Genetically transformed fungal strain for preparing a leather-like bioproduct and methods for obtaining said bioproduct

Abstract

Genetically transformed fungal strains for preparing a leather-like bioproduct and methods for obtaining said bioproduct, wherein the transformed strain has an increased carbohydrate hydrolytic activity as compared to the parent wild type strain and overexpressing genes such as swi6B, glsnf 1, gcn4, and / or nmnat.

Description

[0001] Docket No.: 1063.035

[0002] GENETICALLY TRANSFORMED FUNGAL STRAIN FOR PREPARING A LEATHER-LIKE BIOPRODUCT AND METHODS FOR OBTAINING SAID BIOPRODUCT

[0003] The present invention refers to genetically transformed fungal strains for preparing a leather-like bioproduct and methods for obtaining said bioproduct, where the transformed strain has an increased carbohydrate hydrolytic activity as compared to the parent wild type strain

[0004] BACKGROUND OF THE INVENTION

[0005] Mycelium-based biomaterials, obtained from the vegetative part of filamentous fungi, have garnered significant interest as sustainable alternatives both to animal leather as well as to petroleum-based synthetic leather-like alternatives. Bioleathers are often based on the use of agricultural and industrial residues, aligned with circular economy strategies and responding to the increasing demands of eco-friendly materials for new consumers [1]. In this context, mycelium-based bioleathers have emerged as a promising alternative.

[0006] The number of patents related to these biomaterials has increased significantly since 2019 and are primarily directed to fermentative strategies to generate a fungal biomass intended for use in mycelial materials having leatherlike properties. Three general strategies may be summarized as follows [2]:

[0007] Solid-state surface fermentation (SSSF) involves introducing a fungal organism into a solid growth substrate, allowing for the development of mycelial tissue at the surface of the substrate. It usually employs lignocellulosic substrates, similar to those used in the cultivation of edible mushrooms, due to their affordability and availability [3-5].

[0008] Liquid-state surface fermentation (LSSF) may either employ lignocellulosic fibers mixed into a liquid broth or use a fully dissolved nutrient solution, resulting in the growth of a fungal tissue at the liquid-air interface in a Docket No.: 1063.035 static system [6].

[0009] Stirred submerged liquid fermentation (SSLF), entails the cultivation of submerged fungal biomass in a liquid medium using a bioreactor, a bubble column reactor or a shake flask setup [7].

[0010] The drawback of the first two strategies (SSSF and LSSF) is the use of lignocellulosic residues as substrates which generate solid blocks or a semi-solid sludge as a residual material at the end of the process. These residual materials require composting or other disposal treatments.

[0011] In the case of the third strategy (SSLF), mycelial growth does not form a continuous sheet, but rather a lower cohesion broth with sludge or pellets which requires further processing to form a consistent piece of material.

[0012] In brief, in all cases, the presence of liquid or solid residues implies the need of further treatment operations.

[0013] In addition to the fermentation technique, process efficiency depends on fungal metabolism and on the selection of substrates which may be totally consumed by the end of the cultivation process. Carbon sources not only provide energy, but also act as gene expression, metabolism and growth regulators through signaling networks which allow cells to sense and adapt to changing environmental conditions.

[0014] These pathways prioritize the use of preferred sources and regulate the synthesis of degradative enzymes depending on nutrient availability. More specifically, a detailed knowledge of the enzymatic regulation of carbohydrate degradation is essential in order to optimize the use of a wide range of substrates and to enhance efficiency of mycelial bioleather production.

[0015] BRIEF DESCRIPTION OF THE INVENTION

[0016] Genetically transformed fungal strains or derivatives thereof for preparing a leather-like bioproduct are provided, where the transformed strain has an Docket No.: 1063.035 increased carbohydrate hydrolytic activity as compared to the parent wild type strain. In a preferred embodiment, the strain overexpresses the swi6B, glsnfl, gcn4, and / or nmnat genes when compared to the parent wild type strain. The strain may belong to one of the genera Ganoderma, Pleurotus, Pignoporus, Trametes, Hericium, or any other modified strain that exhibits increased carbohydrate hydrolysis. In a preferred embodiment, the modified swi6B gene comprises the nucleotide sequence set forth in SEQ ID NO. 1 ; the modified glsnfl gene comprises the nucleotide sequence set forth in SEQ ID NO. 2; the modified gcn4 gene comprises the nucleotide sequence set forth in SEQ ID NO. 3; the modified nmnat gene comprises the nucleotide sequence set forth in SEQ ID NO. 4, all of which are included in the genome of the transformed strain.

[0017] Also provided is a method for producing a leather-like bioproduct comprising the following steps: a) growing a transformed strain having an increased carbohydrate hydrolytic activity when compared to the parent wild type strain; b) arranging a sterile solid substrate on a surface; c) contacting an inoculum of the strain grown in step a) with the solid substrate of step b) and incubating it; d) harvesting the mycelium and inactivating the strain; and e) treating the mycelium until a leather-like bioproduct is obtained. The genetically modified strain may belong to the genera Ganoderma, Pleurotus, Pignoporus, Trametes, Hericium, or others.

[0018] The solid substrate may consist of lignocellulose, starch or combinations thereof. For example, and without limitation, the lignocellulosic substrate may comprise: barley scannings, grape lex, beer brewing residue, grape pomace, barley rootlets, soybean hulls, carrot waste, sunflower hulls, peanut hulls, peanut pods. For example, and without limitation, a starch substrate may comprise: edible paper sheets, potato starch-based sheets, cassava starch-based sheets, corn starch-based sheets. When the solid substrate is lignocellulose, the water / substrate ratio is at least 70%.

[0019] In step c) the inoculum may comprise a biomass concentration from 0.8 to Docket No.: 1063.035

[0020] 4 g / L. Step b) comprises providing an amount of solid substrate of from 1 to 3 edible paper sheets. In another embodiment, a fabric may be sandwiched between the edible paper sheets in step b), wherein the fabric comprises at least 90% cotton and has a pore size from 0.0188 to 2.065 pixels2. For example, the fabrics may comprise, without limitation, canvas, LTA80 canvas, raw cotton canvas (Raw 20x20), raw washed linen (Stone Washed), Milano linen, natural linen, combed cotton jersey, pre-washed lightweight tussock, or Gravity gauze.

[0021] In the above method, the incubation time of step c) may comprise from 3 to 10 days and step e) may comprise, for example, a treatment with 10- 30% glycerol for 2-4 hours. Incubation days may vary depending on the strains, medium and growth conditions. Regarding the post-treatment of the mycelial sheet of step e), it may vary according to the desired bioleather, for example, glycerol, polyethylene glycol (PEG), or sorbitol may be used.

[0022] Further provided is a method for producing a leather-like bioproduct, comprising the following steps: a) cultivating a strain selected from the group consisting of Ganoderma, Pleurotus, Pignoporus, Trametes, and Hericium in a liquid medium, b) arranging a sterile solid substrate on a surface; c) contacting an inoculum of the cultivated strain of step a) with the solid substrate of step b) and incubating it; d) harvesting the mycelium and inactivating the strain; and e) treating the mycelium until a leather-like bioproduct is obtained.

[0023] In a preferred embodiment, the strain is Ganoderma and the solid substrate primarily comprises starch (90%) and may be, without limitation, edible paper sheets, and / or a potato starch-based film, and / or cassava starch-based films, and / or films corn starch-based. In step c) the inoculum comprises a biomass concentration from 0.8 and 4 g / L, in step b) the solid substrate is from 1 to 3 edible paper sheets. The method may also comprise sandwhiching a fabric between the edible paper sheets, wherein the fabric is made of cotton with a pore size from 0.0188 and 2.065 pixels2. For example, the fabric may comprise, without limitation, canvas, LTA80 canvas, raw cotton canvas (Raw 20x20), raw Docket No.: 1063.035 washed linen (Stone Washed), Milano linen, natural linen, combed cotton jersey, pre-washed lightweight tussock, or Gravity gauze.

[0024] Regarding post-treatment of the mycelial sheet of step e), it may be selected according to the desired bioleather, for example, from glycerol, polyethylene glycol (PEG), or sorbitol.

