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 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.

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 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 Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Yixuan Zhu ◽  
Jingtao Zhang ◽  
Lang Zhu ◽  
Zefang Jia ◽  
Qi Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Budding Yeast ◽  
Ectopic Expression ◽  
Redox Balance ◽  
Inducible Promoter ◽  
Pentose Phosphate ◽  
Xylose Utilization ◽  
Xylose Metabolism ◽  
Utilization Pathway

Xylose is the second most abundant sugar in lignocellulose, but it cannot be used as carbon source by budding yeast Saccharomyces cerevisiae. Rational promoter elements engineering approaches were taken for efficient xylose fermentation in budding yeast. Among promoters surveyed, HXT7 exhibited the best performance. The HXT7 promoter is suppressed in the presence of glucose and derepressed by xylose, making it a promising candidate to drive xylose metabolism. However, simple ectopic expression of both key xylose metabolic genes XYL1 and XYL2 by the HXT7 promoter resulted in massive accumulation of the xylose metabolic byproduct xylitol. Through the HXT7-driven expression of a reported redox variant, XYL1-K270R, along with optimized expression of XYL2 and the downstream pentose phosphate pathway genes, a balanced xylose metabolism toward ethanol formation was achieved. Fermented in a culture medium containing 50 g/L xylose as the sole carbon source, xylose is nearly consumed, with less than 3 g/L xylitol, and more than 16 g/L ethanol production. Hence, the combination of an inducible promoter and redox balance of the xylose utilization pathway is an attractive approach to optimizing fuel production from lignocellulose.

Growth of yeasts on D-xylulose

1980 ◽  
Vol 26 (9) ◽  
pp. 1165-1168 ◽  
Author(s):  
Patrick Y. Wang ◽  
Henry Schneider
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Schizosaccharomyces Pombe ◽  
Optical Density ◽  
Pentose Phosphate Pathway ◽  
Pentose Phosphate ◽  
Saccharomyces Carlsbergensis ◽  
Step Growth ◽  
Xylulose Kinase ◽  
Xylose Catabolism

Nine of eleven yeasts of different species or genera grew in the presence of air on the intermediate of D-xylose catabolism, D-xylulose (D-threo-pentulose). Growth on this substrate was efficient as judged by the optical density in stationary phase being generally similar to that after growth on glucose. Yeasts which grew on D-xylose also did so on D-xylulose, but among those which grew are included several which utilise neither D-xylose nor xylitol: Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Schizosaccharomyces pombe. Since catabolism of a sugar generally requires an initial phosphorylation step, growth of these strains suggests that they contain an enzyme which can function as a D-xylulose kinase. The D-xylulose-5-phosphate formed thereby is considered to enter the pentose phosphate pathway. Glucose-grown inocula of S. carlsbergensis and Schizosaccharomyces pombe, and of several other yeasts, began to grow logarithmically when placed on D-xylulose with no apparent delay, or one which was minimal, suggesting that the D-xylulose kinase was already present in such cells, or was rapidly induced. Petites of S. cerevisiae did not grow on D-xylulose indicating that, in this species, mitochondria are involved in its utilisation.

scholarly journals Saccharomyces cerevisiae Engineered for Xylose Metabolism Exhibits a Respiratory Response

2004 ◽  
Vol 70 (11) ◽  
pp. 6816-6825 ◽  
Author(s):  
Yong-Su Jin ◽  
Jose M. Laplaza ◽  
Thomas W. Jeffries
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Tricarboxylic Acid Cycle ◽  
Pentose Phosphate ◽  
Oxygen Limitation ◽  
Sugar Uptake ◽  
Xylose Utilization ◽  
Xylose Metabolism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Native Strains

ABSTRACT Native strains of Saccharomyces cerevisiae do not assimilate xylose. S. cerevisiae engineered for d-xylose utilization through the heterologous expression of genes for aldose reductase (XYL1), xylitol dehydrogenase (XYL2), and d-xylulokinase (XYL3 or XKS1) produce only limited amounts of ethanol in xylose medium. In recombinant S. cerevisiae expressing XYL1, XYL2, and XYL3, mRNA transcript levels for glycolytic, fermentative, and pentose phosphate enzymes did not change significantly on glucose or xylose under aeration or oxygen limitation. However, expression of genes encoding the tricarboxylic acid cycle, respiration enzymes (HXK1, ADH2, COX13, NDI1, and NDE1), and regulatory proteins (HAP4 and MTH1) increased significantly when cells were cultivated on xylose, and the genes for respiration were even more elevated under oxygen limitation. These results suggest that recombinant S. cerevisiae does not recognize xylose as a fermentable carbon source and that respiratory proteins are induced in response to cytosolic redox imbalance; however, lower sugar uptake and growth rates on xylose might also induce transcripts for respiration. A petite respiration-deficient mutant (ρ�) of the engineered strain produced more ethanol and accumulated less xylitol from xylose. It formed characteristic colonies on glucose, but it did not grow on xylose. These results are consistent with the higher respiratory activity of recombinant S. cerevisiae when growing on xylose and with its inability to grow on xylose under anaerobic conditions.

scholarly journals Shuffling of Promoters for Multiple Genes To Optimize Xylose Fermentation in an Engineered Saccharomyces cerevisiae Strain

2007 ◽  
Vol 73 (19) ◽  
pp. 6072-6077 ◽  
Author(s):  
Chenfeng Lu ◽  
Thomas Jeffries
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Xylose Fermentation ◽  
Xylose Reductase ◽  
Gene Promoter ◽  
Expression Levels ◽  
Multiple Genes ◽  
Expression Of Genes ◽  
Xylulose Kinase ◽  
Engineered Saccharomyces Cerevisiae

ABSTRACT We describe here a useful metabolic engineering tool, multiple-gene-promoter shuffling (MGPS), to optimize expression levels for multiple genes. This method approaches an optimized gene overexpression level by fusing promoters of various strengths to genes of interest for a particular pathway. Selection of these promoters is based on the expression levels of the native genes under the same physiological conditions intended for the application. MGPS was implemented in a yeast xylose fermentation mixture by shuffling the promoters for GND2 and HXK2 with the genes for transaldolase (TAL1), transketolase (TKL1), and pyruvate kinase (PYK1) in the Saccharomyces cerevisiae strain FPL-YSX3. This host strain has integrated xylose-metabolizing genes, including xylose reductase, xylitol dehydrogenase, and xylulose kinase. The optimal expression levels for TAL1, TKL1, and PYK1 were identified by analysis of volumetric ethanol production by transformed cells. We found the optimal combination for ethanol production to be GND2-TAL1-HXK2-TKL1-HXK2-PYK1. The MGPS method could easily be adapted for other eukaryotic and prokaryotic organisms to optimize expression of genes for industrial fermentation.

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