A method for expressing basidiomycete laccase in high-efficiency homologous recombination in coprinus cinereus

Abstract

The present application relates to a kind of in high-efficiency homologous recombination of Coprinus cinereus method for expressing basidiomycete laccase, belong to genetic engineering and fermentation engineering field.The present application with the laccase enzyme gene lcc5 derived from Coprinus cinereus FA2222 as objective gene, with Coprinus cinereus FA2222 as expression host constructs the homologous recombination expression laccase of Coprinus cinereus recombinant bacteria Cclcc5-13.On this basis, using RNA interference technology to the key chitinase gene ChiE2 in the genome of Coprinus cinereus FA2222 is silenced expression, obtain Coprinus cinereus recombinant bacteria Cclcc5-antiChiE2-5.After 3-L jar fermentation, it is found that its extracellular laccase activity and laccase production efficiency are 38.2U / mL and 0.35U / mL·h, respectively, are 1.5 and 1.8 times of control bacteria, realize the high-efficiency homologous recombination expression of laccase in Coprinus cinereus.

Description

Technical Field

[0001] This invention relates to a method for efficient homologous recombination expression of basidiomycete laccase in *Coprinus comatus*, belonging to the fields of genetic engineering and fermentation engineering. Background Technology

[0002] Lacase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that can oxidize phenols, carboxylic acids, aromatic amines and their derivatives with water as the only byproduct. It is considered a green catalyst with important industrial applications.

[0003] Therefore, laccase is widely used in various industries, from the environmental field to the cosmetics industry, including food processing and textiles (for dye biodegradation and synthesis).

[0004] Laccase genes are widely distributed in nature, existing in the genomes of plants, insects, bacteria, and fungi. To date, more than 80% of the laccases discovered are derived from fungi. Among them, laccases derived from basidiomycetes are the most widely used due to their broad substrate range, high specific activity, and large reduction potential, accounting for more than 90% of the current laccase market share, and the market demand is huge.

[0005] Therefore, the efficient preparation of laccase from basidiomycetes has great application potential.

[0006] However, the yield and efficiency of naturally secreted laccase from basidiomycetes are relatively low. For example, after culturing in mKjalke medium at 25°C for 6 days, the highest extracellular laccase activity of *Coprinus gracilis* was only 3.5 U / mL. Previously, heterologous recombinant expression of basidiomycete laccases in *Ascomycetes* and filamentous ascomycetes (such as *Aspergillus*) has been used to achieve efficient production. However, compared to naturally secreted laccases from basidiomycetes, the recombinant laccases obtained from *Ascomycetes* and filamentous ascomycetes exhibit altered enzymatic properties, including protein molecular weight, redox potential, pH stability, and thermal stability, due to inappropriate post-translational modifications such as incomplete folding and excessive glycosylation. This significantly reduces the practical application value of recombinant laccases obtained from heterologous recombinant expression in *Ascomycetes* and filamentous ascomycetes.

[0007] The aforementioned drawbacks make it difficult for laccase derived from basidiomycetes to be widely used in industry. Summary of the Invention

[0008] This invention provides a method for efficient homologous recombinant expression of laccase in *Coprinopsis cinerea*. The method uses pYSK7 as the expression vector, the laccase gene from *Coprinopsis cinerea* as the target gene, and *Coprinopsis cinerea* FA2222 as the expression host to construct a recombinant *Coprinopsis cinerea* strain for homologous recombinant expression of laccase. Then, the key chitinase gene in the *C. cinerea* FA2222 genome is mined and silenced using RNA interference (RNAi). Finally, the obtained recombinant *Coprinopsis cinerea* strain is optimized for fermentation. Using this engineered *Coprinopsis cinerea* strain, homologous recombinant expression of laccase is highly efficient. When used as a production strain, the laccase activity in the fermentation broth after 5 days of fermentation is 38.2 U / mL.

[0009] This invention provides a recombinant Coprinopsiscinerea that efficiently expresses laccase via homologous recombination. The recombinant Coprinopsiscinerea overexpresses the laccase with the amino acid sequence shown in SEQ ID NO.1, and the laccase is the Lcc5 gene of C. cinerea FA2222.

[0010] SEQ ID NO:1: Amino acid sequence of laccase Lcc5

[0011] MSFAWKALATLAVAIAASRSAFAQSIIGNQDTMTISNINAGPDGFTRPVIAVNGEFPSPLVRANKGDDFRINVVNNLDDDTMLRQTSVHWHGVFQHQSAWADGPDGVTQCPIPQSGQEFEYAFNAGQEAGTFWY HSHYGTQYCDGLRGPLVIYDPEDPHQDLYDVDDENTIITLADWYHLQAPSIQGPAVSQATLINGKGRRPGSTEGDIAVVNVEKDSRYRFRIVSLSCDPDYTFSIDNHTMTIIEADGQNTKPLEVESIRVFAGQ RYSVVVNADQAIGNYWIRAEPNIGDTGLVGTSGGGVNSAILRYATADEVEPDTPRLTNRPALQESNLRALTSGVPGGDGPADITFTFNLGLNFATGTFSMNPGESWVHPDTPVMVQIMNGVPAEDLVPAESLH TITRGQVVVVIPPFGIAGPHPFHLHGHAFSVIKSAGGSPNFVDPVRRDVVAVGTEAGQGDTIIRFVADNPGPWFFHCHIEFHLVTGLAAVFMEAPDEIASSNPPPPSWDALCPAFSALPPSATSIRIVPTPTP

[0012] In one embodiment of the present invention, the expression vector is pYSK7; the expression host is C. cinereaFA2222.

[0013] This invention provides a recombinant Coprinopsiscinerea that efficiently expresses laccase via homologous recombination. The recombinant Coprinopsiscinerea overexpresses the laccase with the amino acid sequence shown in SEQ ID NO.1, while simultaneously suppressing the expression of chitinase in its genome. The suppression is achieved through gene deletion, knockout, or silencing. The laccase is the Lcc5 gene of C. cinerea FA2222.

[0014] In one embodiment of the present invention, the expression vector is pYSK7; the expression host is C. cinereaFA2222.

[0015] In one embodiment of the present invention, the chitinase genes are the chitinase genes ChiEn1 and ChiE2 derived from the C. cinerea FA2222 genome.