[0025] Further provided is a leather-like bioproduct manufactured using any of the methods described herein.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the biomass production (left) and productivity (right) during culture period.

[0028] Figure 2 shows relative biomass of each medium with respect to M1 under the same growth conditions.

[0029] Figure 3 shows ATR-FTIR spectra of the five different types of samples after 10 days of growth. Spectral range 500-3500 cm-1(A) and extended range (B) for the chitin region.

[0030] Figure 4 shows SEM images of mycelium produced with grape pomace, beer brewing residue as WS-SSF substrates and edible paper as TC-SSF substrate.

[0031] Figure 5 shows the relationship between the biomass obtained in liquid culture medium M1 and the thickness of the leather-like biomaterial.

[0032] Figure 6 shows a photograph of mycelium produced with L-like cotton fabric and edible paper as a substrate; (left) upper side, (right) bottom side.

[0033] Figure 7 is a schematic representation of the new designed plasmid pMB. In this plasmid, E1-E8 correspond to restriction enzymes that modularly modify each element to generate new combinations. The gene of interest (G) is cloned between E3 and E4 under the control of the Pgpd promoter. Alternative forms of the plasmid include the presence of sdhB* with introns (1261 pb) or without Docket No.: 1063.035 introns (738 pb), as well as any of the following genes at position “G”: swi6B, gcn4, gslnf 1 , nmnat, or egfp, with or without a gpd (e-i) exon-intron region. The horizontal arrows indicate the location of the primers listed in Table 6 (M&M). Primer 0-4 only hybridizes with sdhB* with introns; 0-3 only hybridizes in versions containing the gpd exon-intron-exon region; 0-5 is specific for the swi6B gene; 0-9 for gcn4', 0-10 for gslnfl , and 0-1 1 for nmnat.

[0034] Figure 8 shows wild type (wt) and pMB06-transformed G. sessile strains, grown in the presence of selected concentrations of carboxin.

[0035] Figure 9 shows microscope images of wild type (wt) and pMB06- transformed G. sessile strains, obtained with phase-contrast and the green fluorescence channel corresponding to eGFP emission.

[0036] Figure 10 shows cellulase activity (U / mL) measured on culture days 7 and 10 for the wild-type strain and three different mutants (SN46, SW06, and NM12). Data are presented as standard error of the mean (SEM).

[0037] Figure 1 1 is a schematic representation of plasmid pMB08 by way of example: -carboxin resistance cassette: PsdhB-sdhB*-TsdhB; expression cassette for a gene of interest: Pgpd-sw / 6S-TsdhB; E1 : Sacl; E2: Not I; E3: Xhol; E4: EcoRI; E5: Bglll; E6: BamHI; E7: Kpnl; E8: Smal.

[0038] DETAILED DESCRIPTION OF INVENTION

[0039] The WS-SSF method of the invention is a solid-state fermentation method in which a mycelium grows on the surface of a solid lignocellulosic substrate containing water in excess of its water-retention capacity (WRC). This excess of moisture promotes surface growth and allows for lifting the mycelial sheet without detaching any material of the underlying solid substrate.

[0040] The TC-SSF method of the invention is a solid-state fermentation method that employs a starch-based substrate, such as edible paper or any other starch film or gel, wherein the substrate is completely consumed by the end of Docket No.: 1063.035 fermentation. Initially, the substrate serves as a support for fungal growth, but it is completely transformed into mycelium, resulting in a method that leaves no solid residues.

[0041] These methods overcome the drawbacks of solid-state surface fermentation (SSSF) and the SSF methods with solid lignocellulosic feedstocks.

[0042] The term starch substrate refers to non-lignocellulosic substrates such as commercial edible paper, starch-based films or substrate films with a high content of starch.

[0043] The terms “bioleather” and “leather-like bioproduct” have the same meaning and are interchangeable.

[0044] In the TC-SSF method, a commercial edible paper or starch film is used as substrate, allowing the mycelium to grow, invade, degrade and completely transform starchy materials, resulting in no solid residues. Furthermore, a liquid culture was used as inoculum instead of mycelial seeds, thus avoiding the problems associated with remnant seeds.

[0045] Liquid cultures of the fungal strains were grown at high density and with high fungal biomass, which later were used as an inoculum for the subsequent solid-state fermentation step, and production of the leather-like bioproduct. The growth of the different fungal strains in the liquid medium formed mycelium pellets. In a preferred embodiment, Ganoderma sessile showed the greatest biomass production in liquid culture, while the other strains (Ganoderma lucidum, Pleurotus ostreatus, Pignoporus sanguineus, Trametes versicolor, and Hericium erinaceu) developed a lesser but usable biomass, of from 0.6 to 2.9 g / L by days 7-10 after starting the culture.

[0046] The Ganoderma sessile inoculum used for liquid fermentation was prepared in two ways: adding 10-20 portions (1 sq cm each) of agar from plate cultures per each 100 mL of medium, or adding 50 g of mycelial seeds with the strain per liter of liquid medium. Biomass monitoring during culture showed a progressive increase, with a maximum peak reached by days 6 and 7, when using Docket No.: 1063.035

[0047] M1 medium at 30 °C (Figure 1 ). A productivity analysis indicated that six culture days were sufficient to obtain optimal results, as shown in Figure 1 .

[0048] In order to optimize the composition of M1 medium, various carbon and nitrogen sources were evaluated, considering both biomass production as well as the cost of raw materials. The results for the newly developed media are summarized in Tables 14 and 15 shown in the Examples. It should be noted that the addition of other alternative carbon sources, or a combination thereof (starch, maltodextrin, glucose, or malt extract), as well as a new nitrogen source, (NH4)2HPO4, significantly improved biomass production by the sixth day of culture, at 30 °C, with respect to M1. Some media, such as M2T, M2NT, and M6T, were supplemented with a 1 :200 dilution of the trace element solution (described in Table 16 of the Examples). However, the impact of adding trace elements on biomass production was negligible.

[0049] Likewise, different mass ratios of yeast extract / (NH4)2HPO4were evaluated: 10:0 in M1 , 5:0 in M2, 1 :5 in M2n and M6n, and 2.5:2.5 in M2N, M2NCa, M2NT, M6Nb and M6NCa. The presence of (NH4)2HPO4generates a good biomass production and a decrease of final pH of the culture with respect to M1 . The initial pH of non-inoculated media was from about 5.5 to 5.8.

[0050] Biomasses and pH after 6 days of fungal growth at 30 °C for all tested media are summarized in Table 1 .

[0051] T able 1 : Biomasses and final pH obtained with the different med ia. Docket No.: 1063.035

[0052] Figure 2 shows the relative biomass of each medium as compared with M1 after 6 days of culture. All culture media listed in Table 1 produced mycelial sheets with favorable features. All tested media exhibited an excellent biomass yield. All alternative media provided advantages in terms of cost-efficiency, culture time, biomass yield and formulation.

[0053] The minimum biomass concentration required for producing bioleather with suitable features was 0.8 g / L, which corresponds to an inoculum of 3.2 g of fungal biomass by square meter of tray surface. All liquid cultures may be stored at 4-8 °C for at least 40 days, without affecting growth or compromising viability. As previously defined, wet solid-state fermentation (WS SSF) involves growth of mycelium on the surface of a solid lignocellulosic substrate with excess water. The high water content (60-80%) prevented the mycelium from penetrating the substrate, thus favoring its growth, primarily on the surface.

[0054] The evaluated substrates included: beer brewing residues from various brewing methods (Stout, Golden, Scottish, and Keinkater), barley scannings, barley rootlets, soybean hulls, grape pomace, grape lex, bran, carrot bagasse, sunflower hulls, peanut hulls, and peanut pods. The performance of each substrate for use in the WS-SSF method was evaluated with different fungal Docket No.: 1063.035 strains, including Ganoderma lucidum, Ganoderma sessile, Pleurotus ostreatus, Pignoporus sanguineus, Trametes versicolor, and Hericium erinaceus.

[0055] All substrates were found to be suitable. However, preferably the most suitable to form the bioleather were: barley scannings, grape lex, beer brewing residues, and grape pomace. The remaining substrates also performed well after modifying some of the growth conditions.

