Co-fermentation Of Xylose Research Articles

Third-generation D-lactic acid production using red macroalgae Gelidium amansii by co-fermentation of galactose, glucose and xylose

Macroalgae biomass has been considered as a promising renewable feedstock for lactic acid production owing to its lignin-free, high carbohydrate content and high productivity. Herein, the D-lactic acid production from red macroalgae Gelidium amansii by Pediococcus acidilactici was investigated. The fermentable sugars in G. amansii acid-prehydrolysate were mainly galactose and glucose with a small amounts of xylose. P. acidilactici could simultaneously ferment the mixed sugars of galactose, glucose and xylose into D-lactic acid at high yield (0.90 g/g), without carbon catabolite repression (CCR). The assimilating pathways of these sugars in P. acidilactici were proposed based on the whole genome sequences. Simultaneous saccharification and co-fermentation (SSCF) of the pretreated and biodetoxified G. amansii was also conducted, a record high of D-lactic acid (41.4 g/L) from macroalgae biomass with the yield of 0.34 g/g dry feedstock was achieved. This study provided an important biorefinery strain for D-lactic acid production from macroalgae biomass.

Engineered Saccharomyces cerevisiae harbors xylose isomerase and xylose transporter improves co-fermentation of xylose and glucose for ethanol production

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.

First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Pretreatment with fermentable and recyclable deep eutectic solvent (DES) for improving resource utilization of biomass

A series of acidic FDES were prepared to pretreat lignocellulose. After pretreatment, solid product containing 82% cellulose, high quality lignin and liquid product containing hemicellulose were obtained, respectively. The enzymatic hydrolysis of the solid product could reach 84.5%. The enzymatic hydrolysate with the was used as feedstock for ethanol fermentation using commercial Saccharomyces cerevisiae live preparation instead of genetically engineered strains that can use 5-carbon sugars. The pretreatment solution was used as a raw material to culture Rhodotorula glutinis for microbial lipid production. phosphoric acid (PA) as a pretreatment catalyst could ensure the enzymatic efficiency compared with other acids, while more polysaccharide fractions could be formed. At the end of fermentation, the biomass could reach 30 g/L, and the lipid concentration could reach 7 g/L. In addition, the lignin prepared in this study has a consistent structure and low molecular weight, showing high potential for bio-based chemicals and materials conversion. According to the results of FDES reutilization, the pretreatment effect and fermentation effect of the FDES prepared here did not decrease significantly with the increase in the reutilization. The biomass pretreatment process developed in this study realized the full utilization of cellulose, hemicellulose, lignin and pretreatment solvent. It is innovative compared with the genetically engineered strain which mainly rely on the co-fermentation of glucose and xylose for cellulosic ethanol fermentation.

Rhizopus oryzae for Fumaric Acid Production: Optimising the Use of a Synthetic Lignocellulosic Hydrolysate

The hydrolysis of lignocellulosic biomass opens an array of bioconversion possibilities for producing fuels and chemicals. Microbial fermentation is particularly suited to the conversion of sugar-rich hydrolysates into biochemicals. Rhizopus oryzae ATCC 20344 was employed to produce fumaric acid from glucose, xylose, and a synthetic lignocellulosic hydrolysate (glucose–xylose mixture) in batch and continuous fermentations. A novel immobilised biomass reactor was used to investigate the co-fermentation of xylose and glucose. Ideal medium conditions and a substrate feed strategy were then employed to optimise the production of fumaric acid. The batch fermentation of the synthetic hydrolysate at optimal conditions (urea feed rate 0.625mgL−1h−1 and pH 4) produced a fumaric acid yield of 0.439gg−1. A specific substrate feed rate (0.164gL−1h−1) that negated ethanol production and selected for fumaric acid was determined. Using this feed rate in a continuous fermentation, a fumaric acid yield of 0.735gg−1 was achieved; this was a 67.4% improvement. A metabolic analysis helped to determine a continuous synthetic lignocellulosic hydrolysate feed rate that selected for fumaric acid production while achieving the co-fermentation of glucose and xylose, thus avoiding the undesirable carbon catabolite repression. This work demonstrates the viability of fumaric acid production from lignocellulosic hydrolysate; the process developments discovered will pave the way for an industrially viable process.

