Xylose Transport Research Articles

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.

Engineering of Saccharomyces cerevisiae for the efficient co-utilization of glucose and xylose.

The rapid co-fermentation of both glucose and xylose is important for the efficient conversion of lignocellulose biomass into fuels and chemicals. Saccharomyces cerevisiae is considered to be a potential cell factory and has been used to produce various fuels and chemicals, but it cannot metabolize xylose, which has greatly limited the utilization of lignocellulose materials. Therefore, numerous studies have attempted to develop xylose fermenting strains in past decades. The simple introduction of the xylose metabolic pathway does not enable yeast to rapidly utilize xylose, and several limitations still need to be addressed, including glucose repression and slow xylose transport, cofactor imbalance in the xylose reductase/xylitol dehydrogenase pathway, functional expression of a heterologous xylose isomerase, the low efficiency of downstream pathways and low ethanol production. In this review, we will discuss strategies to overcome these limitations and the recent progress in engineering xylose fermenting S. cerevisiae strains.

Xylitol production by genetically modified industrial strain of Saccharomyces cerevisiae using glycerol as co-substrate.

Xylitol is commercially used in chewing gum and dental care products as a low calorie sweetener having medicinal properties. Industrial yeast strain of S. cerevisiae was genetically modified to overexpress an endogenous aldose reductase gene GRE3 and a xylose transporter gene SUT1 for the production of xylitol. The recombinant strain (XP-RTK) carried the expression cassettes of both the genes and the G418 resistance marker cassette KanMX integrated into the genome of S. cerevisiae. Short segments from the 5' and 3' delta regions of the Ty1 retrotransposons were used as homology regions for integration of the cassettes. Xylitol production by the industrial recombinant strain was evaluated using hemicellulosic hydrolysate of the corn cob with glucose as the cosubstrate. The recombinant strain XP-RTK showed significantly higher xylitol productivity (212mgL-1h-1) over the control strain XP (81mgL-1h-1). Glucose was successfully replaced by glycerol as a co-substrate for xylitol production by S. cerevisiae. Strain XP-RTK showed the highest xylitol productivity of 318.6mgL-1h-1 and titre of 47gL-1 of xylitol at 12gL-1 initial DCW using glycerol as cosubstrate. The amount of glycerol consumed per amount of xylitol produced (0.47mol mol-1) was significantly lower than glucose (23.7mol mol-1). Fermentation strategies such as cell recycle and use of the industrial nitrogen sources were demonstrated using hemicellulosic hydrolysate for xylitol production.

Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae.

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes d-xylose in the presence of high d-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16 Mutant cells showed increased uptake rates of d-xylose in the presence of d-glucose, as well as elevated maximum rates of metabolism (Vmax) for both d-glucose and d-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders d-xylose metabolism less sensitive to d-glucose inhibition due to an elevated transport rate of d-xylose into the cell.IMPORTANCE The yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.

The low affinity glucose transporter HxtB is also involved in glucose signalling and metabolism in Aspergillus nidulans

One of the drawbacks during second-generation biofuel production from plant lignocellulosic biomass is the accumulation of glucose, the preferred carbon source of microorganisms, which causes the repression of hydrolytic enzyme secretion by industrially relevant filamentous fungi. Glucose sensing, subsequent transport and cellular signalling pathways have been barely elucidated in these organisms. This study therefore characterized the transcriptional response of the filamentous fungus Aspergillus nidulans to the presence of high and low glucose concentrations under continuous chemostat cultivation with the aim to identify novel factors involved in glucose sensing and signalling. Several transcription factor- and transporter-encoding genes were identified as being differentially regulated, including the previously characterized glucose and xylose transporter HxtB. HxtB was confirmed to be a low affinity glucose transporter, localizing to the plasma membrane under low- and high-glucose conditions. Furthermore, HxtB was shown to be involved in conidiation-related processes and may play a role in downstream glucose signalling. A gene predicted to encode the protein kinase PskA was also identified as being important for glucose metabolism. This study identified several proteins with predicted roles in glucose metabolic processes and provides a foundation for further investigation into the response of biotechnologically important filamentous fungi to glucose.

Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9)

Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 & SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.

Enabling glucose/xylose co-transport in yeast through the directed evolution of a sugar transporter.

The capacity to co-transport glucose and xylose into yeast has remained a technical challenge in the field. While significant efforts have been made in transporter engineering to increase xylose transport rates, glucose-based inhibition still limit most of these transporters. To address this issue, we further engineer sugar transporter proteins to remove glucose inhibition and enable glucose/xylose co-transport. Specifically, we start with our previously derived CiGXS1 FIM mutant strain and subjugate it to several rounds of mutagenesis and selection in a hexose metabolism null strain. Through this effort, we identify several mutations including N326H, a truncation in the C-terminal tail, I171F, and M40V as additionally dominant for reducing glucose inhibition. The resulting transporter shows substantially improved xylose transport rates in the presence of high quantities of glucose including up to 70g/L glucose. Moreover, the resulting transporter enables co-utilization of glucose and xylose with glucose rates on par with a wild-type transporter and xylose rates exceeding that of glucose. These results demonstrate that major facilitator superfamily hexose transporters can be rewired into glucose-xylose co-transporters without functional inhibition by either substrate. These results enhance the potential of using lignocellulosic biomass as a feedstock for yeast.

Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans.

BackgroundThe conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae.ResultsThis work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. However, we have shown that the introduction of additional functional copies of CltB increases the growth in the presence of low concentrations of cellobiose, strongly indicating CltB is able to transport cellobiose. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production.ConclusionsThis work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0611-1) contains supplementary material, which is available to authorized users.

Draft genome sequence of Microbacterium oleivorans strain Wellendorf implicates heterotrophic versatility and bioremediation potential

Microbacterium oleivorans is a predominant member of hydrocarbon-contaminated environments. We here report on the genomic analysis of M. oleivorans strain Wellendorf that was isolated from an indoor door handle. The partial genome of M. oleivorans strain Wellendorf consists of 2,916,870bp of DNA with 2831 protein-coding genes and 49 RNA genes. The organism appears to be a versatile mesophilic heterotroph potentially capable of hydrolysis a suite of carbohydrates and amino acids. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, fructose, rhamnose, galactose, xylose, arabinose, alanine, aspartate, asparagine, glutamate, serine, glycine, threonine and cysteine. This is the first detailed analysis of a Microbacterium oleivorans genome.

Production of Acetoin through Simultaneous Utilization of Glucose, Xylose, and Arabinose by Engineered Bacillus subtilis.

Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.

Metabolic engineering of Propionibacterium freudenreichii subsp. shermanii for xylose fermentation

Propionibacterium freudenreichii cannot use xylose, the second most abundant sugar in lignocellulosic biomass. Although Propionibacterium acidipropionici can use xylose as a carbon source, it is difficult to genetically modify, impeding further improvement through metabolic engineering. This study identified three xylose catabolic pathway genes encoding for xylose isomerase (xylA), xylose transporter (xylT), and xylulokinase (xylB) in P. acidipropionici and overexpressed them in P. freudenreichii subsp. shermanii via an expression plasmid pKHEM01, enabling the mutant to utilize xylose efficiently even in the presence of glucose without glucose-induced carbon catabolite repression. The mutant showed similar fermentation kinetics with glucose, xylose, and the mixture of glucose and xylose, respectively, as carbon source, and with or without the addition of antibiotic for selection pressure. The engineered P. shermanii thus can provide a novel cell factory for industrial production of propionic acid and other value-added products from lignocellulosic biomass.

Improving pentose fermentation by preventing ubiquitination of hexose transporters in Saccharomyces cerevisiae.

