Highly efficient lignocellulose-degrading filamentous fungal mutant strains and applications thereof

Abstract

The application discloses a high-efficiency lignocellulose-degrading filamentous fungus mutant strain and application thereof. The high-efficiency lignocellulose-degrading filamentous fungus mutant strain is formed by knocking out a wild-type filamentous fungus Podospora anserina photoperiod gene PaFrq. The application firstly finds that the filamentous fungus Podospora anserina with the knocked-out photoperiod gene PaFrq has the advantages of delayed aging, prolonged life cycle, improved growth speed, higher efficiency in degrading lignocellulose, especially improved cellulase activity in dark conditions, higher efficiency in degrading lignocellulose, and important significance for a fungus fermentation industry, especially for more efficient conversion of fungal biomass.

Description

Technical Field

[0001] This application relates to the field of microbial degradation of lignocellulose technology, and in particular to a filamentous fungal mutant strain that is highly efficient at degrading lignocellulose and its applications. Background Technology

[0002] Light is a ubiquitous environmental signal that can trigger and coordinate the physiological processes of various organisms. Many microorganisms, such as filamentous fungi living in different habitats, can sense and respond to light, using it as a signal to regulate various aspects of fungal life, such as circadian rhythms, vegetative growth, reproductive development, secondary metabolism, stress response, nutrient absorption, and pathogenicity. Fungi sense light through a complex photosensitive system composed of photoreceptors that can distinguish specific wavelengths and intensities of light. Most fungi contain at least two different types of photoreceptors, and some even contain three. These photoreceptors include red, green, and blue photoreceptors. Red photoreceptors use linear tetrapyrrole as their chromophore, green photoreceptors use rhodopsin, and blue photoreceptors use flavin. Some blue photoreceptors are located in the cell nucleus and can directly control gene expression, while red photoreceptors, phytochromes, have both nuclear and cytoplasmic functions and are linked to transcription mechanisms through other signaling modules. Furthermore, blue and red photoreceptors can also control the DNA structure of certain genes, thereby further regulating their transcription. The green light receptor is a transmembrane protein whose signal transduction mechanism is not yet clear. There is also some evidence that a special blue light receptor (a homolog of the white light receptor protein) can sense green light.

[0003] To adapt to the periodic changes in light, many fungi have evolved diurnal clock systems to predict environmental cycles and prepare for the upcoming day or night. Light is a crucial input signal for these clock systems; in Neurospora and other fungi, both the photosensitive and diurnal clock systems contain the blue light sensor WC-1. Studies have shown that much of the genome expression in many fungi is regulated by light; however, fungal gene expression may be independently regulated by light, the biological clock, or both, illustrating the complexity of light sensing and signal transduction, and their crucial roles in regulating fungal life.

[0004] Photocyclins are proteins involved in the regulation of biological rhythms, including various types such as CRY, PER, TIM, and FRQ. These proteins can receive light signals from the environment and influence the physiological behavior of organisms by regulating gene expression. In organisms, photocyclins are mainly involved in the regulation of diurnal rhythms and seasonal physiological rhythms. Diurnal rhythms can be observed in a wide variety of organisms. Almost every organism we see, as well as many tiny, invisible organisms, keeps a daily clock and use this ability to recognize time to regulate their growth and development. Except for some rapidly growing eukaryotic microorganisms, such as baker's yeast (Saccharomyces), and some species living in environments without periodic light (such as trenches or caves), all organisms must cope with periodic environments and adapt to the changing patterns of Earth's light environment. To adapt to the alternation of light and darkness, organisms on Earth have evolved biological clocks.

[0005] The circadian rhythm phenotype of *Neurospora crassa* was reported in 1953, and subsequently, a second circadian clock gene, *frq*, was discovered, encoding a crucial component of the circadian clock cycle. In *Neurospora crassa*, the circadian clock system consists of three parts: an input system, a core oscillator, and an output system. The input system senses environmental signals such as light, temperature, and nutrient conditions and transmits these signals to the core oscillator. The core oscillator processes these external environmental signals, generating an endogenous circadian rhythm, which manifests as the observable circadian rhythm through the output pathway. In this cycle, *frq* encodes two forms of the FRQ protein: a long form containing 989 amino acids and a short form of 890 amino acids, translated starting at the internal ATG codon. Both *frq* RNA and FRQ levels circulate, and FRQ may suppress *frq* transcript levels by interfering with the heterodimer composed of WC-1 and WC-2. In this negative feedback loop, rhythmic variations in *frq* transcription levels may be crucial to the circadian rhythm, while FRQ shifts reset the clock. The biological clock in the filamentous fungus *Neurospora crassa* is highly conserved, similar to the biological clock in animals. Numerous feedback oscillators exist within the cells, a natural result of feedback regulation of metabolic pathways. The biological clock is closely linked to these feedback regulations, thus being related to the entire metabolism of the organism. Besides *Neurospora crassa*, the diurnal rhythmic behavior of *Pilobolus sphaerosporus* has been reported, and its biological clock characteristics have been verified by altering conditions such as temperature and light / dark cycles. *Aspergillus flavus* also exhibits diurnal rhythmic characteristics, while *Aspergillus nidulans* does not show diurnal rhythmic behavior, although the expression of the homologous gene *gpdA* of *frq* does exhibit diurnal rhythmicity.

[0006] The homolog of the FRQ protein is not widely present in fungi, suggesting that while fungal clock genes are conserved, they also evolve independently. An accurate biological clock is crucial for life, controlling circadian rhythms and influencing metabolic processes. Therefore, by altering circadian rhythms, it is possible to screen for industrially valuable engineered strains, which is of great significance to human production and daily life.

[0007] The filamentous fungus *Podospora anserina* (P. anserina) is a fungus with a high capacity for degrading lignocellulose, converting it into bioethanol. As a model organism for lignocellulose degradation, *P. anserina* has been extensively studied, yielding significant results. Experimental studies show that this species can only reproduce sexually and produce fruiting bodies under light conditions; in darkness, the hyphae grow rapidly but can only undergo asexual development. The fruiting body is the spore-producing reproductive body formed during the sexual reproduction stage of higher filamentous fungi. It is an important organ for fungal sexual reproduction and one of the most complex multicellular structures in fungi. The growth and development of *P. anserina* fruiting bodies mark the transition of fungi from vegetative to reproductive growth; blocking fruiting body development can promote better and faster vegetative growth of the hyphae. Reproduction and lifespan of *P. anserina* are vital life characteristics of universal interest and have long been a research hotspot in the life sciences. It is generally believed that organisms, for the sake of population propagation, ultimately prioritize reproduction over individual lifespan. As a model organism for studying aging, exploring the effects of light-responsive genes on growth, development, and secondary metabolites in this species is of great significance. Meanwhile, the biological characteristics of P. anserina make it a powerful model organism for photobiology, enabling a deeper understanding of the regulatory mechanisms of fungal photobiology on fungal production, development, and metabolism. This will help develop more efficient fungal biomass conversion technologies and promote the application of fungi in industrial production, environmental protection, and other fields. Summary of the Invention

[0008] The purpose of this application is to provide a novel mutant strain of filamentous fungus that efficiently degrades lignocellulose and its applications.

[0009] The following technical solution is specifically adopted in this application:

[0010] The first aspect of this application discloses a mutant strain of filamentous fungus that efficiently degrades lignocellulose, which is formed by knocking out the photoperiod gene PaFrq from the wild-type filamentous fungus Podospora anserina.

[0011] It should be noted that, considering the characteristics of *P. anserina*—its ability to degrade cellulose and its relatively short lifespan—the inventors of this application aimed to screen for highly efficient cellulose-degrading strains and shorten the fermentation cycle, thereby reducing production costs, by constructing mutants. This study is the first to perform site-specific knockout of the PaFrq gene in *P. anserina* using homologous recombination, and further investigated the role of the PaFrq gene in the balance between *P. anserina* cell growth and development, sexual and asexual reproduction, as well as in important physiological processes such as reactive oxygen metabolism and biomass utilization. The results of this application show that the PaFrq gene knockout mutant strain (labeled ΔPaFrq) affects the mycelial development and pigment accumulation of *P. anserina*, regulates the development of *P. anserina* fruiting bodies, and exhibits significantly higher cellulase activity (including CMC, β-BG, and CBH) in the dark, which is highly suitable for the closed, dark environment of large-scale industrial fermentation. Therefore, this application preserves a ΔPaFrq strain with high cellulase activity under darkness, with accession number CCTCC M 20231709. In this application, "highly efficient degradation of lignocellulose" means that, compared to the wild-type filamentous fungus *Podospora anserina*, the ΔPaFrq strain of this application can degrade lignocellulose more efficiently, especially under dark conditions, where it exhibits significantly higher lignocellulose degradation performance.

