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The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (k cat /K m ) of 6.6×10 -2 mg -1 ml min -1 and 1.8×10 -2 mg -1 ml min -1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C 1 /C 4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: • Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. • Cello-oligosaccharides strongly downregulated the LPMO1 expression. • Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3., (© 2024. The Author(s).)

The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant Myceliophthora verrucosa was successfully cloned and heterologously expressed in Pichia pastoris . The expressed FE_5DR was purified using UNOsphere™ Q anion exchange chromatography column, exhibiting a homogeneous band of ~ 39 kDa. Its optimum temperature was determined to be 60 °C, with an optimal pH of 6.0. Additionally, the enzyme activity of FE_5DR was significantly enhanced by preincubation in a buffer containing Mg 2+ , Cu 2+ and Ca 2 metal ions. Enzyme kinetic parameters, computed from double reciprocal Lineweaver-Burk plots, yielded observed V max and K m values of 0.758 U/mg and 0.439 mM, respectively. Furthermore, the potential of custom-made cocktails comprising FE_5DR and benchmark cellulase derived from the developed mutant strain of Aspergillus allahabadii MAN 40, as well as the biorefinery-relevant lignocellulolytic enzyme Cellic CTec 3, resulted in improved saccharification of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw when compared to benchmark MAN 40 and Cellic CTec 3., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04013-7., Competing Interests: Conflict of interestIt is declared that no competing interest among all the authors exists., (© King Abdulaziz City for Science and Technology 2024. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

