WO2021199005A1 - Enzyme overexpression for optimized lignocellulosic degradation - Google Patents

Abstract

The invention relates to lignocellulosic degradation. In particular, the invention improves production of cellulolytic enzymes and enzyme complexes for degrading lignocellulosic materials through overexpression of critical enzymes within a recombinant strain of Penicillium funiculosum. In various embodiments, the invention provides transformed fungal strains, integration cassettes, plasmids, and methods for producing a transformed fungal strain – all involving enzyme overexpression for optimized lignocellulosic degradation.

Description

Enzyme Overexpression for Optimized Lignocellulosic Degradation

Field

[001] The present invention relates to lignocellulosic degradation. In particular, the invention improves production of cellulolytic enzymes and enzyme complexes for degrading lignocellulosic materials through overexpression of critical enzymes within a recombinant strain of Penicillium funiculosum.

[002] Conversion of biomass to biofuel is a key focus for industrial and scientific research. Production of biofuels is predicted to serve as a sustainable, low-carbon footprint energy source, that is compatible with existing fuel combustion technologies. Lignocellulosic biomass is believed to present a significant source of sugars that can be fermented to form biofuels such as fuel ethanol. Lignocellulosic biomass includes inter alia hardwoods, softwoods, grasses and agricultural residues.

[003] Conversion of cellulose (from lignocellulosic biomass) to ethanol involves hydrolysis of cellulose molecules to sugars - and subsequent fermentation of said sugars to ethanol. Several microorganisms and fungi are known to secrete enzymes that are able to hydrolyze cellulose. The search for enzymes that are cheaper to produce and more of cellulosic efficient in terms of hydrolyzing efficiencies has resulted in increasingly greater attention on fungi as the subject of bioprospecting research. Such research has resulted in identification of filamentous fungi as an important potential source of lignocellulosic enzymes (cellulases) for hydrolyzing recalcitrant lignocellulosic materials into fermentable sugars. In addition, unprecedented protein secretion abilities of fungi make them a preferred choice for bioprospecting as a source for superior cellulolytic enzymes.

[004] Secretomes of many fungal genus such as Trichoderma, Aspergillus, Neurospora and PeniciUium have been studied for their ability to produce cellulolytic enzymes. While the secretome from T. reesei is commercially used for cellulase production, complete saccharification biomass by its secretome is usually not achievable. This is because the secretome of T. reesei, under cellulase-inducing conditions, secretes mainly cellobiohydrolases (about 85%) while other cellulase components such as b- glucosidase and endoglucanase which participate in biomass hydrolysis are secreted in lower amount and therefore has to be produced in other fungal sources to complement the cellobiohydrolase in commercial cocktails (see (i) Ike, M., Park, )., Tabuse, M., Tokuyasu, K., 2010. Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei. Appl. Microbiol. Biotechnol. 87, 2059-2066. https://doi.org/10.1007/s00253-010-2683-3; (ii) Park, J.-Y., Arakane, M., Shiroma, R., Ike, M., Tokuyasu, K., n.d. Culm in Rice Straw as a New Source for Sugar Recovery via Enzymatic Saccharification, https://doi.org/10.1271/bbb.90535; (iii) Peterson, R., Nevalainen, H., n.d. Trichoderma reesei RUT-C30 - thirty years of strain improvement. https://doi.Org/10.1099/mic.0.054031-0).

[005] Penicillium funiculosum ( P . funiculosum ) has previously been identified as capable of generating secretomes having significantly advantageous biomass hydrolyzing potential (see Ogunmolu, F.E., Kaur, 1., Gupta, M., Bashir, Z., Pasari, N., Yazdani, S.S., 2015. Proteomics Insights into the Biomass Hydrolysis Potentials of a Hypercellulolytic Fungus Penicillium funiculosum. J. Proteome Res. 14, 4342-4358. https://doi.org/10.1021/acs.jproteome.5b00542) - particularly in terms of efficacy of conversion of recalcitrant biomass into fermentable sugars. In particular, a strain of P. funiculosum on deposit with the National Collection of Industrial Microorganisms (NC1M), India and identified by accession no. 1228 (hereinafter “NC1M 1228”) has been found to significantly outperform commercially available products in terms of cellulolytic activity of its secretome. Proteomic studies have indicated that approximately 58% of the total proteins secreted by P. funiculosum under cellulose inducing conditions are carbohydrate-active enzymes (CAZymes) - which are active towards lignocellulosic biomass.

[006] Yet further, a novel recombinant strain of P. funiculosum NC1M1228 has been developed by replacing a Mig1¹³⁴ allele with a null allele Mig1⁸⁸, resulting in a strain named P. funiculosum Mig1⁸⁸ (or PfMig1⁸⁸) - which has been deposited at depository institution Microbial Type Culture Collection (MTCC) and Gene Bank, Chandigarh, India and has been granted accession No. MTCC 25141. [007] It has been discovered, when saccharification potential of the PƒMig1⁸⁸ secretome was evaluated towards different chemically pretreated cellulosic biomass, that PƒMig1⁸⁸ secretome could hydrolyze ~75% of total cellulose into glucose from a nitric acid and ammonium hydroxide pre-treated biomass in 72 hr at 30 mg/g DBW (dry biomass weight) and 20% biomass loading could achieve a benchmark of ~70% hydrolysis of sugars using 20 mg/g DBW at 20% solid loading (see Klein-Marcuschamer, D., Simmons, B.A., Blanch, H.W., 2011. Techno-economic analysis of a lignocellulosic ethanol biorefinery with ionic liquid pre-treatment. Biofuels, Bioprod. Biorefining 5, 562- 569. https://doi.org/10.1002/bbb.303). Contrastingly, pretreatment with either nitric acid or ammonium hydroxide alone resulted in relatively lower release of 60% and 63% sugars respectively. This indicates that the crystallinity of cellulose in the single-step pre- treatment was relatively higher as compared to the two-step pretreatment, eventually leading to lower sugar release.

[008] There is accordingly a need for improving the cellulase content of the secretome of PƒMig1⁸⁸, to improve the digestibility of crystalline cellulose - and to improve the resulting saccharification of cellulosic biomass.

Summary

[009] The invention enables enhanced saccharification of cellulosic biomass (and correspondingly improved lignocellulosic degradation) through a novel recombinant fungal strain developed by overexpressing lytic polysaccharide monooxygenase (LPMO) and Cellobiohydrolase 1 (CBH1) within recombinant P. funiculosum Mig1⁸⁸ (MTCC 25141). The invention also provides novel methods, plasmids and integration cassettes for generating the novel fungal strains.

[0010] In an embodiment the invention provides a transformed fungal strain of Penicillium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25371. [0011] In another embodiment, the invention comprises a fungal strain for production of cellulolytic enzymes or cellulolytic enzyme complexes, comprising a transformed fungal strain of Penicillium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25371.

[0012] In a further embodiment, the invention provides a transformed fungal strain of Penicillium funiculosum, comprising the nucleic acid sequence of SEQ ID No. 28.

[0013] The invention also provides a fungal strain for production of cellulolytic enzymes or cellulolytic enzyme complexes, comprising a transformed strain of PeniciUium funiculosum, wherein said transformed fungal strain of Penicillium funiculosum comprises a nucleic acid sequence of SEQ ID No. 28.

[0014] In a specific embodiment, the invention provides an integration cassette for producing a transformed fungal strain of PeniciUium funiculosum, said integration cassette comprising a nucleic acid sequence of SEQ ID No. 28.

[0015] The invention also provides a plasmid for producing a transformed fungal strain of PeniciUium funiculosum, said plasmid comprising a nucleic acid sequence of SEQ ID No. 26.

[0016] The invention provides a method for producing a transformed fungal strain of PeniciUium funiculosum comprising the nucleic acid sequence of SEQ ID No. 28, the method comprising the steps of (i) synthesizing a plasmid comprising a nucleic acid sequence of SEQ ID No. 26, and (ii) synthesizing a transformant by exposing a fungal strain of PeniciUium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25141, to the synthesized plasmid.

[0017] In a specific embodiment of the method, synthesizing the transformant comprises transforming to the fungal strain of PeniciUium funiculosum, a plasmid comprising a nucleic acid sequence of SEQ ID No. 26.

[0018] In another embodiment of the method, synthesizing the transformant comprises integrating to the fungal strain of PeniciUium funiculosum, an integration cassette comprising a nucleic acid sequence of SEQ ID No. 28. [0019] In a particular method embodiment, wherein the transformant is synthesized based on agrobacterium mediated fungal transformation method. Brief Description of the Accompanying Drawings

[0020] Figure 1 is a vector map for a pOAO1 vector generated for overexpression of P. funiculosum LPMO gene in PƒMig1⁸⁸ fungal strain. [0021] Figure 2 is a vector map for a pOAO2 vector generated for overexpression of

P. funiculosum CBH1 gene in PƒMig1⁸⁸ fungal strain.

[0022] Figure 3 is a vector map for a pOAO5 vector generated for overexpression of LPMO and CBH1 genes in PƒMig1⁸⁸ fungal strain.