[0012] The second aspect of this application discloses the use of the mutant strain of this application in the preparation of reagents for the degradation of lignocellulose.

[0013] In one implementation of this application, the application specifically includes using the mutant strain of this application to degrade lignocellulose and convert lignocellulose into bioethanol.

[0014] It should be noted that the mutant strain of this application can efficiently degrade lignocellulose; therefore, it can be used to prepare reagents for lignocellulose degradation, including but not limited to those for preparing bioethanol.

[0015] A third aspect of this application discloses a method for efficiently degrading lignocellulose, comprising culturing the mutant strain of this application under dark conditions.

[0016] Preferably, the cultivation conditions are as follows: cultivation in the dark for at least 7 days.

[0017] Preferably, the culture temperature is constant at 27℃.

[0018] It should be noted that the method for efficiently degrading lignocellulose in this application is actually to cultivate the mutant strain of this application, thereby achieving efficient lignocellulose degradation by the mutant strain. Specific cultivation conditions can refer to the existing cultivation conditions for wild-type filamentous fungi, *Podospora anserina*, for example, cultivation for at least 7 days under white light, blue light, or darkness. However, considering that the mutant strain of this application exhibits higher lignocellulose degradation performance under darkness, cultivation under darkness is preferred. Constant temperature cultivation at 27°C is merely a conventional cultivation temperature specifically used in one implementation method of this application.

[0019] The fourth aspect of this application discloses a method for increasing the cellulase activity of the filamentous fungus *Podospora anserina*, comprising using gene knockout or gene silencing techniques to prevent the expression of the photoperiod gene *PaFrq* in *Podospora anserina*.

[0020] It should be noted that the mutant strain in this application is able to efficiently degrade lignocellulose because our research found that it can increase the cellulase activity of the filamentous fungus *Podospora anserina*. Therefore, it can be understood that, in principle, as long as the photoperiod gene *PaFrq* is not expressed, the mutant strain in this application can achieve the same or similar effect of increasing cellulase activity. Based on this research and understanding, this application creatively proposes to use gene knockout or gene silencing technology to prevent the expression of the photoperiod gene *PaFrq* in the filamentous fungus *Podospora anserina*, thereby increasing the cellulase activity of *Podospora anserina*.

[0021] The fifth aspect of this application discloses a method for delaying the aging of the filamentous fungus *Podospora anserina*, comprising using gene knockout or gene silencing techniques to prevent the expression of the photoperiod gene *PaFrq* in *Podospora anserina*.

[0022] It should be noted that this application's research found that the mutant strain of this application, after knocking out the photoperiod gene PaFrq, can not only efficiently degrade lignocellulose but also delay fungal senescence. In one implementation of this application, the wild-type filamentous fungus *Podospora anserina* begins to senescent on day 10 of cultivation in the dark, and mycelial growth stops on day 52; the ΔPaFrq strain of this application begins to senescent on day 12 of cultivation in the dark, and mycelial growth has not stopped on day 52, delaying senescence by 33.33%. It can be understood that, in principle, as long as the photoperiod gene PaFrq is not expressed, the same or similar senescence-delaying effect can be achieved with the mutant strain of this application. Based on this research and understanding, this application creatively proposes to delay the senescence of the filamentous fungus *Podospora anserina* by using gene knockout or gene silencing technology to prevent the expression of the photoperiod gene PaFrq.

[0023] The sixth aspect of this application discloses a method for regulating the growth and development, sexual reproduction, orange pigment accumulation, number of male gametes, reactive oxygen species content, or catalase activity of the filamentous fungus *Podospora anserina*. This method includes using gene knockout or gene silencing techniques to prevent the expression of the photoperiod gene *PaFrq* in *Podospora anserina*.

[0024] It should be noted that the study in this application found that the mutant strain of this application, after the photoperiod gene PaFrq was knocked out, was not only able to efficiently degrade lignocellulose, but the deletion of the photoperiod gene PaFrq also had multiple effects on the filamentous fungus Podosporaanserina. For example, 1) it affects the growth and development of *Podospora anserina*; 2) it leads to a decrease in the sexual reproductive capacity of *Podospora anserina*; 3) the photoperiod gene affects pigment accumulation, and the ΔPaFrq mutant showed obvious orange pigment accumulation on the 4th day of culture on GY medium under light; 4) it affects the number of male gametes, and microscopic observation showed that a large number of male gametes differentiated on the hyphae of ΔPaFrq when grown under white light; 5) it affects the content of reactive oxygen species (ROS), and the study in this application showed that in the absence of the PaFrq gene, the imbalance of the intracellular ROS scavenging system led to a reduction in the production and accumulation of ROS, thereby slowing down the rate of oxidative damage to cells and changing the aging time of the strain; 6) it affects catalase activity, and the study in this application showed that under the presence of white light, the CAT enzyme activity of the ΔPaFrq strain was higher than that of the wild type, which indicates that the ΔPaFrq strain may have better antioxidant capacity to a certain extent and be able to better cope with oxidative stress caused by light, thereby reducing cellular oxidative damage and aging. Based on the above research and understanding, this application creatively proposes to regulate the growth and development, sexual reproduction, orange pigment accumulation, number of male gametes, reactive oxygen species content, or catalase activity of the filamentous fungus Podospora anserina by using gene knockout technology or gene silencing technology to prevent the expression of the photoperiod gene PaFrq.

[0025] The beneficial effects of this application are as follows:

[0026] This application presents a mutant strain of filamentous fungus that efficiently degrades lignocellulose, specifically a mutant strain with the photoperiod gene PaFrq knocked out. This application is the first to discover that the filamentous fungus *Podospora anserina* with the photoperiod gene PaFrq knocked out can degrade lignocellulose more efficiently, especially by increasing cellulase activity under dark conditions, thus exhibiting a more efficient lignocellulose degradation capacity. This has significant implications for more efficient fungal biomass conversion. Attached Figure Description

[0027] Figure 1 This is a phylogenetic tree of P. anserina photoperiod proteins in the embodiments of this application;

[0028] Figure 2This is the amplification electrophoresis result of the upstream and downstream fragments of the PaFrq gene and the screening resistance marker gene in the embodiments of this application;

[0029] Figure 3 This is the amplification electrophoresis result of the PaFrq gene fusion fragment in the embodiments of this application;

[0030] Figure 4 These are the amplification electrophoresis results of the ΔPaFrq mutant strain verified by PCR in the embodiments of this application;

[0031] Figure 5 This is a diagram showing the growth observation results of wild-type and mutant strains under white and blue light in the embodiments of this application;

[0032] Figure 6 These are images showing the colony morphology observations of wild-type and mutant strains on M2 medium under red light and dark conditions in the embodiments of this application.

[0033] Figure 7 These are colony morphology observations of WT and ΔPaFrq under different light conditions for 7 days in the embodiments of this application;

[0034] Figure 8 These are microscopic morphological observations of the sub-entities WT and ΔPaFrq under different lighting conditions in the embodiments of this application;

[0035] Figure 9 This is a statistical result of the number of fruiting bodies of WT and ΔPaFrq under different light conditions on the 7th day of cultivation in the embodiments of this application;

[0036] Figure 10 These are colony morphology diagrams of WT and ΔPaFrq on GY medium under white light and dark conditions in the embodiments of this application.

[0037] Figure 11 These are colony morphology diagrams of WT and ΔPaFrq on GY medium supplemented with fluorourea in the embodiments of this application;

[0038] Figure 12 This is an observation diagram of the number of male gametes during mycelial growth and development for 7 days under different light conditions in the embodiments of this application. The black arrows indicate the male gametes.

[0039] Figure 13 This is a fertility analysis diagram of male and female gametes under white light in an embodiment of this application; the small black dots represent fruiting bodies.

[0040] Figure 14 This is a comparison of the aging times of WT and ΔPaFrq in the embodiments of this application;

[0041] Figure 15 This is a graph showing the comparison of ROS content between WT and ΔPaFrq in the embodiments of this application;

[0042] Figure 16 These are the cellulose filter paper enzyme activity (FPase) test results of WT and ΔPaFrq in the embodiments of this application;

[0043] Figure 17 These are the CMCase test results of WT and ΔPaFrq in the embodiments of this application;

[0044] Figure 18 These are the β-glucanase activity (β-BG) test results of WT and ΔPaFrq in the embodiments of this application;

[0045] Figure 19 These are the exoglucanase activity (CBH) test results of WT and ΔPaFrq in the embodiments of this application.

