Efficient Xylose Utilization Research Articles

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization.

High-titer-ethanol production from cellulosic hydrolysate by an engineered strain of Saccharomyces cerevisiae during an in situ removal process reducing the inhibition of ethanol on xylose metabolism

Efficient xylose utilization is critical for the production of fuels from biomass hydrolysates. It is known that xylose catabolism is inhibited by glucose. In this study, we showed that ethanol also inhibits xylose catabolism. By introducing a xylose metabolic pathway into Saccharomyces cerevisiae and using evolutionary engineering, an engineered S. cerevisiae strain, W32N55, was obtained that can anaerobically ferment xylose to ethanol. The effect of ethanol on xylose utilization was investigated. The results showed that xylose catabolism was inhibited upon the addition of ethanol, and it resumed once ethanol was removed. Based on these results, a fermentation–pervaporation coupling process was developed. After the in situ removal of ethanol, 150g/L glucose and 31g/L xylose were consumed in 72h, providing a total of 76g/L ethanol and an overall total sugar yield of 0.42g/g. We believe that this strain will be valuable to the bio-ethanol industry.

Effect of Evolutionary Adaption on Xylosidase Activity in Thermotolerant Yeast Isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3.

Efficient use of xylose along with glucose is necessary for the economic production of lignocellulosic based biofuels. Xylose transporters play an important role in the microorganisms for efficient utilization of xylose. In the present study, a novel method has been developed for a rapid assay of xylose transport activity in the xylose-utilizing isolates and other known yeasts. An assay was conducted to compare the activity of β-xylosidase using p-nitrophenyl-β-D-xylopyranoside (pNPX) in the intact, intracellular, and extracellular yeasts cells showing xylose transporter. Saccharomyces cerevisiae (MTCC 170) showed no xylosidase activity, while little growth was observed in the xylose-containing medium. Although other yeasts, i.e., Kluyveromyces marxianus NIRE-K1 (MTCC 5933), K. marxianus NIRE-K3 (MTCC 5934), and Candida tropicalis (MTCC 230), showed xylosidase activity in intact, intracellular, and extracellular culture. The xylosidase activity in intact cell was higher than that of extracellular and intracellular activity in all the yeast cells. The enzyme activity was higher in case of K. marxianus NIRE-K1 and K. marxianus NIRE-K3 rather than the C. tropicalis. Further, better xylosidase activity was observed in adapted K. marxianus cells which were 2.79-28.46% higher than that of native (non-adapted) strains, which indicates the significant improvement in xylose transportation.

Simultaneous utilization of glucose and xylose for lipid accumulation in black soldier fly

BackgroundLignocellulose is known to be an abundant source of glucose and xylose for biofuels. Yeasts can convert glucose into bioethanol. However, bioconversion of xylose by yeasts is not very efficient, to say nothing of the presence of both glucose and xylose. Efficient utilization of xylose is one of the critical factors for reducing the cost of biofuel from lignocelluloses. However, few natural microorganisms preferentially convert xylose to ethanol. The simultaneous utilization of both glucose and xylose is the pivotal goal in the production of biofuels.ResultsIn this paper, we found that 97.3 % of the glucose and 93.8 % of the xylose in our experiments was consumed by black soldier fly (BSF) simultaneously. The content of lipid reached its highest level (34.60 %) when 6 % xylose was added into the standard feed. 200 g of rice straw was pretreated with 1 % KOH, followed by enzymatic hydrolysis for fermentation of ethanol, the residue from this fermentation was then fed to BSF for lipid accumulation. In total, 10.9 g of bioethanol and 4.3 g of biodiesel were obtained.ConclusionsThe results of this study suggest that BSF is a very promising organism for use in converting lignocellulose into lipid for biodiesel production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0306-z) contains supplementary material, which is available to authorized users.

