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.     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains

Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells^–1 h^–1 compared with 0.833 g g cells^–1 h^–1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 g g cells^–1 h^–1, compared with 0.575 g g cells^–1 h^–1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%–65% of the assimilated carbon could not be accounted for in the products determined.     

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Improving ethanol productivity by modification of glycolytic redox factor generation in glycerol-3-phosphate dehydrogenase mutants of an industrial ethanol yeast

The GPD2 gene, encoding NAD⁺-dependent glycerol-3-phosphate dehydrogenase in an industrial ethanol-producing strain of Saccharomyces cerevisiae, was deleted. And then, either the non-phosphorylating NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Bacillus cereus, or the NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Kluyveromyces lactis, was expressed in the obtained mutant AG2 deletion of GPD2, respectively. The resultant recombinant strain AG2A (gpd2Δ P _PGK -gapN) exhibited a 48.70 ± 0.34% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.60 ± 0.12% (relative to the amount of substrate consumed) increase in ethanol yield, while recombinant AG2B (gpd2Δ P _PGK -GAPDH) exhibited a 52.90 ± 0.45% (relative to the amount of substrate consumed) decrease in glycerol production and a 7.34 ± 0.15% (relative to the amount of substrate consumed) increase in ethanol yield compared with the wild-type strain. More importantly, the maximum specific growth rates (μ _max) of the recombinant AG2A and AG2B were higher than that of the mutant gpd2Δ and were indistinguishable compared with the wild-type strain in anaerobic batch fermentations. The results indicated that the redox imbalance of the mutant could be partially solved by expressing the heterologous genes.     

Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.     

Construction of the industrial ethanol-producing strain of Saccharomyces cerevisiae able to ferment cellobiose and melibiose

The gene mel1, encoding α-galactosidase in Schizosaccharomyces pombe, and the gene bgl2, encoding and α-glucosidase in Trichoderma reesei, were isolated and co-expressed in the industrial ethanolproducing strain of Saccharomyces cerevisiae. The resulting strains were able to grow on cellobiose and melibiose through simultaneous production of sufficient extracellular α-galactosidase and β-glucosidase activity. Under aerobic conditions, the growth rate of the recombinant strain GC1 co-expressing 2 genes could achieve 0.29 OD600 h⁻¹ and a biomass yield up to 7.8 g l⁻¹ dry cell weight on medium containing 10.0 g l⁻¹ cellobiose and 10.0 g l⁻¹ melibiose as sole carbohydrate source. Meanwhile, the new strain of S. cerevisiae CG1 demonstrated the ability to directly produce ethanol from microcrystalline cellulose during simultaneous saccharification and fermentation process. Approximately 36.5 g l⁻¹ ethanol was produced from 100 g of cellulose supplied with 5 g l⁻¹ melibose within 60 h. The yield (g of ethanol produced/g of carbohydrate consumed) was 0.44 g/g, which corresponds to 88.0% of the theoretical yield.     

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

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.     

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.     

Energetics and product formation by Saccharomyces cerevisiae grown in anaerobic chemostats under nitrogen limitation

Anaerobic fermentation of glucose (20 g/l) by Saccharomyces cerevisiae CBS 8066 was studied in a chemostat (dilution rate = 0.05–0.25 h^–1) at different concentrations of the nitrogen source (5.00 g/l or 0.36 g/l ammonium sulphate). The ethanol yield (g ethanol produced/g glucose consumed) was found to be higher and the glycerol yield (g glycerol formed/g glucose consumed) lower during nitrogen limitation than under carbon limitation. The biomass yield on ATP (g dry weight biomass produced/mol ATP consumed) was consequently found to be lower during nitrogen-limited conditions.     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Continuous alcoholic fermentation of glucose/xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae

Viable Saccharomyces cerevisiae and Candida shehatae cells were co-immobilized in a composite agar layer/microporous membrane structure. This immobilized-cell structure was placed in a vertical position between the two halves of a double-chambered, stainless-steel bioreactor of original design and applied to the continuous alcoholic fermentation of a mixture of glucose (35 g dm⁻³) and xylose (15 g dm⁻³). Various dilution rates and initial cell loadings of the gel layer were tested. Simultaneous consumption of the two sugars was always observed. The best fermentation performance was obtained at low dilution rate (0.02 h⁻¹) with an excess of C. shehatae over S. cerevisiae in the initial cell loading of the gel (5.0 mg dry weight and 0.65 mg dry weight cm⁻³ gel respectively): 100% of glucose and 73% of xylose were consumed with an ethanol yield coefficient of 0.48 g g total sugars⁻¹. In these conditions, however, the ethanol production rate per unit volume of gel remained low (0.37 g h⁻¹ dm⁻³). Viable cell counts in gel samples after incubation highlighted significant heterogeneities in the spatial distribution of the two yeast species in both the vertical and the transverse directions. In particular, the overall cell number decreased from the bottom to the top of the agar sheet, which may explain the low ethanol productivity relative to the total gel volume. Received: 26 February 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998     

High-cell-density cultivation for co-production of ergosterol and reduced glutathione by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Two different high-cell-density cultivation processes based on the mutant Saccharomyces cerevisiae GE-2 for simultaneous production of glutathione and ergosterol were investigated. Compared with keeping the ethanol volumetric concentration at a constant low level, feedback control of glucose feeding rate (F) by keeping the descending rate of ethanol volumetric concentration (ΔE/Δt) between −0.1% and 0.15% per hour was much more efficient to achieve a high glutathione and ergosterol productivity. This bioprocess overcomes some disadvantages of traditional S. cerevisiae-based cultivation process, especially shortening cultivation period and making the cultivation process steady-going. A classical on or off controller was used to manipulate F to maintain ΔE/Δt at its set point. The dry cell weight, glutathione yield and ergosterol yield reached 110.0 ± 2.6 g/l, 2,280 ± 76 mg/l, and 1,510 ± 28 mg/l in 32 h, respectively.     

Production of xylitol by Candida peltata

Candida peltata NRRL Y-6888 to ferment xylose to xylitol was evaluated under different fermentation conditions such as pH, temperature, aeration, substrate concentration and in the presence of glucose, arabinose, ethanol, methanol and organic acids. Maximum xylitol yield of 0.56 g g⁻¹ xylose was obtained when the yeast was cultivated at pH 6.0, 28°C and 200 rpm on 50 g L⁻¹ xylose. The yeast produced ethanol (0.41 g g⁻¹ in 40 h) from glucose (50 g L⁻¹) and arabitol (0.55 g g⁻¹ in 87 h) from arabinose (50 g L⁻¹). It preferentially utilized glucose > xylose > arabinose from mixed substrates. Glucose (10 g L⁻¹), ethanol (7.5 g L⁻¹) and acetate (5 g L⁻¹) inhibited xylitol production by 61, 84 and 68%, respectively. Arabinose (10 g L⁻¹) had no inhibitory effect on xylitol production. Received 24 December 1998/ Accepted in revised form 18 March 1999     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

Simultaneous fermentation and isomerization of xylose

Summary Ethanol was produced from xylose by converting the sugar to xylulose, using commercial xylose isomerases, and simultaneously converting the xylulose to ethanol by anaerobic fermentation using different yeast strains. The process was optimized with the yeast strain Schizosaccharomyces pombe (Y-164). The data show that the simultaneous fermentation and isomerization of 6% xylose can produce final ethanol concentrations of 2.1% w/v within 2 days at temperatures as high as 39°C.Nomenclature SFIX simultaneous fermentation and isomerization of xylose - V _p volumetric production (g ethanol·l^-1 per hour) - Q _p specific rate (g ethanol·g^-1 cells per hour) - Y _s yield from substrate consumed (g ethanol, g^-1 xylose) - ET ethanol concentration (% wt/vol) - XT xylitol concentration (% wt/vol) - Glu glucose - Xyl xylose - --m maximum - --f final     

Intermediary Metabolite Concentrations in Xylulose- and Glucose-Fermenting Saccharomyces cerevisiae Cells