[0056] Substrates were also evaluated for their percent composition and waterretention capacity (WRC). The results are summarized in Table 2. Table 2: Percent composition of the residues on a dry base (g / 100 g of dry material) and water-retention capacity (WRC)

[0057] Water content should be in excess as compared to WRC values in order to favor surface growth and allow for lifting the mycelial sheet without dragging any solid substrate. An available water / substrate ratio of at least 70% was used, although in some cases it could reach up to 86 %, depending on the substrate. This amount should be sufficient to allow the mycelium to be harvested as clean as possible, i.e., without any adhering substrate. The substrates were sorted as Docket No.: 1063.035 follows according to the excess water required to obtain a mycelial sheet on the surface: barley rootlets > barley scannings > grape lex > beer brewing residue > grape pomace.

[0058] The appearance and characteristics of the mycelial sheet were outstanding, in a few cases a minimal part of substrate remained adhered to the bottom side of the mycelium along with the mycelial seeds used as inoculum.

[0059] The results of producing leather-like bioproducts by wet solid-state surface fermentation (WS-SSF), using lignocellulosic feedstocks, yielded very good results when appropriate amounts of water were added. Substrates such as grape pomace and beer brewing residue exhibited a low water-retention capacity and formed very resistant leathery mycelia after 10 days.

[0060] As the use of mycelial seeds as an inoculum affects both sensory and mechanical final properties of bioleather the method was also evaluated using an inoculum grown in M1 medium and achieving the water availability ratio previously determined as optimal (an available water / substrate ratio of at least 70%). Similar results were obtained, with the advantage of preventing seeds from adhering to the back of the mycelium.

[0061] Furthermore, it was determined that the substrate may be consumed at proportions exceeding 70%, 75%, or even 80 %, unlike the residual solid blocks typically observed with other methods.

[0062] ATR-FTIR spectroscopy was used for characterizing the chemical composition of the mycelial sheets. The spectra showed significant differences, derived from the different substrates employed. Figure 3 shows typical ATR-FTIR spectra of the five different types of samples after 10 days of growth and fermentation. Mycelium infrared absorption spectra are associated with their composing biomolecules, for example: lipids (3000-2800 cm-1, -1740 cm-1), proteins (amide I in 1700-1600 cm-1, amides II and III in 1575-1300 cm-1), nucleic acids (1255-1245 cm-1) and polysaccharides (1200-900 cm-1) [8]. In particular, the bending band C-H at 1375 cm-1was attributed to chitin, the main Docket No.: 1063.035 molecule responsible for the tensile strength of the mycelium. It is worth noting that, in the case of grape pomace and edible paper, a prominent peak corresponding to the chitin band (1368 cm-1) is observed. Likewise, a more intense peak for the polysaccharide band (1035 cm-1, C-C stretching) was recorded.

[0063] Regarding the proteic bands, a mycelium obtained from barley scannings and grape lex showed higher peaks. Similarly, in the case of lipids, a grape lex mycelium showed a more prominent peak. It was demonstrated that there is a correlation between the proteic and lipid content in the mycelium and its resistance. Among the tested substrates, grape pomace and edible paper produced the best mycelia, consistent with the chitin peak observed in the FTIR spectra. In a preferred embodiment, grape pomace and / or edible paper are used as substrate.

[0064] The mycelial sheets were also characterized by the SEM method. Dry mycelial sheets were covered with gold and observed using a LEITZ AMR1000 scanning electron microscope (SEM). Figure 4 shows SEM images of mycelia produced from lignocellulosic residues (grape pomace and beer brewing residue) and a starch material (edible paper) used as substrate. All the samples were obtained under identical growth conditions. The images reveal that size and morphology of G. sessile hyphae are affected by the type of substrate.

[0065] In the case of grape pomace, hyphae were thin, curved, and continuous, while mycelium grown on beer brewing residue have short, thick, and discontinuous hyphae. When edible paper was used as substrate, the resulting mycelium was denser than with lignocellulosic substrates, showing thin, linear hyphae.

[0066] In another embodiment, a more simple and scalable method was used, defined as totally consumed solid-state fermentation (TC-SSF). The TC-SSF method refers to a method of fermentation and formation of a mycelial sheet wherein the substrate mainly consists of a starch material in the form of a sheet Docket No.: 1063.035 or film; for example, and without limitation, edible paper or any other consumable film or gel. This method employs the substrate as a support for fungal growth, which is then completely transformed into mycelium, without generating any solid residue at the end of the fermentation.

[0067] The TC-SSF methods were carried out by placing one, two, or three commercial edible paper sheets over the entire surface of the trays. Then, the trays were covered with liquid inoculum grown in M1 medium, or other media. Biomass concentration of the inoculum grown in M1 medium was used in the range of 0.8 - 4 g / L, while maintaining an inoculum volume-to-area ratio of 4-4.5 L / m2. The trays were covered with sheets of aluminum foil and incubated in ovens at 30 °C for 7- 10 days in darkness. After harvesting, the mycelium was dehydrated at 90 °C for 4 hours to inactivate the fungus.

[0068] The results showed that, when one or two edible paper sheets were incubated with the liquid inoculum, the substrate was completely consumed and transformed into mycelium, leaving no solid residues. In contrast, when three sheets were used, fragments of unhydrolized edible paper remained at the end of the process, indicative of the need for longer culture periods, for example, for more than 10 days.

[0069] In a preferred embodiment, two sheets of edible paper may be used as a substrate, regardless of the commercial brand. The dry weight results of the mycelial sheet are summarized in Table 3.

[0070] In the TC-SSF method, CO2levels in the oven showed a marked decrease by the fourth day of incubation; and further a complete colonization of the tray by mycelium was observed.

[0071] On day 4, a modification of the TC-SSF method was carried out by transferring the mycelial sheet to a new tray supplemented with two edible paper sheets. Incubation was extended until day 10.

[0072] Although the mycelium was harvested without leaving any remnant unhydrolized substrate, its dry weight did not increase when compared to the Docket No.: 1063.035 method without any tray transfer (see Table 3).

[0073] Table 3: Dry weight of the mycelial sheet expressed as units of mass (g) and mass / area (g / m2).

[0074] In another preferred embodiment, an aliquot of liquid culture grown in medium was collected as inoculum, placed on a film or starch film prepared with potato, cassava, or corn starch, and supplemented, or not, with 2-5% sunflower or corn seed oil. Biomass concentration of the inoculum grown in M1 medium was used in the range of 0.8 - 4 g / L, while maintaining the inoculum volume-to-area ratio of 4-4.5 L / m2. Fermentation was carried out for 7-10 days in a closed oven, at 30 °C, in the dark and without gas addition. Subsequently, the mycelium was harvested and complete consumption of the starch substrate was observed, leaving no residue.

[0075] The mycelial sheets obtained from each type of starch film supplemented with vegetable oil showed a good dry weight, comparable to that obtained using edible paper substrates (see Table 4). Docket No.: 1063.035

[0076] Table 4: Dry weight of mycelium expressed in units of mass (g) and mass / area (g / m2) and thickness (mm) of bioleather obtained from different starch substrates films. In another preferred embodiment, fabric was added to the TC-SSF method. Different cotton fabrics with variations in warp and pore size were selected and added at the beginning of the TC-SSF method. In general, fabrics with larger pore size promoted an enhanced integration of the mycelium with the textile structure. Table 5 summarizes pore sizes of the selected fabrics, determined through analysis of images obtained by optical microscopy.

[0077] Table 5: Analysis of pore sizes obtained by optical microscopy and converted to mm2 by calibration. Docket No.: 1063.035

[0078] The selected fabrics were added to the trays according to the following sequences:

[0079] • Si: Fabric - Edible paper sheet or starch film - liquid inoculum

[0080] • S2: Edible paper sheet or starch film - fabric - liquid inoculum

[0081] • S3: Edible paper sheet or starch film - liquid inoculum - fabric

[0082] Systems S1 and S2 were effective strategies for obtaining bioleathers, with system S3 yielding the best results, in both appearance and mechanical properties of the bioleathers thus produced.

[0083] Notably, in all cases, at the end of the TC-SSF method, following incubation at 30°C, the edible paper substrate was completely hydrolyzed, with no remnant solid residue and the fabric was fully integrated into the mycelial sheet. The S3 strategy was tested with all selected fabrics.

