Method for efficiently expressing laccase by regulating endoplasmic reticulum stress reaction of coprinus cinereus

Abstract

The invention discloses a method for efficiently expressing laccase by regulating endoplasmic reticulum stress reaction of coprinus cinereus, and belongs to the field of gene engineering and fermentation engineering. According to the invention, by regulating endoplasmic reticulum stress reaction action genes der1, Anhac1 and Cchac1 of filamentous fungi coprinus cinereus, recombinant extracellular expression of laccase Lcc5 is finally improved and regulated. Wherein the extracellular laccase activities of the recombinant bacteria can be respectively improved by 18%, 70% and 26% through interference of der1, overexpression of Anhac1 and overexpression of Cchac1, and the obtained extracellular laccase activities are respectively 31.4 U / mL, 45.1 U / mL and 33.6 U / mL, which is the highest level of coprinus cinereus homologous recombination expression laccase Lcc5 reported at present. The invention firstly indicates that the regulation and control of the coprinus cinereus endoplasmic reticulum stress reaction can promote the efficient expression of laccase in coprinus cinereus, and the invention further promotes the development of a coprinus cinereus expression system.

Description

Technical Field

[0001] This invention relates to a method for efficiently expressing laccase by regulating the endoplasmic reticulum stress response of Coprinus gracilistylus, 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 widely found in plants, fungi, bacteria, and insects. It can oxidize phenols, carboxylic acids, aromatic amines, and their derivatives. Due to its extremely high catalytic efficiency and ability to catalyze various in vivo oxidation reactions, with water as the only byproduct, laccase is considered a green and environmentally friendly enzyme catalyst with great potential. Therefore, laccase is widely used in various industries, from environmental applications to cosmetics, including food processing and textiles (for dye biodegradation and synthesis).

[0003] Laccase genes are widely distributed in nature, existing in the genomes of plants, insects, bacteria, and fungi. To date, over 80% of the laccases discovered are derived from fungi. Among these, laccases derived from basidiomycetes are the most widely used due to their broad substrate range, high specific activity, and large reduction potential, accounting for over 90% of the current laccase market share, with substantial market demand. Therefore, the efficient preparation of basidiomycete-derived laccases holds immense application potential.

[0004] Laccases derived from basidiomycetes are often heterologously recombinantly expressed using other hosts such as Aspergillus or yeast to improve their production levels. However, compared to natural laccases, due to their inappropriate post-translational modifications, such as incomplete folding and excessive glycosylation, the enzymatic properties of recombinant laccases obtained using these methods may change in terms of protein molecular weight, redox potential, pH stability, and thermal stability. This significantly reduces the practical application value of the obtained recombinant laccases.

[0005] Grey-capped ghost umbrella ( Coprinopsis cinerea *Coprinus gracilis* is the model strain for basidiomycete development. It possesses advantages such as a clear genetic background, ease of operation, and a short growth cycle. Therefore, *Coprinus gracilis* has the potential to be an excellent host for laccase expression. However, compared to *Aspergillus* and yeast expression systems, the *Coprinus gracilis* expression system is relatively new, and currently lacks effective expression strategies to improve recombinant protein production. This makes it difficult to efficiently express basidiomycete laccases based on *Coprinus gracilis* in the early stages, hindering its widespread industrial application. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for efficiently expressing laccase by regulating the endoplasmic reticulum stress response, aiming to improve the recombinant expression and production level of laccase in *Coprinus comatus*. This invention utilizes genes related to the endoplasmic reticulum stress response process.der 1, Anhac 1 and Cchac 1 is an acting gene. By using RNA interference (RNAi) technology to silence der 1, or by using genome overexpression technology to overexpress Anhac 1, Cchac 1, the laccase activity in the fermentation broth of the recombinant Coprinus cinereus obtained after 7-day fermentation in a flask is 31.4 U / mL, 45.1 U / mL and 33.6 U / mL, respectively, which is increased by 18%, 70% and 26% compared with the control.

[0007] The first technical solution provided by the application is the application of an endoplasmic reticulum stress response-related gene in regulating the laccase expression ability of Coprinus cinereus, wherein the endoplasmic reticulum stress response-related gene comprises genes der 1, Anhac 1, Cchac 1.

