ON THE MECHANISM OF THE INHIBITION OF GROWTH BY XYLULOSE IN CHLOROCOCCUM ECHINOZYGOTUM1,2

We previously established that xylulose inhibits the growth of the green alga Chlorococcum echinozygotum. Utilizing experiments involving exposure of the alga to NaHC¹⁴O₃, it was possible to show by counting the C¹⁴ activity of methanolic extracts of the algal cells that xylulose inhibited CO₂ uptake. Subsequently it was shown that xylulose does not inhibit or otherwise influence the Hill reaction in this alga. Several enzymes related to xylulose metabolism were investigated. It was found that xylulokinase was active in C. echinozygotum while phosphoketolase activity was absent. Transketolase was present but its activity was not notably affected by xylulose. Crude carboxydismutase preparations were found to be inhibited by xylulose and xylulose 5-phosphate. However, as carboxydismutase was purified further, this inhibition was relatively less. When xylulose 1,5-diphosphate was prepared synthetically, this compound was found to be the most effective inhibitor of purified algal carboxydismutase. We conclude that d -xylulose enters the cells of C. echinozygotum where it is converted to d -xylulose 1,5-diphosphate which acts as a competitive inhibitor of carboxydismutase.     

Ethanol Production from Xylose by a Recombinant Candida utilis Strain Expressing Protein-Engineered Xylose Reductase and Xylitol Dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD⁺-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD⁺-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol. Received: 2 July 1999 / Accepted in revised form: 12 October 1999     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

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     

An investigation of d-{1-13C} xylose metabolism in Pichia stipitis under aerobic and anaerobic conditions

Summary The uptake of d-{1-¹³C} xylose, the accumulation of intermediates and the distribution of the label in ethanol in Pichia stipitis under aerobic and anaerobic conditions were investigated by nuclear magnetic resonance spectroscopy. The rate-limiting step of d-xylose metabolism under aerobic conditions appeared to be uptake, whereas under anaerobic conditions it was the conversion of xylitol to xylulose. The yeast showed no preference to either the alpha-or beta-forms of d-xylose. Under anaerobic conditions only {2-¹³C{ ethanol was detected and this suggests that NADH but not NADPH was used as cofactor in the conversion of xylose to xylitol. d-Xylose is most likely metabolised by the pentose phosphate pathway in this yeast.     

XYLULOSE,AN ALGAL GROWTH INHIBITOR12

The growth of Chlorococcum echinozygotum was previously known to be inhibited by an autoclaved solution of xylose in Bold's modified Bristol's medium. We now report the isolation of 2 inhibitory compounds derived from xylose during autoclaving, and the identification of one of them as xylulose. Two strains of C. echinozygotum were isolated, one of which had physiologically adapted to xylulose. The growth rates of both strains were studied using varying amounts of xylulose. Cell counting studies indicated that the zoospores of the unadapted strain were most severely affected by xylulose inhibition and that the zygospores were most resistant.     

D-Xylose fermentation to ethanol bySchizosaccharomyces pombe cloned with xylose isomerase gene

Summary Schizosaccharomyces pombe cloned with the xylose isomerase gene from E. coli is able to grow on YNB and YMP broths containing xylose as the sole carbon source. This yeast can ferment D-xylose to ethanol directly; however, the ethanol production rate and the yield were dependent on the nitrogen source. With the YMP broth as a nitrogen source, the final ethanol concentration can reach 3.7% (w/v), and the ethanol yield was 80% of the theoretical value based on the amount of xylose that was metabolized. The ethanol production is slow, and the xylitol production is still very active; apparently, the limiting step is the isomerization of xylose to xylulose.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Acetone stimulation of ethanol production from d-xylose by Pachysolen tannophilus

Summary Pachysolen tannophilus, a homothallic yeast, converts xylose to ethanol at a yield of 0.3 (g/g xylose). Concomitant with ethanol production, xylitol accumulates in the culture medium at similar yields (0.3 g/g xylose). The addition of the hydrogen-accepting compound, acetone, increases the amount of ethanol produced by this organism by 50–70%. The increase in ethanol is directly correlated with a decrease in xylitol secreted. The results indicate that conversion of acetone to 2-propanol by the cells provides the NAD⁺ used as a cofactor by xylitol dehydrogenase, the enzyme responsible for converting xylitol to xylulose.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

The effects of the oxygen transfer coefficient and substrate concentration on the xylose fermentation by Debaryomyces hansenii

Relevant production of xylitol by Debaryomyces hansenii requires semiaerobic conditions since in aerobic conditions the accumulated reduced adenine dinucleotide coenzyme is fully reoxidized leading to the conversion of xylitol into xylulose. For oxygen transfer coefficient values from 0.24 to 1.88 min^-1, in shake flasks experiments, biomass formation increased proportionally to the aeration rate as shown in the oxygen transfer coefficient and xylose concentration isoresponse contours. The metabolic products under study, xylitol and ethanol were mainly growth associated. However, for oxygen transfer coefficient above 0.5 min^-1 higher initial xylose concentration stimulated the rate of production of xylitol. This fact was less evident for ethanol production. The direct relationship between increased biomass and products formation rate, indicated that the experimental domain in respect to the aeration rate was below the threshold level before the decreasing in metabolic production rates reported in literature for xylose-fermenting yeasts. The fact that ethanol was produced, albeit in low levels, throughout the experimental design indicated that the semiaerobic conditions were always attained. Debaryomyces hansenii showed to be an important xylitol producer exhibiting a xylitol/ethanol ratio above four and a carbon conversion of 54% for xylitol.Abbreviations K_La oxygen transfer coefficient - DO(T) dissolved oxygen (tension) - OUR oxygen uptake rate - NAD(H) oxidised (reduced) nicotinamide adenine dinucleotide - NADP(H) oxidised (reduced) nicotinamide adenine dinucleotide phosphate - CRC catabolic reduction charge - C oxygen concentration in the culture medium - C^* oxygen concentration at saturation conditions - Y_i response from experiment i - parameters of the polynomial model - x experimental factor level (coded units) - R² coefficient of multiple determination - t time     

Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass

An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given. Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation. The resulting hydrolyzsates contain substances inhibitory to fermentation—depending on both the raw material (biomass) and the pre-treatment applied. An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. The furans and phenols generally inhibited growth and ethanol production rate (Q_EtOH) but not the ethanol yields (Y_EtOH) in Saccharomyces cerevisiae. Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P). Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed. Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.     

