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When lignocellulosic biomass is utilized as a fermentative substrate to produce biochemicals, the existence of a yeast strain resistant to inhibitory chemical compounds (ICCs) released from the biomass becomes critical. To achieve the purpose, in this study, Saccharomyces yeast strains from a NBRC yeast culture collection were used for exploration and evaluated in two different media containing ICCs that mimic one another but resemble the hydrolysate of real biomass. Among them, S. cerevisiae F118 strain shows robustness upon the fermentation with unique flocculation trait that was strongly responsive to ICC stress. When this strain was cultured in the presence of ICCs, its cell wall hydrophobicity increased dramatically, and reduced significantly when the ICCs were depleted, demonstrating that cell-surface hydrophobicity can also act as an adaptive response to the ICCs. Cells from the strain with the highest cell-wall hydrophobicity displayed progressively stronger flocculation, indicating that the F118 strain is having unique robustness under ICC stress. Gene expression perturbation analysis revealed that mot3 gene encoding regulatory Mot3p from the F118 strain was expressed in response to the concentration of ICCs. This gene was found to control expression of ygp1 gene that encoding Ygp1p, one of cell wall proteins. Deep sequencing analysis revealed that the Mot3p of the F118 strain features a unique insertion and deletion of nucleotides that encode glutamine or asparagine residues, particularly in N-terminal domain, as determined by comparison to the Mot3p sequence from the S288c strain, which was employed as a control strain. Furthermore, the cell wall hydrophobicity of the S288c strain was greatly enhanced and became ICC-responsive after gene swapping with the mot3 gene from the F118 strain. The gene-swapped S288c strain fermented 6-fold faster than the wild-type strain, producing 14.5 g/L of ethanol from 30 g/L of glucose consumed within 24 h in a medium containing the ICCs. These such modifications to Mot3p in unique locations in its sequence have a potential to change the expression of a gene involved in cell wall hydrophobicity and boosted the flocculation response to ICC stress, allowing for the acquisition of extraordinary robustness.

Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named ‘Vascular Cell Induction Culture System Using Arabidopsis Leaves’ (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation, among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation in cambial cells. Physiological and genetic analyses suggested that sucrose might function through the BRI1-EMS-SUPPRESSOR1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

To synthesize a high-performance biopolyimide bioplastic from lignocellulosic feedstock, Escherichia coli was metabolically engineered to produce 4-amino-L-phenylalanine (4APhe) as a diamine monomer. A high-biomass sorghum cultivar was used as the...

Cellulose has been developed as an alternative to petrochemical materials. By comparison with refined nanofiber (RCNF), lignocellulose nanofiber (LCNF) shows particular promise because it is produced from biomass using only mild pretreatment. The mechanical properties of LCNF depends on the contained lignin. However, the microscopic location of the lignin contained in LCNF has not been determined. Thus, we developed two methods to detect and visualize lignin. One uses a scanning transmission electron microscope (STEM) equipped with an energy dispersive X-ray spectroscopy (EDS) detector. The other method uses an atomic force microscope (AFM) equipped with a cantilever coated with an aromatic molecule. Both methods revealed that the lignin in LCNF covers a thin cellulose fiber and is precipitated in a grained structure. In particular, the AFM system was able to determine the nanoscopic location of lignin-rich areas. The present study establishes a strong tool for analyzing the characteristics of lignin-containing materials.

Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

New enzyme functions exist within the increasing number of unannotated protein sequences. Novel enzyme discovery is necessary to expand the pathways that can be accessed by metabolic engineering for the biosynthesis of functional compounds. Accordingly, various machine learning models have been developed to predict enzymatic reactions. However, the ability to predict unknown reactions that are not included in the training data has not been clarified. In order to cover uncertain and unknown reactions, a wider range of reaction types must be demonstrated by the models. Here, we establish 16 expanded enzymatic reaction prediction models developed using various machine learning algorithms, including deep neural network. Improvements in prediction performances over that of our previous study indicate that the updated methods are more effective for the prediction of enzymatic reactions. Overall, the deep neural network model trained with combined substrate-enzyme-product information exhibits the highest prediction accuracy with Macro F

