Glucose Xylose Research Articles

Cloning and characterization of heterologous transporters in Saccharomyces cerevisiae and identification of important amino acids for xylose utilization

Efficient and specific transporters may enhance pentose uptake and metabolism by Saccharomyces cerevisiae. Eight heterologous sugar transporters were characterized in S. cerevisiae. The transporter Mgt05196p from Meyerozyma guilliermondii showed the highest xylose transport activity among them. Several key amino acid residues of Mgt05196p were suggested by structural and sequence analysis and characterized by site-directed mutagenesis. A conserved aromatic residue-rich motif (YFFYY, position 332–336) in the seventh trans-membrane span plays an important role in d-xylose transport activity. The phenyl ring of the residue at position 336 may take the function to prevent d-xylose from escaping during uptake. F432A and N360S mutations enhanced the d-xylose transport activities of Mgt05196p. Furthermore, mutant N360F specifically transported d-xylose without any glucose-inhibition, high lighting its potential application in constructing glucose–xylose co-fermentation strains for biomass refining.

High-cell-density cultivation of an engineered Rhodococcus opacus strain for lipid production via co-fermentation of glucose and xylose

An engineered strain MITXM-61 of Rhodococcus opacus PD630 capable of utilizing xylose was used to produce biofuel precursor-triacylglycerols (TAG) from mixed glucose–xylose substrates. Optimal nitrogen source and carbon source concentrations were investigated for cell growth and microbial lipid production by MITXM-61 in flask cultures. In a two-stage batch culture, the maximum lipid yield (0.152gTAGperg consumed carbon source) was achieved by utilizing a mixture of glucose and xylose. The fed-batch culture featured intermittent feeding of refined xylose solution during the lipid accumulation stage provided 45gL−1 of cell dry weight, 54% (gTAGperg cell dry weight) of lipid content, 0.179gg−1 (gTAGperg consumed carbon source) of lipid yield, and a 6.9gL−1day−1 of lipid productivity. Fatty acids extracted from microbial lipids produced by MITXM-61 were predominately palmitic and oleic acids, which are the major components in TAG-derived biofuels.

Evaluation of Paecilomyces variotii potential in bioethanol production from lignocellulose through consolidated bioprocessing.

The ascomycete Paecillomyces variotii was evaluated for the first time as a candidate species for the production of bioethanol from lignocellulose through consolidated bioprocessing (CBP) approaches. The examined strain (ATHUM 8891) revealed all the necessary phenotypic characteristics required for 2nd generation biofuel production. The fungus is able to efficiently ferment glucose and xylose to ethanol, with yields close to the theoretical maximum. Nitrogen supplementation greatly affected ethanol production with nitrate-nitrogen presenting the best results. Notably, ethanol yield on xylose fermentation was higher than that of glucose, while in co-fermentation of glucose-xylose mixtures no distinguished diauxic behavior was observed. Furthermore, the fungus seems to possess the necessary enzyme factory for the degradation of lignocellulosic biomass, as it was able to grow and produce ethanol on common agro-industrial derivatives. Overall, the results of our study indicate that P. variotii is a new and possibly powerful candidate for CBP applications.

An integrated process to produce ethanol, vanillin, and xylooligosaccharides from Camellia oleifera shell

This study aims to present an integrated process that can be used to produce ethanol, vanillin, and xylooligosaccharides from Camellia oleifera shell. After the shell was pretreated with NaOH, two fractions were obtained: solid and liquid fractions. The solid fraction was hydrolyzed with cellulase and then fermented with Pichia stipitis to produce ethanol. The liquid fraction was subjected to oxidation to prepare vanillin or hydrolysis with xylanase to prepare xylooligosaccharides. The optimal pretreatment conditions of an orthogonal test were as follows: 12% NaOH concentration; 120°C; 150min; and liquid–solid ratio of 10.0. After pretreatment, the solid fraction containing cellulose and a small part of xylan at 10% substance concentration via enzymatic hydrolysis and glucose–xylose cofermentation could obtain 17.35g/L of ethanol, 80.90% of the theoretical yield. The liquid fraction was initially hydrolyzed with xylanase to produce 1758.63mg/L of xylooligosaccharides (DP2–6) and then oxidized to produce 322.07mg/L of vanillin.

