2-disrupted mutant, BSXDH-3, did not grow on a minimal medium containing d-xylose as a sole carbon source. An enzyme assay experiment indicated that BSXDH-3 lost apparently all XDH activity. Xylitol production by BSXDH-3 was evaluated using a xylitol fermentation medium with glucose as a cosubstrate. As glucose was found to be an insufficient cosubstrate, various carbon sources were screened for efficient cofactor regeneration, and glycerol was found to be the best cosubstrate. BSXDH-3 produced xylitol with a volumetric productivity of 3.23 g liter(-1) h(-1), a specific productivity of 0.76 g g(-1) h(-1), and a xylitol yield of 98%. This is the first report of gene disruption of C. tropicalis for enhancing the efficiency of xylitol production.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

Scale up of the production of xylitol dehydrogenase and alcohol dehydrogenase from Pachysolen tannophilus grown on xylose

Summary Coproduction of two enzymes, xylitol dehydrogenase (XDH) and alcohol dehydrogenase (ADH), was attempted using the yeast Pachysolen tannophilus. Production of both enzymes was scaled up to a volume of 500 l with the yeast growing on xylose a the sole carbon source. Maximal amount of XDH was obtained by harvesting the cells at the end of the logarithmic growth phase. Activity of XDH was induced by imposing anaerobic conditions, thereby yielding 10 times more enzyme than was obtained for aerobic growth conditions. In crude extracts, obtained by passage through a French press (20 000 p.s.i.), XDH activity was 0.057 u/mg protein and ADH was 0.15 u/mg for aerobic growth and 1.5 u/mg for microaerophilic growth. Extraction of intracellular enzymes on a large sale was performed with a combination of cell wall lytic enzymes and an industrial homogenizer (Manton-Gaulin, 3000 p.s.i.). This system enabled a continuous mode of operation (single passage through homogenizer) with a high cell density (100 g/l) and the extracts contained 0.033 u/mg of XDH and 0.45 u/mg protein of ADH.     

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

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.     

Ethanol utilization for metabolite production by Candida utilis strains in liquid medium

Ethanol has been reported to be a gaseous pollutant, originating from the agricultural industry. Interest in its biodegradation has increased over the last two decades. Most of the current studies have focused on its elimination by mixed cultures. This study is part of a broader project intended to utilize Candida utilis strains for gaseous ethanol elimination and to eventually bioconvert them into biomass and/or volatile metabolites. We present here the study of six strains (one from the ATCC and five from the ICIDCA collection) cultivated in a liquid medium, with initial ethanol concentrations of 16 g/l and 32 g/l. At 16 g/l, a maximum ethanol elimination rate of 0.13 g/l × h was obtained in four of the six strains (ATCC 9950, L/375–1, L/375–5 and L/375–10). This rate increased to 0.21 g/l × h with an initial ethanol concentration of 32 g/l. The L/375–5 strain was the best biomass producer (3.3 g/l) at 32 g/l, while the highest ethyl acetate production (0.80 g/l) was obtained with the L/375–1 strain. The L/375–25 and L/375–26 strains which showed very low ethyl acetate production were, by way of contrast, efficient acetaldehyde producers, with 0.54 g/l and 0.66 g/l measured in the broth. While biomass production reached its maximum after two days of culture, the production of acetic acid and ethyl acetate continued during the third day. The results for biomass and metabolite production obtained with the ICIDCA collection strains (L/375–1, L/375–5 and L/375–10) were better than those obtained with the ATCC 9950 strain, although the latter often has been reported to be particularly suitable for metabolite production.     

The effect of xylose reductase genes on xylitol production by industrial Saccharomyces cerevisiae in fermentation of glucose and xylose

The co-production of xylitol and ethanol from agricultural straw has more economic advantages than the production of ethanol only. Saccharomyces cerevisiae, the most widely used ethanol-producing yeast, can be genetically engineered to ferment xylose to xylitol. In the present study, the effects of xylose-specificity, cofactor preference, and the gene copy number of xylose reductase (XR; encoding by XYL1 gene) on xylitol production of S. cerevisiae were investigated. The results showed that overexpression of XYL1 gene with a lower xylose-specificity and a higher NADPH preference favored the xylitol production. The copy number of XYL1 had a positive correlation with the XR activity but did not show a good correlation with the xylitol productivity. The overexpression of XYL1 from Candida tropicalis (CtXYL1) achieved a xylitol productivity of 0.83 g/L/h and a yield of 0.99 g/g-consumed xylose during batch fermentation with 43.5 g/L xylose and 17.0 g/L glucose. During simultaneous saccharification and fermentation (SSF) of pretreated corn stover, the strain overexpressing CtXYL1 produced 45.41 g/L xylitol and 50.19 g/L ethanol, suggesting its application potential for xylitol and ethanol co-production from straw feedstocks.     

酿酒酵母Saccharomyces cerevisiae重组菌株木糖醇发酵的初步研究

木糖还原酶催化木糖为木糖醇的反应,是木糖代谢的第一步。将木糖还原酶的原因ＸＹＬ１引入酿酒酵母中,构建得到儿表达ＸＹＬ１基因的重组酿酒酵母菌株ＨＹＥＸ２,该重组菌株的木糖还原酶比活力为７．４７Ｕ／ｍｇ。研究表明,该菌株获得转化木糖产生木糖醇的能力,当辅助碳源葡萄糖的浓度为２％,并在发酵３０ｈ左右添加木糖,木糖醇的转化率可达到０．９７ｇ／ｇ。     

Characterization of two-substrate fermentation processes for xylitol production using recombinant Saccharomyces cerevisiae containing xylose reductase gene

Fermentation characteristics of recombinant Saccharomyces cerevisiae containing a xylose reductase gene from Pichia stipitis were investigated in an attempt to convert xylose to xylitol, a natural five-carbon sugar alcohol used as a sweetener. Xylitol was produced with a maximum yield of 0.95 g g⁻¹ xylitol xylose consumed in the presence of glucose used as a co-substrate for co-factor regeneration. Addition of glucose caused inhibition of xylose transport and accumulation of ethanol. Such problems were solved by adopting glucose-limited fed-batch fermentations where a high ratio of xylose to glucose was maintained during the bioconversion phase. The optimized two-substrate fed-batch fermentation carried out with S. cerevisiae EH13.15:pY2XR at 30°C resulted in 105.2 g l⁻¹ xylitol concentration with 1.69 g l⁻¹ h⁻¹ productivity.     

Heterologous expression of Aspergillus oryzae xylose reductase and xylitol dehydrogenase genes facilitated xylose utilization in the yeast Saccharomyces cerevisiae

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

Protoplast production from Candida utilis]

The protoplasts of Candida utilis 295 t were produced with the aid of the lytic enzyme from Helix pomatia. If the cell wall of C. utilis 295 t is not treated with SH-compounds (the best effect was found with L-cysteine), it is resistant to the action of the enzyme. The yield of the protoplasts was 100 per cent after 15 minutes of the incubation with the lytic enzyme if the cells were preliminarily treated with L-cysteine. Optimal conditions for the production of the protoplasts are described.     

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD?-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l?1 h?1 xylose consumption rate, 0.25 g l?1 h?1 ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.     

Induction of aldose reductase and xylitol dehydrogenase activities in Candida tenuis CBS 4435

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on d-xylose or l-arabinose as well as on the structurally related sugars d-arabinose or d-lyxose. Mixtures of d-xylose with the more rapidly metabolizable sugar d-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of d-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.     

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37–73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.