Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

Mathematical modelling of ethanol production from glucose/xylose mixtures by recombinant Zymomonas mobilis

A model has been developed for the fermentation of mixtures of glucose and xylose by recombinant Zymomonas mobilis strain ZM4(pZB5), containing additional genes for xylose assimilation and metabolism. A two-substrate model based on substrate limitation, substrate inhibition, and product (ethanol) inhibition was evaluated, and experimental data was compared with model simulations using a Microsoft EXCEL based program and methods of statistical analysis for error minimization. From the results it was established that the model provides good predictions of experimental batch culture data for 25/25, 50/50, and 65/65 g l^–1 glucose/xylose media.     

The utilization of metabolic inhibitors for shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis

Summary Xylose, glucose and xylose/glucose mixtures were fermented with Candida tropicalis ATCC 32113 under aerobic, oxygen limited and anaerobic conditions. Ethanol yields were highest under oxygen limited conditions with xylose and xylose/glucose. Anaerobic conditions were best for glucose fermentations.The effect of four metabolic inhibitors (azide, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), oligomycin A and valinomycin-K⁺) were then studied under oxygen limited conditions. Only azide had a significant influence on ethanol production. At 2¢10^-4 M concentrations, ethanol yield increased up to two times and xylitol levels were repressed by 90% for xylose and glucose/xylose fermentations. 4.2×10^-3 M azide gave highest ethanol yields in glucose fermentations. At this concentration of azide, however, cell growth was inhibited, which seemed to prevent ethanol production in xylose fermentations. The effect of azide is discussed in terms of fine-tuning the respiratory activity necessary for metabolism.     

Over-expression of xylulokinase in a xylose-metabolising recombinant strain of Zymomonas mobilis

The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 x 10(3) CFU per mug of DNA in a recombinant strain of Z. mobilis ZM4/AcR containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/AcR (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/AcR (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/AcR (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/AcR (pZB5) and related strains.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^–1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^–1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis. Nomenclature m Cell maintenance energy coefficient (g g^–1 h^–1)Maximum overall specific growth rate (1 h^–1)Maximum specific ethanol production rate (g g^–1 h^–1)Maximum specific total sugar utilization rate (g g^–1 h^–1)Biomass yield per mole of ATP (g mole^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

Engineering industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for xylose fermentation and comparison for switchgrass conversion

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h⁻¹. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.     

Adaptation yields a highly efficient xylose-fermenting Zymomonas mobilis strain

Zymomonas mobilis is a superb ethanol producer with productivity exceeding yeast strains by several fold. Although metabolic engineering was successfully applied to expand its substrate range to include xylose, xylose fermentation lagged far behind glucose. In addition, xylose fermentation was often incomplete when its initial concentration was higher than 5%. Improvement of xylose fermentation is therefore necessary. In this work, we applied adaptation to improve xylose fermentation in metabolically engineered strains. As a result of adaptation over 80 days and 30 serial transfers in a medium containing high concentration of xylose, a strain, referred as A3, with markedly improved xylose metabolism was obtained. The strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, a total of 9% (w/v) ethanol was obtained from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Further investigation reveals evidence for an altered xylitol metabolism in A3 with reduced xylitol formation. Additionally xylitol tolerance in A3 was increased. Furthermore, xylose isomerase activity was increased by several times in A3, allowing cells to channel more xylose to ethanol than to xylitol. Taken together, these results strongly suggest that altered xylitol metabolism is key to improved xylose metabolism in adapted A3 strain. This work further demonstrates that adaptation and metabolic engineering can be used synergistically for strain improvement.     