Integration of first- and second-generation ethanol production: Evaluation of a mathematical model to describe sucrose and xylose co-fermentation by recombinant Saccharomyces cerevisiae

The use of hydrolysates of lignocellulosic materials and microbial platforms present themselves as viable alternatives to the current system energy. The metabolization of the different carbohydrates present in the hydrolysates by yeasts is a process bottleneck. Genetically modified yeasts can ferment different carbohydrates. Furthermore, mathematical modelling helps to understand the dynamics and interactions during the process and predicts other conditions. The performance of the recombinant yeast Saccharomyces cerevisiae was evaluated for co-fermentation in different carbohydrates proportions (C12 – C5, 30.67 ± 0.71–70.17 ± 2.64%) and initial cell densities (0.28 ± 0.07, 10.95 ± 0.250 and 30.25 ± 0.25 g·L−1) using an unstructured – unsegregated kinetic model aiming the integration of first-and-second generation ethanol production. The method to obtain confidence region, the parameter's correlation, and the convergence of the parameters followed a pipeline previously developed by our researcher group. The mathematical models described the fermentations, and μGlu,max, μFru,max, πGlu,max, πXyl,max, ρXyl,max and ψXyl,max were the parameters with the slightest variation during the convergence process. Also, it was identified that the yeast consumed glucose and fructose simultaneously to form cell, however μGlu,max was higher relative to μFru,max in all cases (0.1909, 0.0341, 0.1815 h−1 for μGlu,max, and 1.00E-5, 2.86E-05, 2.44E-05 h−1 for μFru,max). On the other hand, πGlu,max and πFru,max were higher relative to πXyl,max.

Production of Caffeic Acid with Co-fermentation of Xylose and Glucose by Multi-modular Engineering in Candida glycerinogenes.

Caffeic acid (CA), a natural phenolic compound, has important medicinal value and market potential. In this study, we report a metabolic engineering strategy for the biosynthesis of CA in Candida glycerinogenes using xylose and glucose. The availability of precursors was increased by optimization of the shikimate (SA) pathway and the aromatic amino acid pathway. Subsequently, the carbon flux into the SA pathway was maximized by introducing a xylose metabolic pathway and optimizing the xylose assimilation pathway. Eventually, a high yielding strain CG19 was obtained, which reached a yield of 4.61 mg/g CA from mixed sugar, which was 1.2-fold higher than that of glucose. The CA titer in the 5 L bioreactor reached 431.45 mg/L with a yield of 8.63 mg/g of mixed sugar. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of CA. This is the first report to produce CA in C. glycerinogenes with xylose and glucose as carbon sources, which developed a promising strategy for the efficient production of high-value aromatic compounds.

Integration of First- and Second-Generation Ethanol Production: Evaluation of a Mathematical Model to Describe Sucrose and Xylose Co-Fermentation by Recombinant Saccharomyces Cerevisiae

Integration of First- and Second-Generation Ethanol Production: Evaluation of a Mathematical Model to Describe Sucrose and Xylose Co-Fermentation by Recombinant Saccharomyces Cerevisiae

Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol.

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

BackgroundXylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose.ResultsThe optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively.ConclusionsIsomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

Identification and analysis of sugar transporters capable of co-transporting glucose and xylose simultaneously.

Simultaneous co-fermentation of glucose and xylose is a key desired trait of engineered Saccharomyces cerevisiae for efficient and rapid production of biofuels and chemicals. However, glucose strongly inhibits xylose transport by endogenous hexose transporters of S. cerevisiae. We identified structurally distant sugar transporters (Lipomyces starkeyi LST1_205437 and Arabidopsis thaliana AtSWEET7) capable of co-transporting glucose and xylose from previously unexplored oleaginous yeasts and plants. Kinetic analysis showed that LST1_205437 had lenient glucose inhibition on xylose transport and AtSWEET7 transported glucose and xylose simultaneously with no inhibition. Modelling studies of LST1_205437 revealed that Ala335 residue at sugar binding site can accommodates both glucose and xylose. Docking studies with AtSWEET7 revealed that Trp59, Trp183, Asn145, and Asn179 residues stabilized the interactions with sugars, allowing both xylose and glucose to be co-transported. In addition, we altered sugar preference of LST1_205437 by single amino acid mutation at Asn365. Our findings provide a new mechanistic insight on glucose and xylose transport mechanism of sugar transporters and the identified sugar transporters can be employed to develop engineered yeast strains for producing cellulosic biofuels and chemicals.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae.

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources toward engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, recent advances in metabolic engineering of yeast is summarized to address bottlenecks on xylose assimilation and to enable simultaneous co-utilization of xylose and other substrates in lignocellulosic hydrolysates. Distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals are also highlighted. Although many challenges remain, recent research investments have facilitated the efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding xylose-induced metabolic rewiring in engineered yeast has encouraged the use of xylose as a carbon source for producing various non-ethanol bioproducts. To boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.

