SIDEREAL MYCELIUM FABRICS
20250382734 · 2025-12-18
Inventors
- Leopoldo NARANJO-BRICEÑO (Vitacura, CL)
- Keyla M. FUENTES (Vitacura, CL)
- Melissa GÓMEZ (Vitacura, CL)
- Carlos GIL-DURÁN (Vitacura, CL)
- Vicente OLIVA (Vitacura, CL)
- Maximiliano VENEGAS (Vitacura, CL)
- Hernán Rebolledo De LIMA (Vitacura, CL)
- José Miguel FIGUEROA (Vitacura, CL)
- Pablo ZAMORA (O'Higgins Region, CL)
Cpc classification
D04H1/4266
TEXTILES; PAPER
D06M10/001
TEXTILES; PAPER
D04H1/413
TEXTILES; PAPER
International classification
D04H1/4266
TEXTILES; PAPER
D04H1/413
TEXTILES; PAPER
D06M10/00
TEXTILES; PAPER
Abstract
Methods and compositions (e.g., textiles, as well as components for forming, processing and using such textiles) of mycelium-based materials having superior durability, tensile strength and wear. In some cases, the methods, compositions and apparatuses described herein may include the use of primarily or exclusively dikaryotic fungal strains. These mycelium-based textiles, and methods of making them, may include nanoparticles formed in vivo during growth of the mycelium and/or nanoparticles synthesized in vitro by biogenic synthesis and added to the mycelium mat.
Claims
1. A mycotextile comprising: a support scaffold layer embedded within a crosslinked mycelium matrix, wherein the support scaffold layer is crosslinked to the mycelium matrix; a plurality metallic nanoparticles formed in vivo within the crosslinked mycelium matrix, characterized by a distribution of metallic nanoparticles through the hyphae network within the crosslinked mycelium matrix, wherein the color of the mycotextile is determined by the plurality of metallic nanoparticles.
2. The mycotextile of claim 1, wherein the plurality of metallic nanoparticles are distributed through the hyphae network both over the hyphae and within the hyphae.
3. The mycotextile of claim 1, further comprising a second plurality of ceramic nanoparticles.
4. The mycotextile of claim 3, wherein the ceramic nanoparticles have a different distribution than the metallic nanoparticles of the plurality of nanoparticles.
5. The mycotextile of claim 1, wherein the ceramic nanoparticles are only distributed over the hyphae of the hyphae network.
6. The mycotextile of claim 1, wherein the ceramic nanoparticles are functionalized by the adsorption of a prolamin on their surface.
7. The mycotextile of claim 1, wherein the metallic nanoparticles comprise silver, gold, iron oxide, and/or copper oxide nanoparticles.
8. The mycotextile of claim 1, wherein the mycotextile is coated with a prolamin.
9. The mycotextile of claim 1, wherein the color of the mycotextile is determined by the localized surface plasmon resonance (LSPR) of the metallic nanoparticles.
10. A mycotextile comprising: a support scaffold layer embedded within a crosslinked mycelium matrix, wherein the support scaffold layer is crosslinked to the mycelium matrix; a plurality metallic nanoparticles within the crosslinked mycelium matrix, distributed over the hyphae network, wherein the color of the mycotextile is determined by the localized surface plasmon resonance (LSPR) of the metallic nanoparticles.
11. A method of forming a mycotextile, the method comprising: generating a pre-inoculum substrate seeded with a fungal strain; growing a living mycelium mat using the pre-inoculum substrate, wherein the living mycelium mat is grown around a scaffold layer so that the scaffold layer is incorporated into the living mycelium mat; forming metallic nanoparticles in vivo within the living mycelium mat; and processing the living mycelium mat to crosslink chitin in hypha of the mycelium mat to form the mycotextile.
12. The method of claim 11, wherein the forming metallic nanoparticles in vivo within the living mycelium mat comprises incubating the living mycelium mat with a solution of metallic ions.
13. The method of claim 11, wherein the forming metallic nanoparticles in vivo within the living mycelium mat comprises exposing the living mycelium mat to a solution of metallic ions and reacting metallic ions in the solution of metallic ions with enzymes present in a fungal filtrate of the living mycelium mat.
14. The method of claim 11, further comprising adding functionalized ceramic nanoparticles to the mycelium mat.
15. The method of claim 14, wherein adding the functionalized ceramic nanoparticles to the mycelium mat comprises adding the functionalized ceramic nanoparticles to the living mycelium mat.
16. The method of claim 14, wherein the functionalized ceramic nanoparticles comprise ceramic nanoparticles that are functionalized by the adsorption of a prolamin on their surface.
17. The method of claim 14, further comprising adding in vitro biogenically-synthesized metallic nanoparticles to the living mycelium mat.
18. The method of claim 11, further comprising irradiating the mycelium mat with ultraviolet (UV) light to modify the metallic nanoparticles.
19. The method of claim 11, wherein forming the metallic nanoparticles in vivo within the living mycelium mat comprises forming the metallic nanoparticles extracellularly within the living mycelium mat.
20. The method of claim 11, wherein forming the metallic nanoparticles comprises forming silver, gold, iron oxide, and/or copper oxide nanoparticles.
21. The method of claim 11, wherein forming the nanoparticles in vivo comprises exposing the mycelium mat to a metal ion solution having a concentration of metal ions between 0.1 mM and 50 mM.
22. The method of claim 11, wherein forming the nanoparticles in vivo comprises exposing the mycelium mat to a metal ion solution between about 40 degrees and 90 degrees C.
23. The method of claim 11, wherein forming the nanoparticles in vivo comprises exposing the mycelium mat to a metal ion solution for between 1 hour and 24 hours.
24. The method of claim 11, wherein forming the nanoparticles in vivo comprises immersing the mycelium mat to a metal ion solution.
25. The method of claim 11, wherein forming the metallic nanoparticles in vivo comprises coloring the mycotextile.
26. The method of claim 11, further comprising drying the mycelium mat.
27. The method of claim 11, further comprising coating the mycelium mat with a prolamine protein solution.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] A better understanding of the features and advantages of the methods, compositions, and/or apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:
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[0101] FIGS. 42A1-42A2 show photographs of the Wild-Cu and AgCu mycotextiles, respectively. On the left (FIG. 42A1), the Wild-Cu was obtained by immersion of a wild mycotextile in a dispersion of copper oxide nanoparticles obtained by biosynthesis using an extract of Ilex paraguariensis. On the right (FIG. 42A2), the AgCu mycelium fabric was obtained by immersion of the mycotextile loaded with AgNPs in the same dispersion.
[0102] FIGS. 42B1-42B3 show scanning electron microscopy (SEM) images (FIG. 42B1) of the Wild-Cu mycotextile at 500 and 10 k magnifications that were taken with secondary electrons. The images were collected at the interface of the common hyphae network and the thick cracked deposit. The scale bar of the image at 500 is 50 micrometers, and the image taken at 10 k is 5 micrometers. FIG. 42B2 shows copper mapping and FIG. 42B3 shows an EDS spectrum of the same region that are displayed in the image of FIG. 42B1.
[0103] FIGS. 42C1-42C4 show scanning electron microscopy (SEM) images (FIG. 42C1) of the AgCu mycotextile at 500 and 10 k magnifications, taken with secondary electrons. The images were collected at the interface of the common hyphae network and the thick cracked deposit. The scale bar of the image at 500 is 50 micrometers, and the image taken at 10 k is 5 micrometers. FIGS. 42C2 and 42C3 show silver and copper mapping, respectively, and FIG. 42C4 shows an EDS spectrum of the same region are displayed in the same figure.
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[0105] FIGS. 44A1-44A2 show photographs of the Wild-magnetic IO (FIG. 44A1) and Ag-magnetic IO mycotextiles (FIG. 44A2). The Wild-magnetic IO was obtained by immersion of a wild mycotextile in a dispersion of magnetic iron oxide nanoparticles obtained by biosynthesis using an extract of Aloe vera. The mycotextile was obtained by immersion of the mycotextile loaded with AgNPs in the same dispersion. Scanning electron microscopy (SEM) images of the corresponding Wild-magnetic IO and Ag-magnetic IO mycotextiles at 500. The scale bar is 50 micrometers.
[0106] FIGS. 44B1-44B3 show Iron mapping (FIG. 44B2) and EDS spectrum (FIG. 44B3) of the sample Wild-magnetic IO. A scanning electron microscopy (SEM) image (FIG. 44B1) was used as a reference for the mapping, which was obtained using backscattered electrons.
[0107] FIGS. 44C1-44C4 show Iron (FIG. 44C2) and Silver (FIG. 44C3) mapping and EDS spectra of the sample Ag-magnetic IO. A scanning electron microscopy (SEM) image (FIG. 44C1) was obtained using backscattered electrons as a reference for the mapping.
[0108] FIGS. 45A1 and 45A2 show photographs of the Wild-yellow IO (FIG. 45A1) and Ag-yellow IO mycotextiles (FIG. 45A2). At left, the Wild-yellow IO was obtained by immersion of a wild mycotextile in a dispersion of rod-like IO NPs in a mixture of water, tween 80 and CMC. On the right, the sidereal mycotextile was obtained by immersion of the mycotextile previously loaded with AgNPs in the same dispersion. Scanning electron microscopy (SEM) images of the corresponding Wild-magnetic IO and Ag-magnetic IO mycotextiles at 10 k (BOTTOM). The scale bar is 1 micrometer.
[0109] FIGS. 45B1-45B3 show Iron mapping (FIG. 45B2) and EDS spectrum (FIG. 45B3) of the sample Wild-yellow IO. The scanning electron microscopy (SEM) image (FIG. 45B1) used as a reference for the mapping was obtained using backscattered electrons.
[0110] FIGS. 45C1-45C4 show Iron (FIG. 45C2) and Silver (FIG. 45C3) mapping and EDS spectra (FIG. 45C4) of the sample Ag-yellow IO. A scanning electron microscopy (SEM) image (FIG. 45C1) was used as a reference for the mapping, which was obtained using backscattered electrons.
[0111] FIGS. 46A1-46A2 show photographs of the Wild-red IO (FIG. 46A1) and Ag-red IO mycotextiles (FIG. 46A2). At left, the Wild-red IO was obtained by immersion of a wild mycotextile in a dispersion of spherical IO NPs in a mixture of water, tween 80 and CMC. On the right, the sidereal mycotextile was obtained by immersion in the same dispersion as the mycotextile previously loaded with AgNPs.
[0112] FIGS. 46B1-46B2 show SEM images of the Ag-red IO mycotextile at 2 (FIG. 46B1) and 20 k (FIG. 46B2). The scale bar is 10 micrometers in the image on the left and 1 micrometer in the right image.
[0113] FIGS. 46C1-46C4 show Iron (FIG. 46C2) and Silver (FIG. 46C3) mapping and EDS spectra (FIG. 46C4) of the sample Ag-RED IO. A scanning electron microscopy (SEM) image (FIG. 46C1) was used as a reference for the mapping and obtained using backscattered electrons.
[0114] FIGS. 47A1-47A2 show photographs (top) of the starting IO NPs crosslinked (in situ) mycotextile (FIG. 47A1) and the resulting in situ-Ag mycotextile (FIG. 47A2). FIG. 47A1 is depicted the in-situ cross-linked material used as a base for the sidereal mycelium fabric, and FIG. 47A2 is the prototype obtained by immersion of this base in a silver nitrate solution. Scanning electron microscopy (SEM) images of the corresponding in situ and in situ-Ag mycotextiles are shown at 10 k (bottom). The scale bar is 1 micrometer.
[0115] FIGS. 47B1-47B4 show Iron (FIG. 47B2) and Silver (FIG. 47B3) mapping and EDS spectra (FIG. 47B4) of the sample in situ-Ag. A Scanning Electron Microscopy (SEM) image (FIG. 47B1) was used as a reference for the mapping and obtained using backscattered electrons.
[0116] FIGS. 48A1-48A4 show examples of metallic plates designed with four myco-inspired patterns for conventional embossing approximations.
[0117] FIGS. 48B1-48B4 show examples of the myco-inspired embossing patterns in the material loaded with AgNPs.
[0118] FIG. 48C1-48C4 show examples of myco-inspired embossing patterns in the material loaded with AuNPs.
[0119] FIG. 48D1-48D4 show examples of myco-inspired embossing patterns in the material AgCu NPs.
[0120] FIG. 48E1-48E4 show examples of the myco-inspired embossing patterns in the material Ag-magnetic IO NPs.
[0121] FIG. 48F1-48F4 show examples of the myco-inspired embossing patterns in the material Ag-yellow IO NPs.
[0122] FIG. 48G1-48G4 show examples of the myco-inspired embossing patterns in the material Ag-red IO NPs.
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[0124] FIGS. 49B1-49B2 show examples of photographs of patterned materials loaded with AgNPs (left) and AuNPs (right) using the crude engraving technique.
[0125] FIGS. 49C1-49C3 compare example sections through an un-engraved mycotextile (FIG. 49C1), a mycotextile compressed after crosslinking and drying to form a surface pattern (FIG. 49C2), and a mycotextile engraved into the living mycelium mat (FIG. 49C3) as described herein.
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[0130] FIGS. 52B1-52B3 show photographs of the prototypes wild (FIG. 52B1), AgAu NPs (FIG. 52B2), and Au biosynthesized NPs (FIG. 52B3) before (top) and after (bottom) immersion in the solution of crystal violet for 90 min without photoirradiation with UV light (Adsorption process). The final row corresponds to bleaching experiments in which the adsorbed crystal violet is photodegraded after irradiation with UV light for 150 min.
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[0132] FIGS. 53B1-53B3 show photographs of the prototypes wild (FIG. 53B1), AgAu NPs (FIG. 53B2), and Au biosynthesized NPs (FIG. 53B3) before and after immersion in the solution of crystal violet during 90 min under UV light photoirradiation (Photocatalytic process).
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DETAILED DESCRIPTION
[0136] Described herein are mycotextiles having unique and vibrant colors, textures and/or patterns. As described herein, the color, texture and/or patterns of these materials may result from enzymatic processes and/or treatment steps that take advantage of properties of native and/or engineered strains of the fungal strain forming the mycotextile. For example, in general, the methods and compositions (including mycotextiles) described herein may be used with dikaryon fungal strains, including, but not limited to, engineered dikaryon fungal strains. Thus, described herein are dikaryon fungal strains, including dikaryon Basidomycota fungal strains, and in particular, engineered strains and methods of using them to form and/or process mycotextiles.
[0137] Following cell fusion in some fungi, the two parent nuclei remain distinct. In many fungi, when two mating partners undergo cellular fusion, the resulting bi-nucleate cell grows filamentously, with each filament cell maintaining two independent nuclei that are replicated in a coordinate fashion. This growth stage is referred to as the dikaryon or dikaryotic filament. The largest and most studied group of organisms with a prolonged dikaryotic stage is the basidiomycete fungi. This phylum includes mushrooms, bracket fungi, and many plant pathogens, including the corn smut Ustilago maydis.
[0138] Until the present work, it is widely believed that mycelium-based textiles should preferably be formed of monokaryon forms, and not dikaryon forms of a fungal strain. See, e.g., U.S. Pat. No. 11,576,311 (Monokaryon mycelial material and related method of production). This is because the cells of the dikaryon undergo a complex form of cell division involving the formation of clamp connections to preserve one copy of each haploid nucleus within every dikaryotic cell, and generate fruiting bodies at any stage that are believed to result in genetic variability, variability in thickness and textures, and may result in blemishes in the final fungal mat, which may require additional post-processing. In contrast, the monokaryotic cell does not go through the normal reproductive process and thus does not generate fruiting bodies. Surprisingly, the inventors have found that maintaining the fungal strain used to form the mycotextile in the dikaryotic form may result in materials having particularly beneficial features and may dramatically simplify some processing, particularly in controlling growth and in some cases, in processing the mycotextile to color (or dye) the mycotextile using one or more elements (including metallic elements) synthesized by the fungi itself during processing.
[0139] Thus, in some examples the method and compositions, including textiles, described herein may be limited to the dikaryotic forms of the fungal strains used. Although the methods and examples described herein include engineered dikaryotic strains, described in greater detail below, it should be understood by those of ordinary skill in the art that any appropriate dikaryotic strain may be used, not limited to these specific strains. The benefits and advantages, including improved rates of growth and reproducibility and stability of the resulting textiles, may be achieved with virtually any dikaryotic strain, including strains identified by others as well as the strains explicitly derived herein.
[0140] In some cases, dikaryotic fungal strains may be generated or selected to have desired properties. For example, more than 100 fungal strains with mycotextile potential are maintained in a collection curated by the inventors. Dikaryotic strains may be generated from one or more fungal strains selected for one or more properties. These strains may be manipulated as described herein to form a stable dikaryotic line. Although many of the examples described herein focus on particular strains of fungi that are engineered to be stable dikaryotic strains, the techniques described herein may be generally appliable to other fungal strains, including in particular other Basidomycota fungal strains. In some cases, a strain referred to as the SCC-0006 (e.g., the gold standard) strain, previously referred to as an Unclassified Polyporaceae (see, e.g., U.S. Patent Published Application No. US 2023/0356501 and U.S. patent application Ser. No. 18/595,392) was modified as described herein to form one or more stable dikaryotic strains.
[0141] As part of this process, gene overexpression, genetic engineering, and genome editing techniques were used to obtaining improved fungal strains and ensure high-performance and reproducible mycelium-based textiles at large-scale production. For this, several tools were used, including the genome DNA sequencing of the strain of interest, the determination of the codon usage, promoters useful for constitutive or inducible gene expression, and functional plasmids. In some cases, the validation of the engineered strain's abilities to produce a high-quality mycotextile was performed using plasmids with selectable markers for positive transformants selection, with a robust gene transformation protocol. Further, systems for positive transformants screening by phenotypical, biochemical, microscopic, enzymatic, and molecular approaches were used.
[0142] In some cases, fungal strains having aspects to be improved for mycelium fabric purposes were prepared. For example, to ensure an efficient Sidereal Mycelium Fabric production, stains may be selected and/or engineered having factors directed to enhance the density and homogeneity of mycelium colonization onto the scaffold, the uniformity of the chemical composition of the cell wall facilitating the post-fermentation treatments, and the uniformity of the synthesis of the enzymatic system at the extracellular medium (e.g., oxidoreductase enzymes, etc.).
Processing of Mycelium-Based Fabrics
[0143] It is particularly helpful to use strains of fungi that may be efficiently grown at an industrial scale, without requiring sterile, laboratory conditions, despite environmental contamination. Dikarya, the subkingdom of the fungi kingdom that includes Ascomycota and Basidiomycota phylum was found to be a possible source of candidate strains. One strain used herein (e.g., SCC-0006) is a heterokaryon belonging to the Basidiomycota division, which has beneficial characteristics of its life cycle as compared to other organisms. Basidiomycota are dikaryotic, in which each compartment of a hypha contains two haploid nuclei, each derived from a different parent. A dikaryon strain can be formed by mating two compatible monokaryon strains, resulting in plasmogamy but not karyogamy in the fused compartment. When new hyphae grow, the two nuclei synchronously divide, and each new compartment keeps two nuclei. These strains may not normally be stable. In some cases, only plasmogamy is achieved because only the asexual reproduction of the fungus is harnessed. The karyogamy only occurs before the initiation of sexual reproduction.
