Xylose Fermentation Research Articles

Transcriptional factor Cat8 is involved in regulation of xylose fermentation in engineered Saccharomyces cerevisiae

Aim. The aim of this work is the construction of cat8Δ strain on the base of xylose-fermenting S. cerevisiae strain and evaluation of the xylose fermentation rate. Methods. The CAT8 deletion cassette harboring natNT2 gene flanking with 5’ and 3’ non-coding regions of CAT8 gene has been constructed. After transformation by the cassette the cat8Δ strain was selected on the nourseothricin containing medium. Xylose fermentation experiments of constructed strain was performed in mineral medium supplemented with xylose under oxygen-limited conditions. Results. Xylose-fermenting cat8Δ S. cerevisiae strain has been constructed by homologous recombination of the CAT8 deletion cassette with target sequences in the genome of GS010 strain. The cat8Δ strain possessed increase in ethanol accumulation, ethanol yield, rate of ethanol production and productivity of ethanol synthesis relative to the parental GS010 strain for 9.5, 6, 20 and 12 %, respectively. Conclusions. The mutant of the xylose-fermenting S. cerevisiae strain with knock out of the CAT8 gene coding for transcriptional activator, has been constructed. The cat8Δ mutant showed 9.5 % increase in ethanol production from xylose relative to parental strain.
 Keywords: alcoholic fermentation, xylose, S. cerevisiae, Cat8.

Solvent production from xylose.

Xylose is the second most abundant sugar derived from lignocellulose; it is considered less desirable than glucose for fermentation, and strategies that specifically increase xylose utilization in wild type or engineered cells are goals for biofuel production. Issues arise with xylose utilization because of carbohydrate catabolite repression, which is the preferential utilization of glucose relative to xylose in fermentations with both pure and mixed cultures. Taken together the low substrate utilization rates and solvent yields with xylose compared to glucose, many industrial fermentations ignore the xylolytic portion of the reaction in lieu of methods to maintain high glucose. This is shortsighted given the massive potential for xylose generation from a number of sustainable biomass feedstocks, based on utilization of the hemicellulose fraction(s) that enter pretreatment. A number of strategies have been developed in recent years to address xylose utilization and solvent production from xylose in systems with just xylose, or in systems with mixtures of glucose plus xylose, which are more typical of pretreated lignocellulose. The approaches vary in terms of complexity, stability, and ease of introduction to existing fermentation infrastructure (i.e., so-called drop-in fermentation strategies). Some approaches can be considered traditional engineering approaches (e.g., change the reaction conditions), while others are more subtle cellular approaches to eliminate the impacts of catabolite repression. Finally, genetic engineering has been used to increase xylose utilization, although this can be considered a relatively nascent approach compared to manipulations completed to date for glucose utilization.

Xylitol Production by Candida tropicalis 31949 from Sugarcane Bagasse Hydrolysate

Application of microbial fermentation of xylose to produce xylitol can not only turn waste into useful products and conserve resources, but also protect the environment. Currently, more than 100 kinds of microorganisms are known to ferment xylose, among which Candida tropicalis has strong xylose conversion ability and high tolerance to inhibitors such as furfural, acetate, and phenolics in xylose mother liquor. The present study focused on fermenting xylose hydrolysate from sugarcane bagasse to produce xylitol using C. tropicalis strain 31949. The effects of inoculum age, inoculum quantity, and initial xylose concentration on xylitol fermentation by C. tropicalis 31949 were discussed. The following optimum fermentation conditions were determined: inoculum quantity, 10%; inoculum age, 26 h; and initial xylose concentration, 100 g/L. Under these optimal fermentation conditions, C. tropicalis 31949 could ferment sugarcane bagasse hydrolysate, which was pretreated by vacuum concentration and activated carbon detoxification decoloration, to produce 62.9800 g/L xylitol.