[0046] The mutant strain ΔPaFrq of this application, with the Latin name Podospora anserina, is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on September 18, 2023, with accession number CCTCC M 20231709. Detailed Implementation

[0047] This application focuses on *P. anserina* to investigate the mechanisms by which light regulates the growth, development, fruiting body development, and asexual development of filamentous fungi. The research centers on the molecular mechanisms by which light-responsive genes regulate fruiting body development, senescence rate, reactive oxygen species metabolism, and secondary metabolism. The aim is to construct strains lacking light-responsive genes, screen for strains with abnormal fruiting body development, block fruiting body development, and promote faster and better mycelial growth. This will provide high-quality engineered strains for large-scale industrial production of bioethanol, enhancing the application value of these strains in bioenergy and environmental protection.

[0048] Specifically, the nucleotide sequences of photoperiod genes in *P. anserina* were obtained by indexing the Podospora anserina Genome Project database, and comparative analysis of 16 photoperiod protein sequences was performed to further determine their homologous evolutionary relationships. In ΔPaMus51, a PaFrq gene deletion mutant was constructed using the Split-Marker gene knockout method, and then the PaFrq clone mutant gene was reintroduced into the mutant to restore the wild-type phenotype. Through experiments such as colony morphology recording, pigment deposition, growth rate measurement, strain lifespan measurement, reproductive capacity measurement, and cellulase activity measurement, the role of photoperiod genes in regulating the growth, development, and life cycle of *P. anserina* was confirmed, and the effects of light on its fruiting body development and life cycle were explored. By analyzing the different cellulase activities of mutant and wild-type strains, the superiority of fruiting body development-deficient strains in practical applications was verified, providing high-quality engineered strains for large-scale industrial production of bioethanol and enhancing the application value of this strain in the fields of bioenergy and environmental protection.

[0049] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0050] Example

[0051] I. Materials and Methods

[0052] 1.1 Materials

[0053] 1.1.1 Plasmids and strains: Wild-type filamentous fungus *Podospora anserina* (WT) and Δmus51::phleoR (Δmus51::phleoR is a wild-type strain with the mus51 gene knocked out, which significantly improves homologous recombination efficiency). pBC-Geneticin was used for constructing the ΔPaFrq mutant.

[0054] 1.1.2 Main culture media: The formulation and preparation methods of culture media such as M2 basal medium (M2), germination medium (G) and inoculation medium (Agar) are as follows: refer to the Podospora anserina Genome Project (http: / / podospora.i2bc.paris-saclay.fr / index.php).

[0055] 1.1.3 Main Reagents and Instruments

[0056] The M5 Fungal Genomic DNAKit was purchased from Beijing Jumei Biotechnology Co., Ltd.; Go DNA Polymerase, Ex DNA Polymerase and Max was manufactured by Takara; the BCA Protein Quantification Kit was purchased from Novizan Biotechnology Co., Ltd.; Geneticin was purchased from Nanjing Dulai Biotechnology Co., Ltd.; Hygromycin and other reagents were purchased from Sangon Biotech Co., Ltd.; cell lysing enzymes were purchased from Sigma-Aldrich; NBT, DAB colorimetric reagent kits and superoxide dismutase (SOD) activity assay kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.; the light incubator (S / N: 21004-12938) was purchased from Hybot Co., Ltd.; FM4-64 dye was purchased from Beijing Cooler Technology Co., Ltd.; the LSM710 laser confocal microscope was purchased from Carl Zeiss AG, Germany; and the FastPrep-24 cell disruptor was used. TM Purchased from MP Biomedicals.

[0057] 1.2 Bioinformatics Analysis of Photocyclin

[0058] The putative photoperiod gene Pa_5_9200, named PaFrq, was obtained from the P. anserina genome database. Its corresponding amino acid sequence is PaFrq (XP_001904738.1). Fourteen amino acid sequences from ascomycetes and one from Drosophila were obtained from the NCBI genome database. The Drosophila photoperiod protein sequence was considered an outgroup. These are: Neurospora crassa (AAA57121.1), Plectosphaerella plurivora (KAH6686834.1), Hirsutella rhossiliensis (XP_044722926.1), Fusarium redolens (XP_046049373.1), Trichoderma lentiforme (KAF3068241.1), Sordaria macrospora (KAA8635080.1), and Plectosphaerella. plurivora (KAH6686834.1), Hirsutellarhossiliensis (XP_044722926.1), Lachnellula.hyalina (XP_031003325.1), Lachnellulawillkommii (TVY93613.1), Trichoderma.spinulosum (AAA6 8072.1), Leptosphaeria.australiensis (AAB96844.1), Colletotrichum.fructicola (KAF4938655.1), Lachnellula.suecica (TVY71232.1), Lachnellula.cervina (TVY55205.1), Beauveria bassiana (KAF1737372.1), Drosophila melanogaster (NP_001259675.1). The CLUSTALW algorithm in MEGA 11.0 software was used to align the sequences of 15 proteins. The maximum likelihood method in MEGA 11.0 was used to construct a phylogenetic tree for the 15 protein sequences and analyze their homologous evolutionary relationships.

[0059] 1.3 Genomic DNA Extraction

[0060] (1) Genomic amplification and extraction

[0061] 1. After activating the strain for 2 days, take a 1cm sample. 2The mycelia were placed into 2 mL EP tubes containing 400 μL ddH2O. The EP tubes were then placed in a cell disruptor, and the parameters were set to 5.0 m / s and a running time of 20 s. The disruption was repeated twice.

[0062] 2. Take 100 μL of the above-mentioned broken bacterial culture and add it to 150 mL of sterilized M2 liquid culture medium. Incubate at 28°C under white light for 2 days at a rotation speed of 150 rpm.

[0063] 3. Filter the above mycelium using a vacuum filter, grind the mycelium, and add 0.5g of the ground mycelium into a 2mL EP tube.

[0064] 4. Extract total fungal DNA using the M5 Fungal Genomic DNA Kit.

[0065] 5. Determine the concentration of total DNA and perform electrophoresis identification. Store high-quality DNA at -20℃.

[0066] (2) Genomic extraction for PCR validation

[0067] Inoculate the culture blocks onto M2 medium and allow them to grow for 2 days. Scrape the mycelia (using as little agar as possible) and place them in a 2 mL EP tube containing glass beads. Add 20 μL of 0.5 mol / L NaOH solution to the EP tube, place it in a cell disruptor, set the speed to 5.0 m / s (20 s), and shake twice. Centrifuge the shaken EP tube at 13000 rpm for 30 s, then boil it in water for 1 min. Add 100 μL of TE solution to the boiled EP tube, cool it on ice, and it is ready for use as a PCR template.

[0068] 1.4 Construction of photoperiodic gene knockout expression cassette

[0069] Primers were designed using NCBI online software, and the primer sequences are shown in Table 1. The 5' end (PaFrq-5') and 3' end (PaFrq-3') of the PaFrq gene were amplified using PCR technology. Gene gene fragments were then amplified from the pBC-Geneticin plasmid, and the PCR products were purified and recovered. Specifically, a purification and recovery kit purchased from OMEGA was used to purify and recover the PCR products. The specific purification and recovery steps were followed according to the kit's instruction manual.

[0070] Experimental strains: Wild-type P. anserina (WT), ΔPaMus51::phleomycinR mutant strain (a strain with a phleomycin resistance marker replacing the mus51 gene sequence; deletion of mus51 effectively enhances homologous grouping efficiency to over 90%), and Escherichia coli competent cells DH5α were purchased from Shenzhen Shanghai Weidi Biotechnology Co., Ltd. Experimental plasmids: pBC expression vector containing a phleomycin resistance marker.

[0071] The PCR amplification primers are shown in Table 1. PCR amplification was performed using Go. DNA polymerase, reaction system: Green Buffer 10 μL, dNTPs 2 μL, MgCl2 4 μL, upstream primer 2 μL, downstream primer 2 μL, genomic DNA 2 μL, Polymerase 0.5 μL, ddH2O 28.5 μL. PCR reaction conditions: 95℃ pre-denaturation for 5 min, followed by 35 cycles: 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 3 min, after which 72℃ extension for 7 min, and then standby at 4℃.

[0072] Fusion PCR was used to fuse the two flanking fragments of PaFrq purified in the first round with the resistance selection marker gene.

[0073] Fusion PCR using Prime MAX DNA polymerase, reaction system: PaFrq-5' / PaFrq-3' 2μL, Geneticin R 2μL, PaFrq-1F / PaFrq-MkF 2μL, PaFrq-MkR / PaFrq-4R 2μL, Prime MAX 25μL, ddH2O 17μL. The reaction conditions were: pre-denaturation at 98℃ for 2 min, followed by 35 cycles: denaturation at 98℃ for 10 s, annealing at 58℃ for 5 s, extension at 72℃ for 25 s, and after the cycle, extension at 72℃ for 7 min, followed by standby at 4℃.

