Xylitol Dehydrogenase Activities Research Articles

Development of engineered Candida tropicalis strain for efficient corncob-based xylitol-ethanol biorefinery

BackgroundXylitol has a wide range of applications in the pharmaceuticals, cosmetic, food and beverage industry. Microbial xylitol production reduces the risk of contamination and is considered as environment friendly and sustainable compared to the chemical method. In this study, random mutagenesis and genetic engineering approaches were employed to develop Candida tropicalis strains with reduced xylitol dehydrogenase (XDH) activity to eliminate co-substrate requirement for corn cob-based xylitol-ethanol biorefinery.ResultsThe results suggest that when pure xylose (10% w/v) was fermented in bioreactor, the Ethyl methane sulfonate (EMS) mutated strain (C. tropicalis K2M) showed 9.2% and XYL2 heterozygous (XYL2/xyl2Δ::FRT) strain (C. tropicalis K21D) showed 16% improvement in xylitol production compared to parental strain (C. tropicalis K2). Furthermore, 1.5-fold improvement (88.62 g/L to 132 g/L) in xylitol production was achieved by C. tropicalis K21D after Response Surface Methodology (RSM) and one factor at a time (OFAT) applied for media component optimization. Finally, corncob hydrolysate was tested for xylitol production in biorefinery mode, which leads to the production of 32.6 g/L xylitol from hemicellulosic fraction, 32.0 g/L ethanol from cellulosic fraction and 13.0 g/L animal feed.ConclusionsThis work, for the first time, illustrates the potential of C. tropicalis K21D as a microbial cell factory for efficient production of xylitol and ethanol via an integrated biorefinery framework by utilising lignocellulosic biomass with minimum waste generation.

Fermentation performance of a Mexican native Clavispora lusitaniae strain for xylitol and ethanol production from xylose, glucose and cellobiose

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 gethanol/gglucose and 0.344 gethanol/gcellobiose, when grown on 1% glucose or cellobiose, respectively. In mixtures (0.5% each) of glucose:xylose and glucose:xylose:cellobiose the yields were 0.367 gethanol/gGX and 0. 380 gethanol/gGXC, respectively. Likewise, in identical conditions, C. lusitaniae produced xylitol from xylose with a yield of 0.421 gxylitol/gxylose. In 5% glucose or xylose, this yeast had better ethanol and xylitol titers and yields, respectively. However, glucose negatively affected xylitol production in the mixture of both sugars (3% each), producing only ethanol. Xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were evaluated in cultures growing on xylose or glucose, obtaining the highest values in cultures on xylose at 8 h (25.9 and 6.22 mU/mg, respectively). While in glucose cultures, XR and XDH activities were detected once this substrate was consumed (4.06 and 3.32 mU/mg, respectively). Finally, the XYL1 and XYL2 genes encoding xylose reductase and xylitol dehydrogenase, respectively, were up-regulated by xylose, whereas glucose down-regulated their expression.

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae.

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

Influence of glucose on xylose metabolization by Spathaspora passalidarum

The yeast Spathaspora passalidarum is able to produce ethanol from D-xylose and D-glucose. However, it is not clear how xylose metabolism is affected by D-glucose when both sugars are available in the culture medium. The aims of this work were to evaluate the influence of D-glucose on D-xylose consumption, ethanol production, gene expression, and the activity of key xylose-metabolism enzymes under both aerobic and oxygen-limited conditions. Ethanol yields and productivities were increased in culture media containing D-xylose as the sole carbon source or a mixture of D-xylose and D-glucose. S. passalidarum preferentially consumed D-glucose in the co-fermentations, which is consistent with the reduction in expression of genes encoding the key xylose-metabolism enzymes. In the presence of D-glucose, the specific activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) were lower. Interestingly, in accordance with other studies, the presence of 2-deoxyglucose (2DG) did not inhibit the growth of S. passalidarum in culture medium containing D-xylose as the sole carbon source. This indicates that a non-canonical repression pathway is acting in S. passalidarum. In conclusion, the results suggest that D-glucose inhibits D-xylose consumption and prevents the D-xylose-mediated induction of the genes encoding XR, XDH, and XK.