[0023] Figure 4 illustrates schematic representation of P. funiculosum LPMO gene cassette used for overexpression.

[0024] Figure 5 shows the restriction digestion analysis of pBIF (1) and pOAO1 (2) by BamHI restriction enzyme.

[0025] Figure 6 shows the transformants of pOAO1 after agrobacterium-mediated transformation in PƒMig1⁸⁸ fungal strain. [0026] Figure 7 shows the integration of P. funiculosum LPMO in the genome of PƒMig1⁸⁸ transformants by PCR.

[0027] Figure 8 is the Southern blot of transformants confirmed by PCR. [0028] Figure 9 shows the results for LPMO and FPU activities in the fermentation broth of PƒMig1⁸⁸ and five transformants.

[0029] Figure 10 illustrates the construction cassette for dual overexpression of LPMO and CBH1 genes in P. funiculosum. [0030] Figure 11 describes the restriction digestion analysis of pBIF (1) and pOAO3 (2) by AƒI II and Spel. [0031] Figure 12 shows the transformants of pOAO3 after agrobacterium-mediated transformation in PƒMig1⁸⁸.

[0032] Figure 13 shows the integration of LPMO/CBH1 cassette in the genome of PƒMig1⁸⁸ transformants by PCR.

[0033] Figure 14 shows the Southern blot of transformants confirmed by PCR.

[0034] Figure 15 shows the enzymatic profile of the overexpressed enzymes in the fermentation broth of PƒMig1⁸⁸ and six transformants.

[0035] Figure 16 illustrates the growth and phenotypic characterization of PƒMig1⁸⁸ strain and the corresponding transformants of PƒOAO1, PƒOAO2 and PƒOAO3 on different polymeric carbon sources after 5 days of incubation. [0036] Figure 17 describes the transcriptional expression of LPMO, cellobiohydrolase, endoglucanase, b-glucosidase, and xylanase in NC1M1228, PƒMig1⁸⁸ and all engineered strains measured by quantitative real-time PCR after growing for 48 h in the presence of 4% Avicel. [0037] Figure 18 shows the CBH1 activity as determined using Avicel as the substrate.

[0038] Figure 19 shows the LPMO activity as determined using Amplex red. [0039] Figure 20 shows the β-glucosidase activity using pNPG as substrate.

[0040] Figure 21 shows the overall cellulase activity on filter paper. [0041] Figure 22 is the endoglucanase activity determined using CMC as the substrate.

[0042] Figure 23 shows the xylanase activity measured using beech wood xylan as the substrate.

[0043] Figure 24 is the total secreted proteins of all the strains.

[0044] Figure 25 shows the protein profile for the secretome of all strains separated on SDS-PAGE.

[0045] Figure 26 shows the MUG-zymogram assay for identification of b-glucosidase in the secretome of all strains.

[0046] Figure 27 shows the time course profile for the saccharification of nitric acid pre-treated wheat straw by the secretome of NC1M1228, PƒMig1⁸⁸ and the all engineered strains at 20% solid loading and protein concentration of 30 mg/g biomass.

[0047] Figure 28 shows the total fermentable sugar obtained at 72h saccharification time point.

[0048] The invention enables enhanced saccharification of cellulosic biomass (and correspondingly improved lignocellulosic degradation) through a novel recombinant fungal strain developed by overexpressing lytic polysaccharide monooxygenase (LPMO) and Cellobiohydrolase 1 (CBH1) within recombinant P. funiculosum Mig1⁸⁸ (MTCC 25141). The invention also provides novel methods, plasmids and integration cassettes for generating the novel fungal strain.

[0049] Research on fortifying the secretome of cellulase producing strains for better biomass hydrolysis, has identified significant roles of certain synergistic or auxiliary proteins in promoting cellulase activity. Effective utilization of these synergistic proteins such as expansins, swollenin, hydrophobins, and lytic polysaccharide monooxygenase (LPMO) have been found to significantly reduce the total cellulase loading required to achieve about 80 - 90 % cellulose conversion required for bioethanol production.

[0050] In particular, LPMOs (which comprise a family of recently discovered auxiliary proteins) have been found to be unique in terms of their capability of catalyzing the cleavage of glycosidic bonds of polysaccharides via an oxidative mechanism (instead of through hydrolytic means). The mechanism of cellulose cleavage achieved by LPMOs involves the reduction of Cu²⁺ at the active site by some redox partners. This has been found to result in an attack on the pyranose ring of the glucose moieties at the C-1 or C-4 position, thereby destabilizing the adjacent glycosidic bond and breaking it by an elimination reaction (see (i) Kim, I.J., Seo, N., An, H.J., Kim, J.-H., Harris, P. V., Kim, K.H., 2017. Type-dependent action modes of TtAA9E and TaAA9A acting on cellulose and differently pretreated lignocellulosic substrates. Biotechnol. Biofuels 10, 46. https://doi.org/10.1186/s13068-017-0721-4, and (ii) Kittl, R., Kracher, D., Burgstaller, D., Haltrich, D., Ludwig, R., 2012. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay. Biotechnol. Biofuels 5, 79. https://doi.org/10.1186/1754-6834-5-79).

[0051] LPMOs have been found to target crystalline regions of the cellulose surface, which are typically more recalcitrant to cellulase action and introduce nicks on the substrate surface which provide extra chain ends for glycoside hydrolases (GHs) to act (see Vaishnav, N., Singh, A., Adsul, M., Dixit, P., Sandhu, S.K., Mathur, A., Puri, S.K., Singhania, R.R., 2018. Penicillium: The next emerging champion for cellulase production. Bioresour. Technol. Reports 2, 131-140. https://doi.Org/10.1016/J.BITEB.2018.04.003). This action has been found to improve reaction kinetics as well as enhance enzymatic synergy during saccharification (see (i) Beeson, W.T., Vu, V. V., Span, E.A., Phillips, C.M., Marietta, M.A., 2015. Cellulose Degradation by Polysaccharide Monooxygenases. Annu. Rev. Biochem. 84, 923-946, https://doi.org/10.1146/annurev-biochem-060614- 034439, and (ii) Vaishnav et al. 2018).

[0052] More particularly, in connection with P. funiculosum (NCIM 1228), proteomic analysis of its secretome revealed that about 58% of the total proteins secreted under cellulase inducing conditions belongs to the Carbohydrate-Active Enzymes (CAZymes) family. When the ratio of the Cellobiohydrolases (CBH1and CBH2) was assessed, it was found that the proportion of CBHs in the secretome of P. funiculosum was only 15% of the total proteins which is contrary to the secretome of T. reesei where the CBHs represented 95% of the total proteins (see (i) Gusakov, A. V, 2011. Alternatives to Trichoderma reesei in biofuel production. Trends Biotechnol. 29, 419-425. https://doi.Org/10.1016/j.tibtech.2011.04.004, and (ii) Ogunmolu et al. 2015).

[0053] Additionally, during structure-function characterization of the CBH1 enzyme of P. funiculosum, it has been found that CBH1 of P. funiculosum can hydrolyse crystalline biomass with approximately five-fold higher efficiency than its counterpart from Trichoderma reesei (see Ogunmolu, F.E., Jagadeesha, N.B.K., Kumar, R., Kumar, P., Gupta, D., Yazdani, S.S., 2017. Comparative insights into the saccharification potentials of a relatively unexplored but robust Penicillium funiculosum glycoside hydrolase 7 cellobiohydrolase. Biotechnol. Biofuels 10, 1-17. https://doi.org/10.1186/s13068-017- 0752-x).

[0054] The enhancement in enzymatic activity of CBH1 of P. funiculosum has been found to be mainly caused by the catalytic domain of PfCBH1 when the subdomains of PfCBH1 and TrCBH1 were swapped (see Taylor, L.E., Knott, B.C., Baker, J.O., Alahuhta,

P.M., Hobdey, S.E., Linger, J.G., Lunin, V. V, Amore, A., Subramanian, V., Podkaminer, K., Xu,

Q., VanderWall, T.A., Schuster, L.A., Chaudhari, Y.B., Adney, W.S., Crowley, M.F., Himmel, M.E., Decker, S.R., Beckham, G.T., 2018. Engineering enhanced cellobiohydrolase activity. Nat. Commun. 9, 1186. https://doi.org/10.1038/s41467-018-03501-8).

