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.     

Kinetic modelling reveals current limitations in the production of ethanol from xylose by recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks the ability to ferment the pentose sugar xylose that is the second most abundant sugar in nature. Therefore two different xylose catabolic pathways have been heterologously expressed in S. cerevisiae. Whereas the xylose reductase (XR)-xylitol dehydrogenase (XDH) pathway leads to the production of the by-product xylitol, the xylose isomerase (XI) pathway results in significantly lower xylose consumption. In this study, kinetic models including the reactions ranging from xylose transport into the cell to the phosphorylation of xylulose to xylulose 5-P were constructed. They were used as prediction tools for the identification of putative targets for the improvement of xylose utilization in S. cerevisiae strains engineered for higher level of the non-oxidative pentose phosphate pathway (PPP) enzymes, higher xylulokinase and inactivated GRE3 gene encoding an endogenous NADPH-dependent aldose reductase. For both pathways, the in silico analyses identified a need for even higher xylulokinase (XK) activity. In a XR-XDH strain expressing an integrated copy of the Escherichia coli XK encoding gene xylB about a six-fold reduction of xylitol formation was confirmed under anaerobic conditions. Similarly overexpression of the xylB gene in a XI strain increased the aerobic growth rate on xylose by 21%. In contrast to the in silico predictions, the aerobic growth also increased 24% when the xylose transporter gene GXF1 from Candida intermedia was overexpressed together with xylB in the XI strain. Under anaerobic conditions, the XI strains overexpressing xylB gene and the combination of xylB and GFX1 genes consumed 27% and 37% more xylose than the control strain.     

Cloning of the glucose isomerase (D-xylose isomerase) and xylulose kinase genes of Streptomyces violaceoniger

Summary Xylose utilization mutants of Streptomyces violaceoniger were isolated lacking one or both of the enzymes, glucose isomerase (xylose isomerase) and xylulose kinase. Using pUT206 as a cloning vector, complementation of the glucose isomerase negative phenotype with fragments of the S. violaceoniger chromosome permitted isolation of two recombinant plasmids, designated pUT220 and pUT221, which contained 10.6 and 10.1 kb of chromosomal DNA, respectively. Both of these plasmids complemented all three different classes of xylose negative mutants and also provoked an increase of glucose isomerase and xylulose kinase activity in the mutant and wild-type strains. Plasmid pUT220 was chosen for detailed study by subcloning experiments. The putative glucose isomerase gene was localized to a 2.1 kb segment of the 10.6 kb chromosomal DNA fragment. The putative xylulose kinase gene resides nearby. Thus both genes seem to be clustered at a single chromosomal localization. This organization appears similar to that of the xylose utilization pathway in Escherichia coli, Salmonella typhimurium and Bacillus subtilis.     

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     

Breeding of a xylose-fermenting hybrid strain by mating genetically engineered haploid strains derived from industrial Saccharomyces cerevisiae

The industrial Saccharomyces cerevisiae IR-2 is a promising host strain to genetically engineer xylose-utilizing yeasts for ethanol fermentation from lignocellulosic hydrolysates. Two IR-2-based haploid strains were selected based upon the rate of xylulose fermentation, and hybrids were obtained by mating recombinant haploid strains harboring heterogeneous xylose dehydrogenase (XDH) (wild-type NAD⁺-dependent XDH or engineered NADP⁺-dependent XDH, ARSdR), xylose reductase (XR) and xylulose kinase (XK) genes. ARSdR in the hybrids selected for growth rates on yeast extract-peptone-dextrose (YPD) agar and YP-xylose agar plates typically had a higher activity than NAD⁺-dependent XDH. Furthermore, the xylose-fermenting performance of the hybrid strain SE12 with the same level of heterogeneous XDH activity was similar to that of a recombinant strain of IR-2 harboring a single set of genes, XR/ARSdR/XK. These results suggest not only that the recombinant haploid strains retain the appropriate genetic background of IR-2 for ethanol production from xylose but also that ARSdR is preferable for xylose fermentation.     

Identification of a xylulokinase catalyzing xylulose phosphorylation in the xylose metabolic pathway of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NBRC1777

Xylulokinase is one of the key enzymes in xylose metabolism and fermentation, and fine-tuned expression of xylulokinase can improve xylose fermentation in yeast. To improve the efficiency of xylose fermentation in Kluyveromyces marxianus, the gene KmXYL3, which encodes a d-xylulokinase (E.C. 2.7.1.17), was isolated from K. marxianus NBRC1777. KmXYL3 was expressed in Escherichia coli BL21 (DE3) cells, and the specific activity of the resulting recombinant purified xylulokinase was 23.5 mU/mg. Disruption of KmXYL3 resulted in both loss of xylitol utilization and marked decrease in xylose utilization, proving that KmXYL3 encodes a xylulokinase that catalyzes the reaction from xylulose to xylulose 5-phosphate in the xylose metabolic pathway. The slow assimilation of xylose observed in the KmXYL3-disrupted strain indicates that KmXYL3 is critical for xylose and xylitol utilization; however, K. marxianus utilizes a bypass pathway for xylose assimilation, and this pathway does not involve xylitol or xylulose.     