Biorefinery Platform for Spathaspora passalidarum NRRL Y-27907 in the Production of Ethanol, Xylitol, and Single Cell Protein from Sugarcane Bagasse

Spathaspora passalidarum is a naturally pentose-fermenting yeast with the potential to be applied for biotransformation of sugars from lignocellulosic biomasses. Despite being mostly investigated for ethanol production from sugarcane bagasse sugars (mainly xylose and glucose), this microorganism is also capable of producing high xylitol concentrations as a by-product of the ethanol fermentation. The integration of ethanol and xylitol production can improve the economic viability of the process due to the lower sale price of ethanol and the higher added-value of xylitol, a sucrose-substitute sugar with healthier properties. Considering the metabolic pathways interaction from glucose and xylose, it is essential to understand the effect of different glucose and xylose concentrations in the production of ethanol and xylitol by S. passalidarum. In this way, a simultaneous production of both products for the development of an integrated platform for food and chemical and biofuel industries can be accessed. Yeast biomass can also be recovered and applied as a protein source. Therefore, in this study, the fermentative performance of S. passalidarum was investigated in batch fermentations with different xylose and glucose concentrations, using synthetic substrate. The ATP levels and the enzymatic activities of xylose reductase (XR), xylitol dehydrogenase (XDH), and alcohol dehydrogenase (ADH) were also determined for each condition. The results indicated that low amounts of glucose (35%) were necessary to promote a higher xylitol production (10.58 ± 0.29 g/L) without losses on ethanol yield (78.99 ± 4.41%), being the most interesting condition for simultaneous formation of both products.

Enhanced xylitol production using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse by newly isolated Pichia fermentans

BackgroundIntegrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable lignocellulosic biorefineries. In this aspect, microbial cell factories are harnessed for the sustainable production of commercially viable biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, in the terrestrial ecosystem, microbial systems can efficiently consume glucose. On the contrary, pentose sugars are less preferred carbon source as most of the microbes lack metabolic pathway for their utilization. The effective utilization of both pentose and hexose sugars is key for economical biorefinery.ResultsBioprospecting the food waste and selective enrichment on xylose-rich medium led to screening and isolation of yeast which was phylogenetically identified as Pichia fermentans. The newly isolated xylose assimilating yeast was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with > 40% conversion yield. Chemical mutagenesis of isolated yeast with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min EMS exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titer and yield of 34.0 g/L and 0.68 g/g, respectively. However, under the same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of the mutant for xylitol accumulation. The xylitol titer and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively. In comparison, xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g.ConclusionThis study demonstrates the potential of newly isolated P. fermentans in successfully valorising the hemicellulosic fraction for the sustainable xylitol production.

Valorization of apple pomace using bio-based technology for the production of xylitol and 2G ethanol.

Apple pomace was studied as a raw material for the production of xylitol and 2G ethanol, since this agroindustrial residue has a high concentration of carbohydrate macromolecules, but is still poorly studied for the production of fermentation bioproducts, such as polyols. The dry biomass was subjected to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and fermented. The hydrolyzate after characterization was submitted to submerged fermentations, which were carried out in Erlenmeyer flasks using, separately, the yeasts Candida guilliermondii and Kluyveromyces marxianus. High cellulose (32.62%) and hemicellulose (23.60%) contents were found in this biomass, and the chemical hydrolysis yielded appreciable quantities of fermentable sugars, especially xylose. Both yeasts were able to metabolize xylose, but Candida guilliermondii produced only xylitol (9.35gL-1 in 96h), while K. marxianus produced ethanol as the main product (10.47gL-1 in 24h) and xylitol as byproduct (9.10gL-1 xylitol in 96h). Maximum activities of xylose reductase and xylitol dehydrogenase were verified after 24h of fermentation with C. guilliermondii (0.23 and 0.53U/mgprot, respectively) and with K. marxianus (0.08 e 0.08U/mgprot, respectively). Apple pomace has shown potential as a raw material for the fermentation process, and the development of a biotechnological platform for the integrated use of both the hemicellulosic and cellulosic fraction could add value to this residue and the apple production chain.

