For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has 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 XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 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. To obtain an economically feasible industrial process for ethanol production from lignocellulose, it is necessary to ferment all sugars present with high yields and productivities (53). The commonly used Saccharomyces cerevisiae has many advantages as an ethanol producer, such as fast sugar consumption, high ethanol yield from hexoses, and high resistance to inhibitory compounds that are present in the hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose to xylulose is a two-step process catalyzed by xylose reductase (XR) and xylitol dehydrogenase (XDH) (10), whereas bacteria perform the conversion in one step with xylose isomerase (XI) (23).Xylose fermentation by recombinant S. cerevisiae carrying heterologous XYL1 and XYL2 genes from Pichia stipitis, which encode XR and XDH, respectively, has resulted mainly in xylitol formation (24,44,48). Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then only limited xylose fermentation is observed (47). Limited xylose fermentation by recombinant S. cerevisiae has been ascribed to poor xylose uptake (9, 24, 25), a cofactor imbalance generated by the discrepancy in cofactor usage by XR and XDH (8,24,49), limitations in the pentose phosphate pathway (12,24,38,48), and insufficient induction or activation of ethanologenic enzymes (5,17,20,29). When homologous XKS1, which encodes xylulokinase (XK), was overexpressed in a Saccharomyces sp. strain carrying XYL1 and XYL2, the ethanol yield and the xylose uptake rate increased under oxygenlimited conditions, but xylitol was still a major by-product (22).Although the shortcomings of xylose fermentation by recombinant S. cerevisiae have been investigated in several studies, data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and concentrations of substrates and products are constant enable quantitative determinations of metabolic fluxes. Analysis o...

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The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.

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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 K s 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.

A metabolic flux model was constructed for the yeast Saccharomyces cerevisiae comprising the most important reactions during anaerobic metabolism of xylose and glucose. The model was used to calculate the intracellular fluxes in a recombinant, xylose‐utilizing strain of S. cerevisiae (TMB 3001) grown anaerobically in a defined medium at dilution rates of 0.03, 0.06, and 0.18 h−1. The feed concentration was varied from 0 g/L xylose and 20 g/L glucose to a mixture of 15 g/L xylose and 5 g/L glucose, so that the total concentration of carbon source was kept at 20 g/L. The specific uptake of xylose increased with the xylose concentration in the feed and with increasing dilution rate. The excreted xylitol was less than half of the xylose consumed. With increasing xylose concentration in the feed, the fluxes in the pentose phosphate pathway increased, whereas the flux through glycolysis decreased. Under all cultivation conditions, nicotinamide adenine dinucleotide (NADH) was the preferred cofactor for xylose reductase. The model showed that the flux through the reaction from ribulose 5‐phosphate to xylulose 5‐phosphate was very low under all cultivation conditions. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 289–296, 2001.

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.