[0074] The fusion fragment was transformed into protoplasts of the ΔPaMus51 strain, and resistant transformants were screened using resistance medium containing Geneticin.

[0075] Specific conversion methods include:

[0076] Preparation of P. anserina protoplasts

[0077] After thawing the ΔPaMus51::nourseoR(+) strain on ice for 20 min, a piece was picked and inoculated onto M2 medium and activated at 27°C for 2 days.

[0078] (1) Take a piece with an area of ​​approximately 1 cm² 2 The mycelia were added to an EP tube containing 500 μL ddH2O. The EP tube was then placed in a cell disruptor and shaken at 5.0 m / s for 20 s, with a 10 s interval, for a total of 2 shakes to obtain the bacterial culture.

[0079] (2) Add tetracycline to 1L of fungal liquid culture medium (final concentration 10μg / mL), and fill each cell culture flask with 200mL. Prepare 5 clean and sterilized cell culture flasks in advance, and add 200μL of bacterial solution to each cell culture flask.

[0080] (3) After incubating in the dark at 27℃ for 2-3 days, filter the mycelium with two layers of sterile medical gauze. Wash the filtered mycelium multiple times with TPS1 solution to thoroughly remove any residual fungal liquid culture medium.

[0081] (4) Transfer the washed mycelia to an Erlenmeyer flask and weigh the mycelia. Add 20 mg / mL cell wall-breaking enzyme to the Erlenmeyer flask. Dissolve the cell wall-breaking enzyme in TPS1. The volume (mL) of the added TPS1 solution should be equal to the mass (g) of the mycelia. Note that the liquid should completely cover the mycelia.

[0082] (5) The Erlenmeyer flask was lysed and cultured on a shaker at 37°C and 70 rpm for 4 hours. The mycelium was observed under an optical microscope to see if it was enzymatically broken down into spherical protoplasts.

[0083] (6) Four layers of sterilized gauze were fixed on the sterilized funnel. A 50mL centrifuge tube was inserted into the lower end of the funnel. The mycelium was washed multiple times with TPS1 and the protoplasts were collected.

[0084] (7) Place the 50mL centrifuge tube in a centrifuge and centrifuge at 20℃ for 10min at a speed of 3,200rpm.

[0085] (8) Discard the supernatant, add 5 mL of TPS1 solution to resuspend the precipitate, gently blow it with a pipette, add TPS1 to 30 mL, centrifuge at 20°C for 10 min at 3,200 rpm.

[0086] (9) Pour out the supernatant, add 5 mL of TPC solution to resuspend the precipitate, gently pipette it, then add TPC solution to 30 mL, centrifuge at 20°C for 10 min at 3,200 rpm.

[0087] (10) After centrifugation, discard the TPC supernatant, leaving approximately 1 mL of supernatant. Mix the supernatant and precipitate thoroughly, then count the protoplasts using a hemocytometer to ensure a final concentration of approximately 2 × 10⁻⁶. 8 per mL.

[0088] (11) Aliquot the protoplasts into 1.5mL EP tubes, and dispense 5 vials, each containing approximately 200μL. After aliquoting, the protoplasts can be stored at -80℃ or used directly.

[0089] Fungal transformation

[0090] (1) Thaw the protoplasts on ice or use them directly, incubate at 48°C for 5 min, immediately transfer them to ice for 30 s, and then let them stand at room temperature for 5 min.

[0091] (2) Add the amplified DNA fragments to the tube containing protoplasts and let it stand at room temperature for 15 minutes to ensure that the mass of each fragment is more than 5 μg.

[0092] (3) Add 2 mL of Tampon D solution to a 50 mL centrifuge tube in advance, and transfer the mixture from step (2) to a 50 mL centrifuge tube, mix well, and let stand at room temperature for 15 min.

[0093] (4) Centrifuge at 20℃ for 10 min at a speed of 3,200 rpm, slowly aspirate and discard all supernatant, and retain the precipitate.

[0094] (5) Add 5 mL of RG liquide to a 50 mL centrifuge tube, gently mix the precipitate and liquid, and incubate in the dark at 27 °C for 12 h.

[0095] (6) Sorbitol and RG Top were mixed in a 3:1 ratio beforehand and placed in a 60°C oven to prevent the mixture from solidifying. 30 mL of the mixture was then mixed with the protoplasts from step (5), resulting in a final volume of 35 mL. Different antibiotics were then added to the mixture, with the final concentration of Norseoticin being 40 μg / mL (i.e., adding 14 μL of Norseoticin at a concentration of 100 mg / mL) and the final concentration of Geneticin being 100 μg / mL (i.e., adding 35 μL of Geneticin at a concentration of 100 mg / mL).

[0096] (7) Turn on the water bath in advance and adjust the temperature to 47℃. Quickly transfer the above mixture into the water bath and incubate for 7 minutes. Pour 7 mL of the mixture into an M2 culture dish containing the corresponding antibiotic and prepare 5 antibiotic culture media. Incubate at 27℃ for 2-5 days, wait for the transformants to grow, and then inoculate the transformants again onto antibiotic plates for secondary verification.

[0097] It is understandable that after the transformants grow on the resistance plate, the transformant colonies are inoculated again onto the resistance plate for a second resistance verification. Colonies that can grow on both resistance plates are considered to be resistant and stable transformants.

[0098] Wild-type strains were hybridized with transformants, and microspores with resistance marker genes but without Δmus51::phleoR protoplast resistance were screened to obtain stably heritable mutant strains. Specifically, transformants that had undergone two resistance verifications were inoculated with wild-type strains of opposite mating types and co-cultured on the same M2 medium at 27°C. The mycelia of the two colonies crossed over after 3 days of growth, and after 7 days of growth, the hybrid mycelia could fertilize to form mature fruiting bodies and release spores.

[0099] The mutant strain ΔPaFrq was verified by PCR and gene sequencing. The PCR amplification products were sequenced by BGI Genomics, specifically including:

[0100] (1) Transformants that have undergone two-stage resistance plate verification were inoculated onto M2 medium and cultured at 27°C under white light for 2 days.

[0101] (2) Add 20 μL of 0.5 mol / L NaOH and a small amount of fine glass beads to a 1.5 mL EP tube, collect the hyphae into the EP tube, put the tube into a cell disruptor, shake at 5.0 m / s for 20 s, and shake twice.

[0102] (3) Centrifuge at room temperature for 30 seconds at a speed of 12,000 rpm to allow the glass beads and mycelium to settle to the bottom, and then boil in boiling water for 1 minute.

[0103] (4) Add 100 μL of a mixture of 1 mol / L Tris-HCl and 1×TE to the EP tube, mix well, and then cool in ice water. The cooled mixture is used as a reaction template.

[0104] (5) Verify primers PaFrq-5'Test and PaFrq-3'Test, as shown in Table 1, according to Ex The DNA polymerase PCR reaction system was used for amplification, Ex The DNA polymerase extension rate is 1,000 bp / min, and the extension time is adjusted according to the length of the target fragment.

[0105] Ex The PCR reaction system for DNA polymerase was as follows: template, PaFrq-5' Test 1 μL, PaFrq-3' Test 1 μL, Ex DNA Polymerase 12.5μL, ddH2O 9.5μL.

[0106] The reaction conditions were: pre-denaturation at 94℃ for 10 min, followed by 30 cycles: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 2 min, extension at 72℃ for 5 min after the cycle, and standby at 4℃.

[0107] (6) After performing agarose gel electrophoresis on the PCR products, comparative analysis was performed.

[0108] (7) Purification of transformants: After obtaining the target gene knockout transformants, at least three transformants were hybridized with wild-type strains of opposite mating type. After culturing for 7 days, the erupted spores were collected, and microspores were picked under a microscope. The microspores were inoculated onto GY medium and cultured for 2 days. Then, the mating type and resistance of the germinating microspores were verified. The germinating microspore hyphae were inoculated onto resistance plates containing protoplast resistance and resistance selection markers, respectively. Strains that could grow on the resistance selection markers but not on resistance plates containing protoplast resistance were screened. Strains that could only grow on resistance plates containing resistance markers were inoculated between strains containing opposite mating types and cultured for 7 days. The resistance of the strains was determined based on whether fruiting bodies could form between adjacent colonies. Then, the strains of opposite mating types were stored at -80℃ to obtain the target gene deletion mutant ΔPaFrq.

