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16 p.-6 fig.-2 tab., The synthesis of customized glycoconjugates constitutes a major goal for biocatalysis. To this end, engineered glycosidases have received great attention and, among them, thioglycoligases have proved useful to connect carbohydrates to non-sugar acceptors. However, hitherto the scope of these biocatalysts was considered limited to strong nucleophilic acceptors. Based on the particularities of the GH3 glycosidase family active site, we hypothesized that converting a suitable member into a thioglycoligase could boost the acceptor range. Herein we show the engineering of an acidophilic fungal β-xylosidase into a thioglycoligase with broad acceptor promiscuity. The mutant enzyme displays the ability to form O-, N-, S- and Se- glycosides together with sugar esters and phosphoesters with conversion yields from moderate to high. Analyses also indicate that the pKa of the target compound was the main factor to determine its suitability as glycosylation acceptor. These results expand on the glycoconjugate portfolio attainable through biocatalysis., This work has been funded by Projects RTI2018-093683-Β-I00, RTI2018-094751-B-C22, and PID2019-107476GB-I00 from MCIU/AEI/FEDER, UE, and S2018/EMT-4459 from Comunidad de Madrid

The study of endoxylanases as catalysts to valorize hemicellulosic residues and to obtain glycosides with improved properties is a topic of great industrial interest. In this work, a GH10 β-1,4-endoxylanase (XynSOS), from the ascomycetous fungus Talaromyces amestolkiae, has been het- erologously produced in Pichia pastoris, purified, and characterized. rXynSOS is a highly glycosylated monomeric enzyme of 53 kDa that contains a functional CBM1 domain and shows its optimal activity on azurine cross-linked (AZCL)–beechwood xylan at 70 ◦C and pH 5. Substrate specificity and kinetic studies confirmed its versatility and high affinity for beechwood xylan and wheat arabi- noxylan. Moreover, rXynSOS was capable of transglycosylating phenolic compounds, although with low efficiencies. For expanding its synthetic capacity, a glycosynthase variant of rXynSOS was developed by directed mutagenesis, replacing its nucleophile catalytic residue E236 by a glycine (rXynSOS-E236G). This novel glycosynthase was able to synthesize β-1,4-xylooligosaccharides (XOS) of different lengths (four, six, eight, and ten xylose units), which are known to be emerging prebiotics. rXynSOS-E236G was also much more active than the native enzyme in the glycosylation of a broad range of phenolic compounds with antioxidant properties. The interesting capabilities of rXynSOS and its glycosynthase variant make them promising tools for biotechnological application., This research was funded by the MICIU/AEI/FEDER [RTI2018-093683-B-I00, RTI2018- 094751-B-C22, PID2019-107476GB-I00], Comunidad de Madrid [RETOPROSOST-2-CM P2018/EMT- 4459], and CIBERES (an initiative from the Spanish Institute of Health Carlos I

20 p.-7 fig.-1 tab., The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated, This work has been funded by Projects RTI2018-093683-Β-I00 and PID2019-105838RB-C31 from the Spanish Ministry of Science and Innovation (MCIU/AEI/FEDER, UE), S2018/EMT-4459 from the Comunidad de Madrid, Grant and EU-H2020 H2020-NMBP-TR-IND-2018-2020.

52 p.-2 fig.-2 tab., The term hemicellulose groups different polysaccharides with heterogeneous structures, mannans, xyloglucans, mixed-linkage β-glucans and xylans, which differ in their backbone and branches, and in the type and distribution of glycosidic linkages. The enzymatic degradation of these complex polymers requires the concerted action of multiple hemicellulases and auxiliary enzymes. Most commercial enzymes are produced by Trichoderma and Aspergillus species, but recent studies have disclosed Penicillium and Talaromyces as promising sources of hemicellulases. In this review, we summarize the current knowledge on the hemicellulolytic system of these genera, and the role of hemicellulases in the disruption and synthesis of glycosidic bonds. In both cases, the enzymes from Penicillium and Talaromyces represent an interesting alternative for valorization of lignocellulosic biomass in the current framework of circular economy., This work was funded by MICIU/AEI/FEDER (GLYSUS RTI2018-093683-B-I00) and Comunidad de Madrid (RETOPROSOST-2-CM, P2018/EMT-4459) projects.

