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 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 Food Grade Ethanol Production Process of Sorghum Stem Juice Using Immobilized Cells Technique

2015 ◽  
Vol 9 (7) ◽  
pp. 8 ◽  
Author(s):  
Tri Widjaja ◽  
Ali Altway ◽  
Arief Widjaja ◽  
Umi Rofiqah ◽  
Rr Whiny Hardiyati Erlian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Sieve ◽  
Zymomonas Mobilis ◽  
Immobilized Cells ◽  
Ethanol Yield ◽  
Continuous Fermentation ◽  
Continuous Process ◽  
Pichia Stipitis ◽  
Batch Process ◽  
Food Grade

One form of economic development efforts for waste utilization in rural communities is to utilize stem sorghum to produce food grade ethanol. Sorghum stem juice with 150 g/L of sugar concentration was fermented using conventional batch process and cell immobilization continuous process with K-carrageenan as a supporting matrix. The microorganism used was Mutated Zymomonas Mobilis to be compared with a mixture of Saccharomyces Cerevisiae and Pichia Stipitis, and a mixture of Mutated Zymomonas Mobilis and Pichia Stipitis. Ethanol in the broth, result of fermentation process, was separated in packed distillation column. Distilate of the column, still contain water and other impurities, was flown into molecular sieve for dehydration and activated carbon adsorption column to remove the other impurities to meet food grade ethanol specification. The packing used in distillation process was steel wool. For batch fermentation, the fermentation using a combination of Saccharomyces Cerevisiae and Pichia Stipitis produced the best ethanol with 12.07% of concentration, where the yield and the productivity were 63.49%, and 1.06 g/L.h, respectively. And for continuous fermentation, the best ethanol with 9.02% of concentration, where the yield and the productivity were 47.42% and 174.27 g/L.h, respectively, is obtained from fermentation using a combination of Saccharomyces Cerevisiae and Pichia Stipitis also. Fermentation using combination microorganism of Saccharomyces Cerevisiae and Pichia Stipitis produced higher concentration of ethanol, yield, and productivity than other microorganisms. Distillation, molecular sieve dehydration and adsorption process is quite successful in generating sufficient levels of ethanol with relatively low amount of impurities.

scholarly journals Ethanol Production from Nondetoxified Dilute-Acid Lignocellulosic Hydrolysate by Cocultures of Saccharomyces cerevisiae Y5 and Pichia stipitis CBS6054

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Ping Wan ◽  
Dongmei Zhai ◽  
Zhen Wang ◽  
Xiushan Yang ◽  
Shen Tian
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ethanol Production ◽  
Ethanol Concentration ◽  
Ethanol Yield ◽  
Synthetic Medium ◽  
Pichia Stipitis ◽  
Dilute Acid ◽  
Acid Hydrolysate ◽  
Issatchenkia Orientalis ◽  
Final Ethanol Concentration

Saccharomyces cerevisiae Y5 (CGMCC no. 2660) and Issatchenkia orientalis Y4 (CGMCC no. 2159) were combined individually with Pichia stipitis CBS6054 to establish the cocultures of Y5 + CBS6054 and Y4 + CBS6054. The coculture Y5 + CBS6054 effectively metabolized furfural and HMF and converted xylose and glucose mixture to ethanol with ethanol concentration of 16.6 g/L and ethanol yield of 0.46 g ethanol/g sugar, corresponding to 91.2% of the maximal theoretical value in synthetic medium. Accordingly, the nondetoxified dilute-acid hydrolysate was used to produce ethanol by co-culture Y5 + CBS6054. The co-culture consumed glucose along with furfural and HMF completely in 12 h, and all xylose within 96 h, resulting in a final ethanol concentration of 27.4 g/L and ethanol yield of 0.43 g ethanol/g sugar, corresponding to 85.1% of the maximal theoretical value. The results indicated that the co-culture of Y5 + CBS6054 was a satisfying combination for ethanol production from non-detoxified dilute-acid lignocellulosic hydrolysates. This co-culture showed a promising prospect for industrial application.

Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent xylose reductase-encoding gene (XYL1) from the xylose-assimilating yeast Pichia stipitis

Gene ◽  
1991 ◽  
Vol 109 (1) ◽  
pp. 89-97 ◽  
Author(s):  
René Amore ◽  
Peter Kötter ◽  
Christina Küster ◽  
Michael Ciriacy ◽  
Cornelis P. Hollenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Cloning And Expression ◽  
Encoding Gene

scholarly journals Decreased Xylitol Formation during Xylose Fermentation in Saccharomyces cerevisiae Due to Overexpression of Water-Forming NADH Oxidase

2011 ◽  
Vol 78 (4) ◽  
pp. 1081-1086 ◽  
Author(s):  
Guo-Chang Zhang ◽  
Jing-Jing Liu ◽  
Wen-Tao Ding
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Xylose Fermentation ◽  
Nadh Oxidase ◽  
Xylose Reductase ◽  
Control Strain ◽  
Content Type ◽  
Ethanol Yields ◽  
Glycerol Yield ◽  
Set Up ◽  
And Control

ABSTRACTThe recombinant xylose-fermentingSaccharomyces cerevisiaestrain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) fromScheffersomyces stipitisrequires NADPH and NAD+, creates cofactor imbalance, and causes xylitol accumulation during growth ond-xylose. To solve this problem,noxE, encoding a water-forming NADH oxidase fromLactococcus lactisdriven by thePGK1promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD+by XDH and the formation of NAD+by water-forming NADH oxidase. Overexpression ofnoxEsignificantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initiald-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing tonoxEoverexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due tonoxEoverexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression ofnoxE.

scholarly journals A Modified Saccharomyces cerevisiae Strain That Consumes l-Arabinose and Produces Ethanol

2003 ◽  
Vol 69 (7) ◽  
pp. 4144-4150 ◽  
Author(s):  
Jessica Becker ◽  
Eckhard Boles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Yeast Strain ◽  
Dna Microarrays ◽  
Dry Weight ◽  
Cellulosic Biomass ◽  
Pentose Sugar ◽  
Ethanol Yields ◽  
Ethanol Production Rate ◽  
Efficient Fermentation ◽  
Utilization Pathway

ABSTRACT Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar l-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial l-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the l-arabinose-transporting yeast galactose permease, we were able to select an l-arabinose-utilizing yeast strain by sequential transfer in l-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of l-arabinose is a lowered activity of l-ribulokinase. Moreover, high l-arabinose uptake rates and enhanced transaldolase activities favor utilization of l-arabinose. With a doubling time of about 7.9 h in a medium with l-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) · h−1 under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.

scholarly journals Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

2004 ◽  
Vol 70 (5) ◽  
pp. 2892-2897 ◽  
Author(s):  
Marco Sonderegger ◽  
Michael Schümperli ◽  
Uwe Sauer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plant Material ◽  
Xylose Fermentation ◽  
Ethanol Yield ◽  
Limited Capacity ◽  
Fermentation Rate ◽  
Phosphoketolase Pathway ◽  
Ethanol Yields ◽  
Acetate Accumulation ◽  
Acetate Formation

ABSTRACT Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.

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 Endogenous Xylose Pathway in Saccharomyces cerevisiae

2004 ◽  
Vol 70 (6) ◽  
pp. 3681-3686 ◽  
Author(s):  
Mervi H. Toivari ◽  
Laura Salusj�rvi ◽  
Laura Ruohonen ◽  
Merja Penttil�
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Dry Mass ◽  
Genes Encoding ◽  
Main Carbon Source ◽  
Endogenous Genes ◽  
Endogenous Enzymes ◽  
Main Carbon

ABSTRACT The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on d-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter−1 h−1 and a xylitol yield of 55% when xylose was the main carbon source.

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