Effect of glucose on xylose utilization in Saccharomyces cerevisiae harboring the xylose reductase gene

2011 ◽  
Author(s):  
Ji-Hye Han ◽  
Ju-Yong Park ◽  
Kye Sang Yoo ◽  
Hyun Woo Kang ◽  
Gi-Wook Choi ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Reductase ◽  
Xylose Utilization ◽  
Reductase Gene ◽  
Effect Of Glucose

scholarly journals Limitations in Xylose-Fermenting Saccharomyces cerevisiae, Made Evident through Comprehensive Metabolite Profiling and Thermodynamic Analysis

2010 ◽  
Vol 76 (22) ◽  
pp. 7566-7574 ◽  
Author(s):  
Mario Klimacek ◽  
Stefan Krahulec ◽  
Uwe Sauer ◽  
Bernd Nidetzky
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Thermodynamic Analysis ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Xylose Utilization ◽  
Quantitative Metabolomics ◽  
Liquid Chromatography Tandem Mass ◽  
Potential Factors ◽  
Xylulose Kinase ◽  
Reduction Charge

ABSTRACT Little is known about how the general lack of efficiency with which recombinant Saccharomyces cerevisiae strains utilize xylose affects the yeast metabolome. Quantitative metabolomics was therefore performed for two xylose-fermenting S. cerevisiae strains, BP000 and BP10001, both engineered to produce xylose reductase (XR), NAD+-dependent xylitol dehydrogenase and xylulose kinase, and the corresponding wild-type strain CEN.PK 113-7D, which is not able to metabolize xylose. Contrary to BP000 expressing an NADPH-preferring XR, BP10001 expresses an NADH-preferring XR. An updated protocol of liquid chromatography/tandem mass spectrometry that was validated by applying internal 13C-labeled metabolite standards was used to quantitatively determine intracellular pools of metabolites from the central carbon, energy, and redox metabolism and of eight amino acids. Metabolomic responses to different substrates, glucose (growth) or xylose (no growth), were analyzed for each strain. In BP000 and BP10001, flux through glycolysis was similarly reduced (∼27-fold) when xylose instead of glucose was metabolized. As a consequence, (i) most glycolytic metabolites were dramatically (≤120-fold) diluted and (ii) energy and anabolic reduction charges were affected due to decreased ATP/AMP ratios (3- to 4-fold) and reduced NADP+ levels (∼3-fold), respectively. Contrary to that in BP000, the catabolic reduction charge was not altered in BP10001. This was due mainly to different utilization of NADH by XRs in BP000 (44%) and BP10001 (97%). Thermodynamic analysis complemented by enzyme kinetic considerations suggested that activities of pentose phosphate pathway enzymes and the pool of fructose-6-phosphate are potential factors limiting xylose utilization. Coenzyme and ATP pools did not rate limit flux through xylose pathway enzymes.

scholarly journals Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

2010 ◽  
Vol 76 (23) ◽  
pp. 7796-7802 ◽  
Author(s):  
David Runquist ◽  
B�rbel Hahn-H�gerdal ◽  
Maurizio Bettiga
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Parent Strain ◽  
Selection Process ◽  
Xylose Reductase ◽  
Dry Weight ◽  
Batch Cultivation ◽  
Genetically Engineered ◽  
Ethanol Productivity ◽  
Anaerobic Growth ◽  
Xylose Utilization

ABSTRACT Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V max of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased ∼40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] � h versus 0.007 g/g [DW] � h), and the anaerobic growth rate was increased from ∼0 h−1 to 0.08 h−1. The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.

scholarly journals Molecular Analysis of a Saccharomyces cerevisiae Mutant with Improved Ability To Utilize Xylose Shows Enhanced Expression of Proteins Involved in Transport, Initial Xylose Metabolism, and the Pentose Phosphate Pathway

2003 ◽  
Vol 69 (2) ◽  
pp. 740-746 ◽  
Author(s):  
C. Fredrik Wahlbom ◽  
Ricardo R. Cordero Otero ◽  
Willem H. van Zyl ◽  
Bärbel Hahn-Hägerdal ◽  
Leif J. Jönsson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pentose Phosphate Pathway ◽  
Specific Activity ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Percentage Error ◽  
Xylose Utilization ◽  
Xylose Metabolism ◽  
Physiological Characterization ◽  
Cell Extracts

