High Concentrations Of Xylose Research Articles

Deficiency of β-xylosidase activity in Aspergillus luchuensis mut. kawachii IFO 4308.

In this study, we investigated a deleterious mutation in the β-xylosidase gene, xylA (AkxylA), in Aspergillus luchuensis mut. kawachii IFO 4308 by constructing an AkxylA disruptant and complementation strains of AkxylA and xylA derived from A. luchuensis RIB2604 (AlxylA), which does not harbor the mutation in xylA. Only the AlxylA complementation strain exhibited significantly higher growth and substantial β-xylosidase activity in medium containing xylan, accompanied by an increase in XylA expression. This resulted in lower xylobiose and higher xylose concentrations in the mash of barley shochu. These findings suggest that the mutation in xylA affects xylose levels during the fermentation process. Because the mutation in xylA was identified not only in the genome of strain IFO 4308 but also the genomes of other industrial strains of A. luchuensis and A. luchuensis mut. kawachii, these findings enhance our understanding of the genetic factors that affect the fermentation characteristics.

Effect of carbonyl-amino condensation, non-covalent cross-linking and conformational changes induced by ultrasound-assisted Maillard reaction on lysinoalanine formation in silkworm pupa protein

The inhibition of cross-linked lysinoalanine (LAL) formation in silkworm pupa protein isolates (SPPI) by Maillard reaction (using varying xylose concentration) and ultrasound treatment was studied. Results showed that sonicated SPPI was effectively grafted with high concentration of xylose (5 %), resulting in the lowest LAL content, which was 48.75 % and 30.64 % lower than the control and ultrasound-treated samples, respectively. Chemical bond analysis showed that the combined treatment destroyed the ionic bonds, intrachain (g-g-t), and interchain (g-g-g) disulfide bonds, but stimulated the polymerization of hydrogen and hydrophobic bonds between SPPI and xylose, and as well enhanced the net negative charge between SPPI/Xylose complexes. The particles of the complexes were more loose, dispersed and rough, and had a stronger hydrophilic microenvironment, accompanied by alterations in microscopic, secondary and tertiary structures. Ultrasound treatment induced the breakdown of the oxidative cross-linking in SPPI, and promoted the sulfhydryl group-dehydroalanine binding and the carbonyl-amino condensation of the protein and xylose, and thus inhibited the formation of cross-linked LAL. Furthermore, the physicochemical and structural parameters were highly interrelated with cross-linked LAL content (|r| > 0.9). The outcomes provided a novel avenue and theoretical basis for minimizing LAL formation in SPPI and improving the nutrition and safety of SPPI.

Solid feeding and co-culture strategies for an efficient enzymatic hydrolysis and ethanol production from sugarcane bagasse

The feasibility of second-generation (2G) processes still highly depends on improvements in the hydrolysis and fermentation steps, ensuring the whole use of biomass sugars in an integrated approach. The main bottlenecks are the differences between the optimal conditions for both steps, limitations for high solid load (HSL) hydrolysis, and hexoses and pentose co-fermentation. To overcome these problems, the present work explored a feeding strategy to improve HSL enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse (PSB), at optimal and suboptimal temperatures, further integrated with a non-recombinant yeast co-culture (Saccharomyces cerevisiae and Kluyveromyces marxianus) for sugar co-fermentation. The entire PSB fraction was used without conditioning. Efficient enzymatic hydrolysis was achieved using a feeding strategy in a helical stirred bioreactor (64 and 75% cellulose conversion with 22.5% of solids, 72 h at 35 and 50 °C, respectively), obtaining hydrolysates with high glucose and xylose concentrations (100 g/L). Simultaneous saccharification and co-fermentation (SSCF) provided high glucose (98%) and xylose (52%) conversion in 24 deep-well plates, with ethanol yield of 0.23 getOH/gS, but faced problems of cell viability loss in the bioreactor. Sequential SCF (SqSCF) co-culture stood out as a promising strategy for the use of biomass sugars. The results of this study provide significant contribution to the intensification and feasibility of 2G production, in addition to highlighting the difficulties faced for process integration aiming at the full conversion of sugars coming from high solid load enzymatic hydrolysis.