Background: Metal nanoparticles have been proposed as potential radiosensitizers. Among those, titanium nanoparticles are attractive candidates for application as radiosensitizers. We have newly established and evaluated titanium peroxide nanoparticles (TiOxNPs). The TiOxNPs are generated from titanium dioxide nanoparticles (TiO2NPs), manufactured via direct reaction of TiO2NPs with hydrogen peroxide. Distinguished characteristics of the TiOxNPs are continuous generation of reactive oxygen species (ROS) by X-ray irradiation. Bio-availabilities and safety was previously reported (Nanoparticles, 2020; 10(6): 1125). Materials and Methods: Characteristics of TiOxNPs were determined following experiments. Dynamic light scattering analysis was used to measure the diameter and Z-potential of the NPs. Transmission electron microscope (TEM) observation revealed the detailed topology of the NPs. X-ray irradiation was performed using the X-ray generator at a voltage of 150 kV and a current of 5 mA with a 1-mm thick aluminum filter in vitro and in vivo. Reactive oxygen species (ROS) generation was evaluated using human pancreatic cancer cells, named MIAPaCa-2. The cells were stained with 50 μM carboxy-20,70-dichlorofluorescein (C-H2DCF) incubated for 45 min, and then stained with Hoechst for nuclear staining. The degree of fluorescence of C-H2DCF was detected using the fluorescence microscope. To evaluate cytotoxic effects of TiOxNPs, a colony-formation assay was performed. For xenograft experiments, the MIAPaCa-2 cells (2 x 106 cells) were injected subcutaneously into the hind legs of the BALB/cAJcl nude mice. Once the tumor volume reached 100-200 mm3, mice were randomly assigned into six groups: the control group, TiOxNPs alone, 5 Gy alone, and TiOxNPs combined with 5 Gy. Tumor size, body weight, and health condition of all mice were followed every two to three days for 55 days post-treatment. Results: Primary particles smaller than 10 nm gathered and formed secondary particles that were approximately 50 nm in diameter, and their Z-potential was -30 mV. TEM observation revealed the detailed topology of the NPs. The TiOxNPs enhanced H2O2 production more than 7-folds compared to X-ray irradiation alone in MIA PaCa-2 cells. In xenografts, combination effects of the TiOxNPs and X-ray irradiation were significantly greater than X-ray irradiation (5 Gy) alone. Conclusions: The TiOxNPs revealed to be powerful radiosensitizers in pancreatic cancer model. Clinical tests are warranted to clarify the application of TiOxNPs in the nearest future. Citation Format: Ryohei Sasaki, Hiroaki Akasaka, Masao Nakayama, Yoshiko Fujita, Hikaru Kubota, Kenta Morita, Mennaallah Hassan, Mohammed Salah, Yuya Nishimura, Naritoshi Mukumoto, Takeaki Ishihara, Daisuke Miyawaki, Chiaki Ogino. A novel radiosensitizer of titanium peroxide nanoparticles (TiOxNPs) through continuous ROS generation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2837.