[0016] In one embodiment of the present invention, the amino acid sequences of the chitinase genes ChiEn1 and ChiE2 are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0017] SEQ ID NO.2: Amino acid sequence of the chitinase gene ChiEn1

[0018] MQFKTSFFALLAGFLASSTLAAYVPVADSVPDNDSLTAPDCTHDGSTTTTAPAGDAVVPVPAEPDSEVRLNATQVLASLDAGGKVATAWYPSWQAAAHPPESLSWDKYNAMTFAFA TTTSDPANPLALDAESQALLPKFVEQAKQHNVKALLSLGGWTGSIYFSDHVSTPERRTAFVKAVVDLATQYNLDGIDFDWEFPNKQGIGCNHISNADSANFLAFLQELRQDPTGGK LMLTAAVGLLPFVGSDGQPMSDVSGFAEVFDFIAIMAYDVWGAWSPTVGPNAPLQDSCAANGVGSVASSVAAWTGAGFPANKLVLGVPAYGRSYYVDPANALSAAGELTPYAQFDK SKQPLGEGETGEQTVDQCGVASGPSGLFNFAGLVDAGYLNPDGTAAEGMVYLCDQCSETPFVYQKDTGTMITYDDAESTAAKGNFIAEQGLKGFAIWHGIGDYNDILLDAVSRGMGQ

[0019] SEQ ID NO.3: Amino acid sequence of the chitinase gene ChiE2

[0020] MGTALWLFLSFALVATSSFADHPPSGKKHRRRAEIPFRIEEPPFHPLSSSPAPSIISTSGLDSPTSTSLSPVTFTAAHQRSTAPGRPDLPVVIQGDSLKILPHPGSGTQRSDSITGSGAQATDTPSNPLPLFMGYYPDWAADTFP PESLDYGRYDWIDFAFAVPTAEFGLEWDSDDAPNVLTRLVTSAHLSRCKVKLSIGGWTGSKYFSTAVSTSQSRQLFASNILSVYSRYNLDGIDLDWEYPGHEGNRGNIYDHSDTLNFLDFLILLRDTLPPSARITVAAQTFPFTD QNGEPLSDSSPFASVLDWVLIMNYDTWGSSTSPGPNAPLYDACGNSTQPEASALSSYRSWTKSGFPASKLVLGVPAYGYVQRSVAQRLRNRWYGEDDGGRPRLAYGGYRHSPDHFGHGHWGLQDSDDGWDEHWGGADGHTGWSGH SRNGNDGKGDWPDGDDNDDQGSVSPPGSPSPGRPIEVVDDEEQIQFRDLVKQGALVLAPSSSNETYPRFLASGGFERRWDFCSETPFLRSASAGQIITYDDPESLALKSRFAKEAGMLGVNMFDIHGDTDDYHLADSIRKALGLS

[0021] In one embodiment of the present invention, the expression of the chitinase gene in the C. cinerea FA2222 genome is inhibited by RNAi.

[0022] In one embodiment of the present invention, the expression host is C. cinerea FA2222 or a genetically engineered bacterium whose chitinase gene is silenced.

[0023] The present invention also provides the application of chitinase with an amino acid sequence as shown in SEQ ID NO.2 or SEQ ID NO.3 in increasing the expression level of laccase in recombinant Coprinus fusiforme, wherein the recombinant Coprinus fusiforme overexpresses laccase with an amino acid sequence as shown in SEQ ID NO.1; and simultaneously prevents the expression of chitinase on its genome; wherein the non-expression is due to gene deletion, knockout or silencing.

[0024] The present invention also provides a method for efficient homologous recombination expression of basidiomycete laccase in Coprinus gravidus, wherein the method comprises overexpressing laccase with an amino acid sequence as shown in SEQ ID NO.1, or overexpressing laccase with an amino acid sequence as shown in SEQ ID NO.1, in Coprinus gravidus host cells, while simultaneously preventing the expression of chitinase on its genome; wherein the non-expression is due to gene deletion, knockout, or silencing.

[0025] In one embodiment of the present invention, the amino acid sequence of the chitinase is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0026] This invention provides a method for producing laccase, wherein the method involves fermentation of a recombinant Coprinus grayis expressing laccase using the aforementioned highly efficient homologous recombinant method.

[0027] In one embodiment of the present invention, the method is to first inoculate the above-mentioned highly efficient homologous recombinant expression of laccase into a seed culture plate to obtain seed mycelial blocks, then inoculate the seed mycelial blocks into a seed liquid culture medium to obtain seed mycelium, then homogenize the seed mycelium to obtain seed liquid, and finally inoculate the seed liquid into a fermentation medium for culture.

[0028] In one embodiment of the present invention, the method involves first inoculating the recombinant *Coprinus comatus* expressing laccase with the aforementioned high-efficiency homologous recombinant culture into a seed culture plate and incubating it statically at 35–38°C for 6–10 days to obtain seed mycelial blocks. Then, the seed mycelial blocks are inoculated into seed liquid culture medium and cultured at 35–38°C and 100–140 rpm for 3–5 days to obtain seed mycelia. The seed mycelia are then homogenized at 3000–4000 rpm for 10–20 seconds to obtain seed liquid. Finally, the seed liquid is inoculated into fermentation medium and cultured at 35–38°C and 100–140 rpm for 4–6 days.

[0029] In one embodiment of the present invention, the seed culture plate is a seed liquid culture medium with 1-3% agar powder added.

[0030] In one embodiment of the present invention, the seed liquid culture medium comprises 3-5 g / L of yeast extract, 3-5 g / L of malt extract and 8-12 g / L of glucose.

[0031] In one embodiment of the present invention, the fermentation medium comprises 18-22 g / L glucose, 8-12 g / L yeast extract, 0.4-0.6 g / L calcium chloride, 1-3 g / L potassium dihydrogen phosphate, and 48-52 g / L magnesium sulfate.

[0032] In one embodiment of the present invention, the fermentation optimization refers to the optimization of the fermentation stirring speed of the 3-L tank.

[0033] This invention provides laccase prepared by using the above-described highly efficient homologous recombination expression of laccase by *Coprinus gracilis* or by the above-described method for producing laccase.

[0034] This invention provides the above-mentioned highly efficient homologous recombinant expression of laccase by the engineered strain *Coprinus gracilis* or the above-mentioned method for producing laccase, and its application in the treatment and degradation of oxidized phenols, carboxylic acids and aromatic amines and their derivatives.

[0035] Beneficial effects

[0036] (1) This invention uses pYSK7 as the expression vector, the laccase Lcc5 gene from C. cinerea as the target gene, and C. cinerea FA2222 as the expression host to obtain an engineered strain of Coprinus comatus that can efficiently express laccase through homologous recombination.