Yeast Intracellular Staining (yICS): Enabling High-Throughput, Quantitative Detection of Intracellular Proteins via Flow Cytometry for Pathway Engineering.

The complexities of pathway engineering necessitate screening libraries to discover phenotypes of interest. However, this approach is challenging when desirable phenotypes cannot be directly linked to growth advantages or fluorescence. In these cases, the ability to rapidly quantify intracellular proteins in the pathway of interest is critical to expedite the clonal selection process. While Saccharomyces cerevisiae remains a common host for pathway engineering, current approaches for intracellular protein detection in yeast either have low throughput, can interfere with protein function, or lack the ability to detect multiple proteins simultaneously. To fill this need, we developed yeast intracellular staining (yICS) that enables fluorescent antibodies to access intracellular compartments of yeast cells while maintaining their cellular integrity for analysis by flow cytometry. Using the housekeeping proteins β actin and glyceraldehyde 3-phophate dehydrogenase (GAPDH) as targets for yICS, we demonstrated for the first time successful antibody-based flow cytometric detection of yeast intracellular proteins with no modification. Further, yICS characterization of a recombinant d-xylose assimilation pathway showed 3-plexed, quantitative detection of the xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) enzymes each fused with a small (6-10 amino acids) tag, revealing distinct enzyme expression profiles between plasmid-based and genome-integrated expression approaches. As a result of its high-throughput and quantitative capability, yICS enabled rapid screening of a library created from CRISPR-mediated XDH integration into the yeast δ site, identifying rare (1%) clones that led to an 8.4-fold increase in XDH activity. These results demonstrate the utility of yICS for greatly accelerating pathway engineering efforts, as well as any application where the high-throughput and quantitative detection of intracellular proteins is desired.

High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD+- or protein-engineered NADP+-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD+-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.

Physiological and comparative genomic analysis of new isolated yeasts Spathaspora sp. JA1 and Meyerozyma caribbica JA9 reveal insights into xylitol production.

Xylitol is a five-carbon polyol of economic interest that can be produced by microbial xylose reduction from renewable resources. The current study sought to investigate the potential of two yeast strains, isolated from Brazilian Cerrado biome, in the production of xylitol as well as the genomic characteristics that may impact this process. Xylose conversion capacity by the new isolates Spathaspora sp. JA1 and Meyerozyma caribbica JA9 was evaluated and compared with control strains on xylose and sugarcane biomass hydrolysate. Among the evaluated strains, Spathaspora sp. JA1 was the strongest xylitol producer, reaching product yield and productivity as high as 0.74 g/g and 0.20 g/(L.h) on xylose, and 0.58 g/g and 0.44 g/(L.h) on non-detoxified hydrolysate. Genome sequences of Spathaspora sp. JA1 and M. caribbica JA9 were obtained and annotated. Comparative genomic analysis revealed that the predicted xylose metabolic pathway is conserved among the xylitol-producing yeasts Spathaspora sp. JA1, M. caribbica JA9 and Meyerozyma guilliermondii, but not in Spathaspora passalidarum, an efficient ethanol-producing yeast. Xylitol-producing yeasts showed strictly NADPH-dependent xylose reductase and NAD+-dependent xylitol-dehydrogenase activities. This imbalance of cofactors favors the high xylitol yield shown by Spathaspora sp. JA1, which is similar to the most efficient xylitol producers described so far.