Glucose and xylulose fermentation and product formation by Saccharomyces cerevisiae were compared in batch culture under anaerobic conditions. In both cases the main product was ethanol, with glycerol, xylitol, and arabitol produced as by-products. During glucose and xylulose fermentation, 0.74 and 0.37 g of cell mass liter⁻¹, respectively, were formed. In glucose-fermenting cells, the carbon balance could be closed, whereas in xylulose-fermenting cells, about 25% of the consumed sugar carbon could not be accounted for. The rate of sugar consumption was 3.94 mmol g of initial biomass⁻¹ h⁻¹ for glucose and 0.39 mmol g of initial biomass⁻¹ h⁻¹ for xylulose. Concentrations of the intermediary metabolites fructose-1,6-diphosphate (FDP), pyruvate (PYR), sedoheptulose 7-phosphate (S7P), erytrose 4-phosphate, citrate (CIT), fumarate, and malate were compared for both types of cells. Levels of FDP, PYR, and CIT were lower, and levels of S7P were higher in xylulose-fermenting cells. After normalization to the carbon consumption rate, the levels of FDP were approximately the same, whereas there was a significant accumulation of S7P, PYR, CIT, and malate, especially of S7P, in xylulose-fermenting cells compared with in glucose-fermenting cells. In the presence of 15 μM iodoacetate, an inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), FDP levels increased and S7P levels decreased in xylulose-assimilating cells compared with in the absence of the inhibitor, whereas fermentation was slightly slowed down. The specific activity of transaldolase (EC 2.2.1.2), the pentose phosphate pathway enzyme reacting with S7P and glyceraldehyde-3-phosphate, was essentially the same for both glucose- and xylulose-fermenting cells. It was, however, several orders of magnitude lower than that reported for a Torula yeast and Candida utilis. The presence of iodoacetate did not influence the activity of transaldolase in xylulose-fermenting cells. The results are discussed in terms of a competition between the pentose phosphate pathway and glycolysis for the common metabolite, glyceraldehyde-3-phosphate, which would explain the low rates of xylulose assimilation and ethanol production from xylulose by S. cerevisiae.     

The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001

Saccharomyces cerevisiae is able to ferment xylose, when engineered with the enzymes xylose reductase (XYL1) and xylitol dehydrogenase (XYL2). However, xylose fermentation is one to two orders of magnitude slower than glucose fermentation. S. cerevisiae has been proposed to have an insufficient capacity of the non-oxidative pentose phosphate pathway (PPP) for rapid xylose fermentation. Strains overproducing the non-oxidative PPP enzymes ribulose 5-phosphate epimerase (EC 5.1.3.1), ribose 5-phosphate ketol isomerase (EC 5.3.1.6), transaldolase (EC 2.2.1.2) and transketolase (EC 2.2.1.1), as well as all four enzymes simultaneously, were compared with respect to xylose and xylulose fermentation with their xylose-fermenting predecessor S. cerevisiae TMB3001, expressing XYL1, XYL2 and only overexpressing XKS1 (xylulokinase). The level of overproduction in S. cerevisiae TMB3026, overproducing all four non-oxidative PPP enzymes, ranged between 4 and 23 times the level in TMB3001. Overproduction of the non-oxidative PPP enzymes did not influence the xylose fermentation rate in either batch cultures of 50 g l(-1) xylose or chemostat cultures of 20 g l(-1) glucose and 20 g l(-1) xylose. The low specific growth rate on xylose was also unaffected. The results suggest that neither of the non-oxidative PPP enzymes has any significant control of the xylose fermentation rate in S. cerevisiae TMB3001. However, the specific growth rate on xylulose increased from 0.02-0.03 for TMB3001 to 0.12 for the strain overproducing only transaldolase (TAL1) and to 0.23 for TMB3026, suggesting that overproducing all four enzymes has a synergistic effect. TMB3026 consumed xylulose about two times faster than TMB30001 in batch culture of 50 g l(-1) xylulose. The results indicate that growth on xylulose and the xylulose fermentation rate are partly controlled by the non-oxidative PPP, whereas control of the xylose fermentation rate is situated upstream of xylulokinase, in xylose transport, in xylose reductase, and/or in the xylitol dehydrogenase.