[0144] Basidiomycota fungi germinate from a spore to form a primary monokaryotic mycelium, where each cell compartment contains a single haploid nucleus. During fertilization, the nuclei from one compatible mycelium migrate to the other and are incorporated, forming a secondary mycelium.
[0145] The dikaryon is characterized by the formation of clamp connections, where a regular association of non-fused haploid nuclei occurs, as shown in
[0146] Once the strain establishes itself as a dikaryotic organism, it can no longer incorporate another nucleus, leading to a characteristic phenotype. The gene expression patterns in dikaryons may be similar to that of diploids during vegetative growth. However, the mycelium can still fertilize monokaryons by donating one of its nuclei. These dikaryon-monokaryon (di-mon) matings were first described by Buller in 1930 and are called the Buller phenomenon. This mating in basidiomycetes is regulated by two unlinked mating type loci (matA and matB), which must be different to ensure compatibility. Because many species have multiple alleles at each mating type locus, in most cases, both nuclei in a dikaryon are, in principle, capable of donating nuclei to a monokaryon. However, only one of the two nuclei will successfully fertilize the mycelium for each mating. Thus, the two nuclei of a dikaryon compete to fertilize the monokaryon they encounter.
[0147] Normally, fungi do not remain as a dikaryotic organism indefinitely, and it may be difficult to control these organisms in this state. The dikaryon mycelium stage tends to break down, forming monokaryotic hyphae; this phenomenon is called dedikaryotization. This process occurs frequently and can be attributed to environmental factors, such as changes in the surroundings, variations in nutrient availability, temperature, or other conditions, which can influence the stability of the dikaryotic state. If the environment changes in a way that no longer favors the existence of a dikaryotic state, fungi can undergo dedikaryotization. Recombination events during the life cycle of fungi, especially under specific conditions, can lead to genetic recombination between dikaryotic nuclei. This event may result in the fusion of the two nuclei, leading to the loss of dikaryotization and the formation of a monokaryotic mycelium. Finally, genetic instability, as some fungal strains can be genetically unstable, meaning they are more prone to the loss of the dikaryotic condition. Described herein are methods and techniques in which a fungal strain may be maintained as a dikaryotic by maintaining a continuous selection pressure favoring its continued existence, such as antibiotic resistance or auxotrophy.
[0148] SCC-0006 is a Basidiomycota heterokaryon strain that is highly promising for mycelium fabric production. However, the homogeneity of mycelium growth, the uniformity of the chemical composition of the cell wall, and the uniformity of the exoenzymes secretion are important to obtaining an efficient Sidereal Mycelium Fabric. Therefore, a dikaryotic SCC-0006 strain was obtained to maintain its phenotypic characteristics for a controllable and defined period of time (e.g., the time that occurs in a batch of mycelium fabric production).
[0149] As used herein, maintaining a fungal fidelity in a dikaryon can generally be achieved by an infallible selective pressure through antibiotic resistance or auxotrophy strategies. Nuclear divorce may be avoided in the dikaryon strain (e.g., a dikaryon strain of SCC-0006 or other appropriate strain), e.g., regeneration into a monokaryon mycelial stage. Note that although the examples described herein include the SCC-0006 strain and derived dikaryon strains, other strains may be used, including other stable dikaryon strains (and specifically other engineered dikaryon strains).
[0150] Different selectable markers to identify positive transformants have been described in the genetic engineering of fungi. In some cases, the strains may be engineered to have an antibiotic resistance to a known antibiotic, such as hygromycin, neomycin, geneticin, phleomycin, blasticidin, and nourseothricin. Within the chemical agents are ammonium glufosinate, carboxin, etc. In this case, antibiotic resistance as selective pressure may be used as a strategy to build a stable dikaryon strain (e.g., a stable dikaryon strain of SCC-0006).
[0151] Besides enhancing homogeneity, this strategy may also help control of environmental contamination by incorporating a gene that confers resistance to an antibiotic in the working fungal strain. This can favor the development and optimization of production because it may provide greater ease in selecting, maintaining, and growing a resistant strain and better genetic stability. Finally, in the production process, an antibiotic-resistant working strain provides an alternative to creating a selective environment where only the engineered strain can develop and without other contaminants.
[0152] Genetic engineering applied to mycelium-based textile development must consider developing an appropriate pipeline to obtain improved strains (see, e.g.,
Whole Genome Sequencing of SCC-0006 Strain and Analysis:
[0153] The whole genome DNA of the SCC-0006 was sequenced to understand the capabilities of this gold standard strain from a genomic perspective. The primary objective was to discern key genes, promoters, and terminators, and to provide the background for subsequent genetic transformation. The sequence of the strain (e.g., the SCC-0006 strain) allowed the implementation of sophisticated genetic transformation strategies, ensuring the precision and efficacy of introducing selection markers. The SCC-0006 strain had not previously been sequenced.
[0154] Genomic DNA (gDNA) extraction was performed using the Quick-DNA HMW MagBead Kit from Zymo Research (Zymo Research, Irvine, California). This technique ensured the procurement of gDNA characterized by high molecular weight and integrity and subsequent genome sequencing with Nanopore and Illumina technologies. The resulting reads were strategically harnessed for assembly through Flye 2.8v, subsequently annotated with funannotate. The culmination of these processes resulted in the metrics detailed in Table 1.
TABLE-US-00001 TABLE 1 General Metrics of the wild strain SCC-0006 genome assembly. Number of contigs 502 GC (%) 55.59 N50 383,699 L50 24 Largest contig 2,463 Mb Proteins 11,973 BUSCO: euk_genome_met 96.60% tRNAs 371
[0155] In this example, the assembly, resolving in 502 contigs, reflects a GC content of 55.59%. The L50 index, denoting 24, while the contig itself boasts a substantial length of 383 Kb (N50). The predictive prowess of the assembly pipeline foretells 11,973 CDS or proteins, and a thorough BUSCO completeness analysis, tethered to the euk_genome_met database, yielded a commendable completeness score of 96.60% ([S:95.7%, D:0.9%], F:0.6%, M:2.8%, n:1764). Delving deeper into the genomic landscape reveals 371 tRNA-encoding sequences, where cysteine and tryptophan emerge as the less-represented entities with five copies each. In contrast, the amino acids alanine and arginine dominate, each boasting a presence of 30 copies each. The 11,973 CDS obtained were systematically subjected to codon usage analysis utilizing CodonU.
[0156] Genomic DNA sequencing was used for the in silico identification of candidate genes, promoters, and terminators that could work suitably as selectable markers. This allowed the consequent in vivo gene expression analysis and the design and synthesis of the functional plasmids carrying the cassettes of resistance markers' expression. Particular attention was given to optimizing codon usage, facilitating the efficient expression of heterologous genes in the strain of interest. The use of advanced technologies in this comprehensive exploration expanded the understanding of the intricate fungal genome and strategically positions for redesign and refined genetic manipulations within the patented methodologies. This emphasis on codon usage for efficient homologous or heterologous gene overexpression enhanced the translational impact of genetic modifications in various scientific applications described herein.
Selection of a Proper Antibiotic as a Selectable Marker in the Transformation Experiments:
[0157] Before designing and synthesizing functional plasmids containing the cassettes of resistance marker expression, the first step was to assess the fungus's resistance/tolerance to a specific antibiotic. Different selectable markers to identify positive transformants have been described in the genetic engineering of fungi. The most knowledgeable antibiotics are hygromycin B, neomycin, geneticin, phleomycin, blasticidin, and nourseothricin. The SCC-0006's resistance/tolerance to growth in different concentrations of diverse antibiotics was evaluated. The screening results show a high sensitivity to Hygromycin B, which is widely used as a selectable marker in both prokaryotic and eukaryotic cells. It acts by inhibiting polypeptide synthesis and thereby inhibiting translation in non-resistant strains. The inhibition curve of SCC-0006 with hygromycin B, ranging from 0 g/mL to 25 g/mL, was done in darkness at 28 C. for 7 days (
Selection of Native Promoters in the SCC-0006 Genome for Hph Gene Expression:
[0158] To determine the best promoter to be used for the optimized hph6 gene expression in the SCC-0006 strain, the transcription levels of six (6) different genes related to the central metabolism, previously described having high levels of constitutive expression in Basiodiomycota species, were qualitatively analyzed using RT-PCR and visualization in agarose gel. For this, previously, an in silico analysis of genome mining was used to identify the chromosomal localization of these genes in the genome of SCC-0006 (
[0159] RNA was extracted from samples of living aerial mycelia obtained once the mycotextiles were harvested from the fermentation stage (approx. 7 to 10 days of incubation), using the Quick-RNA Plant Miniprep Kit (Zymo Research, Irvine, California) following the manufacturer instructions. Total RNA was treated with DNAse (New England Biolabs, Ipswich, Massachusetts). Then, cDNA was obtained using an All-In-One 5 RT MasterMix (Applied Biological Materials, Richmond, Canada) following the manufacturer's instructions. RT-PCR was performed using GoTaq Green Master Mix (Promega, Madison, WI, USA), the cDNA obtained before as a template and the corresponding primers (Table 2). Each primer was designed around an intronic region, and different amplification sizes were obtained from cDNA and gDNA. PCR program: Initial denaturation: 95 C., 3 m; Cycle Denaturation; 95 C., 40 s; Annealing: 58 C., 40 s; Cicle Elongation: 72 C., 1 m; Final Elongation: 72 C., 10 min. The visualization of the PCR products was done in agarose gel 1.5% stained with Safe View Plus.
[0160] As shown in
TABLE-US-00002 TABLE2 Genomiccontextinformationofsevengenepromotersanalyzedforthe hph6geneexpressioninSCC-0006.PrimersforRT-PCRareshownwiththeexpectedamplicon sizeusinggDNAorcDNAasatemplate. Genome Genomic Primer DNAsequence gDNA CDNA Gene annotation localization Name 5...3 (bp) (bp) Beta-tubulin FUN_007094 contig_270 183424. RT_ CGCAAGTGAT 507 294 (BTB) 181739 tubulin_FW GTGTGGTGTT (SEQIDNO.1) RT_ GTTCCGCCTC tubulin_RV TTGTGACG (SEQIDNO.2) Fructose-1,6- FUN_002805 contig_135 22870.. RT_FBA_ CCTTCCCAAC 537 430 bisphosphate 24559 FW GACAAGCAG aldolase(FBA) (SEQIDNO.3) RT_FBA_ GAAGTTGGGG RV GAGATCTTGG (SEQIDNO.4) Glucose-6- FUN_000156 contig_105 424145.. RT_PGI_ CAAGTTCGCG 572 501 Phosphate 421878 FW GACTACTTCC Isomerase(PGI) (SEQIDNO.5) RT_PGI_ ATCAACGAGC RV CGAGCGTAG (SEQIDNO.6) Phospho- FUN_000628 contig_107 55777.. RT_PGK_ GGAAGGAAA 559 449 glycerate 53949 FW GAAGGTCAAG Kinase(PGK) G (SEQIDNO.7) RT_PGK_ CCACGGGGAA RV CACAATCTTG (SEQIDNO.8) Triosephosphate FUN_000234 contig_105 657304.. RT_TPI_ CTCCGCAAGG 633 483 isomerase(TPI) 656255 FW AAGTCCAG (SEQIDNO.9) RT_TPI_ GGTAACCGAG RV CCACCATAG (SEQIDNO.10) Glutamate FUN_005514 contig_210 92241.. RT_GDH_ ACTTACCGGT 596 490 dehydrogenase 96629 FW CCCAAGG (GDH) (SEQIDNO.11) RT_GDH_ AGCAGTAGAG RV CAGCGAC (SEQIDNO.12) Glyceraldehyde- FUN_002634 contig_122 14229.. Pgpd-start- GAATTGGGTA 1378 N/A 3- 147173 FW CCTGAGTTGA phosphate AT dehydrogenase (SEQIDNO.13) (GPD) Pgpd-end- AAGCTTGGTT RV GAGAGGATG GGATG (SEQIDNO.14)
Design and Synthesis of the Cassette for Hph6 Gene Expression Functional in SCC-0006:
[0161] Construction of the pGDH-HPH6-GFP6-tGDH plasmid. Once the pGDH promoter and the tGDH terminator were selected and the hph gene was optimized, the Hygromycin B resistance cassette functional in SCC-0006 using the GDH promoter was designed as follows (
[0162] pGDH_0006 module: pGDH promoter (1589 bp) with a KpnI restriction enzyme site added upstream from ATG. HPH6 module: hph6 optimized gene functional in SCC-0006 (1023 bp) flanked by SpeI and EcoRI restriction enzyme sites at 5 and 3 ends, respectively. The original coding DNA sequence of the hph gene was obtained from the commercial plasmid pAN7.1. Next, a DNA sequence with Gly-link8 (SEQ ID NO. 39) polylinker (24 bp) was included. fuGFP6 module: Green fluorescent protein (GFP) encoding gene optimized with the SCC-0006's usage codon (723 bp) and flanked with EcoRI and BamHI restriction enzyme sites at 5 and 3 ends, respectively. tGDH_0006 module: tGDH terminator (326 bp) flanked by BamHI and HindIII at 5 and 3 ends, respectively. The whole construct was synthesized (Genscript, Piscataway, NJ, USA) and cloned in the KpnI restriction enzyme site of the plasmid pBluescript II SK(+), giving rise to the pGDH-HPH6-GFP6-tGDH plasmid (