Systematic Approach To Engineer Escherichia coli Pathways for Co-utilization of a Glucose–Xylose Mixture

Glucose and xylose are two major sugars of lignocellulosic hydrolysate. The regulatory program of catabolite repression in Escherichia coli dictates the preferred utilization of glucose over xylose, which handicaps the development of the lignocellulose-based fermentation process. To co-utilize a glucose-xylose mixture, the E. coli strain was manipulated by pathway engineering in a systematic way. The approach included (1) blocking catabolite repression, (2) enhancing glucose transport, (3) increasing the activity of the pentose phosphate pathway, and (4) eliminating undesirable pathways. Moreover, the ethanol synthetic pathway from Zymomonas mobilis was introduced into the engineered strain. As a consequence, the resulting strain was able to simultaneously metabolize glucose and xylose and consume all sugars (30 g/L each) in 16 h, leading to 97% of the theoretical ethanol yield. Overall, this indicates that this approach is effective and straightforward to engineer E. coli for the desired trait.

Genetic engineering of yeasts to improve ethanol production from xylose

Saccharomyces cerevisiae, expressing xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) genes from Pichia stipitis, has the ability to produce ethanol from xylose. To improve ethanol production, three recombinant strains of F102X (fps1Δ), F104X (fps1Δ gpd2Δ) and F106X (fps1Δ gpd2Δ GLN1) with TKL1, TAL1, RKI1, RPE1, XYL1, XYL2 and XKS1 overexpression were constructed in this study. Compared to the control strain FB3X, ethanol production from xylose in strains F102X, F104X and F106X was increased by 4.3, 9.5 and 17.2%, respectively. F106X produced 18.2% less glycerol from a glucose–xylose mixture medium than FB3X, whereas no glycerol production was detected in these four strains when grown on a xylose medium. In addition, the xylitol production and ethanol yield in strain F106X did not differ significantly. These results suggested that the improvement in ethanol production in strain F106X was mainly due to increased xylose consumption.

Functional expression of Burkholderia cenocepacia xylose isomerase in yeast increases ethanol production from a glucose–xylose blend

This study presents results regarding the successful cloning of the bacterial xylose isomerase gene (xylA) of Burkholderia cenocepacia and its functional expression in Saccharomyces cerevisiae. The recombinant yeast showed to be competent to efficiently produce ethanol from both glucose and xylose, which are the main sugars in lignocellulosic hydrolysates. The heterologous expression of the gene xylA enabled a laboratorial yeast strain to ferment xylose anaerobically, improving ethanol production from a fermentation medium containing a glucose–xylose blend similar to that found in sugar cane bagasse hydrolysates. The insertion of xylA caused a 5-fold increase in xylose consumption, and over a 1.5-fold increase in ethanol production and yield, in comparison to that showed by the WT strain, in 24h fermentations, where it was not detected accumulation of xylitol. These findings are encouraging for further studies concerning the expression of B. cenocepacia xylA in an industrial yeast strain.

Techno-economic evaluation of membrane cascades relative to simulated moving bed chromatography for the purification of mono- and oligosaccharides

In this paper the McCabe–Thiele method, previously used for the design of gas separation membrane cascades, was adapted for membrane cascades for solute–solute separations. This method was applied here on three different sugar separations: raffinose–sucrose, fructose–glucose and xylose–glucose. The state of the art for all these separations is simulated moving bed (SMB) chromatography. For all separations a membrane cascade could be designed to reach the same specifications as the reference SMB. This is especially remarkable for the very challenging glucose–fructose separation where starting from 50% fructose purity a 94% fructose purity could be reached. Due to the high number of required stages, for this separation the cascade cost was several times higher than the cost of the reference SMB. However, for the raffinose–sucrose and glucose–xylose separation the cascade cost was similar or lower than the cost of the SMB. Moreover from the raffinose–sucrose separation and purification it could be concluded that the competitiveness of membrane cascades over SMB chromatography increases with the plant size. Also if the purity requirement becomes less stringent the competitiveness increases as could be seen from the glucose–xylose separation. As a result membrane cascades seem most promising for large scale continuous processes for producing pure but depending on the selectivity not ultrapure products. A hybrid membrane cascade SMB process could be envisaged to also cover the ultrapure products range in a cost effective way.