Candida intermedia CBS 141442: A Novel Glucose/Xylose Co-Fermenting Isolate for Lignocellulosic Bioethanol Production

The present study describes the isolation of the novel strain Candida intermedia CBS 141442 and investigates the potential of this microorganism for the conversion of lignocellulosic streams. Different C. intermedia clones were isolated during an adaptive laboratory evolution experiment under the selection pressure of lignocellulosic hydrolysate and in strong competition with industrial, xylose-fermenting Saccharomyces cerevisiae cells. Isolates showed different but stable colony and cell morphologies when growing in a solid agar medium (smooth, intermediate and complex morphology) and liquid medium (unicellular, aggregates and pseudohyphal morphology). Clones of the same morphology showed similar fermentation patterns, and the C. intermedia clone I5 (CBS 141442) was selected for further testing due to its superior capacity for xylose consumption (90% of the initial xylose concentration within 72 h) and the highest ethanol yields (0.25 ± 0.02 g ethanol/g sugars consumed). Compared to the well-known yeast Scheffersomyces stipitis, the selected strain showed slightly higher tolerance to the lignocellulosic-derived inhibitors when fermenting a wheat straw hydrolysate. Furthermore, its higher glucose consumption rates (compared to S. stipitis) and its capacity for glucose and xylose co-fermentation makes C. intermedia CBS 141442 an attractive microorganism for the conversion of lignocellulosic substrates, as demonstrated in simultaneous saccharification and fermentation processes.

Production of cellulase by Microbulbifer hydrolyticus through co-fermentation of glucose and xylose from lignocellulose

Cellulase is a compound enzyme that catalyzes cellulose into monosaccharides or oligosaccharides. Large amounts of cellulase are needed with the development of the lignocellulose processing industry, which necessitates faster methods to produce cellulase. In this work, the marine bacterium Microbulbifer hydrolyticus IRE-31-192 was selected to produce cellulase, due to its fast growth rate and short high space-time yield. Co-fermentation of glucose and xylose to produce cellulase was investigated on the basis of previous work. When the ratio of glucose/xylose was 2:1 (w/w), 294 U/L cellulase activity with highest space-time yield of 12.2 U/L h was obtained. The hydrolytic liquid of lignocellulose prepared from dried distiller’s grains with solubles (DDGS) with the similar ratio of glucose/xylose was used as medium to produce cellulase. The efficiency of cellulase production from processed and unprocessed hydrolysates of DDGS was compared. Unprocessed hydrolysates were more beneficial for the production of cellulase, such that its activity was 261 U/L with a space-time yield of 14.5 U/L h. Thus, commonly used pure glucose and xylose could be replaced by hydrolysates of DDGS, and marine bacteria has potential application for cellulase production.

Adaptive laboratory evolution induced novel mutations in Zymomonas mobilis ATCC ZW658: a potential platform for co-utilization of glucose and xylose.

A systematic adaptive laboratory evolution strategy was employed to develop a potential Zymomonas mobilis strain with the ability to co-utilize glucose and xylose. Z. mobilis ATCC ZW658, a recombinant xylose fermenting strain, was subjected to adaptive laboratory evolution over a period of 200days under strict selection pressure of increasing concentration of xylose. The evolved strain exhibited 1.65 times increase in the overall specific xylose utilization rate when compared with the parent strain. Furthermore, the strain displayed significantly improved performance in terms of co-fermentation of xylose in the presence of glucose with specific glucose and xylose utilization rate of 1.24gg-1 h-1 and 1.34gg-1 h-1, respectively. Altered phenotypic response of the evolved strain, in terms of improved xylose utilization, co-utilization of mixed sugars, enhanced growth, ethanol production, and reduced xylitol production has been explained by novel mutations, identified using next-generation sequencing, in xylose assimilating, metabolizing, and crucial regulatory pathway genes and key enzyme activity assays.

Improved simultaneous co-fermentation of glucose and xylose by Saccharomyces cerevisiae for efficient lignocellulosic biorefinery

BackgroundLignocellulosic biorefinery offers economical and sustainable production of fuels and chemicals. Saccharomyces cerevisiae, a promising industrial host for biorefinery, has been intensively developed to expand its product profile. However, the sequential and slow conversion of xylose into target products remains one of the main challenges for realizing efficient industrial lignocellulosic biorefinery.ResultsIn this study, we developed a powerful mixed-sugar co-fermenting strain of S. cerevisiae, XUSEA, with improved xylose conversion capacity during simultaneous glucose/xylose co-fermentation. To reinforce xylose catabolism, the overexpression target in the pentose phosphate pathway was selected using a DNA assembler method and overexpressed increasing xylose consumption and ethanol production by twofold. The performance of the newly engineered strain with improved xylose catabolism was further boosted by elevating fermentation temperature and thus significantly reduced the co-fermentation time by half. Through combined efforts of reinforcing the pathway of xylose catabolism and elevating the fermentation temperature, XUSEA achieved simultaneous co-fermentation of lignocellulosic hydrolysates, composed of 39.6 g L−1 glucose and 23.1 g L−1 xylose, within 24 h producing 30.1 g L−1 ethanol with a yield of 0.48 g g−1.ConclusionsOwing to its superior co-fermentation performance and ability for further engineering, XUSEA has potential as a platform in a lignocellulosic biorefinery toward realizing a more economical and sustainable process for large-scale bioethanol production.