[0084] In another preferred embodiment, trays were assembled according to sequence S3 but a transfer of mycelium was carried out on the fourth day of growth to a new tray without adding substrate (edible paper) or additional liquid inoculum but inverting the mycelium. In this way, the mycelium-covered surface was placed in contact with the tray. Then, fermentation continued until day 10 as previously described at 30 °C on the sheet surface that had previously been in contact with the tray. This allowed the mycelium to develop on both sides of the piece. This embodiment resulted in mycelial sheets with uniform surfaces, concealing the fabric inside. The resulting bioleather showed two similar sides and an improved aesthetics; furthermore thickness of the harvested mycelium increased significantly, reaching values of 3-5 mm.

[0085] In addition to starch films, substrates, such as guar gum, xanthan gum, Docket No.: 1063.035 agar-agar, other polysaccharides and gelatin, may be prepared.

[0086] Once the mycelium was harvested after the 7-10 incubation days, a drying method was applied in order to inactivate the strains, thereby preventing further growth and / or production of undesirable secondary metabolites. In all cases, drying was carried out in a forced-convection oven at 90 °C for 4 hours. After this stage, the mycelial sheets were submerged into a 12.5% glycerol solution (within the tested range of 10-30%) for 3 hours at room temperature. Subsequently, the sheets were dried again in a forced-convection oven at 40 °C for 1 hour to obtain the “bioleather”. This glycerol post-treatment improves appearance and plasticity of the bioleather.

[0087] Variations of biomass concentration in the inoculum did not show a significant effect on the dry weight per area unit of bioleather when these are within the range from 1 .1 to 4.4 g / L, as shown in Figure 5.

[0088] The bioleather thus obtained does not require to be treated with tannins, as is the case of animal leather; the results show that this step is not necessary when starch-based substrates are used, without compromising the mechanical properties of the resulting material. The production method is thus simplified, thereby reducing both time and costs, while also improving overall sustainability of the bioleather. Figure 7 shows the two sides of a bioleather produced with cotton canvas, liquid inoculum in M1 , and edible paper as TC-SSF substrate.

[0089] The results revealed marked differences in the mechanical properties, thickness, and appearance of the bioleathers depending on the substrate. The bioleathers derived from barley scannings, beer brewing residue, and grape pomace showed better resistance to mechanical testing (see Table 6). Based on the above, it was demonstrated that the percent composition of the substrates affects the mechanical properties of bioleathers. Docket No.: 1063.035

[0090] Table 6: Mechanical properties of bioleathers using agroindustrial residues as substrates in the WS-SSF method

[0091] Regarding tensile strength, bioleathers obtained from different starch films (potato, cassava, and corn) reached comparable results. Table 7 summarizes the mechanical properties of bioleathers produced with starch-based substrates using the TC-SSF method without incorporating fabric.

[0092] Table 7: Mechanical properties of bioleathers produced from starch substrates by TC-SSF, without fabric. Docket No.: 1063.035

[0093] The method that employed edible paper as substrate with incorporation of fabric produced a bioleather which significantly improved tensile strength when compared with the bioleather obtained using the same TC-SSF method but with no fabric. The mechanical properties of bioleathers with fabric were evaluated for

[0094] 5 two fabric orientations: parallel and perpendicular.

[0095] Table 8 summarizes the mechanical properties of bioleathers produced from starch-based substrates using the TC-SSF method with fabric, employing two edible paper sheets as substrate following sequence S3.

[0096] 10 Table 8: Mechanical properties of bioleathers produced from starch substrates by TC-SSF, with fabric. Docket No.: 1063.035 Docket No.: 1063.035

[0097] The addition of fabric according to sequence S3 for producing bioleather, substantially improved the mechanical properties of bioleather (see Table 7). While all tested fabrics yielded good results, three bioleathers were selected whose fabrics provided the best results regarding aesthetics, as the mycelium 5 penetrates the fabric, thereby completely and homogenously covering the surface. The tear resistance of fabric-containing bioleathers was analyzed using the “trouser” tear type method (trouser tear method), evaluating both maximum strength (Fmax) and average strength (Fave). As may be seen in Table 9, bioleathers with raw washed linen (SWL) fabrics and LTA80 canvas improved io tear resistance in both directions (parallel and perpendicular). However, the results indicated that all three fabrics performed well against tearing in the bioleather production. The results are summarized in Table 9.

[0098] Table 9: Bioleather tear resistance using three fabrics selected according Docket No.: 1063.035

[0099] Construction of strains overexpressing genes that enhance both the production method and the properties of bioleathers: a plasmid based on the available gene sequences of G. lucidum, the modular and minimalist plasmid, pMB (Figure 8), in which each element such as promoters, terminators, and a resistance gene were all flanked by unique restriction sites were constructed, to generate new combinations. The following elements were included: cassette of fungal resistance: sdhB* with its own promoter and terminator sequences, which confer resistance to carboxin (sdhB* may be used with or without introns).

[0100] The gene encoding the iron-sulfur succinate dehydrogenase protein (sdhB) of strain CGMCC 5.616 of Ganoderma lucidum was taken from the complete CDS sequence available at GenBank: JN37741 1.1. The histidine at position 216 (His216) was replaced by leucine to obtain the gene corresponding to the protein conferring resistance to carboxin. From this sequence the promoter (PsdhB) and terminator (TsdhB) regions were obtained, along with the sequence, with or without introns. cassette for overexpressing the gene of interest: promoter Pgpd and terminator sdhB sequences were included. The promoter region of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from of Ganoderma lucidum was taken from the complete CDS sequence available at GenBank: AH015702.2. The eGFP {enhanced green fluorescent protein) coding sequence, was obtained from GenBank: AAB02572.1 . The original introns of the gene of interest may be included or not. Additionally, a region spanning the first exon, the first intron and part of the second exon of gpd may be included, since it has been reported that it increases expression of the gene under the control of Pgpd

[0101] The plasmid pMB06 was constructed, bearing the egfp gene cloned into Docket No.: 1063.035 the E3-E4 site.

[0102] Although all the genetic constructs were based on gene sequences from G. lucidum, the strain of G. sessile was transformed, as it showed the best performance in producing the leather-like bioproduct. The transformation of G. sessile was performed using the polyethylene glycol-mediated protoplast transformation method, and transformants were selected using carboxin in a range of 5-10 pg / mL. The plasmids were efficiently transformed into G. sessile, and DNA insertion in the chromosomal DNA was confirmed by amplification from the genomic DNA of the target regions.

[0103] Strains overexpressing the glswi6B, glsnfl, and glnmnat genes, which show effect on carbon metabolism, were prepared. The resistant strains were maintained in successive subcultures (Figure 9), and the expression was confirmed using egfp as a reporter gene (Figure 10).

[0104] The plasmid pMB08 was constructed, which bears the swi6B gene in its B isoform, inserted between the E3 and E4 sites of plasmid pMB. G. sessile cells were transformed by the polyethylene glycol-mediated protoplasto transformation method, and the transformed strains were selected using carboxin in a range of 5-10 pg / mL. The strain with the best growth, designated SW06, was selected. The introduction of the construct in SW06 was confirmed by amplification, from the genomic DNA of the target regions. Furthermore, mitotic stability of the transformed strain SW06 was evaluated and it showed that it was stable.

[0105] Similarly, plasmids pMB1 1 and pMB12, bearing the genes glsnfl and glnmnat, respectively, were used for transforming Ganoderma sessile cells and the transformed strains, designated SN46 and NM12, were selected.

[0106] Cellulose enzyme production was evaluated on days 7 and 10 of the submerged liquid culture using wheat bran as substrate, showing consistently higher cellulase production compared to the untransformed wild type strain (Figure 10).

[0107] The impact on mycelium-based bioleather production was evaluated Docket No.: 1063.035 compared to the untransformed wild type strain (wt) using the TC-SSF method with edible paper as substrate, and an improvement in tensile strength of the final material obtained with the modified strain SW06 was observed (Table 10).

[0108] Table 10: Mechanical properties of bioleather obtained with the modified 5 strain SW06 using the TC-SSF method with edible paper as substrate.