Recombinant Diploid Saccharomyces cerevisiae Strain Development for Rapid Glucose and Xylose Co-Fermentation

Cost-effective production of cellulosic ethanol requires robust microorganisms for rapid co-fermentation of glucose and xylose. This study aims to develop a recombinant diploid xylose-fermenting Saccharomyces cerevisiae strain for efficient conversion of lignocellulosic biomass sugars to ethanol. Episomal plasmids harboring codon-optimized Piromyces sp. E2 xylose isomerase (PirXylA) and Orpinomyces sp. ukk1 xylose (OrpXylA) genes were constructed and transformed into S. cerevisiae. The strain harboring plasmids with tandem PirXylA was favorable for xylose utilization when xylose was used as the sole carbon source, while the strain harboring plasmids with tandem OrpXylA was beneficial for glucose and xylose cofermentation. PirXylA and OrpXylA genes were also individually integrated into the genome of yeast strains in multiple copies. Such integration was beneficial for xylose alcoholic fermentation. The respiration-deficient strain carrying episomal or integrated OrpXylA genes exhibited the best performance for glucose and xylose co-fermentation. This was partly attributed to the high expression levels and activities of xylose isomerase. Mating a respiration-efficient strain carrying the integrated PirXylA gene with a respiration-deficient strain harboring integrated OrpXylA generated a diploid recombinant xylose-fermenting yeast strain STXQ with enhanced cell growth and xylose fermentation. Co-fermentation of 162 g L−1 glucose and 95 g L−1 xylose generated 120.6 g L−1 ethanol in 23 h, with sugar conversion higher than 99%, ethanol yield of 0.47 g g−1, and ethanol productivity of 5.26 g L−1·h−1.

Constitutive display of Candida antarctica lipase B on the cell surface of Aspergillus niger and regulation of its fermentation

Displaying Candida antarctica lipase B (CALB) on the cell surface of Aspergillus niger is effectively applied for the industries of food, cosmetics, pharmaceutical and so on. Displaying CALB using induced promoter of glucoamylase on the cell surface of A. niger SH-1 has some problems such as inhibiting its expression under high concentration of glucose, mycelium cleavage and decreasing enzyme activity in the later period of fermentation process. Displaying CALB manipulated by constitutive promoter from glyceraldehyde-3-phosphate dehydrogenase instead of glucoamylase on the cell surface of A. niger SH-1, called AN-GpdA, could solve the above problems effectively. Furthermore, it can not only use glucose, but also xylose as a sole carbon source. Enzyme activity of AN-GpdA using xylose for fermentation reached 1 100.28 U/g of dry cell. We also used lignocellulose such as the hydrolysate of bagasse for fermentation with good performance. The result would provide a novel strategy for the utilization of bagasse.

Optimizing the coordinated transcription of central xylose-metabolism genes in Saccharomyces cerevisiae.

The efficient fermentation of xylose can improve biofuel production. We previously developed a two-stage transcriptional reprogramming (TSTR) strategy (including a glucose fermentation stage and a xylose fermentation stage) and demonstrated its application for the construction of Saccharomyces cerevisiae strains with efficient xylose utilization. In this study, we used these as initial strains to assess the effects of copy number variation (CNV) on optimal gene expression and rewiring the redox balance of the xylose utilization pathway. We obtained strains that contained several integrated copies of XYL1, XYL2, and XKS1 and showed increased ethanol yields. An examination of the individual and combined effects of CNVs of key genes and the redox balance pathway revealed that the TSTR strategy improves ethanol production efficiency. Furthermore, XYL1 or XYL2 overexpression was related to improved xylose utilization. These results showed that strains with faster growth and/or higher ethanol production produced more ethanol from xylose via the synthetic xylose-assimilation pathway. Accordingly, TSTR is an effective strategy to improve xylose metabolism in industrial yeast strains.

Co-fermentation of cellobiose and xylose by mixed culture of recombinant Saccharomyces cerevisiae and kinetic modeling.