Role of xylose transporters in xylitol production from engineered Escherichia coli

Escherichia coli W3110 was previously engineered to co-utilize glucose and xylose by replacing the wild-type crp gene with a crp* mutant encoding a cAMP-independent CRP variant (Cirino et al., 2006 [Cirino, P.C., Chin, J.W., Ingram, L.O., 2006. Engineering Escherichia coli for xylitol production from glucose-xylose mixtures. Biotechnol. Bioeng. 95, 1167-1176.]). Subsequent deletion of the xylB gene (encoding xylulokinase) and expression of xylose reductase from Candida boidinii (CbXR) resulted in a strain which produces xylitol from glucose-xylose mixtures. In this study we examine the contributions of the native E. coli xylose transporters (the d-xylose/proton symporter XylE and the d-xylose ABC transporter XylFGH) and CRP* to xylitol production in the presence of glucose and xylose. The final batch xylitol titer with strain PC09 (Delta xylB and crp*) is reduced by 40% upon deletion of xylG and by 60% upon deletion of both xyl transporters. Xylitol production by the wild-type strain (W3110) expressing CbXR is not reduced when xylE and xylG are deleted, demonstrating tight regulation of the xylose transporters by CRP and revealing significant secondary xylose transport. Finally, plasmid expression of XylE or XylFGH with CbXR in PC07 (Delta xylB and wild-type crp) growing on glucose results in xylitol titers similar to that achieved with PC09 and provides an alternative strategy to the use of CRP*.     

Expression of protein engineered NADP<Superscript>+</Superscript>-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD⁺-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP⁺. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP⁺-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP⁺-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.     

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.     

Growth characteristics and metabolic flux analysis of Candida milleri

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.     

High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Xylose fermentation performance was studied of a previously developed Saccharomyces cerevisiae strain TMB 3057, carrying high xylose reductase (XR) and xylitol dehydrogenase (XDH) activity, overexpressed non-oxidative pentose phosphate pathway (PPP) and deletion of the aldose reductase gene GRE3. The fermentation performance of TMB 3057 was significantly improved by increased ethanol production and reduced xylitol formation compared with the reference strain TMB 3001. The effects of the individual genetic modifications on xylose fermentation were investigated by comparing five isogenic strains with single or combined modifications. All strains with high activity of both XR and XDH had increased ethanol yields and significantly decreased xylitol yields. The presence of glucose further reduced xylitol formation in all studied strains. High activity of the non-oxidative PPP improved the xylose consumption rate. The results indicate that ethanolic xylose fermentation by recombinant S. cerevisiae expressing XR and XDH is governed by the efficiency by which xylose is introduced in the central metabolism.     

Metabolic engineering for improved fermentation of pentoses by yeasts

The fermentation of xylose is essential for the bioconversion of lignocellulose to fuels and chemicals, but wild-type strains of Saccharomyces cerevisiae do not metabolize xylose, so researchers have engineered xylose metabolism in this yeast. Glucose transporters mediate xylose uptake, but no transporter specific for xylose has yet been identified. Over-expressing genes for aldose (xylose) reductase, xylitol dehydrogenase and moderate levels of xylulokinase enable xylose assimilation and fermentation, but a balanced supply of NAD(P) and NAD(P)H must be maintained to avoid xylitol production. Reducing production of NADPH by blocking the oxidative pentose phosphate cycle can reduce xylitol formation, but this occurs at the expense of xylose assimilation. Respiration is critical for growth on xylose by both native xylose-fermenting yeasts and recombinant S, cerevisiae. Anaerobic growth by recombinant mutants has been reported. Reducing the respiration capacity of xylose-metabolizing yeasts increases ethanol production. Recently, two routes for arabinose metabolism have been engineered in S. cerevisiae and adapted strains of Pichia stipitis have been shown to ferment hydrolysates with ethanol yields of 0.45 g g^–1 sugar consumed, so commercialization seems feasible for some applications.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Reduction of furan derivatives by overexpressing NADH-dependent Adh1 improves ethanol fermentation using xylose as sole carbon source with Saccharomyces cerevisiae harboring XR–XDH pathway