[0109] Strain SW06 was also evaluated under WS-SSF conditions using grape pomace as a substrate, with M1 medium for inoculum preparation, as described in the examples.

[0110] When compared to the wild type strain, the transformed strain produced a o thicker bioleather, with improved mechanical properties (Table 11 ). This was consistent with the effect observed with siv / 6B gene overexpression, which led to an increased expression of I ignocell ulolytic enzymes. Consequently, the modified strain also exhibited superior characteristics compared to the wild type strain, for example, for growth in lignocellulosic substrates, thereby achieving an improved 5 mycelium. Docket No.: 1063.035

[0111] Table 1 1 : Mechanical properties of the bioleather produced with the WS- SSF method using grape pomace as substrate, comparing the wild-type strain (wt) with the modified SW06 strain.

[0112] Furthermore, plasmid pMB10, bearing the gcn4 gene, was used to transform cells and transformants were selected. One outstanding transformant was isolated, designated GC07 (Figure 13A). Mitotic stability was also evaluated and confirmed. Integration of the construct was verified by amplification from genomic DNA of the target regions. The GC07 variant showed accelerated growth until day 10 under the conditions of the TC-SSF method. All the resulting strains were dikaryotic; however, it should be noted that it is possible to obtain monokaryotic strains overexpressing each of the genes. These may then be crossed to produce dikaryons with all the possible combinations of gene overexpression.

[0113] The TC-SSF method, which employs commercial edible paper or a starch film as substrate, promotes mycelium growth, invasion, degradation, and complete transformation of starchy materials, resulting in a mycelial sheet without solid residues. Furthermore, a liquid culture was used as inoculum instead of mycelial seeds, thus avoiding the problems associated with remnant seeds. The TC-SSF method completely transforms the substrate and therefore it is a highly Docket No.: 1063.035 efficient method. This method does not require external inputs of CO2, controlled airflow, or complex tray arrangements. The resulting bioleather components are biodegradable, and the method requires low amounts of water and energy.

[0114] When cotton fabrics were added to edible paper substrates, they were integrated with the mycelium, resulting in a bioleather with significantly improved mechanical properties.

[0115] When modified fungal strains were employed, the resulting bioleather showed improvements in quality, consistency and mechanical properties.

[0116] This invention is better illustrated in the following examples, which should not be construed as a limitation of the scope thereof. On the contrary, it should be clearly understood that other embodiments, modifications and equivalents thereof may be possible after reading the present description, which may be suggested to a person of skill without departing from the spirit of the present invention and / or the scope of the appended claims.

[0117] Examples

[0118] Example 1 : Strains, culture media, growth, and biomass

[0119] The following strains were selected for bioleather production: Ganoderma lucidum, Ganoderma sessile, Pleurotus ostreatus, Pignoporus sanguineus, Trametes versicolor, and Hericium erinaceus. All were commercially available.

[0120] The selected strains were grown on a potato dextrose agar (PDA) medium with or without oat, on plates for 7 days at 30 °C. These cultures were used for preparing mycelial seeds to be used as inoculum and as storage medium of the strains. In a first step, the horse oats seeds were boiled for 8 minutes for hydration. Subsequently, they were drained and sterilized at 121 °C for 20 minutes. Once cooled, 70 g of seeds were inoculated with two 1 cm x 1 cm squares from starter culture plates, obtained by scalpel cutting. Finally, incubation was performed at 30 °C for 10 days, followed by storage at 4 °C to preserve the fungal strains until further use.

[0121] Fermentation in liquid medium: the container with the liquid culture Docket No.: 1063.035 medium was autoclaved for 20 minutes at 121 °C. The liquid fermentations were carried out under stirring at 135-150 rpm, at 30 °C, for 5-7 days. Different compositions of culture media were tested (see Table 12).

[0122] Systems containing a fixed volume of sterile liquid medium (0.25 - 5 L) were inoculated in three different ways comprising: a) adding pieces of mycelium grown on 1 cm2plates for 4 days in PDA, at a ratio of 10-20 pieces / 100 ml_. b) adding 50 g / L of mycelial seeds previously incubated at 30 °C for 5-7 days. c) an aliquot of 5-10% v / v of a liquid culture of 5-10 days of growth at 30 °C, previously inoculated as described in items “a” or “b”."

[0123] Table 12: Composition of the tested media Docket No.: 1063.035

[0124] Although all tested media allowed good fungal growth, medium “D” produced a larger fungal biomass. The culture with medium “D” required 3 to 10 days to develop under stirring at 135-150 rpm and at a temperature of 27-30 °C. This medium was designated “M1 The composition of M1 and maximum fungal biomass achieved are set forth in Table 13 and Table 1 , respectively.

[0125] Table 13: Composition of the M1 medium

[0126] Other carbon and nitrogen sources were also evaluated as alternatives to improve the M1 medium. The results are summarized in Tables 14 and 15.

[0127] Table 14: Culture media with alternative carbon sources Docket No.: 1063.035

[0128] Table 15: Composition of alternative media to M2 and M6

[0129] All liquid media were inoculated with Ganoderma sessile and the fungal biomass was quantified for each medium after 5-7 days of growth. Before their use as an inoculum in the solid state fermentation method on edible paper, lignocellulosic substrate or other, biomass concentration is adjusted by adding water or culture medium. The media were evaluated based on yield, minimum composition, low costs, and biomass production.

[0130] Composition of the trace elements solution: Table 16 shows the composition of elements and molecules of the trace solution. The culture medium may include, or not, a 1 :200 dilution of this solution. Docket No.: 1063.035

[0131] Table 16: Composition of the trace elements solution:

[0132] The biomass present in the liquid culture was quantified by transferring 50 mL of liquid culture of G. sessile to a Falcon tube. The sample was centrifuged at 9000 rpm for 18 minutes and the supernatant was discarded. The resulting pellet was centrifuged at 9000 rpm for 18minutes, and resuspended in 50 mL of distilled water. This method is repeated three times. After the final wash, was supernatant carefully removed, only leaving the pellet at the bottom of the tube.

[0133] To determine biomass dry weight, filter papers were weighed after previously being dried in an oven at 105 °C for 30 minutes and stored in a desiccator to determine their dry weight. The pellet was then transferred to the filter paper placed in a Buchner funnel where excess liquid was filtered using a vacuum pump. Docket No.: 1063.035

[0134] The sample on the filter paper was placed on a watch glass and dried in a convection oven at 105 °C for 3 hours. After cooling in a desiccator, the dry sample was weighed and biomass dry weight was calculated as the difference between total weight (sample + filter paper) and the initial weight of the filter paper. Finally, biomass concentration was calculated as g / L (equation 1 ):

[0135] Equation 1 :

[0136] Bsomass dry weight

[0137] Biomass

[0138] Biomass was determined for each tested condition in the liquid medium and measured daily in duplicate throughout the cultivation period. Biomass productivity in culture was calculated as the ratio between the amount of generated biomass (AX) and the time (t) in which it was produced (equation 2):

[0139] " "" T Eq. 2

[0140] All tested liquid cultures were stored at 4-8 °C for 40 days. Aliquots were taken weekly and grown on PDA-oat plates to analyze stability over time.

[0141] Example 2: Methods of producing bioleather by solid-state fermentations

[0142] General aspects: to perform the solid-state fermentation (SSF) methods, aluminum trays and lids were used, which were sterilized using 70% ethanol. The solid substrates were autoclaved during 1 hour at 121 °C. Sterilization was not necessary in the case of edible paper substrates.

[0143] The systems were assembled placing the solid substrate on the trays and which were inoculated with mycelial seeds or with liquid culture, as appropriate. Dimensions of the aluminum trays were: 19x12x4.5 cm (F100), 27x17x4.5 cm (F200), and of stainless steel: 30x40 cm and 40x60 cm.

[0144] The trays, covered with aluminum foil, were incubated for 7-10 days at 28- 35 °C. Fungal metabolic activity was monitored by measuring CO2levels throughout the culture period. Mycelial sheets were harvested after 7-10 days or incubation after full consumption of the substrate or when they reached a thickness of 0.5-1 .4 cm. Docket No.: 1063.035

[0145] A) “WS-SSF” method (Wet Surface Solid-State Fermentation).