As the main decomposers and recyclers in nature, fungi secrete complex mixtures of extracellular enzymes for degradation of plant biomass, which is essential for mobilization of the organic carbon fixed by the photosynthesis in vegetal cells. Biotechnology can emulate the closed natural biological cycles, using lignocellulosic biomass as a renewable resource and lignocellulolytic fungal enzymes as catalysts to sustainably produce consumer goods. Cellulose and hemicellulose are the major polysaccharides on Earth, and the main enzymes involved in their hydrolytic depolymerization are cellulases (endoglucanases, cellobiohydrolases, and β-glucosidases) and hemicellulases (mainly endoxylanases and β-xylosidases). This work will focus on the enzymes secreted by the filamentous ascomycete Talaromyces amestolkiae and on some of their biotechnological applications. Their excellent hydrolytic activity was demonstrated by the partial degradation of xylans to prebiotic oligosaccharides by the endoxylanase XynN, or by the saccharification of lignocellulosic wastes to monosaccharides (fermentable to ethanol) either by the whole secretomes or by isolated enzymes used as supplements of commercial cocktails. However, apart from their expected hydrolytic activity, some of the β-glycosidases produced by this strain catalyze the transfer of a sugar molecule to specific aglycons by transglycosylation. As the synthesis of customized glycoconjugates is a major goal for biocatalysis, mutant variants of the β-xyloxidase BxTW1 and the ß-glucosidases BGL-1 and BGL-2 were obtained by directed mutagenesis, substantially improving the regioselective production yields of bioactive glycosides since they showed reduced or null hydrolytic activity., This work was funded by projects GLYSUS RTI2018-093683-B-I00 (MICIU/AEI/FEDER) and RETOPROSOST-2-CM, S2018/EMT-4459 (Comunidad de Madrid). Preliminary investigations were funded by MINECO or Comunidad de Madrid in collaboration with Abengoa Research and Bioenergy.

[Background] Genomic and proteomic analysis are potent tools for metabolic characterization of microorganisms. Although cellulose usually triggers cellulase production in cellulolytic fungi, the secretion of the different enzymes involved in polymer conversion is subjected to different factors, depending on growth conditions. These enzymes are key factors in biomass exploitation for second generation bioethanol production. Although highly effective commercial cocktails are available, they are usually deficient for β-glucosidase activity, and genera like Penicillium and Talaromyces are being explored for its production., [Results] This article presents the description of Talaromyces amestolkiae as a cellulase-producer fungus that secretes high levels of β-glucosidase. β-1,4-endoglucanase, exoglucanase, and β-glucosidase activities were quantified in the presence of different carbon sources. Although the two first activities were only induced with cellulosic substrates, β-glucosidase levels were similar in all carbon sources tested. Sequencing and analysis of the genome of this fungus revealed multiple genes encoding β-glucosidases. Extracellular proteome analysis showed different induction patterns. In all conditions assayed, glycosyl hydrolases were the most abundant proteins in the supernatants, albeit the ratio of the diverse enzymes from this family depended on the carbon source. At least two different β-glucosidases have been identified in this work: one is induced by cellulose and the other one is carbon source-independent. The crudes induced by Avicel and glucose were independently used as supplements for saccharification of slurry from acid-catalyzed steam-exploded wheat straw, obtaining the highest yields of fermentable glucose using crudes induced by cellulose., [Conclusions] The genome of T. amestolkiae contains several genes encoding β-glucosidases and the fungus secretes high levels of this activity, regardless of the carbon source availability, although its production is repressed by glucose. Two main different β-glucosidases have been identified from proteomic shotgun analysis. One of them is produced under different carbon sources, while the other is induced in cellulosic substrates and is a good supplement to Celluclast in saccharification of pretreated wheat straw., This work has been funded by projects RTC-2014-1777-3 and BIO2015-68387-R from MINECO and S2013/MAE-2907 from Comunidad de Madrid. M. Nieto and J.A. Méndez thank their fellowships, FPU of the MINECO and Tatiana Pérez de Guzmán el Bueno Foundation, respectively.

[Background] Transglycosylation represents one of the most promising approaches for obtaining novel glycosides, and plant phenols and polyphenols are emerging as one of the best targets for creating new molecules with enhanced capacities. These compounds can be found in diet and exhibit a wide range of bioactivities, such as antioxidant, antihypertensive, antitumor, neuroprotective and anti-inflammatory, and the eco-friendly synthesis of glycosides from these molecules can be a suitable alternative for increasing their health benefits., [Results] Transglycosylation experiments were carried out using different GH3 β-glucosidases from the fungus Talaromyces amestolkiae. After a first screening with a wide variety of potential transglycosylation acceptors, mono-glucosylated derivatives of hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were detected. The reaction products were analyzed by thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry. Hydroxytyrosol and vanillyl alcohol were selected as the best options for transglycosylation optimization, with a final conversion yield of 13.8 and 19% of hydroxytyrosol and vanillin glucosides, respectively. NMR analysis confirmed the structures of these compounds. The evaluation of the biological effect of these glucosides using models of breast cancer cells, showed an enhancement in the anti-proliferative capacity of the vanillin derivative, and an improved safety profile of both glucosides., [Conclusions] GH3 β-glucosidases from T. amestolkiae expressed in P. pastoris were able to transglycosylate a wide variety of acceptors. Between them, phenolic molecules like hydroxytyrosol, vanillin alcohol, 4-hydroxybenzyl alcohol, and hydroquinone were the most suitable for its interesting biological properties. The glycosides of hydroxytyrosol and vanillin were tested, and they improved the biological activities of the original aglycons on breast cancer cells.