[0109] 1.5 Construction of the complement strain of ΔPaFrq

[0110] The complete coding region of the PaFrq gene of wild-type P. anserina strain and 500 bp upstream and downstream of the target gene were amplified by PCR. Specifically, primers were designed at 500 bp upstream and downstream of the PaFrq gene fragment using WT genomic DNA as a template, and named PaFrq-1 and PaFrq-2, as shown in Table 1.

[0111] The target gene PaFrq fragment and the enzyme-digested pBC-Hygromycin plasmid vector were transformed into ΔPaFrq protoplasts. Subsequently, the resistance of the transformants was verified to obtain the complement strains, and it was observed whether the phenotype of the complement strains was restored.

[0112] Specifically, in this experiment, the PaFrq gene fragment and the pBC-Hygromycin plasmid vector were digested with a single enzyme overnight at 37°C. The band size was confirmed by agarose gel electrophoresis analysis.

[0113] The enzyme digestion reaction system consisted of: 2 μL Buffer, 0.2 μL BSA, more than 1 μg template, 0.5 μL EcoRV, and ddH2O added to bring the total volume to 20 μL.

[0114] Transformation of the pBC-Hygromycin plasmid vector into ΔPaFrq protoplasts specifically includes:

[0115] (1) Thaw the protoplasts on ice or use them directly, incubate at 48°C for 5 min, immediately transfer them to ice for 30 s, and then let them stand at room temperature for 5 min.

[0116] (2) Add the enzyme-digested pBC-Hygromycin plasmid vector, PaFrq-1 and PaFrq-2 DNA fragments to the tube containing protoplasts.

[0117] (3) Add 2 mL of Tampon D solution to a 50 mL centrifuge tube in advance, and transfer the mixture from step (2) to a 50 mL centrifuge tube, mix well, and let stand at room temperature for 15 min.

[0118] (4) Centrifuge at 20℃ for 10 min at a speed of 3,200 rpm, slowly aspirate and discard all supernatant, and retain the precipitate.

[0119] (5) Add 5 mL of RG liquide to a 50 mL centrifuge tube, mix the precipitate and liquid, and incubate in the dark at 27 °C for 12 h.

[0120] (6) Sorbitol and RG Top were mixed in a 3:1 ratio beforehand and placed in a 60°C oven to prevent the mixture from solidifying. 30 mL of the mixture was then mixed with the protoplasts from step (5), resulting in a final volume of 35 mL. The antibiotic Hygromycin was then added to the mixture to a final concentration of 75 μg / mL.

[0121] (7) Turn on the water bath in advance and adjust the temperature to 47℃. Quickly transfer the above mixture into the water bath and incubate for 7 minutes. Pour 7 mL of the mixture into an M2 culture dish containing Hygromycin antibiotic and prepare 5 resistance culture media. Incubate at 27℃ for 2-5 days and wait for transformants to grow.

[0122] The resistance verification of transformants included: taking 1 cm of transformants grown on M2 medium containing the antibiotic Hygromycin. 2 The mycelium was then inoculated again onto M2 medium containing the antibiotic Hygromycin, which served as a test of the transformant's resistance.

[0123] Table 1 Primer sequences

[0124]

[0125]

[0126] In Table 1, PaFrq_1F and PaFrq_2R amplify the 5' flanking region, PaFrq_MkF and PaFrq_MkR amplify the gene sequence, PaFrq_3F and PaFrq_4R amplify the 3' flanking region, PaFrq-5'Test and PaFrq-3'Test amplify mutant detection, and PaFrq-1 and PaFrq-2 amplify PaFrq complementation.

[0127] 1.6 Determination of ΔPaFrq biophenotype under different light conditions

[0128] Wild-type strain (WT) and mutant strain (ΔPaFrq) were inoculated onto M2 medium and activated at 27°C for 2 days. A 1cm section was scraped off. 2 The mycelium was added to a 2 mL centrifuge tube containing 500 μL of sterile water, placed in a cell disruptor, and shaken at 5.0 m / s for 20 s, and shaken twice to prepare the bacterial culture.

[0129] (1) Growth and development of strains under different light conditions

[0130] 5 μL of WT and ΔPaFrq bacterial suspensions were inoculated onto M2 and GY media, respectively, and placed under white light, blue light, red light, and darkness, and incubated at a constant temperature of 27°C. The growth, development, and morphological changes of the mycelium were observed and recorded daily.

[0131] (2) Sexual reproduction assay of strains under different light conditions

[0132] Mixed bacterial suspensions of WT and ΔPaFrq mating types were prepared separately. 5 μL of each suspension was inoculated onto basal medium M2 and incubated at 27°C for 7 days under white light, blue light, red light, and darkness, respectively. The microstructure of fruiting bodies and gametes was observed under a microscope, and the number of fruiting bodies for WT and ΔPaFrq strains was quantified.

[0133] (3) Determination of the life cycle and senescence time of strains under different light conditions

[0134] The same mating type strains of WT and ΔPaFrq were inoculated onto a 15cm diameter culture medium and cultured under white light, blue light, and darkness, respectively. The time taken for obvious melanin accumulation to appear at the tip of the hyphae was the time when the colony began to senescent, and the time taken for the hyphae to stop growing was the life cycle. The senescence time and life cycle were observed and recorded.

[0135] 1.7 Determination of ROS content under different light conditions

[0136] The WT and ΔPaFrq strains activated for 2 days were inoculated onto M2 medium and cultured at 27°C in the dark for 3 days. The mycelia were stained using the DAB chromogenic kit and NBT reagent, respectively. After incubation in the dark for 2 hours, the staining reagents were removed, and the mycelia were washed with sterile water. The staining of the mycelia was then observed.

[0137] 1.8 Determination of catalase activity under different light conditions

[0138] Catalase (CAT), ubiquitous in tissues and cells, is one of the important enzymes protecting the body from oxidation. Its function is to remove hydrogen peroxide produced during metabolism, preventing its accumulation and subsequent oxidative damage to cells. Its activity is related to the body's environmental resistance. Hydrogen peroxide absorbs at 240 nm. When a protein extract containing CAT is added to a reaction system containing hydrogen peroxide, CAT catalyzes the decomposition of hydrogen peroxide to produce water and oxygen. The decomposition of hydrogen peroxide causes the absorbance of the reaction solution to decrease with increasing reaction time. The rate of change in absorbance can be used to calculate the CAT activity. Enzyme activity calculation formula:

[0139] CAT (nmol / min / mg) = [ΔA 240 ×V1÷(ε×d)×10 9 ]÷(V2×C pr )÷T

[0140] V1: Total volume of the reaction system (L); ε: Molar extinction coefficient of H2O2, 4.36 × 10⁻⁶ 4 L / mol / cm; d: optical path length of the 96-well microplate (cm); V2: volume of sample added (mL); C pr : Sample protein concentration, mg / mL; T: Reaction time (min).

[0141] (1) Required reagents

[0142] 1.50 mM phosphate buffer (pH = 7.8);

[0143] 2.0.05mM phosphate buffer (pH=8), 50mM PBS (pH=7.8);

[0144] 3.1.044g K2HPO4:3H2O and 0.0578g KH2PO4, diluted to 1L;

[0145] 4. 200mM H2O2 solution: Dissolve 11.36mL of 30% H2O2 in phosphate buffer; bring 50mM phosphate buffer (pH=7) to 250mL; 50mM Tris-HCl buffer (pH7.0): Dissolve 0.7g of Tris-HCl to 100mL.

[0146] (2) Experimental steps

[0147] 1. Preparation of mycelial protein extract: Wild-type and mutant strains were cultured in shake flasks on 100 mL of M2 liquid medium for 9 days at 150 rpm and 27°C. The mycelia were then filtered, washed twice with 1×PBS, and filtered again to obtain relatively pure mycelia. The mycelia were ground into a fine powder using liquid nitrogen, and the protein was lysed using a filamentous fungal protein extraction kit to prepare the mycelial protein extract. The protein concentration was determined using a BCA protein concentration assay kit and denoted as C. pr .

[0148] 2. Inactivate the crude enzyme solution: Take 50 μL of the enzyme solution and place it in a PCR instrument. Incubate at 100℃ for 10 min to obtain the inactivated enzyme solution as a blank control.

[0149] 3. Prepare a 96-well quartz microplate, add samples in 200 μL increments, with 3 replicates per sample, as follows:

[0150] Mix 14 μL of protein extract, 100 μL of 1×PBS, and 80 μL of ddH2O. Incubate at 25°C for 3 min. Then add 6 μL of 200 mM H2O2, for a total volume of 200 μL.