Efficient conversion of cellulosic sugars in cellulosic hydrolysates is important for economically viable production of biofuels from lignocellulosic biomass, but the goal remains a critical challenge. The present study reports a new approach for simultaneous fermentation of cellobiose and xylose by using the co-culture consisting of recombinant Saccharomyces cerevisiae specialist strains. The co-culture system can provide competitive advantage of modularity compared to the single culture system and can be tuned to deal with fluctuations in feedstock composition to achieve robust and cost-effective biofuel production. This study characterized fermentation kinetics of the recombinant cellobiose-consuming S. cerevisiae strain EJ2, xylose-consuming S. cerevisiae strain SR8, and their co-culture. The motivation for kinetic modeling was to provide guidance and prediction of using the co-culture system for simultaneous fermentation of mixed sugars with adjustable biomass of each specialist strain under different substrate concentrations. The kinetic model for the co-culture system was developed based on the pure culture models and incorporated the effects of product inhibition, initial substrate concentration and inoculum size. The model simulations were validated by results from independent fermentation experiments under different substrate conditions, and good agreement was found between model predictions and experimental data from batch fermentation of cellobiose, xylose and their mixtures. Additionally, with the guidance of model prediction, simultaneous co-fermentation of 60 g/L cellobiose and 20 g/L xylose was achieved with the initial cell densities of 0.45 g dry cell weight /L for EJ2 and 0.9 g dry cell weight /L SR8. The results demonstrated that the kinetic modeling could be used to guide the design and optimization of yeast co-culture conditions for achieving simultaneous fermentation of cellobiose and xylose with improved ethanol productivity, which is critically important for robust and efficient renewable biofuel production from lignocellulosic biomass.

A non-linear model of hydrogen production by Caldicellulosiruptor saccharolyticus for diauxic-like consumption of lignocellulosic sugar mixtures

BackgroundCaldicellulosiruptor saccharolyticus is an attractive hydrogen producer suitable for growth on various lignocellulosic substrates. The aim of this study was to quantify uptake of pentose and hexose monosaccharides in an industrial substrate and to present a kinetic growth model of C. saccharolyticus that includes sugar uptake on defined and industrial media. The model is based on Monod and Hill kinetics extended with gas-to-liquid mass transfer and a cybernetic approach to describe diauxic-like growth.ResultsMathematical expressions were developed to describe hydrogen production by C. saccharolyticus consuming glucose, xylose, and arabinose. The model parameters were calibrated against batch fermentation data. The experimental data included four different cases: glucose, xylose, sugar mixture, and wheat straw hydrolysate (WSH) fermentations. The fermentations were performed without yeast extract. The substrate uptake rate of C. saccharolyticus on single sugar-defined media was higher on glucose compared to xylose. In contrast, in the defined sugar mixture and WSH, the pentoses were consumed faster than glucose. Subsequently, the cultures entered a lag phase when all pentoses were consumed after which glucose uptake rate increased. This phenomenon suggested a diauxic-like behavior as was deduced from the successive appearance of two peaks in the hydrogen and carbon dioxide productivity. The observation could be described with a modified diauxic model including a second enzyme system with a higher affinity for glucose being expressed when pentose saccharides are consumed. This behavior was more pronounced when WSH was used as substrate.ConclusionsThe previously observed co-consumption of glucose and pentoses with a preference for the latter was herein confirmed. However, once all pentoses were consumed, C. saccharolyticus most probably expressed another uptake system to account for the observed increased glucose uptake rate. This phenomenon could be quantitatively captured in a kinetic model of the entire diauxic-like growth process. Moreover, the observation indicates a regulation system that has fundamental research relevance, since pentose and glucose uptake in C. saccharolyticus has only been described with ABC transporters, whereas previously reported diauxic growth phenomena have been correlated mainly to PTS systems for sugar uptake.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Distinct effects of furfural, hydroxymethylfurfural and its mixtures on dark fermentation hydrogen production and microbial structure of a mixed culture