[0055] Yet further, proteomic analysis of the secretome under cellulase inducing condition has also detected oxidative enzyme LPMO (formerly GH61) belonging to the AA9 family.

[0056] Analysis of the genome sequence of P. funiculosum NC1M1228 has established that the fungal strain has only a single copy of gene coding for LPMO, and only a single copy of gene coding for CBH1, while the same fungal strain has more than one copy of genes coding for other cellulase components such as b-glucosidase, endoglucanase and xylanase (see Ogunmolu et al., 2017 and Ogunmolu et al., 2015).

[0057] Based on research, it has been surprisingly discovered that the efficiency of the secretome of PƒMig1⁸⁸ in terms of its saccharification performance towards highly recalcitrant biomass, can be significantly enhanced to enable high substrate loading with a minimal amount of protein - by fortifying the secretome of PƒMig1⁸⁸ through engineering of the key hydrolytic and oxidative enzymes which participate in saccharification of crystalline cellulosic biomass.

In particular, it has been discovered that overexpression of LPMO or CBH1, and even more particularly, co-overexpression of both LPMO and CBH1, has a surprising, significant and synergistic impact on the efficiency of the secretome of PƒMig1⁸⁸ for the purposes oflignocellulosic degradation, particularly towards highly recalcitrant biomass.

[0058] The term “overexpressing” or “overexpression” or “enhancing the expression” used in the present description in connection with an enzyme (or protein) in a P. funiculosum strain, shall be understood as referring to artificially increasing the quantity of said enzyme produced in the fungal strain compared to a reference (control) fungal strain wherein said enzyme is not overexpressed. This term also encompasses expression of an enzyme (or protein) in a P. funiculosum strain which does not naturally contain a gene encoding said enzyme.

[0059] The term “co-overexpressing” or “co-overexpression” or “enhancing the expression of both of”, when used in the present description in connection with two enzymes (or proteins) in a P. funiculosum, shall be understood as referring to artificially increasing the quantity of both enzymes produced in the fungal strain compared to a reference (control) fungal strain wherein said enzymes are not overexpressed. This term also encompasses expression of two enzymes (or proteins) in a P. funiculosum strain which does not naturally contain a gene encoding for either one or both of said enzymes.

[0060] In various embodiments of the invention the LPMO and CBH1 genes were overexpressed in the catabolite derepressed strain of P. funiculosum (PƒMig1⁸⁸) independently, and also overexpressed in the catabolite derepressed strain of P. funiculosum (PƒMig1⁸⁸) simultaneously. The secretome from each of the resulting transformants was assessed for improved efficiency of the secretome of P. funiculosum strains towards highly recalcitrant biomass. Briefly, the resulting transformants showed about 66% and 200% increase in activities of CBH1 and LPMO, respectively. Secretomes of transformant strains of PƒMig1⁸⁸ where either LPMO and CBH1 were overexpressed were found to result in increase of the saccharification of acid pretreated wheat straw (PWS) by 6% and 12% respectively, over PƒMig1⁸⁸ when compared at the same enzyme concentrations. Importantly, co-overexpression of both LPMO and CBH1 increased the saccharification efficiency by 20%. In working examples, discussed in more detail below, at 20% solid loading and 30 mg protein/g dry biomass, 82% saccharification was achieved with the secretome, (i.e. DICzyme-3), of the transformant strain of PƒMig1⁸⁸ that co-overexpresses both CBH1 and LPMO, as compared to about 64% obtained with the secretome (i.e., DlCzyme-1), of PƒMig1⁸⁸.

Plasmids and Microbial strains

[0061] Strains and plasmids referred to in this written description have been listed in Table 1 below.

Table 1 : Plasmids and fungal strains referred to in the written description

*NCIM (National Culture Collection Centre, Pune, India)

**MTCC (Microbial Type Culture Collection and Gene Bank, Chandigarh, India) [0062] Escherichia coli DH5α was used for plasmid propagation throughout the experiments.

[0063] Agrobacterium tumefaciens LBA4404 strain used for fungal transformation was maintained on low sodium LB medium (10 g/L tryptone 5 g/L yeast extract, 5 g/L sodium chloride) containing 100 μg/mL kanamycin and 30 μg/mL rifampicin.

[0064] The pBIF vector which was used as a backbone vector for fungal transformation contains hygromycin and kanamycin resistance genes which were used as selective markers for selection of transformants. [0065] The pBluescript (pBSK+) vector containing ampicillin resistance gene was used as a shuttle vector for simultaneous cloning of LPMO and CBH1.

[0066] Penicillium funiculosum NC1M1228 and its derivative P. funiculosum Mig¹⁸⁸, which comprise the fungal strains used for this study were routinely cultivated on Petri dishes containing low malt extract-peptone (LMP) agar for about 14 days until full sporulation.

[0067] For selection and maintenance of fungal transformants, LMP medium supplemented with Hygromycin B at 100 μg/mL was used.

Cellulosic substrate and pretreatment

[0068] Nitric acid treated wheat straw was used as a substrate for the purposes of the working examples described below. Wheat straw biomass was comminuted in a cutting mill and sieved through 1.5 mm mesh to obtain particles of uniform size prior to chemical pretreatment.

[0069] Pretreatment was carried out using 0.5% nitric acid in a bioreactor for 30 mins at 120 °C temperature and 18 bars pressure. The pretreated straw was subsequently washed repeatedly until the pH was neutral (see for example, the method disclosed in Lali, A., Nagwekar, P., Varavadekar, Wadekar, P., Gujarathi, S., Valte, R., Birhade, S., Odaneth, A., 2012. Method for Production of Fermentable Sugars from Biomass. https://doi.org/10.1039/C2CS35165). Compositional analysis conducted according to the NREL/TP510-42618 procedure (see for example, the procedure described in Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., Crocker, D., 2008, Determination of Structural Carbohydrates and Lignin in Biomass: Laboratory Analytical Procedure (LAP) (Revised July 2011), on the pretreated wheat straw yielded a cellulose content of 61.3%, 6.1% hemicellulose, 15.6% lignin, as well as 6% ashes.

Engineering of Penicillium funiculosum Migl⁸⁸for overexpression of LPMO and CBH1 [0070] All vectors for fungal expression described in this study were constructed on the backbone of the pBIF vector that has been constructed using the binary vector pCAMBIA1300 as a backbone (see for example the disclosure in Nizam, S., Singh, K., Verma, P.K., 2010. Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei. Curr. Genet. 56, 391-399. https://doi.org/10.1007/s00294-010-0305-3).

[0071] Primers referred to in this written description have been listed in Table 2 below.

Table 2 : Primers used for the purposes of the present written description

^*The underlined text indicates the site of the restriction enzyme used for cloning.

[0072] Binary vectors for overexpression of LPMO and CBH1 genes from P. funiculosum were constructed in the manner described below.

[0073] The endogenous gene coding for LPMO was amplified from the genome of P. funiculosum NCIM1228 using the primers PfLPMO-F (SEQ ID No. 1) and PfLPMO-R (SEQ ID No. 2). The primers were designed according to LPMO sequence containing both its native promoter and terminator which were obtained from the draft genome sequence of the strain. The PCR product obtained was digested with Sbfl and Spel restriction enzymes before ligating unto the pBIF vector earlier digested with Pstl and Spel enzymes to generate the pBIF/LPMO (pOAO1) vector. Figure 1 illustrates the vector map for the pOAO1 vector that is thus generated (i.e. the vector map for overexpression of P. funiculosum LPMO gene in PƒMig1⁸⁸ fungal strain). SEQ ID NO. 25 provides the DNA sequence of the pOAO1 vector that is generated for overexpression of P. funiculosum LPMO gene in PƒMig1⁸⁸ fungal strain.

[0074] The ligated product of pOAO1 was then transformed in E. coli DH5α cells and selected on 50 μg/ml kanamycin. Resulting colonies were screened for positive transformants by colony PCR, followed by restriction digestion of the corresponding plasmids.

[0075] To create a strain for co-overexpression of both LPMO and CBH1 genes, sequential cloning of the two genes was adopted using pBluescript (pBSK+) as a shuttle vector.

[0076] First, the LPMO gene which had earlier been amplified from the genome was cloned into the pBSK vector between the Pstl and Spel site of its multiple cloning sites (MCS) to obtain the plasmid pBSK/LPMO (pOA03). [0077] Second, CBH1 gene with its native promoter and terminator was further amplified from the genome of the P. funiculosum NC1M1228 using the primers PfCBH1- F2 (SEQ ID No. 3) and PfCBH1-R2 (SEQ ID No. 4) containing Aflll and Spel restriction sites, respectively. The PCR product obtained was digested with Aflll and Spel restriction enzymes before ligating at the corresponding sites of pOAO3 vector to obtain a pOAO4 vector.