Cellobiose와 xylose를 동시에 대사하는 Pichia stipitis 돌연변이를 활용한 동시당화발효

In order to economically produce biofuels in simultaneous saccharification and fermentation (SSF) of cellulosic biomass, it is required to construct microbial host efficiently utilizing various sugars. Previously, a mutant Pichia stipitis YN14 strain co-fermenting cellobiose and xylose was isolated. In this study, ethanol production performance of P. stipitis YN14 was compared with the parental P. stipitis CBS 6054 through the series of SSF experiments. In SSF of cellulose, the mutant strain employing Celluclast 1.5 L as the cellulolytic enzyme produced 6.1 g/L of ethanol, which was 5.5-times and 1.2-times higher than the parental strain with Celluclast 1.5 L and Cellic CTec2, respectively. In addition, the mutant YN14 strain with Celluclast 1.5 L showed 26.6 g/L of ethanol production in simultaneous saccharification and cofermentation (SSCF) of cellulose and xylose, which was 75% and 60% higher than the parental CBS 6054 strain with Celluclast 1.5 L and Cellic CTec2, respectively. Our results propose that efficient ethanol production from the cellulosic biomass would be accomplished by utilization of yeast strain fermenting cellobiose and xylose simultaneously.

Improved production of 3-hydroxypropionic acid in engineered Escherichia coli by rebalancing heterologous and endogenous synthetic pathways

To improve 3-hydroxypropionic acid (3-HP) production by Escherichia coli, glycerol accumulation needs to be reduced. To accomplish this, we constructed a novel E. coli strain that overexpresses the endogenous aldehyde dehydrogenase gene (puuC) under the control of a strong promoter. The fermentation performance of the engineered strain was significantly improved compared to that of the parental control strain in the presence of glucose and xylose. We also inactivated the puu operon repressor gene, puuR, which resulted in a decrease in glycerol accumulation and an increase in 3-HP production through the co-fermentation of glucose and xylose. Through fed-batch fermentation by utilizing glucose and xylose, the engineered strain, JHS_Δgypr-PT7, produced 53.7 g/L 3-HP and accumulated 1.5 g/L glycerol. This combination strategy, wherein we overexpressed the endogenous puuC gene from a strong promoter and eliminate its transcriptional repression, may be extended to rebalance another biochemical pathway.

Evaluating the Pathway for Co-fermentation of Glucose and Xylose for Enhanced Bioethanol Production Using Flux Balance Analysis

Two novel thermotolerant yeasts, Kluyveromyces marxianus NIRE-K1.1 and Kluyveromyces marxianus NIRE-K3.1, evolutionarily adapted for fermentation of glucose and xylose were analyzed for their metabolic fluxes with an objective of maximum ethanol production. Metabolic fluxes were analyzed for these thermotolerant yeasts by co-fermenting glucose/xylose mixture in two different ratios (1:1 and 4:1). Flux balance analysis revealed the active role of pentose phosphate pathway for effective xylose utilization in both yeasts. A comparison between co-fermentation of glucose/xylose mixtures in the ratio of 1:1 and 4:1 (g/L) reveals that the flux from glucose-6-phosphate to ribulose-5-phosphate was approximately 2.56-fold and 3.75-fold higher in 1:1 mixture in K. marxianus NIRE-K1.1 and K. marxianus NIRE-K3.1, respectively. Overall, flux towards pyruvate (for ethanol production) was found to be higher in both glucose/xylose mixtures 1:1 (1.87%) and 4:1 (0.89%) for K. marxianus NIRE-K3.1 than K. marxianus NIRE-K1.1. Tricarboxylic Acid (TCA) cycle was also found to be incomplete for both the isolates which signify that most of the available substrates were utilized for ethanol production rather than biomass formation. Moreover, it was also observed that in both, the ethanol yields were found to be higher in case of K. marxianus NIRE-K3.1 than K. marxianus NIRE-K1.1, however, xylose uptake rates were higher in the later as compared to the former. Thus, this study concludes with the capable potential of both the yeasts for the production of bioethanol from glucose/xylose mixtures with higher yield and is highly correlated to the relative concentration of both xylose and glucose in a mixture.