[0037] (2) This invention first points out that chitinase genes ChiEn1 and ChiE2 are key chitinase genes in the C. cinerea FA2222 genome, and that silencing chitinase gene ChiE2 can increase the homologous recombination expression level of laccase Lcc5 in Coprinus comatus.

[0038] (3) Using the engineered strain of Coprinus gracilistylus of the present invention to produce laccase, the laccase activity in the fermentation broth after 5 days of fermentation in a 3-L tank can reach as high as 38.2 U / mL, which is the highest level of laccase Lcc5 expressed by homologous recombination of Coprinus gracilistylus to date. Attached Figure Description

[0039] Figure 1 Flowchart for constructing recombinant plasmid pYSK7-lcc5.

[0040] Figure 2 Fermentation curves for laccase production by recombinant Coprinus grayis Cclcc5.

[0041] Figure 3 The transcription level of the chitinase gene in the genome of Coprinus fabricus FA2222.

[0042] Figure 4 Fermentation curves for laccase production by recombinant strains Cclcc5-antiChiEn1 and Cclcc5-antiChiE2 of Coprinus gracilistylus.

[0043] Figure 5Fermentation curves for laccase production in 3-L tanks using recombinant *Coprinus gracilis* strains Cclcc5-13 and Cclcc5-antiChiE2-5; where hollow sections represent recombinant *Coprinus gracilis* strain Cclcc5-13 and solid sections represent recombinant *Coprinus gracilis* strain Cclcc5-antiChiE2-5. Detailed Implementation

[0044] The present invention will be further described below through specific embodiments.

[0045] The C. cinerea FA2222 strain involved in the following examples has been published in Rühl M, Lange K, Kües U (2018) Laccase production and pellet morphology of Coprinopsis cinerea transformants in liquid shake flask cultures. Appl Microbiol Biotechnol 102:7849-7863 doi:10.1007 / s00253-018-9227-7.

[0046] The detection methods involved in the following embodiments are as follows:

[0047] Enzyme activity assay: Take 950 μL of sodium tartrate solution (100 mM; pH 4.0), 33 μL of ABTS (concentration 15 mM), and 17 μL of enzyme solution, and add each system to a 2 mL centrifuge tube using a pipette. Vortex to mix, then incubate at 30℃ for 3 min, followed by an ice bath for 30 s. Measure the absorbance at 420 nm (OD420). Count the enzyme activity based on the absorbance (before measuring laccase activity, the absorbance OD420 needs to be adjusted to zero using a solution without enzyme solution).

[0048] Under the above conditions, the amount of enzyme required to catalyze 1 μmol of guaiacol substrate per minute is defined as one enzyme activity unit (U).

[0049] The method used for sample preparation in atomic force microscopy is as follows:

[0050] First, wash the mycelium 3-5 times in pre-cooled PBS buffer (0.2% NaN3), then resuspend it in fresh PBS buffer to dilute the biomass to an appropriate concentration to prevent cell overlap. Next, add 40 μL of poly-L-lysine solution (0.1%) to a round coverslip, let it stand for half an hour, then immediately rinse the coverslip with deionized water and allow it to air dry naturally, allowing the mycelium to settle. Finally, gently wash with deionized water to remove surface salt residue, and dry before use.

[0051] The working conditions for atomic force microscopy to determine the samples were as follows: the hyphae were scanned in contact mode to locate the position of the indentation experiment, and the force curve was collected at a scan rate of 0.5 μm / sz, a scan size of 250 nm z, and a cantilever beam of approximately 40 nm.

[0052] The culture media and buffer solutions involved in the following examples are as follows:

[0053] Seed culture plate: 3-5 g / L yeast extract, 3-5 g / L malt extract, 8-12 g / L glucose, 1-3% agar powder.

[0054] Seed liquid culture medium: 3-5 g / L yeast extract, 3-5 g / L malt extract, 8-12 g / L glucose.

[0055] Fermentation medium: 18-22 g / L glucose, 8-12 g / L yeast extract, 0.4-0.6 g / L calcium chloride, 1-3 g / L potassium dihydrogen phosphate, 48-52 g / L magnesium sulfate.

[0056] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 0.2 g / L agar powder.

[0057] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0058] Alkaline lysis buffer I (80 mL): 0.72 g glucose, 0.24 g Tris, 0.232 g EDTA.

[0059] Alkaline lysis buffer II (40 mL): 0.32 g sodium hydroxide, 0.4 g SDS.

[0060] Alkaline lysis buffer III (80 mL): 23.52 g potassium acetate, 9.2 mL glacial acetic acid.

[0061] 1M mannitol solution (80mL): 14.5728g mannitol.

[0062] 0.2M maleate buffer (60mL): 0.464g maleic acid, 1.28g disodium maleate, dissolved in pure water, and adjusted to pH 5.5 with sodium hydroxide.

[0063] STC buffer (50 mL): 9.1 g sorbitol, 0.060 g Tris, 0.1388 g calcium chloride.

[0064] 1M CaCl2 solution (50mL): 7.35g calcium chloride.

[0065] 1M Tris solution (10mL): 1.2114g Tris.

[0066] PEG solution (40mL): 10g PEG 4000, 0.4mL Tris (1M, pH 8.0), 1mL calcium chloride (1M).

[0067] MM Buffer (25mL): Pipette 12.5mL of prepared sterile 1M mannitol solution, 6.25mL of 0.2M sterile maleate buffer, and 12.5mL of sterile water, mix thoroughly before use.

[0068] MMC Buffer (25 mL): Pipette 12.5 mL of prepared sterile 1 M mannitol solution, 6.25 mL of maleate buffer (0.2 M), 0.625 mL of CaCl2 solution (1 M), and 5.625 mL of sterile water. Mix thoroughly before use.

[0069] Cellulase solution (10mL): Weigh 400mg cellulase and 1mg chitinase, add MM buffer, dissolve and bring to a final volume of 10mL, centrifuge at 3000×g for 30min at 4℃, filter under low temperature and aliquot into 2mL sterile centrifuge tubes.

[0070] Stock solution A (80mL): Weigh 2.3g KH2PO4, 7.2g Na2HPO4, 0.928g Na2SO3, and 1.6g ammonium tartrate. Dissolve in pure water and bring to a final volume of 80mL. Add 400μL of chloroform.

[0071] Original solution B (80mL): 2.3mg Vitamin B1.

[0072] Stock solution C (50mL): Weigh 1.25g MgSO4·7H2O, dissolve it in pure water and make up to 50mL, and add 200μL chloroform.