[0008] In some embodiments, the application is any one of the following: (1) by interfering with or inhibiting the expression of Coprinus cinereus gene der 1, or overexpressing gene Anhac 1 or Cchac 1 in Coprinus cinereus, to improve the laccase expression ability of Coprinus cinereus; (2) by overexpressing Coprinus cinereus gene der 1, or interfering with or inhibiting the expression of Coprinus cinereus gene Cchac 1, to reduce the expression of laccase of Coprinus cinereus.

[0009] In some embodiments, the nucleotide sequences of the genes der 1, Anhac 1, Cchac 1 are shown in SEQ ID NO. 2-4, respectively.

[0010] In some embodiments, the Coprinus cinereus is Coprinus cinereus FA2222 strain or Coprinus cinereus Cclcc 5-13 strain.

[0011] In some embodiments, the Coprinus cinereus Cclcc 5-13 strain is Coprinus cinereus FA2222 overexpressing laccase, wherein the laccase is laccase Lcc5 gene derived from Coprinus cinereus FA2222, and the nucleotide sequence of the laccase Lcc5 gene is shown in SEQ ID NO. 1.

[0012] The second technical solution provided by the application is a method for efficiently expressing laccase of Coprinus cinereus, wherein the method comprises the following steps: overexpressing Coprinus cinereus gene der1interfering expression, or overexpressing genes in Coprinus cinereus Anhac 1or Cchac 1.

[0013] In some embodiments, the gene der 1, Anhac 1, Cchac The nucleotide sequences of the genes

[0014] In some embodiments, the gene der 1and Cchac 1is derived from the genome of Coprinus cinereus FA2222, Anhac 1is derived from the genome of Aspergillus niger (A. niger) Aspergillus niger ).

[0015] In some embodiments, the der 1interfering sequence, Cchac 1overexpression sequence and Anhac 1overexpression sequence are shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

[0016] In some embodiments, the Coprinus cinereus is Coprinus cinereus FA2222 strain or Coprinus cinereus Cclcc 5-13 strain.

[0017] In some embodiments, the Coprinus cinereus Cclcc 5-13 strain is Coprinus cinereus FA2222 overexpressing laccase, which is laccase Lcc5 gene derived from Coprinus cinereus FA2222, and the nucleotide sequence of the laccase Lcc5 gene is shown in SEQ ID NO. 1.

[0018] The third technical solution provided by the present application is a genetically engineered bacterium, which uses Coprinus cinereus as a host cell, interferes with the gene der 1of the host cell; or overexpresses the gene Anhac 1or Cchac 1in the host cell.

[0019] In some embodiments, the gene der 1, Anhac 1, Cchac 1The nucleotide sequences of the genes

[0020] In some embodiments, the host cell is Coprinus cinereus FA2222 strain or Coprinus cinereus Cclcc 5-13 strain.

[0021] In some embodiments, the Coprinus cinereusCclcc 5-13 strain is Coprinus cinereus FA2222 overexpressing laccase, which is laccase Lcc5 gene derived from Coprinus cinereus FA2222, and the nucleotide sequence of the laccase Lcc5 gene is shown in SEQ ID NO. 1.

[0022] The fourth technical solution provided by the present application is a method for producing laccase, which is fermentation of the genetically engineered bacteria of the third technical solution to obtain recombinant expression of laccase.

[0023] In some embodiments, the method is to inoculate the genetically engineered bacteria of the third technical solution into a seed culture plate for cultivation to obtain a seed mycelial block, then inoculate the seed mycelial block into a seed liquid medium for cultivation to obtain a seed mycelium, then homogenize the seed mycelium to obtain a seed liquid, and finally inoculate the seed liquid into a fermentation medium for cultivation.

[0024] In some embodiments, the method is to inoculate the genetically engineered bacteria of the third technical solution into a seed culture plate, and incubate at 37°C for 6-10 days to obtain a seed mycelial block. Then inoculate the seed mycelial block into a seed liquid medium, and cultivate at 37°C, 100-140 rpm for 3-5 days to obtain a seed mycelium. Then homogenize the seed mycelium at 3000-4000 rpm for 10-20 s to obtain a seed liquid, and finally inoculate the seed liquid into a fermentation medium, and cultivate at 37°C, 100-140 rpm for 4-6 days.