Abstract: Release and presentation of tumor antigens are the preconditions of cellular immune response. Radiotherapy have been reported to release endogenous tumor antigens and enhanced the adjuvanticity from dying cancer cells via endoplasmic reticulum (ER) stress, known as immunogenic cell death. We developed a novel radiotherapy using radiosensitizer (TiOxNPs) that could drive effective anti-tumor responses. The TiOxNPs were robustly radiation-induced reactive oxygen species (ROS) generation. This burst therapy could enhance to release and presentation of endogenous tumor antigens, subsequently cross-presentation of antigens by Dendritic cells (DCs) for T-cell activation in secondary lymphoid organs (SLO). Methods: B16-F10 murine melanoma cells (5 × 105) were subcutaneously injected into the right flank of C57BL/6J respectively. Mice were divided into five treatment groups: 1) untreated; 2) XRT; 3) XRT+ TiOxNPs; 4) XRT+anti-PD-1Ab; and 5) XRT+ TiOxNPs +anti-PD-1Ab. B16-F10 tumors were intratumorally injected with TiOxNPs (8mg/kg) on days 10 and received 5Gy or 10Gy X-ray radiation approximately 1 hour after the injection. Anti-PD-1 antibody (200µg) intraperitoneally administered to the mice on the first day of X-ray irradiation, days 13 and 16. The tumor volume were assessed every 2-3 days after treatment. Mice were sacrificed after days 17 and isolated spleen and tumor. Results: TiOxNPs acted as a potent radiosensitizer, which enhanced cytotoxic ROS-mediated cell death. This combination effect could not only a local treatment that directly kills cancer cells but also could enhance the adjuvanticity from dying cancer cells by DAMPs, and significantly upregulate expression of MHC class I on tumor cells. Treatment of B16-F10 melanoma tumor model with burst therapy resulted in tumor shrinkage and extended overall survival. Burst therapy facilitated the recruitment and maturation of DCs into SLO, and improved antigen presentation to naive T cells. Infiltration into the tumor site of CTL were correlated with increasing of migration DCs into SLO. Conclusion: A novel radiotherapy combining with TiOxNPs played an important role in release of endogenous tumor antigens and DAMPs. The upregulation of MHC class I expression on tumor cells after X-ray irradiation facilitated the recognition of the tumor cells by T cells, leading to significantly enhanced antitumor immune response compared to exisiting immunoradiotherapy. This novel nanoparticles exposed to X-ray irradiation could trigger of antigen-specific immune responses and transform a tumor into a site for activation of antitumor immune response. Citation Format: Yoshiko Fujita, Hikaru Kubota, Chiaki Ogino, Ryohei Sasaki. Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6408.

Background: Radiotherapy is widely used as the main treatment for multiple malignancies. However, several types of cancers, including pancreatic cancer, show resistance to radiation therapy. The presence of cancer stem cells (CSCs) are the major cause of radiation resistance in pancreatic cancer. Whereas, titanium peroxides nanoparticles (TiOxNPs) which are a modified form of titanium oxide nanoparticles promote enhanced remarkable efficacy of radiation therapy. Therefore, we examined the efficacy of TiOxNPs as radiosensitizers to eradicate pancreatic cancer stem cells. Methods and materials: In vitro, Sphere-forming assay, survival assay, migration, and invasion assay were evaluated after using TiOxNPs prior to radiation exposure to pancreatic cancer cell line. In vivo, tumor-bearing nude mice were injected by TiOxNPs either intratumoral or intravenous one hour prior to radiation treatment, and the tumor volume, body weight, and mice survival was calculated. In addition, proteins-related stemness were measured in vitro and in vivo to evaluate the usage of TiOxNPs as radiosensitizers to pancreatic CSCs. Moreover, we planned to evaluate the mechanism beyond the efficacy of TiOxNPs as radiosensitizers to CSCs by detecting reactive oxygen species (ROS) production, mitochondrial function, and the phosphorylation of some signaling proteins. We found that TiOxNPs combined with ionizing radiation showed anti-cancer effects on radioresistant CSCs both in vitro and in vivo. Results: In vitro, a marked reduction in growth was detected after exposing the TiOxNPs-treated cells to radiation therapy, specifically with a 5Gy dose compared with 2Gy dose. TiOxNPs exhibited a synergistic effect with radiation on pancreatic CSC-enriched spheres by downregulating self-renewal regulatory factors and CSC surface markers. In vivo, we first established an aggressive xenograft by injecting MIA PaCa-2 sphere cells into the flank region of BALB/c nude mice, and found that animals treated with combined TiOxNPs and irradiation showed a dramatic reduction in tumor volume and weight compared to the untreated group. Moreover, combined treatment suppressed epithelial-mesenchymal transition, migration, and invasion properties in primary and aggressive pancreatic cancer cells by reducing the expression of proteins relevant to these processes. Radiosensitizing TiOxNPs suppressed pancreatic xenograft outgrowth after primary or dissociating sphere MIA PaCa-2 cell implantation. It is assumed that synergy is created by inactivating the AKT signaling pathway and producing unbearable amounts of ROS. Conclusion: Our findings showed that using TiOxNPs in combination with radiation might be a favorable therapeutic approach to eradicate pancreatic CSCs. Citation Format: Mohammed Salah, Hiroki Kawaguchi, Hiroaki Akasaka, Yasuyuki Shimizu, Kenta Morita, Yuya Nishimura, Hikaru Kubota, Tomomi Sogawa, Naritoshi Mukumoto, Chiaki Ogino, Ryohei Sasaki. Combatting pancreatic cancer stem cells by novel titanium peroxides nanoparticles combined with X-ray radiation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5048.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