Separation of furfural from monosaccharides by nanofiltration

Furfural, found in the lignocellulosic prehydrolyzates at high concentration, is a strong inhibitor of growth and ethanol fermentation of Saccharomyces cerevisiae. Removal of furfural and concentration of monosaccharides were investigated by using two commercial nanofiltraton (NF) membranes with synthetic glucose–xylose–furfural solution as model. The effects of main operating parameters such as feed pH, permeation flux, temperature and feed concentration on the rejections of the three solutes, were studied. Results showed that rejections of the three solutes decreased with increasing feed pH and temperature, and increased with increasing permeation flux for both membranes. The concentrations of the three solutes had interaction effect on the rejection of furfural by NF90 membrane and rejections of the three solutes by NF270 membrane. Furthermore, the effects of two filtration modes, concentration and diafiltration, on the separation of furfural from monosaccharides were also investigated. With the two commercial NF membranes, concentration and purification of monosaccharides in the model solution can be accomplished.

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose–xylose mixtures

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

Impact of recycling stillage on conversion of dilute sulfuric acid pretreated corn stover to ethanol

Both the current corn starch to ethanol industry and the emerging lignocellulosic biofuels industry view recycling of spent fermentation broth or stillage as a method to reduce fresh water use. The objective of this study was to understand the impact of recycling stillage on conversion of corn stover to ethanol. Sugars in a dilute-acid pretreated corn stover hydrolysate were fermented to ethanol by the glucose-xylose fermenting bacteria Zymomonas mobilis 8b. Three serial fermentations were performed at two different initial sugar concentrations using either 10% or 25% of the stillage as makeup water for the next fermentation in the series. Serial fermentations were performed to achieve near steady state concentration of inhibitors and other compounds in the corn stover hydrolysate. Little impact on ethanol yields was seen at sugar concentrations equivalent to pretreated corn stover slurry at 15% (w/w) with 10% recycle of the stillage. However, ethanol yields became progressively poorer as the sugar concentration increased and fraction of the stillage recycled increased. At an equivalent corn stover slurry concentration of 20% with 25% recycled stillage the ethanol yield was only 5%. For this microorganism with dilute-acid pretreated corn stover, recycling a large fraction of the stillage had a significant negative impact on fermentation performance. Although this finding is of concern for biochemical-based lignocellulose conversion processes, other microorganism/pretreatment technology combinations will likely perform differently.

Multi-stage continuous culture fermentation of glucose–xylose mixtures to fuel ethanol using genetically engineered Saccharomyces cerevisiae 424A

Multi-stage continuous (chemostat) culture fermentation (MCCF) with variable fermentor volumes was carried out to study the utilization of glucose and xylose for ethanol production via mixed sugar fermentation (MSF). Variable fermentor volumes were used to enable enhanced sugar utilization, accounting for differences in glucose and xylose utilization rates. Saccharomyces cerevisiae 424A-LNH-ST was used for fermentation of glucose–xylose mixtures. The dilution rates employed for continuous fermentation were based on earlier batch kinetic studies of ethanol production and sugar utilization. With a feed containing approximately 30 g L −1 glucose and 15 g L −1 xylose, cell washout was observed at a dilution rate of 0.8 h −1. At dilution rates below 0.5 h −1, complete glucose utilization was observed. Xylose consumption in the first-stage 1 L reactor was only 37% at the lowest dilution rate studied, 0.05 h −1. At this same flow rate, xylose consumption rose to 69% after subsequently passing through 3 and 1 L reactors in series, primarily due to the longer residence time in the 3 L reactor (0.0167 h −1 dilution rate).

Nonlinear analysis of the effect of maintenance in continuous cell cultures

The effect of maintenance on steady-state behavior in continuous cell cultures is studied theoretically with particular emphasis on stability and multiplicity characteristics. Bifurcation theory and stability analysis is applied to determine how the number and stability of steady-states depend on dilution rate and feed composition for Klebsiella pneumoniae feeding on mixed glucose–xylose substrates. The biological system is analyzed using a cybernetic modeling approach. We propose a numerical strategy which enables us to determine all possible steady-states for a given set of operating conditions. The algorithm exploits the polynomial nature of the equations. Results for a model without maintenance are compared to the predictions of a model with maintenance. It is shown that the model without maintenance effects and constitutive enzyme synthesis rate admits spurious steady-state solutions, which disappear when these effects are taken into account. In both cases multiple steady-states are observed over a range of dilution rates. In addition, the model with maintenance also predicts dynamic instability in the form of self-sustained oscillations. We have also investigated the effect of various parameters on the region of multiplicity to determine operating conditions which would enable experimental verification of our predictions.

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 (Δ 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 (Δ 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*.

Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on d-xylose

Overexpression of d-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Δ mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Δ mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose–xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Δ mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.

