Xylose Uptake Rate Research Articles

Scaling up xylitol bioproduction: Challenges to achieve a profitable bioprocess

Xylitol is a GRAS (Generally Recognized as Safe) polyol commonly used in the food industry and able to promote several benefits to the health. In addition, it can also be used as a building block molecule for the manufacture of different high-value chemicals. Currently, the commercial production of xylitol occurs by chemical route through the catalytic hydrogenation of xylose from lignocellulosic biomass. Since this is an expensive process due to the severe reactional conditions employed, the biotechnological route for xylitol production, which comprises the biological conversion of xylose into xylitol, emerges as a potential lower-cost alternative to obtain this polyol due to the milder process conditions required. However, the biotechnological route still presents important bottlenecks and challenges that impairs the process scaling up. Modern strategies and technologies that can potentially improve xylitol bioproduction include adaptive evolution of microbial strains to enhance their tolerance to inhibitors and the xylose uptake rate during the fermentation step; development of engineered microorganisms to result in higher xylose-to-xylitol bioconversion yields; as well as xylitol purification techniques to improve the recovery yields. Moreover, techno-economic analysis of the overall production chain is essential to identify the process viability for large-scale implementation as well as the steps requiring improvements. These are some key factors discussed in this review, which aims to provide insights for the development of a more economically competitive, less energy demanding and scalable new technology for xylitol production.

Lipid Accumulation by Xylose Metabolism Engineered Mucor circinelloides Strains on Corn Straw Hydrolysate.

Previously, we presented a novel approach for increasing the consumption of xylose and the lipid yield by overexpressing the genes coding for xylose isomerase (XI) and xylulokinase (XK) in Mucor circinelloides. In the present study, an in-depth analysis of lipid accumulation by xylose metabolism engineered M. circinelloides strains (namely Mc-XI and Mc-XK) using corn straw hydrolysate was to be explored. The results showed that the fatty acid contents of the engineered M. circinelloides strains were, respectively, increased by 19.8% (in Mc-XI) and 22.3% (in Mc-XK) when compared with the control strain, even though a slightly decreased biomass in these engineered strains was detected. Moreover, the xylose uptake rates of engineered strains in the corn straw hydrolysate were improved significantly by 71.5% (in Mc-XI) and 68.8% (in Mc-XK), respectively, when compared with the control strain. Maybe the increased utilization of xylose led to an increase in lipid synthesis. When the recombinant M. circinelloides strains were cultured in corn straw hydrolysate medium with the carbon-to-nitrogen ratio (C/N ratio) of 50 and initial pH of 6.0, at 30 °C and 500 rpm for 144 h, a total biomass of 12.6-12.9 g/L with a lipid content of 17.2-17.7% (corresponding to a lipid yield of 2.17-2.28 g/L) was achieved. Our study provides a foundation for the further application of the engineered M. circinelloides strains to produce lipid from lignocelluloses.

Construction of industrial xylose-fermenting Saccharomyces cerevisiae strains through combined approaches

Saccharomyces cerevisiae strain with excellent xylose-fermenting capacity and inhibitor tolerance is crucial for lignocellulosic ethanol production. In this study, a combined strategy including site-directed mutagenesis, mating, evolutionary engineering, and haploidization was applied to obtain strains with ideal xylose fermentabilities. Haploid industrial strain KFG4-6B was engineered to overexpress endogenous xylulokinase (XK) and heterologous native or mutated xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis. The XR-mutated strain HX57D showed over 12% increase in both xylose consumption rate and ethanol yield compared with the XR-native strain. To improve the xylose uptake, the HX57D-derived diploids were subjected to evolutionary engineering. In comparison with HX57D, evolved diploid Z4X-21-18 achieved 4.5-fold increases in rates of xylose consumption and ethanol production when fermenting xylose. When fermenting mixed sugars, the glucose and xylose uptake rates were 1.4-fold and 8.3-fold, respectively, higher. H18s28, a haploid of Z4X-21-18, enabled a further 10% increase in xylose consumption rate when fermenting xylose only. However, it was inferior to its diploid parent when fermenting mixed sugars. In the presaccharification-simultaneous saccharification and fermentation (P-SSF) of the whole pretreated wheat straw slurry with high contents of multiple inhibitors, Z4X-21-18 produced approximately 42 g/L ethanol with a yield of 0.38 g/g total sugars.

Improving glucose and xylose assimilation in Azotobacter vinelandii by adaptive laboratory evolution.

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8gL-1) and glucose (2gL-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.

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.