[0073] 100mM sodium tartrate solution (pH 4.0, 3L): Weigh 90g of L-tartaric acid, dissolve it in pure water, adjust the pH to 4.0 with sodium hydroxide, and then bring the volume to 3L.

[0074] 15mM ABTS (100mL): Weigh 0.822g ABTS, dissolve it in 10mL of anhydrous ethanol using a graduated cylinder, add water and bring the volume to 100mL.

[0075] Regenerated solid culture medium (1L): Weigh 25mL of the prepared stock solution A, 1mL of stock solution B, 10mL of stock solution C, 172g of sucrose, 2g of soluble starch, 2g of asparagine, and 10g of agar powder, then bring the volume to 1L with pure water. Separately, weigh 5g of glucose and dissolve it in 10mL of pure water, then autoclave at 115℃ for 30min. Mix the two substances thoroughly in a laminar flow hood and pour the mixture into plates.

[0076] Basic solid culture medium (1L): Weigh 25mL of the prepared stock solution A, 1mL of stock solution B, 10mL of stock solution C, 2g of asparagine, and 10g of agar powder, and then dilute to 1L with pure water.

[0077] Example 1: Construction of the laccase recombinant expression vector pYSK7-lcc5

[0078] The specific steps are as follows (the construction process can be referenced). Figure 1 ):

[0079] 1. Primers containing homologous arms, lcc5-F / lcc5-R, were designed. Using the C. cinerea FA2222 genome as a template, lcc5 with homologous arms was amplified by PCR (amino acid sequence shown in SEQ ID NO.1). C. cinerea FA2222 has been published in Rühl M, Lange K, Kües U (2018) Laccase production and pellet morphology of Coprinopsis cinerea transformants in liquid shake flask cultures. Appl Microbiol Biotechnol 102:7849-7863 doi:10.1007 / s00253-018-9227-7.

[0080] Primer sequences are shown in Table 1:

[0081] Table 1 Primer sequences

[0082]

[0083]

[0084] The underlined parts are homologous arm sequences.

[0085] The PCR system is shown in Table 2:

[0086] Table 2 PCR reaction system

[0087] 5xPhusion HF Reaction Buffer 10.0 μL dNTP 4.0 μL Genome of C. cinerea FA2222 0.5 μL lcc5-F 0.5 μL lcc5-R 0.5 μL Primerstar DNA 0.5 μL <![CDATA[ddH2O]]> Up to 50 μL

[0088] PCR conditions: 94℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 1 min 30 s / 5 min 55 s, 30 cycles, PCR products were recovered by gel extraction.

[0089] 2. The vector pYSK7 was treated with restriction endonucleases BamHI and HpaI to obtain a linearized vector fragment. The vector pYSK7 has been published in Kilaru S, Hoegger PJ, Majcherczyk A, et al. (2006) Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basic cycetous promoters. Appl Microbiol Biotechnol 71:200-210 doi:10.1007 / s00253-005-0128-1.

[0090] The enzyme digestion system is shown in Table 3:

[0091] Table 3 BamH I and Hpa I enzyme digestion systems

[0092] <![CDATA[QuickCut TM English III]]> 0.2 μL 10×QuickCut Green Buffer 1.0 μL pYSK7 2.0 μL <![CDATA[ddH2O]]> 6.8 μL

[0093] Enzyme digestion conditions: 37℃ for 30 min.

[0094] 3. The two fragments amplified and recovered in steps 1 and 2, containing homologous arms, are then processed according to... The H Cloning Kit (purchased from Novizan Biotechnology Co., Ltd.) requires mixing the insert fragment and vector at a molar ratio of 2:1. After ligation using the following ligation system, the ligation product is transformed into Escherichia coli JM109 competent cells, plated on LB agar plates, and incubated overnight at 37°C.

[0095] The connection system is shown in Table 4:

[0096] Table 4 HD Cloning Kit Reagent Connection System

[0097]

[0098]

[0099] Connection conditions: 50℃, 25min.

[0100] 4. Pick a single colony from an overnight LB agar plate and inoculate it into LB liquid medium. Incubate at 37°C and 200 rpm for 8–10 hours, then extract plasmids for QuickCut. TM BamH I and QuickCut TM HpaI enzyme digestion verification; successful enzyme digestion verification yields the recombinant vector pYSK7-lcc5.

[0101] The enzyme digestion system is shown in Table 5:

[0102] Table 5 QuickCut TM BamH I and QuickCut TM HpaI enzyme digestion system

[0103] <![CDATA[QuickCut TM English III]]> 0.2 μL 10×QuickCut Green Buffer 1.0 μL pYSK7-lcc5 2.0 μL <![CDATA[ddH2O]]> 6.8 μL

[0104] Enzyme digestion conditions: 37℃ for 20 min.

[0105] Example 2: Construction and fermentation of recombinant *Coprinus grayis* strain expressing laccase

[0106] The specific steps are as follows:

[0107] 1. Preparation of C. cinerea protoplasts

[0108] (1) In a clean bench, use an inoculation loop to inoculate C. cinerea blocks onto a seed culture plate containing tryptophan and place it at 37°C for about 8-10 days. Continue culturing for another day when the mycelium just touches the edge.

[0109] (2) Add 8 mL of sterile water to the plate and gently scrape the mycelium on the surface with a spatula.

[0110] (3) Collect the suspension using a spore filter, trapping the mycelium on the glass fibers in the filter. Filter out the spore suspension and dispense it into 10 mL Eppendorf tubes. Centrifuge at 2600 × g, 4 °C for 5 min and discard the supernatant.

[0111] (4) Resuspend the precipitate with 8 mL of freshly prepared MM Buffer, centrifuge at 2600×g and 4℃ for 10 min, and discard the supernatant.

[0112] (5) Add 1.5 mL of lysozyme solution to an Eppendorf tube containing spore suspension, resuspend, and incubate at 37°C for about 3-5 hours. After 3 hours of incubation, take samples every 25 minutes to prepare slides and observe the degree of protoplast incubation. The degree of incubation observed under a microscope should be about 40-70%.

[0113] (6) Add 8 mL of MMC Buffer to terminate the enzymatic hydrolysis reaction, centrifuge at 640×g and 4℃ at low speed for 15 min and discard the supernatant.

[0114] (7) Add 300-500 μL of MMC Buffer to dilute the protoplast concentration to approximately 2 × 10⁻⁶. 8 per mL.

[0115] 2. Extraction of plasmids to be transformed using SDS alkaline lysis method.