Lignocellulose is resistant to degradation and requires pretreatment before hydrolytic enzymes can release fermentable sugars. Sulfuric acid has been widely used for biomass pretreatment, but high amount of degradation products usually occurred when using this method. To enhance accessibility to cellulose, we studied the performances of several dilute organic acid pretreatments of sugarcane bagasse and oil palm empty fruit bunch fiber. The results revealed that pretreatment with maleic acid yields the highest xylose and glucose release among other organic acids. The effects of concentration, duration of heating and heating temperature were further studied. Dilute maleic acid 1 % (w/w) pretreatment at 180 °C was the key to its viability as a substitute for sulfuric acid. Moreover, maleic acid did not seem to highly promote the formation of either furfural or 5-HMF in the liquid hydrolysate after pretreatment.

The interaction between negatively charged all-inorganic silicon quantum dots (Si QDs) and bovine serum albumin (BSA) is studied. It is shown that a small difference in the size of Si QDs affects the structure of Si QD–BSA composites significantly. When the diameter of Si QDs is 4 nm, a heterodimer (~20 nm) composed of one Si QD and one BSA molecule is a preferable and stable structure. On the other hand, when the diameter is 7 nm, the size of the composites increases to ~50 nm. The Si QD–BSA composites exhibit stable photoluminescence in the near-infrared range in phosphate-buffered saline.

Radiosensitizing therapy for cancer treatment that enhances the effect of existing radiation therapy and enables noninvasive therapy has attracted attention. In this study, to achieve target cell-s...

Pyruvate is a central metabolite for the biological production of various chemicals. In eukaryotes, pyruvate produced by glycolysis is used in conversion to ethanol and lactate and in anabolic metabolism in the cytosol, or is transported into the mitochondria for use as a substrate in the tricarboxylic acid (TCA) cycle. In this study, we focused on controlling pyruvate metabolism in aerobic microorganisms for the biological production of various chemicals. We successfully improved productivity by redirecting pyruvate metabolism in the aerobic filamentous fungus Aspergillus oryzae via the deletion of two genes that encode pyruvate decarboxylase and mitochondrial pyruvate carriers. Production of ethanol as a major byproduct was completely inhibited, and the limited translocation of pyruvate into the mitochondria shifted the metabolism from respiration for energy conversion to the effective production of lactate or 2,3-butandiole, even under aerobic conditions. Metabolomic and transcriptomic analyses showed an emphasis on glycolysis and a repressed TCA cycle. Although the dry mycelial weights of the deletion mutants were reduced compared with those of wild type, the titer and yields of the target products were drastically increased. In particular, the redirection of pyruvate metabolism shifted from anabolism for biomass production to catabolism for the production of target chemicals. Conclusively, our results indicate that the redirection of pyruvate metabolism is a useful strategy in the metabolic engineering of aerobic microorganisms.

Palm oil mill effluent (POME), a liquid waste from the palm oil industry, presents an alternative source for biodiesel production without interfering with food supply. This study attempted to produce biodiesel from untreated POME with aqueous ethanol using Thermomyces lanuginosus lipase as a biocatalyst. The effects of enzyme concentration, alcohol to oil ratio, and ethanol concentration were considered in the transesterification reaction. The optimum conditions were 2100 U lipase loading, 4 : 1 ethanol to oil molar ratio, and 45% (v/v) ethanol concentration at 40 °C reaction, and under 24 hours. The maximum fatty acid ethyl ester (FAEE) yield reached 97.43% (w/w) under these conditions. Integration of dilute ethanol for the conversion of POME to biodiesel could be promising as both feedstocks could be obtained from the same location, and thus reduce the logistical burden on biodiesel production.