[0146] Preparation of the mycelial sheet using the WS-SSF method and use of agroindustrial residues as substrates: several lignocellulosic residues were evaluated as substrates for WS-SSF, including grape pomace, beer brewing residues, barley screenings (Barley screenings), barley rootlets, soybean hulls, grape lex, carrot waste, sunflower hulls, peanut hulls, and peanut pods.

[0147] Prior to use, the residues were oven-dried until a moisture content of 6-7 % was reached, and then they were milled.

[0148] Percent composition of the lignocellulosic substrates: the percent composition of each lignocellulosic substrate, previously dried and milled, was determined at the Servicio de la Facultad de Ciencias Bioquimicas and Farmaceuticas [Services Department of the School of Biochemical and Pharmaceutical Sciences] of the Universidad National de Rosario [National University of Rosario], Argentina. The following standardized methods were used: moisture, AOAC 925.10; fat content, AOAC 920.85; proteins, AOAC 920.87, factor: 6,25; mineral content, AOAC 923.03; fiber, AOAC 973.18; carbohydrates, by difference; iron, AOAC 985.35.

[0149] Determination of water-retention capacity: To determine the waterretention capacity (WRC), 2.5 g (Po) of substrates of each sample were weighed into Falcon tubes and then 50 mL of distilled water were added. Samples were stirred for 10 minutes and then the mixtures were left to stand during 24 hours at room temperature. Subsequently, the samples were centrifuged at 6000 rpm during 10 minutes, the supernatant was removed and the sediment was weighed (P . The water-retention capacity (WRC) was calculated using the following Formula (equation 3): Eq. 3

[0150] Preparation of mycelial sheets: All the pretreated solid residues were placed on trays and inoculated with 0.1 g of mycelial seeds per gram of solid Docket No.: 1063.035 substrate. Fermentation was carried out for 10 days in a closed oven at 30 °C, in the dark and without gas addition. Different amounts of water were added according to the WRC of each substrate, in order to maintain an “available water / substrate” ratio of at least 7:3, depending on the substrate. This resulted in excess water, providing sufficient moisture for surface mycelium growth.

[0151] Seeding using seeds was replaced by inoculation of a liquid culture at a concentration from 0.8 to 4 g / L and an amount of 0.40-0.45 mL / cm2of substrate, where fermentation and development of mycelial sheet is carried out under the same conditions described in the previous paragraph.

[0152] B) “TC-SSF” method (Totally Consumed Solid-State Fermentation).

[0153] Preparation of mycelial sheet by the TC-SSF method using a commercial edible paper substrate: Commercial edible papers were used as substrate for TC- SSF. Their main component is starch (>90%), which was used without pretreatment or sterilization. Three brands of edible paper were evaluated. Regardless the provider, edible paper generally consists of corn starch, cassava starch and hydrogenated vegetable oil.

[0154] One, two, or three sheets of commercial edible paper were used as nutrients and substrate on aluminum trays. The edible paper substrate was inoculated with an aliquot of 0.40-0.45 mL / cm2of liquid culture with a biomass concentration from 0.8 and 4 g / L, without supplementation. Fermentation was carried out in a closed oven at 28-32 °C, in darkness, for 7-10 days. After this period, the mycelial sheet was harvested and subsequently dried at 90 °C for 4 hours to inactivate the fungus.

[0155] As an alternative to the above method, on the fourth day of culture, the mycelial sheet was transferred to a new tray supplemented with one or two additional sheets of edible paper. Incubation continued until a 10-day cultivation was completed. The mycelial sheet was harvested and dried at 90 °C for 4 hours to inactivate the fungus.

[0156] Several substrates were prepared using various plant starch sources such Docket No.: 1063.035 as corn, potato, and cassava, supplemented or not with 2-5% sunflower or corn oil, to be used in the TC-SSF method. To this end, 30 g of sterile starch powder were hydrated with 255 mL of water at 50-80 °C with continuous stirring until gelation occurred. The resulting gel was poured onto a tray and left to cool at 30 °C until a film formed and then it was inoculated with a 0.40-0.45 mL / cm2aliquot of liquid culture at a biomass concentration of 0.8 and 4 g / L as described above. Fermentation was carried out on trays placed in a closed oven at 30 °C, in complete darkness, for 7 to 10 days, maintaining 40% of minimum relative humidity. After this period, the mycelial sheet was harvested and subsequently dried at 90 °C for 4 hours to inactivate the fungus.

[0157] Another variant of the TC-SSF method was carried out, which included the addition of fabrics: different cotton fabrics were selected to be incorporated to the trays along with starch substrates (edible paper). Prior to use, the fabrics were washed and autoclaved at 121 °C for 20-40 minutes.

[0158] Differences in warp and woof structures of the different fabrics were analyzed by optical microscopy and were initially determined in pixels2. These values were converted into mm2by calibration with a micrometer using a factor of 7225.43. The results are listed in Table 17.

[0159] Table 17: Pore size calculated in pixels2 and code assigned to each selected fabric Docket No.: 1063.035

[0160] The selected fabrics were added to the trays according to the following sequences:

[0161] • Si: Fabric - Edible paper sheet or starch film - liquid inoculum

[0162] • S2: Edible paper sheet or starch film - fabric - liquid inoculum

[0163] • S3: Edible paper sheet or starch film - liquid inoculum - fabric

[0164] The TC-SSF method was performed as previously described. Among the tested fabrics, NL is the only one which is not 100% cotton.

[0165] Another variant incorporating mycelium inversion: System S3 was prepared following the method described above. In this case, on the fourth day of incubation, the mycelial sheet was transferred onto a new tray, and inverted upside down without adding any additional substrate or liquid. Fermentation continued until day 10, after which the mycelial sheet was harvested and dried at 90 °C for 4 hours to inactivate the fungus.

[0166] Carbon dioxide (CO2) levels were monitored in all variants of the method using a non-dispersive infrared (NDIR) sensor. Measurements were recorded throughout the fermentation period of the procedure. CO2monitoring made it possible to identify the point at which fungal metabolic activity began to decrease. Example 3:

[0167] Post-treatment of mycelial sheets and production of leather-like bioproducts: As mentioned above, at the end of the process, the mycelial sheets underwent heat treatment in an oven at 90 °C for 4 hours. A post-treatment was then performed, consisting of submerging it in a 10- 30% glycerol solution for 2- 4 hours, followed by an additional drying at 40 °C for 1 hour. After this treatment, Docket No.: 1063.035 the mycelial sheet was designated “leather-like bioproduct” Alternatively, before the treatment with glycerol, an immersion method was applied to the mycelial sheet in a 0.5% tannic acid solution for 24 hours.

[0168] Example 4: Physico-chemical analysis of the mycelial sheets

[0169] Analysis of chemical properties by FT-IR: The dehydrated mycelial sheets were analyzed using a Shimadzu FTIR Prestige-21 with an ATR accessory. The FTIR spectrum of the mycelium was recorded in the range of 500-3.500 cm’1at room temperature, with a resolution of 2.0.

[0170] For spectral analysis, corrections were made using the Shimadzu IR Solution 1.10 software, including an ATR correction, a baseline correction and smoothing (with a factor of 10). Additionally, due to potential variability in the optical path length caused by the solid and heterogeneous nature of the sample, and in the absence of a consistent peak for normalization, the Standard Normal Variate (SNV) correction method was applied.

[0171] Scanning Electron Microscopy (SEM) for mycelial sheet characterization: the dry mycelial sheets were gold-coated and observed using a LEITZ AMR1000 scanning electron microscope (SEM). Images were processed with the Imaged software to measure hyphal diameters. This technique allowed analysis of mycelium surface images, measurement of hyphal size and shape and observation of mycelial vegetative structures, such as chlamydospores.

[0172] Measurement of mycelial dry weight and thickness: Each dry mycelial sheet was weighed using a precision balance. Thickness was determined using a Vernier caliper, with an average of 3 measurements in different areas of each mycelial sheet.

[0173] Example 5: Mechanical properties of the leather-like bioproduct:

[0174] After the post-treatments, the resulting leather-like bioproducts were characterized based on their weight, thickness, and mechanical properties, according to ISO 3376, by INTI (Institute Nacional de Tecnologia Industrial [National Institute of Industrial Technology], Argentina) and / or by INTEMA- Docket No.: 1063.035

[0175] CONICET (Institute de Investigaciones en Ciencia y Tecnologia de Materiales, Consejo Nacional de Investigaciones Cientificas y Tecnicas [Institute of Materials Science and Technology Research, National Scientific and Technical Research Council], Mar del Plata, Argentina). In the case of bioproducts containing fabrics, the mechanical properties were evaluated in two directions: parallel and perpendicular to fabric orientation. For tensile strength measurement, the reported values represent the average of at least five dog-bone specimens.

[0176] • Tensile strength (MPa)

[0177] Wherein: o = tensile strength (MPa); Fmax = maximum applied strength (N); and Ao= original cross-sectional area (m2).

[0178] • Maximum deformation %, cmax (%)

[0179] The maximum deformation represents relative elongation before rupture:

[0180] Wherein: Lf = final length after stretching (mm) and Lo= initial length (mm).

[0181] • Elastic modulus (MPa)

[0182] The Elastic modulus, or Young’s modulus, is obtained from the initial linear portion of the Stress-Strain curve.

[0183] Wherein: E = elastic modulus (MPa), Ao = change in stress in the elastic region (MPa), Ac = change in strain (dimensionless).

[0184] • Maximum strength (N)

[0185] Maximum strength supported by the specimen during the test. It is obtained directly from the load-elongation curve, as the maximum load recorded before rupture.

[0186] Wherein: Fmax = maximum force (Newton, N); omax = maximum stress (MPa or N / m2) and A = original cross-sectional area (m2). Docket No.: 1063.035

[0187] Tear resistance (N / mm)

[0188] A “trouser” type tear method was used which consisted in preparing a specimen shaped like trousers, with a central notch dividing the lower end into two legs. The specimen was mounted in a Universal Testing Machine, holding each leg with a separate clamp. During the test, the machine applied a vertical tension at a constant speed, causing the material to tear progressively from the notch through the specimen body. The force required to propagate the tear was continuously recorded. Tear resistance was calculated as the average force measured during tear propagation and it is expressed as N / mm (force per unit of thickness) and it may also be expressed using the maximum force.

[0189] Fave Fmax

[0190] Tear resistance = — — — or -- — — x x

[0191] Wherein : F ave : average force during the tear propagation ; F max, maximum force (N), and x: specimen thickness (mm).

[0192] Example 6: Genetic modification of fungal strains:

[0193] The following genes were used for the genetic constructs:

[0194] The gene encoding the iron-sulfur succinate dehydrogenase protein (sdhB) of strain CGMCC 5.616 of Ganoderma lucidum was taken from the complete CDS sequence available at GenBank: JN377411 .1 [9], The histidine at position 216 (His216) was replaced by leucine to obtain the gene corresponding to the protein conferring resistance to carboxin. From this sequence the promoter (PsdhB) and terminator (TsdhB) regions were obtained, along with the sequence, with or without introns.

[0195] The promoter region of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene from of Ganoderma lucidum was taken from the complete CDS sequence available at GenBank: AH015702.2

[0010] . The egfp coding sequence (enhanced green fluorescent protein, obtained from GenBank: AAB02572.1 .

[0196] The coding sequence of one of the isoforms of the Swi6 regulator from Ganoderma lucidum, Swi6B, was taken from the partial Swi6B mRNA of strain Docket No.: 1063.035

[0197] ACCC53264 from G. lucidum, available at GenBank: MF770573.1 [1 1 ,12], The coding sequence of glsnfl, an AMP-dependent serine-threonine protein kinase homologous to snf1 (sucrose-non-fermenting protein kinase 1 ) of G. lucidum, was taken from GenBank: AUN37949.1

[0013] .

[0198] The coding sequence of gcn4 from strain ACCC53264 of G. lucidum, a transcription factor that plays a key role in nitrogen metabolism, was taken from GenBank: MN380309

[0014] , The coding sequence of nicotinamide mononucleotide adenylyltransferase (nmnat) of G. lucidum was taken from GenBank:MH394247

[0015] .

[0199] The synthetic genes were optimized for expression in G. lucidum according to codon usage and were ordered from Twist Biosciences.

[0200] The modified coding sequences used herein were:

[0201] Swi6B gene: SEQ ID NO. 1

[0202] Glsnfl gene: SEQ ID NO. 2

[0203] Gcn4 gene: SEQ ID NO. 3

[0204] Nmnat gene'. SEQ ID NO. 4

[0205] Assembly of the constructs was performed by standard molecular biology techniques, using restriction enzymes and cloning into Escherichia coli, following routine protocols for plasmid ligation, transformation, and isolation.

[0206] Protoplasts were obtained and transformed following and adjusting previously disclosed protocols [16-18]. The strain from Ganoderma was grown in PDA plates for 7 days at 30 °C. For protoplast isolation, all steps were performed under sterile conditions. Two hundred mL of liquid medium (M1 ) with 7-8 agar plugs from the Ganoderma plate were inoculated and incubated for 4 days at 30 °C, with stirring at 200 rpm. The culture was then centrifuged in 50 mL Falcon tubes at 4500 g for 15 min at 4 °C, after which the supernatant was discarded and the pellet was divided into two tubes. The pellets were resuspended in 15 mL of 0.6 M mannitol, gently mixing by tapping the bottom of the tube, and then they were centrifuged at 4500 g for 10 min. This washing step was repeated twice. Docket No.: 1063.035

[0207] Subsequently, the pellet was resuspended in 5 mL of 0.6 M mannitol, 0.1 M phosphate buffer (pH 5.8), and 250 pL of 5% Viscozyme were added. The mixture was incubated at 30 °C for 3 h, with gentle stirring in horizontal shaker.

[0208] After incubation, the solution was filtered through a 40 pm cell strainer. From this point on, protoplasts were kept on ice. The filtrate was transferred to microcentrifuge tubes and centrifuged. The protoplast suspensions were washed twice with STC buffer (pH 7.5; 0.6 M sorbitol; 10 mM Tris-HCI; 10 mM CaCI2) by centrifugation at 2000 g for 15 min. Finally, the protoplasts were resuspended in 500-900 pL of STC buffer. Protoplasts were counted using a Neubauer chamber, yielding expected values of about ~107cells / mL.

[0209] For the transformation, 170 pL of protoplast suspension were mixed with DNA (2.5-10 pg) or with H2O (negative control). The sample was incubated on ice during 10 min. Then, 400 pL of cold PTC buffer (40 % PEG4000, 50 mM CaCI2, 10 mM Tris-HCI, pH 7.5) were added to the sample, followed by incubation on ice for 20 min. Subsequently, 600 pL of cold PTC were added and the mixture was incubated at 28 °C for 30 min. Finally, the sample was centrifuged at 3500 g for 10 min at room temperature.

[0210] After the centrifugation, the pellet was resuspended in 1 mL of YSMB broth , and the resulting suspension was transferred to a Petri dish. Next, 10 mL of YSMB regeneration broth (0.4% yeast extract; 0.4% peptone; 0.8% malt extract; 1 ,2% glucose; 0.6 M sucrose) were added to the Petri dish, and the mixture was gently homogenized.

[0211] The suspension was incubated at 30 °C for two days. After this period, 10 mL of molten PDA which contained carboxin (at a concentration from 2.5 to 10 pg / mL) were added to the transformation plate. A viability control of the protoplasts was performed by plating them on PDA without carboxin, and a negative control was included by adding water instead of DNA. Finally, the plates were homogenized and incubated at 30 °C until colonies appeared.

[0212] Modified strains were subcultured five times in PDA medium without Docket No.: 1063.035 carboxin and they were grown for 7 days. Subsequently, they were grown again in the presence of carboxin (2.5-10 pg / mL).

[0213] The genomic DNA was extracted using the Quick-DNA Fungal / Bacterial Miniprep kit (Zymo Research), following the manufacturer’s instructions. Detection of genes inserted in the genomic DNA from transformants was performed using the primers listed in Table 18. The genomic DNA was purified and then diluted 1 / 100 to 1 / 1000 in order to use it as a template in a PCR reaction. Positive controls were performed using each plasmid transformant as a template. The amplification method included an initial denaturation at 98 °C for 5 min, followed by 35 denaturation cycles at 98 °C for 30 s, annealing at temperatures from 55 °C for 30 s and elongation at 72 °C for 1 min. Finally, a final elongation step was performed at 72 °C for 5 min.