[0116] (1) E. coli JM109 strains containing the plasmids pYSK7-lcc5 and pBD5 (containing the tryptophan marker gene for subsequent screening of positive transformants of Coprinus comatus) were inoculated into 5 mL of LB liquid medium and cultured at 37°C and 200 rpm for approximately 12 h. pBD5 has been published in [Journal Name - 2000 rpm]. B,Kües U(2016) Paradoxical performance of tryptophan synthase gene trp1 + in transformations of thebasidiomycete Coprinopsis cinerea.Appl Microbiol Biotechnol 100:8789-8807doi:10.1007 / s00253-016-7693-3.

[0117] (2) Take an appropriate amount of fermentation broth and centrifuge at 2000×g for 10 min at 4℃ to collect the cells and discard the supernatant culture medium.

[0118] (3) Take 250 μL of alkaline lysis buffer I to resuspend the bacterial cells, and transfer the suspension to a 2 mL Ep centrifuge tube.

[0119] (4) Add 450 μL of alkaline lysis buffer II, invert and mix well, then place on ice.

[0120] (5) Add 350 μL of alkaline lysis buffer III, invert and mix well, then place on ice for about 4 min.

[0121] (6) After centrifuging at 12000×g for 12 min at 4℃, transfer 650 μL of supernatant to a centrifuge tube, add 700 μL of a mixture of phenol and chloroform (the volume ratio of phenol to chloroform is 16:9), centrifuge at 12000×g for 2 min at 4℃, transfer the uppermost aqueous phase to an Ep centrifuge tube, add 700 μL of isopropanol, mix well, and let stand for 5 min.

[0122] (7) Centrifuge at 12000×g at 4℃ for 5 min and collect the precipitate. Add 1 mL of 70% ethanol and centrifuge at 12000×g for 1 min to remove the ethanol. Open the Eppendorf tube and let it stand for about 2 min to allow the ethanol to evaporate completely.

[0123] (8) Redissolve the nucleic acid in 30 μL of sterile water, detect it by electrophoresis, and store it at -20℃ until it is digested by protoplasts before use.

[0124] 3. Genetic transformation of C. cinerea.

[0125] (1) Add 1 μg plasmid DNA, 12.5 μL PEG solution and 50 μL protoplasts to a pre-cooled centrifuge tube.

[0126] (2) After standing on ice for 20 min, add 500 μL of PEG solution, mix well and incubate at room temperature for 4 min.

[0127] (3) Add 1 mL of STC buffer, mix well, take 380 μL of solution, spread evenly on the regeneration medium solid plate, and perform initial screening.

[0128] (4) Incubate the plates of regenerated medium at 37°C for about 3 days until transformants appear.

[0129] (5) Pick out the transformants and transfer them to a solid plate of basic culture medium for re-screening.

[0130] (6) The screened transformants were inoculated onto seed culture plates, cultured at 37°C and then stored at 4°C.

[0131] 4. Screening and validation of positive transformants

[0132] A portion of the mycelium growing on the seed culture plate was scraped into a 2 mL Eppendorf tube, and the genome of the transformants was extracted. An appropriate amount of the transformant genome was used as a template for PCR using primers YF / YR. The primers and PCR system are shown in Tables 6 and 7, respectively. Nucleic acid electrophoresis was used to detect the PCR amplification results. The positive transformants were identified as recombinant laccase-expressing *Coprinus cinerea* FA2222 / pYSK7-lcc5 (Cclcc5). After verification, a total of 8 positive transformants were obtained, labeled Cclcc5-13, Cclcc5-16, Cclcc5-29, Cclcc5-34, Cclcc5-48, Cclcc5-63, Cclcc5-76, and Cclcc5-77, respectively.

[0133] Primer sequences are shown in Table 6:

[0134] Table 6 Primer sequences

[0135]

[0136]

[0137] The PCR system is shown in Table 7:

[0138] Table 7 PCR Reaction System

[0139] <![CDATA[ddH2O]]> 29 μL 5×PS buffer 10 μL dNTP mix 4 μL YF 1 μL YR 1 μL Template 1 μL PrimeStar 1 μL

[0140] PCR conditions: 94℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 1 min 30 s / 5 min 55 s, 30 cycles.

[0141] 4. Shake-flask fermentation of recombinant *Coprinus grayis* strain expressing laccase

[0142] The eight positive transformants obtained above, Cclcc5-13, Cclcc5-16, Cclcc5-29, Cclcc5-34, Cclcc5-48, Cclcc5-63, Cclcc5-76 and Cclcc5-77, were first inoculated into seed culture plates and cultured statically at 35-38℃ for 6-10 days to obtain seed mycelial blocks.

[0143] Then, the seed mycelium blocks were inoculated into seed liquid culture medium and cultured at 35-38℃ and 100-140 rpm for 3-5 days to obtain seed mycelium.

[0144] The seed mycelium was then homogenized at 3000–4000 rpm for 10–20 seconds to obtain seed liquid.

[0145] Finally, the seed culture was transferred to the fermentation medium at an inoculation rate of 5% (v / v) and cultured at 35–38°C and 100–140 rpm for 6 days.

[0146] During this period, laccase activity was measured in the supernatant from shake-flask fermentation at different fermentation time points. For example... Figure 2 As shown, all transformed positive strains overexpressed laccase lcc5, but the laccase activity varied considerably among different strains. The extracellular laccase activities of transformants Cclcc5-13, Cclcc5-16, Cclcc5-29, Cclcc5-34, Cclcc5-48, Cclcc5-63, Cclcc5-76, and Cclcc5-77 were 25, 9.01, 8.72, 10.95, 7.9, 8.6, 8.3, and 9.7 U / mL, respectively. Among them, transformant Cclcc5-13 exhibited the highest extracellular laccase activity after shake-flask fermentation, at 25 U / mL.

[0147] Moreover, batch experiments revealed that the enzyme activity of the positive recombinant transformants of the present invention is stable.

[0148] Example 3: Discovery of key chitinase genes in the C. cinerea FA2222 genome

[0149] The specific steps are as follows:

[0150] 1. Collection of mycelium from C. cinerea FA2222

[0151] (1) Pick out a block of C. cinerea FA2222 mycelium from the glycerol tube of the preserved strain using an inoculation loop, inoculate it onto a seed culture plate, and culture it at 37°C for about 6 to 10 days.