Xylitol Production From Byproducts Generated During Sequential Acid-/Alkali-Pretreatment of Empty Palm Fruit Bunch Fiber by an Adapted Candida tropicalis

Xylose is a pentose sugar with the potential to convert a variety of valuable chemical products. In this study, acidic pretreatment wastewater generated during a sequential acid-/alkali-pretreatment process was recycled to increase the hydrolyzed hemicellulose fraction from empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass. The xylose in the reused wastewater was subjected to overliming and an activated charcoal column was used to remove inhibitory compounds, for xylitol fermentation using the adapted C. tropicalis strain. The cell growth and xylose uptake rates in the adapted strain were 1.7- and 5-fold higher, respectively, compared to the wild-type strain. During batch fermentation using the adapted yeast strain and the post-pretreated xylose solution, 35.2 ± 0.8 g/L xylitol was obtained within 61 h for a production yield of 0.44 g xylitol/g xylose. These results indicate that xylose in the byproducts produced in the bioethanol process could be recovered for production of xylitol.

Sugarcane Syrup Improves Xylitol Bioproduction from Sugarcane Bagasse and Straw Hemicellulosic Hydrolysate

To evaluate the supplementation of the sugarcane bagasse and straw hemicellulosic hydrolysate with sugarcane syrup, which is a sucrose-rich co-product, for xylitol production by Candida guilliermondii FTI 20037. Recently, we demonstrated that sucrose supplementation to sugarcane straw hemicellulosic hydrolysate favoured this bioprocess. A mixture 1:1 of sugarcane bagasse and straw was submitted to dilute-acid hydrolysis with H2SO4 to obtain the hemicellulosic hydrolysate, which was concentrated, detoxified and autoclaved. Three sugarcane syrup concentrations (0.83, 2.50 and 3.33% v/v) were added to the hemicellulosic hydrolysate, which was also supplemented with rice bran extract, (NH4)2SO4, CaCl2·2H2O. Next, a full factorial design was performed to assess the replacement of conventional nutrients by sugarcane syrup. Fermentations were performed in 125 mL Erlenmeyer flasks containing 50 mL of fermentation medium, for 48 h at 30 °C and 200 rpm. Improvements of 16.85% and 12.24% in xylose uptake rate (1.04 ± 0.01 g L−1 h−1) and xylitol volumetric productivity (0.55 ± 0.02 g L−1 h−1), respectively, were achieved with syrup supplementation (2.50% v/v), which led to a final xylitol titer of 26.19 ± 0.02 g L−1. Conventionally employed nutrients (rice bran extract, (NH4)2SO4 and CaCl2·2H2O) did not have significant influence on xylitol production when sugarcane syrup was added to the fermentation medium. Sugarcane syrup as a nutritional supplement improved xylitol production from a mixture of bagasse and straw, becoming an alternative to reduce costs in the formulation of the fermentation medium and contributing to the integration of this bioprocess in a sugarcane biorefinery.

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.

Deletion of four genes in Escherichia coli enables preferential consumption of xylose and secretion of glucose

Overcoming carbon catabolite repression presents a significant challenge, largely due to the complex regulatory networks governing substrate catabolism, even in microbial cells. In this work, we have engineered an E. coli strain, which we have named X2G, that not only exhibits a reversed substrate preference for xylose over glucose, but also demonstrates an unusual ability to produce significant amounts of glucose. We obtained this non-intuitive phenotype by deleting four genes in upper central metabolism: ptsI, glk, pfkA, and zwf, which respectively encode Enzyme I of the phosphotransferase system, glucokinase, the dominant isozyme of phosphofructokinase, and glucose-6-phosphate dehydrogenase. The deletion of ptsI and glk blocks glucose uptake in E. coli, while the deletion of pfkA and zwf prevents the reassimilation of carbons through glycolysis and the oxidative pentose phosphate pathway, respectively. Our strain X2G is capable of converting 34% of the carbon it takes up as xylose into exported glucose. This corresponds to a glucose production rate of 1.4 ± 0.3 mmol/gDW/h at a specific growth rate of 0.25 ± 0.03 h−1, or about 1.8 ± 0.1 mM of glucose accumulated for every unit increase in OD600. Despite a 22% decrease in xylose uptake rate, a 33% decrease in biomass yield, and a 52% decrease in acetate production rate relative to the wild-type, the intracellular flux profile and cofactor allocation of X2G remain largely unperturbed, as elucidated through 13C-metabolic flux analysis. Further quantification of the pool sizes of key intracellular metabolites revealed that glucose secretion by X2G is likely driven by the substantial accumulation of intracellular glucose 6-phosphate, fructose 6-phosphate, glucose and fructose at levels greater than 20x of that in wild-type E. coli. Combined, our results shed light on the flexibility of central metabolism, and the opportunities this affords for producing value-added pentose- and hexose-derived products from lignocellulosic feedstocks.

