scholarly journals Xylulokinase Overexpression in Two Strains ofSaccharomyces cerevisiae Also Expressing Xylose Reductase and Xylitol Dehydrogenase and Its Effect on Fermentation of Xylose and Lignocellulosic Hydrolysate

2001 ◽  
Vol 67 (9) ◽  
pp. 4249-4255 ◽  
Author(s):  
Björn Johansson ◽  
Camilla Christensson ◽  
Timothy Hobley ◽  
Bärbel Hahn-Hägerdal
Keyword(s):  
Ethanol Yield ◽  
Xylose Reductase ◽  
Xylitol Dehydrogenase ◽  
Host Strain ◽  
Lignocellulosic Hydrolysate ◽  
Xylose Consumption ◽  
Sugar Mixture ◽  
Pentose Sugar ◽  
Ethanol Yields ◽  
Ethanol Producer

ABSTRACT 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) andXYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The geneXKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect ofXKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressingXYL1 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, XKS1overexpression 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.

scholarly journals Deletion ofFPS1, Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

2013 ◽  
Vol 79 (10) ◽  
pp. 3193-3201 ◽  
Author(s):  
Na Wei ◽  
Haiqing Xu ◽  
Soo Rin Kim ◽  
Yong-Su Jin
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Plant Biomass ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Enzymatic Reactions ◽  
Xylose Metabolism ◽  
Xylose Consumption ◽  
Content Type ◽  
Ethanol Yields

ABSTRACTAccumulation of xylitol in xylose fermentation with engineeredSaccharomyces cerevisiaepresents 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-producingS. cerevisiaestrains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, whenFPS1was 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 thatFPS1deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion ofFPS1decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion ofFPS1resulted 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 inS. cerevisiaeand present a new gene deletion target,FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.

scholarly journals Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

2006 ◽  
Vol 72 (5) ◽  
pp. 3418-3428 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Alain A. Vert�s ◽  
Shohei Okino ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Xylose Isomerase ◽  
Mineral Medium ◽  
Gene Encoding ◽  
Xylose Consumption ◽  
E Coli ◽  
High Copy Number Plasmid ◽  
Sugar Mixture ◽  
Pentose Sugar

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vert�s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

scholarly journals Efficient Bioethanol Production by a Recombinant Flocculent Saccharomyces cerevisiae Strain with a Genome-Integrated NADP+-Dependent Xylitol Dehydrogenase Gene

2009 ◽  
Vol 75 (11) ◽  
pp. 3818-3822 ◽  
Author(s):  
Akinori Matsushika ◽  
Hiroyuki Inoue ◽  
Seiya Watanabe ◽  
Tsutomu Kodaki ◽  
Keisuke Makino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Yield ◽  
Xylitol Dehydrogenase ◽  
Wood Chips ◽  
Bioethanol Production ◽  
Xylose Consumption ◽  
Acid Hydrolysate ◽  
A Genome ◽  
Dehydrogenase Gene ◽  
Flocculent Yeast

ABSTRACT The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP+-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

scholarly journals Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, andXKS1 in Mineral Medium Chemostat Cultures

2000 ◽  
Vol 66 (8) ◽  
pp. 3381-3386 ◽  
Author(s):  
Anna Eliasson ◽  
Camilla Christensson ◽  
C. Fredrik Wahlbom ◽  
Bärbel Hahn-Hägerdal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Dry Weight ◽  
Pichia Stipitis ◽  
Feed Ratio ◽  
Gene Encoding ◽  
Chemostat Cultures ◽  
Glucose Ratio ◽  
Ethanol Yields

ABSTRACT For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiaehas been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenousXKS1 gene, encoding xylulokinase (XK), under control of thePGK1 promoter were integrated into the chromosomalHIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h−1 g (dry weight) of cells−1 (0.24 to 0.30 g h−1 g [dry weight] of cells−1) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h−1. The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h−1 g (dry weight) of cells−1 when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h−1 g (dry weight) of cells−1 when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.

scholarly journals Improving Acetic Acid and Furfural Resistance of Xylose-Fermenting Saccharomyces cerevisiae Strains by Regulating Novel Transcription Factors Revealed via Comparative Transcriptomic Analysis

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Bo Li ◽  
Li Wang ◽  
Ya-Jing Wu ◽  
Zi-Yuan Xia ◽  
Bai-Xue Yang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acetic Acid ◽  
Transcription Factors ◽  
Ethanol Yield ◽  
Regulation Mechanism ◽  
Lignocellulosic Hydrolysate ◽  
Inhibitor Tolerance ◽  
Xylose Consumption ◽  
Content Type ◽  
Beneficial Effects