[0152] (2) Use an inoculation loop to divide the bacteria on the plate into mycelial blocks with a diameter of about 1 cm. Select 4 blocks and place them in seed liquid culture medium. Incubate them in a shaker at 37℃ and 120 rpm for 4 days.

[0153] (3) Homogenize the mycelium at 3500 rpm for 15 s using a homogenizer, then inoculate it into the new seed liquid medium at an inoculation rate of 5% (v / v) and incubate it in a shaker at 37℃ and 120 rpm. Take samples every 12 h, centrifuge at 12000×g for 25 min at 4℃ to collect the mycelium, and store it at -80℃ for later use.

[0154] 2. Total RNA extraction from C. cinerea FA2222

[0155] (1) After thawing the mycelium at different fermentation times frozen in a -80℃ freezer, add 1mL of RNAisoPlus (purchased from Baori Biotechnology (Beijing) Co., Ltd.).

[0156] (2) After standing on ice for 6 minutes, seal the RNase-free centrifuge tube tightly and place it in a fully automatic sample cryogenic grinder for grinding (12000×g, 5 minutes). Then transfer the supernatant to a new RNase-free centrifuge tube.

[0157] (3) Add 300 μL of chloroform, vortex and mix well, and let stand on ice for 5 minutes after it is fully mixed.

[0158] (4) After centrifuging at 4℃ and 12000×g for 15 min, transfer the supernatant to a new RNase-free centrifuge tube.

[0159] (5) Add 800 μL of isopropanol again and place on ice for 10 min.

[0160] (6) After centrifuging at 4℃ and 12000×g for 10 min, discard the supernatant and add 1 mL of 75% ethanol.

[0161] (7) After centrifuging at 12000×g for 5 min at 4℃, discard the supernatant, let stand for about 2 min, dissolve the precipitate with 30μL DEPC water, and store at -80℃ for later use.

[0162] 3. Removal of genomic DNA from C. cinerea FA2222

[0163] Following the instructions for use of the Evo M-MLV reverse transcription kit (purchased from Acrel Biotech Co., Ltd.), the reaction conditions and reaction system were set and adjusted to remove genomic DNA, with the total RNA sample concentration uniformly adjusted to 800 ng. The experimental reaction system is shown in Table 8.

[0164] Table 8 Reaction system for removing genomic DNA

[0165] gDNA Clean Reagent 2 μL 5×gDNA Clean Buffer 4 μL RNase free water up to 20 μL Total RNA 800 ng

[0166] 4. cDNA generation of C. cinerea FA2222

[0167] The reverse transcription reaction system is shown in Table 9. The reaction conditions were 37℃ for 15 min and 85℃ for 5 s.

[0168] Table 9. Reaction system for RNA reverse transcription

[0169]

[0170]

[0171] 5. qRT-PCR reaction assay and transcriptional level analysis

[0172] First, the gene sequence of chitinase in C. cinerea FA2222 was found using NCBI. Then, the sequence was aligned using DNAMAN and the intron sequence was located. Next, a gene sequence segment spanning two introns was selected, and primers were designed using the cDNA obtained in step 4 above as a template. The primer sequences are shown in Table 10 below.

[0173] Table 10 Primer sequences for qRT-PCR

[0174] qRT-ChiE1-F AGAGGGAGAACGGCGAAGGATGCAA qRT-ChiE1-R CAGGTTGGCTTGGTGGTTCGCAGT qRT-ChiE2-F TCTTCACCGTTCGCTTCTGTACTGG qRT-ChiE2-R TAACTAGAAAGAGCGCTGGCTTCCG qRT-ChiIII-F TTGGGTGGTCGATCGGATATGAGGC qRT-ChiIII-R GACTTGGTTGAGGACCTCTCCCAG qRT-ChiB1-F ACCGGCAAAGCCAACTTCGCCTACT qRT-ChiB1-R ATAAAGCTGCTTCAGGCAGCCGTAG qRT-ChiEn1-F CCTGGGACAAGTATAATGCCATGACGTT qRT-ChiEn1-R AGATCCAGTCCATCCACCAAGCGAA qRT-ChiEn2-F AGATCCCTTGGGAGAAGTACACCGA qRT-ChiEn2-R AACGGCCGGAGAGAAGTATTGGGAA qRT-ChiEn3-F AACGGCCGGAGAGAAGTATTGGGAA qRT-ChiEn3-R AGTCCATCCACCAATCGAAATCAAGGC qRT-ChiEn4-F TGGTACACTGGCTGGCATTCGAGA qRT-ChiEn4-R CAACGTCGAGGAGGGTGTTCACAAA

[0175] according to Follow the instructions for the Green Premix Pro Taq HS qPCR kit (purchased from Acery Biotech Co., Ltd.) to set up the qRT-PCR reaction system and conditions. The experiment used a quantitative PCR instrument. Detection was performed using a 96-real-time PCR system, and transcription levels were determined based on 2... -ΔΔCT Formula calculation. The reaction conditions for qRT-PCR are: 95℃, 30s (x35) and 95℃, 5s (x35); 60℃, 30s, as shown in Table 11. Among them, 2 -ΔΔCT The formula calculation method has been reported in Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 -ΔΔCT method. Methods 25(4):402-408doi:10.1006 / meth.2001.1262.

[0176] Table 11 qRT-PCR reaction system

[0177]

[0178]

[0179] The results of the relative transcription levels of each gene are as follows: Figure 3 As shown.

[0180] like Figure 3 As shown, the relative transcription levels of ChiB1, ChiE1, ChiE2, ChiEn1, ChiEn2, ChiEn3, ChiEn4, and ChiIII at 24h were 0.977, 0.143, 2.280, 7.441, 0.310, 0.264, 0.088, and 0.223, respectively.

[0181] like Figure 3 As shown, the relative transcription levels of ChiB1, ChiE1, ChiE2, ChiEn1, ChiEn2, ChiEn3, ChiEn4, and ChiIII at 48h were 0.104, 0.107, 0.802, 4.564, 0.211, 0.122, 0.014, and 0.198, respectively.

[0182] like Figure 3As shown, the relative transcription levels of ChiB1, ChiE1, ChiE2, ChiEn1, ChiEn2, ChiEn3, ChiEn4, and ChiIII at 72h were 0, 0.008, 1.946, 46.135, 0.033, 0.235, 0.131, and 0.015, respectively.

[0183] It is evident that during fermentation, the transcription levels of chitinase genes ChiEn1 and ChiE2 in the C. cinerea FA2222 genome were significantly higher than those of other chitinase genes, ranging from tens to hundreds of times higher. This suggests that chitinases ChiEn1 (amino acid sequence shown in SEQ ID NO.2) and ChiE2 (amino acid sequence shown in SEQ ID NO.3) may play a major role in the C. cinerea cell wall remodeling process.