Evolutionary Adaptation of Kluyveromyces marxianus NIRE-K3 for Enhanced Xylose Utilization

The evolutionary adaptation was approached on the thermotolerant yeast Kluyveromyces marxianus NIRE-K3 at 45oC on xylose as a sole source of carbon for enhancement of xylose uptake. After 60 cycles, evolved strain K. marxianus NIRE-K3.1 showed comparatively 3.75-fold and 3.0-fold higher specific growth and xylose uptake rates, respectively than that of native strain. Moreover, the short lag phase was also observed on adapted strain. During batch fermentation with xylose concentration of 30 g l-1, K. marxianus NIRE-K3.1 could utilize about 96 % of xylose in 72 h and produced 4.67 and 15.7 g l-1 of ethanol and xylitol, respectively, which were 9.72-fold and 4.63-fold higher than that of native strain. Similarly, specific sugar consumption rate, xylitol and ethanol yields were 5.07-fold, 1.15-fold and 2.44-fold higher as compared to the native strain, respectively. The results obtained after evolutionary adaptation of K. marxianus NIRE-K3 show the significant improvement in the xylose utilization, ethanol and xylitol yields, and productivities. By understanding the results obtained, the significance of evolutionary adaptation has been rationalized, since the adapted culture could be more stable and could enhance the productivity.

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.

A design\u2013build\u2013test cycle using modeling and experiments reveals interdependencies between upper glycolysis and xylose uptake in recombinant S. cerevisiae and improves predictive capabilities of large-scale kinetic models

BackgroundRecent advancements in omics measurement technologies have led to an ever-increasing amount of available experimental data that necessitate systems-oriented methodologies for efficient and systematic integration of data into consistent large-scale kinetic models. These models can help us to uncover new insights into cellular physiology and also to assist in the rational design of bioreactor or fermentation processes. Optimization and Risk Analysis of Complex Living Entities (ORACLE) framework for the construction of large-scale kinetic models can be used as guidance for formulating alternative metabolic engineering strategies.ResultsWe used ORACLE in a metabolic engineering problem: improvement of the xylose uptake rate during mixed glucose–xylose consumption in a recombinant Saccharomyces cerevisiae strain. Using the data from bioreactor fermentations, we characterized network flux and concentration profiles representing possible physiological states of the analyzed strain. We then identified enzymes that could lead to improved flux through xylose transporters (XTR). For some of the identified enzymes, including hexokinase (HXK), we could not deduce if their control over XTR was positive or negative. We thus performed a follow-up experiment, and we found out that HXK2 deletion improves xylose uptake rate. The data from the performed experiments were then used to prune the kinetic models, and the predictions of the pruned population of kinetic models were in agreement with the experimental data collected on the HXK2-deficient S. cerevisiae strain.ConclusionsWe present a design–build–test cycle composed of modeling efforts and experiments with a glucose–xylose co-utilizing recombinant S. cerevisiae and its HXK2-deficient mutant that allowed us to uncover interdependencies between upper glycolysis and xylose uptake pathway. Through this cycle, we also obtained kinetic models with improved prediction capabilities. The present study demonstrates the potential of integrated “modeling and experiments” systems biology approaches that can be applied for diverse applications ranging from biotechnology to drug discovery.

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol.

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Sequence analysis of KmXYL1 genes and verification of thermotolerant enzymatic activities of xylose reductase from four Kluyveromyces marxianus strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.

Enhancement in xylose utilization using Kluyveromyces marxianus NIRE-K1 through evolutionary adaptation approach.

The evolutionary adaptation was carried out on the thermotolerant yeast Kluyveromyces marxianus NIRE-K1 at 45 °C up to 60 batches to enhance its xylose utilization capability. The adapted strain showed higher specific growth rate and 3-fold xylose uptake rate and short lag phase as compared to the native strain. During aerobic growth adapted yeast showed 2.81-fold higher xylose utilization than that of native. In anaerobic batch fermentation, adapted yeast utilized about 91% of xylose in 72 h and produced 2.88 and 18.75 g l⁻¹ of ethanol and xylitol, respectively, which were 5.11 and 5.71-fold higher than that of native. Ethanol yield, xylitol yield and specific sugar consumption rate obtained by the adapted cells were found to be 1.57, 1.65 and 4.84-fold higher than that of native yeast, respectively. Aforesaid results suggested that the evolutionary adaptation will be a very effective strategy in the near future for economic lignocellulosic ethanol production.

Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose

BackgroundAn efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease.ResultsWe have engineered and evolved a cellobiose-negative and xylose-negative C. glutamicum that utilizes cellobiose as sole carbon and co-ferments cellobiose and xylose. NGS-genomic and DNA microarray-transcriptomic analysis revealed the multiple genetic mutations for the evolved cellobiose-utilizing strains. As a result, a consortium of mutated transporters and metabolic and auxiliary proteins was responsible for the efficient cellobiose uptake. Evolved and engineered strains expressing an intracellular bG showed a better rate of growth rate on cellobiose as sole carbon source than did other bG-secreting or bG-displaying C. glutamicum strains under aerobic culture. Our strain was also capable of co-fermenting cellobiose and xylose without a biphasic growth, although additional pentose transporter expression did not enhance the xylose uptake rate. We subsequently assessed the strains for simultaneous saccharification and fermentation of cellulosic substrates derived from Canadian Ponderosa Pine.ConclusionsThe combinatorial strategies of metabolic engineering and adaptive evolution enabled to construct C. glutamicum strains that were able to co-ferment cellobiose and xylose. This work could be useful in development of recombinant C. glutamicum strains for efficient lignocellulosic-biomass conversion to produce value-added chemicals and fuels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0420-z) contains supplementary material, which is available to authorized users.

Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae.

Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min−1•mg−1) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation.

De Novo Assembly of Candida sojae and Candida boidinii Genomes, Unexplored Xylose-Consuming Yeasts with Potential for Renewable Biochemical Production.

Candida boidinii and Candida sojae yeasts were isolated from energy cane bagasse and plague-insects. Both have fast xylose uptake rate and produce great amounts of xylitol, which are interesting features for food and 2G ethanol industries. Because they lack published genomes, we have sequenced and assembled them, offering new possibilities for gene prospection.

Novel thermostable clostridial strains through protoplast fusion for enhanced biobutanol production at higher temperature—preliminary study

The objective of this study is to improve the thermal stability of clostridium strains for enhanced biobutanol production. Thermostable clostridia species were developed through protoplast fusion between mesophilic clostridial species (i.e., Clostridium beijerinckii and Clostridium acetobutylicum) and thermophilic clostridial species (i.e., Clostridium thermocellum). Production of biobutanol was examined in the present preliminary study using the clostridium strains and their protoplast fusants using sugar mixture with composition identical to that of wheat straw acid hydrolysate. Maximum biobutanol production of 9.4 g/L was achieved by a fused strain at 45 °C with total sugar consumption of 66% compared to that at 35 °C (i.e., 8.4 g/L production and 64% total sugar consumption). Glucose and xylose uptake rates were generally higher compared to all other individual sugars in the feedstock. In general, average cell concentrations were in close proximity for all parenting and fused strains at 35 °C; i.e., in the range of 5.12 × 107 to 5.49 × 107 cells/mL. Average cell concentration of fusants between the mesophilic clostridial species and the thermophilic clostridial species slightly increased to ~ 5.62 × 107 cells/mL at a higher temperature of 45 °C. These results, in addition to the ones obtained for the butanol production, demonstrate enhanced thermal stability of both fusants at a higher temperature (45 °C).

Biochemical conversion of sugarcane straw hemicellulosic hydrolyzate supplemented with co-substrates for xylitol production

Biotechnological production of xylitol is an attractive route to add value to a sugarcane biorefinery, through utilization of the hemicellulosic fraction of sugarcane straw, whose availability is increasing in Brazil. Herein, supplementation of the sugarcane straw hemicellulosic hydrolyzate (xylose 57gL−1) with maltose, sucrose, cellobiose or glycerol was proposed, and their effect as co-substrates on xylitol production by Candida guilliermondii FTI 20037 was studied. Sucrose (10gL−1) and glycerol (0.7gL−1) supplementation led to significant increase of 8.88% and 6.86% on xylose uptake rate (1.11gL−1h−1 and 1.09gL−1), respectively, but only with sucrose, significant increments of 12.88% and 8.69% on final xylitol concentration (36.11gL−1) and volumetric productivity (0.75gL−1h−1), respectively, were achieved. Based on these results, utilization of complex sources of sucrose, derived from agro-industries, as nutritional supplementation for xylitol production can be proposed as a strategy for improving the yeast performance and reducing the cost of this bioprocess by replacing more expensive nutrients.