As the third most plentiful biopolymer after other lignocellulosic derivates such as cellulose and hemicellulose, lignin carries abundant potential as a substitute for petroleum-based products. However, the efficient, practical, value-added product valorization of lignin remains quite challenging. Although several studies have reviewed the valorization of lignin by microorganisms, this present review covers recent studies on the valorization of lignin by employing yeast to obtain products such as single-cell oils (SCOs), enzymes, and other chemical compounds. The use of yeasts has been found to be suitable for the biological conversion of lignin and might provide new insights for future research to develop a yeast strain for lignin to produce other valuable chemical compounds.

Lignocellulosic biomass has great potential as an inedible feedstock for bioplastic synthesis, although its use is still limited compared to current edible feedstocks of glucose and starch. This review focuses on recent advances in the production of biopolymers and biomonomers from lignocellulosic feedstocks with downstream processing and chemical polymer syntheses. In microbial production, four routes composed of existing poly (lactic acid) and polyhydroxyalkanoates (PHAs) and the emerging biomonomers of itaconic acid and aromatic compounds were presented to review present challenges and future perspectives, focusing on the use of lignocellulosic feedstocks. Recently, advances in purification technologies decreased the number of processes and their environmental burden. Additionally, the unique structures and high-performance of emerging lignocellulose-based bioplastics have expanded the possibilities for the use of bioplastics. The sequence of processes provides insight into the emerging technologies that are needed for the practical use of bioplastics made from lignocellulosic biomass.

Nanoparticles (NPs) are useful for radiotherapy. Currently, efforts are underway globally for the development of novel titanium dioxide NPs (TiO2-NPs) that exhibit both contrast effects and anti-tumor effects. In this study, the image contrast properties of TiO2-NPs were evaluated using a clinical magnetic resonance imaging (MRI) system and a clinical computed tomography (CT) scanner, as the use of TiO2-NPs as an anti-cancer agent has been reported in several reports. An obvious difference in visualization was observed between the control and TiO2-NP samples on T2-weighted images. These results suggest that TiO2 can potentially be used as a novel theranostic drug with radiosensitizing ability and radiological diagnostic ability, through modification of chemical groups on its surface, and as a component of drug delivery systems.

An organosolv pretreatment consisting of an H2SO4/n-butanol biphasic system was designed to separate lignocellulosic biomass in three distinct phases: a cellulose-rich solid residue, hydrolyzed hem...

The long revolution of nitrile rubber in the rubber industries, especially for lightly crosslinked products has been reviewed in this paper. The advancement is remarkable from the first invention of acrylonitrile butadiene rubber and till date, the physical and chemical properties were elevated. Recent rubber chemists are mainly focus in modifying the nature of the synthetic latex based on butadiene and crosslinker to enhance the performability for rubber product to be used in broad range of applications. Besides, the different types of rubber had contribute to different sectors of usage due to their speciality properties. All belong to the effort of various types of crosslinking methods which making the liquid latex to suit in plenty of applications and usage from space shuttle (O-ring) to food hawker hygiene gloves. This paper summarized all sort of gloves products in comparison with nitrile rubber glove. Natural rubber is a superior latex glove which show high elasticity and tactility however not suitable for medical usage due to its protein allergic issues. Moreover, current development of natural rubber by synthesis through, in vitro and in vivo method, with the aim to produce equivalent properties of rubber product with lower materials cost. In fact, the glove manufacturing process have various parameters to monitor to produce a accepted quality of gloves. A quality of nitrile glove may need to pay close attention on latex compounding formulation and choice of the value-added process options. Metal ions crosslinker shows an exceptional cost and time saving for crosslinking especially on nitrile rubber. The discussion on the bonding characteristic which contribute for the tensile strength and durability of the nitrile rubber glove.