13C metabolic flux analysis of the extremely thermophilic, fast growing, xylose-utilizing Geobacillus strain LC300

Thermophiles are increasingly used as versatile hosts in the biotechnology industry. One of the key advantages of thermophiles is the potential to achieve high rates of feedstock conversion at elevated temperatures. The recently isolated Geobacillus strain LC300 grows extremely fast on xylose, with a doubling time of less than 30min. In the accompanying paper, the genome of Geobacillus LC300 was sequenced and annotated. In this work, we have experimentally validated the metabolic network model using parallel 13C-labeling experiments and applied 13C-metabolic flux analysis to quantify precise metabolic fluxes. Specifically, the complete set of singly labeled xylose tracers, [1-13C], [2-13C], [3-13C], [4-13C], and [5-13C]xylose, was used for the first time. Isotopic labeling of biomass amino acids was measured by gas chromatography mass spectrometry (GC–MS). Isotopic labeling of carbon dioxide in the off-gas was also measured by an on-line mass spectrometer. The 13C-labeling data was then rigorously integrated for flux elucidation using the COMPLETE-MFA approach. The results provided important new insights into the metabolism of Geobacillus LC300, its efficient xylose utilization pathways, and the balance between carbon, redox and energy fluxes. The pentose phosphate pathway, glycolysis and TCA cycle were found to be highly active in Geobacillus LC300. The oxidative pentose phosphate pathway was also active and contributed significantly to NADPH production. No transhydrogenase activity was detected. Results from this work provide a solid foundation for future studies of this strain and its metabolic engineering and biotechnological applications.

Inverse metabolic engineering of Bacillus subtilis for xylose utilization based on adaptive evolution and whole-genome sequencing.

Efficient utilization of xylose by bacteria is essential for production of fuels and chemicals from lignocellulosic biomass. In this study, Bacillus subtilis 168 was subjected to laboratory adaptive evolution, and a mutant E72, which could grow on xylose with a maximum specific growth rate of 0.445 h(-1), was obtained. By whole-genome sequencing, 16 mutations were identified in strain E72. Through further analysis, three of them, which were in the coding regions of genes araR, sinR, and comP, were identified as the beneficial mutations. The reconstructed strain 168ARSRCP harboring these three mutations exhibited similar growth capacity on xylose to the evolved strain E72, and the average xylose consumption rate of this strain is 0.530 g/l/h, much higher than that of E72 (0.392 g/l/h). Furthermore, genes acoA and bdhA were deleted and the final strain could utilize xylose to produce acetoin at 71 % of the maximum theoretical yield. These results suggested that this strain could be used as a potential platform for production of fuels and chemicals from lignocellulosic biomass.

Overcoming inefficient cellobiose fermentation by cellobiose phosphorylase in the presence of xylose.

BackgroundCellobiose and xylose co-fermentation holds promise for efficiently producing biofuels from plant biomass. Cellobiose phosphorylase (CBP), an intracellular enzyme generally found in anaerobic bacteria, cleaves cellobiose to glucose and glucose-1-phosphate, providing energetic advantages under the anaerobic conditions required for large-scale biofuel production. However, the efficiency of CBP to cleave cellobiose in the presence of xylose is unknown. This study investigated the effect of xylose on anaerobic CBP-mediated cellobiose fermentation by Saccharomyces cerevisiae.ResultsYeast capable of fermenting cellobiose by the CBP pathway consumed cellobiose and produced ethanol at rates 61% and 42% slower, respectively, in the presence of xylose than in its absence. The system generated significant amounts of the byproduct 4-O-β-d-glucopyranosyl-d-xylose (GX), produced by CBP from glucose-1-phosphate and xylose. In vitro competition assays identified xylose as a mixed-inhibitor for cellobiose phosphorylase activity. The negative effects of xylose were effectively relieved by efficient cellobiose and xylose co-utilization. GX was also shown to be a substrate for cleavage by an intracellular β-glucosidase.ConclusionsXylose exerted negative impacts on CBP-mediated cellobiose fermentation by acting as a substrate for GX byproduct formation and a mixed-inhibitor for cellobiose phosphorylase activity. Future efforts will require efficient xylose utilization, GX cleavage by a β-glucosidase, and/or a CBP with improved substrate specificity to overcome the negative impacts of xylose on CBP in cellobiose and xylose co-fermentation.

Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae.