BackgroundEngineering of the yeast Saccharomyces cerevisiae for improved utilization of pentose sugars is vital for cost-efficient cellulosic bioethanol production. Although endogenous hexose transporters (Hxt) can be engineered into specific pentose transporters, they remain subjected to glucose-regulated protein degradation. Therefore, in the absence of glucose or when the glucose is exhausted from the medium, some Hxt proteins with high xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely xylose.ResultsThe low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant), and Hxt5 are subjected to catabolite degradation as evidenced by a loss of GFP fused hexose transporters from the membrane upon glucose depletion. Catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. Therefore, N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination, were replaced for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely xylose concomitantly with markedly stimulated growth on xylose. The mutagenesis also improved the late stages of sugar fermentation when cells are grown on both glucose and xylose.ConclusionsSubstitution of N-terminal lysine residues in the endogenous hexose transporters Hxt1 and Hxt36 that are subjected to catabolite degradation results in improved retention at the cytoplasmic membrane in the absence of glucose and causes improved xylose fermentation upon the depletion of glucose and when cells are grown in d-xylose alone.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0573-3) contains supplementary material, which is available to authorized users.

Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei.

BackgroundGlobal climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes.ResultsA hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization.ConclusionsThis study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0564-4) contains supplementary material, which is available to authorized users.

Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

Xylitol production was compared in fed batch fermentation by Saccharomyces cerevisiae strains overexpressing xylose reductase (XR) genes from Candida tropicalis, Pichia stipitis, Neurospora crassa, and an endogenous gene GRE3. The gene encoding a xylose specific transporter (SUT1) from P. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate NADPH. Xylitol yield was near theoretical for all the strains in fed batch fermentation. The highest volumetric (0.28 gL−1 h−1) and specific (34 mgg−1 h−1) xylitol productivities were obtained by the strain overexpressing GRE3 gene, while the control strain showed 7.2 mgg−1 h−1 specific productivity. The recombinant strains carrying XR from C. tropicalis, P. stipitis, and N. crassa produced xylitol with lower specific productivity of 14.3, 6.8, and 6.3 mgg−1 h−1, respectively, than GRE3 overexpressing strain. The glucose fed as cosubstrate was converted to biomass and ethanol, while xylose was only converted to xylitol. The efficiency of ethanol production was in the range of 38–45 % of the theoretical maximum for all the strains. Xylitol production from the non-detoxified corncob hemicellulosic hydrolysate by recombinant S. cerevisiae was reported for the first time. Xylitol productivity was found to be equivalent in the synthetic xylose as well as hemicellulosic hydrolysate-based media showing no inhibition on the S. cerevisiae due to the inhibitors present in the hydrolysate. A systematic evaluation of heterologous XRs and endogenous GRE3 genes was performed, and the strain overexpressing the endogenous GRE3 gene showed the best xylitol productivity.

Inhibitor Discovery for the Human GLUT1 from Homology Modeling and Virtual Screening.

The human Glucose Transporter 1 (hGLUT1 or SLC2A1) is a facilitative membrane transporter found in the liver, intestines, kidney, and brain, where it transports sugars such as d-glucose and d-galactose. Genetic variations in hGLUT1 are associated with a broad range of diseases and metabolic disorders. For example, hGLUT1 is upregulated in various cancer types (e.g., breast carcinoma) to support the increased anaerobic glycolysis and the Warburg effect. Thus, hGLUT1 is an emerging therapeutic target, which also transports commonly used cancer biomarkers (e.g., (18)F-DG). In this study, we use computational prediction followed by experimental testing, to characterize hGLUT1. We construct homology models of hGLUT1 in a partially occluded outward open ("occluded") conformation based on the X-ray structure of the E. coli xylose transporter, XylE. Comparison of the binding site of the occluded models to experimentally determined hGLUT structures revealed a hydrophobic pocket adjacent to the sugar-binding site, which was tested experimentally via site-directed mutagenesis. Virtual screening of various libraries of purchasable compounds against the occluded models, followed by experimental testing with cellular assays revealed seven previously unknown hGLUT1 ligands with IC50 values ranging from 0.45 μM to 59 μM. These ligands represent three unique chemotypes that are chemically different from any other known hGLUT1 ligands. The newly characterized hydrophobic pocket can potentially be utilized by the new ligands for increased affinity. Furthermore, the previously unknown hGLUT1 ligands can serve as chemical tools to further characterize hGLUT1 function or lead molecules for future drug development.