[0025] In some embodiments, the seed culture plate is a seed liquid medium added with 1-3% agar powder.

[0026] In some embodiments, the composition of the seed liquid medium comprises 8-10 g / L malt extract, 2-4 g / L glucose, and 2-4 g / L yeast extract.

[0027] In some embodiments, the composition of the fermentation medium comprises 8-10 g / L yeast extract, 18-20 g / L glucose, 1-2 g / L dipotassium hydrogen phosphate, 0.2-0.5 g / L calcium chloride dihydrate, and 40-50 mg / L magnesium sulfate heptahydrate.

[0028] The fifth technical solution provided by the present application is the method of the second technical solution, or the genetically engineered bacteria of the third technical solution, or the application of the method of the fourth technical solution in the preparation of laccase or products containing laccase.

[0029] The sixth technical solution provided by the application is the method of the second technical solution, or the genetically engineered bacteria of the third technical solution, or the application of the method of the fourth technical solution in the treatment and degradation of oxidized phenols, carboxylic acids and arylamine compounds and derivatives thereof.

[0030] Compared with the prior art, the application has the following beneficial effects: 1. The application interferes with or overexpresses the endoplasmic reticulum stress response related genes of Coprinus cinereus strains der1 , Anhac1 and Cchac1 , and the laccase activity in the fermentation broth of the obtained recombinant bacteria after shaking flask is 31.4 U / mL, 45.1 U / mL, and 33.6 U / mL, respectively, which is increased by 18%, 70% and 26% compared with the control.

[0031] 2. The application overexpresses the endoplasmic reticulum stress response related genes of Coprinus cinereus strains Anhac1 , and the laccase activity in the fermentation broth of the obtained recombinant bacteria after shaking flask is the highest level reported about the homologous recombinant expression of laccase Lcc5 of Coprinus cinereus.

[0032] 3. The application first points out that regulating the endoplasmic reticulum stress response of Coprinus cinereus can efficiently improve the recombinant expression level of laccase in Coprinus cinereus, which not only accelerates the industrial application process of laccase, but also promotes the development of the expression system of Coprinus cinereus. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 is a qRT-PCR result graph of the endoplasmic reticulum stress response related genes. Among them, figures A-D are the expression levels of genes der 1、 hrd 1、 pdi 1 and bip 1.

[0034] Figure 2 is a schematic diagram of vector construction of the application.

[0035] Figure 3 is a positive transformant PCR verification electrophoretogram of the recombinant strain of the application. Among them, M is a DNA Maeker, figure A is a positive transformant screening of the interference strain der1 , figure B is a positive transformant screening of the Anhac 1 overexpression strain Cchac , and figure C is a positive transformant screening of the 1 overexpression strain.

[0036] Figure 4 is a hygromycin resistance concentration screening result graph of Coprinus cinereus of the application.

[0037] Figure 5Figure A is the qRT-PCR verification result of the recombinant strain of the present application. Figure A is the gene expression level of the interference strain Anti- der 1-2; Figure B is the gene expression level of the overexpression strain OE- Anhac 1-8; and Figure C is the gene expression level of the overexpression strain OE- Cchac 1-8.

[0038] Figure 6 Figure is the fermentation curve of the laccase produced by the recombinant strain of the present application. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explanation of the present application and are not used to limit the present application.

[0040] Test method: Method for determining total laccase enzyme activity: Take 950 μL of Sodium tartrate solution (100 mmol / L; pH 4.0), 33 μL of ABTS (concentration of 15 mmol / L), and 17 μL of enzyme solution, and add the above system to a 2 m sterile EP tube with a pipette. Vortex for 10 s, place in a water bath at 30°C for 3 min, then quickly take out, ice bath on ice for 30 s, then immediately measure the value of the absorbance OD420, and count the enzyme activity according to the absorbance value (before measuring the absorbance of laccase enzyme activity, the absorbance value OD420 needs to be adjusted to zero with a solution system without adding enzyme solution. When using the above system with ABTS as the substrate, the calculation formula of enzyme activity is: E (U / L) = OD420 x 555.56.