Past research has suggested that furfural and hydroxymethylfurfural (HMF) present in lignocellulosic hydrolyzates exert a synergistic effect on dark fermentative H2 production, which has not adequately proven in microbial consortia. It is possible that some members of a consortium experience less inhibition than others, helping to the entire consortium to overcome the inhibition. To elucidate the type of inhibitory effect that these agents exert, the objective of this study was to contrast the individual impacts of furfural and HMF with the corresponding mixtures at the same concentration threshold on the hydrogen production and the relative abundance of different members in the consortium. Heat-treated anaerobic granules served as inoculum to ferment xylose for hydrogen production in presence of furfural (0.10, 0.50, and 1.00 g/L), HMF (0.02, 0.09, 0.19 g/L) and the corresponding mixtures in comparison with a control in absence of these agents. Furfural alone did not inhibit the hydrogen production; indeed, the inoculum completely degraded furfural at all its concentrations with the presence of furoic acid.HFM was partially degraded in the treatments with the lowest/middle concentrations, resulting in higher hydrogen production than the control. In contrast, at the highest HMF concentration, the inoculum was unable to remove it resulting in the strongest inhibition of hydrogen production. All the F/HMF mixtures had an inhibitory effect on the hydrogen production. A log2 fold change analysis of pyrosequencing reads evidenced that these agents promoted the growth of Lactobacillus, and clostridia species such as Clostridium butyricum, Clostridium bifermentans, and Clostridium sartagoforme, suggesting their active participation in the detoxification process.

Kinetic modelling and simulation of batch, continuous and cell-recycling fermentations for acetone-butanol-ethanol production using Clostridium saccharoperbutylacetonicum N1-4

A kinetic model describing acetone-butanol-ethanol (ABE) production applicable to both single substrate fermentations of glucose and xylose as well as co-fermentation of the substrates has been developed. The model accounts for carbon catabolite repression as well as the inhibition of kinetic rates at high substrate concentrations (∼90 g l−1). Model parameters were obtained through kinetic fitting to previously report experimental data. The model was used to study the design and operation of a continuous ABE fermentation process (with and without recycle of biomass). For continuous operation, it was shown that multiple steady-states exist at low dilution rates. For operation with recycle of biomass, the influence of recycle rate on both biomass concentration and ABE productivity were studied. The results indicate that for a range of recycle and dilution rates, ABE productivity can increase to 16 g l−1 h−1 (10 times higher than that without biomass recycling), consistent with experimental results.

Laboratory evolution for forced glucose-xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae.

Simultaneous fermentation of glucose and xylose can contribute to improved productivity and robustness of yeast-based processes for bioethanol production from lignocellulosic hydrolysates. This study explores a novel laboratory evolution strategy for identifying mutations that contribute to simultaneous utilisation of these sugars in batch cultures of Saccharomyces cerevisiae. To force simultaneous utilisation of xylose and glucose, the genes encoding glucose-6-phosphate isomerase (PGI1) and ribulose-5-phosphate epimerase (RPE1) were deleted in a xylose-isomerase-based xylose-fermenting strain with a modified oxidative pentose-phosphate pathway. Laboratory evolution of this strain in serial batch cultures on glucose–xylose mixtures yielded mutants that rapidly co-consumed the two sugars. Whole-genome sequencing of evolved strains identified mutations in HXK2, RSP5 and GAL83, whose introduction into a non-evolved xylose-fermenting S. cerevisiae strain improved co-consumption of xylose and glucose under aerobic and anaerobic conditions. Combined deletion of HXK2 and introduction of a GAL83G673T allele yielded a strain with a 2.5-fold higher xylose and glucose co-consumption ratio than its xylose-fermenting parental strain. These two modifications decreased the time required for full sugar conversion in anaerobic bioreactor batch cultures, grown on 20 g L−1 glucose and 10 g L−1 xylose, by over 24 h. This study demonstrates that laboratory evolution and genome resequencing of microbial strains engineered for forced co-consumption is a powerful approach for studying and improving simultaneous conversion of mixed substrates.

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.