BackgroundEfficient utilization of both glucose and xylose is necessary for a competitive ethanol production from lignocellulosic materials. Although many advances have been made in the development of xylose-fermenting strains of Saccharomyces cerevisiae, the productivity remains much lower compared to glucose. Previous transcriptional analyses of recombinant xylose-fermenting strains have mainly focused on central carbon metabolism. Very little attention has been given to other fundamental cellular processes such as the folding of proteins. Analysis of previously measured transcript levels in a recombinant XR/XDH-strain showed a wide down-regulation of genes targeted by the unfolded protein response during xylose fermentation. Under anaerobic conditions the folding of proteins is directly connected with fumarate metabolism and requires two essential enzymes: FADH2-dependent fumarate reductase (FR) and Ero1p. In this study we tested whether these enzymes impair the protein folding process causing the very slow growth of recombinant yeast strains on xylose under anaerobic conditions.ResultsFour strains over-expressing the cytosolic (FRD1) or mitochondrial (OSM1) FR genes and ERO1 in different combinations were constructed. The growth and fermentation performance was evaluated in defined medium as well as in a complex medium containing glucose and xylose. Over-expression of FRD1, alone or in combination with ERO1, did not have any significant effect on xylose fermentation in any medium used. Over-expression of OSM1, on the other hand, led to a diversion of carbon from glycerol to acetate and a decrease in growth rate by 39% in defined medium and by 25% in complex medium. Combined over-expression of OSM1 and ERO1 led to the same diversion of carbon from glycerol to acetate and had a stronger detrimental effect on the growth in complex medium.ConclusionsIncreasing the activities of the FR enzymes and Ero1p is not sufficient to increase the anaerobic growth on xylose. So additional components of the protein folding mechanism that were identified in transcription analysis of UPR related genes may also be limiting. This includes i) the transcription factor encoded by HAC1 ii) the activity of Pdi1p and iii) the requirement of free FAD during anaerobic growth.

Metabolomic and Transcriptomic Analysis for Rate-Limiting Metabolic Steps in Xylose Utilization by RecombinantCandida utilis

We have reported that a recombinant Candida utilis strain expressing a Candida shehatae xylose reductase K275R/N277D, a C. shehatae xylitol dehydrogenase, and xylulokinase from Pichia stipitis produced ethanol from xylose, but its productivity was low. In the present study, metabolomic (CE-TOF MS) and transcriptomic (microarray) analyses were performed to characterize xylose metabolism by engineered C. utilis and to identify key genetic changes contributing to efficient xylose utilization. The metabolomic analysis revealed that the xylose-fermenting strain accumulated more pentose phosphate pathway intermediates, more NADH, and more glycolytic intermediates upstream of glyceraldehyde 3-phosphate than the wild-type. Transcriptomic analysis of the strain grown on xylose indicated a significant increase in expression of the genes encoding tricarboxylic acid cycle enzymes, respiratory enzymes, and enzymes involved in ethanol oxidation. To decrease the NADH/NAD(+) ratio and increase the ethanol yield of the fermentation of xylose, ADH1 encoding NADH-dependent alcohol dehydrogenase was overexpressed. The resulting strain exhibited a 17% increase in ethanol production and a 22% decrease in xylitol accumulation relative to control.

Investigating xylose metabolism in recombinant Saccharomyces cerevisiae via 13C metabolic flux analysis.

BackgroundTo engineer Saccharomyces cerevisiae for efficient xylose utilization, a fungal pathway consisting of xylose reductase, xylitol dehydrogenase, and xylulose kinase is often introduced to the host strain. Despite extensive in vitro studies on the xylose pathway, the intracellular metabolism rewiring in response to the heterologous xylose pathway remains largely unknown. In this study, we applied 13C metabolic flux analysis and stoichiometric modeling to systemically investigate the flux distributions in a series of xylose utilizing S. cerevisiae strains.ResultsAs revealed by 13C metabolic flux analysis, the oxidative pentose phosphate pathway was actively used for producing NADPH required by the fungal xylose pathway during xylose utilization of recombinant S. cerevisiae strains. The TCA cycle activity was found to be tightly correlated with the requirements of maintenance energy and biomass yield. Based on in silico simulations of metabolic fluxes, reducing the cell maintenance energy was found crucial to achieve the optimal xylose-based ethanol production. The stoichiometric modeling also suggested that both the cofactor-imbalanced and cofactor-balanced pathways could lead to optimal ethanol production, by flexibly adjusting the metabolic fluxes in futile cycle. However, compared to the cofactor-imbalanced pathway, the cofactor-balanced xylose pathway can lead to optimal ethanol production in a wider range of fermentation conditions.ConclusionsBy applying 13C-MFA and in silico flux balance analysis to a series of recombinant xylose-utilizing S. cerevisiae strains, this work brings new knowledge about xylose utilization in two aspects. First, the interplays between the fungal xylose pathway and the native host metabolism were uncovered. Specifically, we found that the high cell maintenance energy was one of the key factors involved in xylose utilization. Potential strategies to reduce the cell maintenance energy, such as adding exogenous nutrients and evolutionary adaptation, were suggested based on the in vivo and in silico flux analysis in this study. In addition, the impacts of cofactor balance issues on xylose utilization were systemically investigated. The futile pathways were identified as the key factor to adapt to different degrees of cofactor imbalances and suggested as the targets for further engineering to tackle cofactor-balance issues.