Crabtree Effect on Rhodosporidium toruloides Using Wood Hydrolysate as a Culture Media

The interest in microorganisms to produce microbial lipids at large-scale processes has increased during the last decades. Rhodosporidium toruloides-1588 could be an efficient option for its ability to simultaneously utilize five- and six-carbon sugars. Nevertheless, one of the most important characteristics that any strain needs to be considered or used at an industrial scale is its capacity to grow in substrates with high sugar concentrations. In this study, the effect of high sugar concentrations and the effect of ammonium sulfate were tested on R. toruloides-1588 and its capacity to grow and accumulate lipids using undetoxified wood hydrolysates. Batch fermentations showed a catabolic repression effect on R. toruloides-1588 growth at sugar concentrations of 120 g/L. The maximum lipid accumulation was 8.2 g/L with palmitic, stearic, oleic, linoleic, and lignoceric acids as predominant fatty acids in the produced lipids. Furthermore, R. toruloides-1588 was able to utilize up to 80% of the total xylose content. Additionally, this study is the first to report the effect of using high xylose concentrations on the growth, sugar utilization, and lipid accumulation by R. toruloides-1588.

Effects of ultrasonic-assisted alkaline pretreatment on xylose production from pineapple peel waste

The pineapple industry generates large amounts of unusable waste (peel and core) with adverse environmental impacts. This experimental study aims to systemize the potential of ultrasonic-assisted alkaline pretreatment for xylose production from pineapple peel waste. The best condition for single alkaline pretreatment (1 % NaOH w/v, 100 °C, 60 min) has obtained hemicellulose, cellulose, and lignin composition at 34.80 %, 32.16 %, and 8.66 %, respectively, retained in the biomass. Meanwhile, a combination of alkaline (1 % NaOH, w/v) and ultrasonic (frequency 40 kHz, 45 min) pretreatment has obtained the percentage yield of hemicellulose and lignin at 51.15 % and 7.15 %, respectively. Both single alkaline and ultrasonic-assisted alkaline pretreated samples were subsequently hydrolyzed with 2 % H2SO4 (w/v). After acid hydrolysis for 30 min, the maximum xylose concentration of 48.85 g.L-1 was achieved by using ultrasonic-assisted alkaline pretreatment, while single alkaline pretreatment contributed to the lowest yield of xylose (37.11 g.L-1). It is shown that the ultrasonic-assisted alkaline treatment is more favorable than single alkaline pretreatment as it can produce high xylose concentration after the subsequent hydrolysis. These results indicated that ultrasonic-assisted alkaline pretreatment and its subsequent acid hydrolysis were appropriate for producing xylose from pineapple peel waste.

Enterococcus faecium s6 Enabled Efficient Homofermentative Lactic Acid Production from Xylan-Derived Sugars

A thermotolerant Enterococcus faecium s6 strain exhibited homoferementative lactic acid (LA) production at high xylose concentration (≥50 g/L). In batch fermentation at 45 °C and controlled pH of 6.5, strain E. faecium s6 produced up to 72.9 g/L of LA with a yield of 0.99 g/g-consumed xylose and productivity of 1.74 g/L.h from 75 g/L xylose. An increased LA concentration and productivities with high yields were obtained in repeated batch fermentation that was conducted for 24 runs. In this mode, the strain could produce LA up 81.1 g/L within 5 h fermentation at a high productivity of 13.5 g/L.h and a yield of 1.02 g/g-consumed xylose. The strain was unable to consume birchwood xylan as sole carbon source. However, it could efficiently utilize xylooligosaccharides of xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose. The intracellular xylosidase activity was induced by xylose. Using xylooligosaccharide (50 g/L)/xylose (5 g/L) mixtures, the strain was able to produce maximum LA at 48.2 g/L within 120 h at a yield of 1.0 g/g-consumed sugar and productivity of 0.331 g/L.h. These results indicate that E. faecium s6 is capable of directly utilizing xylan-hydrolyzate and will enable the development of a feasible and economical approach to the production of LA from hemicellulosic hydrolysate.