[0078] The resulting Sbfl/Spel fragment obtained in the pOAO4 vector was excised and cloned between the Pstl/Spel sites of pBIF vector thereby generating the pBIF/LPMO/CBH1 ( pOAO5) vector. Figure 3 illustrates the vector map for the pOAO5 vector that is thus generated (i.e. the vector map for overexpression of LPMO/CBH1 genes in PƒMig1⁸⁸ fungal strain). SEQ ID NO. 26 provides the DNA sequence of the pOAO5 vector that is generated for overexpression of P. funiculosum LPMO/CBH1 gene in PƒMig1⁸⁸ fungal strain.

[0079] Thereafter, positive plasmids of pOAO1 and pOA05 were transformed to PfMig⁸⁸ fungal strain following the agrobacterium-mediated fungal transformation method (AMTM) as described by in Michielse, C.B., Hooykaas, P.J.J., van den Hondel, C.A.M.J J., Ram, A.F.J., 2008. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat. Protoc. 3, 1671-1678. https://doi.org/10.1038/nprot.2008.154.

Molecular analysis of transformants

[0080] After transformation, the resulting hygromycin resistant transformants were screened following the method described in Fang, H., Xia, L, 2013. High activity cellulase production by recombinant Trichoderma reesei ZU-02 with the enhanced cellobiohydrolase production. Bioresour. Technol. 144, 693-697. https://doi.Org/10.1016/j.biortech.2013.06.120.

[0081] The transformants were verified both by PCR and Southern blot hybridization for the integration of a LPMO and CBH1 gene expression cassette. For PCR analysis, the primers PgpdA-F (SEQ ID No. 5) and TrpC-R (SEQ ID No. 6) were used to screen for LPMO and CBH1 integration while transformants of LPMO/CBH1 were screened using PgpdA IR-F (SEQ ID No. 7) and PfCBH1-IR R (SEQ ID No. 8) primer set.

[0082] Southern hybridization was performed using standard procedures described in Southern, E., 2006, Southern blotting. Nat. Protoc. 1, 518-525. https://doi.org/10.1038/nprot.2006.73.

[0083] Genomic DNAs (8 μg) of PfMig¹⁸⁸ and transformants of LPMO and LPMO/CBH1 were digested with Xhol, Hindlll and Apal restriction enzymes. The digested gDNAs were then size-fractionated by electrophoresis on 0.8% agarose gel in 1x TAE (Tris-acetate) buffer. After depurination using 250 mM HC1, and denaturation (1.5 M NaCl and 0.5 M NaOH) and neutralization (1.5 M NaCl and 1.0 M Tris-HCl pH 8.0) steps, the gel was capillary-blot onto a positively charged HybondTM-N+ membranes (Amersham Biosciences, USA).

[0084] The 621-bp fragment of LPMO gene was PCR-amplified with the primers LPMO-Probe F (SEQ ID No. 9) and LPMO-Probe R (SEQ ID No. 10) and labelled as probe to detect the LPMO integration.

[0085] Similarly, a fragment of CBH1 gene (605-bp long) was amplified using the primers CBH1-Probe F (SEQ ID No. 11) and CBH1-Probe R (SEQ ID No. 12) and used to confirm the insertion of CBH1 expression cassette in LPMO/CBH1 strains. The LPMO and CBH1 amplicons for detection were radio-labelled with [α-32P] dCTP using the NEBlot Kit (NEB, USA) according to the manufacturer’s instructions and used as probe to determine the copy number of T-DNA integrations in all the transformants.

Growth conditions of transformants and microscopic examination

[0086] For culturing respective strains of P. funiculosum that have been obtained pursuant to the above, on agar plates, a uniform inoculum of 5 μl of fungal spore suspension (1 x 10⁶ spores/ml) was directly placed onto the centre of Petri dishes containing Yeast Nitrogen Base (YNB) supplemented with different polymeric carbon sources. The carbon sources used include carboxymethyl cellulose (CMC), xylan, Avicel, potato dextrose and wheat bran and were added at a final concentration of 2% according to the method described in Randhawa, A., Ogunyewo, O.A., Eqbal, D., Gupta, M., Yazdani, S.S., 2018, Disruption of zinc finger DNA binding domain in catabolite repressor Mig1 increases growth rate, hyphal branching, and cellulase expression in hypercellulolytic fungus Penicillium funiculosum NC1M1228. Biotechnol. Biofuels 11. https://doi.org/10.1186/s13068-018-1011-5.

[0087] The inoculated plates were incubated at 28 °C for five days after which the growth pattern of the different strains in response to the different polymeric carbon sources was observed. For observing colony structures and sizes, the diameter of the colonies of the different stains on all the carbon substrate tested was measured and recorded. The morphology of all the strains after each genetic engineering was checked using high-resolution fluorescence microscope (Nikon Eclipse, Japan). For microscopy, cultures were grown in potato dextrose broth (PDB) for 24 hours before being stained with lactophenol blue for examination. Transverse sections of all strains were observed under 20x and IOO^c magnifications.

Expression analysis of cellulolytic enzymes via real-time PCR

[0088] For real-time PCR experiments, cultures of all strains were grown in Minimal Mandel's media containing 4% Avicel for 48 hours Mandels, M. Reese, E.T., 1957, Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J. Bacteriol. 73, 269-78. Mycelia were harvested by filtration and lyophilized in liquid nitrogen. RNA was extracted using RNeasy kit (Qiagen) according to the manufacturer’s instructions. RNA was DNase treated (Invitrogen) before cDNA synthesis. One microgram of RNA was used as template in each quantitative real-time PCR (qRT-PCR) reaction. cDNA synthesis control was performed to ensure the absence of DNA contamination. qRT-PCR was carried out using iTaq™ Universal SYBR® Green Supermix (Bio-Rad) and Bio-Rad CFX96 qPCR detection system. Primers for test transcripts were designed using boundary sequence of two exons to avoid any amplification from genomic DNA contamination. To check for LPMO transcripts, primers LPMO RT F (SEQ ID No. 13) and LPMO RT R (SEQ ID No. 14) were utilized. Also, to evaluate the transcripts of cellobiohydrolase, endoglucase, glucosidase, xylanase and tubulin, the following primer sets were used CBH1 F (SEQ ID No. 15) and CBH1 R (SEQ ID No. 16); EG-GH5 F (SEQ ID No. 17) and EG-GH5 R (SEQ ID No. 18); BGL-GH3 F (SEQ ID No. 19) and BGL-GH3 R (SEQ ID No. 20); XYL-(GHIO-CBMI) F (SEQ ID No. 21) and XYL-(GHIO-CBMI) R (SEQ ID No. 22). Tubulin F (SEQ ID No. 23) and Tubulin R (SEQ ID No. 24). qRT-PCR was done in biological triplicates with tubulin as the endogenous control. Relative expression levels were normalized to tubulin, and fold changes in RNA level were the ratios of the relative expression level of PfMig¹⁸⁸ and the corresponding transformants of LPMO and CBH1 to NC1M1228 under cellulase inducing conditions (see Kitchen, R.R., Kubista, M., Tichopad, A., 2010. Statistical aspects of quantitative real-time PCR experiment design. Methods 50, 231-236. https://doi.Org/10.1016/J.YMETH.2010.01.025).

Cellulolytic secretome preparation

[0089] Penicillium funiculosum NCIM1228, P. funiculosum Mig1⁸⁸ (PfMig1⁸⁸) and the resulting transformants of LPMO and LPMO/CBH1 were cultivated on Petri dishes containing low malt extract agar until there was full sporulation. After 14 days of incubation, spores were recovered with sterile water, filtered through sterile Mira cloth and quantified using a hemocytometer. The primary culture of each strain was prepared by culturing 10⁷ conidiophores in potato dextrose broth (PDB) for 36 h.

[0090] Primary cultures of the strains were added to the cellulase inducing media in

Erlenmeyer flasks at a final concentration of 10%.

[0091] Cellulase inducing media contains soya peptone (24 g/1), wheat bran (21.4 g/1), microcrystalline cellulose (MCC) (24 g/1), KH2P04 (12.4 g/L), K2HP04 (2.68 g/1) (NH4)2S04 (0.28 g/1), CaC03 (2.5 g/1), corn steep liquor (1%), urea (0.52 g/1), and yeast extract (0.05 g/1) with the final pH adjusted to 5.0. The flasks were kept at 28 °C for five days with orbital shaking at 150 rpm (Innova 44, Eppendorf AG, Germany). Induced cultures were centrifuged at 9000 rpm for 10 min at 4 °C, and the cellulolytic supernatants were collected and stored at 4 °C until use.