[0041] Materials used in the examples: The strain Coprinus cinereus FA2222 (5-13) involved in the following examples is disclosed in: Yao, Dongbang, et al. "Enhanced extracellular production of laccase in C. cinerea by silencing chitinase gene." The laccase overexpression strain of Coprinus cinereus 5-13 (5-13) involved in the following examples is disclosed in: Yao, Dongbang, et al. "Enhanced extracellular production of laccase in Cclcc by silencing chitinase gene." Coprinopsis cinerea by silencing chitinase gene." Applied Microbiology and Biotechnology 108.1 (2024): 324. The expression vector pYSK involved in the following examples is disclosed in: Zhang, Z., Zhou, G., Liu, J., Fang, Z., Xiao, Y. Homologous overexpression of laccase Lcc9 in Coprinus cinereus [J]. Journal of Biology, 2022, 39(06): 47-51. The molecular biology experimental methods not specifically described in the following examples are all carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) J. Sambrook, or according to the kit and product instructions.

[0042] The reagents, culture media and buffers involved in the following experimental examples are as follows: 1. Biochemical reagents: Green Premix Pro Taq HS qPCR Kit3 purchased from Hunan Aikuo Biological Engineering Co., Ltd., and others are domestic reagents (all can be purchased from ordinary biochemical reagent companies).

[0043] 2. Culture medium: (1) Strain culture base medium YMG: 8-10 g / L malt extract, 2-4 g / L glucose, 2-4 g / L yeast extract in 1 L deionized water, sterilized at 115°C for 30 min.

[0044] (2) E. coli culture medium LB: 8-10 g / L sodium chloride, 8-10 g / L tryptone, 1-2 g / L yeast extract, diluted to 1000 mL with pure water, sterilized at 121°C for 20 min.

[0045] (3) Enzyme production medium mKjalke: 8-10 g / L yeast extract, 18-20 g / L glucose, 1-2 g / L potassium phosphate dibasic, 0.2-0.5 g / L calcium chloride dihydrate, 40-50 mg / L magnesium sulfate heptahydrate, in 1 L deionized water, sterilized at 115°C for 30 min.

[0046] 3. Buffer: MM Buffer (25 mL): pipette 12.5 mL of prepared sterile 1 M mannitol solution, 6.25 mL of 0.2 M sterile maleate buffer, 12.5 mL of sterile water, mix well and use.

[0047] 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), 5.625 mL of sterile water, mix well and use.

[0048] Alkaline lysis solution I (80 mL): 0.72 g glucose, 0.24 g Tris, 0.232 g EDTA.

[0049] Alkaline lysis solution II (40 mL): 0.32 g sodium hydroxide, 0.4 g SDS.

[0050] Alkaline lysis solution III (80 mL): 23.52 g potassium acetate, 9.2 mL glacial acetic acid.

[0051] 1 M Mannitol solution (80 mL): Mannitol 14.5728 g.

[0052] 0.2 M Maleate buffer (60 mL): 0.464 g maleic acid, 1.28 g disodium maleate, dissolved in pure water, and adjusted to pH 5.5 with sodium hydroxide.

[0053] STC buffer (50 mL): 9.1 g sorbitol, 0.060 g Tris, 0.1388 g calcium chloride.

[0054] 1 M CaCl2solution (50 mL): 7.35 g calcium chloride.

[0055] 1 M Tris solution (10 mL): 1.2114 g Tris.

[0056] PEG solution (40 mL): 10 g PEG 4000, 0.4 mL Tris (1 M, pH 8.0), 1 mL calcium chloride (1 M).

[0057] Cellulase solution (10 mL): Take 400 mg cellulase and 1 mg chitinase, add MM Buffer buffer, dissolve and dilute to 10 mL, centrifuge at 3000 x g for 30 min at 4°C, and after filtration under low temperature, aliquot in 2 mL sterile centrifuge tubes. g

[0058] Stock solution A (80 mL): Take 2.3 g KH2PO4, 7.2 g Na2HPO4, 0.928 g Na2SO3, and 1.6 g ammonium tartrate. Dissolve in pure water and dilute to 80 mL, and add 400 μL chloroform.

[0059] Stock solution B (80 mL): 2.3 mg Vitamin B1.