Several alcohol dehydrogenase (ADH)-related genes have been identified as enzymes for reducing levels of toxic compounds, such as, furfural and/or 5-hydroxymethylfurfural (5-HMF), in hydrolysates of pretreated lignocelluloses. To date, overexpression of these ADH genes in yeast cells have aided ethanol production from glucose or glucose/xylose mixture in the presence of furfural or 5-HMF. However, the effects of these ADH isozymes on ethanol production from xylose as a sole carbon source remain uncertain. We showed that overexpression of mutant NADH-dependent ADH1 derived from TMB3000 strain in the recombinant Saccharomyces cerevisiae, into which xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway of Pichia stipitis has been introduced, improved ethanol production from xylose as a sole carbon source in the presence of 5-HMF. Enhanced furan-reducing activity is able to regenerate NAD⁺ to relieve redox imbalance, resulting in increased ethanol yield arising from decreased xylitol accumulation. In addition, we found that overexpression of wild-type ADH1 prevented the more severe inhibitory effects of furfural in xylose fermentation as well as overexpression of TMB3000-derived mutant. After 120 h of fermentation, the recombinant strains overexpressing wild-type and mutant ADH1 completely consumed 50 g/L xylose in the presence of 40 mM furfural and most efficiently produced ethanol (15.70 g/L and 15.24 g/L) when compared with any other test conditions. This is the first report describing the improvement of ethanol production from xylose as the sole carbon source in the presence of furan derivatives with xylose-utilizing recombinant yeast strains via the overexpression of ADH-related genes.     

Anaerobic Xylose Fermentation by Recombinant Saccharomyces cerevisiae Carrying XYL1, XYL2, and XKS1 in Mineral Medium Chemostat Cultures

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. 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.     

Comparison between Escherichia coli K-12 strains W3110 and MG1655 and wild-type E. coli B as platforms for xylitol production

Escherichia coli W3110 was previously engineered to produce xylitol from a mixture of glucose plus xylose by expressing xylose reductase (CbXR) and deleting xylulokinase (DeltaxylB), combined with either plasmid-based expression of a xylose transporter (XylE or XylFGH) (Khankal et al., J Biotechnol, 2008) or replacing the native crp gene with a mutant (crp*) that alleviates glucose repression of xylose transport (Cirino et al., Biotechnol Bioeng 95:1167-1176, 2006). In this study, E. coli K-12 strains W3110 and MG1655 and wild-type E. coli B were compared as platforms for xylitol production from glucose-xylose mixtures using these same strategies. The engineered strains were compared in fed-batch fermentations and as non-growing resting cells. Expression of CRP* in the E. coli B strains tested was unable to enhance xylose uptake in the presence of glucose. Xylitol production was similar for the (crp*, DeltaxylB)-derivatives of W3110 and MG1655 expressing CbXR (average specific productivities of 0.43 g xylitol g cdw(-1 )h(-1) in fed-batch fermentation). In contrast, results varied substantially between different DeltaxylB-derivative strains co-expressing either XylE or XylFGH. The differences in genetic background between these host strains can therefore profoundly influence metabolic engineering strategies.     

Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.     

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.     

The influence of cosubstrate and aeration on xylitol formation by recombinantSaccharomyces cerevisiae expressing theXYL1 gene

Xylitol formation by a recombinantSaccharomyces cerevisiae strain containing theXYL1 gene fromPichia stipitis CBS 6054 was investigated under three sets of conditions: (a) with glucose, ethanol, acetate, or glycerol as cosubstrates, (b) with different oxygenation levels, and (c) with different ratios of xylose to cosubstrate. With both glucose and ethanol the conversion yields were close to 1 g xylitol/g consumed xylose. Decreased aeration increased the xylitol yield on the basis of consumed cosubstrate, while the rate of xylitol formation decreased. The xylitol yield based on consumed cosubstrate also increased with increased-xylose:cosubstrate ratios. The transformant utilized the cosubstrate more efficiently than did a reference strain in terms of utilization rate and growth rate, implying that the regeneration of NAD(P)⁺ during xylitol formation by the transformant balanced the intracellular redox potential.