Determination of enzyme activities of the engineered secretome [0092] All enzymatic activities performed in this study were routinely determined following standard assay procedures. Cellobiohydrolase activity was determined by incubating appropriate dilution of the enzyme with 1% Avicel® PH-101 (Sigma) for 120 mins. Endoglucanase, xylanase and b-glucosidase activities were determined by incubating appropriate dilution of enzyme with 2% CMC (Sigma), 2% beechwood xylan (HiMedia) and p-nitrophenyl-β-D-glucopyranoside (Sigma), respectively, for 30 mins, after which the amount of reducing sugars released was measured as previously reported (see Randhawa et al., 2018). One unit of CMCase, Avicelase and xylanase activity is defined as the amount of enzyme releasing 1 μmol of reducing sugar per min, while one unit of b-glucosidase activity was defined as the amount of protein that released 1 prnol of p-nitrophenol per min. LPMO activity was assayed using the Amplex red assay as described earlier (see Kittl et al., 2012). The reaction mixture was composed of 20 μL of LPMO source (enzyme) and 180 μL assay solution, which comprised 18 μL of 300 pM ascorbate, 18 μL of 500 pM Amplex Red, 18 μL of 71.4 units/mL Horse Radish Peroxidase (HRP), 18 μL of 1 M sodium phosphate buffer pH 6.0, and 108 μL of HPLC-grade water. Resorufin fluorescence was taken at an excitation wavelength of 530 nm and emission wavelength 580 nm after 10 min of incubation at 22 °C using a multimode plate reader (Spectra Max M3, USA).

[0093] In reference experiments without LPMO, the background signal was measured and subtracted from the assays. A standard curve obtained with various dilutions of H₂O₂ was used for the calculation of an enzyme factor to convert the fluorimeter readout (counts min^-1) into enzyme activity. LPMO activity is defined as 1 pmol of H₂O₂ generated per minute under the defined assay conditions.

[0094] Total cellulase activity in the secretome was measured in terms of “filter paper units” (FPU) per millilitre of original (undiluted) enzyme solution. The assay requires a fixed degree of conversion of substrate, from 50 mg of filter paper within 60 min at 50 °C. The FPU is defined as the amount of enzyme required to produce 2 mg of glucose from 50 mg of filter paper within 60 min of incubation. Total protein of each secretome was estimated by the Bicinchoninic acid (BCA) using bovine serum albumin (BSA) as standard. SDS-PAGE and Zymogram Analysis

[0095] Sodium dodecyl sulfate (SDS) -polyacrylamide gels (12%) were prepared, and proteins obtained following the culture supernatant preparation were separated via SDS- polyacrylamide gel electrophoresis (PAGE) according to the method earlier described (see Laemmli, U.K., 1970, Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0).

[0096] Mini-PROTEAN Tetra Cell (Bio-Rad) with gel size of 8.6 x 6.7 cm2 was used for protein separation. After protein separation, the gels were first washed with lx PBS buffer to remove the SDS. The Zymogram analysis was carried out using 5 mM 4-methyl umbelliferyl β-D-glucopyranoside (MUG) as substrate following standard procedures in 50 mM citrate-phosphate buffer pH 4.0 for 10 mins before visualization under UV light. Proteins of the gel were then stained with Coomassie blue R-250 (Sigma-Aldrich, USA) and the molecular mass of the proteins was determined with reference to standard proteins (Thermo Scientific, USA).

Biomass saccharification and quantification of fermentable sugars

[0097] The saccharification efficiency of the secretome of all the strains used in the study towards pretreated wheat straw was carried out according to the method described by Ogunmolu et al., 2015, with some modifications. Performance of the secretomes towards nitric acid treated wheat straw was evaluated at 20% dry weight of biomass using enzyme concentrations of 30 mg/g biomass. Saccharification was performed in 50 ml screw capped Falcon tubes in an incubator shaker at 50 °C for 96 h. The reaction mixture included the nitric acid-treated wheat straw at 20% dry weight loading in a 5 ml final reaction volume. Total protein content in the secretome of all the fungal strains tested was measured, and the appropriate volume of desired protein concentration (30 mg/g DBW) was added to the reaction mixture.

[0098] The reactions were set up in 100 mM citrate -phosphate buffer (pH 4.0) and incubated at 50 °C with constant shaking at 300 rpm for 96 hr. Samples were collected every 24 h and analyzed for production of fermentable sugars. Control experiments were carried out under the same conditions using substrates without enzymes (enzyme blank) and enzymes without substrates (substrate blank); a substrate-free negative control was set up by filling the Falcon tubes with 100 mM citrate-phosphate buffer, pH 4.0, and the background of soluble sugars present in the respective biomass was determined by incubating each biomass in the absence of enzyme.

[0099] Following completion of hydrolysis at each time point, the Falcon tubes were centrifuged at 3500 rpm for 10 min in a swinging bucket centrifuge (Eppendorf, Germany) to separate the solid residue from the digested biomass. Supernatants which were recovered after enzymatic hydrolysis of the pretreated wheat straw were analyzed by high-performance liquid chromatography equipped with Aminex HPX-87H anion exchange column (Bio-Rad, USA) and a refractive index detector to analyze released monosaccharides (glucose and xylose) by anion exchange chromatography. The filtered mobile phase (4 mM H2S04) was used at a constant rate of 0.3 ml/min with the column and RI detector temperatures maintained at 35 °C. The concentration of each monosaccharide was calculated from calibration curves of external standards (xylose and glucose) purchased from Absolute Standards Inc. The following equations provided in NREL's LAP TP- 510-43630 was then used to determine percent theoretical conversions of cellulose and hemicellulose into monomeric sugars;

where [Glucose] is the glucose concentration (g/L), [Xylose] is the xylose concentration (g/L), [Pretreated Biomass] is the dry pretreated biomass concentration at the beginning of enzymatic hydrolysis (g/L), and f is the cellulose or hemicellulose fraction in the dry pretreated biomass (g/g) (see (i) Avila, P.F., Forte, M.B.S., Goldbeck, R., 2018. Evaluation of the chemical composition of a mixture of sugarcane bagasse and straw after different pretreatments and their effects on commercial enzyme combinations for the production of fermentable sugars. Biomass and Bioenergy 116, 180-188. https://doi.Org/10.1016/j.biombioe.2018.06.015, and (ii) Resch, M.., Baker, J.O., Decker, S.R., 2015. Low Solids Enzymatic Saccharification of Lignocellulosic Biomass Laboratory Analytical Procedure (LAP). Tech. Rep. NREL/TP- 5100-63351, Lab. Anal. Proced. 1-9. https: //doi.org/10.1016/j.jallcom.2018.03.019).

Vector construction and overexpression of PfLPMO in PfMig1⁸⁸ strain

[00100] Figure 4 illustrates the P. funiculosum LPMO gene integration cassette. SEQ ID NO. 27 provides the DNA sequence of the P. funiculosum LPMO gene integration cassette that is used to transform the pOAO1 vector (See SEQ ID NO. 25) to a PfMig⁸⁸ fungal strain exhibiting overexpression of the P. funiculosum LPMO gene, through the agrobacterium- mediated fungal transformation method.

[00101] Preliminary analysis of the P. funiculosum LPMO nucleotide and the encoded protein sequence showed that it consists of 1020 bp, including two introns, which encodes 310 amino acids. To overexpress the LPMO gene in PfMig1⁸⁸, the expression vector containing the endogenous gene along with its promoter and terminator was constructed using the backbone of pBIF binary vector with primers PfLPMO-F (SEQ ID No. 1) and PfLPMO-R (SEQ ID No. 2). The 3.0 kb gene was amplified from the genome of the parent strain and cloned into the pBIF vector to generate the pOAO1 plasmid (see Figure 1). To confirm the presence of the LPMO gene in the pOAO1 plasmid, the plasmid and pBIF vector used for cloning were both digested with BamHl restriction enzyme.

[00102] Figure 5 shows the restriction digestion analysis of pBIF (1) and pOAO1 (2) by BamHl restriction enzyme. Referring to Figure 5, the resulting 2.1 kb fragment generated in the pOAO1 plasmid and not in the pBIF vector confirms the presence of the gene in the vector.

[00103] The pOAO1 recombinant plasmid of Figure 1 was thereafter transformed into the PfMig¹⁸⁸ fungal strain using the AMTM procedure, and the hygromycin-resistant transformants obtained were analyzed subsequently (as shown in Figure 6 - which illustrates the transformants of pOAO1 after agrobacterium-mediated transformation in PƒMig1⁸⁸ fungal strain). PCR was first performed to confirm the integration of LPMO cassette into the genome. Keeping in mind that the host strain has the native LPMO gene, the primers used for PCR detection were particularly designed to capture some regions on the T-DNA of the pOAO1 plasmid in order to evade the disturbing effect of the native LPMO gene on PCR result.