ABSTRACT Acetic acid and furfural are the two prevalent inhibitors coexisting with glucose and xylose in lignocellulosic hydrolysate. The transcriptional regulations of Saccharomyces cerevisiae in response to acetic acid (Aa), furfural (Fur), and the mixture of acetic acid and furfural (Aa_Fur) were revealed during mixed glucose and xylose fermentation. Carbohydrate metabolism pathways were significantly enriched in response to Aa, while pathways of xenobiotic biodegradation and metabolism were significantly enriched in response to Fur. In addition to these pathways, other pathways were activated in response to Aa_Fur, i.e., cofactor and vitamin metabolism and lipid metabolism. Overexpression of Haa1p or Tye7p improved xylose consumption rates by nearly 50%, while the ethanol yield was enhanced by nearly 8% under acetic acid and furfural stress conditions. Co-overexpression of Haa1p and Tye7p resulted in a 59% increase in xylose consumption rate and a 12% increase in ethanol yield, revealing the beneficial effects of Haa1p and Tye7p on improving the tolerance of yeast to mixed acetic acid and furfural. IMPORTANCE Inhibitor tolerance is essential for S. cerevisiae when fermenting lignocellulosic hydrolysate with various inhibitors, including weak acids, furans, and phenols. The details regarding how xylose-fermenting S. cerevisiae strains respond to multiple inhibitors during fermenting mixed glucose and xylose are still unknown. This study revealed the transcriptional regulation mechanism of an industrial xylose-fermenting S. cerevisiae strain in response to acetic acid and furfural. The transcription factor Haa1p was found to be involved in both acetic acid and furfural tolerance. In addition to Haa1p, four other transcription factors, Hap4p, Yox1p, Tye7p, and Mga1p, were identified as able to improve the resistance of yeast to these two inhibitors. This study underscores the feasibility of uncovering effective transcription factors for constructing robust strains for lignocellulosic bioethanol production.

scholarly journals Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

2002 ◽  
Vol 68 (4) ◽  
pp. 1604-1609 ◽  
Author(s):  
Marie Jeppsson ◽  
Björn Johansson ◽  
Bärbel Hahn-Hägerdal ◽  
Marie F. Gorwa-Grauslund
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Xylitol Production ◽  
Xylose Consumption ◽  
Xylitol Yield ◽  
Pathway Flux

ABSTRACT In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD+. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g−1) and the lowest xylitol yield (0.05 g g−1) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.

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

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Cai-Yun Xie ◽  
Bai-Xue Yang ◽  
Qing-Ran Song ◽  
Zi-Yuan Xia ◽  
Min Gou ◽  
...  
Keyword(s):  
Ethanol Yield ◽  
Protein Biosynthesis ◽  
Xylose Reductase ◽  
Mapk Signaling ◽  
Xylose Metabolism ◽  
Alcohol Fermentation ◽  
Transcriptional Responses ◽  
Major Barrier ◽  
Xylose Consumption ◽  
Underlying Basis

Abstract 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.

scholarly journals New approaches for improving the production of the 1st and 2nd generation ethanol by yeast.

2015 ◽  
Vol 63 (1) ◽  
Author(s):  
Olena Kurylenko ◽  
Marta Semkiv ◽  
Justyna Ruchala ◽  
Orest Hryniv ◽  
Barbara Kshanovska ◽  
...  
Keyword(s):  
Elevated Temperatures ◽  
Ethanol Yield ◽  
Hansenula Polymorpha ◽  
Xylose Reductase ◽  
Biomass Accumulation ◽  
Xylose Metabolism ◽  
Gene Coding ◽  
Lignocellulosic Feedstocks ◽  
Pentose Sugar ◽  
Futile Cycles

Increase in the production of 1st generation ethanol from glucose is possible by the reduction in the production of ethanol co-products, especially biomass. We have developed a method to reduce biomass accumulation of Saccharomyces cerevisiae by the manipulation of the intracellular ATP level due to overexpression of genes of alkaline phosphatase, apyrase or enzymes involved in futile cycles. The strains constructed accumulated up to 10% more ethanol on a cornmeal hydrolysate medium. Similar increase in ethanol accumulation was observed in the mutants resistant to the toxic inhibitors of glycolysis like 3-bromopyruvate and others. Substantial increase in fuel ethanol production will be obtained by the development of new strains of yeasts that ferment sugars of the abundant lignocellulosic feedstocks, especially xylose, a pentose sugar. We have found that xylose can be fermented under elevated temperatures by the thermotolerant yeast, Hansenula polymorpha. We combined protein engineering of the gene coding for xylose reductase (XYL1) along with overexpression of the other two genes responsible for xylose metabolism in yeast (XYL2, XYL3) and the deletion of the global transcriptional activator CAT8, with the selection of mutants defective in utilizing ethanol as a carbon source using the anticancer drug, 3-bromopyruvate. Resulted strains accumulated 20-25 times more ethanol from xylose at the elevated temperature of 45°C with up to 12.5 g L(-1) produced. Increase in ethanol yield and productivity from xylose was also achieved by overexpression of genes coding for the peroxisomal enzymes: transketolase (DAS1) and transaldolase (TAL2), and deletion of the ATG13 gene.