Sustainable and green approach of chitosan production from Penicillium citrinum biomass using industrial wastewater as a cheap substrate

Marine sources especially crustaceans have been extensively used worldwide for the production of chitosan. However, limited availability as well as variations in the properties of the derived chitosan is a serious drawback of utilizing marine sources for chitosan production. This study investigated sustainable and green approach of fungal chitosan production using paper mill wastewater as a cheap and easily available substrate. The fungus Penicillium citrinum IITG_KP1 used in this study was initially isolated from an infected bamboo shoot. Addition of acetic acid at low levels led to a 150% increase in the yield of chitosan from 95 g/kg to 138 g/kg of dry fungal biomass. This result correlated well with an increase in xylose uptake rate due to acetic acid addition that was confirmed by enhanced activity of xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes in the presence of acetic acid. Very high COD removal efficiency (75%) along with 70% phenolic reduction and 84% decolourization efficiency of the raw paper mill wastewater without any prior pre-treatment was further achieved by carrying out the fungal fermentation using a bioreactor under batch mode of operation. The fungal chitosan showed properties comparable with those of a commercially available standard.

Augmenting Pentose Utilization and Ethanol Production of Native Saccharomyces cerevisiae LN Using Medium Engineering and Response Surface Methodology.

Economics of ethanol production from lignocellulosic biomass depends on complete utilization of constituent carbohydrates and efficient fermentation of mixed sugars present in biomass hydrolysates. Saccharomyces cerevisiae, the commercial strain for ethanol production uses only glucose while pentoses remain unused. Recombinant strains capable of utilizing pentoses have been engineered but with limited success. Recently, presence of endogenous pentose assimilation pathway in S. cerevisiae was reported. On the contrary, evolutionary engineering of native xylose assimilating strains is promising approach. In this study, a native strain S. cerevisiae LN, isolated from fruit juice, was found to be capable of xylose assimilation and mixed sugar fermentation. Upon supplementation with yeast extract and peptone, glucose (10%) fermentation efficiency was 78% with ~90% sugar consumption. Medium engineering augmented mixed sugars (5% glucose + 5% xylose) fermentation efficiency to ~50 and 1.6% ethanol yield was obtained with concomitant sugar consumption ~60%. Statistical optimization of input variables Glucose (5.36%), Xylose (3.30%), YE (0.36%), and peptone (0.25%) with Response surface methodology led to improved sugar consumption (74.33%) and 2.36% ethanol within 84 h. Specific activities of Xylose Reductase and Xylitol Dehydrogenase exhibited by S. cerevisiae LN were relatively low. Their ratio indicated metabolism diverted toward ethanol than xylitol and other byproducts. Strain was tolerant to concentrations of HMF, furfural and acetic acid commonly encountered in biomass hydrolysates. Thus, genetic setup for xylose assimilation in S. cerevisiae LN is not merely artifact of xylose metabolizing pathway and can be augmented by adaptive evolution. This strain showed potential for commercial exploitation.

Ethanol production from xylose is highly increased by the Kluyveromyces marxianus mutant 17694-DH1.

Directed evolutionary approach and random mutagenesis were performed on thermotolerant yeast Kluyveromyces marxianus KCTC17694 for isolating a yeast strain producing ethanol from xylose efficiently. The isolated mutant strain, K. marxianus 17694-DH1, showed 290% and 131% improvement in ethanol concentration and ethanol production yield from xylose, respectively, as compared with the parental strain. Sequencing of the KmXYL1 gene of K. marxianus 17694-DH1 revealed substitutions of arginine and tryptophan with lysine and leucine at positions 25 and 202, respectively, as compared to the parental strain. In addition, sequencing of the KmXYL2 gene uncovered a substitution of glutamate with leucine at position 232. When enzymatic assays of xylose reductase (XR) and xylitol dehydrogenase (XDH) from the parental strain and K. marxianus 17694-DH1 were performed, XR activities were not significantly different whereas XDH activities were significantly improved in the mutant strain up to 50°C of reaction temperatures. RNA-Seq based transcriptome analysis showed that alcohol dehydrogenases and glucose transporters were up-regulated while TCA cycle involved enzymes were down-regulated in K. marxianus 17694-DH1.