[0151] 4. Measure the absorbance at 240nm, repeating every 30 seconds for a total of 3 minutes. Since H2O2 decomposes readily upon exposure to light, it is recommended to adjust the microplate reader parameters before quickly adding H2O2 to the plate.

[0152] 1.9 Determination of cellulase activity under different light conditions

[0153] To select strains that efficiently degrade cellulose, the effect of light on the ability of P. anserina to degrade lignocellulose after the deletion of the photoperiod gene was investigated. Strains that efficiently degrade lignocellulose were screened, and the cellulose filter paper enzyme activity (FMase), endoglucanase activity (CMCase), β-glucanase activity (β-BG), and exoglucanase activity (CBH) of ΔPaFrq were measured under different light conditions.

[0154] Wild-type and mutant strains were inoculated onto M2 medium and cultured at 27°C under white light for 2 days. Subsequently, 1 cm samples were collected. 2Mycelia were added to 2 mL centrifuge tubes containing 500 μL of the mycelium and placed in a cell disruptor. The mixture was shaken twice at 5.0 m / s for 20 s to prepare a bacterial suspension. 100 μL of the bacterial suspension was inoculated into 50 mL of cellulose induction medium and cultured on a shaker at 27°C and 150 rpm for 5 days. After 5 days of culture, 1.8 mL of the culture supernatant was transferred to 2 mL centrifuge tubes. All supernatants were centrifuged at 4°C for 10 min at 12,000 rpm. The resulting supernatant was the crude enzyme solution. The enzyme activity, endo-β-1,4-glucanase activity, β-glucosidase activity, and exo-glucanase activity were measured according to the modified enzyme activity assay methods reported by Eveleigh et al., as per the national standard QB2583-2003. For specific operational methods, please refer to the graduation thesis: Qiu Yanling. Study on the mechanism of NADPH dehydrogenase regulating energy metabolism and fatty acid metabolism of filamentous fungus Podospora anserina and its influence on fruiting body formation [D]. Shenzhen University, 2020.

[0155] II. Results and Analysis

[0156] 2.1 Bioinformatics Analysis of Photocyclin

[0157] The protein PaFrq (XP_001904738.1) encoded by the PaFrq gene of *P. anserina* shows a similarity of over 56.45% to rhythmic proteins in other fungi such as *Neurospora crassa* and *Sordaria macrospora*, with the highest similarity (57.14%) to FRQ (AAA57121.1) in *Neurospora crassa*. Therefore, it is speculated that the protein encoded by PaFrq is a photoperiodic protein similar to FRQ in *Neurospora crassa*. A phylogenetic tree was constructed using MEGA11.0 for photoperiodic proteins from 15 ascomycetes and one Drosophila. Phylogenetic analysis showed that, as... Figure 1 As shown, PaFrq and the FRQ of the ascomycete *Neurospora crassa* have a higher phylogenetic relationship, suggesting they may have similar mechanisms of action in photoperiod regulation. Furthermore, the FRQ of *Neurospora crassa* has been identified as absorbing blue light, indicating that the PaFrq protein of *P. anserina* may also have a similar function. Therefore, these findings provide an important foundation for further research into the photoperiod regulation mechanism of *P. anserina*.

[0158] 2.2 Construction of photoperiod gene knockout mutants and identification of transformants

[0159] Using WT genomic DNA as a template, the upstream fragment PaFrq-5' and the downstream fragment PaFrq-3' (approximately 1000 bp) of the PaFrq gene were amplified. Using pBC-Geneticin plasmid DNA as a template, the resistance selection marker Geneticin gene fragment was amplified. The results are as follows: Figure 2 As shown in the figure. The PaFrq knockout expression cassettes PaFrq-5'-Geneticin and Geneticin-PaFrq-3' were constructed by fusion PCR, and the results are shown in the figure. Figure 3 As shown.

[0160] Figure 2 The results show the amplification of upstream and downstream fragments of the PaFrq gene and the selection of resistance marker genes. Figure 3 The amplification results are for the fusion fragment. Figure 2 Agarose gel electrophoresis of PaFrq-5', Gene, and PaFrq-3' (1,2,3). Figure 3 Agarose gel electrophoresis of PaFrq-5'-Gene and Gene-PaFrq-3'(1,2); M is a DNA molecular marker (bp).

[0161] After transforming the knockout expression cassette into protoplasts of the Δmus51::phleoR strain, transformants containing geneticin resistance but not phleomycin resistance were screened. Homologous recombination fragments were amplified by validating primers, such as... Figure 4 As shown in Table 1, the verification primer sequences were obtained. The purified amplification product was sequenced, and the sequencing results were consistent with the target sequence, indicating that the screening resistance marker gene was successfully integrated into the PaFrq locus. Figure 4 The PCR validation results for the ΔPaFrq mutant strain are shown in the agarose gel electrophoresis images of PaPaFrq-vF / valid5' and (1,3) and PaPaFrqvalid3' / vR(2,4), where M represents the DNA molecular marker (bp).

[0162] 2.3 Identification of Photoperiod Gene Complementation Strains

[0163] Agarose gel electrophoresis was used to confirm band sizes, and the enzyme digestion products were recovered by gel extraction. The two digested fragments were transformed into protoplasts, and transformants containing the corresponding resistance were selected for PCR verification. The amplified target gene fragment matched the expectations. The bacterial morphology was observed under white light; the phenotype of the supplemented strain was consistent with that of the wild-type strain. Figure 5 As shown, this indicates that the PaFrq gene replacement was successful. Figure 5 The images show the growth of wild-type and mutant strains under white and blue light. Figure (A) shows the colony morphology under white light, and Figure (B) shows the colony morphology under blue light.

[0164] 2.4 Photoperiod genes affect the growth and development of P. anserina

[0165] To investigate the effects of PaFrq on asexual development and morphogenesis of P. anserina under white light, blue light, red light, and darkness, WT, ΔPaFrq, and ΔPaFrq were compared. c The strains were cultured on M2 and GY media under constant light conditions, respectively. ΔPaFrq c It is a complement strain. On M2 medium, the colony morphology of the ΔPaFrq mutant was unaffected in the dark. However, compared to the wild-type and complement strains, ΔPaFrq showed abnormal colony morphology and a significantly slower growth rate on both media under constant white and blue light. Figure 5 As shown.

[0166] Unlike the fluffy colony surface of the wild-type strain, the ΔPaFrq strain formed only sparse aerial hyphae on its ribbon-like colonies when grown under white and blue light. On M2 medium, ΔPaFrq showed significant over-accumulation of pink pigment after 2 days of culture. The central region of the ΔPaFrq colonies underwent a pigment transition from pink to brownish-green on day 3, and after 4 days of culture, ΔPaFrq showed even more pronounced pigment deposition, such as... Figure 5 As shown. There was no difference in colony morphology between wild-type and mutant under red light, as... Figure 6 As shown, it is therefore speculated that this species is insensitive to red light, and red light cannot effectively induce changes in the physiological processes of this bacterium. All strains grown under white and blue light showed more pronounced pigmentation compared to their counterparts cultured in the dark, indicating that white light can induce pigment synthesis in P. anserina, and that the loss of function of the PaFrq gene in P. anserina leads to severe growth defects and morphological changes, which are entirely induced by blue light. Figure 6 Colony morphology of wild-type and mutant strains on M2 medium under red light and dark conditions.

[0167] 2.5 Deletion of photoperiod genes leads to decreased sexual reproductive capacity.

[0168] The fruiting body is the spore-producing reproductive body formed during the sexual reproduction stage of higher filamentous fungi. It is a crucial organ for sexual reproduction in fungi and the most complex multicellular structure in fungi. The reproductive growth of the fruiting body requires significantly more energy. Studies on the sexual reproductive capacity of *P. anserina* revealed that it only produces fruiting bodies and engages in sexual reproduction under white and blue light. Under red light and darkness, neither the wild-type nor the mutant can reproduce sexually and cannot produce fruiting bodies with sexual reproductive structures. Figures 7 to 9 As shown. Figure 7These are the colony morphologies of WT and ΔPaFrq cells after 7 days of growth and development under different light conditions. Figure 8 This is an image showing the results of microscopic morphological observation of the fruiting body. Figure 9 The values ​​represent the number of fruiting bodies on day 7 of WT and ΔPaFrq cultures, where *: P < 0.05, **: P < 0.01, ***: P < 0.001. This indicates that light signal is one of the key factors controlling the sexual reproduction of *P. anserina*. Blue light can effectively induce the sexual reproduction process of *P. anserina*, while red light does not have this function. The physiological and biochemical processes of wild-type strains require a specific light environment to regulate and support the formation of fruiting bodies. This result also suggests the possible existence of phytochromes or other photoreceptor mechanisms that regulate the reproductive process of strains by sensing light signals. In summary, this result provides an important direction for further research on the physiological and biochemical mechanisms of strains. Unlike the annular asci produced after complete fertilization of wild-type strains, more rings were observed on the surface of the banded colonies of ΔPaFrq under white light. Subsequent quantification of fruiting bodies revealed that ΔPaFrq produced fewer fruiting bodies, such as... Figure 7 and Figure 9 As shown in the figure, compared with the wild type, the number of fruiting bodies produced by ΔPaFrq decreased by 43.02% under white light, 40.52% under combined blue and red light, and 28.75% under blue light. The microstructure of ΔPaFrq fruiting bodies was not different from that of the wild type. These results indicate that the sexual reproduction process of ΔPaFrq is severely affected under white and blue light conditions, but the sexual reproduction pathway is not completely blocked. The PaFrq gene plays an important regulatory role in the reproductive process of organisms, and its deletion has a negative impact on reproduction.