Activation of an Otherwise Silent Xylose Metabolic Pathway in Shewanella oneidensis.

Shewanella oneidensis is unable to metabolize the sugar xylose as a carbon and energy source. In the present study, an otherwise silent xylose catabolic pathway was activated in S. oneidensis by following an adaptive evolution strategy. Genome-wide scans indicated that the S. oneidensis genome encoded two proteins similar to the xylose oxido-reductase pathway enzymes xylose reductase (SO_0900) and xylulokinase (SO_4230), and purified SO_0900 and SO_4230 displayed xylose reductase and xylulokinase activities, respectively. The S. oneidensis genome was missing, however, an Escherichia coli XylE-like xylose transporter. After 12 monthly transfers in minimal growth medium containing successively higher xylose concentrations, an S. oneidensis mutant (termed strain XM1) was isolated for the acquired ability to grow aerobically on xylose as a carbon and energy source. Whole-genome sequencing indicated that strain XM1 contained a mutation in an unknown membrane protein (SO_1396) resulting in a glutamine-to-histidine conversion at amino acid position 207. Homology modeling demonstrated that the Q207H mutation in SO_1396 was located at the homologous xylose docking site in XylE. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass. The activation of an otherwise silent xylose metabolic system in Shewanella oneidensis is a powerful example of how accidental mutations allow microorganisms to adaptively evolve. The expansion of the S. oneidensis metabolic repertoire to xylose expands the electron donors whose oxidation may be coupled to the myriad of terminal electron-accepting processes catalyzed by S. oneidensis Since xylose is a lignocellulose degradation product, this study expands the potential substrates to include lignocellulosic biomass.

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

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

Improvement of ectoine productivity by using sugar transporter-overexpressing Halomonas elongata.

We successfully enhanced the productivity of ectoine with Halomonas elongata by improvement of the transport of sugar. First, we carried out screening for sugar transporters capable of improving glucose and xylose consumption. We found two transporters: b3657 from Escherichia coli, which is capable of improving glucose consumption, and HEO_0208 from H. elongata, which is capable of improving xylose consumption. Using transporter-overexpressing strains, the productivity of ectoine was improved. These results indicate that sugar consumption is important for efficient ectoine production. As result of phenotypic analysis of a HEO_0208 deletion strain, we discovered that HEO_0208 is the major xylose transporter in H. elongata. This is the first report demonstrating improvement of ectoine productivity by enhancing the transport of sugar.

Mechanistic Study of Human Glucose Transport Mediated by GLUT1.

The glucose transporter 1 (GLUT1) belongs to the major facilitator superfamily (MFS) and is responsible for the constant uptake of glucose. However, the molecular mechanism of sugar transport remains obscure. In this study, homology modeling and molecular dynamics (MD) simulations in lipid bilayers were performed to investigate the combination of the alternate and multisite transport mechanism of glucose with GLUT1 in atomic detail. To explore the substrate recognition mechanism, the outward-open state human GLUT1 homology model was generated based on the template of xylose transporter XylE (PDB ID: 4GBZ), which shares up to 29% sequence identity and 49% similarity with GLUT1. Through the MD simulation study of glucose across lipid bilayer with both the outward-open GLUT1 and the GLUT1 inward-open crystal structure, we investigated six different conformational states and identified four key binding sites in both exofacial and endofacial loops that are essential for glucose recognition and transport. The study further revealed that four flexible gates consisting of W65/Y292/Y293-M420/TM10b-W388 might play important roles in the transport cycle. The study showed that some side chains close to the central ligand binding site underwent larger position changes. These conformational interchanges formed gated networks within an S-shaped central channel that permitted staged ligand diffusion across the transporter. This study provides new inroads for the understanding of GLUT1 ligand recognition paradigm and configurational features which are important for molecular, structural, and physiological research of the MFS members, especially for GLUT1-targeted drug design and discovery.

Modular Optimization of a Hemicellulose-Utilizing Pathway in Corynebacterium glutamicum for Consolidated Bioprocessing of Hemicellulosic Biomass.

Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass.