Evaluation of endoglucanase and xylanase production by Aspergillus tamarii cultivated in agro-industrial lignocellulosic biomasses.

To better understand the production of enzymes of industrial interest from microorganisms with biotechnological potential using lignocellulosic biomass, we evaluated the production of endoglucanase and xylanase from Aspergillus tamarii. CAZymes domains were evaluated in the genome, and a screening of the enzymatic potential of A. tamarii in various agricultural biomasses was done. The enzymatic profile could be associated with the biomass complexity, with increased biomass recalcitrance yielding higher activity. A time-course profile defined 48 h of cultivation as the best period for cultivating A. tamarii in sugarcane bagasse reached 12.05 IU/mg for endoglucanase and 74.86 IU/mg for xylanase. Using 0.1% (w/v) tryptone as the only nitrogen source and 12 µmol/L CuSO4 addition had an overall positive effect on the enzymatic activity and protein production. A 22 factorial central composite design was used then to investigate the simultaneous influence of tryptone and CuSO4 on enzyme activity. Tryptone strongly affected enzymatic activity, decreasing endoglucanase activity but increasing xylanase activity. CuSO4 supplementation was advantageous for endoglucanases, increasing their activity, and it had a negative effect on xylanases. But overall, the experimental design increased the enzymatic activity of all biomasses used. For the clean cotton residue, the experimental design was able to reach the highest enzyme activity for endoglucanase and xylanase, with 1.195 IU/mL and 6.353 IU/mL, respectively. More experimental studies are required to investigate how the biomass induction effect impacts enzyme production.

The enzyme interactome concept in filamentous fungi linked to biomass valorization.

Biomass represents an abundant and inexpensive source of sugars and aromatic compounds that can be used as raw materials for conversion into value-added bioproducts. Filamentous fungi are sources of plant cell wall degrading enzymes in nature. Understanding the interactions between enzymes is crucial for optimizing biomass degradation processes. Herein, the concept of the interactome is presented as a holistic approach that depicts the interactions among enzymes, substrates, metabolites, and inhibitors. The interactome encompasses several stages of biomass degradation, starting with the sensing of the substrate and the subsequent synthesis of hydrolytic and oxidative enzymes (fungus-substrate interaction). Enzyme-enzyme interactions are exemplified in the complex processes of lignocellulosic biomass degradation. The enzyme-substrate-metabolite-inhibitor interaction also provides a better understanding of biomass conversion, allowing bioproduct production from recalcitrant agro-industrial residues, thus bringing greater value to residual biomass. Finally, technological applications are presented for optimizing the interactome at various levels.

Polymer ultrastructure governs AA9 lytic polysaccharide monooxygenases functionalization and deconstruction efficacy on cellulose nano-crystals.

Lytic Polysaccharide MonoOxygenases display great variability towards cellulose ultrastructure while performing oxidative functionalization of the polymers. Aiming at employing AA9-LPMOs for isolation of cellulose nano-crystals (CNCs), the ratio between functionalization/crystalline degradation became a crucial parameter. Here are reported the constraints posed by the substrate ultrastructure on the activity of seven different AA9 LPMOs representative of various regioselectivity and substrate affinity: TtAA9E, TaAA9A, PcAA9D, MtAA9A, MtAA9D, MtAA9I-CBM and MtAA9J. The substrate crystallinity and dry matter loading greatly affected the seven AA9s in an enzyme-specific manner, impacting their efficiency for CNCs functionalization purposes. X-ray diffraction pattern analyses were used to assess the cracking efficacy of the enzymatic treatment to de-crystallize CNCs, revealing that those AA9s with minor efficiency in releasing oligosaccharides resulted instead the most disruptive towards the crystal lattice and in reducing the particle sizes. These non-catalytic effects were found comparable with the one caused by the expansin BsEXLX1 enzyme.

A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.

A review on the potential of filamentous fungi for microbial self-healing of concrete.

Concrete is the most used construction material worldwide due to its abundant availability and inherent ease of manufacturing and application. However, the material bears several drawbacks such as the high susceptibility for crack formation, leading to reinforcement corrosion and structural degradation. Extensive research has therefore been performed on the use of microorganisms for biologically mediated self-healing of concrete by means of CaCO3 precipitation. Recently, filamentous fungi have been recognized as high-potential microorganisms for this application as their hyphae grow in an interwoven three-dimensional network which serves as nucleation site for CaCO3 precipitation to heal the crack. This potential is corroborated by the current state of the art on fungi-mediated self-healing concrete, which is not yet extensive but valuable to direct further research. In this review, we aim to broaden the perspectives on the use of fungi for concrete self-healing applications by first summarizing the major progress made in the field of microbial self-healing of concrete and then discussing pioneering work that has been done with fungi. Starting from insights and hypotheses on the types and principles of biomineralization that occur during microbial self-healing, novel potentially promising candidate species are proposed based on their abilities to promote CaCO3 formation or to survive in extreme conditions that are relevant for concrete. Additionally, an overview will be provided on the challenges, knowledge gaps and future perspectives in the field of fungi-mediated self-healing concrete.

LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea.

Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

Characterization of two family AA9 LPMOs from Aspergillus tamarii with distinct activities on xyloglucan reveals structural differences linked to cleavage specificity.

Aspergillus tamarii grows abundantly in naturally composting waste fibers of the textile industry and has a great potential in biomass decomposition. Amongst the key (hemi)cellulose-active enzymes in the secretomes of biomass-degrading fungi are the lytic polysaccharide monooxygenases (LPMOs). By catalyzing oxidative cleavage of glycoside bonds, LPMOs promote the activity of other lignocellulose-degrading enzymes. Here, we analyzed the catalytic potential of two of the seven AA9-type LPMOs that were detected in recently published transcriptome data for A. tamarii, namely AtAA9A and AtAA9B. Analysis of products generated from cellulose revealed that AtAA9A is a C4-oxidizing enzyme, whereas AtAA9B yielded a mixture of C1- and C4-oxidized products. AtAA9A was also active on cellopentaose and cellohexaose. Both enzymes also cleaved the ß-(1→4)-glucan backbone of tamarind xyloglucan, but with different cleavage patterns. AtAA9A cleaved the xyloglucan backbone only next to unsubstituted glucosyl units, whereas AtAA9B yielded product profiles indicating that it can cleave the xyloglucan backbone irrespective of substitutions. Building on these new results and on the expanding catalog of xyloglucan- and oligosaccharide-active AA9 LPMOs, we discuss possible structural properties that could underlie the observed functional differences. The results corroborate evidence that filamentous fungi have evolved AA9 LPMOs with distinct substrate specificities and regioselectivities, which likely have complementary functions during biomass degradation.

Xylanase from Aspergillus tamarii shows different kinetic parameters and substrate specificity in the presence of ferulic acid.

A 22 kDa xylanase (AtXyl1) from Aspergillus tamarii was purified by two chromatographic steps and presented preference for oat spelt (OSX), birchwood (BrX) and beechwood (BeX) xylans respectively, as substrates. AtXyl1 displays the highest activity at pH 5.5 and 55 °C and showed tolerance over a range of different phenolic compounds. The activity of AtXyl1 was not inhibited when the enzyme was incubated with ferulic acid (FA) using OSX or BrX as substrate. On the other hand, the incubation of AtXyl1 with BeX and FA resulted in an increase in enzyme activity. The molecular docking of a GH11 xylanase from Aspergillus niger with FA showed the preference for binding within the catalytic site. The position of FA was based on the presence or absence of a complexed substrate. When the enzyme from A. niger was docked in the absence of xylan in its crystal structure, FA interacted with Tyr164 and a water molecule. For the enzyme socked with xylo-oligosaccharides, FA interacted with Ser94, Tyr89 and the xylo-oligosaccharide present in the catalytic site. Thermodynamic parameters from the reaction of AtXyl1 with different xylans and FA indicate that FA can cause a conformational change in the enzyme, and this can influence the substrate fitting and makes the enzyme tolerant or active toward the substrate. Our findings suggest that enzyme activation or tolerance to phenolic compounds can be correlated to subtle changes in enzyme conformation due to the presence of the phenolic compound.

Fungal lytic polysaccharide monooxygenases from family AA9: Recent developments and application in lignocelullose breakdown.

Fungal lytic polysaccharide monooxygenases (LPMOs) from family AA9 are oxidative enzymes that, in the past few years, have changed the paradigm of cellulose conversion. They are key factor in the lignocellulose breakdown and are widely distributed among fungi. This review focuses on LPMOs from family AA9 and gives an overview of recent discoveries relative to their structure, mode of action, and synergism with other enzymes. Finally, several aspects regarding their potential applications toward deconstruction of biomass and biorefinery processes are discussed.

Time-of-day effect on a food-induced conditioned place preference task in monkeys.

Time can be an important contextual cue for cognitive performance, with implications for reward-associated learned behaviors such as (drug and food) addiction. So, we analyzed: (1) if marmoset monkeys develop a place preference that is conditioned to previous pairings with a highly-palatable food reward; (2) if the response is strongest when training and testing times match - time stamp effect; and (3) if there is an optimal time of the day (morning vs. afternoon) when this preference occurs - time-of-day effect. Subjects were first habituated to a two-compartment conditioned-place-preference (CPP) box. Then, during six training sessions held either in the morning or afternoon, a mixture of jellybeans and live mealworms was made available in a specific compartment. Marmosets were subsequently tested for preferring the food-paired context at the circadian time that either matched or was different from that of training. Compared to baseline levels, only subjects trained and tested in the afternoon made significantly longer and more frequent visits to the food-paired context and with a shorter latency to first entry. Thus, highly-palatable food rewards induced a CPP response. This behavior was exhibited only when training and testing times overlapped and during a restricted circadian timeframe (afternoon), consistent with a time-stamp and time-of-day effect, respectively. In this case, time may have been an internal circadian contextual cue. Whether due to circadian-mediated oscillations in memory and/or reward processes, such findings may be applied to addiction and other learned behaviors.