[0060] ​Stock solution C (50 mL): Weigh 1.25 g MgS04·7H20, dissolve in pure water and make up to 50 mL, and add 200 μL chloroform.

[0061] 100 mM sodium tartrate solution (pH 4.0, 3 L): Weigh 90 g L-tartaric acid, dissolve in pure water and adjust the pH to 4.0 with sodium hydroxide, and then make up to 3 L.

[0062] 15 mM ABTS (100 mL): Weigh 0.822 g ABTS, dissolve in 10 mL anhydrous ethanol with a measuring cylinder, add water and make up to 100 mL.

[0063] Regenerated solid medium (1 L): Weigh 25 mL of the above prepared stock solution A, 1 mL of stock solution B, 10 mL of stock solution C, 172 g sucrose, 2 g soluble starch, 2 g asparagine, and 10 g agar powder, and then make up to 1 L with pure water. In addition, separately weigh 5 g glucose in 10 mL pure water, sterilize at 115°C for 30 min under high pressure. Mix the above two substances in a clean bench and pour into a flat plate.

[0064] Example 1: Mining of key genes of endoplasmic reticulum stress response in Coprinus cinereus The specific steps are as follows: 1、 Cclcc 5-13 mycelium collection (1) Use a loop to pick up Cclcc 5-13 mycelial blocks from the glycerol tube of the preserved strain, inoculate them on seed culture plates, and incubate them at 37°C for about 6-10 days.

[0065] (2) Use a loop to divide the mycelial blocks on the plate into mycelial blocks with a diameter of about 1 cm, select 4 blocks and place them in seed liquid medium, and incubate them at 37°C, 120 rpm.

[0066] (3) Use a homogenizer to homogenize at 3500 rpm for 15 s, inoculate in new seed liquid medium at a 5% (v / v) inoculation amount, and incubate at 37°C, 120 rpm. Take samples every 12 h, centrifuge at 4°C, 12000 x g for 25 min, collect the mycelium, and store at -80°C for use.

[0067] 2、 Cclcc Extraction of total RNA of 5-13 (1) Thaw the mycelium at different fermentation times frozen in the -80°C refrigerator, add 1 mL RNAisoPlus (purchased from Baori Medical Biotechnology (Beijing) Co., Ltd.).

[0068] (2) After standing on ice for 6 min, seal the RNase free centrifuge tube tightly, put it into the automatic sample freezer grinder for grinding (12000 x g, 5 min), and then transfer the supernatant to a new RNase free centrifuge tube.

[0069] (3) Add 300 μL of chloroform, mix well on a vortex shaker, and stand on ice for 5 min after mixing.

[0070] (4) After centrifugation at 4°C, 12000 x g for 15 min, transfer the supernatant to a new RNase free centrifuge tube.

[0071] (5) Add 800 μL of isopropanol again, and stand on ice for 10 min.

[0072] (6) After centrifugation at 4°C, 12000 x g for 10 min, discard the supernatant, add 1 mL of 75% ethanol (7) After centrifugation at 4°C, 12000 x g for 5 min, discard the supernatant, stand for about 2 min, dissolve the precipitate with 30 μL of DEPC water, and store it in a -80°C refrigerator for standby.

[0073] 3、 Cclcc Removal of genomic DNA of 5-13 According to the instruction manual of Evo M-MLV reverse transcription kit (purchased from Aikuer Biological Engineering Co., Ltd.), the reaction conditions and reaction system were set and adjusted to remove genomic DNA, and the total RNA sample concentration was uniformly adjusted to 800 ng. The experimental reaction system is shown in Table 1.

[0074] Table 1 Genomic DNA removal reaction system

[0075] The reaction conditions for removing genomic DNA are 42°C, 2 min; 4°C.

[0076] 4、 Cclcc cDNA generation of 5-13 According to the contents of the following table, the reaction solution was configured, and the reverse transcription reaction was carried out.

[0077] Table 2 RNA reverse transcription reaction system

[0078] The reaction conditions are 37°C, 15 min; 85°C, 5 s.

[0079] 5, qRT-PCR reaction determination of endoplasmic reticulum stress related genes First, use NCBI to locate the Grey Coat Ghost Umbrella FA2222. der 1 (CC1G-14444) hrd 1 (CC1G-02491) pdi 1 (CC1G-00344) and bip The gene sequence of 1 (CC1G-06790) was obtained, and then the sequence was aligned using SnapeGene. A gene sequence fragment spanning two or more introns was selected for primer design. The primer sequences are shown in Table 3 below.