Multi-response optimization of acid hydrolysis in sugarcane bagasse to obtain high xylose concentration

Multi-response optimization of acid hydrolysis in sugarcane bagasse to obtain high xylose concentration

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

BackgroundEfficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.ResultsHeterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.ConclusionsThis study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Characterization of a highly xylose tolerant \u03b2-xylosidase isolated from high temperature horse manure compost

BackgroundThere is a continued need for improved enzymes for industry. β-xylosidases are enzymes employed in a variety of industries and although many wild-type and engineered variants have been described, enzymes that are highly tolerant of the products produced by catalysis are not readily available and the fundamental mechanisms of tolerance are not well understood.ResultsScreening of a metagenomic library constructed of mDNA isolated from horse manure compost for β-xylosidase activity identified 26 positive hits. The fosmid clones were sequenced and bioinformatic analysis performed to identity putative β-xylosidases. Based on the novelty of its amino acid sequence and potential thermostability one enzyme (XylP81) was selected for expression and further characterization. XylP81 belongs to the family 39 β-xylosidases, a comparatively rarely found and characterized GH family. The enzyme displayed biochemical characteristics (KM—5.3 mM; Vmax—122 U/mg; kcat—107; Topt—50 °C; pHopt—6) comparable to previously characterized glycoside hydrolase family 39 (GH39) β-xylosidases and despite nucleotide identity to thermophilic species, the enzyme displayed only moderate thermostability with a half-life of 32 min at 60 °C. Apart from acting on substrates predicted for β-xylosidase (xylobiose and 4-nitrophenyl-β-D-xylopyranoside) the enzyme also displayed measurable α-L-arabainofuranosidase, β-galactosidase and β-glucosidase activity. A remarkable feature of this enzyme is its ability to tolerate high concentrations of xylose with a Ki of 1.33 M, a feature that is highly desirable for commercial applications.ConclusionsHere we describe a novel β-xylosidase from a poorly studied glycosyl hydrolase family (GH39) which despite having overall kinetic properties similar to other bacterial GH39 β-xylosidases, displays unusually high product tolerance. This trait is shared with only one other member of the GH39 family, the recently described β-xylosidases from Dictyoglomus thermophilum. This feature should allow its use as starting material for engineering of an enzyme that may prove useful to industry and should assist in the fundamental understanding of the mechanism by which glycosyl hydrolases evolve product tolerance.

Bioconversion of Untreated Corn Hull into L-Malic Acid by Trifunctional Xylanolytic Enzyme from Paenibacillus curdlanolyticus B-6 and Acetobacter tropicalis H-1.

L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

Dynamic Modeling Application To Evaluate the Performance of Spathaspora passalidarum in Second-Generation Ethanol Production: Parametric Dynamics and the Likelihood Confidence Region

Lignocellulosic materials and agro-industrial residues are essential in a sustainable biorefinery. The effective fermentation of renewable sugars is still an important challenge for an economically viable production of biofuels and biochemicals. This challenge involves biological aspects, such as choosing the microbial platform, and industrial aspects. These aspects can be assessed employing mathematical models to understand the complex synergies between the microorganism and processes. In this work, the performance of the yeast Spathaspora passalidarum was evaluated for the fermentation of xylose and glucose at different proportions, initial concentrations, and initial cell densities using an unstructured–unsegregated kinetic model. The likelihood confidence region, correlations between the parameters, and the convergence of the parameters through the optimization process were identified. It was also possible to identify that πXyl, max, μGlu, max, K’S, Xyl, Y’PEth/SGlu, Y’PEth/SXyl, and Y’X/SGlu were the most representative parameters, due to the high correlation (positive and negative) they have with the other model parameters. It was also possible to identify how initial substrate concentrations affected the specific uptake substrate and ethanol production rates; in this sense, the increase in the glucose concentration causes a decrease in the specific ethanol formation rate at a high concentration of xylose for S. passalidarum cultivated in the conditions proposed by this study.