[00104] Figure 7 shows the integration of P. funiculosum LPMO in the genome of PƒMig1⁸⁸ transformants by PCR. In Figure 7, lane M is DNA molecular mass marker; lane (+) is the pOAO1 plasmid which served as positive control, lanes 1-10 are the transformants of pOAO1, lane (-) is the gDNA of PƒMig1⁸⁸ which was the negative control. As shown in Figure 7, the reactions with primers PgpdA-F (SEQ ID No. 5) and TrpC-R (SEQ ID No. 6) generated the anticipated amplification products (4.8 kb fragment) in all the ten transformants selected but not in the control PfMig1⁸⁸, which indicated the integration of the LPMO cassette. To further confirm the integration and copy number, Southern blot was carried out for five out of the ten transformants confirmed by PCR. Genomic DNA from each strain was digested with Xhol and Hindlll and probed with a 621-bp LPMO gene fragment - as shown in Figure 8 (which is the Southern blot of transformants confirmed by PCR). In Figure 8, M is HindIII lambda DNA size marker used, lane C indicates genomic DNA of non-transformed parental strain, and lanes 1-5 represent genomic DNA from transformants with LPMO cassette integration. The results shown in Figure 8 establish that all the transformants possessed more than one bands in contrast with the control (PfMig¹⁸⁸) which had only one single band, further verifying the insertion of an additional copy of the LPMO cassette in the genome of PfMig1⁸⁸.

[00105] In order to evaluate the expression level of the overexpressed LPMO gene in the LPMO-overexpressing transformants, the five-fast growing transformants confirmed by Southern hybridization were cultivated in cellulase inducing media for five days. The resulting secretome from the strains was used to screen for LPMO activity. The LPMO activity of the transformants was measured using the culture supernatant and was compared to the parent strain, PfMig¹⁸⁸ and is shown in Figure 9 (which shows the results for LPMO and FPU activities in the fermentation broth of PƒMig1⁸⁸ and five transformants). The results showed remarkable increase in LPMO activity in all the transformants when compared to the parent strain in different magnitude. [00106] Specifically, the increase in activity was found to be in the range of 155 - 203%, in which transformant T4 showed the maximum activity of 2.68 U/ml which was more than 200% increase over that of PfMig¹⁸⁸ strain (0.88 U/ml). Variation in enzyme activity across the transformants as determined by the Amplex red assay is believed to be due to the difference in integration loci of the expression cassette in PfMig188 genome as earlier reported in (i) Dashtban, M., Qin, W., 2012, Overexpression of an exotic thermotolerant b-glucosidase in Trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw. Microb. Cell Fact. 11, 1. https://doi.org/10.1186/1475-2859-11-63 and (ii) Zhang, J., Zhong, Y., Zhao, X., Wang, T., 2010, Development of the cellulolytic fungus Trichoderma reesei strain with enhanced b-glucosidase and filter paper activity using strong artificial cellobiohydrolase 1 promoter. Bioresour. Technol. 101, 9815-9818. https://doi.Org/10.1016/J.biortech.2010.07.078.

[00107] However, when the total cellulase activity of the transformants was evaluated using the filter paper assay to see if there would be any corresponding impact on the total cellulase activity, there was no significant difference in the activity of the transformants when compared to that of the parent strain (see Figure 9). It is believed that the observed indifference may be a consequence of the absence of reducing agents which serves as electron donors required by the LPMO enzyme to act on pure cellulose in the filter paper assay mixture (see Kim et al., 2017).

[00108] However, the remarkable increase in LPMO activity recorded in all the transformants over that of the parent strain indicates that the gene was successfully overexpressed and the LPMO amount in the PfMig1⁸⁸ cellulase system was significantly improved.

Simultaneous overexpression of LPMO and CBH1 in PfMig1⁸⁸ strain results in an improved cellulase system in its secretome

[00109] The above data establishes that individual overexpression of LPMO or CBH1 genes increased their corresponding enzyme component in the cellulase mixtures of the PfMig1⁸⁸ strain remarkably. Thereafter, to establish whether overexpression of these target genes simultaneously could produce a more versatile cellulase system in PfMig1⁸⁸, a double overexpression strain was constructed.

[00110] To this end, as illustrated in Figure 10 (which illustrates the construction cassette for dual overexpression of LPMO and CBH1 genes in P. funiculosum), the LPMO/CBH1 expression cassette was first constructed in the pBSK vector by fusing the CBH1 gene at the C-terminal of the LPMO gene to facilitate good expression and secretion of both enzymes. For fungal transformation, the LPMO/CBH1 cassette was then excised with restriction enzymes and cloned into the pBIF vector (the pOA05 vector as shown in Figure 3). SEQ ID NO. 28 provides the DNA sequence of the P. funiculosum LPMO/CBH1 gene integration cassette that is used to transform the pOA05 vector (See SEQ ID NO. 26) to a PfMig⁸⁸ fungal strain exhibiting co-overexpression of the P. funiculosum LPMO/CBH1 genes, through the agrobacterium-mediated fungal transformation method.

[00111] The resulting pOA05 plasmid was confirmed by restriction digestion before transformation into PfMig¹⁸⁸ strain - as shown in Figure 11 (which describes the restriction digestion analysis of pBIF (1) and pOA03 (2) by A/711 and Spel).

[00112] Figure 12 shows the transformants of pOA03 after agrobacterium-mediated transformation in PƒMig1⁸⁸. Figure 13 shows the integration of LPMO/CBH1 cassette in the genome of PƒMig1⁸⁸ transformants by PCR. Lane M is DNA molecular mass marker, lane (+) is the pOA03 plasmid which served as positive control, lanes 1-6 are the transformants of pOA03, lane (-) is the gDNA of PƒMig1⁸⁸ which was the negative control.

[00113] Figures 12 and 13 show the PCR analysis (and results) of the obtained Hygromycin-resistant transformants to confirm the integration of LPMO/CBH1 cassette into the genome. The internal primers PgpdA 1R-F (SEQ ID No. 7) and PfCBH1-lR R (SEQ ID No. 8) in the expression cassette were used to screen for positive transformants containing the fused LPMO/CBH1 cassette so as to avoid the interfering effect of the native copy for each of the individual genes in the transformants on PCR results. [00114] PCR reactions with these primers generated the anticipated amplification products of 5.0 kb fragment in all the six fast-growing transformants selected but not in the control PfMig188 - which is shown in Figure 19. Similarly, to further confirm the integration and copy number, Southern blot was carried out for all selected transformants. gDNAs from each strain were digested with Xhol and Apal and then probed with a 621-bp CBH1 gene fragment - which is shown in Figure 20. Figure 14 shows the Southern blot of transformants confirmed by PCR. M is Hindlll lambda DNA size marker used, lane C indicates genomic DNA of non-transformed parental strain, lanes 1-5 represent genomic DNA from transformants with LPMO/CBH1 cassette integration. The results establish that all the transformants possessed more than one band in contrast with the control (PfMig¹⁸⁸) strain which had a single prominent band, further verifying the insertion of an additional copy of the LPMO/CBH1 cassette in the genome of PfMig188. Transformant T5 showed two faint bands at the similar locations is believed to have occurred due to experimental error leading to lower loading of digested genomic DNA. Four out of the five transformants had only one additional band, suggesting a single copy insertion of LPMO/CBH1 cassette in the genome, while one of the transformants (Tl) had two copies of the integrated cassette.

[00115] To further assess the effect of this simultaneous overexpression on the expression level of cellulolytic enzymes by the transformants, the six transformants confirmed positive by PCR for integration of the LPMO/CBH1 expression cassette were cultivated in cellulase inducing media for five days. The resulting secretome from the strains was recovered and used for enzyme assays. The LPMO, cellobiohydrolase and total cellulase activities of the transformants were measured and compared with that of parent Pf Mig¹⁸⁸ strain. As expected, based on the results from the specific overexpression of the enzymes, all the transformants screened demonstrated an enhancement in all the activities when compared with the parent strain - which data is presented in Figure 15 (Figure 15 shows the enzymatic profile of the overexpressed enzymes in the fermentation broth of PƒMig1⁸⁸ and six transformants). The results show that there was about 130 - 212% increment in LPMO activity over that of PfMig¹⁸⁸ across the transformants. Similarly, the data establishes between 40 - 66% increase in Avicelase activity and between 14 - 20% increase in filter paper unit when compared with PfMig1⁸⁸. Although there was variation in enzyme activities tested across all the transformants, the highest production of all the enzymes was found with transformants T6 in which activities of LPMO, Avicelase and FPU were 3.3 U/ml, 3.51 U/ml and 5.2 U/ml respectively. It was surprising to discover that there was no difference between the enzyme activities of the transformants with single copy integration and that of transformant T1 which had two additional copies of the LPM0/CBH1 cassette. It is believed that this suggests that the integration locus other than the gene copy numbers might have also affected the expression of the cellulolytic enzymes (see Xue, X., Wu, Y., Qin, X., Ma, R., Luo, H., Su, X., Yao, B., 2016. Revisiting overexpression of a heterologous b- glucosidase in Trichoderma reesei: Fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities. Microb. Cell Fact. 15, 1-13. https://doi.org/10.1186/s12934-016-0520-9).