[0169] 2.6 Photoperiod genes affect pigment accumulation

[0170] The ΔPaFrq mutant showed significant orange pigment accumulation on day 4 of culture on GY medium under light, such as... Figure 10 As shown, Figure 10The colony morphology of WT and ΔPaFrq on GY medium under white light and dark conditions is shown. Given that the deletion of the vvd gene in the ascomycete *Neurospora crassa* leads to increased carotenoid production, resulting in significant orange pigment deposition, it is hypothesized that the orange pigment in the ΔPaFrq strain may also be caused by carotenoid accumulation. To identify whether the orange pigment is a carotenoid, WT and ΔPaFrq were cultured on GY medium supplemented with fluorouracil. Fluorouracil effectively inhibits the production of the first enzyme in the carotenoid biosynthesis pathway—phytopene dehydrogenase (PDS). If the pink pigment were a carotenoid, ΔPaFrq would not have accumulated orange pigment on GY medium supplemented with fluorouracil. However, in the presence of fluorouracil (concentrations of 10 μM, 100 μM, and 500 μM), the pigment deposition in *P. anserina* was not reduced, and the orange pigment synthesis pathway was not blocked. Figure 11 As shown, Figure 11 The colony morphology of WT and ΔPaFrq on GY medium supplemented with fluorouracil; Figure 11 The results showed that increasing the concentration of fluorourea did not block the synthesis pathway of orange pigment. Therefore, it is speculated that, in addition to carotenoids, other pigments may be induced in ΔPaFrq, and that the accumulation of these pigments is dependent on white light irradiation.

[0171] 2.7 Deletion of photoperiod genes leads to an increase in the number of male gametes.

[0172] Given the decrease in the number of sexual reproductive structures—fruiting bodies—of *ΔPaFrq*, the fertility of male and female gametes was analyzed using optical microscopy and a propagation experiment to investigate the impact of male and female gametes on the reduced sexual reproductive capacity. Microscopic observation showed that under white light, a large number of male gametes differentiated from the hyphae of *ΔPaFrq*, such as... Figure 12 As shown, Figure 12 The number of male gametes after 7 days of mycelial growth and development under different light conditions is shown, with black arrows indicating male gametes. In contrast, there was no significant difference in the number of male gametes between the wild-type strain and the ΔPaFrq strain in the dark. The data indicate that ΔPaFrq can act as a positive regulator of male gamete formation under light conditions. Then, the fertility of male and female gametes of ΔPaFrq was studied through a propagation experiment. When ΔPaFrq acted as the female parent and the wild-type strain as the male parent, the formation of fruiting bodies in ΔPaFrq decreased, as shown in the figure. Figure 13 As shown, Figure 13 This is a fertility analysis of male and female gametes under white light; the small black dots represent fruiting bodies. Figure 13The results showed that PaFrq deficiency led to sac dysfunction or defects in fertilization process in P. anserina, resulting in a decrease in the number of fruiting bodies produced by ΔPaFrq and exhibiting abnormal sexual reproductive capacity.

[0173] 2.8 Deletion of photoperiod genes delays bacterial senescence

[0174] Reproduction is the link between the short lifespan of an individual and the long lifespan of a population, and is the foundation for the existence and continuation of a species. It is generally believed that organisms, for the sake of population propagation, will ultimately prioritize reproduction rather than extending individual lifespan and delaying aging. Aging is a complex biological process closely related to external environmental stimuli. In investigating the effect of light on the sexual reproduction of *P. anserina*, it was found that the fruiting bodies of WT and ΔPaFrq matured at different times. WT fruiting bodies matured and released ascospores on day 7, while ΔPaFrq fruiting bodies matured and released ascospores on day 9. Therefore, it is speculated that the life cycle of phytochrome-deficient strains is affected, and their aging time and lifespan may be altered. To investigate whether the PaFrq gene is involved in the aging mechanism of *P. anserina*, leading to an impact on the life cycle of ΔPaFrq and potentially altering its aging time, WT and ΔPaFrq were cultured in darkness, and the distance of hyphal tip extension was observed and recorded daily. Figure 14 As shown, WT began to age on day 10 of culture in the dark, and hyphae stopped growing on day 52; ΔPaFrq began to age on day 12 of culture in the dark, and hyphae continued to grow on day 52, delaying aging by 33.33%. This result indicates that the PaFrq gene deletion mutant has a prolonged lifespan, suggesting that PaFrq plays an important regulatory role in the aging process of *P. anserina*. Specifically, the PaFrq gene may be involved in various physiological and biochemical processes such as metabolic regulation, cell proliferation and apoptosis, and DNA repair, thus affecting the aging process.

[0175] 2.9 Deletion of photoperiod genes leads to reduced mycelial ROS content.

[0176] Previous studies have shown that fungal growth from vegetative to reproductive requires certain physiological conditions to be met by the mycelium, and the physiological state of the mycelium is usually related to nutrient and energy metabolism. Based on previous research, the PaFrq gene may participate in the metabolic regulation of organisms, especially reactive oxygen species (ROS) metabolism, thereby affecting the aging rate of the strain. ROS has certain biological functions under normal physiological conditions, but excessive ROS can cause oxidative damage to cell structures, nucleic acids, proteins, and other biomolecules, thus affecting cell function. To investigate whether PaFrq affects the ROS metabolism of *P. anserina*, and the relationship between ROS production and ΔPaFrq in delaying aging, the secretion of peroxides and superoxides in WT and ΔPaFrq strains was detected. The strains were inoculated on M2 medium and cultured in the dark for 3 days. Afterward, the strains were stained with DAB and NBT reagents, respectively. DAB stained the mycelium red-brown, and NBT stained it blue-purple. The experimental results are as follows: Figure 15 As shown, NBT staining revealed a deeper blue color in WT hyphae compared to ΔPaFrq hyphae; DAB staining showed that the brown color of WT hyphae was darker than that of the mutant. These results indicate that WT hyphae have the highest ROS content; ΔPaFrq hyphae are lighter in color than WT hyphae, indicating a relatively lower ROS content. Therefore, it is speculated that the PaFrq gene plays a role in regulating the production and clearance of ROS within cells. This may be because the PaFrq gene regulates ROS production pathways, such as the cellular respiratory chain and the activity of oxidoreductases like NOx; or it may be involved in ROS clearance pathways, such as the expression and activity of superoxide dismutase (SOD) and peroxidase (CAT). In the absence of the PaFrq gene, the imbalance of the intracellular ROS clearance system leads to reduced ROS production and accumulation, thereby slowing down the rate of oxidative damage to cells and altering the aging time of the strain.

[0177] 2.10 Increased ΔPaFrq catalase activity under white and blue light

[0178] Catalase (CAT) is a common antioxidant enzyme that breaks down accumulated hydrogen peroxide (H2O2) in cells into water and oxygen, thus preventing damage to cellular structure and function. CAT effectively removes excess hydrogen peroxide from cells, protecting them from oxidative damage. Measuring CAT activity is one method for assessing an organism's antioxidant capacity; changes in CAT activity in bacterial strains can reflect the degree of oxidative stress and the strength of antioxidant capacity. The previous study concluded that the PaFrq gene may be involved in ROS scavenging pathways, such as the expression and activity of superoxide dismutase (SOD) and catalase (CAT). Therefore, CAT activity was measured under different light conditions to explore the correlation between the reduced ROS content caused by PaFrq deficiency, CAT activity, and delayed senescence in ΔPaFrq strains. The results showed that under both white and blue light conditions, the CAT enzyme activity of the ΔPaFrq strain was higher than that of the wild type. This suggests that the ΔPaFrq strain may possess better antioxidant capacity to some extent, enabling it to better cope with light-induced oxidative stress and thus reduce cellular oxidative damage and aging. This finding also indicates that PaFrq deficiency may lead to changes in cellular antioxidant defense mechanisms, thereby affecting cellular oxidative stress responses.