A novel isolate of Clostridium butyricum for efficient butyric acid production by xylose fermentation

Bacterial fermentation of lignocellulose has been regarded as a sustainable approach to butyric acid production. However, the yield of butyric acid is hindered by the conversion efficiency of hydrolysate xylose. A mesophilic alkaline-tolerant strain designated as Clostridium butyricum B10 was isolated by xylose fermentation with acetic and butyric acids as the principal liquid products. To enhance butyric acid production, performance of the strain in batch fermentation was evaluated with various temperatures (20–47 °C), initial pH (5.0–10.0), and xylose concentration (6–20 g/L). The results showed that the optimal temperature, initial pH, and xylose concentration for butyric acid production were 37 °C, 9.0, and 8.00 g/L, respectively. Under the optimal condition, the yield and specific yield of butyric acid reached about 2.58 g/L and 0.36 g/g xylose, respectively, with 75.00% butyric acid in the total volatile fatty acids. As renewable energy, hydrogen was also collected from the xylose fermentation with a yield of about 73.86 mmol/L. The kinetics of growth and product formation indicated that the maximal cell growth rate (μ m ) and the specific butyric acid yield were 0.1466 h−1 and 3.6274 g/g cell (dry weight), respectively. The better performance in xylose fermentation showed C. butyricum B10 a potential application in efficient butyric acid production from lignocellulose.

RETRACTED: Corn stover pretreatment by metal oxides for improving lignin removal and reducing sugar degradation and water usage

Five metal oxides, Fe2O3, CuO, NiO, ZnO, and MgO, were investigated as catalysts to improve lignin removal and reduce sugar degradation during corn stover pretreatment. Liquid hot water (LHW) pretreatment was used as control. Among the five metal oxides, MgO was the most suitable for biomass pretreatmen. The optimal pretreatment condition was the solid/liquid ratio of 1/10 with 0.10 mol/L MgO at 190 °C for 40 min. The fermentable xylose (85%) and glucose (97%) from MgO pretreatment were equivalent to those (89 and 95%) from LHW pretreatment, and lignin removal was 1.5-fold more than that from LHW pretreatment. The pH of the resulting biomass slurry was close to 7.0 and without furfural and 5-hydroxymethylfurfural formation. Thus, the water-washing step for inhibitor removal can be omitted. The biomass liquor can be used directly for downstream hydrolysis and fermentation. Acid-resistant equipment is not required due to the absence of acids.

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.

Xylose fermentation to ethanol by new Galactomyces geotrichum and Candida akabanensis strains.

The conversion of pentoses into ethanol remains a challenge and could increase the supply of second-generation biofuels. This study sought to isolate naturally occurring yeasts from plant biomass and determine their capabilities for transforming xylose into ethanol. Three yeast strains with the ability to ferment xylose were isolated from pepper, tomato and sugarcane bagasse. The strains selected were characterized by morphological and auxanographic assays, and they were identified by homology analysis of 5.8 S and 26 S ribosomal RNA gene sequences. The identities of two lineages of microrganism were associated with Galactomyces geotrichum, and the other was associated with Candida akabanensis. Fermentative processes were conducted with liquid media containing only xylose as the carbon source. YP/S values for the production of ethanol ranging between 0.29 and 0.35 g g−1 were observed under non-optimized conditions.

Inoculum pretreatment differentially affects the active microbial community performing mesophilic and thermophilic dark fermentation of xylose

The influence of different inoculum pretreatments (pH and temperature shocks) on mesophilic (37 °C) and thermophilic (55 °C) dark fermentative H2 production from xylose (50 mM) and, for the first time, on the composition of the active microbial community was evaluated. At 37 °C, an acidic shock (pH 3, 24 h) resulted in the highest yield of 0.8 mol H2 mol−1 xylose. The H2 and butyrate yield correlated with the relative abundance of Clostridiaceae in the mesophilic active microbial community, whereas Lactobacillaceae were the most abundant non-hydrogenic competitors according to RNA-based analysis. At 55 °C, Clostridium and Thermoanaerobacterium were linked to H2 production, but only an alkaline shock (pH 10, 24 h) repressed lactate production, resulting in the highest yield of 1.2 mol H2 mol−1 xylose. This study showed that pretreatments differentially affect the structure and productivity of the active mesophilic and thermophilic microbial community developed from an inoculum.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production.