[0184] Example 4: Construction of a chitinase gene silencing vector

[0185] The specific steps are as follows:

[0186] 1. Primer pairs antiChiEn1-F / antiChiEn1-R and antiChiE2-F / antiChiE2-R were designed respectively. Using the cDNA of C. cinerea FA2222 in Example 3 as a template, the RNA interference fragments of ChiEn1 and ChiE2 were amplified by PCR respectively.

[0187] The RNA interference fragments antiChiEn1 and antiChiE2 of ChiEn1 are shown in Table 12.

[0188] Table 12 Primer sequences

[0189]

[0190]

[0191] Primer sequences are shown in Table 13:

[0192] Table 13 Primer sequences

[0193]

[0194] The underlined part is the homologous arm sequence.

[0195] The PCR system is shown in Table 14:

[0196] Table 14 PCR Reaction System

[0197] 5xPhusion HF Reaction Buffer 10.0μL dNTP 4.0μL cDNA of C. cinerea FA2222 0.5μL LCC5-F 0.5μL LCC5-R 0.5μL Primerstar DNA 0.5μL <![CDATA[ddH2O]]> Up to 50μL

[0198] PCR conditions: 94℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 1 min 30 s / 5 min 55 s, 30 cycles, PCR products were recovered by gel extraction.

[0199] 2. Following the method in Example 1, the vector pYSK7 was treated with restriction endonucleases BamHI and HpaI to obtain a linearized vector fragment. Then, using... The H Cloning Kit fuses linearized pYSK7 with ChiEn1 and ChiE2 interference fragments. After plasmid extraction and enzyme digestion verification, the positive transformants are the silencing vectors pYSK-antiChiEn1 and pYSK-antiChiE2 for ChiEn1 and ChiE2, respectively.

[0200] The connection system is shown in Table 15:

[0201] Table 15 HD Cloning Kit Reagent Connection System

[0202] 5X In-Fusion HD Enzyme Premix 2.0μL pYSK7 linearized fragments 1.2μL ChiEn1 or ChiE2 3.5μL <![CDATA[ddH2O]]> 3.3μL

[0203] Connection conditions: 50℃, 25min.

[0204] Example 5: Construction and fermentation of recombinant *Coprinus gracilistylus* strain with chitinase gene silencing.

[0205] The specific steps are as follows:

[0206] 1. Construction of recombinant *Coprinus grayis* strain with chitinase gene silencing

[0207] Following the method in Example 2, the chitinase silencing vectors pYSK-antiChiEn1 and pYSK-antiChiE2 constructed in Example 4 were co-transformed with vector pCRII (containing the hygromycin resistance gene, used for subsequent resistance screening of Coprinus pulmonarius recombinant transformants) into the Coprinus pulmonarius recombinant strain Cclcc5-13 obtained in Example 2, thereby obtaining Coprinus pulmonarius recombinant strains Cclcc5-13 / pYSK-antiChiEn1 (named Cclcc5-antiChiEn1) and Cclcc5-13 / pYSK-antiChiE2 (named Cclcc5-antiChiE2) with the chitinase genes ChiEn1 and ChiE2 silenced, respectively.

[0208] After screening and verification, 4 and 5 positive transformants were obtained from the recombinant strains of Coprinus macrocephala Cclcc5-antiChiEn1 and Cclcc5-antiChiE2, respectively, and were labeled as Cclcc5-antiChiEn1-1, Cclcc5-antiChiEn1-2, Cclcc5-antiChiEn1-3, Cclcc5-antiChiEn1-4, Cclcc5-antiChiE2-1, Cclcc5-antiChiE2-2, Cclcc5-antiChiE2-3, Cclcc5-antiChiE2-4 and Cclcc5-antiChiE2-5.

[0209] Among them, the pCRII vector has been published in B,Peng C,Fang Z,Kamran A,Yulvizar C,KüesU(2020)Selection markers for transformation of the sequenced referencemonokaryon Okayama 7 / #130and homokaryon AmutBmut of Coprinopsiscinerea.Fungal Biol and Biotech 7(1):1-18doi:10.1186 / s40694-020-00105-0.

[0210] The PCR primer pairs used to screen and verify the silence of chitinase genes ChiEn1 and ChiE2 in recombinant Coprinus grayis were antiChiEn1-F / antiChiEn1-R and antiChiE2-F / antiChiE2-R, respectively. The specific sequences are shown in Table 13.

[0211] 2. Fermentation of recombinant *Coprinus gravidarum* strain with chitinase gene silencing

[0212] Following the method in Example 2, the recombinant *Coprinus pulcherrima* strains Cclcc5-antiChiEn1-1, Cclcc5-antiChiEn1-2, Cclcc5-antiChiEn1-3, Cclcc5-antiChiEn1-4, Cclcc5-antiChiE2-1, Cclcc5-antiChiE2-2, Cclcc5-antiChiE2-3, Cclcc5-antiChiE2-4, and Cclcc5-antiChiE2-5 obtained in step 1 above, as well as the recombinant *Coprinus pulcherrima* strain Cclcc5-13 (control strain, without chitinase gene silencing) from Example 2, were subjected to shake-flask fermentation. The results are as follows: Figure 4 As shown.

[0213] The results showed that the extracellular laccase activities of Cclcc5-antiChiEn1-1, Cclcc5-antiChiEn1-2, Cclcc5-antiChiEn1-3, and Cclcc5-antiChiEn1-4 were 10.1, 15, 11.9, and 13.2 U / mL, respectively.

[0214] The extracellular laccase activities of Cclcc5-antiChiE2-1, Cclcc5-antiChiE2-2, Cclcc5-antiChiE2-3, Cclcc5-antiChiE2-4, and Cclcc5-antiChiE2-5 were 13.3, 11.4, 11.4, 8.5, and 22 U / mL, respectively.

[0215] The extracellular laccase activity of recombinant Coprinus grayis Cclcc5-13 was 25 U / mL.

[0216] It is evident that among the recombinant strains of Coprinus grayis with the silenced chitinase gene ChiEn1, the recombinant strain Cclcc5-antiChiEn1-2 exhibited the highest extracellular laccase activity, at 15 U / mL.

[0217] Among the recombinant strain Cclcc5-antiChiE2 containing the silenced chitinase gene ChiE2, the recombinant strain Cclcc5-antiChiE2-5 exhibited the highest extracellular laccase activity, at 22 U / mL.