[0179] 2.11 Deletion of photoperiod genes increased cellulase activity in the dark.

[0180] P. anserina contains multiple genes for proteases that degrade lignocellulose, exhibiting a strong ability to degrade lignocellulose and convert it into bioethanol, playing a crucial role in the degradation and utilization of cellulose. Therefore, breeding P. anserina mutant strains with high efficiency in degrading lignocellulose has significant application value.

[0181] To determine the effect of PaFrq on the cellulase production pathway of P. anserina, industrial application strains were screened, and the activities of four cellulases in WT and ΔPaFrq strains were measured. The results are as follows: Figures 16 to 19 As shown. Figure 16 The results are for cellulose filter paper enzyme activity (FPase) assays of WT and ΔPaFrq. Figure 17 The results are for the endoglucanase activity (CMCase) assay of WT and ΔPaFrq. Figure 18 The results are for the β-glucanase activity (β-BG) assay of WT and ΔPaFrq. Figure 19 The results are for the exoglucanase activity (CBH) assays of WT and ΔPaFrq, where *: P<0.05, **: P<0.01, ***: P<0.001.

[0182] Figures 16 to 19 The results showed that under dark conditions, the activities of all four cellulases in ΔPaFrq were higher than those of the wild type and also higher than those under white light conditions. Significant differences were observed in filter paper enzyme activity, β-BG enzyme activity, and CBH enzyme activity. This indicates that the PaFrq-deficient strain can better adapt to dark environments and utilize available carbon sources more effectively. This provides a new approach for industrial cellulose biodegradation. ΔPaFrq can be used to produce enzyme preparations for further application in cellulose biodegradation and other industrial applications. Furthermore, utilizing ΔPaFrq for biomass fermentation can effectively improve biomass conversion rate and ethanol yield.

[0183] III. Discussion and Conclusion

[0184] Among ascomycetes, *Neurospora crassa* has been the subject of relatively in-depth research on the function of photocyclin, with its FRQ defined as a blue light receptor. This study, through genome mining of *P. anserina*, discovered a photocyclin (PaFrq protein). Amino acid sequence alignment analysis showed that the PaFrq protein shares a 57.14% similarity with the *Neurospora crassa* FRQ, and both possess the characteristic PAS domain of blue light receptors. Therefore, it is hypothesized that the photocyclin in *P. anserina* may function as a blue light receptor, located in the cell nucleus, and directly control gene expression.

[0185] Based on our laboratory research, light, as an important environmental signal, is indispensable for the development of the fruiting bodies of the model organism *P. anserina*. However, the mechanisms by which this fungus senses and responds to light are not yet fully understood. In this study, we demonstrated that the light-dependent colony morphological changes induced by the deletion of the *PaFrq* gene in *P. anserina* are similar to those in *Neurospora crassa*, *Aspergillus nidus*, and *Trichoderma reesei*. For example, in *Trichoderma reesei*, light stimulates the expression of cellulase genes, a stimulation regulated by the blue light receptor ENVOY. However, some differences also exist. For instance, under constant white light, the deletion of the *PaFrq* gene in *P. anserina* leads to slower colony growth and a reduction in the number of male gametes. Figure 6 and 12As shown. Therefore, it is believed that, as previously reported in other fungi, PaFrq is a major regulator in *P. anserina*, balancing asexual development and male gamete differentiation in response to white light. Studies have reported that light is a stimulant promoting fungal pigment production; under constant white and blue light, the PaFrq deletion mutant exhibits a bright orange color. To our knowledge, *P. anserina* is closely related to *Neurospora crassa* and *Dictyophora foetida*, all of which contain carotenoid genes (also known as albino genes, denoted as al-1, al-2, and al-3), and thus can synthesize carotenoids such as β-carotene, γ-carotene, and neuroastaxanthin. In *Neurospora crassa*, the deletion of the blue light receptor VVD leads to a more intense response to light and a more pronounced orange pigment deposition. Given that the ΔPaFrq mutant grown on GY medium exhibits the same orange pigment accumulation as the vvd deletion mutant in *Neurospora crassa*, it is hypothesized that the orange pigment appearing in the ΔPaFrq mutant may be a carotenoid. Therefore, a chemical inhibitor, fluorourea, was selected. As reported in *Neurospora crassa*, *Pseudococcus nicotine*, and *Pseudococcus puddleii*, this inhibitor can block carotenoid biosynthesis and ultimately induce the accumulation of colorless carotenoid precursor phytoenes. However, no significant color change was observed in the wild-type *P. anserina* strain under fluorourea treatment, and no reduction in orange pigment accumulation was observed in the ΔPaFrq mutant. These results suggest that, in addition to carotenoids, other pigments may be induced in ΔPaFrq, and that the accumulation of these pigments may be dependent on white or blue light irradiation. To identify these pigments and their functions, further extraction using physical, chemical, or biological methods is required. For example, solvent extraction methods or preliminary identification of the pigments using gas chromatography or high-performance liquid chromatography can be used; these methods can help determine the chemical properties and composition of the pigments. Subsequently, techniques such as gas chromatography-mass spectrometry and nuclear magnetic resonance (NMR) can be used to further determine the chemical structure and composition of the pigments; finally, their biological activity is determined, and the effects of the pigments on cells, tissues, or organisms are assessed. These tests can reveal whether the pigment possesses biological activity, such as antioxidant, antibacterial, or antitumor properties. Based on the pigment's color, structure, and biological activity, the fungal pigment can be identified as a known type of fungal pigment or a novel type of pigment.

[0186] The reduced ROS content and increased free radical scavenging enzyme activity in mutant strains may both weaken the oxidative damage of free radicals to cells, thus delaying strain aging. Aging is a complex life process caused by the combined effects of multiple factors. By reducing ROS levels, the ΔPaFrq strain can effectively slow down the aging process of cells, providing new possibilities for cellulose degradation applications. Since aging is one of the main causes of decreased metabolic activity in organisms, extending the lifespan will allow the ΔPaFrq strain to maintain its effective cellulose degradation capacity for a longer period. Based on the characteristics of the ΔPaFrq strain, it can be considered as an engineered strain for cellulose degradation. ΔPaFrq has the characteristics of low ROS levels, slowed aging process, extended lifespan, and high cellulase activity under dark conditions; applying these characteristics to cellulose degradation processes can help improve efficiency, reduce costs, and play an important role in the field of sustainable bioenergy.

[0187] In summary, PaFrq participates in the growth, orange pigment deposition, and sexual reproduction of *P. anserina*, providing a research foundation for the photobiology of the model fungus *P. anserina*. Meanwhile, ΔPaFrq can efficiently degrade lignocellulose under dark conditions and has a longer lifespan, making it suitable as an engineered strain. Therefore, this application deposits the obtained ΔPaFrq strain, which has a long lifespan and can efficiently degrade lignocellulose under dark conditions, with accession number CCTCC M20231709.

[0188] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A mutant strain of filamentous fungus which efficiently degrades lignocellulose, characterized in that: The mutant strain is derived from a wild-type filamentous fungus Podospora anserina by knocking out a photoperiod gene PaFrq formed, the PaFrq The corresponding amino acid sequence is XP_001904738.

1.

2. The mutant strain according to claim 1, characterized in that: The preservation number of the mutant strain is CCTCC M20231709.

3. Use of the mutant strain of claim 1 or 2 in preparing a fermentation process of lignocellulose degradation.

4. Use according to claim 3, characterized in that: Degradation of lignocellulose by using the mutant strain of claim 1 or 2, and conversion of lignocellulose into bioethanol.

5. A method for efficient degradation of lignocellulose, characterized by: The mutant strain of claim 1 or 2 is cultured under dark condition.

6. The method of claim 5, wherein: The culture condition is culturing under dark condition for at least 7 days.

7. The method according to claim 5 or 6, characterized in that: The culture temperature is constant temperature culturing at 27℃.

8. A method of increasing the cellulase activity of a filamentous fungus Podospora anserina characterized in that: Using gene knockout techniques to make filamentous fungi Podospora anserina photoperiodic genes PaFrq non-expressing, said PaFrq The corresponding amino acid sequence is XP_001904738.

1.

9. A method of delaying senescence of filamentous fungi Podospora anserina characterized in that: Gene knockout or gene silencing techniques are used to induce filamentous fungi Podospora anserina Photoperiodic genes PaFrq Not expressed, the stated PaFrq The corresponding amino acid sequence is XP_001904738.1.

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