Promiscuous activities of heterologous enzymes lead to unintended metabolic rerouting in Saccharomyces cerevisiae engineered to assimilate various sugars from renewable biomass

BackgroundUnderstanding the global metabolic network, significantly perturbed upon promiscuous activities of foreign enzymes and different carbon sources, is crucial for systematic optimization of metabolic engineering of yeast Saccharomyces cerevisiae. Here, we studied the effects of promiscuous activities of overexpressed enzymes encoded by foreign genes on rerouting of metabolic fluxes of an engineered yeast capable of assimilating sugars from renewable biomass by profiling intracellular and extracellular metabolites.ResultsUnbiased metabolite profiling of the engineered S. cerevisiae strain EJ4 revealed promiscuous enzymatic activities of xylose reductase and xylitol dehydrogenase on galactose and galactitol, respectively, resulting in accumulation of galactitol and tagatose during galactose fermentation. Moreover, during glucose fermentation, a trisaccharide consisting of glucose accumulated outside of the cells probably owing to the promiscuous and transglycosylation activity of β-glucosidase expressed for hydrolyzing cellobiose. Meanwhile, higher accumulation of fatty acids and secondary metabolites was observed during xylose and cellobiose fermentations, respectively.ConclusionsThe heterologous enzymes functionally expressed in S. cerevisiae showed promiscuous activities that led to unintended metabolic rerouting in strain EJ4. Such metabolic rerouting could result in a low yield and productivity of a final product due to the formation of unexpected metabolites. Furthermore, the global metabolic network can be significantly regulated by carbon sources, thus yielding different patterns of metabolite production. This metabolomic study can provide useful information for yeast strain improvement and systematic optimization of yeast metabolism to manufacture bio-based products.

Improving Xylose Utilization of Saccharomyces cerevisiae by Expressing the MIG1 Mutant from the Self-Flocculating Yeast SPSC01.

The major carbohydrate components of lignocellulosic biomass are cellulose and hemicelluloses. Saccharomyces cerevisiae cannot efficiently utilize xylose derived upon the hydrolysis of hemicelluloses. Although engineering the yeast with xylose metabolic pathway has been intensively studied, challenges are still ahead for developing robust strains for lignocellulosic bioethanol production. The main objective of this study was to reveal the role of the MIG1 mutant isolated from the self-flocculating S. cerevisiae SPSC01 in xylose utilization, glucose repression and ethanol fermentation by S. cerevisiae. The MIG1 mutant was amplified from S. cerevisiae SPSC01 by PCR and MIG1- overexpression-cassette was transformed into S. cerevisiae S288c and xylose-metabolizing strain YB-2625-T through homologous recombination. Yeast growth was measured by colony assay on plates with or without xylose supplementation. Then xylose utilization and ethanol production were further evaluated through flask fermentation when mixed sugars of glucose and xylose at 3:1 and 2:1, respectively, were supplied. Fermentation products were detected by HPLC, and activities of xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) were also measured. The transcription of genes regulated by the expression of the MIG1 mutant was analyzed by RTqPCR. Evolutionary relationship of various MIG1s was developed by gene sequencing and sequence alignment. No difference was observed for S288c growing with xylose when it was engineered with the overexpression or deletion of its native MIG1, but its growth was enhanced when overexpressing the MIG1 mutant from SPSC01. The submerged culture of YB-2625-T MIG1-SPSC engineered with xylose-metabolic pathway and the MIG1 mutant indicated that xylitol accumulation was decreased, and consequently, more biomass was accumulated. Furthermore, improved activities of the key enzymes such as XR, XDH and XK were detected in YB-2625-T MIG1-SPSC. Evolutionary analysis of MIG1s amplified from S. cerevisiae strains commonly used for ethanol production revealed a close relationship of SPSC01 and YB-2625. Our results demonstrated the effect of the overexpression of the MIG1 mutant from SPSC01 on xylose utilization of S. cerevisiae. This study could be an alternative strategy for engineering S. cerevisiae with improved xylose utilization.