[0080] Table 3 Primer sequences for qRT-PCR

[0081] The qRT-PCR reaction system and conditions were set up according to the instructions of the SYBR® Green Premix Pro Taq HS qPCR kit (purchased from Acrel Biotech Co., Ltd.). The experiment was conducted using a quantitative real-time PCR instrument (PCR LightCycler® 96 real-time PCR system), and transcription levels were calculated using the 2-ΔΔCT formula. The specific qRT-PCR reaction system is shown in Table 4.

[0082] Table 4 qRT-PCR reaction system

[0083] The reaction conditions for qRT-PCR were: 95℃, 30 s (x35) and 95℃, 5 s (x35); 60℃, 30 s. The calculation method for the 2-ΔΔCT formula 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):402-408 doi:10.1006 / meth.2001.1262.

[0084] The relative transcriptional levels of each gene are as follows: Figure 1 As shown: like Figure 1 As shown in Figure A, the grey-capped *Coprinus comatus* FA2222 at 4, 8, and 12 h... der The relative transcription levels were: 0, 0.121, and 0.195. Cclcc 5-13 at 4, 8 and 12 h der The relative transcription levels were 0.112, 0.165, and 0.332.

[0085] like Figure 1 As shown in B, the grey-capped *Coprinus comatus* FA2222 at 4, 8, and 12 h... hrd The relative transcription levels were: 0.008, 0.006, and 6.526. Cclcc 5-13 at 4, 8 and 12 h hrd The relative transcription levels were 0.008, 0.328, and 9.211.

[0086] like Figure 1 As shown in C, the grey-capped *Coprinus comatus* FA2222 at 4, 8, and 12 h... pdi The relative transcription levels were: 0, 0.003, 5.291; Cclcc 5-13 at 4, 8 and 12 h pdi The relative transcription levels were 0.004, 0.0352, and 3.4.

[0087] like Figure 1 As shown in D, the gray-capped *Coprinus comatus* FA2222 at 4, 8, and 12 h... bip The relative transcription levels were: 0.051, 0.003, and 53.321. Cclcc 5-13 at 4, 8 and 12 h bip The relative transcription levels were: 0.001, 0.379, and 23.001.

[0088] 6. Gray-covered Ghost Umbrella Cclcc 5-13 hac 1 gene and homology in Aspergillus niger hac 1. Gene mining This invention selects endogenous gray-capped Coprinus comatus. hac 1 ( Cchac 1) And Aspergillus niger, which is also a filamentous fungus hac 1 ( Anhac 1) The nucleotide sequences of its gene are SEQ ID NO.3 and SEQ ID NO.4.

[0089] Example 2: Construction of interference vectors and overexpression vectors The specific steps are as follows ( Figure 2 ): 1. Gene fragment amplification design der The amplification primers for the interference sequence are Fi- der 1 and Ri- der 1, Anhac The amplification primers for the overexpressed sequence were Fe- Anhac 1 and Re- Anhac 1, Cchac The amplification primers for the overexpressed sequence were Fe-Cchac 1 and Re- Cchac 1 extraction Cclcc 5-13 and total RNA of Aspergillus niger, then reverse transcription into cDNA, using PCR amplification to obtain interfering sequences and overexpression sequences with cDNA as template. The primer sequences of gene amplification are shown in Table 5.

[0090] Table 5 Primer sequences

[0091] The PCR system is shown in Table 6.

[0092] Table 6 PCR reaction system

[0093] PCR conditions: 94℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 1 min 3, 30 cycles, and the PCR product was recovered by gel.

[0094] 2, vector amplification The pYSK vector was amplified using primers L22 and L24, and after linearization of the vector, the PCR amplification product was subjected to nucleic acid gel electrophoresis, and then recovered for ligation.

[0095] Table 7 Primer for PCR amplification of vector fragments

[0096] The PCR system is shown in Table 8.

[0097] Table 8 PCR amplification reaction system

[0098] The PCR reaction program is as follows: 94℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 10 min, 30 cycles, and the PCR product was recovered by gel.