TABLE-US-00003 >pGDH_0006module:(SEQIDNO.15): cgccagaaaacatcgatatcgggcccgaatgaaccgcgacatgctcccaagtgactggctggaa ggtggctccaccgctattctgtcaatgtcacttccaaggagccggcagatctatgcacaagtat ttgtgactggattgtgagccgtaaatgtggatagcaccaaaatgcgaggccactatgtaagaaa acctttcggtgggacctgttgagagaatcggcgcttccactcacgcctcccttctgccggaacg acagtaacaccaacttagcacattcacagctgagatttggagatagaaggtcgcaaagatgtgc gaatcactgtgagggtggccggcttcatgagcttcagaagtagtctccgcagcgcggggagcta gcttcgttgcataacggttgtgcgacatcccgcggaggatctacgttgcgcaagatggccccaa cgttggccgaggacggcacaaggcagcagtctgcacataacgtacgttgtacgttgtacatatg ccattgggcactcgcaagcgttccccacaaacatacggcttcgagaccggggtaatgtggtggc acttcgcatgcctccgtgcacagatgcacacgttcgactctagagcagccccttcaaacgttca ccgcgaacaactaacgctgcagctgactgggcacgccgctccttacgcacgctcggtagggtcg gtacgagagccgatgaaggggcggacattggggccgtcgggcgtggtgggcaggtgacgcggag gaagagacgttttgggtgtttggcgagcacgcacctgcgtcgcgagcgtctgggtaattgcgac cgtcaccaagcatttctcgggggtacacatccgcctggagaggctgtggcatgggacgacatcg ggcagcttggtatttgtcgcggccagggcagcggcgggggcgccctggagagatcgtggtccga ccggcggctgaggcggggtcacaagacggatggagagagggagggcatggaccggccgagggag agacacgtatgtcggggaggggacatcggatcttctgaccctctcaccgcggaaagcaatgatc gcgagggtattcttgggtgggccaatccatcgcggaggaatgcaagaaattgttgggggccaaa cgcaagacccggacaggcgcacgcgagcggcgtgcacgggactcgtgagcgcgcgcacgggagt atggcaacacgaaaggcggtgagaacagggccgtgctcgctgtgctgcgtggtgagaaacgcgc tggttgcgacgtcctcggccgacggagaatggcatcggacgggtacgaagacgaggatgggatg caaacgggaggaaggattatcactccgcccaggcaaggacgggcgtctgtgcgattgtctagat ctggaccataacccaaacgttaatgcagtctcctccgcttgacgtatataaaatgccagggcct ccccattccattcacttctccctcttccatctcccccatatcttcctcgcgcctctccacactc acctcacctccgcatagcacacaacgagcgcggtcacatcccccgacgcgacc >HPH6-module:(SEQIDNO.16): atgaagaagcccgagctcacggcgacgtcggtcgagaagttcctcatcgagaagttcgactcgg tctcggacctcatgcagctctcggagggcgaggagtcgcgcgcgttctcgttcgacgtcggcgg ccgcggctacgtcctccgcgtcaactcgtgcgcggacggcttctacaaggaccgctacgtctac cgccacttcgcgtcggcggcgctccccatccccgaggtcctcgacatcggcgagttctcggagt cgctcacgtactgcatctcgcgccgcgcgcagggcgtcacgctccaggacctccccgagacgga gctccccgcggtcctccagcccgtcgcggaggcgatggacgcgatcgcggcggcggacctctcg cagacgtcgggcttcggccccttcggcccccagggcatcggccagtacacgacgtggcgcgact tcatctgcgcgatcgcggacccccacgtctaccactggcagacggtcatggacgacacggtctc ggcgtcggtcgcgcaggcgctcgacgagctcatgctctgggcggaggactgccccgaggtccgc cacctcgtccacgcggacttcggctcgaacaacgtcctcacggacaacggccgcatcacggcgg tcatcgactggtcggaggcgatgttcggcgactcgcagtacgaggtcgcgaacatcttcttctg gcgcccctggctcgcgtgcatggagcagcagacgcgctacttcgagcgccgccaccccgagctc gcgggctcgccccgcctccgcgcgtacatgctccgcatcggcctcgaccagctctaccagtcgc tcgtcgacggcaacttcgacgacgcggcgtgggcgcagggccgctgcgacgcgatcgtccgctc gggcgcgggcacggtcggccgcacgcagatcgcgcgccgctcggcggcggtctggacggacggc tgcgtcgaggtcctcgcggactcgggcaaccgccgcccctegacgcgcccccgcgcgaagaag >fuGFP6module:(SEQIDNO.17): atggtctcgtcgggcgaggacatcttctcgggcctcgtccccatcctcatcgagctcgagggcg acgtcaacggccaccgcttctcggtccgcggcgagggctacggcgacgcgtcgaacggcaagct cgagatcaagttcatctgcacgacgggccgcctccccgtcccctggcccacgctcgtcacgacg ctctcgtacggcgtccagtgcttcgcgaagtaccccgagcacatgcgccagaacgacttcttca agtcggcgatgcccgacggctacgtccaggagcgcacgatctcgttcaaggaggacggcacgta caagacgcgcgcggaggtcaagttcgagggcgaggcgctcgtcaaccgcatcgacctcaagggc ctcgagttcaaggaggacggcaacatcctcggccacaagctcgagtactcgttcaactcgcact acgtctacatcacggcggacaagaaccgcaacggcctcgaggcgcagttccgcatccgccacaa cgtcgacgacggctcggtccagctcgcggaccactaccagcagaacacgcccatcggcgagggc cccgtcctcctccccgagcagcactacctcacgacgaactcggtcctctcgaaggacccccagg agcgccgcgaccacatggtcctcgtcgagttcgtcacggcggcgggcctctcgctcggcatgga cgagctctacaagtcgtga >tGDH_0006module:(SEQIDNO.18): ggcgcagacgcacccgtcggccctcacgcgtcatagtcacacaaaagctatcgggagactcgtc gaattgaatacagtgtggtaaaaggcaagaatcagcagtaaacctaatacggagccagccgctg ctccgctgtgtaccaagctcccatgtagaagaattgtatgataatataaatagtagattttcgt catcgagattcattcgttagattgtcagtgaggatgtccgacatacaaacgatcgacattgtat gtgccgagggtccaacgttgtgcatatattgtgctcactgctcgtgcgctgaatggaatcgaag ttcaca >GDH-HPH6-GFP6-tGDH_cassette:(SEQIDNO.19): cgccagaaaacatcgatatcgggcccgaatgaaccgcgacatgctcccaagtgactggctggaa ggtggctccaccgctattctgtcaatgtcacttccaaggagccggcagatctatgcacaagtat ttgtgactggattgtgagccgtaaatgtggatagcaccaaaatgcgaggccactatgtaagaaa acctttcggtgggacctgttgagagaatcggcgcttccactcacgcctcccttctgccggaacg acagtaacaccaacttagcacattcacagctgagatttggagatagaaggtcgcaaagatgtgc gaatcactgtgagggtggccggcttcatgagcttcagaagtagtctccgcagcgcggggagcta gcttcgttgcataacggttgtgcgacatcccgcggaggatctacgttgcgcaagatggccccaa cgttggccgaggacggcacaaggcagcagtctgcacataacgtacgttgtacgttgtacatatg ccattgggcactcgcaagcgttccccacaaacatacggcttcgagaccggggtaatgtggtggc acttcgcatgcctccgtgcacagatgcacacgttcgactctagagcagccccttcaaacgttca ccgcgaacaactaacgctgcagctgactgggcacgccgctccttacgcacgctcggtagggtcg gtacgagagccgatgaaggggcggacattggggccgtcgggcgtggtgggcaggtgacgcggag gaagagacgttttgggtgtttggcgagcacgcacctgcgtcgcgagcgtctgggtaattgcgac cgtcaccaagcatttctcgggggtacacatccgcctggagaggctgtggcatgggacgacatcg ggcagcttggtatttgtcgcggccagggcagcggcgggggcgccctggagagatcgtggtccga ccggcggctgaggcggggtcacaagacggatggagagagggagggcatggaccggccgagggag agacacgtatgtcggggaggggacatcggatcttctgaccctctcaccgcggaaagcaatgatc gcgagggtattcttgggtgggccaatccatcgcggaggaatgcaagaaattgttgggggccaaa cgcaagacccggacaggcgcacgcgagcggcgtgcacgggactcgtgagcgcgcgcacgggagt atggcaacacgaaaggcggtgagaacagggccgtgctcgctgtgctgcgtggtgagaaacgcgc tggttgcgacgtcctcggccgacggagaatggcatcggacgggtacgaagacgaggatgggatg caaacgggaggaaggattatcactccgcccaggcaaggacgggcgtctgtgcgattgtctagat ctggaccataacccaaacgttaatgcagtctcctccgcttgacgtatataaaatgccagggcct ccccattccattcacttctccctcttccatctcccccatatcttcctcgcgcctctccacactc acctcacctccgcatagcacacaacgagcgcggtcacatcccccgacgcgaccactagtatgaa gaagcccgagctcacggcgacgtcggtcgagaagttcctcatcgagaagttcgactcggtctcg gacctcatgcagctctcggagggcgaggagtcgcgcgcgttctcgttcgacgtcggcggccgcg gctacgtcctccgcgtcaactcgtgcgcggacggcttctacaaggaccgctacgtctaccgcca cttcgcgtcggcggcgctccccatccccgaggtcctcgacatcggcgagttctcggagtcgctc acgtactgcatctcgcgccgcgcgcagggcgtcacgctccaggacctccccgagacggagctcc ccgcggtcctccagcccgtcgcggaggcgatggacgcgatcgcggcggcggacctctcgcagac gtcgggcttcggccccttcggcccccagggcatcggccagtacacgacgtggcgcgacttcatc tgcgcgatcgcggacccccacgtctaccactggcagacggtcatggacgacacggtctcggcgt cggtcgcgcaggcgctcgacgagctcatgctctgggcggaggactgccccgaggtccgccacct cgtccacgcggacttcggctcgaacaacgtcctcacggacaacggccgcatcacggcggtcatc gactggtcggaggcgatgttcggcgactcgcagtacgaggtcgcgaacatcttcttctggcgcc cctggctcgcgtgcatggagcagcagacgcgctacttcgagcgccgccaccccgagctcgcggg ctcgccccgcctccgcgcgtacatgctccgcatcggcctcgaccagctctaccagtcgctcgtc gacggcaacttcgacgacgcggcgtgggcgcagggccgctgcgacgcgatcgtccgctcgggcg cgggcacggtcggccgcacgcagatcgcgcgccgctcggcggcggtctggacggacggctgcgt cgaggtcctcgcggactcgggcaaccgccgcccctcgacgcgcccccgcgcgaagaaggaattc ggcggtggcggtggcggtggcggtatggtctcgtcgggcgaggacatcttctcgggcctcgtcc ccatcctcatcgagctcgagggcgacgtcaacggccaccgcttctcggtccgcggcgagggcta cggcgacgcgtcgaacggcaagctcgagatcaagttcatctgcacgacgggccgcctccccgtc ccctggcccacgctcgtcacgacgctctcgtacggcgtccagtgcttcgcgaagtaccccgagc acatgcgccagaacgacttcttcaagtcggcgatgcccgacggctacgtccaggagcgcacgat ctcgttcaaggaggacggcacgtacaagacgcgcgcggaggtcaagttcgagggcgaggcgctc gtcaaccgcatcgacctcaagggcctcgagttcaaggaggacggcaacatcctcggccacaagc tcgagtactcgttcaactcgcactacgtctacatcacggcggacaagaaccgcaacggcctcga ggcgcagttccgcatccgccacaacgtcgacgacggctcggtccagctcgcggaccactaccag cagaacacgcccatcggcgagggccccgtcctcctccccgagcagcactacctcacgacgaact cggtcctctcgaaggacccccaggagcgccgcgaccacatggtcctcgtcgagttcgtcacggc gggggcctctcgctcggcatggacgagctctacaagtcgtgaggatccggcgcagacgcaccc gtcggccctcacgcgtcatagtcacacaaaagctatcgggagactcgtcgaattgaatacagtg tggtaaaaggcaagaatcagcagtaaacctaatacggagccagccgctgctccgctgtgtacca agctcccatgtagaagaattgtatgataatataaatagtagattttcgtcatcgagattcattc gttagattgtcagtgaggatgtccgacatacaaacgatcgacattgtatgtgccgagggtccaa cgttgtgcatatattgtgctcactgctcgtgcgctgaatggaatcgaagttcaca >HPH6-GFP6chimericprotein(aa)(SEQIDNO.20): MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDRYVY RHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETELPAVLOPVAEAMDAIAAADLS QTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQALDELMLWAEDCPEVR HLVHADFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPEL AGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDG CVEVLADSGNRRPSTRPRAKKEFGGGGGGGGMVSSGEDIFSGLVPILIELEGDVNGHRFSVRGE GYGDASNGKLEIKFICTTGRLPVPWPTLVTTLSYGVQCFAKYPEHMRQNDFFKSAMPDGYVQER TISFKEDGTYKTRAEVKFEGEALVNRIDLKGLEFKEDGNILGHKLEYSFNSHYVYITADKNRNG LEAQFRIRHNVDDGSVQLADHYQQNTPIGEGPVLLPEQHYLTTNSVLSKDPQERRDHMVLVEFV TAAGLSLGMDELYKS*
[0163] Construction of the pPGPD-HPH6-tGPD plasmid. The Hygromycin B resistance cassette functional in SCC-0006 using the GPD promoter was designed as follows (see
[0164] pGPD_0006 module: pGPD promoter (1329 bp) with a KpnI restriction enzyme site added upstream from ATG. HPH6 module: hph6 optimized gene functional in SCC-0006 (1026 bp) flanked by HindIII and BamHI restriction enzyme sites at 5 and 3 ends, respectively. The original coding DNA sequence of the hph gene was obtained from the commercial plasmid pAN7.1. tGPD_0006 module: tGPD terminator (230 bp) flanked by BamHI and XbaI at 5 and 3 ends, respectively. The whole construct was synthesized (Genscript, Piscataway, NJ, USA) and cloned in the plasmid pBluescript II SK(+), giving rise to the pGPD-HPH6-tGPD plasmid (see
TABLE-US-00004 >pgpd_0006module:(SEQIDNO.21): tgagttgaatggtattatggttgcgcatattatgttgggtttatccgtcgatacttc gaagatatatgtgaacgcttggaatggagagcactgaggatgcaatttcgatgctacggcatca caacgatatgcggacgcaactgctataggggagacacattccctagacagtcaacacatcagcg gtgccataagtagtcaagcaacatatgcgatttcatgacgtacggtcgttgaaagtgattcacg gataccaatgtgcatgtgaaggttcagggcctgggagagttagtagtctagtctgtctgaagag ctgacctggcctacaaatggctgtcatgattgacgtaggaggaggctagtacctacagtgaacg aggcaccggaagagcagagctataagtgggatatgacggatggaaggaattatactgggccact cacgctcgacgcatccaagccgtgtatttcgagaggccgtctccgtctccagcggcagcggaca caagcacacgaacggcgtctgattggtggagtgggattagacgctcgtatgacgtcgtgggatt ggagagcagacacggagaacggatcttgctctcagcattcgagtcagaaaaaacatggggagca tatgtcgcgatatgtacgaacagcgacctgtgcattgctgacgaggacgagacaagtcgctgac agagagggcgcgtacggatctactgtgtcctattagcagtacacttgctagaaggtgccgtcga gacgcgcgaacggaataaggcgattgggagggcgcgtcgcgtccgagtcgcgtccagaccagac ccccgtcaattcggagaacgggccttcggcagccttgcgcgtacattctctctggtaatacacc ccttgcagcccttcgcgccgttcgtcctatcgattgatggccgttggcgagagagagagaggca gtgtgtgccccaaagggcgtcgcgactggtggggagacgcgtcgtacgctcctcctcaaagcaa ccaatcccgggcgggtaaattctggatcggtcgatggctgcgtatcgcccgcacgttcatccct ttcgcccgacggtcaccgcattccgtacagcgcgctgtgccctccgtatggatttgtaacgtgt tcggtctcattatgaacgtctcgccgtgggcaaatacgccgaagaatgtggagagagggaggag agtaggagaggaaagaggcgccaggagaggacgaggacgagacgacgcggcgatcgaacatctg agatgagataacaatcatccgccccttataaaccctcctcctctccctcctccttc >HPH6-module:(SEQIDNO.22): atgaagaagcccgagctcacggcgacgtcggtcgagaagttcctcatcgagaagttcgactcgg tctcggacctcatgcagctctcggagggcgaggagtcgcgcgcgttctcgttcgacgtcggcgg ccgcggctacgtcctccgcgtcaactcgtgcgcggacggcttctacaaggaccgctacgtctac cgccacttcgcgtcggcggcgctccccatccccgaggtcctcgacatcggcgagttctcggagt cgctcacgtactgcatctcgcgccgcgcgcagggcgtcacgctccaggacctccccgagacgga gctccccgcggtcctccagcccgtcgcggaggcgatggacgcgatcgcggcggcggacctctcg cagacgtcgggcttcggccccttcggcccccagggcatcggccagtacacgacgtggcgcgact tcatctgcgcgatcgcggacccccacgtctaccactggcagacggtcatggacgacacggtctc ggcgtcggtcgcgcaggcgctcgacgagctcatgctctgggcggaggactgccccgaggtccgc cacctcgtccacgcggacttcggctcgaacaacgtcctcacggacaacggccgcatcacggcgg tcatcgactggtcggaggcgatgttcggcgactcgcagtacgaggtcgcgaacatcttcttctg gcgcccctggctcgcgtgcatggagcagcagacgcgctacttcgagcgccgccaccccgagctc gcgggctcgccccgcctccgcgcgtacatgctccgcatcggcctcgaccagctctaccagtcgc tcgtcgacggcaacttcgacgacgcggcgtgggcgcagggccgctgcgacgcgatcgtccgctc gggcgcgggcacggtcggccgcacgcagatcgcgcgccgctcggcggcggtctggacggacggc tgcgtcgaggtcctcgcggactcgggcaaccgccgcccctcgacgcgcccccgcgcgaagaagt ga tGPD_0006module:(SEQIDNO.23): gccaacgacgatggacttgtgtggtctgtacccgatgatcagtaggaacggaagaagtatctcg tgtgtatgacgcctgtgtggaaacgtgtacatgtacttgtactctgctgcgtgcgtcgtgtagc gagacgattgtaatcataaaatgccatagacgtgcctcgtatctgtccctcgtgtgcgatgcga gactttcgagtacaagacaagcatggtgccagtggagg >GDH-HPH6-GFP6-tGDH_cassette:(SEQIDNO.24): tgagttgaatggtattatggttgcgcatattatgttgggtttatccgtcgatacttcgaagata tatgtgaacgcttggaatggagagcactgaggatgcaatttcgatgctacggcatcacaacgat atgcggacgcaactgctataggggagacacattccctagacagtcaacacatcagcggtgccat aagtagtcaagcaacatatgcgatttcatgacgtacggtcgttgaaagtgattcacggatacca atgtgcatgtgaaggttcagggcctgggagagttagtagtctagtctgtctgaagagctgacct ggcctacaaatggctgtcatgattgacgtaggaggaggctagtacctacagtgaacgaggcacc ggaagagcagagctataagtgggatatgacggatggaaggaattatactgggccactcacgctc gacgcatccaagccgtgtatttcgagaggccgtctccgtctccagcggcagcggacacaagcac acgaacggcgtctgattggtggagtgggattagacgctcgtatgacgtcgtgggattggagagc agacacggagaacggatcttgctctcagcattcgagtcagaaaaaacatggggagcatatgtcg cgatatgtacgaacagcgacctgtgcattgctgacgaggacgagacaagtcgctgacagagagg gcgcgtacggatctactgtgtcctattagcagtacacttgctagaaggtgccgtcgagacgcgc gaacggaataaggcgattgggagggcgcgtcgcgtccgagtcgcgtccagaccagacccccgtc aattcggagaacgggccttcggcagccttgcgcgtacattctctctggtaatacaccccttgca gcccttcgcgccgttcgtcctatcgattgatggccgttggcgagagagagagaggcagtgtgtg ccccaaagggcgtcgcgactggtggggagacgcgtcgtacgctcctcctcaaagcaaccaatcc cgggcgggtaaattctggatcggtcgatggctgcgtatcgcccgcacgttcatccctttcgccc gacggtcaccgcattccgtacagcgcgctgtgccctccgtatggatttgtaacgtgttcggtct cattatgaacgtctcgccgtgggcaaatacgccgaagaatgtggagagagggaggagagtagga gaggaaagaggcgccaggagaggacgaggacgagacgacgcggcgatcgaacatctgagatgag ataacaatcatccgccccttataaaccctcctcctctccctcctccttccgcatctcctcctct ctccatcccatcctctcaaccatgtctgtgcgtaccatccatacccgtctcctttccgtgtcgc atgctgatcgccctccacatgcagcaagtcaaggtcggaagcttcggatgaagaagcccgagct cacggcgacgtcggtcgagaagttcctcatcgagaagttcgactcggtctcggacctcatgcag ctctcggagggcgaggagtcgcgcgcgttctcgttcgacgtcggcggccgcggctacgtcctcc gcgtcaactcgtgcgcggacggcttctacaaggaccgctacgtctaccgccacttcgcgtcggc ggcgctccccatccccgaggtcctcgacatcggcgagttctcggagtcgctcacgtactgcatc tcgcgccgcgcgcagggcgtcacgctccaggacctccccgagacggagctccccgcggtcctcc agcccgtcgcggaggcgatggacgcgatcgcggcggcggacctctcgcagacgtcgggcttcgg ccccttcggcccccagggcatcggccagtacacgacgtggcgcgacttcatctgcgcgatcgcg gacccccacgtctaccactggcagacggtcatggacgacacggtctcggcgtcggtcgcgcagg cgctcgacgagctcatgctctgggcggaggactgccccgaggtccgccacctcgtccacgcgga cttcggctcgaacaacgtcctcacggacaacggccgcatcacggcggtcatcgactggtcggag gcgatgttcggcgactcgcagtacgaggtcgcgaacatcttcttctggcgcccctggctcgcgt gcatggagcagcagacgcgctacttcgagcgccgccaccccgagctcgcgggctcgccccgcct ccgcgcgtacatgctccgcatcggcctcgaccagctctaccagtcgctcgtcgacggcaacttc gacgacgcggcgtgggcgcagggccgctgcgacgcgatcgtccgctcgggcgcgggcacggtcg gccgcacgcagatcgcgcgccgctcggcggcggtctggacggacggctgcgtcgaggtcctcgc ggactcgggcaaccgccgcccctcgacgcgcccccgcgcgaagaagtgaggatccgccaacgac gatggacttgtgtggtctgtacccgatgatcagtaggaacggaagaagtatctcgtgtgtatga cgcctgtgtggaaacgtgtacatgtacttgtactctgctgcgtgcgtcgtgtagcgagacgatt gtaatcataaaatgccatagacgtgcctcgtatctgtccctcgtgtgcgatgcgagactttcga gtacaagacaagcatggtgccagtggagg >HPH6protein(aa):(SEQIDNO.25): MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYVLRVNSCADGFYKDRYVY RHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPETELPAVLQPVAEAMDAIAAADLS QTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTVMDDTVSASVAQALDELMLWAEDCPEVR HLVHADFGSNNVLTDNGRITAVIDWSEAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPEL AGSPRLRAYMLRIGLDQLYQSLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDG CVEVLADSGNRRPSTRPRAKK*
[0165] Obtaining transformant strains and initial selection: A genetic transformation technique was designed and validated. This technique allowed the insertion of DNA fragments (e.g., promoters, genes, terminators, cassettes, constructions, sgRNAs, etc.) in an aleatory or directed way in the genome (e.g., using integrative plasmids). It is also possible to introduce genetic information to be expressed in a non-integrated way into the genome, acting as an extrachromosomal element (e.g., using replicating plasmids with the useful AMA1 region).
[0166] The genetic transformation protocol developed for the SCC-0006 gold standard strain allowed various strategies to improve product performance and/or scaling-up bioprocess: I) homologous or heterologous gene overexpression (e.g., synthetic biology), II) genetic engineering, and III) genome editing using the CRISPR/Cas9 system. The transformation protocol described herein was used for the nonreferenced Unclassified Polyporaceae SCC-0006 strain, resulting in improvements in transformation efficiency, as described below.