Directed Evolution of Xylose Isomerase for Improved Xylose Catabolism and Fermentation in the Yeast Saccharomyces cerevisiae

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.

Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption

Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source.

Reconstruction and analysis of a genome-scale metabolic model for Scheffersomyces stipitis

BackgroundFermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes.ResultsWe present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation.ConclusionThe genome-scale metabolic model developed for Scheffersomyces stipitis successfully predicted substrate utilization and anaerobic growth requirements. Useful insights were drawn on xylose metabolism, cofactor recycling and mechanism of mitochondrial respiration from model simulations. These insights can be applied for efficient xylose utilization and cofactor recycling in other industrial microorganisms. The developed model forms a basis for rational analysis and design of Scheffersomyces stipitis metabolic network for the production of fuels and chemicals from lignocellulosic biomass.

Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum

BackgroundBiochemical conversion of lignocellulose hydrolysates remains challenging, largely because most microbial processes have markedly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal to improving this process.ResultsIn this study, we found that the oleaginous yeast strain Trichosporon cutaneum AS 2.571 assimilated glucose and xylose simultaneously, and accumulated intracellular lipid up to 59 wt% with a lipid coefficient up to 0.17 g/g sugar, upon cultivation on a 2:1 glucose/xylose mixture in a 3-liter stirred-tank bioreactor. In addition, no classic pattern of diauxic growth behavior was seen; the microbial cell mass increased during the whole culture process without any lag periods. In shake-flask cultures with different initial glucose:xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. Simultaneous utilization of glucose and xylose was also seen during fermentation of corn-stover hydrolysate with a lipid content and coefficient of 39.2% and 0.15 g/g sugar, respectively. The lipid produced had a fatty-acid compositional profile similar to those of conventional vegetable oil, indicating that it could have potential as a raw material for biodiesel production.ConclusionEfficient lipid production with simultaneous consumption of glucose and xylose was achieved in this study. This process provides an exciting opportunity to transform lignocellulosic materials into biofuel molecules, and should also encourage further study to elucidate this unique sugar-assimilation mechanism.

Developing a thermophilic hydrogen-producing microbial consortia from geothermal spring for efficient utilization of xylose and glucose mixed substrates and oil palm trunk hydrolysate

Xylose and glucose are the major sugar components of lignocellulosic hydrolysate. This study aims to develop thermophilic hydrogen-producing consortia from eight sediments-rich samples of geothermal springs in Southern Thailand by repeated batch cultivation at 60 °C with glucose, xylose and xylose–glucose mixed substrates. Significant hydrogen production potentials were obtained from thermophilic enriched cultures encoded as PGR and YLT with the maximum hydrogen yields of 241.4 and 231.6 mL H 2/g sugar consumed, respectively. After repeated batch cultivation the hydrogen yield from xylose–glucose mixed substrate of PGR increased to 375 mL H 2/g sugar consumed which was 30% higher than that of YLT (287 mL H 2/g sugar consumed). Soluble metabolites from xylose–glucose mixed substrates were composed mostly of butyric acid (20.6–21.8 mM), acetic acid (7.2–13.5 mM), lactic acid (8.2–11.7 mM) and butanol (4.4–13.0 mM). Denaturing gradient gel electrophoresis (DGGE) profiles illustrated small difference in microbial patterns of PGR enriched with glucose, xylose–glucose mixed substrate and xylose. The dominant populations were affiliated with low G + C content Gram-positive bacteria, Thermoanaerobacterium sp., Thermoanaerobacter sp., Caloramater sp. and Anoxybacillus sp. based on the 16S rRNA gene. Cultivation of the enriched culture PGR in oil palm trunk hydrolysate, the maximum hydrogen yield of 301 mL H 2/g sugar consumed was achieved at hydrolysate concentration of 40% (v/v). At higher concentration to 80% (v/v), the hydrogen fermentation process was inhibited. Therefore, the efficient thermophilic hydrogen-producing consortia PGR has successfully developed and has great potential for production of biohydrogen from mixed sugars hydrolysate.