Heterologous expression of Spathaspora passalidarum xylose reductase and xylitol dehydrogenase genes improved xylose fermentation ability of Aureobasidium pullulans

BackgroundAureobasidium pullulans is a yeast-like fungus that can ferment xylose to generate high-value-added products, such as pullulan, heavy oil, and melanin. The combinatorial expression of two xylose reductase (XR) genes and two xylitol dehydrogenase (XDH) genes from Spathaspora passalidarum and the heterologous expression of the Piromyces sp. xylose isomerase (XI) gene were induced in A. pullulans to increase the consumption capability of A. pullulans on xylose.ResultsThe overexpression of XYL1.2 (encoding XR) and XYL2.2 (encoding XDH) was the most beneficial for xylose utilization, resulting in a 17.76% increase in consumed xylose compared with the parent strain, whereas the introduction of the Piromyces sp. XI pathway failed to enhance xylose utilization efficiency. Mutants with superior xylose fermentation performance exhibited increased intracellular reducing equivalents. The fermentation performance of all recombinant strains was not affected when glucose or sucrose was utilized as the carbon source. The strain with overexpression of XYL1.2 and XYL2.2 exhibited excellent fermentation performance with mimicked hydrolysate, and pullulan production increased by 97.72% compared with that of the parent strain.ConclusionsThe present work indicates that the P4 mutant (using the XR/XDH pathway) with overexpressed XYL1.2 and XYL2.2 exhibited the best xylose fermentation performance. The P4 strain showed the highest intracellular reducing equivalents and XR and XDH activity, with consequently improved pullulan productivity and reduced melanin production. This valuable development in aerobic fermentation by the P4 strain may provide guidance for the biotransformation of xylose to high-value products by A. pullulans through genetic approach.

Saccharomyces cerevisiae strain comparison in glucose\u2013xylose fermentations on defined substrates and in high-gravity SSCF: convergence in strain performance despite differences in genetic and evolutionary engineering history

BackgroundThe most advanced strains of xylose-fermenting Saccharomyces cerevisiae still utilize xylose far less efficiently than glucose, despite the extensive metabolic and evolutionary engineering applied in their development. Systematic comparison of strains across literature is difficult due to widely varying conditions used for determining key physiological parameters. Here, we evaluate an industrial and a laboratory S. cerevisiae strain, which has the assimilation of xylose via xylitol in common, but differ fundamentally in the history of their adaptive laboratory evolution development, and in the cofactor specificity of the xylose reductase (XR) and xylitol dehydrogenase (XDH).ResultsIn xylose and mixed glucose–xylose shaken bottle fermentations, with and without addition of inhibitor-rich wheat straw hydrolyzate, the specific xylose uptake rate of KE6-12.A (0.27–1.08 g gCDW−1 h−1) was 1.1 to twofold higher than that of IBB10B05 (0.10–0.82 g gCDW−1 h−1). KE6-12.A further showed a 1.1 to ninefold higher glycerol yield (0.08–0.15 g g−1) than IBB10B05 (0.01–0.09 g g−1). However, the ethanol yield (0.30–0.40 g g−1), xylitol yield (0.08–0.26 g g−1), and maximum specific growth rate (0.04–0.27 h−1) were in close range for both strains. The robustness of flocculating variants of KE6-12.A (KE-Flow) and IBB10B05 (B-Flow) was analyzed in high-gravity simultaneous saccharification and co-fermentation. As in shaken bottles, KE-Flow showed faster xylose conversion and higher glycerol formation than B-Flow, but final ethanol titres (61 g L−1) and cell viability were again comparable for both strains.ConclusionsIndividual specific traits, elicited by the engineering strategy, can affect global physiological parameters of S. cerevisiae in different and, sometimes, unpredictable ways. The industrial strain background and prolonged evolution history in KE6-12.A improved the specific xylose uptake rate more substantially than the superior XR, XDH, and xylulokinase activities were able to elicit in IBB10B05. Use of an engineered XR/XDH pathway in IBB10B05 resulted in a lower glycerol rather than a lower xylitol yield. However, the strain development programs were remarkably convergent in terms of the achieved overall strain performance. This highlights the importance of comparative strain evaluation to advance the engineering strategies for next-generation S. cerevisiae strain development.