scholarly journals Combining Xylose Reductase from Spathaspora arborariae with Xylitol Dehydrogenase from Spathaspora passalidarum to Promote Xylose Consumption and Fermentation into Xylitol by Saccharomyces cerevisiae

Fermentation ◽  
2020 ◽  
Vol 6 (3) ◽  
pp. 72
Author(s):  
Adriane Mouro ◽  
Angela A. dos Santos ◽  
Denis D. Agnolo ◽  
Gabriela F. Gubert ◽  
Elba P. S. Bon ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Reductase ◽  
Xylitol Dehydrogenase ◽  
Gene Encoding ◽  
Xylose Consumption ◽  
Reductase Gene ◽  
Food And Beverage ◽  
Spathaspora Passalidarum ◽  
Commercial Applications ◽  
Specific Growth Rates

In recent years, many novel xylose-fermenting yeasts belonging to the new genus Spathaspora have been isolated from the gut of wood-feeding insects and/or wood-decaying substrates. We have cloned and expressed, in Saccharomyces cerevisiae, a Spathaspora arborariae xylose reductase gene (SaXYL1) that accepts both NADH and NADPH as co-substrates, as well as a Spathaspora passalidarum NADPH-dependent xylose reductase (SpXYL1.1 gene) and the SpXYL2.2 gene encoding for a NAD+-dependent xylitol dehydrogenase. These enzymes were co-expressed in a S. cerevisiae strain over-expressing the native XKS1 gene encoding xylulokinase, as well as being deleted in the alkaline phosphatase encoded by the PHO13 gene. The S. cerevisiae strains expressing the Spathaspora enzymes consumed xylose, and xylitol was the major fermentation product. Higher specific growth rates, xylose consumption and xylitol volumetric productivities were obtained by the co-expression of the SaXYL1 and SpXYL2.2 genes, when compared with the co-expression of the NADPH-dependent SpXYL1.1 xylose reductase. During glucose-xylose co-fermentation by the strain with co-expression of the SaXYL1 and SpXYL2.2 genes, both ethanol and xylitol were produced efficiently. Our results open up the possibility of using the advantageous Saccharomyces yeasts for xylitol production, a commodity with wide commercial applications in pharmaceuticals, nutraceuticals, food and beverage industries.

scholarly journals Hemicellulosic Bioethanol Production from Fast-Growing Paulownia Biomass

Processes ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 173
Author(s):  
Elena Domínguez ◽  
Pablo G. del Río ◽  
Aloia Romaní ◽  
Gil Garrote ◽  
Lucília Domingues
Keyword(s):  
Ethanol Production ◽  
Ethanol Concentration ◽  
Ethanol Yield ◽  
Xylose Reductase ◽  
Energy Crop ◽  
Scheffersomyces Stipitis ◽  
Fractionation Process ◽  
Renewable Source ◽  
Fast Growing ◽  
Engineered Strain

In order to exploit a fast-growing Paulownia hardwood as an energy crop, a xylose-enriched hydrolysate was obtained in this work to increase the ethanol concentration using the hemicellulosic fraction, besides the already widely studied cellulosic fraction. For that, Paulownia elongata x fortunei was submitted to autohydrolysis treatment (210 °C or S0 of 4.08) for the xylan solubilization, mainly as xylooligosaccharides. Afterwards, sequential stages of acid hydrolysis, concentration, and detoxification were evaluated to obtain fermentable sugars. Thus, detoxified and non-detoxified hydrolysates (diluted or not) were fermented for ethanol production using a natural xylose-consuming yeast, Scheffersomyces stipitis CECT 1922, and an industrial Saccharomyces cerevisiae MEC1133 strain, metabolic engineered strain with the xylose reductase/xylitol dehydrogenase pathway. Results from fermentation assays showed that the engineered S. cerevisiae strain produced up to 14.2 g/L of ethanol (corresponding to 0.33 g/g of ethanol yield) using the non-detoxified hydrolysate. Nevertheless, the yeast S. stipitis reached similar values of ethanol, but only in the detoxified hydrolysate. Hence, the fermentation data prove the suitability and robustness of the engineered strain to ferment non-detoxified liquor, and the appropriateness of detoxification of liquor for the use of less robust yeast. In addition, the success of hemicellulose-to-ethanol production obtained in this work shows the Paulownia biomass as a suitable renewable source for ethanol production following a suitable fractionation process within a biorefinery approach.

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