Kinetics of growth and ethanol production on different carbon substrates using genetically engineered xylose-fermenting yeast

Saccharomyces cerevisiae 424A (LNH-ST) strain was used for fermentation of glucose and xylose. Growth kinetics and ethanol productivity were calculated for batch fermentation on media containing different combinations of glucose and xylose to give a final sugar concentration of 20 ± 0.8 g/L. Growth rates obtained in pure xylose-based medium were less than those for media containing pure glucose and glucose–xylose mixtures. A maximum specific growth rate μ max of 0.291 h −1 was obtained in YPD medium containing 20 g/L glucose as compared to 0.206 h −1 in YPX medium containing 20 g/L xylose. In media containing combinations of glucose and xylose, glucose was exhausted first followed by xylose. Ethanol production on pure xylose entered log phase during the 12–24 h period as compared to the 4–10 h for pure glucose based medium using 2% inoculum. When glucose was added to fermentation flasks which had been initiated on a pure xylose-based medium, the rate of xylose usage was reduced indicating cosubstrate inhibition of xylose consumption by glucose.

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting 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.

Fermentation performance and intracellular metabolite profiling of Fusarium oxysporum cultivated on a glucose–xylose mixture

The influence of aeration on the fermentation of a mixture of glucose and xylose by the naturally xylose fermenting fungus Fusarium oxysporum was studied in batch cultivations. The aeration level had considerable influence on the co-metabolism of glucose and xylose. Under anaerobic conditions xylose consumption was limited and xylitol was the main product of xylose metabolism. When the artificial electron acceptor acetoin was added xylose consumption increased by 95% with a significant improvement of ethanol yield from 0.96 to 1.52 mol /mol, mainly due to a 72% reduction of xylitol excretion. The profile of the phosphorylated intermediates during the fermentation suggests that a high glycolytic flux occurred during the oxygen-limited conditions, characterized by high efflux of glyceraldehyde-3-P (G3P) and fructose-6-P (F6P) from Pentose Phosphate Pathway (PPP) to Embden-Meyerhof-Parnas Pathway (EMP) resulting in the higher ethanol production. An accumulation of sedoheptulose-7-P (S7P) was also observed indicating a block in the PPP which affects the production of NADPH and results in the production of acetate and the reduced xylose consumption.

Isolation of two anthocyanin sambubiosides from bilberry ( Vaccinium myrtillus) by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was used for the separation of anthocyanins on a preparative scale from bilberry fruit crude extract ( Vaccinium myrtillus, Ericaceae) with a biphasic solvent system composed of methyl tert-butyl ether– n-butanol–acetonitrile–water–trifluoroacetic acid (1:4:1:5:0.01, v/v). Each injection of 500 mg crude extract yielded 130 mg of pure delphinidin-3-O-sambubioside, and 77 mg of pure cyanidin-3-O-sambubioside. The two anthocyanin disaccharides with a glucose (2 → 1) xylose unit are novel compounds for bilberry fruit, and were elucidated by means of electrospray ionization MS–MS, 1 H , 13 C , distortion less enhancement by polarization transfer NMR, and two-dimensional HC-correlation experiments (heteronuclear multiple bond correlation and heteronuclear multiple quantum correlation).

Symplastic and apoplastic sugar contents in gall tissues and callus of the sumac (Rhus chinensis MILL.)

To elucidate the role of cell wall in interaction with gall-inducing organisms, symplastic and apoplastic sugar contents in different shapes of gall tissue of the sumac (Rhus chinensis Mill.) were compared with those of the callus. The gall tissues with vascular cylinders, intercellular spaces and callus were fractionated into symplastic [methanol (MeOH), hot water (HW), and starch] fractions and apoplastic [pectin, hemicellulose, trifluoroacetic acid (TFA)-soluble, and cellulose] fractions. Symplastic sugar content of gall tissues was higher than that of callus. In apoplastic (cell wall) fractions, the cellulose content of gall tissues was lower than that of callus, due to large amount of pectin with high ratio of uronic acid (UA) and hemicellulose with low ratio of UA. Analysis of neutral sugar component of the hemicellulosic, TFA-soluble fraction showed that arabinose (side chain) and galactose (backbone) of arabinogalactan were rich in gall tissues and callus. The gall tissues had higher glucose and lower xylose contents than the callus. These results suggest that the structure of cell wall polysaccharides of gall changed during its development with an increase in symplastic sugar contents. The feeding activities occuring in gall by the gall-inducers were discussed.