Genetic Transformation Protocol:
[0167] Mycelial growth: Two 500 mL flasks with 100 mL of modified PDB media were inoculated with 4-8 blocks of mycelium grew for 5-7 days in agar medium (1 cm.sup.2 each) and incubated for 48-72 h at 25-28 C. and 200-250 rpm. The grown mycelium was double-filtered with a sterile nylon cloth (35 m) and Miracloth (Millipore). Then, the filtered mycelium was collected and homogenized in a sterile mortar (
[0168] Protoplast liberation: To prepare the lytic enzymes, a solution of Vinotaste Pro (Novozymes) was prepared in MgOsm buffer (0.5-1M MgSO.sub.4, 15-25 mM MES, pH 4.2-6.2). The lytic enzymes solution was sterilized by filtration with two syringe filters (0.45 m and then 0.22 m) compatible with aqueous solutions. The regenerated mycelium was filtered with a sterile nylon cloth (35 m) or Miracloth (Millipore) and washed with 400 mL of NaCl 0.9%. The mycelium was collected with a spatula and transferred to a 50 mL tube. The mycelium was suspended in 20 mL of MgOsm buffer and vortexed until disaggregated (see, e.g.,
[0169] Filtration of protoplasts and washing: Once the mycelium was incubated with the lytic enzymes, a sample was taken to confirm the protoplast liberation by optical microscope (
[0170] Protoplast transformation: the DNA mix was prepared in sterile 2 mL Eppendorf tubes containing: 10 L 0.01-0.1 M aurintricarboxylic acid (ATA) solution (Sigma-Aldrich, USA); 10 L PCM buffer (20-40% PEG 3350, 25-50 mM CaCl.sub.2), 5-10 mM MES, pH 5-6); 10-20 L concentrated plasmid (2-10 g plasmidic DNA).
[0171] 100 L of the protoplast solution was added to the DNA mix and homogenized using gentle finger touches. It was then incubated at 4 C. for 40-60 min. 50 L of protoplast was separated for positive and negative controls.
[0172] 500 L of PCM buffer was added to the previous mixture, homogenized using gentle finger touches and inversion, and then incubated at room temperature for 30 min.
[0173] 600 L of Sorb/CaOsm buffer was added to the previous mixture and homogenized using gentle finger touches and inversion.
[0174] For the recuperation step, the protoplast solution was mixed with 2 mL of PDB 1 M Sorbitol in a 15 mL tube and incubated horizontally for 1 hour at 30 C. in a rotator.
[0175] A two-layer method was used to plate the transformed protoplasts. The bottom layer was prepared with PDA 1M Sorb (2% agar) and supplemented with antibiotics (60 g/mL hygromycin B). For the top layer, PDA 1M Sorb (1% agar) was melted and tempered in a water bath at 55 C. for at least 1 hour. Once completed the protoplast's recuperation time, the whole 3 mL of protoplast mixtures were mixed with melted PDA 1M Sorb (1% agar), supplemented with antibiotics when needed (e.g., 60 g/mL hygromycin B), and distributed in petri dish with PDA base (2% agar).
[0176] As a control, the same protocol was used without the plasmidic DNA. Only 2 Petri dishes were used to plate the control, one with antibiotics (e.g., 60 g/mL hygromycin B) and the other without them. Instead of using the whole 3 mL of the protoplast's mixture, as shown above, 1.2 mL was mixed with 20 mL of PDA and then plated. The resulting plates were incubated at 28 C. for 1 week or until the appearance of transformant colonies (protoplast regeneration step).
[0177] Phenotypic screening of positive transformants HPH.sup.+: From several rounds of transformation experiments and later preselection of transformants obtained, nine colonies were obtained using the pGDH-HPH6-GFP6-tGDH plasmid, achieving a transformation efficiency of 0.9 transformants/g plasmidic DNA). These strains underwent three growth rounds under selective pressure (e.g., 60 g/mL hygromycin B), and finally, only three transformants were able to grow in the presence of hygromycin B without losing the cassette of hygromycin resistance (
[0178] Microscopic analysis of the selected positive transformants HPH.sup.+: The hyphal architecture of these three strains, SCC-0006/3.1, SCC-0006/3.6, and SCC-0006/3.9, were analyzed by optical microscopy and compared to the parental strain (wild strain). As a result, it was found that the SCC-0006/3.6 transformant best kept the hyphal architecture observed in the native SCC-0006 strain (
[0179] As discussed above, it is known that fungi possess different states concerning their ploidy. Monokaryotic strains possess one nucleus, and their hyphal architecture is characterized by septa that do not have clamp connections. On the other hand, dikaryotic strains (e.g., SCC-0006 and SCC-0006/3.6) possess two nuclei and their hyphal architecture is characterized by presenting septa with clamp connections. Based on this, it was proposed that the parental and SCC-0006/3.6 may form stable dikaryotic strains, and SCC-0006/3.1 and SCC-0006/3.9 are primarily monokaryotic strains. The proposal was tested by staining micro-cultures of each strain with DAPI to visualize the nuclei in the hyphal structure. Both the parental strain SCC-0006 and the SCC-0006/3.6 transformant presented cells delimited by septa with clamps and two visible nuclei (
Molecular Screening of Positive Transformants HPH+
[0180] To determine if the construction (e.g., GDH-HPH6-GFP6-tGDH cassette) was adequately integrated into the genome of the SCC-0006/3.6 transformant, PCR amplification and sequencing of the amplified DNA fragments was carried out using the parental strain SCC-0006 as the negative control, and the pyrG gene encoding orotidine-5-decarboxylase (FUN_004118) as an internal positive control.
[0181] In some cases, it may be desirable for a fungal strain used for the large-scale production of mycotextiles to allow traceability to the identity of the strain during and after the industrial process. This traceability can be performed using a unique DNA barcode inserted into the fungal strains' genomic DNA, as shown in
Effect of the Monokaryon and Dikaryon Fungal Strains on Mycelium Fabrics Development
[0182] To evaluate the abilities of the monokaryon and dikaryon strains to obtain well-colonized, dense and homogeneous mycelium fabrics, spawn of the transformants SCC-0006/3.1, SCC-0006/3.6, and SCC-0006/3.9 were used in the fermentation process as previously described, using the parental strain SCC-0006 as control. As observed, the mycelium of the monokaryon strains is relatively weak, fragmented (forming a fine powder), and easily detached from the scaffold. In the SCC-0006/3.6 dikaryon strain, the mycelium showed greater density, more vigorous, homogeneous growth, and the most adherence to the scaffold (
[0183] One possible theory to explain these results is represented in
[0184]
[0185] This mycelial stage in fungi can be preserved for long periods. Finally, two events can be observed: the first is to continue with the sexual reproduction cycle by the fusion of nuclei (karyogamy), culminating in the formation of fruiting bodies, and the second is to undergo a process called dedikaryotization, which converts dikaryotic cells into monokaryotic cells. This dedikaryotization process can be favored by environmental factors such as stress, nutrient availability, temperature, humidity, and CO.sub.2, among others, influencing the stability of the dikaryotic state in fungi and, therefore, changes in phenotype.
[0186] In the case of mycotextiles, dedikaryotization affects the homogeneity of the materials, as it can be observed that these will be composed of hyphae with different nuclear characteristics (dikaryotic and monokaryotic), each with its characteristic phenotype and therefore heterogeneity in the material. To regulate this heterogeneity in the SCC-0006 strain, the engineered strain SCC-0006/3.6 was created, which, thanks to the introduction of a resistance marker, exerts a selective pressure that prevents hyphae from undergoing dedikaryotization processes, thus resulting in nuclear fidelity, reflected in the maintenance of the dikaryotic genotype (diploid) and a more homogeneous phenotype during mycelial development.
[0187] Wild-type Basidiomycete fungi typically undergo de-dikaryotization processes. This phenomenon can be attributed to environmental factors such as nutrient availability, pH, temperature and other factors that can trigger transitions between dikaryotic and monokaryotic states. Another factor that may be responsible for this process is mutation or epigenetic changes within the nuclei, which may cause a loss of heterokaryotic compatibility, leading to dedication. When comparing the mycotextiles generated by the SCC-0006 parental strain, the mycelium may lose homogeneity due to dedikaryotization processes; this has been verified by the presence of arthroconidia, a reproduction structure of monokaryotic cells. The mycelium of the stable dikaryotic strain SC0006/3.6 tends to be more homogeneous, robust and vigorous, which when observed under a microscope shows the presence of generative hyphae with fibulas which is an indicator of a dikaryotic state.
[0188] In general, mycotextiles formed using a dikaryotic strain, and particularly a strain which is grown under conditions maintaining the dikaryotic state, may result in superior properties, and further the resulting mycotextile may be readily identified as arising from a dikaryotic strain by the presence of one or more easily identified markers, including clamp connections, and/or an enrichment for molecules related to clamp connections.
[0189] The mechanical properties of the resulting mycelia, hence the overall quality of the final mycotextile product, depend on hyphal architecture. SCC-006 possesses a trimitic hyphal system composed of generative hyphae, skeletal hyphae and ligative hyphae, the latter two being the most relevant for better mechanical properties. In monokaryotic mycelium, generative hyphae is predominant, and it is also prone to reproduce asexually by generating arthroconidia, fractioning generative hyphae and generating mycelia that is easily washed out during post-fermentation processes when growing the fungus to produce the mycotextile.
[0190] In contrast, dikaryotic mycelia may possess higher proportions of skeletal hyphae and ligative hyphae (e.g., greater than 50%, of the hyphal architecture may be skeletal hyphae and ligative hyphae, 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, etc.), which may be related to natural processes of fruiting bodies generation where their outstanding mechanical properties are due to the combination and interaction of the three types of hyphae. Dikaryotic strains may be better suited than a non-dikaryotic strain to obtain high-quality mycotextiles.
[0191] One advantage mentioned above is that the dikaryotic hypha may be produced by the actions of two nuclei on one phenotype. If, for example, one of the nuclei has a defective gene, the other can replace the function and if this type of genetic variation is not present, the phenotype is due to the additive action of the gene products of two nuclei.
[0192] The dedikaryotization phenomenon has been widely observed in fungi, but the environmental cues that trigger this event are not completely understood, making it particularly difficult to reliably control in manufacturing processes. It is unknown whether there is a basal dedikaryotization rate in the growing hyphae or whether that is influenced by conditions such as temperature, CO.sub.2 levels, humidity, etc. Further, such factors or conditions may be different for different fungal strains or species and may need to be determined for each fungal species.
[0193] For example, initial work has shown that the native strain SCC-006 is prone to developing monokaryotic growth areas during both pre-fermentation and fermentation processes. These areas usually coexist with dikaryotic mycelia, which may result in irregular growth. In the pre-fermentation stage, especially during plate propagation, the dedikaryotization phenomenon may be observed even when incubation conditions are maintained constant. Thus it may be particularly helpful to provide and/or use strains (dikaryotic fungal strains) that include a selective driver (such as antibiotic resistance) to help maintain selective pressure to remain dikaryotic. In fermentation processes, such as mycelia growth on mats, higher proportions of monokaryotic mycelium may result in deficient materials that do not endure post-fermentation treatments, resulting in loss of mycelia and poor-quality mycotextiles.
[0194] Although the fermentation processes may have growth conditions that are optimized to ensure fast substrate and mat colonization, these conditions have also been found to be suitable for dedikaryotization. At low-scale fermentation, environmental conditions are easier to control than large-scale processes, resulting in more regular-quality mats at low-scale. Concerning large-scale fermentation, complete control of this phenomenon is hard to ensure only by addressing environmental growth conditions. Thus, large-scale growth needed for mycotextiles may require the use of stable dikaryotic fungal strains and/or strains including a driver (such as chemical resistance and growth in the chemical) to help maintain the fungal strain in the dikaryotic state. As described above, the two separate haploid nuclei of the dikaryon are genetically similar to a diploid by having two copies of the nuclear genome per cell, so all gene products are duplicated, which gives an advantage in the phenotype, homogeneous behavior in growth, homogeneous behavior in the cell wall chemical composition, and homogeneous behavior related to the extracellular synthesis of enzymes associated to the nanoparticles mycosynthesis processes.
Detection of Dikaryotic Fungi in Mycotextile
[0195] A mycotextile formed using the methods and compositions described herein in which the fungal strain used is dikaryotic may include one or more characteristics indicating or confirming the use of the dikaryotic strain. For example, these compositions (mycotextiles) may have an ultrastructure visible by microscopy (including electron microscopy) unique to dikaryotic strains. In some cases, the mycotextile may include clamp connections or residue (e.g., proteins) correlated with clamp connections such as septins (e.g., one or more of CcCdc3, CcCdc10, CcCdc11a, CcCdc11b, and CcCdc12), cell end markers (e.g., TeaA, TeaB, Formin SepA, etc.), and proteins associated with mating and cell polarity (e.g., Pec1, Prf1, etc.) or other proteins (e.g., MAK-1, Thn1, etc.). Proteins/protein residues may be identified using, e.g., antibodies or immunohistochemical means.
[0196] In the final mycotextile, a genetic test may be used to verify the mycelium is dikaryotic. For example, genomic DNA may be extracted from the mycotextile and amplified (e.g., by PCR amplification) and sequenced to confirm the identity of the fungal strain used to form the material (e.g., the use of one of the strains identified herein), and in some cases, to identify that the stain include a sequence corresponding to resistance to the compound (e.g., drug, antibiotic, etc.). In any of these compositions the fungal strain may be identified by sequencing of a key gene (e.g., transcription factors, etc.) correlated with the fungal strain. For example, it may be reasonably concluded that the fungal strain used to form the mycotextile was in a dikaryotic state by confirming that the strain corresponds to SCC-006/3.6, e.g., by identifying the HPH:GFP cassette described above based on its sequence, e.g., using molecular markers such as ITS or RPB2. HPH:GFP cassette can allow differentiation between the SCC_0006/3.6 transformant from the SCC-0006 parental strain because the genes that code for resistance to hygromycin and the Green Fluorescent protein are naturally absent in the wild strain.
[0197] For example, the ITS region sequence of SCC-0006/3.6 (Seq. ID no.: 30) may be used to identify this and related fungal strains configured to be dikaryotic:
TABLE-US-00005 TTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGA AGGATCATTAACGAGTTCTGACATGGGTTGTAGCTGGCCTCACGAGGCA TGTGCACGCCCTGCTCATCCACTCTACACCTGTGCACTTACTGTAGGTT TGGCGTGGGCTTCGAGGGCCTTCACGGGCTTTTGAGGCATTCTGCCTGC CTATGTATCACTACAAACACTATAAAGTAACAGAATGTAATCGCGTCTA ACGCATCTTAATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCG ATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAG TGAATCATCGAATCTTTGAACGCACCTTGCGCTCCTTGGTATTCCGAGG AGCATGCCTGTTTGAGTGTCATGGTATTCTCAACCCACACATCCTTGTG ATGCTTGTGAGGCTTGGACTTGGAGGCTTGCTGGCCCGTCGCGGTCGGC TCCTCTTGAATGCATTAGCTTGGTTCCTTGCGGATCGGCTCTCAGTGTG ATAATTGTCTACGCTGTGACCGTGAAGCGTTTGGCGAGCTTCTAACCGT CCTGCTAGGGACAACTTACTTGACATCTGACCTCAAATCAGGTAGGACT ACCCGCTGAACTTAAGCATATCAATAA
[0198] All or a region of this sequence may be identified (e.g., 10 bp or more, 12 bp or more, 13 bp or more, 15 bp or more, etc.).
[0199] As mentioned, the strain used to form the mycotextile may include a gene for resistance to a drug or other agent. As mentioned, the resistance may be to Hygromycin B (hph gene) as the antibiotic resistance to allow selection of the dikaryotic strain. Other resistances may include Nourseothricin, Carboxin are other antibiotics that have an inhibitory effect on the growth of the native fungal strain (e.g., a native SCC-006 strain) in an acceptable concentration range. For Nourseothricin the fungal strain may include a plasmid for the expression of the nat gene (Nourseothricin N-acetyl transferase) that confers resistance to the antibiotic.
[0200] In some cases, an engineered dikaryotic fungal strain, such as, but not limited to SCC-0006/3.6, may include genetic material conferring resistance to a drug (e.g., an antibiotic). In some examples the resistance results from integrating the pGDH-HPH6-GFP6-tGDH plasmid in its genome (see in the Annex). Therefore, it is possible to amplify by PCR and sequence a fragment of this unique DNA barcode that is only present in this engineered strain (e.g., the resistance cassette carrying the HPH6 and/or GFP6 gene, which were designed and synthesized using the own usage codon of the SCC-0006 strain).
[0201] In one example a molecular marker may be used to verify that the mycelium of the mycotextile was in the dikaryotic state. In this example, genetic material from the formed and completed mycotextile (which may be present but fragmented) may be amplified from the mycotextile by PCR and sequenced to identify the Hom2 gene (transcription factor). In some cases, the presence of a single nucleotide polymorphism (SNP) can feasibly indicate if the strain is in a dikaryotic state. For example, genomic DNA may be isolated from the mycotextile (fabric), and multiple (e.g., four) PCR reactions can be performed with different combinations of primer pairs (forward and reverse) as indicated: i) M13F+M13R: Amplification with universal primers; ii) M13F+HPH_int_R: Amplification of a region of the pHPH_GFP V1 plasmid for the identification of the transformant; iii) gfpF+M13R: Amplification of a region of the pHPH_GFP V1 plasmid for the identification of the transformant; and iv) ITS1 and ITS4: Amplification of the intergenic spacer sequence (ITS), as a positive control. Also, the parental SCC-0006 strain is used as a control in all the PCR reactions. The conditions for amplification using the PCR technique (e.g., thermocycler conditions) may include: Initial Denaturation: 95 C.3; Denaturation: 95 C.45; Annealing: 58 C.45; Extension: 72 C.1; Final Extension: 72 C.10.