Phenotypic and morphological characterization of parent and all engineered strains

[00116] Since genetic transformation may result in phenotypic variation between the mutants and the parent strains, therefore the phenotype of the transformants generated was studied.

[00117] Figure 16 illustrates the growth and phenotypic characterization of PƒMig1⁸⁸ strain and the corresponding transformants of PƒOAO1, PƒOAO2 (expressing only CBH1) and PƒOAO3 on different polymeric carbon sources after 5 days of incubation. As shown in Figure 22, to investigate the influence of overexpression of the selected enzymes under study on growth and colonization of Pf Mig¹⁸⁸, an equal number of spores of PfMig¹⁸⁸ along with the best performing transformants of LPMO (PfOAOl), CBH1 (PfOAO2) and LPMO/CBH1 (PfOAO3) were spotted on SC agar plates which contains yeast nitrogen base and 2% polymeric carbon sources, i.e., Avicel, wheat bran, potato dextrose, xylan and CMC. P. funiculosum NC1M1228 which was the parent strain for the PfMig¹⁸⁸ strain was also taken as a control. The results showed a more compact organization of the colonies in PfMig¹⁸⁸ on all the carbon sources when compared with NC1M1228 where the colonies were found to be spreading broadly after incubation at 28 °C for five days. However, a slight reduction in the sizes of the colonies for the transformants of PfOAOl and PfOAO2 was observed when overexpressed individually in PfMig¹⁸⁸ on all the carbon tested.

Comparative analysis of cellulase expression in NCIM1228, PfMig¹⁸⁸ and the engineered strains

[00118] Conventionally, the transcriptional regulation of protein synthesis is key to understanding the mechanism of enzyme secretion by microorganisms. Since all the mutants generated in this study were earlier verified by PCR using specific primers for integration of the overexpressed enzymes in the genome, the transcript abundance of each overexpressed genes at the mRNA level was also examined to understand the overall regulation of the cellulase system. Additionally, due to the increase in cellulase activity observed in the enzymatic screening of the different transformants obtained, transcription of some of the major cellulases known to be produced both by NC1M1228 and PfMig¹⁸⁸ was effected under a de-repressing condition in all the mutants under study. Cultures of NC1M1228, PfMig1⁸⁸ and the best performing transformants of PfOAO1, PfOAO2 (only CBH1 expressing strain) and PfOAO3 were grown in 4% Avicel for 48 hours to get good mycelia growth. Transcript levels of all the five strains under de-repressing conditions were determined by real-time PCR with tubulin as control, and the relative fold change was normalized to NC1M1228 since it was the original strain from which all other mutants were generated. The results of the study are summarized in Table 3 - which identifies specific activities (U/mg protein) of selected biomass hydrolyzing enzymes of P.funiculosum NC1M1228, PƒMig1⁸⁸ and its resulting transformants

Table 3 : Specific Activities (U/mg protein) of selected biomass hydrolyzing enzymes of P.funiculosum NCIM1228, PƒMig1 ⁸⁸ and its resulting transformants

*Values represent mean ± standard deviations of triplicate determinations. Means with different superscript are significantly different (P < 0.05) across the column. Data were evaluated by one-way AN OVA and Duncan’s multiple range test with graphPad prism version8.1

[00119] For LPMO overexpression, a 5-fold increase in LPMO transcript for PfMig1⁸⁸ was observed while there was 18 and 26-fold increase in transcripts for PfOAOl and PfOAO3 strains.

[00120] Figure 17 describes the transcriptional expression of LPMO, cellobiohydrolase, endoglucanase, b-glucosidase, and xylanase in NC1M1228, PƒMig1⁸⁸ and all engineered strains measured by quantitative real-time PCR after growing for 48 h in the presence of 4% Avicel. As expected, there was no difference in the transcript level in LPMO gene for PfOAO2 transformant when compared to that of PfMig1⁸⁸ (see Figure 35). Likewise, for CBH1 overexpressing strains, about 4, 30 and 39- fold increase in CBH1 transcript was seen for PfMig¹⁸⁸, PfOAO2 and PfOAO3 strains respectively while there was no change in the CBH1 transcripts of PfOAOl (see Figure 35). When the transcript level for b-glucosidase (BGL) expression in all the strains was checked, the results showed a 5-fold change in BGL expression for PfMig1⁸⁸ and PfOAOl while there was 10 and 11-fold changes recorded for PfOAO2 and PfOAO3 mutants, respectively. The increase in the expression level of BGL in the two CBH1 transformants could be as a result of the increase in cellobiohydrolase level in the transformants. The increased cellobiohydrolase level in the media may yield more cellobiose, thereby providing signal to the cells to produce higher level of BGL to hydrolyze these cellobiose into glucose. However, no significant difference between the parent strain and all the mutants was observed in the expression level of endoglucanase (EG) and xylanase (XYL) (see Figure 35).

[00121] To examine and compare the influence of LPMO and CBH1 overexpression on the overall cellulase system, cultures of NC1M1228, PfMig¹⁸⁸ and the best performing transformants of all engineered strains were grown in cellulase-inducing medium (CIM). The resulting supernatants containing the secreted enzymes were recovered from the mycelia and used for all enzyme assays. Individual cellulase activities of LPMO, CBH, EG, BGL, XYL and FPU for all strains were assayed, compared and presented in Figures 20 to 26. PfMig¹⁸⁸ strain showed a 2-fold increase in all enzyme activities evaluated including the total extracellular protein. It was found that PfOAOl possessed the EG, XYL, CBH and FPU activities comparable to PfMig¹⁸⁸, which were much higher than that of the original strain NC1M1228. As expected, PfOAOl exhibited higher LPMO activity, which was 200% higher than that of PfMig¹⁸⁸. While there was no significant change in the activities of LPMO, EG and XYL in PfOAO2 strain, the activities of CBH1, BGL and FPU increased by 61%, 18% and 25%, respectively.

[00122] Figure 18 shows the CBH1 activity as determined using Avicel as the substrate. Figure 19 shows the LPMO activity as determined using Amplex red. Figure 20 shows the b-glucosidase activity using pNPG as substrate. Figure 21 shows the overall cellulase activity on filter paper. Figure 22 is the endoglucanase activity determined using CMC as the substrate. Figure 23 shows the xylanase activity measured using beechwood xylan as the substrate. Figure 24 is the total secreted proteins of all the strains. When all the activities of the LPMO/CBH1 double overexpression strain were checked, the strain showed 210%, 65% and 27% increment in LPMO, CBH and FPU activities while there was no difference in activities of EG and XYL when compared with PfMig¹⁸⁸ (see Figures 18 to 24).

[00123] In the course of evaluating the activities of all cellulolytic enzymes present in the secretome of all the engineered and parent strains, about 18% increase in BGL activity was observed in the secretome of CBH1 overexpressing strains. The increase in BGL production in the two transformants is believed to be a result of the increase in cellobiohydrolase abundance as earlier seen at the mRNA level. Further, the increase in BGL production by PfOAO2 and PfOAO3 transformants relative to PfMig1⁸⁸ was further confirmed by zymography analysis (see Figures 25 to 26). Figure 25 shows the protein profile for the secretome of all strains separated on SDS-PAGE. Lane M, molecular weight marker. Lanes 1, 2, 3, 4, 5 were secretome of NCIM 1228, PƒMig1⁸⁸, PƒOAO1, PƒOAO2 and Pf DA03, respectively. Figure 26 shows the MUG-zymogram assay for identification of b- glucosidase in the secretome of all strains. Lane M, molecular weight marker. Lanes 1, 2, 3, 4, 5 were secretome of NCIM 1228, PƒMig1⁸⁸, PƒOAO1, PƒOAO2 and PƒOAO3, respectively.

[00124] In addition, reference to Table 3 above establishes that the specific activities of the overexpressed enzymes also increased in addition to volumetric activities. The result summarized in Table 3 show a significant increase in the specific activity of LPMO in PfOAOl and PfOAO3 strains relative to that of PfMig¹⁸⁸. Similarly, there was a significant increase in specific activities of CBH1, BGL and FPU in PfOAO2 and PfOAO3 overexpressing strains. However, there was no significant difference in the specific activities of EG and XYL across all the strains. The increase in specific activities of the key hydrolytic and oxidative enzymes establishes that the performance of the enzyme mixture produced by the overexpressed strains have been enhanced when compared with PfMig¹⁸⁸ and is believed to enable reduction of the enzyme load required for biomass saccharification.