[0214] Table 18: Primers used for validating the insertion Docket No.: 1063.035

[0215] # See Figure 7 for primer location

[0216] The endo-p-1 ,4-glucanase activity of cellulases was measured by the DNS method (3,5-dinitrosalicylic acid), based on the amount of reducing sugars released during hydrolysis

[0019] . A 1 % CMC solution was prepared in 1 M citrate buffer (pH 5.0), considered as substrate. One hundred pL of sample were added to 1 mL of CMC solution and 1 mL of citrate buffer. The mixture was incubated at 45 °C for 30 min. Then, DNS was added to the solution to stop the reaction. The treated samples were boiled for 10 min, cooled in water to stabilize color and optical density was measured at 540 nm. One enzyme unit (U) is defined as the amount of cellulase that releases 1 pmol glucose from cellulose during 1 min, at pH 5.0 and at 37 °C

[0020] .

[0217] Figure 1 1 shows as an example, a schematic of plasmid pMB08 which overexpresses the swi6B gene.

[0218] References

[0219]

[0001] Rahim Khan. The Microbe, 8 (2025) 100439.

[0220] [2] Elsacker E, et al., Front. Bioeng. Biotechnol. 1 1 (2023) 1204861 .

[0221] [3] Bentangan, M. A. et al., (2020) Patent WO2020136448A1 .

[0222] [4] Ross P. et al., (2020) Patent US10687482B2.

[0223] [5] Greetham, L, et al. (2022) Patent US1 1277979B2.

[0224] [6] Gandia, A., et al. (2022) Patent US20220025318A1 .

[0225] [7] Szilvay, G. et al., (2021 ). Patent WO2021136883A1 .

[0226] [8] Haneef, M. et al., Scientific Reports, 7 (2017) 41292.

[0227] [9] Jun-Wei X. et al., Journal Applied Environmental Microbiology, 78 Docket No.: 1063.035

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[0010] Xu X., et aL, Journal of Microbiology, 44 (2006) 515.

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[0011] Lingdan L. et al, Environmental Microbiology 23 (2021 ) 4405.

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[0012] Ling-Dan L, et aL, Journal of Fungi, 8 (2022) 187.

[0013] Yanru H, et al, Environmental Microbiology, 22 (2020) 107.

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[0014] Lingdan L. et al, Applied Environmental Microbiology, 87 (2021 ).

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[0015] Shengli W. et al, Applied Microbiology and Biotechnology, 104 (2020) 7079.

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[0016] Chou, T. et al, Annals of Microbiology, 66 (2016) 1 1 1.

[0017] Fook-Hwa L. et al, Folia Microbioliogica, 66 (2021 ) 677.

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[0018] Ab W. et al, Malaysian Applied Biology, 51 (2022) 43.

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[0019] Miller GL. Analytical Chemistry. 31 (1959) 426

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[0020] Meini, M. et aL, Food Chemistry, 283, (2019) 257.

Claims

Docket No.: 1063.035CLAIMS:Having thus specifically described and determined the nature and the best mode of carrying out the present invention, the following is declared as claimed property and exclusive right:

1. A genetically transformed fungal strain, or a derivative thereof, for preparing a leather-like bioproduct, wherein the transformed strain has an increased carbohydrate hydrolytic activity as compared to the parent wild type strain.

2. The strain according to claim 1 , wherein one or more genes selected from the group consisting of swi6B, glsnf 1 , gcn4, nmnat, and combinations thereof are overexpressed, as compared to the parent wild type strain.

3. The strain according to claim 1 , wherein the strain is selected from the genera Ganoderma, Pleurotus, Pignoporus, Trametes, and Hericium.

4. The strain according to claim 3, wherein the strain belongs to the Ganoderma genus.

5. The strain according to claim 1 , comprising in its genome the modified swi6B gene having the nucleotide sequence set forth in SEQ ID NO. 1 .

6. The strain according to claim 1 , comprising in its genome the modified glsnf 1 gene having the nucleotide sequence set forth in SEQ ID NO. 2.

7. The strain according to claim 1 , comprising in its genome the modified gcn4 gene having the nucleotide sequence set forth in SEQ ID NO. 3.

8. The strain according to claim 1 , comprising in its genome the modified nmnat gene having the nucleotide sequence set forth in SEQ ID NO. 4.

9. A method for producing a leather-like bioproduct, comprising the following steps: a) growing the strain of claim 1 in a liquid medium,Docket No.: 1063.035 b) arranging a sterile solid substrate on a surface; c) contacting an inoculum of the strain grown in step a) with the solid substrate of step b) and incubating it; d) harvesting the mycelium and inactivating the strain; and e) treating the mycelium until a leather-like bioproduct is obtained.

10. The method according to claim 9, wherein the genetically modified strain of a genus selected from Ganoderma, Pleurotus, Pignoporus, Trametes, and Hericium.

11. The method according to claim 9, wherein the solid substrate is selected from the group of substrates consisting of lignocellulose, starch and combinations thereof.

12. The method according to claim 1 1 , wherein the solid substrate is lignocellulose and has a water / substrate ratio of at least 70%.

13. The method according to claim 1 1 , wherein the lignocellulosic substrate is selected from the group consisting of barley screenings, grape lex, beer brewing residue, grape pomace, barley rootlets, soybean hulls, carrot waste, sunflower hulls, peanut hulls, peanut pods, and combinations thereof.

14. The method according to claim 11 , wherein the starch substrate is selected from the group consisting of edible paper sheets, potato starch-based sheets, cassava starch-based sheets, corn starch-based sheets, and mixtures thereof.

15. The method according to claim 9, wherein, in step c), the inoculum comprises a biomass concentration from 0.8 to 4 g / L.

16. The method according to claim 9, wherein step b) comprises arranging as a solid substrate an amount from 1 to 3 edible paper sheets.

17. The method according to claim 9, wherein, in step b), the solid substrate comprises a combination of edible paper sheets and fabric, wherein the fabric is a cotton fabric with a pore size from 0.0188 to 2.065 pixels2.

18. The method according to claim 9, wherein the incubation time inDocket No.: 1063.035 step c) comprises from 3 to 10 days.

19. The method according to claim 9, wherein step e) comprises a treatment with 10-30% glycerol for 2-4 hours.

20. A method for producing a leather-like bioproduct, comprising the following steps: a) cultivating a strain selected from the group consisting of Ganoderma, Pleurotus, Pignoporus, Trametes, and Hericium in a liquid medium, b) arranging a sterile solid substrate on a surface; c) contacting an inoculum of the strain grown in step a) with the solid substrate of step b) and incubating it; d) harvesting the mycelium and inactivating the strain; and e) treating the mycelium until a leather-like bioproduct is obtained.

21. The method according to claim 20, wherein the strain is selected from the group consisting of Ganoderma sessile, Ganoderma lucidum, Pleurotus ostreatus, Pignoporus sanguineus, Trametes versicolor, and Hericium erinaceu.

22. The method according to claim 20, comprising a solid starch substrate selected from the group consisting of edible paper sheets, potato starch-based film, cassava starch-based films, corn starch-based films, and mixtures thereof.

23. The method according to claim 20, wherein, in step c), the inoculum comprises a biomass concentration from 0.8 to 4 g / L.

24. The method according to claim 20, wherein step b) comprises arranging as a solid substrate from 1 to 3 edible paper sheets.

25. The method according to claim 24, wherein, in step b), the solid substrate comprises a combination of edible paper sheets and fabric, wherein the fabric is a cotton fabric with a pore size from 0.0188 to 2.065 pixels2.

26. The method according to claim 20, wherein the incubation time in step c) comprises from 3 to 10 days.

27. The method according to claim 20, wherein step e) comprises aDocket No.: 1063.035 treatment with 10- 30% glycerol for 2-4 hours.

28. A leather-like bioproduct manufactured with the method of claim 9.

29. A leather-like bioproduct manufactured with the method of claim 20.

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