Comparative Xylose Metabolism among the Ascomycetes C. albicans,S. stipitis and S. cerevisiae

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.     

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.     

<Emphasis Type="Italic">Pichia stipitis</Emphasis> xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

Background  

Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.     

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.     

Some aspects of xylose isomerase constitutive biosynthesis in <Emphasis Type="Italic">Arthrobacter nicotianae</Emphasis>

The specific features of biosynthesis of the cell-bound xylose isomerase by the actinobacterium Arthrobacter nicotianae BIM V-5 were studied. It was demonstrated that the constitutive synthesis of this enzyme in the studied bacteria, not subject to catabolite repression, was inhibited by xylulose, an intermediate product of xylose utilization and the final product of its enzymatic isomerization. Short-term experiments demonstrated that xylulose at a concentration of 0.005% almost completely repressed the xylose isomerase synthesis in A. nicotianae. This effect was independent of the time moment when the repressor was added to the cultivation medium and was not associated with its influence on the catalytic activity of the enzyme.     

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     

Xylitol production from xylose by two yeast strains: Sugar tolerance

The kinetics and enzymology ofd-xylose utilization are studied in micro-, semi-, and aerobic batch cultures during growth ofCandida guilliermondii andCandida parapsilosis in the presence of several initial xylose concentrations. The abilities of xylitol accumulation by these two yeast strains are high and similar, although observed under various growth conditions. WithCandida parapsilosis, optimal xylitol production yield (0.74 g/g) was obtained in microaerobiosis with 100 g/L of xylose, whereas optimal conditions to produce xylitol byCandida guilliermondii (0.69 g/g) arose from aerobiosis with 300 g/L of sugar. The different behavior of these yeasts is most probably explained by differences in the nature of the initial step of xylose metabolism: a NADPH-linked xylose reductase activity is measured with a weaker NADH-linked activity. These activities seem to be dependent on the degree of aerobiosis and on the initial xylose concentration and correlate with xylitol accumulation.     

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.     

Co-expression of xylose reductase gene from <Emphasis Type="Italic">Candida shehatae</Emphasis> and endogenous xylitol dehydrogenase gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and the effect of metabolizing xylose to ethanol

The inability oft Saccharomyces cerevisiae to utilize xylose is attributed to its inability to convert xylose to xylulose. Low xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in S. cerevisiae are regarded as the reason of blocking the pathway from xylose to xylulose. We had found that Candida shehatae could also be another source for XR gene except Pichia stipitis in the previous study. In this study, we tried to investigate if the expressed XR from C. shehatae could work with the over-expressed endogenous XDH together to achieve the same goal of converting xylose to ethanol in S. cerevisiae. The XR gene (XYL1) from C. shehatae and endogenous XDH gene (XYL2) were both cloned and over-expressed in host S. cerevisiae cell. The specific enzyme activities of XR and XDH were measured and the result of fermentation revealed that the new combination of two enzymes from different sources other than P. stipitis could also coordinate and work with each other and confer xylose utilization ability to S. cerevisiae.     

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.     

Metabolism of d-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene

Summary The Escherichia coli xylose isomerase gene was transformed into Schizosaccharomyces pombe for direct d-xylose utilization. In order to understand d-xylose metabolism and determine the limiting factors on d-xylose utilization by the transformed yeast, d-xylose transport, xylose isomerization, and xylulose phosphorylation were investigated. The results indicated that low activity of xylose isomerization in the cloned yeast was the limiting step for d-xylose fermentation. An in vitro study showed that yeast proteases decreased xylose isomerase activity. Xylitol, a by-product of d-xylose fermentation, had no effect on the activity of xylose isomerase.     

Transfer of genes fromPseudomonas saccharophila to construct xylose-utilizing strains ofAlcaligenes eutrophus

About 1500 hybrid broad-host-range plasmids from a genomic library ofPseudomonas saccharophila were individually transferred by conjugation fromEscherichia coli toAlcaligenes eutrophus. Direct selection for pentose-utilizing transconjugants yielded three clones capable of growth on xylose. Growth ofP. saccharophila as well as the transconjugants ofA. eutrophus on xylose was relatively slow, exhibiting doubling times of about 9.5 h. Plasmid pGN3 harbored by one transconjugant contained a 28-kb DNA insert, 16.4 kb of which comprised the minimal information required for xylose utilization byA. eutrophus. At least thexyl genes encoding xylose isomerase and xylulokinase were located within this region, as indicated by their induction during growth ofA. eutrophus (pGN3) on xylose. Southern hybridizations with a heterologous gene probe confirmed the presence of thesexyl genes. In bothP. saccharophila andA. eutrophus (pGN3), low activities of several enzymes operating in the pentose phosphate and Entner-Doudoroff pathways might limit the rate of xylose catabolism.