[Background] Currently, industrial societies are seeking for green alternatives to conventional chemical synthesis. This demand has merged with the efforts to convert lignocellulosic biomass into value-added products. In this context, xylan, as one of main components of lignocellulose, has emerged as a raw material with high potential for advancing towards a sustainable economy., [Results] In this study, the recombinant endoxylanase rXynM from the ascomycete Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris and used as one of the catalysts of an enzyme cascade developed to synthesize the antiproliferative 2-(6-hydroxynaphthyl) β-d-xylopyranoside, by transglycosylation of 2,6-dihydroxynaphthalene. The approach combines the use of two fungal xylanolytic enzymes, rXynM and the β-xylosidase rBxTW1 from the same fungus, with the cost-effective substrate xylan. The reaction conditions for the cascade were optimized by a Central Composite Design. Maximal productions of 0.59 and 0.38 g/L were reached using beechwood xylan and birchwood xylan, respectively. For comparison, xylans from other sources were tested in the same reaction, suggesting that a specific optimization is required for each xylan variety. The results obtained using this enzyme cascade and xylan were similar or better to those previously reported for a single catalyst and xylobiose, an expensive sugar donor., [Conclusions] Beechwood and birchwood xylan, two polysaccharides easily available from biomass, were used in a novel enzyme cascade to synthetize an antiproliferative agent. The approach represents a green alternative to the conventional chemical synthesis of 2-(6-hydroxynaphthyl) β-d-xylopyranoside using a cost-effective substrate. The work highlights the role of xylan as a raw material for producing value-added products and the potential of fungal xylanolytic enzymes in the biomass conversion., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work has been funded by projects BIO2015-68387-R, CTQ2015-64597-C2-2-P and RTI2018-093683-B-I00 from MINECO and S2018/EMT-4459 from Comunidad de Madrid. M. Nieto-Domínguez and Beatriz Fernández de Toro thank the MINECO for an FPU and an FPI fellowship, respectively.

49 p.-7 fig.-3 tab., This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu2+ tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and Km and Vmax values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-D-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries., This work was carried out with funding from the MINECO projects PRI-PIBAR-2011-1402 and RTC-2014-1777-3 and the Comunidad de Madrid Program RETO-PROSOT S2013/MAE-2907.

The eco-friendly synthesis of non-natural glycosides from different phenolic antioxidants was carried out using a fungal β-xylosidase to evaluate changes in their bioactivities. Xylosides from hydroquinone and catechol were successfully formed, although the best results were obtained for hydroxytyrosol, the main antioxidant from olive oil. The formation of the new products was followed by thin-layer chromatography, liquid chromatography, and mass spectrometry. The hydroxytyrosyl xyloside was analyzed in more detail, to maximize its production and evaluate the effect of glycosylation on some hydroxytyrosol properties. The synthesis was optimized up to the highest production reported for a hydroxytyrosyl glycoside. The structure of this compound was solved by two-dimensional nuclear magnetic resonance and identified as 3,4-dihydroxyphenyl-ethyl-O-β-d-xylopyranoside. Evaluation of its biological effect showed an enhancement of both its neuroprotective capacity and its ability to ameliorate intracellular levels of reactive oxygen species.

34 p.-4 fig.-1 tab., A fungal endoxylanase belonging to the glycoside hydrolase gene family 11 (GH11) was obtained from the ascomycete Talaromyces amestolkiae. The enzyme was purified, characterized and used to produce a mixture of xylooligosaccharides (XOS) from birchwood xylan. A notable yield of neutral XOS was obtained (28.8%) upon enzyme treatment and the mixture contained a negligible amount of xylose, having xylobiose, xylotriose and xylotetraose as its main components. The prebiotic potential of this mixture was demonstrated upon analyzing the variations in microorganisms’ composition and organic acids profile in breast-fed child faeces fermentations. The strong production of acetic and lactic acid, the decrease of potentially pathogenic bacteria and the increase of bifidobacteria, and possible beneficial commensals, confirmed the prebiotic value of these xylooligosaccharides., This work was carried out with funding from projects BIO2013-48779-C4-1-R, BIO2016-76601-C3-1-R and RTC-2014-1777-3 from MINECO, BIO2015-68387-R from MINECO/FEDER and S2013/MAE-2972 from Comunidad de Madrid.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al., [Background]: Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal ß-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated. [Results]: The ß-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of ß-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-ß-d-xylopyranoside of 0.20 mM and 69.3 s¿1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) ß-d-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR. [Conclusions]: A ß-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) ß-d-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest., This work was carried out with funding from projects BIO2015-68387-R, RTC-2014-1777-3 and CTQ2015-64597-C2 from MINECO and S2013/MAE2972 from Comunidad de Madrid, as well as from the Natural Sciences and Engineering Research Council of Canada. M. Nieto-Domínguez thanks the MINECO for an FPU fellowship., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).