Cellulase enzyme complex with LPMO and CBH1 overexpression showed higher hydrolysis efficiency against nitric acid treated biomass at high substrate loading

[00125] To assess the relevant industrial application of overexpression of these two key enzymes, the secretome from ah the engineered strains as well as the parent strains was used to investigate their saccharification performance on acid pretreated wheat straw (PWS). The saccharification reaction was setup using 20% substrate loading of PWS, and secretome of NCIM1228, PfMig1⁸⁸, PfOAOl, PfOAO2 and PfOAO3 at same enzyme loading of 30 mg/gDBW, and incubated at 50 °C for 96 hr. Samples were collected every 24 hr and analyzed for production of fermentable sugars. The results for the time course of the PWS hydrolysis are presented in Figures 27 and 28. Figure 27 shows the time course profile for the saccharification of nitric acid pre-treated wheat straw by the secretome of NCIM1228, PƒMig1⁸⁸ and the all engineered strains at 20% solid loading and protein concentration of 30 mg/g biomass - and shows the percentage sugar release measured at 24-hour interval over 96 h saccharification period. Figure 28 shows the total fermentable sugar obtained at 72h saccharification time point.

[00126] From the results, a linear increase in the concentration of reducing sugars and hydrolysis efficiency was seen in all the strains with increasing time until the 72h after which no appreciable increase in sugar concentration was observed. However, since the time point usually recommended for efficient and complete saccharification of cellulosic biomass is 72 hours (see Klein-Marcuschamer et al., 2011), the data obtained in this study at the 72 hour time point was thus used to compare the saccharification performance of all the strains (see Figures 27 and 28). About 27% holocellulose (cellulose + hemicellulose) conversion was obtained in case of NC1M1228 secretome whereas more than 64% of the pretreated biomass was hydrolyzed by PfMig¹⁸⁸ secretome where the total reducing sugars (glucose + xylose) recorded were 37.5 g/L and 90 g/L, respectively (see Figure 27 - which shows the time course profile for the saccharification of nitric acid pre-treated wheat straw by the secretome of NC1M1228, PƒMig1⁸⁸ and the all engineered strains at 20% solid loading and protein concentration of 30 mg/g biomass, (a) Percentage sugar release measured at 24-hour interval over 96 h saccharification period). The remarkable increase in biomass hydrolyzing capacity of PfMig¹⁸⁸ secretome could majorly be attributed to an increased proportion of CBH, EG and BGL being secreted due to the absence of functional CCR in PfMig¹⁸⁸. The 64% hydrolysis achieved with the secretome of PfMig¹⁸⁸ at 20% solid loading was highly significant.

[00127] When the hydrolysis ability of the cellulases produced by the LPMO overexpressing strain on PWS relative to PfMig¹⁸⁸ was evaluated, a marginal increase in the concentration of reducing sugars and cellulose conversion was observed. It was found that the amount of reducing sugars released by PfOAOl secretome was 99 g/L, which corresponded to 70% holocellulose conversion, after a total enzymatic reaction of 72 h which was about 6% increase over that of PfMig¹⁸⁸ (Figures 27 and 28). The observed marginal increase is believed to be due to the synergistic cooperation between the hydrolytic cellulases and the overexpressed accessory enzyme. Similarly, the total reducing sugar released by secretome of CBH1 overexpressing strain was 107 g/L which corresponded to 77% holocellulose conversion and 12% increase over that of PfMig¹⁸⁸. The enhanced saccharification obtained with the PfOAO2 strain could not only be due to the increase in cellobiohydrolase level in the system but also linked to the enhancement in the production of b-glucosidase as seen earlier (see Figure 40). This is believed to have facilitated the enhanced enzymatic degradation of PWS since BGL has the capacity to hydrolyze cellobiose to glucose in the final step and relieve the feedback inhibition of cellobiose on the activities of CBH and EG (see Qian, Y., Zhong, L, Gao, J., Sun, N., Wang, Y., Sun, G., Qu, Y., Zhong, Y., 2017. Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues Microbial Cell Factories. Microb Cell Fact 16, 207. https://doi.org/10.1186/s12934-017-0825-3).

[00128] However, it was observed that there was no significant increase in hydrolysis of hemicellulose in all the engineered strains when compared with that of PfMig1⁸⁸ - which is established by the data in below Table 4.

Table 4 : Yield of fermentable sugars obtained at 72h saccharification of nitric acid treated wheat straw by the secretome of NCIM1228, PƒMig1 ⁸⁸ and all engineered strains

*Values represent mean ± standard deviations of triplicate determinations. Means with different superscript are significantly different (P < 0.05) across the column. Data were evaluated by one-way AN OVA and Duncan’s multiple range test with graphPad prism version 8.1 [00129] When both LPMO and CBH1 were co-overexpressed, the total reducing sugar released by the secretome of the strain increased to 112 g/L corresponding to 82% holocellulose conversion (see Figures 27 and 28). This shows that the simultaneous over- expression of LPMO and CBH1 provided a highly significant increase in saccharification efficiency compared to the individual ones. The results, therefore indicate that co- overexpression of major cellulase components and its accessory enzyme could facilitate the creation of a more efficient cellulolytic system for optimal hydrolysis of cellulosic biomass at high substrate loading.

[00130] The present invention accordingly provides a significantly more efficient cellulase cocktail with an enhanced saccharification performance on lignocellulosic biomass at high substrate loading, by combining overexpression of LPMO and CBH1 in PfMig1⁸⁸. The LPMO/CBH1 engineered cellulase system has been found to exhibit a significantly enhanced cellulose conversion after 72 hour enzymatic saccharification of PWS at 20% loading, in comparison with PfMig1⁸⁸.

Deposit of Biological Material at Depository Institution [00131] The details ofbiological materials disclosed in the present invention, that have been deposited at the depository institution Microbial Type Culture Collection (MTCC) and Gene Bank, Chandigarh, India are provided in Table 5 below.

Table 5: Details of engineered strains of Penicillium funiculosum generated in accordance with the present invention, along with their respective accession numbers

[00132] While the exemplary embodiments of the present invention are described and illustrated herein, it will be appreciated that they are merely illustrative. It will be understood by those skilled in the art that various modifications in form and detail may be made therein without departing from or offending the spirit and scope of the invention as defined by the appended claims. Additionally, the invention illustratively disclose herein suitably may be practiced in the absence of any element which is not specifically disclosed herein - and in a particular embodiment that is specifically contemplated, the invention is intended to be practiced in the absence of any one or more element which are not specifically disclosed herein.

Claims

We Claim:

1. A transformed fungal strain of Penicillium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25371.

2. A fungal strain for production of cellulolytic enzymes or cellulolytic enzyme complexes, comprising a transformed fungal strain of Penicillium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25371.

3. A transformed fungal strain of Penicillium funiculosum, comprising the nucleic acid sequence of SEQ ID No. 28.

4. A fungal strain for production of cellulolytic enzymes or cellulolytic enzyme complexes, comprising a transformed strain of Penicillium funiculosum, wherein said transformed fungal strain of Penicillium funiculosum comprises a nucleic acid sequence of SEQ ID No. 28.

5. An integration cassette for producing a transformed fungal strain of Penicillium funiculosum, said integration cassette comprising a nucleic acid sequence of SEQ ID No. 28.

6. A plasmid for producing a transformed fungal strain of Penicillium funiculosum, said plasmid comprising a nucleic acid sequence of SEQ ID No. 26.

7. A method for producing a transformed fungal strain of Penicillium funiculosum comprising the nucleic acid sequence of SEQ ID No. 28, the method comprising the steps of: synthesizing a plasmid comprising a nucleic acid sequence of SEQ ID No. 26; and synthesizing a transformant by exposing a fungal strain of Penicillium funiculosum bearing accession number Microbial Type Culture Collection (MTCC) 25141, to the synthesized plasmid.

8. The method as claimed in claim 7, wherein synthesizing the transformant comprises transforming to the fungal strain of Penicillium funiculosum , a plasmid comprising a nucleic acid sequence of SEQ ID No. 26.

9. The method as claimed in claim 7, wherein synthesizing the transformant comprises integrating to the fungal strain of Penicillium funiculosum , an integration cassette comprising a nucleic acid sequence of SEQ ID No. 28.

10. The method as claimed in claim 7, wherein the transformant is synthesized based on agrobacterium mediated fungal transformation method.