Engineering of Klebsiella oxytoca for the Production of 2,3-Butanediol from High Concentration of Xylose

Engineering of <i>Klebsiella oxytoca</i> for the Production of 2,3-Butanediol from High Concentration of Xylose

L-malic acid production from xylose by engineered Saccharomyces cerevisiae.

L-malic acid is widely used in the food, chemical, and pharmaceutical industries. Here, we report on production of malic acid from xylose, the second most abundant sugar in lignocellulosic hydrolysates, by engineered Saccharomyces cerevisiae. To enable malic acid production in a xylose-assimilating S. cerevisiae, we overexpressed PYC1 and PYC2, coding for pyruvate carboxylases, a truncated MDH3 coding for malate dehydrogenase, and SpMAE1, coding for a Schizosaccharomyces pombe malate transporter. Additionally, both the ethanol and glycerol-producing pathways were blocked to enhance malic acid production. The resulting strain produced malic acid from both glucose and xylose, but it produced much higher titers of malic acid from xylose than glucose. Interestingly, the engineered strain had higher malic acid yield from lower concentrations (10g L-1 ) of xylose, with no ethanol production, than from higher xylose concentrations (20and 40g L-1 ). As such, a fed-batch culture maintaining xylose concentrations at low levels was conducted and 61.2g L-1 of malic acid was produced, with a productivity of 0.32g L-1 h. These results represent successful engineering of S. cerevisiae for the production of malic acid from xylose, confirming that that xylose offers the efficient production of various biofuels and chemicals by engineered S. cerevisiae.

Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform.

ABSTRACTThe capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

A Comparative Study of Maize and Miscanthus Regarding Cell-Wall Composition and Stem Anatomy for Conversion into Bioethanol and Polymer Composites

Due to an increasing demand for environmentally sustainable products, miscanthus and maize stover represent interesting lignocellulosic resources for conversion into biofuels and biomaterials. The overall purpose was to compare miscanthus and maize regarding cell-wall composition and stem anatomy for conversion into bioethanol and polymer composites using partial least squares regressions. For each of the two crops, six contrasted genotypes were cultivated in complete block design, and harvested. Internodes below the main cob for maize, and on the first aboveground internode for miscanthus, were analyzed for biochemistry and anatomy. Their digestibility was predicted using crop-specific near infrared calibrations, and the mechanical properties were evaluated in stem-based composites. On average, the internode cross-section of miscanthus anatomy was characterized by a thick rind (26.2%) and few but dense pith-bundles (3.5 nb/mm2), while cell-wall constituted 95.2% of the dry matter with high lignin (243.2 mg/g) and cellulose concentrations (439.7 mg/g). Maize internode-anatomy showed large cross-sections (397.5 mm2), pith with the presence of numerous bundles and non-lignified-pith fractions (22.3% of the section). Its cell-wall biochemistry displayed high concentrations of hemicelluloses, galactose, arabinose, xylose, and ferulic acid. Cell-wall, lignin, and cellulose concentrations were positively correlated with rind-fraction and pith-bundle-density, which explained strong mechanical properties as shown in miscanthus. Hemicelluloses, galactose, arabinose, and ferulic acid concentrations were positively correlated with pith fraction and stem cross-section, revealing high digestibility as shown in maize. This underlines interesting traits for further comparative genetic studies, as maize represents a good model for digestibility and miscanthus for composites.