[0202] The amplification of the PCR product with the universal primers M13 Forward and M13 Reverse will only be positive in the pHPH-GFP V1 plasmid used as a control, yielding the expected sizes, as shown in Table 2:
TABLE-US-00006 TABLE I PCR reaction results as shown in FIG. 1. gDNA SCC- gDNA SCC- pHPH- Expected Primers Pairs 0006 (A) 0006/3.6 (A) GFP V1 size M13F + M13R (i) Not amplified Not amplified 3938 bp 3938 bp M13F + hph_int_R (ii) Not amplified 1824 bp 1824 bp 1824 bp gfp_F + M13R (iii) Not amplified 652 bp 652 bp 652 bp ITS1 + ITS4 (control) 643 bp 643 bp Not amplified 643 bp
Table 2: PCR Reaction Results
[0203] In one example, amplification with the universal primers M13 (Forward or Reverse)+specific primers (ii and iii) was positive in the SCC-0006/3.6 strain and the pHPH-GFP V1 plasmid. Notably, to obtain PCR amplifications using primer pairs ii and iii using genomic DNA of SCC-0006/3.6 DNA as a template, the internal DNA sequence of the hygromycin resistance cassette (HPH) is known (see above). The PCR products may be generated using primers ITS1 and ITS4, used as an internal control, were positive in the genomic DNA samples from both the parental strain SCC-0006 and SCC-0006/3.6. Table 2 presents the expected sizes in base pairs for each of the PCR products, which are consistent with the results obtained and shown in
[0204] Also described herein are methods and apparatuses (e.g., systems, including reagents) for detecting that the mycotextile was formed using a dikaryotic fungal strain. For example, in some cases, an analysis of a mycotextile may include determining if the mycotextile can be sequenced to identify the presence of a drug resistance gene; in some cases, the test may sequence genetic material (residual genetic material) from the mycotextile including the Hom2 (a transcription factor gene) which is correlated with the presence of dikaryotic fungal strains. Dikaryotic strains in fungi, especially in basidiomycetes, are characterized by having two haploid nuclei (n+n) coexisting within the same cell without fusion. Unlike monokaryotic strains, which contain a single haploid nucleus (n) per cell and represent the initial phase of mycelium after spore germination, dikaryotic strains form when two compatible monokaryotic mycelium fuse, sharing their nuclei but without immediate karyogamy. This dikaryotic condition allows for developing advanced reproductive and differentiating structures for the mycelial network. To differentiate monokaryotic from dikaryotic strains, tools such as Single Nucleotide Polymorphism (SNP) identification can be employed, distinguishing specific genetic sequences of each nucleus in dikaryotic. Due to the presence of these polymorphisms in specific genes between heterokaryons, it is possible to distinguish them molecularly. In the SCC-0006 species, a polymorphism has been found in the FUN_010251 gene that encodes a putative transcription factor Hom2. This transcription factor is crucial for regulating genes involved in fungal development and morphogenesis. The SNP identified in the Hom2 gene can be used as an effective tool to distinguish between monokaryotic and dikaryotic strains, as is shown in
TABLE-US-00007 Forthisexample,thistechniquemayuseprimersincluding: M13F,e.g., SEQIDNo.:31 (5...GTAAAACGACGGCCAGT...3; M13R, SEQIDNo.:32 (5...CAGGAAACAGCTATGAC...3); gfpF, SEQIDNo.:33 (5...GGACCACTACCAGCAGAACA...3); hph_int_R, SEQIDNo.:34 (5...CGTAGCGGTCCTTGTAGAA...3); ITS1, SEQIDNo.35 (5...TCCGTAGGTGAACCTGCGG...3); ITS4, SEQIDNo.36 (5...TCCTCCGCTTATTGATATGC...3) >gDNA_Hom2 (SEQIDNo.:SEQIDNo.37 ATGCTACCCACCACTCACGATCCTTCCTCCTCCGCACTCAACCACCAGCCCTCCCTCTACCCCG TCGTCCCCCACCACTCCCTCCCACAGAACCCCGTTCACCACCATCCTTATCCTATTCCCCCTCA GAACCCTCCCTTTGATCACCACCAGCAACTCCAGCAGCTGCACCCCGCCCTCTCCCATCCTCAC AACCACCACCCACACATCCACCACCTCCAGCAGAACCAGACTTGCGTACCCGCTCTCATCCCAC CAGGCATGGACCCTTCCCAGGTCGACATCCGCACCTTCTATCCCTACACCCCAAACGAAGTAAA GCACAGAAAGCGCACCACCCGCGCTCAGCTCAAGGTCCTCGAGGGTGTCTACAAATATGACACA AAGCCCAACGCCTCCCTTCGCAAGAAACTCGCGGCAGAGCTCGACATGACTCCCCGCGGTGTCC AGGTACTTGCCTTCCTCGATTGCAACCCGTTCCCTTCTATCTCCTCTCCCTCTGTCATTGTCCC GTGCTTATCTGGTCCTTCCTTCCTCTCTTCCTCCAGGTCTGGTTCCAGAATCGCCGTGCCAAAA CAAAACAGCAAGCCAAGAAGGCGGAGGCCGCCAACGCCGCCAAGGGCTCCCCGTCTGACCCCTC ATC(G/A)TCGGCGACGGCAGCAGCAGCCGCCTCCAACCCCCCGATAGCACCTCGTCCTACCGA AGACGCGGCAGACGACGACGACAACGACAACGACGATCCAGACGAGCCATGTGCATTGGCGCCT CCGTCTCCCAAGCAATCGGAGGACGCAACACCGGAAAACGCTGCAACAGCTGACGGCACCGTTC CCGCTTCCTCAAACGCAAACCCGAACGCCAGTGCACAGCAGGACAACGAACAGGCCCACCTAAC GCCTGATAGCCGCCGCTCCTCCATGGCCCCTCCCCTTCCTCGGTCCCCCGCTTGGTCATCGTCT TCTCCCTCGGTCTCCACGGCGCCTGCCCCCTCTTCAACCGCTCCCTCGTCCGCACTCAGCAGTC CCAATCCCGCGAATCATGCGAACAGTTCCCACGTTCGTCTCTCCACTCACCCGCCCTCCGCCAC CTCTTCCTACTCGCACAACCACCTCGCGCCCACCGACATTTACTCGCAGCGACGCACCTCTCTC CCGCCTTCCCTTTCCTCCACATCTGGTCATGCCCCCGGCATGCTGAGTACCCTCCGTCGACGCG GCTATGACGACACCCGCCGACGCTCGACAGACATGGGCGGTCACCGCATCGTCGCCCATCCGTA CATCTCTGTCGCCCAGAGCGCCAACGGCCCGCATAATATATACGGCGATGGTGACGACGCTCCG CCTGCCAGGCGTCCCATGCTCATGCAGCGTATGACCGCACCCTACGCTTCGAACCACCACTCGC AAGGGACGCAAGCGATGCACTCTCCGCAGGCGCATTCTCAACATGCCCACTCCCAGTCGATCTT GCCCTCCCAGGCGCAGCAACAGCGAGCGCAGCACCTGGGACAGCAGCGCCAGCACTACGACATC TCGCCCATTCAGGTGTCCATCTCCCATTCGCAGGGCTACAACCAGCCGCCAAACCACCCGGGCA GTCACGGCTACGATCTGTTCGCGCCCCGACATTCGATCGACGGCAGCGCGCTCGGCCTGACCCA AGCGCACGCACAGATGAGCATGGGCCTGTCTCCCTTGCACCCTGCGCCTGGCACAGGAATGGAC AACGACGCGTTTAGCATGGGTCTCGGGATGAGCAGCCACTACGCTGTGTCGCAGCGTCCACTAC CCCCTACCATACCTGGACCGCTTCCCTCTCCCAACTATCAGTTCGGGAACCCGTTCGTACCTGC CCCGAACGACTCGTCTTCTAGCTCTTCCATGAGCAACTCTGCGTCGGGCACGCCTCCCAATGGC GCTTCGCCGCCATTGCTCAGCCTGCGGCGTACAAGCGAGAGCGCATTGTCAGATGGAGACACGG AGGAGAGCAGCGGCGCGCCTCTGTCCAGATTTGGCAGTATTGCGAGTATCAACGGC >Hom2protein (SEQIDNO.38) MLPTTHDPSSSALNHQPSLYPVVPHHSLPQNPVHHHPYPIPPQNPPFDHHQQLQQLHPALSHPH NHHPHIHHLQQNQTCVPALIPPGMDPSQVDIRTFYPYTPNEVKHRKRTTRAQLKVLEGVYKYDT KPNASLRKKLAAELDMTPRGVQVWFQNRRAKTKQQAKKAEAANAAKGSPSDPSSSATAAAAASN PPIAPRPTEDAADDDDNDNDDPDEPCALAPPSPKQSEDATPENAATADGTVPASSNANPNASAQ QDNEQAHLTPDSRRSSMAPPLPRSPAWSSSSPSVSTAPAPSSTAPSSALSSPNPANHANSSHVR LSTHPPSATSSYSHNHLAPTDIYSQRRTSLPPSLSSTSGHAPGMLSTLRRRGYDDTRRRSTDMG GHRIVAHPYISVAQSANGPHNIYGDGDDAPPARRPMLMQRMTAPYASNHHSQGTQAMHSPQAHS QHAHSQSILPSQAQQQRAQHLGQQRQHYDISPIQVSISHSQGYNQPPNHPGSHGYDLFAPRHSI DGSALGLIQAHAQMSMGLSPLHPAPGTGMDNDAFSMGLGMSSHYAVSQRPLPPTIPGPLPSPNY QFGNPFVPAPNDSSSSSSMSNSASGTPPNGASPPLLSLRRTSESALSDGDTEESSGAPLSRFGS IASINGSEASWTSAYVSDSAAEGEEGDGSASRKMSCASEFLGMFSGLDVGSNGGTPAPHGLQES HPLRQSRSSSHLSPNAFVPHHMQAPAPTPPDSMGADQQQQQHIHMQAPSDADGYPSPSSASTVS AGSNHGNGGSHTHILSHDTTPSHTTVGNGNGGHLQQQGANSMRNHSSSELAYALQGEPEPPRGY QQHPPAGAKEELQYPMYVSQEASETDAGDAAAYGYAQEQGHHDAREYAKAQMGHFPTLYEGYVY PHGNVNDQIPEEDASAMSEAYAAGAIELSHMCVPASEGVHFMGGYMQYS*
[0205] In any these cases, microscopy (e.g., light microscopy, electron microscopy, etc.) may be used to analysis the mycotextiles, including newly harvested mycotextiles. For example, in basidiomycete fungi, monokaryotic organisms are characterized by having a single nucleus in their hyphae; therefore, these cells are haploid. They originate from a basidiospore or arthrospore that germinates to form a hypha, which expands and grows. The monokaryotic mycelium generally cannot form fruiting bodies by itself. It needs to be fused with another compatible mycelium to generate a dikaryotic (diploid) organism and advance in its life cycle. Therefore, asexual reproduction mediated by arthroconidia predominates in them. Dikaryotic hyphae contain two haploid nuclei (n+n) of different origins in each cell. These nuclei do not fuse immediately and coexist in the cell. One of the distinguishing characteristics between dikaryotic and monokaryotic mycelia is the presence of clamp connections in the dikaryotic mycelium, as discussed above. This structure ensures that each daughter cell receives one of the two haploid nuclei, maintaining the dikaryotic state in the hyphae. Optical microscopy was used to observe the dikaryotic state in the transformant strain SCC-0006/3.6. The search for clamp connections in the final product (mycotextile) is complicated due to post-harvest treatments applied to the material, which hinder the observation of this structure. However, during the fermentation stages, its identification is clear and serves as an indicator of the dikaryotic composition of the hyphae.
Redox Potential of Different Fungal Strains
[0206] Also described herein are methods, compositions and systems for forming nanoparticles as part of the fungal growth. Nanomaterials are synthesized using a variety of methods, both physical and chemical. In the chemical method, the synthesis of nanoparticles is based on an oxidation/reduction process, in which metal ions present in the precursor are reduced by certain compounds, generating nucleation centers, from where the nanoparticles emerge and grow until reaching a size at the nanoscale. In this method, the reducing agent is crucial in the synthesis process because it delivers the electrons to the ions to form (e.g., to accumulate) atoms. It is known that the reducing power of a compound is related to its electron transfer capacity, which is linked to its potential antioxidant activity. It is known that certain natural products with high antioxidant ability can synthesize metallic nanoparticles because they not only effectively reduce metal salts but also act as stabilizing agents. In order to evaluate the antioxidant capability of certain compounds, some spectrophotometrically quantifiable model molecules could be used. In this case, the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH.sup., C.sub.18H.sub.12N.sub.5O.sub.6) was used to evaluate different fungal strains and determine their potential to synthesize metal nanoparticles. DPPH.sup. is characterized as a stable free radical because of the delocalization of the unpaired electron on the whole molecule. Antioxidants (AH) or other radical species (R) can react with this stable radical, providing an electron or a hydrogen atom, thus reducing it to 2,2-diphenyl-1-hydrazine (DPPH-H) or a substituted hydrazine analog (DPPH-R) characterized by the color change from dark violet to colorless or pale yellow. The resulting discoloration is stoichiometric concerning the number of electrons absorbed that could be followed spectrophotometrically.
Materials and Methods for Determining Antioxidant Activity by the DPPH.SUP. Method
[0207] A solution of DPPH.sup. (600 M) in 80% ethanol was prepared and diluted until an absorbance of 0.70.02 units was obtained at 517 nm. A 0.1 g mycelium mass from each strain was weighed and introduced in an amber Eppendorf tube. Then, 1000 l of the DPPH.sup. solution was added. In parallel, a control sample (without mycelium) was prepared. Each tube was incubated in the dark, and samples were taken every 15 minutes for 1 hour to measure the absorbance at 517 nm. The % of DPPH.sup. consumed was determined as follows: DPPH.sup. consumed (%)=[(Absorbance.sub.ControlAbsorbance.sub.Sample/Absorbance.sub.Control)100]. Knowing the initial amount of DPPH and the calculated consumed percentage, the mg of DPPH.sup. consumed was normalized by the mg of mycelium.
[0208] Results of the antioxidant activity of selected fungal strains:
TABLE-US-00008 TABLE 3 The list of fungal strains selected to determine antioxidant activity by the DPPH Method. SCC No. Family Genus Species Source 0002 Polyporaceae N.A N.A MYCOTEXTILES INCLUDING 0006 N.A N.A ACTIVATED SCAFFOLDS 0013 Lentinus sp. AND NANO-PARTICLE 0028 N.A N.A CROSS-LINKERS AND 0046 Polyporus sp. METHODS OF MAKING THEM U.S. patent publication No. US 2023/0356501-A1 0053 Ganodermataceae Ganoderma resinaceum LARGE-SCALE PRODUCTION 0066 Ganoderma cf. australe OF MYCELIUM-BASED 0067 Ganoderma cf. australe TEXTILES AT MUSHROOM 0075 Ganoderma cf. australe FARM FACILITIES 0076 Ganoderma cf. australe U.S. patent application 0078 Ganoderma cf. australe Ser. No. 18/595,392 0079 Ganoderma cf. australe 0080 Ganoderma cf. australe 0054 Fomitopsidaceae Fomitopsis abieticola 0059 Meruliaceae Bjerkandera adusta
[0209] The figures are discussed according to the families to which fungal strains belong. These results refer to the capacity of each of these strains for the biosynthesis of metallic nanoparticles since it also requires the transfer of electrons from the biological entity to the metallic ion dissolved in water.
[0210] The antioxidant activities measured in the five fungal strains belonging to the Polyporaceae family are displayed in
[0211] Likewise, eight fungal strains from the Ganodermataceae family, one of them belongs to G. resinaceum (e.g., SCC-0053) and seven to G. australe (e.g., SCC-0066, SCC-0067, SCC-0075, SCC-0076, SCC-0078, SCC-0079 and SCC-0080) species, were evaluated according to their antioxidant capabilities. It is essential to mention that G. australe native strains were isolated from different sampling sites and do not correspond to the same strain. As shown in
[0212] In
[0213] Regarding the comparison between the transformant SCC-0006/3.6 and its parental strain SCC-0006, the transformant shows an increase that almost doubles the antioxidant activity of its parental strain (
[0214] As mentioned above, the SCC-0002 and SCC-0013 strains in the Polyporaceae group showed a higher antioxidant activity after 45 and 60 minutes of reaction, respectively, reaching a plateau of around 0.075 mg of DPPH.sup./mg mycelium. In addition, the Ganoderma australe SCC-0079 was the Ganodermataceae strain with higher antioxidant activities after 30 minutes of reaction with 0.068 mg of DPPH.sup./mg mycelium and Fomitopsis abieticola SCC-0054 belonging to the Fomitopsidaceae family showed a uniform behavior over time, reaching an average of 0.063 mg DPPH/mg mycelium during all reactions. Also, the SCC-0006/3.6 transformant, a stable dikaryon transformant derived from SCC-0006, reached an average of 0.06 mg DPPH/mg mycelium from the beginning and during the entire reaction time (from 15 to 60 minutes).
[0215] It is clear that the high antioxidant activity, the speed of the enzymatic reaction from the beginning, and its uniformity during the time (e.g., without decay or decrease during the treatment) are aspects that evidence the adequate presence of reductase enzymes in the extracellular medium, which may promote a more efficient synthesis of nanoparticles throughout the whole treatment and, finally, ensuring a sidereal mycotextile with a modulated homogeneous and uniform pattern.
[0216] Considering the improved and homogenized properties of the dikaryon SCC-0006/3.6 regarding its high antioxidant activity, speed, and stability of the enzymatic reaction, as well as its high skills, the reproducible and scalable potential to produce a mycotextile with appropriate quality and performance, it was selected as a promising strain to continue the experiments, however the methods and compositions for in vivo formation of metallic nanoparticles (and subsequent coloring of the mycotextile) described herein may be used (and adapted for use) with virtually any fungal strain.
Sidereal Mycotextiles and Method of Forming them
[0217] Described herein are sidereal mycotextiles including at least some of the nanoparticles formed by an in vitro process and methods to obtain sideral mycelium fabrics according to the metallic species employed in each approximation. Mainly three approaches are described in this patent and may include the in vivo mycosynthesis of nanoparticles, including nanoparticles of silver, gold and iron oxide by directly incubating a freshly harvested crude mycotextile with the solution of each metallic salt made from raw mycotextile. In this case, the same living fungus performs the NP's mycosynthesis as well as forming the bulk of the mycotextile. Thus, additional nanoparticles (referred to as in vitro nanoparticles, typically ceramic nanoparticles, and/or ex vivo nanoparticles, typically metallic nanoparticles) optionally be included. The in vivo formation of nanoparticles by the activity of the fungus itself may be related to the antioxidant potential of the fungus used. The in vitro NPs biosynthesis methods described herein may be based on the biological synthesis of nanoparticles mediated by an organism different from the fungus (e.g., plants, prokaryotic or eukaryotic microalgae, etc.) and the addition of these NPs to the mycotextiles (containing the inactivated fungus), e.g., at a post-fermentation stage. Also described herein is the combination of both in vivo NPs mycosynthesis and in vitro NPs biosynthesis (e.g., ex vivo nanoparticles), which may include a first step of NP's mycosynthesis made by the same living fungus and a subsequent treatment with NPs biosynthesized by other organisms and its application in the post-fermentation stage. A graphical guide of the examples developed herein is displayed in
The In Vivo Mycosynthesis of Silver, Gold and Iron Oxide Nanoparticles
[0218] Silver nanoparticles (AgNPs) exhibit various biological activities, including antibacterial, antifungal, antiviral, hepatoprotective, and hypotensive properties. Extracts from the fungal fruiting body and mycelium may be used for AgNP mycosynthesis. In contrast, biomass, live cultures, and fungal metabolites of different purity (including enzymes, polysaccharides, and phenolic compounds) have been less explored. Moreover, additional external physical influences combined with the mycosynthesis methods have been developed to improve the characteristics of the nanoparticles. Likewise, gold nanoparticles (AuNPs) have been used in chemical and biological sensing, bioimaging, nonlinear optics, catalysis, targeted drug delivery, as antimicrobial and antioxidant agents, and in treating cancer, Alzheimer's, and cardiovascular diseases. On the other hand, iron oxides (IO NPs) have one of the most outstanding biocompatibilities because of their magnificent physical characteristics, such as superparamagnetism, low susceptibility to oxidation and long half-lives in blood. These nanoparticles have a wide range of applications, such as antibiotic degradation, dye adsorption, food-related processes, biomedical (drug delivery, magnetic cell sorting, magnetic resonance imaging, immunoassays, tissue engineering, tracking of stem cells, and cancer hyperthermia treatment).
Methods for the In Vivo NPS Mycosynthesis of Ag, Au, and IO NPs
[0219] In general, any of the methods and compositions described herein may include the formation of in vivo nanoparticles by incubating the live mycelium mat with a solution including the metallic salt (salts of one or more of: Au, Ag, and iron oxide) so that endogenous activity of the fungi within the mycelium mat may form nanoparticles by a redox reaction to synthesize nanoparticles. The time, temperature and concentration of the metallic salt solution may be adjusted within the parameters described herein in order to effect the final concentration, localization/distribution and size of the resulting nanoparticles, which may in turn modulate the color of the resulting mycotextile. Any of the mycotextiles described herein may be colored (dyed) by the inclusion of one or more nanoparticle (e.g., metallic nanoparticles) without the addition of a chemical dye, such as an alcohol-based and/or oil-based dye (as may be used with traditional leathers) or synthetic dyes, pigment dyes or water-based dyes. Thus the methods and compositions (mycotextiles) may be substantially free of these dyes. However, in some cases, it may be desirable to include one or more additional dyes (e.g., water based dyes, pigment dyes, alcohol-based dyes, and/or oil-based dyes).
[0220] When generating materials loaded with silver nanoparticles (AgNPs), the concentration of the precursor may affect the synthesis of AgNPs, as described herein. For this, a freshly harvested mycotextile was incubated with a specific volume of silver nitrate (AgNO.sub.3) solution with different concentrations (e.g., 1, 2, 3, 5, 10 and 15 mM). In some examples every 3040 cm mycotextile was completely immersed in the dark in a container with 500 ml of the corresponding solution. The temperature may affect parameters such as the speed of synthesis and the size and stability of the nanoparticles; incubations at 50, 60 and 70 C. were also evaluated. The incubation may be between about 40 degrees and 90 degrees C. (e.g., between about 45 degrees C. and 75 degrees C., between about 40 degrees C. and 80 degrees C., etc.). Thus, these methods may include incubating at between 0.1 mM and 50 mM of the metallic ion in solution. the mycelium mat may be incubated for between about 1 hour and 36 hours. For example, after an overnight incubation (e.g., between 2 hours and 30 hours, between 4 hours and 24 hours, between 12 and 24 hours, etc.), the mycotextiles may be removed from the metallic salt (e.g., AgNO.sub.3) solution, and the excess of liquid drained off (at this point, for AgNO.sub.3 salt solutions, the mycotextile will acquire a brownish color). The mycelium mat may be crosslinked (in some cases, including the application of a ceramic nanoparticle). The mycotextiles may then be dried in an oven, e.g., at 70 C. In any of these examples, once dry, each mycotextile may be immersed, e.g., for 10 min, in 1% nanoemulsion as described in U.S. patent application No. US 2023/0356501, International patent application No. PCT/IB2023/053847, U.S. provisional patent No. 63/520,933 and U.S. provisional patent No. 63/590,397, each of which is herein incorporated by reference in its entirety. The putative mycotextiles may then be drained (e.g., at room temperature) and then dried, e.g., at 70 C. Once dried, a solution of prolamine protein (e.g., a zein solution) in glycerol (e.g., 30% glycerol, pH 12) may be applied, e.g., by spraying, and the final material may be allowed to dry at room temperature. Finally, each prototype was exposed to ultraviolet light (8 W, 254 nm) for 10 min. The timing and temperature of the incubation may be adjusted, as will the use of UV irradiation (energy applied, duration, etc.) to adjust the final color of the material. Similar processes may be used for any of these in vivo metallic nanoparticles.
[0221] To obtain materials loaded with gold nanoparticles (AuNPs), two main strategies were followed: (i) direct mycosynthesis from a crude mycotextile (freshly harvested) and (ii) from the spontaneous redox reduction of AgNPs present in a mycotextile prepared as described above. In any of the cases, the gold starting solution was prepared as follows: lg of pure metallic gold was weighed and digested with 20 ml of boiling aqua regia (HCl with HNO.sub.3 in a 3:1 ratio) for 30 min. Upon completion of the chemical digestion, the solution was allowed to cool to room temperature and analytically transferred to a 100 ml volumetric flask and completed with distilled water (this mother solution remains at a concentration of 1 mM). For each reaction, the proper volume was diluted using a 1:100 ratio. For the first approximation, each 3040 cm mycotextile was fully immersed in a glass container containing 600 ml of gold solution (0.01 mM) and incubated overnight (e.g., between 12 to 24 h) at 60 C. After the incubation, the mycotextiles were removed from the gold solution, and the excess liquid was drained off (at this point, the mycotextile will turn purple). The resulting material was processed as described above for the mycosynthesis of AgNPs: immersion in the nanoemulsion, drying, coated with a prolamin:glycerol (e.g., zein:glycerol) solution. For the second approximation, the resulting material of the AgNPs mycosynthesis was dipped in 600 ml of gold solution (0.01 mM) and allowed to react for 5, 10 and 20 min. A color change from brownish to purple was observed here and in the remaining liquid. The evidence of this chemical transformation was recorded in the remaining liquid, employing the analysis of the plasmon light absorption using UV-Vis spectroscopy in solutions containing both silver and gold.
[0222] Regarding the myco-synthesis of iron oxide (IO NPs), a determined concentration of iron salts solutions. Iron (II) chloride (FeCl2) and iron (II) sulfate (FeSO.sub.7*7H.sub.2O) were tested. For these experiments, iron starting concentrations were 10, 15, 20 and 25 mM. The mycotextiles were completely immersed in a container with 500 ml of the corresponding solution with the precaution that the mycelium remained in contact with them. Each container was sealed and incubated at 60 C. overnight. The next day, the mycotextiles were removed from the solutions, and the excess was drained off (at this point, the mycotextile will acquire a yellowish color). The resulting materials were processed as described above for silver and gold mycosynthesis.
Results on A2NP Mycosynthesis
[0223] There are several factors determining the effectiveness of mycosynthesis, mainly the initial concentration of the precursor, the incubation time, and the incubation temperature. Exploratory tests using incubations in stirred liquid medium (e.g., potato dextrose broth) revealed that the range of the parameters that could be tested are the following: silver nitrate concentration between 1 and 20 mM; incubation times between 4 and 24 h and temperatures between 25 to 80 C. The effect of the concentration of the silver nitrate was visually observed that the brownish color developed in the mycotextile, which increases the intensity with the silver concentration when evaluated from 0 to 15 mM (
[0224]
[0225] It is known that higher concentrations of silver nitrate may lead to larger nanoparticles. The nucleation and growth of nanoparticles are influenced by the availability of silver ions, and higher concentrations result in increased nucleation events. Higher nucleation sites lead to a higher density of nanoparticles. High silver concentrations also increase the reduction reaction's overall yield, which was observed in the intensification of the color. Reaction kinetics may also be affected by higher concentrations of the precursors because the reduction process occurs faster. Finally, the shape and size dispersion of the nanoparticles may also be affected by concentration, leading to variations in shape (spherical, triangular, hexagonal, etc.) and agglomeration.
[0226] Regarding the effect of the temperature,
[0227] The processed mycotextiles were exposed to UV irradiation for an appropriate period of time (e.g., between 1 minute and 120 minutes, between 1 minutes and 60 minutes, between 3 minutes and 30 minutes, between 5 minutes and 14 minutes, between 7 minutes and 12 minutes, about 8 minutes, about 10 min, about 12 minutes, etc.) to complete the process. The light source may be placed on the top of the surface at a distance of, e.g., between 1 cm and 30 cm (e.g., between 1 cm and 25 cm, between 5 cm and 25 cm, between 10 cm and 20 cm, about 15 cm, etc.). After the photoirradiation, an increase in the intensity of the brownish color of the material was observed (
[0228] The in vivo nanoparticles formed as described herein may have a range of particle diameters, e.g., between about 10 nm and 900 nm, between about 50 nm and 800 nm, etc. In some cases, the use of UV irradiation may reduce the size range to approximately half (or less) than the original size range (e.g., between 5 nm and 500 nm, etc.).
Comparison of the Homogeneity and Entropy Derives from the AgNPs Mycosynthesis Capabilities Between the Wild Type SCC-0006 vs. SCC-0006/3.6 Improved Strain
[0229] As discussed above, the heterokaryon wild-type SCC-0006 strain has an inherent ability to biosynthesize metallic nanoparticles such as AgNPs and AuNPs. To evaluate the effect of the introduced dikaryon condition into SCC-0006/3.6 on the homogeneity of the AgNPs mycosynthesis, the photographs of several prototypes loaded with AgNPs obtained using both SCC-0006 and SCC-0006/3.6 strains were analyzed using the processing software ImageJ (public domain programmed in java). The nine images shown in
[0230] Under the naked eye view, an astonishing effect on the homogeneity was observed in those prototypes obtained with the modified strain, with the SCC-006/3.6 (dikaryotic) strain having a much more homogeneous appearance. To evaluate the qualitative change observed, the homogeneity was analyzed quantitatively. The images shown above were transformed into 8-Bit images. Subsequently, they underwent digital analysis using the Gray Level Co-occurrence Matrix (GLCM) toolkit, a statistical method employed in image processing and computer vision to analyze spatial relationships among pixel intensities in a grayscale image. The central areas of each material for strains of study were delineated for this purpose, demarcated by rectangles shown in
Where, P(i,j) represents the probability of co-occurrence for the pixel value pair (i,j) in the GLCM matrix. This calculation is performed for each pair of pixel values in the image. The Entropy function quantitatively measures the variability (or disorder) in the co-occurrence relationships of pixel value pairs in the image. A higher entropy value indicates a minor uniform and disorderly distribution of value pairs, suggesting greater textural complexity. Conversely, lower entropy reflects a more organized and predictable distribution, indicating a more homogeneous texture. Entropy, derived from the GLCM, emerges as a sensitive and valuable indicator for characterizing image textural complexity.
[0231] Additionally, the Contrast was used as a secondary parameter to analyze the images, whose calculation formulae run as follows:
[0232] Contrast measures the intensity difference between adjacent pixels. A higher contrast value indicates a more significant variation between pixel values in the image, suggesting a more pronounced texture. Conversely, a lower contrast value indicates smoother variation in pixel values, indicating a more uniform texture.
[0233] Both values were graphed using GraphPad Prism version 10.0.0, and the results are displayed in
[0234] To compare the homogeneity and the distribution of the AgNPs, the strains with the highest antioxidant activities in the Polyporaceae family (SCC-0002 and SCC-0013), were used to obtain silver-loaded mycotextiles using the direct mycosynthesis. The photographs of the obtained prototype and the corresponding SEM images (magnification 20k) are shown in
[0235] The specific mechanisms involved in the biogenic synthesis of silver nanoparticles using fungi have not been fully elucidated. It is known that extracellular synthesis of nanoparticles occurs according to reactions in which the enzymes present in the fungal filtrate act to reduce silver ions, producing elemental silver (Ag.sup.0) at a nanometric scale. Many biomolecules can react with silver ions, such as those associated with the complex pathways involving electron transfer during the conversion of NADPH/NADH to NADP.sup.+/NAD.sup.+. Nicotinamide adenine dinucleotide (NADH) and NADH-dependent nitrate reductase enzymes are considered the most important in the biogenic synthesis of metallic nanoparticles.
Results on AuNPs Mycosynthesis
[0236] The ability to biosynthesize silver and gold nanoparticles was studied mainly in two groups of fungi: Ascomycota and Basidiomycota. Mycogenic AuNPs are usually spherical, but other shapes have also been found. For the obtention of AuNPs loaded mycotextiles, two strategies were used: (i) direct biosynthesis and (ii) the spontaneous redox reaction between the as-synthetized AgNPs and chloroauric ions.
[0237] A prototype obtained by direct mycosynthesis requires an overnight incubation in the gold solution (pH below 1). After that, the material is removed from the acidic solution and then treated with the nanoemulsion and prolamine protein (e.g., zein) coating, as mentioned above, for AgNPs.
[0238] A strategy to avoid aggregation is the spontaneous redox reactions of metallic silver and gold ions. A spontaneous redox reaction is associated with positive cell potential that imparts a negative Gibbs free energy change during a chemical reaction. Hence, a positive cell potential determines the spontaneity of a redox reaction. In the case of the gold reduction at the expense of silver oxidation, the standard cell potential is equal to 0.5 V. It could be calculated from the following net redox reaction: Au.sup.3+(aq)+3Ag(s).fwdarw.Au(s)+3Ag.sup.+(aq) (E.sup.0=0.5 V).
[0239] As observed in
[0240] Nanosized gold has some unexpected optical properties. For example, Au NPs colloids may look red, orange, or even blue. The color depends on the size and shape of the nanoparticles and also on the distance between them. The different colors of Au NPs arise from a physical phenomenon named localized surface plasmon resonance (LSPR). When light interacts with the surface of a metal, it creates a surface plasmon, which is a group of electrons moving back and forth in a synchronized feature across the surface of the metal. When the electrons are moving at the same frequency as the incident light, the plasmon is said to be in resonance. At this stage, the electrons absorb and scatter light (according to Mie's theory), producing the colors we can see. The LSPR of 5-10 nm spherical Au NPs ranges from 520 to 580 nm, which means they absorb green or yellow light. As a result, they show the complementary color of green or yellow, which is red or purple. In the case of AgNPs, the LSPR peak is exhibited at 380 to 460 nm for monodispersed colloidal dispersion with spherical particles with an average diameter of less than 100 nm. The occurrence of the LSPR phenomena could be measured utilizing UV-visible spectroscopy. As observed in the UV-visible spectrum (
[0241] SEM images taken with magnifications of 5 and 50 k (
Results on IO NPs Mycosynthesis0260
[0242] The materials resulting from the incubation with Fe.sup.2+ salts are shown in
[0243] In the case of iron species, it is well known that extracellular enzymes produced by some fungal species can catalyze the breakdown of anionic iron complexes of ferric and ferrous salts. Moreover, under aerobic and moderate pH conditions, ferrous iron (Fe.sup.2+) is oxidized spontaneously to the ferric (Fe.sup.3+) form and is hydrolyzed (abiotically) to insoluble ferric hydroxide (Fe(OH).sub.3). This process is the opposite of the one observed with silver and gold, where electrons are needed to transform the oxidized ions to zero valent atoms and, therefore, metallic nanoparticles. In this example, (Fe.sup.2+) ions must gain one electron to transform into (Fe.sup.3+) ions that further precipitate as a hydroxide or an oxyhydroxide; therefore, the metabolic pathway and involved proteins in this case should be different.
In Vitro Biosynthesis and Combination with In Vivo Mycosynthesis
[0244] In an example of an ex situ approach, nanoparticles were synthesized using biological methods since the reducing agents derived from natural resources such as plants, algae, microalgae, and microorganisms are more efficient and environmentally friendly. For instance, plant extracts contain different phytochemicals that stabilize or reduce metal ions to metal nanoparticles (e.g., phenolic compounds, terpenoids, flavones, ketones, amides, aldehydes and carboxylic acids). The resulting colloidal suspension can be incorporated either by immersion or spray-dry techniques. In the case of immersion, the mycotextiles were dipped in the colloidal dispersion of the nanoparticles for 15 min, ensuring that the side with the mycelium was facing up to induce the penetration of the liquid dispersion mechanically. The prototype is disposed horizontally for the spray-dry method, controlling the liquid flow and flow speed to ensure the mycelium is well-soaked. Then, the mycotextiles were dried at room temperature. For these examples, copper oxide, magnetic iron oxide, and yellow and red iron oxide pigments were used in several combinations as described below:
Biosynthesis of Copper Oxide Nanoparticles
[0245] Copper can be considered an important antimicrobial agent because they are effective against many microorganisms, including fungi, viruses, and multidrug-resistant bacteria. An extract of Ilex paraguariensis (yerba mate) was prepared at 5 w/v % in distilled water at 85 C. for 10 min with constant stirring. The extract is filtered by gravity at room temperature using a proper porous material. A 0.1M copper sulfate (CuSO.sub.4 5H.sub.2O) solution was prepared in parallel. The yerba mate extract was slowly added dropwise to copper sulfate solution in a volume 1:5 ratio and kept under constant stirring; after adding the extract, the pH was adjusted to 9 units using a 3M NaOH solution. Finally, the synthesis was left under stirring for 1 h, and the resulting deep grass green colored suspension was reserved for its application. This synthesis process can be carried out using extracts from different plants that have high antioxidant activities, including native trees, such as Ilex guayusa, Peumus boldus, Schinus molle, Quillaja saponaria, Cryptocarya alba, among others. In addition, macro and microalgae, such as Ulva compressa, Spirulina platensis, Spirulina maxima, Arthrospira platensis, Chrorella sp., Euglena gracilis, Euglena sp., Nannochloropsis sp., Phaeodactylum tricornutum, Asterarcys sp., Scenedesmus sp., Uronema sp., etc.
Biosynthesis of Magnetic Iron Oxide Nanoparticles
[0246] Iron oxide nanoparticles have attracted great interest in recent times due to their magnetic nature, biocompatibility, of these metal oxide nanoparticles, one of them, low susceptibility to oxidation, immunoassays and antimicrobial activity against various pathogens such as fungi and bacteria, and viral particles. Furthermore, iron oxide nanoparticles have been used for their different coloring tissues. An Aloe vera extract was prepared at 1% w/v in distilled water at 85 C. for 10 min with constant stirring. The extract is filtered by gravity at room temperature using a proper porous material. A solution of 0.1 M of iron sulfate (FeSO.sub.4*7H.sub.2O) was prepared in parallel. Aloe vera extract was slowly added to the FeSO.sub.4 7H.sub.2O solution in a volume ratio of 1:2 (with constant stirring). Once this was finished, the pH was adjusted to 12 units with a 3M NaOH solution. Finally, the synthesis was left stirring for 2 h, and the resulting deep brownish suspension was reserved at room temperature for the application.
Formulations of Yellow and Red Nano Pigments of Iron Oxide
[0247] Iron oxides have been widely used in the textiles industry to impart novel properties, such as antibacterial and antifungal activity, removal of adsorbed dyes, thermal stability, mechanical properties and other functions. Moreover, iron oxides are the most abundant minerals in the earth's crust, allowing its utilization as pigments in prehistoric ages. Iron oxides exist in a wide range of colors, from yellow to black. The most common are yellow, corresponding to goethite (FeOOH); red, corresponding to hematite (Fe.sub.2O.sub.3); and black, corresponding to hematite (Fe.sub.3O.sub.4). The colloidal dispersion of yellow and red iron oxides was obtained by dispersing the proper amount of the corresponding solid in water (the concentration of yellow IO was 0.125% w/v, and the red IO was 0.25% w/v). Also, a dispersing agent such as polysorbates (e.g., tween 20, tween 80), polyacrylic acid (PAA), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), ionic liquids (IL), among others; in concentrations around 1 and 10% w/v; and, a thickener such as polyvinyl alcohol (PVA), carboxymethylcellulose (CMC), Xanthan Gum (XG), Guar Gum (GG), alginate (A), among others in concentrations around 0.01 and 0.1% w/v. These mixtures were formulated to reach viscosities between 100 to 1000 cps.
Starting Mycomaterials and Combinations
[0248] The bases for these approximations are any of the following materials: 1) a wild mycotextile, 2) an Ag NPs loaded mycotextile or, 3) an in-situ crosslinked mycotextile (for a graphic reference see
[0249] First, a Wild mycotextile is obtained by adding the nanoparticles as part of the post-processing steps (see, e.g., the post-treatment described in U.S. patent application No. US 2023/0356501-A1, and U.S. provisional patent No. U.S. 63/590,397). The mycelium is grown onto the scaffold for 7-10 days, preferably in fermentation conditions as encountered in a mushroom farm. Then, the harvested material is dried and immersed in an O/W nanoemulsion. The greased and dried material is immersed in the colloidal suspension of functionalized (e.g., ceramic) nanoparticles. Finally, the mechanically reinforced material is dried and coated with a prolamine protein (e.g., zein) solution. From this point, copper or iron oxide nanoparticles are incorporated.
[0250] Any of these apparatuses may include one or more types of ceramic nanoparticles. Ceramic nanoparticles may include oxide and non-oxide types. Common examples of oxide ceramic nanoparticles include silica (SiO.sub.2), alumina (Al.sub.2O.sub.3), titania (TiO.sub.2), and zirconia (ZrO.sub.2), while non-oxide examples include silicon carbide (SiC) and boron nitride (BN). Hydroxyapatite (HA), calcium phosphate (CP), and calcium carbonate (CaCO.sub.3) are also ceramic nanoparticles.
[0251] Second, the Ag or Au NPs (nanoparticle) loaded mycotextiles may be obtained as described above by mycosynthesis, e.g., forming the nanoparticles in vivo, following fermentation but prior to post-fermentation processing, by incubating in the appropriate (e.g., metallic ion) solution.
[0252] Third, in-situ crosslinked materials may be obtained by adding nanoparticles during growth of the mycelium mat (e.g., see U.S. patent application Ser. No. 18/595,392, field Mar. 4, 2024). Here, the ceramic nanoparticles used for the reinforcement are the yellow-iron oxide (yellow IO). The nanoparticles were added when the mycelium was self-generated in the fermentation room. Therefore, the harvested material already possesses the functionalized nanoparticles. The harvested material may be dried and greased with the O/W nanoemulsion. Finally, the material is coated with a prolamine protein (e.g., zein-protein) and is ready to incorporate the biosynthesized nanoparticles.
Results of the Reinforcement with Copper Oxide Nanoparticles
[0253] The advantage of combining different types of nanomaterials includes the possibility of achieving a synergistic activity. For example, combinations of silver and copper nanoparticles increase antibacterial efficacy against a wide range of bacteria, including antibiotic-resistant strains such as the gram-negative Escherichia coli and Pseudomonas aeruginosa, as well as the gram-positive Staphylococcus aureus, Enterococcus faecalis, and Streptococcus disgalactiae. Likewise, nanostructures based on zinc, copper, aluminum and titanium could be assembled into the collagen fibers of leather or sprayed to improve rub fastness, color fastness and adhesion strength. As seen in FIG. 42A1-42A2, either when the starting mycotextile is the wild prototype (left, FIG. 42A1) or the AgNPs loaded mycotextile (right, FIG. 42A2), the copper oxide nanoparticles exist at the surface in the form of deep green grass-like regions that apparently did not penetrate the mycelium network. SEM images in FIGS. 42B1 and 42C1 show the microstructure of the corresponding materials at the interface of these green spots at 500 magnification for each material. In both cases, two regions are clearly observed: a region on the left corresponding to the common hyphae network and a region at the right of the image that could be described as a thick cracked deposit. Furthermore, when zooming in on both sides at 10k magnification, it could be observed the ceramic nanoparticles used for the mechanical reinforcement during the post-fermentation stage in the wild-Cu prototype and the cracked thick coating is composed of aggregates of tiny spheroidal nanoparticles. In the case of the EDS spectrum and copper mapping, it was confirmed that these agglomerates on the mycelium surface are copper nanoparticles. The approximate concentration in the analyzed area was 4.3 and 10.9 wt. % for wild-Cu and AgCu, respectively. This difference could be explained by the aggregation that does not provide a homogenous distribution of the copper at the surface. This accumulation in the surface could result from the previous loading with AgNPs, limiting the interaction between the hyphae and the nanoparticles introduced in this step.
Results of the Material Reinforcement with Magnetic Iron Oxide Nanoparticles
[0254] Magnetic properties are highly desirable for novel applications in the textile industry. For instance, they provide conductive or antistatic properties, electromagnetic shielding, radar-absorbing capabilities, soft keyboards, magnetic heating, etc. Different types of magnetic nanostructures, such as iron oxides (e.g., magnetite, maghemite), cobalt, nickel, ferrites, and metallic alloys, could be obtained by biological routes. The biosynthesis of magnetite has been reported. An efficient approach is using Aloe vera extract as a stabilizing agent as it provides chromones, anthrones and phenolic compounds, leading a green and environmentally friendly synthetic route. The brownish colloidal suspension in the aqueous media of the stabilized magnetic-IO nanoparticles presents a viscosity slightly higher than that of pure water at the same temperature due to the mucilaginous substances existing in Aloe vera. However, dried magnetite separated by centrifugation is black-colored. The obtained solid exhibits a magnetic response close to the magnetic field of a magnet, as illustrated in the photographs in
Results of Yellow and Red Nanopigments
[0255] The wild and Ag-loaded prototypes immersed in a suspension of the yellow-IO nanopigments are shown in FIG. 45A1-45A2. They appear more homogenous than previous examples. When the initial mycotextile is the wild-yellow IO (FIG. 45A1), iron oxide nanoparticles are present on the surface, giving the prototypes an intense yellow color. However, in the case of the mycotextile loaded with Ag-yellow IO (FIG. 45A2), this intense yellow coloration was not observed. The microstructure observed using the SEM images (FIG. 45A1) at 10k magnification for each material), reveals that the iron oxide nanopigments comprise nanorods around 200500 nm, more extensive than copper and magnetic IO nanoparticles. They tend to agglomerate instead of forming a continuous coating on the material. In the case of the EDS spectrum and iron mapping (FIG. 45B1-45B3), the presence of iron oxide nanoparticles distributed homogeneously throughout the hyphae was confirmed; the approximate concentration in the analyzed area was 2.5% by weight. In the case of mycotextile, which has silver nanoparticles, it was confirmed that the rod structures incorporated in the hyphae of the mycelium are iron oxide nanoparticles. The approximate concentration in the analyzed area was 9.9% and 0.4% in weight for silver and iron, respectively (FIG. 45C1-45C4). This difference regarding the iron loading in the wild prototype could indicate some incompatibility between the surface charge of these particles and the surface of the materials, leading to a lower distribution of the nanopigments. In the case of the resulting materials by immersion in the red IO NPs formulation (shown in FIG. 46A1-46A2), these nanoparticles remain on the surface of the hypha in a similar way to what was observed with the yellow IO nanoparticles but turning the surface of the mycotextile into red color. As observed in both SEM images shown in FIG. 46B1-46B2 (taken at 2 and 20 Kx, respectively) for the Ag-red IO prototype, these nanoparticles show a more heterogeneous distribution in size and yield a higher covering of the hyphae network than those obtained with the same concentration of yellow IO NPS. A closer look at a single hypha shows that these particles are heterogeneous in shape and range around 100 nm. Elemental mapping in a given region using EDS reveals that the aggregates on the surface are composed of iron and oxygen, confirming the presence of iron oxide nanoparticles. The iron and silver content in the analyzed region is 8.2 and 1.7 wt. %, respectively. See FIGS. 46C1-46C4.
In-Situ Nano Crosslinking and Silver Nanoparticles
[0256] Prolamine protein (e.g., zein-) functionalized IO NPs nanoparticles were dispersed in a BIOrganic CHAI solution; the mixture was sonicated (60 Hz for 1 h) and sterilized before application onto the growing mycelium during the fermentation process using a spraying gun. The details of formulations and application schemes for this colloidal dispersion are described in the provisional patent No. U.S. 63/520,933.
[0257] FIGS. 47A1-47A2 show at the left (FIG. 47A1) the starting prototype obtained with yellow IO NPs where a yellowish surface is observed, being characteristic of the ocher-colored solid used as a crosslinker. The observation of the SEM image at 10 k magnification, present in the same figure (bottom), revealed that the nanoparticles are in intimate contact with the hyphae but also tend to be more aggregated in some areas of the network; however, the morphology of each hypha could be clearly distinguished. After immersing this material in the silver nitrate solution overnight at 60 C., the material took the expected brownish color given by the silver, and the microstructure changed. Here, the hyphae appear highly interconnected and also covered by a dense layer, which agrees with the result in
[0258] The procedure's versatility allows it to introduce many other functional elements and compounds. For instance, other precious metals may be incorporated as described herein, including platinum, palladium, iridium, osmium, rhenium and non-precious metals such as iron, aluminum, titanium, tin, cobalt, manganese, molybdenum, nickel, vanadium, niobium, zirconium and/or, rare earth such as lanthanum, cerium, dysprosium, europium, gadolinium, erbium, holmium, and/or metal compounds or composites such as oxides, oxyhydroxides, salts, carbonates, aluminates, ferrites, silicates, iron phosphates, titanates, perovskites, pyroxenes, carbides, nitrides, carbonitrides.
Mechanical Characterization of Sidereal Mycotextiles
[0259] The industrial application of the described sidereal materials depends on their mechanical properties. It arises from a combination of the characteristics provided by the fungal mycelium layer, their nano-mycosynthesis skills, and the scaffold selected for its growth. Also, it arises from the combination of the methods described here because it yields at least twelve combinations of sidereal mycotextiles.
[0260] For instance, as reported before, fungal mycelium alone typically has tensile strength values below 1 MPa while, in cowhide leather, this parameter varies between 8 and 20 MPa. Moreover, synthetic leather can have values of 10-15 MPa. Other characteristics, such as elongation after a break or tear strength, must be similar to those expected in industrial applications, such as leather goods, shoe manufacturing or upholstery. The preliminary tests carried out in several of the sidereal mycelium fabrics fabricated as described herein were: i) tensile strength (TS), ii) elongation percentage before rupture (E), both according to the ASTM D2209 standard and tear strength (TeS) according to ASTM D4704-13 standard. These tests were conducted using a GESTER GT-C02-1A Universal Tensile Strength Tester (Single Column) with a 2.5 kN load cell at 254 mm/min speed. The specimens of each sample were obtained by a laser-cutting machine of the corresponding geometry requested by each standard. The rubbing color fastness test, which refers to a test in which colored samples are rubbed with dry rubbing cloth and wet rubbing cloth, respectively, was determined using a GESTER GT-D05 Crockmeter/Rubbing fastness tester. The values presented correspond to a mean value of three replicates for every experiment.
[0261] To be measured, the selected scaffold has two directions regarding the elongation and tensile strength, considering the direction of the applied force. Different from woven textiles that show a weft and warp direction, the direction of the non-woven textile is organized by the magnitude of the elongation. The results will be discussed regarding the strategy of obtention of the sidereal mycotextile. First, the control material (wild) will be compared to those resulting from AgNPs mycosynthesis (in vivo approach). Second, those obtained from the in vitro approach and the corresponding combination of AgNPs loaded mycotextile. Finally, the in situ-Ag mycotextile will be compared with the corresponding control (in-situ crosslinked material).
[0262] Table 3 lists the tensile strength (TS) and elongation (E) before break and tear strength (TeS) for the materials obtained by AgNPs mycosynthesis. As observed, in the direction of the higher elongation, the tensile strength does not show a difference statistically representative after the mycosynthesis of silver nanoparticles regardless of the fungal strain. However, the elongation at break increased by around 5 and 15%. Likewise, the tear strength increased in all the examples (between 28 and 38%), meaning that the reaction conditions changed the material's mechanical properties. In the direction of the lower elongation, TS, E % and TeS showed a slight increase in all the experiments. These results indicate that the process of obtaining AgNPs does not significantly affect the mechanical properties of the crude mycotextile. However, the mycotextile generated with the SCC-0006/3.6 strain used to obtain the AuNPs by chemical reduction showed a significant decrease of all the properties; for instance, TS and elongation decreased by around 30%, while TeS diminished by 44%. Therefore, the synthesis of AuNPs changes these characteristics, probably due to the immersion in the acidic solution that causes partial hydrolysis of the scaffold fibers.
[0263] Regarding the in vitro approaches comparing the materials wild-Cu, wild-magnetic IO, and wild-red IO, it is possible to observe the effect of the incorporation of the nanoparticles in the mechanical properties. The results are listed in Table 4. In the direction of the highest elongation, TS increases after immersion of the mat in the dispersion of copper and magnetic IO NPs obtained by biosynthesis. The change with the wild-red IO was negligible. Likewise, the elongation before the break and the tear strength increased in all the experiments. Remarkably, the tear strength shows enhancements between 24 and 44% in this direction. On the other hand, analyzing the results in the direction of the lower elongation, it was observed that the elongation in this direction did not change in these experiments. The most relevant changes were observed in the TS and TeS values: TS increased between 30 and 46%, while TeS increased between 7 and 38%.
[0264] Table 4 shows the mechanical properties of the materials obtained by combining the Ag NPs-loaded mycotextile with the different oxides tested herein. In this case, the results differ significantly from those observed using the wild mycotextile as starting material, because the properties of these materials are not predictable. Therefore, the effect of the oxides in these examples will fulfill two leading roles, one related to the unique look and feel of the resulting combinations and the other regarding the functionality imparted by them. For instance, it could be observed that in the case of the incorporation of biosynthesized oxides in the AgNPs loaded mycotextile, all the parameters diminished after the process in both directions of the material. After immersion, in the formulation containing the nanopigments, the mycotextiles do not show a predictable behavior. These differences could be explained by the deficient distribution of the nanoparticles in the materials previously loaded with Ag NPs, as observed in the SEM images above. Thus, this variability in the distribution also causes variability in the mechanical properties.
[0265] The example of silver biosynthesis (in vivo) using the material crosslinked during the fermentation (e.g., in situ nano-crosslinking) showed a behavior similar to the one described before for obtaining a prototype (see Table 5). In this case, a slight increase in the tensile strength and the tear strength was obtained, while the elongation showed an increase of around 34 and 41%.
[0266] All the metrics compared herein confirm the viability of the application of these materials in the fashion industry, where they could comprise all the mechanical and esthetic requirements to become a useful material as an alternative to other materiality in the market based on animal hides or plastic-based leather.
TABLE-US-00009 TABLE 3 Maximum stress, Tensile strength (TS), elongation percentage (E) and tear strength (TeS) for the sidereal mycotextiles obtained by mycosynthesis of silver. TS E TeS Prototype (MPa) (%) (N/mm) Control 1 (wild) 5.83 1.05 70 1 3.91 0.56 6.04 0.93 41 1 5.06 0.43 AgNPs (SCC-0006/3.6) 6.34 0.93 74 4 6.31 0.72 6.34 0.57 47 2 5.07 0.49 AgNPs (SCC-0002) 6.23 0.79 82 4 5.59 0.84 6.21 0.44 49 4 5.12 0.74 AgNPs (SCC-0013) 6.39 0.10 80 2 5.41 0.11 6.15 0.77 50 2 5.26 0.59 AgAu NPs 4.23 0.11 49 3 3.53 0.84 4.27 0.56 34 4 2.84 0.15
TABLE-US-00010 TABLE 4 Maximum stress, Tensile strength (TS), elongation percentage (E), and tear strength (TeS) for the sidereal mycotextiles obtained by combining metal nanoparticles at the post-fermentation stage. TS E TeS Prototype (MPa) (%) (N/mm) Cu 7.37 0.32 78 1 5.15 0.10 11.16 0.55 47 5 7.00 0.09 Magnetic IO 7.77 0.32 72 2 5.41 0.75 10.77 0.55 41 6 6.73 0.76 red IO 5.46 0.55 85 5 6.13 0.88 8.64 0.36 46 1 5.47 0.69 AgCu 4.50 0.41 52 4 3.26 0.58 5.07 0.58 41 4 3.34 0.51 Ag-magnetic IO 4.60 0.20 60 3 5.02 0.36 4.57 0.38 42 1 3.98 0.20 Ag-red IO 5.96 0.31 73 4 6.49 0.31 9.32 0.40 50 3 5.40 0.98 Ag-yellow IO 6.70 0.54 81 3 4.25 0.38 7.45 0.38 53 3 5.14 0.47
TABLE-US-00011 TABLE 5 Maximum stress, Tensile strength (TS), elongation percentage (E), and tear strength (TeS) for the in-situ-AgNPs mycotextile compared to the control. TS E TeS Prototype (MPa) (%) (N/mm) Control 2 (In-Situ 6.84 0.46 39 7 4.23 0.88 nano-crosslinking) 6.58 1.05 31 4 4.85 0.53 In situ-AgNPs 7.66 1.06 66 7 5.59 0.70 7.43 0.95 47 2 4.60 1.06
[0267] In general, the method, compositions and apparatuses described herein may include nanoparticles, which may be added (e.g., functionalized NP) and/or may be formed by an in vivo formation of metallic NP (e.g., silver, gold, iron oxide) by adding metallic salts, and/or in vitro (e.g., ex vivo) formation of metallic NP using extracts (plant or fungal) that are then added as previously described. Using in vitro nanoparticles may have a final distribution of the nanoparticles within the material that is particularly distinct as compared with the distribution identified when adding nanoparticles (including ex vivo addition) during growth of the mycelium mat. In some cases, the mycotextile may include nanoparticles added to the mycelium mat, e.g., an in vitro approach, and nanoparticles formed in vivo. For example, the in vitro approach may be performed on materials previously reinforced with SiO.sub.2 nanoparticles. These added nanoparticles (in vitro) may be derived from a plant and/or fungal-based source. The examples shown here were done with plant extracts. These nanoparticles, based on the in vitro source, may be deposited in the surface of the mycotextile because they were added, e.g., after the post-fermentation stage, as a type of pigment or functional coating. SEM analysis on transversal sections confirms that they may penetrate the mycelium network.
[0268] In variations in which, as described herein, the nanoparticles are synthesized, in vivo, by the fungal strain, the resulting nanoparticles do not need to be separately functionalized. In ex vivo embodiments, the added nanoparticles may be synthesized using plant extracts and one or more metabolites may act as organic capping agents to stabilize them, thus adding a barrier between the NPs and the surface of the hypha. This layer could be equivalent to a functionalizing agent. The ex vivo nanoparticles may not include a crosslinker (because they may be added after the crosslinking stage of the post-fermentation process), and the mechanical properties of the resulting materials may be increased in comparison to a material reinforced only with SiO.sub.2.
[0269] Nanoparticles (e.g., metallic nanoparticles) synthesized in vivo in the fungal strain during fabrication of the mycotextile may be distributed through the final mycotextile in a manner that is distinct from nanoparticles (including ex vivo formed and added nanoparticles) added during fabrication. For example, all of the SEM images and elemental mapping shown in
[0270] In practice the in vivo process may include the addition of a metallic salt solution to the freshly harvested mycotextile. This in vivo approach uses living fungus for the incubation in the metallic salts just before the post-fermentation. After incubating, the material is processed by immersion in the nanoemulsion, e.g., in a formulation of an oil-in-water nanoemulsions such as (but not limited to): 1% of coconut oil, 4.66% of a non-ionic surfactant (hydrogenated castor oil) and 94.34% of water. After immersion for 10 minutes all the materials are dried until losing between 80-90% of humidity.