Lignocellulolytic enzymes from a novel Myceliophthora verrucosa (5DR) strain was found to potentiate the efficacy of benchmark cellulase during saccharification of acid/alkali treated bagasse by ~ 2.24 fold, indicating it to be an important source of auxiliary enzymes. The De-novo sequencing and analysis of M. verrucosa genome (31.7 Mb) revealed to encode for 7989 putative genes, representing a wide array of CAZymes (366) with a high proportions of auxiliary activity (AA) genes (76). The LC/MS QTOF based secretome analysis of M. verrucosa showed high abundance of glycosyl hydrolases and AA proteins with cellobiose dehydrogenase (CDH) (AA8), being the most prominent auxiliary protein. A gene coding for lytic polysaccharide monooxygenase (LPMO) was expressed in Pichia pastoris and CDH produced by M. verrucosa culture on rice straw based solidified medium were purified and characterized. The mass spectrometry of LPMO catalyzed hydrolytic products of avicel showed the release of both C1/C4 oxidized products, indicating it to be type-3. The lignocellulolytic cocktail comprising of in-house cellulase produced by Aspergillus allahabadii strain spiked with LPMO & CDH exhibited enhanced and better hydrolysis of mild alkali deacetylated (MAD) and unwashed acid pretreated rice straw slurry (UWAP), when compared to Cellic CTec3 at high substrate loading rate., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The present study reports a highly thermostable β-glucosidase (GH3) from Rasamsonia emersonii that was heterologously expressed in Pichia pastoris. Extracellular β-glucosidase was purified to homogeneity using single step affinity chromatography with molecular weight of ~ 110 kDa. Intriguingly, the purified enzyme displayed high tolerance to inhibitors mainly acetic acid, formic acid, ferulic acid, vanillin and 5-hydroxymethyl furfural at concentrations exceeding those present in acid steam pretreated rice straw slurry used for hydrolysis and subsequent fermentation in 2G ethanol plants. Characteristics of purified β-glucosidase revealed the optimal activity at 80 °C, pH 5.0 and displayed high thermostability over broad range of temperature 50-70 °C with maximum half-life of ~ 60 h at 50 °C, pH 5.0. The putative transglycosylation activity of β-glucosidase was appreciably enhanced in the presence of methanol as an acceptor. Using the transglycosylation ability of β-glucosidase, the generated low cost mixed glucose disaccharides resulted in the increased induction of R. emersonii cellulase under submerged fermentation. Scaling up the recombinant protein production at fermenter level using temporal feeding approach resulted in maximal β-glucosidase titres of 134,660 units/L. Furthermore, a developed custom made enzyme cocktail consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant β-glucosidase resulted in significantly enhanced hydrolysis of pretreated rice straw slurry from IOCL industries (India). Our results suggest multi-faceted β-glucosidase from R. emersonii can overcome obstacles mainly high cost associated enzyme production, inhibitors that impair the sugar yields and thermal inactivation of enzyme., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The glycoside hydrolase family 39 (GH39) proteins are renowned for their extremophilic and multifunctional enzymatic properties, yet the molecular mechanisms underpinning these unique characteristics continue to be an active subject of research. In this study, we introduce WsuXyn, a GH39 protein with a molecular weight of 58 kDa, originating from the thermophilic Geobacillus sp. WSUCF1. Previously reported for its exceptional thermostable β-xylosidase activity, WsuXyn has recently demonstrated a significant endoxylanase activity (3752 U·mg -1 ) against beechwood xylan, indicating towards its bifunctional nature. Physicochemical characterization revealed that WsuXyn exhibits optimal endoxylanase activity at 70 °C and pH 7.0. Thermal stability assessments revealed that the enzyme is resilient to elevated temperatures, with a half-life of 168 h. Key kinetic parameters highlight the exceptional catalytic efficiency and strong affinity of the protein for xylan substrate. Moreover, WsuXyn-mediated hydrolysis of beechwood xylan has achieved 77 % xylan conversion, with xylose as the primary product. Structural analysis, amalgamated with docking simulations, has revealed strong binding forces between xylotetraose and the protein, with key amino acid residues, including Glu278, Tyr230, Glu160, Gly202, Cys201, Glu324, and Tyr283, playing pivotal roles in these interactions. Therefore, WsuXyn holds a strong promise for biodegradation and value-added product generation through lignocellulosic biomass conversion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Rohit Rai reports financial support was provided by Science and Engineering Research Board. Rajesh K Sani reports financial support was provided by National Science Foundation. Rohit Rai reports financial support was provided by Lovely Professional University. Kian Mau Goh reports financial support was provided by University of Technology Malaysia., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed., Results: A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3., Conclusions: Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The global cannabis ( Cannabis sativa ) market was 17.7 billion in 2019 and is expected to reach up to 40.6 billion by 2024. Canada is the 2nd nation to legalize cannabis with a massive sale of $246.9 million in the year 2021. Waste cannabis biomass is managed using disposal strategies (i.e., incineration, aerobic/anaerobic digestion, composting, and shredding) that are not good enough for long-term environmental sustainability. On the other hand, greenhouse gas emissions and the rising demand for petroleum-based fuels pose a severe threat to the environment and the circular economy. Cannabis biomass can be used as a feedstock to produce various biofuels and biochemicals. Various research groups have reported production of ethanol 9.2-20.2 g/L, hydrogen 13.5 mmol/L, lipids 53.3%, biogas 12%, and biochar 34.6% from cannabis biomass. This review summarizes its legal and market status (production and consumption), the recent advancements in the lignocellulosic biomass (LCB) pre-treatment (deep eutectic solvents (DES), and ionic liquids (ILs) known as "green solvents") followed by enzymatic hydrolysis using glycosyl hydrolases (GHs) for the efficient conversion efficiency of pre-treated biomass. Recent advances in the bioconversion of hemp into oleochemicals, their challenges, and future perspectives are outlined. A comprehensive insight is provided on the trends and developments of metabolic engineering strategies to improve product yield. The thermochemical processing of disposed-off hemp lignin into bio-oil, bio-char, synthesis gas, and phenol is also discussed. Despite some progress, barricades still need to be met to commercialize advanced biofuels and compete with traditional fuels., Competing Interests: Conflict of interestThe authors declare no competing interests., (© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022.)

Two lignocellulolytic accessory enzymes, feruloyl esterase D (FAED_SCYTH) and β-xylosidase (XYL43B_SCYTH) were cloned and produced in the Pichia pastoris X33 as host. The molecular weight of recombinant enzymes FAED_SCYTH and XYL43B_SCYTH were ~ 31 and 40 kDa, respectively. FAED_SCYTH showed optimal activity at pH 6.0, 60 °C; and XYL43B_SCYTH at pH 7.0, 50 °C. FAED_SCYTH and XYL43B_SCYTH exhibited t 1/2 : 4 and 0.5 h, respectively (50 °C, pH 5.0). The β-xylosidase was bi-functional with pronounced activity against pNP-α-arabinofuranoside besides being highly xylose tolerant (retaining ~ 97% activity in the presence of 700 mM xylose). Cocktails prepared using these enzymes along with AA9 protein (PMO9D_SCYTH) and commercial cellulase CellicCTec2, showed improved hydrolysis of the pre-treated lignocellulosic biomass. Priming of pre-treated lignocellulosic biomass with these accessory enzymes was found to further enhance the hydrolytic potential of CellicCTec2 promising to reduce the enzyme load and cost required for obtaining sugars from biorefinery relevant pre-treated substrates., (Copyright © 2021 Elsevier Ltd. All rights reserved.)