Removal of fermentation inhibitors from pre-hydrolysis liquor using polystyrene divinylbenzene resin

BackgroundThe presence of soluble lignin, furfural and hydroxymethylfurfural (HMF) in industrial pre-hydrolysis liquor (PHL) from the pulping process can inhibit its bioconversion into bioethanol and other biochemicals. Although various technologies have been developed to remove these inhibitors, certain amounts of sugars are also inevitably removed during the treatment process. Hence, polystyrene divinylbenzene (PS-DVB) resin was used as an adsorptive material to simultaneously remove fermentation inhibitors while retaining sugars with high yields to improve the fermentability of PHL after acid hydrolysis by enriching its xylose concentration. The fermentability of acid-hydrolyzed PHL (A-PHL) was evaluated by the bioconversion into ethanol and xylosic acid (XA) after treatment with PS-DVB resin.ResultsThe results showed that the highest xylose concentration (101.1 g/L) in PHL could be obtained by acid hydrolysis at 100 °C for 80 min with 4% acid, while the concentration of fermentation inhibitors (furfural, HMF and lignin) in PHL could also be significantly improved during the acid-hydrolysis process. After treatment with PS-DVB resin, not only were 97% of lignin, 92% of furfural, and 97% of HMF removed from A-PHL, but also 96% of xylose was retained for subsequent fermentation. With resin treatment, the fermentability of A-PHL could be improved by 162–282% for ethanol production from A-PHL containing 30–50 g/L xylose and by 18–828% for XA production from A-PHL containing 90–150 g/L xylose.ConclusionsThese results confirmed that PS-DVB resin can remove inhibitors from PHL before producing value-added products by bioconversion. In addition, this work will ideally provide a concept for producing value-added chemicals from pre-hydrolysis liquor, which is regarded as the waste stream in the pulping process.

Novel Synergistic Mechanism for Lignocellulose Degradation by a Thermophilic Filamentous Fungus and a Thermophilic Actinobacterium Based on Functional Proteomics.

Effective artificial microbial consortia containing microorganisms with desired biological functions have the potential to optimize the lignocellulose-based bioindustry. Thermobifida fusca was a dominant actinobacterium in high-temperature corn stalk composts, but it was unable to grow alone in corn stalk solid medium. Interestingly, T. fusca showed good growth and secreted enzymes when cocultured with Thermomyces lanuginosus. T. lanuginosus grew firstly during the initial stage, whereas T. fusca dominated the system subsequently during cocultivation. The secretome indicated that T. lanuginosus mainly degraded xylan by expressing a GH11 xylanase (g4601.t1, GenBank AAB94633.1; with relative secretion of 4.95 ± 0.65%). T. fusca was induced by xylan mainly to secrete a xylanase from GH11 family (W8GGR4, GenBank AHK22788.1; with relative secretion of 8.71 ± 3.83%) which could rapidly degrade xylan to xylo-oligosaccharide (XOS) and xylose within 2 min, while high concentrations (>0.5%, w/v) of XOS or xylose suppressed the growth of T. fusca; which may be the reason why T. fusca unable to grow alone in corn stalk solid medium. However, T. lanuginosus could utilize the XOS and xylose produced by xylanases secreted by T. fusca. During the synergistic degradation of lignocellulose by T. lanuginosus and T. fusca, xylan was rapidly consumed by T. lanuginosus, the residual cellulose could specifically induced T. fusca to express a GH10 xylanase with a CBM2 domain (Q47KR6, GenBank AAZ56956.1; with relative secretion of 5.03 ± 1.33%) and 6 cellulases (2 exocellulases and 4 endocellulases). Moreover, T. lanuginosus increased the secretion of cellulases from T. fusca by 19–25%. The order of T. lanuginosus and T. fusca was consistent with the multilayered structures of lignocellulose and could be regulated by different concentrations of XOS and xylose. The novel synergism of T. lanuginosus and T. fusca gave a new sight for revealing more synergetic relationships in natural environments and exploring efficient microbial inoculants and enzyme cocktails for lignocellulose degradation.