Construction of a xylose-fermenting Saccharomyces cerevisiae strain by combined approaches of genetic engineering, chemical mutagenesis and evolutionary adaptation

Recombinant Saccharomyces cerevisiae strains constructed by metabolic engineering approaches can ferment xylose but with low efficiency. We constructed a S. cerevisiae strain via combined approaches of recombinant DNA technology, chemical mutagenesis and evolutionary adaptation for an efficient xylose utilization and ethanol fermentation. A haploid derivative of an industrial ethanol-fermenting S. cerevisiae strain was first engineered to express 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). This recombinant strain, LEK122, was then subjected to EMS mutagenesis followed by adaptive evolution, resulting in a single isolate, LEK513, which displayed significantly improved xylose-utilizing property. The specific growth rate of the LEK513 strain was 0.225 h−1 under aerobic condition (0.205 h−1 under oxygen-limited condition) with xylose as the sole carbon source, while that of the LEK122 was 0.055 h−1. During 100 h batch cultivation, the optical density of LEK513 reached 60, while LEK122 only grew to 7.5. In the same time period, LEK513 consumed 95% of the xylose in the medium, while LEK122 only consumed 20% of that. The LEK513 strain produced 11% more ethanol in oxygen-limited fermentation than it did in aerobic fermentation.

Use of population genetics to derive nonrecombinantSaccharomyces cerevisiaestrains that grow using xylose as a sole carbon source

According to scientific dogma, Saccharomyces cerevisiae cannot grow utilizing xylose as a sole carbon source. Although recombinant DNA technology has overcome this deficiency to some degree, efficient utilization of xylose appears to require complex global changes in gene expression. This complexity provides a significant challenge to the development of yeasts suitable for the utilization of xylose-rich lignocellulosic substrates. In contrast to the dogma, we have found that native strains of S. cerevisiae can grow on xylose as a sole carbon source, albeit very slowly. This observation provided the basis for a new approach using natural selection to develop strains of S. cerevisiae with improved ability to utilize xylose. By applying natural selection and breeding over an extended period, we have developed S. cerevisiae strains that can double in less than 6 h using xylose as a sole carbon source. Strains with improved growth rate possessed increased xylose reductase and xylitol dehydrogenase activities, with the latter showing the greater improvement. This unique, completely nonrecombinant approach to developing xylose-utilizing strains of S. cerevisiae opens an alternative route to the development of yeast that can fully utilize lignocellulosic substrates.

Structure-Guided Engineering of Xylitol Dehydrogenase Cosubstrate Specificity

Xylitol dehydrogenase (XDH) is one of several enzymes responsible for assimilating xylose into eukaryotic metabolism and is useful for fermentation of xylose contained in agricultural byproducts to produce ethanol. For efficient xylose utilization at high flux rates, cosubstrates should be recycled between the NAD+-specific XDH and the NADPH-preferring xylose reductase, another enzyme in the pathway. To understand and alter the cosubstrate specificity of XDH, we determined the crystal structure of the Gluconobacter oxydans holoenzyme to 1.9 angstroms resolution. The structure reveals that NAD+ specificity is largely conferred by Asp38, which interacts with the hydroxyls of the adenosine ribose. Met39 stacked under the purine ring and was also located near the 2' hydroxyl. Based on the location of these residues and on sequence alignments with related enzymes of various cosubstrate specificities, we constructed a double mutant (D38S/M39R) that was able to exclusively use NADP+, with no loss of activity.

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.

Complete Reversal of Coenzyme Specificity of Xylitol Dehydrogenase and Increase of Thermostability by the Introduction of Structural Zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).