[0271] In general, any of these methods of forming, particularly but not limited to in vivo nanoparticle formation, may include a UV treatment, e.g., breaking up the larger particles formed from the metallic salt solution. This may be done before other post-fermentation steps (e.g., addition of nanoemulsion). The application of UV radiation may help to desegregate the NPs to homogenize the nanoparticle distribution and may thereby provide a more intense color. This is the last (or one of the last) steps of the process, may be performed after the nanoemulsion and the application of a prolamine protein solution (e.g., zein:glycerol mixture).
[0272] When adding nanoparticles to a formulation, these particles may be added after the post-fermentation process. Either starting from an in vitro mycotextile or from an Ag or Au in vivo loaded mycotextile. For example, in some cases, the in vitro addition of nanoparticles in the mycotextile may be obtained by: (i) harvesting, (ii) inactivation, (iii) nano-emulsion, (iv) functionalized-SiO.sub.2, (v) zein. In contrast, the in vivo Ag or Au loaded mycotextile may be obtained by: (i) harvesting, (ii) in vivo Ag or Au synthesis, (iii) nano-emulsion, (iv) zein, (v) UV-irradiation.
[0273] In general, in any of the mycotextiles described herein, the mycotextile may include both in vivo nanoparticles (metallic nanoparticles, such as silver or gold nanoparticles which may color/dye the final mycotextile) as well as ceramic nanoparticles (such as SiO.sub.2 nanoparticles) added in vitro, which may provide additional structural reinforcement.
Engraving of Mycofabrics
[0274] Also described herein are methods, compositions and apparatuses that may beneficially allow the imprinting of a pattern, design and/or texture to the material in a manner that is significantly more environmentally favorable, requiring less resources, including energy, producing less waste, and resulting in a product having superior properties. These methods and compositions may include mycotextiles in which a pattern, design or texture are engraved onto the living mycelium mat prior to post-processing.
[0275] In general, the search for crafting innovative materials, it is crucial that such material may be imbued with distinctive personality and characteristics. It is particularly desirous to forge new embossing patterns that seamlessly align with the conceptual essence of sidereal mycelium fabrics. The sidereal space described herein may provide an inexhaustible wellspring of inspiration. While conventional embossing patterns for texturing leather and its alternatives often draw from the intricate patterns found in the skins of animals (e.g., live or death), such as cows, crocodiles, snakes, and ostriches, a notable sense persists in deriving textures from other elements. Thus, pioneering the integration of such organic motifs into materials akin to leather or its substitutes represents uncharted territory, brimming with potential for groundbreaking innovation.
[0276] Adding surface patterns and textures to the mycofabrics as described herein may be of particular use. Traditionally leather and similar fabrics are embossed by the application of high pressure to transfer a pattern. Described herein are methods, compositions and apparatuses for performing an engraving procedure on the living mycelium mat that may require significantly less pressure and other resources and may result in a more robust pattern transfer in the material, without weaking (or generating compressed regions of weaker/stronger domains) of the material. These methods and compositions may also be significantly easier, less expensive and require fewer additional components, including chemicals and energy. Although these materials and techniques may be referred to herein as embossing, they are more similar to an engraving technique and are otherwise distinguished from traditional embossing of fabric materials. The patterning of the sidereal mycelium fabrics described herein may be inspired in the natural world and universe to ground an awareness of environmental responsibility and raise a profound sense that we are not alone in the universe and that humanity's interconnectedness within the vast cosmos by extending our conception far beyond the limits of our body, our niche of life and the planet where we live. This expanded cosmovision serves as a catalyst for inspiring collective action and fostering a sense of expanded unity (e.g., totality) and empathy towards both our planet and the incommensurable sidereal space. For example, our muse can be born from the elements of nature and the universe, such as morphological structures of mushrooms (Basiodiomycota division), as possible alien inhabitants, geological and orographic impressions, volcanoes, craters and surface marks on planets and satellites, falling meteorites and comets, as well as constellations of stars, black holes and galaxies.
[0277] The Fungi Kingdom offers great bioinspiration, including the lower part of the mushroom cap, lamellar or porous structures that are precisely ordered. In the lamellar structures, thin sheets or plates extend from the center towards the edges of the cap where basidia are located. These specialized cells produce the spores (Basidiospores), allowing the sexual reproduction and dispersion of the fungus. These sheets can develop in tight, separate, very separated or anastomosed structures that tend to interconnect. They can also be bifurcated as they approach the edges of the cap. Bioinspired patterns on these mushroom-ordered structures refer to applying principles observed in nature to the design of artificial structures that could be imprinted or engraved either in vitro embossing or in vivo engraving.
[0278] Taking as inspiration the mushroom structures (lamellar or porous), four types of embossing have been designed to differentiate the look and feel of the sidereal mycelium fabrics from any related material existing in the global market. The patterns shown in FIGS. 48A1-48A4 correspond to embossing plates patterned with myco-inspired designs. The patterns used for the print were bioinspired by the hymnenia of some fruiting bodies that are part of a culture collection. However, hymenia structures from all Basiodiomycota fungi can be used as bioinspiration. The emulated patterns are as follows: pattern A (shown in FIGS. 48A1, 48B1, 48C1, 481, 48E1, 48F1 and 48G1) corresponds to the hymenium of laminae, which can be found in fungi of the Agaricales order, Pattern B (shown in FIGS. 48A2, 48B2, 48C2, 48D2, 48E2, 48F2 and 48G2) has a folded shape present in fungi of the Auriculariales order. Pattern C (shown in FIGS. 48A3, 48B3, 48C3, 48D3, 48E3, 48F3 and 48G3) is shaped like stingers and is present in fungi of the Cantharellales order, and finally, pattern D (shown in FIGS. 48A4, 48B4, 48C4, 48D4, 48E4 and 48F4) is shaped like tubes or channels, bioinspired by the Polyporales order.
[0279] AI-based simulations of the resulting materials applying these myco-inspired embossing are shown in FIGS. 48B1-48B4, 48C1-48C4, 48D1-48D4, 48E1-48E4, 48F1-48F4, and 48G1-48G4 for the following sidereal mycelium fabrics: Ag; AgAu: AgCu, Ag-magnetic IO, Ag-yellow IO, and Ag-red IO, respectively. These patterns could be applied to any type of mycelium-based articles since they could be adapted to the conventional embossing technique using machines with high pressures and temperatures, as applied for leather.
[0280] The examples by in vivo engraving are most profoundly related to the nature of the fungal growth process. This technique must be applied in the process in which the fungal mycelium is still alive (this treatment may be performed before the mycosynthesis of the nanoparticles). In this case, manual engraving, 3D printed patterns, or any other artistic intervention is allowed, as the raw mycelium mat acts as a living canvas. The in vivo engraving considers a raw mycelium fabric with the living mycelium; this avoids the energetic costs associated with conventional embossing processes that require high pressures and temperatures, considerably diminishing the post-fermentation stage's carbon footprint. As outlined in FIG. 49A, this engraving process could be used to artistically intervene in the sidereal mycelium fabric, allowing consumers unique pieces of art and more meaningful pieces. As shown in
[0281] In general, the engraving techniques described herein, in which the mycotextile is engraved by applying pressure to the harvested mycotextile, may apply pressure within a range from about 1-50 kg/ft.sup.2 (e.g., 2 to 30 kg/ft.sup.2, 4 to 20 kg/ft.sup.2, 5 to 15 kg/ft.sup.2, about 10 kg/ft.sup.2, etc.). The time period the pressure is applied may be, e.g., between about 1 second to about 500 seconds (e.g., 5 seconds to 400 seconds, 10 seconds to 300 seconds, 60 seconds to 180 seconds, 90 seconds to 150 seconds, about 90 seconds, about 120 seconds, about 150 seconds, etc.). The pressure may be applied immediately after the harvesting while the fungus are alive during the in vivo synthesis (which is the following step). The engraving depth will depend on the mycelium thickness. However, a fully defined pattern may have, at least, about half of the depth of the mycelium thickness (e.g., between about 30%-70% of the depth of the mycelium, about 35%-65%, about 40%-60%, etc.). For instance, with a mycelium layer of around 4-5 mm a plate with a 2 mm pattern-depth may be used for the engraving. Regarding the manual engraving, modeling tools used for ceramics and clay pottery may be used. In any of these examples, a flat press may be used to perform the engraving, taking into consideration the parameters described above.
[0282] The final structure of the engraved mycotextile may be distinct from other fabrics, including embossed fabrics. For example, the mycotextile that has been engraved as described herein may have compressed regions where it was engraved. The resulting look and structure is different from what is seen in conventional embossing processes in which it is mandatory to add a PU coating to avoid die-cutting of the material. This coating layer exhibits the pattern and also contains pigments and finishing resins used for different looks (Matte, Satin, Shiny, Glossy, Patent, etc.), all equivalent to the finishing strategies used in the leather industry. Also, the patterns used to resemble different animal skins (crocodile, snake, ostrich, etc.). By using the proposed engraving, the mycelium layer receives all the pressure and therefore, the pattern is visualized as a combination of compressed areas and less compressed regions, where both the in vivo nanoparticles and the in vitro nanoparticles may be differentially deposited, increasing the contrast between both regions.
[0283] The differences between the technique for engraving the living mycelium mat as described herein and other embossing a cross-linked mycotextile (in which the mycelium mat has been cross-linked) is shown schematically in FIGS. 49C1-49C3. FIG. 49C1 shows a section through an example of a mycotextile including a cross-linked hyphae matrix embed over a scaffold layer 4902. The mycotextile has an upper surface 4904 and a lower surface 4906. The upper surface is separated from the scaffold layer 4902 by a distance, d, and the scaffold layer extends flat, essentially in the same plane as the mycotextile. The thickness of the upper region (above the scaffold layer) may be equal to or different from the thickness of the lower region of the hyphae matrix.
[0284] Embossing the mycotextile, e.g., after cross-linking (and in some cases, drying) the hyphae matrix may result in a sectional profile similar to that shown in FIGS. 49B1-49B2, in which the upper surface 4904 is compressed to form regions of different thicknesses (in this example, relative to the bottom surface 4906) and the scaffold layer 4902 is displaced from the horizontal by a proportion of the compression of the upper layer. In this example the scaffold layer remains relatively centered between the upper and lower layers, as shown.
[0285] In FIG. 49C3, the mycotextile is engraved by compressing (e.g., using a tool, plate, etc.) the upper region of the living mycelium mat as described above. In this example although the scaffold layer 4902 may be compressed somewhat, the relative amount of compression (as compared with embossing, FIG. 49C2) is substantially less. For example, the scaffold layer may be displaced relative to the plane of the mycotextile (in this example, parallel to the bottom surface 4906) by less than, e.g., 25%, 20%, 15%, 10%, 5%, etc. This may be true so long as the depth of the engraving remains less than the thickness of the upper region. In this example, the scaffold layer also does not remain centered between the upper and lower surfaces, so that the distance between the scaffold layer and the up surface is different along the length and width of the mycotextile underlying the engraved region, as compared to a non-engraved region. This may be the result of engraving into the living mycelium mat.
Functional Properties of the Sidereal Mycelium Fabrics:
[0286] The incorporation of the nanoparticles not only provides novel characteristics regarding the material reinforcement and the esthetic (look and feel) and avoids the use of petrochemical-based dyes, but it provides a wide option of smart functionalities. For example, specific properties include antibacterial, antifungal, magnetic, adsorptive and photocatalytic activity, among others. Nanoparticles possess unique physical and chemical properties due to their large surface area in the nanoscale range. Its optical properties are known to be size-dependent, revealing different colors due to absorption in the visible region. Its reactivity, hardness and other properties also depend on its unique size, shape and structure.
[0287] Antibacterial activity: Antibacterial textiles provide resistance to bacterial growth, improve hygiene, reduce odors, and potentially prevent the spread of infections and diseases. This property is beneficial in consumer products like clothing and shoes as they can help to maintain freshness and cleanliness. It has been observed that different nanoparticles have antibacterial activity that mainly depends on the size of the particles, and the smaller ones have the best activity. Metallic nanoparticles destabilize the outer membrane of bacteria, producing ruptures in the plasma membrane and reducing the synthase activity of the intracellular adenosine triphosphate (ATP) depletion layer, thus reducing the metabolic process. Furthermore, they destroy the ribosome subunit by binding to the tRNA and, thus, ultimately, the total collapse of the biological mechanism. Nanoparticles based on metals and metal oxides have been widely studied for their antimicrobial efficacy against various bacterial strains. For example, silver nanoparticles have received special attention due to their application as an antimicrobial device coating. Similarly, gold, copper oxide, gallium, zinc oxide, and magnesium oxide nanoparticles have shown promising antimicrobial effects. Furthermore, combining different nanoparticles to enhance their efficacy in a synergistic approach has been widely investigated.
[0288] An antibacterial assay was made using the disk diffusion technique in agar plates inoculated with a Gram-negative control bacteria (Escherichia coli). In this test, the size of an area around the disc where bacteria cannot grow (called the zone of inhibition) indicates the effectiveness of the antimicrobial compounds. Initially, the bacterial inoculum of ampicillin-resistant E. coli was prepared in Lisogenia broth (LB medium), which was left to incubate overnight at 37 C. with constant shaking. The turbidity of the inoculum was adjusted to the bacterial suspension obtained until reaching the standard turbidity of 0.5 on the Mc scale Farland. The inoculum (70 L) was plated on Mueller Hinton agar plates. Then, discs of wild-Ag, Wild-Cu, AgCu, wild-magnetic IO, and Ag-magnetic IO mycotextiles were carefully placed on the inoculated medium and allowed to incubate overnight at 37 C.
[0289] Results of the antibacterial assay by disk diffusion:
[0290] Magnetic properties: Magnetic nanostructures are attracting considerable interest due to their unique potential in storage, sensing, spintronics, and optoelectronics in flexible devices. Several factors, such as composition, shape, size, surface morphology, anisotropy, layer thickness, and molecular interactions, govern the magnetic properties of nanostructures. Furthermore, the bactericidal action of magnetic iron oxide nanoparticles could be enhanced by the magnetic properties producing local hyperthermia and several cell wall damages induced by vibrations such as membrane rupture, fusion of different cells with each other, and death. Magnetic nanoparticles are nanomaterials composed of elements such as iron, nickel, cobalt, chromium, manganese, gadolinium and some of their compounds. Nanostructured magnetite (Fe.sub.3O.sub.4) and metallic ferrites (M.sub.xFe.sub.3-xO.sub.4) are the most explored magnetic nanoparticles due to their high magnetic response and the possibility of being biosynthesized. Likewise, magnetic mycotextiles can be used as elements of magnetic cores and as parts of meters, transmitters and textile actuators. They can be used in electronic circuits and smart clothing.
[0291]
[0292] Photocatalytic properties: Textile washing requires a lot of water, and the concept of self-cleaning is very concerning for reducing the consumption of this vital substance. In addition to self-cleaning materials, they clean the surface, degrade the stain, and inhibit microbial growth. The photocatalytic activity of the nanocomposites is carried out by submerging the mycotextiles in an aqueous solution of a cationic organic dye. The experiments were carried out using an 8 W UV lamp (.sub.max=254 nm) located in a zenithal plane along the prototype's surface at a distance of 10 cm. For the experiment, 55 cm pieces of the wild, AgAu, and Au mycosynthesized prototypes were incubated in containers with 25 ml of crystal violet (CV), (at 10 mg/ml). Each container was exposed to ultraviolet light for 90 minutes and 5 mL aliquots were withdrawn every 30 min. The remaining solutions after 90 min of reaction were measured by UV-Vis spectroscopy. Likewise, the results were compared with the adsorption experiments (keeping all the conditions without light).
[0293] The adsorption experiments in
[0294] When the same experiment was carried out in the presence of UV irradiation, the discoloration process was accelerated because of a photocatalytic effect driven by the Ag and or AuNPs. As observed in
[0295] As observed in FIGS. 53B1-53B3, the resulting materials after these photocatalysis experiments are less dyed than those obtained during the adsorption experiments, suggesting that the dye molecules were bleached through a photocatalytic process during the incubation with light. Several enhancements could be further tested to increase the effectiveness of these techniques. For instance, hybrid photocatalysts can be fabricated, including combinations with semiconductors such as TiO.sub.2/Au, ZnO/Au, Fe.sub.2O.sub.3/Au, or composites.
[0296]
[0297] Considering the versatility of these protocols the incorporation of chemical elements such as silver, gold, platinum, palladium, iridium, osmium, rhenium and, non-precious metals such as iron, aluminum, titanium, tin, cobalt, manganese, molybdenum, nickel, vanadium, niobium, titanium, zirconium and/or, rare earths such as lanthanum, cerium, dysprosium, europium, gadolinium, erbium, holmium, and/or metal compounds or composites such as oxides, oxyhydroxides, salts, carbonates, aluminates, ferrites, silicates, iron phosphates, titanates, perovskites, pyroxenes, carbides, nitrides, carbonitrides, these methods and compositions may provide a wide range of applications including: (i) catalysis for pharmaceuticals, petrochemicals, and environmental remediation, (ii) medical diagnostics and therapeutics, drug delivery, imaging, and photothermal therapy, (iii) as sensors for detecting gases, biomolecules, and environmental pollutants, (iv) in flexible electronics and displays for solar cells, (v) catalytic converters and fuel cells among others.
[0298] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, it should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.
[0299] A person of ordinary skill in the art will recognize that any process or method disclosed herein can be modified in many ways. The process parameters and sequence of the steps described and/or illustrated herein are given by way of example only and can be varied as desired. For example, while the steps illustrated and/or described herein may be shown or discussed in a particular order, these steps do not necessarily need to be performed in the order illustrated or discussed.
[0300] The various exemplary methods described and/or illustrated herein may also omit one or more of the steps described or illustrated herein or comprise additional steps in addition to those disclosed. Further, a step of any method as disclosed herein can be combined with any one or more steps of any other method as disclosed herein.
[0301] The processor as described herein can be configured to perform one or more steps of any method disclosed herein. Alternatively or in combination, the processor can be configured to combine one or more steps of one or more methods as disclosed herein.
[0302] When a feature or element is herein referred to as being on another feature or element, it can be directly on the other feature or element, or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being directly on another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being connected, attached or coupled to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being directly connected, directly attached or directly coupled to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed adjacent another feature may have portions that overlap or underlie the adjacent feature.
[0303] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms comprises and/or comprising, when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items and may be abbreviated as /.
[0304] Spatially relative terms, such as under, below, lower, over, upper and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as under, or beneath other elements or features would then be oriented over the other elements or features. Thus, the exemplary term under can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms upwardly, downwardly, vertical, horizontal and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.
[0305] Although the terms first and second may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.
[0306] In general, any of the apparatuses, compositions and/or methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive and may be expressed as consisting of or alternatively consisting essentially of the various components, steps, sub-components or sub-steps.
[0307] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word about or approximately, even if the term does not expressly appear. The phrase about or approximately may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/0.1% of the stated value (or range of values), +/1% of the stated value (or range of values), +/2% of the stated value (or range of values), +/5% of the stated value (or range of values), +/10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value 10 is disclosed, then about 10 is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that less than or equal to the value, greater than or equal to the value and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value X is disclosed the less than or equal to X as well as greater than or equal to X (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point 10 and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
[0308] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.
[0309] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term invention merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.