Novel xylose transporter Cs4130 expands the sugar uptake repertoire in recombinant Saccharomyces cerevisiae strains at high xylose concentrations

BackgroundThe need to restructure the world’s energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes.ResultsAssessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters.ConclusionsConsidering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast.

Microwave-Assisted Degradation of Biomass with the Use of Acid Catalysis

The aim of the study was to assess the effectiveness of microwave pretreatment combined with acid catalysis in the decomposition of various types of biomass (pine and beech chips and hemp stems). It was clearly demonstrated that sulfuric acid was a catalyst enabling the most effective decomposition of the tested plant biomass, guaranteeing the highest concentrations of simple sugars released. Acid catalysis with 1% v/v sulfuric acid combined with microwave radiation provided high glucose concentrations of 89.8 ± 3.4, 170.4 ± 2.4 and 164.6 ± 4.6 mg/g for pine chips, beech chips and hemp stems, respectively. In turn, the use of nitric acid promoted the degradation of hemicellulose, which resulted in high concentrations of galactose and xylose, i.e., 147.6 ± 0.6, 163.6 ± 0.4 and 134.9 ± 0.8 mg/g of pine chips, beech chips and hemp stems, respectively, while glucose levels remained relatively low. It was also demonstrated that the undesirable dehydration of sugars such as glucose and xylose is more pronounced in sulfuric acid than nitric acid processes. The use of H2SO4 and increased pressure generated 5-hydroxymethylfurfural (5-HMF) and furfural at a concentration of ca. 12 and 6 mg/g, 10 and 45 mg/g and 14 and 30 mg/g, of pine chips, beech chips and hemp shoots, respectively. Our studies confirmed the usefulness of the combined use of microwaves and acid catalysis in the degradation of softwood, hardwood and non-wood plant biomass. It should be emphasized that obtaining high concentrations of released simple sugars (as potential substrates in biosynthesis), while maintaining low levels of toxic by-products (inhibitors), requires precise selection of process parameters such as pressure, exposition time and type of acid catalyst.

Genomic and transcriptomic analysis of Candida intermedia reveals the genetic determinants for its xylose-converting capacity

BackgroundAn economically viable production of biofuels and biochemicals from lignocellulose requires microorganisms that can readily convert both the cellulosic and hemicellulosic fractions into product. The yeast Candida intermedia displays a high capacity for uptake and conversion of several lignocellulosic sugars including the abundant pentose d-xylose, an underutilized carbon source since most industrially relevant microorganisms cannot naturally ferment it. Thus, C. intermedia constitutes an important source of knowledge and genetic information that could be transferred to industrial microorganisms such as Saccharomyces cerevisiae to improve their capacity to ferment lignocellulose-derived xylose.ResultsTo understand the genetic determinants that underlie the metabolic properties of C. intermedia, we sequenced the genomes of both the in-house-isolated strain CBS 141442 and the reference strain PYCC 4715. De novo genome assembly and subsequent analysis revealed C. intermedia to be a haploid species belonging to the CTG clade of ascomycetous yeasts. The two strains have highly similar genome sizes and number of protein-encoding genes, but they differ on the chromosomal level due to numerous translocations of large and small genomic segments. The transcriptional profiles for CBS 141442 grown in medium with either high or low concentrations of glucose and xylose were determined through RNA-sequencing analysis, revealing distinct clusters of co-regulated genes in response to different specific growth rates, carbon sources and osmotic stress. Analysis of the genomic and transcriptomic data also identified multiple xylose reductases, one of which displayed dual NADH/NADPH co-factor specificity that likely plays an important role for co-factor recycling during xylose fermentation.ConclusionsIn the present study, we performed the first genomic and transcriptomic analysis of C. intermedia and identified several novel genes for conversion of xylose. Together the results provide insights into the mechanisms underlying saccharide utilization in C. intermedia and reveal potential target genes to aid in xylose fermentation in S. cerevisiae.