The biomass-derived source, low-cost and hydrophobicity/oleophilic advantages of activated carbon (AC) were explored for the immobilization of Aspergillus oryzae whole-cells expressing Fusarium heterosporum lipase. The adsorptive influence of AC favored growth of the cells into its porous interfaces with paralleled exterior dense film formation. Increasing AC weights hindered extracellular lipase activity. Cell aggregation of 0.34 ± 0.02 mg/BSP was found to be effective in catalyzing an industrially challenging feedstock (68.77% w/w free fatty acids, 20.48% w/w triglycerides) to 98% fatty acid methyl esters (FAME). In a comparative investigation with polyurethane as matrix, higher trans/esterification facilitation was observed with AC. Benefiting from the oleophilicity of AC; denaturation effect from methanol on the lipases was reduced. Surface characterization with FE-SEM, XPS and FT-IR evidenced effective cell-matrix adhesion and a retention of the AC’s intrinsic properties. The advantageous tribology o...

5-hydroxymethylfurfural (5-HMF), a key precursor to biochemicals has been produced from simple sugars. We report the production of 5-HMF directly from sustainable Chlorella sorokiniana cultivated in a saline medium supplied with CO2 as the sole carbon source. 43% carbohydrate and 16% lipid production in C. sorokiniana were achieved with an optimized CO2 concentration of 1%. 5-HMF yield of 52% was produced in a one-pot acid dehydration reactor at 150 °C in 2 h. 5-HMF yield from C. sorokiniana was almost a 40-fold higher than that from lignocellulose sugar cane bagasse – a highly abundant biomass. SEM images revealed that, the cells of C. sorokiniana could easily be broken in this one-pot reactor to liberate the built-up carbohydrate from the plastids of the cells for subsequent conversion to 5-HMF. The presence of lipids in C. sorokiniana was found to promote the conversion of starch to 5-HMF. Ultimately, the addition of LiCl was found to produce more than 2-fold 5-HMF from C. sorokiniana when compared to the absence of metal salts, with a 5-HMF selectivity of 98.7%. These findings suggest that, starch accumulating microalgae could be potential sustainable feedstock for 5-HMF production.

International audience; Xylitol is a major commodity chemical widely used in both the food and pharmaceutical industries. Although the worldwide demand for xylitol is constantly growing, its industrial production from purified D-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production of xylitol from biomass is a promising strategy to establish an environmentally friendly sustainable conversion process. In this study, xylitol was produced from woody Kraft pulp (KP) by using an engineered strain of Saccharomyces cerevisiae (YPH499-XR-BGL-XYL-XYN) expressing cytosolic xylose reductase (XR), along with beta-D-glucosidase (BGL), xylosidase (XYL) and xylanase (XYN) enzymes co-displayed on the cell surface. All these enzymes contributed to the consolidated bioprocessing of KP to xylitol with a yield of 2.3 g L-1 (28% conversion) after 96 hours, along with a significantly reduced amount of commercial enzymes required for pre-treatment (commercial hemicellulase cocktail (CHC), [CHC] = 0.02 g-DW per g). Further improvement of the cell surface display of XYL and XYN was obtained by using a SED1 "SSS" cassette, containing the coding sequences of the SED1 promoter, the SED1 secretion signal, and the SED1 anchoring domain, to generate the improved strain YPH499-XR-BGL-XYLsss-XYNsss. This improved strain showed a significantly enhanced xylitol production capacity reaching a yield of 3.7 g L-1 (44% conversion) after 96 hours. The cellulosic content of KP residues was also significantly increased, from 78% to 87% after 96 hours of fermentation, and nanofibrillation of KP residues was observed by scanning electron microscopy. Pre-treatment and fermentation were successfully performed as a proof of concept to further scale up bio-refinery industrial production of xylitol from lignocellulose.

A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-β-xylanase with a ricin-type β-trefoil type of domain and XYN11Ks_480 endo-1,4-β-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.