The yeast Scheffersomyces amazonensis is an efficient xylitol producer.

This study assessed the efficiency of Scheffersomyces amazonensis UFMG-CM-Y493T, cultured in xylose-supplemented medium (YPX) and rice hull hydrolysate (RHH), to convert xylose to xylitol under moderate and severe oxygen limitation. The highest xylitol yields of 0.75 and 1.04gg-1 in YPX and RHH, respectively, were obtained under severe oxygen limitation. However, volumetric productivity in RHH was ninefold decrease than that in YPX medium. The xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in the YPX cultures were strictly dependent on NADPH and NAD+ respectively, and were approximately 10% higher under severe oxygen limitation than under moderate oxygen limitation. This higher xylitol production observed under severe oxygen limitation can be attributed to the higher XR activity and shortage of the NAD+ needed by XDH. These results suggest that Sc. amazonensis UFMG-CM-Y493T is one of the greatest xylitol producers described to date and reveal its potential use in the biotechnological production of xylitol.

Production of xylitol by a Coniochaeta ligniaria strain tolerant of inhibitors and defective in growth on xylose.

In conversion of biomass to fuels or chemicals, inhibitory compounds arising from physical-chemical pretreatment of the feedstock can interfere with fermentation of the sugars to product. Fungal strain Coniochaeta ligniaria NRRL30616 metabolizes the furan aldehydes furfural and 5-hydroxymethylfurfural, as well as a number of aromatic and aliphatic acids and aldehydes. Use of NRRL30616 to condition biomass sugars by metabolizing the inhibitors improves their fermentability. Wild-type C. ligniaria has the ability to grow on xylose as sole source of carbon and energy, with no accumulation of xylitol. Mutants of C. ligniaria unable to grow on xylose were constructed. Xylose reductase and xylitol dehydrogenase activities were reduced by approximately two thirds in mutant C8100. The mutant retained ability to metabolize inhibitors in biomass hydrolysates. Although C. ligniaria C8100 did not grow on xylose, the strain converted a portion of xylose to xylitol, producing 0.59 g xylitol/g xylose in rich medium and 0.48 g xylitol/g xylose in corn stover dilute acid hydrolysate. 2016 American Institute of Chemical Engineers Biotechnol. Prog., 2016 © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:606-612, 2016.

Metabolic properties of Pachysolen tannophilus mutants producing xylitol and ethanol from D-xylose

Activity of the major enzymes of D-xylose metabolism in the mutants of xylose-utilizing yeasts Pachysolen tannophilus selectively producing xylitol or ethanol was studied. The xylitol-producing strain exhibited low activities of xylitol dehydrogenase, xylose reductase with preferential affinity to NADPH, NAD+-dependent malate dehydrogenase, and cytochrome c oxidase (4.40, 4.80, 1.87, and 0.28 μmol mg−1 min−1, respectively). The cells of the ethanol-producing mutants exhibited elevated activity of NADH/NADPH-xylose reductase, xylitol dehydrogenase, 1-glycerophosphate dehydrogenase, and lactate dehydrogenase to 6.80, 8.60, 4.68, and 16.48 μmol mg−1 min−1, respectively. Effect of the NADPH/NADH imbalance on ethanol production accumulation and xylitol accumulation is discussed.