[0218] The extracellular laccase activity of the recombinant strain Cclcc5-antiChiE2-5 was higher than that of Cclcc5-antiChiEn1-2, but lower than that of the control strain Cclcc5-13 (25 U / mL).

[0219] 3. To further verify the effect of chitinase gene silencing on the homologous recombinant expression of laccase Lcc5 in Coprinus pulmonarius, and considering that the recombinant strain Cclcc5-antiChiE2-5 of Coprinus pulmonarius with chitinase gene silencing showed the highest extracellular laccase activity, subsequent analysis and research will be conducted using the recombinant strain Cclcc5-antiChiE2-5 of Coprinus pulmonarius.

[0220] Cause analysis:

[0221] To investigate why the extracellular laccase activity of the recombinant strain Cclcc5-antiChiE2-5 was lower than that of the control strain Cclcc5-13, force analysis was performed on the recombinant strain Cclcc5-antiChiE2-5 and the control strain Cclcc5-13 using atomic force microscopy.

[0222] The results showed that the pressure tolerance range of the control strain Cclcc5-13 was 0.8-1.2 GPa, while that of the recombinant strain Cclcc5-antiChiE2-5 was 2-3 GPa, significantly higher than that of the control strain Cclcc5-13. Therefore, it is reasonable to infer that the shaker speed at this point is unsuitable for the fermentation of the recombinant strain Cclcc5-antiChiE2-5, and is more suitable for the fermentation of the control strain Cclcc5-13.

[0223] Therefore, the recombinant strain Cclcc5-antiChiE2-5 and the control strain Cclcc5-13 were subsequently scaled up and fermented in a 3-L tank for verification, and the effect of stirring speed on the fermentation of recombinant strain Cclcc5-antiChiE2-5 and control strain Cclcc5-13 on laccase production was investigated.

[0224] Example 6: 3-L tank fermentation and fermentation optimization of recombinant *Coprinus comatus* strain.

[0225] The specific steps are as follows:

[0226] 1. Following the seed culture preparation method for shake-flask fermentation in Example 2, 3-L fermentation seed cultures of recombinant *Coprinus comatus* Cclcc5-13 and Cclcc5-antiChiE2-5 were prepared respectively:

[0227] The above-mentioned Cclcc5-13 and Cclcc5-antiChiE2-5 were first inoculated into seed culture plates and then statically cultured at 35-38℃ for 6-10 days to obtain seed mycelial blocks.

[0228] Then, the seed mycelium blocks were inoculated into seed liquid culture medium and cultured at 35-38℃ and 100-140 rpm for 3-5 days to obtain seed mycelium.

[0229] The seed mycelium was then homogenized at 3000–4000 rpm for 10–20 s to obtain seed solutions.

[0230] 2. Transfer the seed culture prepared in step 1 above to a 3-L fermenter containing 1L of fermentation medium at an inoculation rate of 5% (v / v).

[0231] The fermentation medium consisted of 20 g / L glucose, 10 g / L yeast extract, 0.5 g / L calcium chloride, 2 g / L potassium dihydrogen phosphate, and 50 g / L magnesium sulfate.

[0232] Additionally, Cu was added to a final concentration of 0.1. 2+ The strain was cultured and fermented at 37℃, 100–300 rpm, and an initial pH of 5.8, with the dissolved oxygen (DO) in the fermentation broth maintained at approximately 40%. During the fermentation process in a 3-L tank, samples were taken at specific times, centrifuged at 12000 x g at 4℃ for 10 min, and the laccase activity in the fermentation supernatant was measured.

[0233] Extracellular laccase fermentation curves of recombinant *Coprinus comatus* strains Cclcc5-13 and Cclcc5-antiChiE2-5 in a 3-L tank are shown below. Figure 5 As shown in Table 16, the corresponding enzyme activities are shown in the table.

[0234] Table 16: Extracellular laccase activity of recombinant *Coprinus gracilistylus* under different rotation speeds

[0235]

[0236] The results showed that the extracellular laccase activities of recombinant *Coprinus gracilis* strains Cclcc5-13 and Cclcc5-antiChiE2-5 reached their maximum at stirring speeds of 150 and 250 rpm, respectively.

[0237] Among them, the extracellular laccase activity of the recombinant strain Cclcc5-13 of Coprinus grayis reached its maximum of 25.5 U / mL when fermented at 150 rpm for 132 h, with a corresponding laccase production rate of 0.19 U / mL·h.

[0238] The extracellular laccase activity of the recombinant strain Cclcc5-antiChiE2-5, grown at 250 rpm for 108 h, reached a maximum of 38.2 U / mL, with a corresponding laccase production rate of 0.35 U / mL·h.

[0239] After chitinase is silenced, the mycelium is more tolerant to the external tangential force of the stirring shaft, which can increase mass transfer, gas transfer and heat transfer under high speed conditions. Therefore, under the fermentation conditions of the upper tank, the extracellular laccase activity and laccase productivity of the recombinant strain Cclcc5-antiChiE2-5 of Coprinus grayis are 1.5 and 1.8 times that of the control strain Cclcc5-13, respectively.

[0240] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for producing laccase, characterized in that, The method involves preparing laccase using recombinant Coprinus fusiforme fermentation; the recombinant Coprinus fusiforme uses Coprinus fusiforme FA2222 as the host cell to overexpress the laccase with the amino acid sequence shown in SEQ ID NO.1, while simultaneously preventing the expression of chitinase in its genome; the non-expression is achieved through gene deletion, knockout, or silencing; the amino acid sequence of the chitinase is shown in SEQ ID NO.3; The method involves first inoculating the recombinant *Coprinus comatus* onto a seed culture plate and incubating it statically at 35-38°C for 6-10 days to obtain seed mycelial blocks. Then, the seed mycelial blocks are inoculated into seed liquid culture medium and cultured at 35-38°C and 100-140 rpm for 3-5 days to obtain seed mycelium. The seed mycelium is then homogenized at 3000-4000 rpm for 10-20 seconds to obtain seed liquid. Finally, the seed liquid is inoculated into fermentation medium and cultured at 35-38°C and 200-250 rpm for 4-6 days.

2. The method according to claim 1, characterized in that, The fermentation medium comprises 18-22 g / L glucose, 8-12 g / L yeast extract, 0.4-0.6 g / L calcium chloride, 1-3 g / L potassium dihydrogen phosphate, and 48-52 g / L magnesium sulfate.

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