ABSTRACT Differences between the recombinant xylose-utilizing Saccharomyces cerevisiae strain TMB 3399 and the mutant strain TMB 3400, derived from TMB 3399 and displaying improved ability to utilize xylose, were investigated by using genome-wide expression analysis, physiological characterization, and biochemical assays. Samples for analysis were withdrawn from chemostat cultures. The characteristics of S. cerevisiae TMB 3399 and TMB 3400 grown on glucose and on a mixture of glucose and xylose, as well as of S. cerevisiae TMB 3400 grown on only xylose, were investigated. The strains were cultivated under chemostat conditions at a dilution rate of 0.1 h−1, with feeds consisting of a defined mineral medium supplemented with 10 g of glucose liter−1, 10 g of glucose plus 10 g of xylose liter−1 or, for S. cerevisiae TMB 3400, 20 g of xylose liter−1. S. cerevisiae TMB 3400 consumed 31% more xylose of a feed containing both glucose and xylose than S. cerevisiae TMB 3399. The biomass yields for S. cerevisiae TMB 3400 were 0.46 g of biomass g of consumed carbohydrate−1 on glucose and 0.43 g of biomass g of consumed carbohydrate−1 on xylose. A Ks value of 33 mM for xylose was obtained for S. cerevisiae TMB 3400. In general, the percentage error was <20% between duplicate microarray experiments originating from independent fermentation experiments. Microarray analysis showed higher expression in S. cerevisiae TMB 3400 than in S. cerevisiae TMB 3399 for (i) HXT5, encoding a hexose transporter; (ii) XKS1, encoding xylulokinase, an enzyme involved in one of the initial steps of xylose utilization; and (iii) SOL3, GND1, TAL1, and TKL1, encoding enzymes in the pentose phosphate pathway. In addition, the transcriptional regulators encoded by YCR020C, YBR083W, and YPR199C were expressed differently in the two strains. Xylose utilization was, however, not affected in strains in which YCR020C was overexpressed or deleted. The higher expression of XKS1 in S. cerevisiae TMB 3400 than in TMB 3399 correlated with higher specific xylulokinase activity in the cell extracts. The specific activity of xylose reductase and xylitol dehydrogenase was also higher for S. cerevisiae TMB 3400 than for TMB 3399, both on glucose and on the mixture of glucose and xylose.

Stable expression of xylose reductase gene enhances xylitol production in recombinant Saccharomyces cerevisiae

2002 ◽  
Vol 30 (6) ◽  
pp. 809-816 ◽  
Author(s):  
Yun-Seung Chung ◽  
Myoung-Dong Kim ◽  
Woo-Jong Lee ◽  
Yeon-Woo Ryu ◽  
Ji-Hyeon Kim ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Reductase ◽  
Stable Expression ◽  
Xylitol Production ◽  
Recombinant Saccharomyces Cerevisiae ◽  
Reductase Gene

scholarly journals Construction of a Xylan-Fermenting Yeast Strain through Codisplay of Xylanolytic Enzymes on the Surface of Xylose-Utilizing Saccharomyces cerevisiae Cells

2004 ◽  
Vol 70 (9) ◽  
pp. 5407-5414 ◽  
Author(s):  
Satoshi Katahira ◽  
Yasuya Fujita ◽  
Atsuko Mizuike ◽  
Hideki Fukuda ◽  
Akihiko Kondo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Surface ◽  
Yeast Strain ◽  
High Performance ◽  
Surface Engineering ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Direct Conversion ◽  
Engineering System ◽  
Xylose Utilization

ABSTRACT Hemicellulose is one of the major forms of biomass in lignocellulose, and its essential component is xylan. We used a cell surface engineering system based on α-agglutinin to construct a Saccharomyces cerevisiae yeast strain codisplaying two types of xylan-degrading enzymes, namely, xylanase II (XYNII) from Trichoderma reesei QM9414 and β-xylosidase (XylA) from Aspergillus oryzae NiaD300, on the cell surface. In a high-performance liquid chromatography analysis, xylose was detected as the main product of the yeast strain codisplaying XYNII and XylA, while xylobiose and xylotriose were detected as the main products of a yeast strain displaying XYNII on the cell surface. These results indicate that xylan is sequentially hydrolyzed to xylose by the codisplayed XYNII and XylA. In a further step toward achieving the simultaneous saccharification and fermentation of xylan, a xylan-utilizing S. cerevisiae strain was constructed by codisplaying XYNII and XylA and introducing genes for xylose utilization, namely, those encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis and xylulokinase from S. cerevisiae. After 62 h of fermentation, 7.1 g of ethanol per liter was directly produced from birchwood xylan, and the yield in terms of grams of ethanol per gram of carbohydrate consumed was 0.30 g/g. These results demonstrate that the direct conversion of xylan to ethanol is accomplished by the xylan-utilizing S. cerevisiae strain.

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.

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