Ethanol production from xylose by a recombinant Candida utilis strain expressing protein-engineered xylose reductase and xylitol dehydrogenase

The industrial yeast Candida utilis can grow on media containing xylose as sole carbon source, but cannot ferment it to ethanol. The deficiency might be due to the low activity of NADPH-preferring xylose reductase (XR) and NAD(+)-dependent xylitol dehydogenase (XDH), which convert xylose to xylulose, because C. utilis can ferment xylulose. We introduced multiple site-directed mutations in the coenzyme binding sites of XR and XDH derived from the xylose-fermenting yeast Candida shehatae to alter their coenzyme specificities. Several combinations of recombinant and native XRs and XDHs were tested. Highest productivity was observed in a strain expressing CsheXR K275R/N277D (NADH-preferring) and native CsheXDH (NAD(+)-dependent), which produced 17.4 g/L of ethanol from 50 g/L of xylose in 20 h. Analysis of the genes responsible for ethanol production from the xylose capacity of C. utilis indicated that the introduction of CsheXDH was essential, while overexpression of CsheXR K275R/N277D improved efficiency of ethanol production.     

Xylose isomerase improves growth and ethanol production rates from biomass sugars for both Saccharomyces pastorianus and Saccharomyces cerevisiae

The demand for biofuel ethanol made from clean, renewable nonfood sources is growing. Cellulosic biomass, such as switch grass (Panicum virgatum L.), is an alternative feedstock for ethanol production; however, cellulosic feedstock hydrolysates contain high levels of xylose, which needs to be converted to ethanol to meet economic feasibility. In this study, the effects of xylose isomerase on cell growth and ethanol production from biomass sugars representative of switch grass were investigated using low cell density cultures. The lager yeast species Saccharomyces pastorianus was grown with immobilized xylose isomerase in the fermentation step to determine the impact of the glucose and xylose concentrations on the ethanol production rates. Ethanol production rates were improved due to xylose isomerase; however, the positive effect was not due solely to the conversion of xylose to xylulose. Xylose isomerase also has glucose isomerase activity, so to better understand the impact of the xylose isomerase on S. pastorianus, growth and ethanol production were examined in cultures provided fructose as the sole carbon. It was observed that growth and ethanol production rates were higher for the fructose cultures with xylose isomerase even in the absence of xylose. To determine whether the positive effects of xylose isomerase extended to other yeast species, a side-by-side comparison of S. pastorianus and Saccharomyces cerevisiae was conducted. These comparisons demonstrated that the xylose isomerase increased ethanol productivity for both the yeast species by increasing the glucose consumption rate. These results suggest that xylose isomerase can contribute to improved ethanol productivity, even without significant xylose conversion.     

Aeration alleviates ethanol inhibition and glycerol production during fed-batch ethanol fermentation

In this study, we investigated the effects of aeration on ethanol inhibition and glycerol production during fed-batch ethanol fermentation. When aeration was conducted at 0.13, 0.33, and 0.8 vvm, the ethanol productivity, specific ethanol production rate, and ethanol yield in the presence of greater than 100 g/L of ethanol were higher than when aeration was not conducted. In addition, estimation of the parameters (α and β) in a model equation of ethanol inhibition kinetics indicated that aeration alleviated ethanol inhibition against the specific growth rate and the specific ethanol production rate. Specifically, when aeration was conducted, the glycerol yield and specific glycerol production rate decreased approximately 50 and 70%, respectively. Finally, the results of this study indicated that aeration during fed-batch ethanol fermentation may improve the ethanol concentration in the final culture broth, as well as the ethanol productivity.     

Discovery and characterization of a xylose reductase from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> ZM4

Formation of xylitol, a byproduct from xylose fermentation, is a major limiting factor in ethanol production from xylose in engineered Zymomonas strains, yet the postulated xylose reductase remains elusive. We report here the discovery of xylose reductase in Zymomonas mobilis and, for the first time, to associate the enzyme function with its gene. Besides xylose and xylulose, the enzyme was active towards benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, exhibiting nearly 150-times higher affinity with benzaldehyde than xylose. The discovery of xylose reductase paves the way for further improvement of xylose fermentation in Z. mobilis. The enzyme may also be used to mitigate toxicity of furfural and other inhibitors from plant biomass.     

Influence of magnesium concentration on growth,ethanol and xylitol production by Pichia stipitis NRRL Y-7124

Summary The effect of Mg⁺² on Pichia stipitis growth and ethanol production was studied under condition of constant oxygen uptake rate (OUR) . Biomass/xylose and biomass/Mg⁺² yields increased with Mg⁺² concentration with a maximum value at Mg⁺² 4mM, ethanol being the main product obtained. At low Mg⁺² levels (ImM) 49 % of carbon flux to ethanol was redirected to xylitol production, accomplished through NADH intracellular accumulation.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.