[0099] 3, connection and transformation of fragments and vectors In vitro ligation was performed using the method of in vitro homologous recombination, and the ligation product was transformed into E. coli (DH5α) competent cells to amplify and construct pYSK-anti- Escherichia coli 1, pYSK- der 1 and pYSK- Anhac 1 and pYSK- Cchac1 Vector. The recovered three fragments of the derl, Anhac 1 and Cchac 1 fragments with homologous arms were mixed with the pYSK vector fragment according to the required molar ratio of insert to vector of 2: 1, respectively, and the following ligation system was used to ligate the products, which were then transferred into DH5a competent cells, coated on LB solid plates and incubated at 37°C overnight. The ligation system is shown in Table 9.

[0100] Table 9 Homologous recombination ligation reaction system

[0101] Ligation reaction conditions: 37°C water bath heating, 30 min.

[0102] 4. PCR verification of positive monoclonal The positive monoclonal obtained by overnight culture was picked into sterile water as a template for PCR amplification verification to avoid false positives.

[0103] The PCR amplification primers are shown in Table 10.

[0104] Table 10 Monoclonal PCR amplification primers

[0105] The PCR amplification system is shown in Table 11: Table 11 Monoclonal PCR system

[0106] PCR conditions: 94°C pre-denaturation for 10 min; 98°C denaturation for 10 s, 55°C annealing for 5 s, 72°C extension for 1 min, 30 cycles, and the PCR product was detected by agarose gel electrophoresis.

[0107] Obtaining recombinant strains E. coli DH5a / pYSK-anti- der 1, E. coli DH5a / pYSK- Anhac 1 and E. coli DH5a / pYSK- Cchac 1.

[0108] Example 3: Hygromycin resistance activity experiment The specific steps are as follows: (1) Prepare the protoplasts of strain Cclcc5-13 and plate them on the regeneration medium plate.

[0109] (2) Incubate in an incubator at 37°C for 24 h.

[0110] (3) Under sterile conditions, a certain amount of hygromycin B was added to the culture plate of the above-mentioned strain, i.e. a regeneration medium containing hygromycin B was added to the surface of the plate. Lightly spread, and set different hygromycin B gradients (0-80 μg / mL).

[0111] (4) The plate with hygromycin B was placed back in a 37 ℃ constant temperature incubator for culture, and after 3-4 days, the growth of the protoplasts was observed.

[0112] The results are shown in Table 1. Figure 4 As shown in Table 1, the growth of Cclcc5-13 protoplasts can be completely inhibited by 80 μg / mL hygromycin B.

[0113] Example 4: Construction and screening of a recombinant strain of Coprinus cinereus The specific steps are as follows: 1. Protoplast preparation (1) Cclcc 5-13 spore collection: A culture dish of a laccase-overexpressing strain in good growth condition was selected, 5 mL sterile water was added to the culture dish in a clean bench, and the mycelium was lightly scraped off with a sterilized medicine spoon burned by an alcohol lamp, filtered through a spore filter, and the filtrate was taken. Centrifugation at 2600xg, 4 ℃ for 10 min, discard the supernatant under sterile conditions, add 8 mL MM buffer to resuspend and wash the spores, centrifuge again at 2600xg under the above conditions, discard the supernatant.

[0114] (2) Cclcc 5-13 protoplast preparation: 1 mL cell lysis solution was added to the collected spore suspension to resuspend the spores, and incubated in a 37 ℃ constant temperature incubator for 3.5-4 h. Sample under sterile conditions every 15 min and observe the spore lysis effect under a microscope, when the spore lysis effect reaches more than 60%, add 5 mL of freshly prepared MMC buffer. Terminate the enzymolysis reaction. Centrifugation at 640xg, 4 ℃ for 10 min, discard the supernatant under sterile conditions. Add appropriate amount of MMC buffer to adjust the concentration of protoplasts.

[0115] 2. Extraction of plasmid by alkaline lysis method (1) Recombinant strain of Example 2 E. coli DH5α / pYSK-anti- der 1, E. coli DH5α / pYSK- Anhac 1 and E. coli DH5α / pYSK- Cchac 1, inoculate into the corresponding liquid LB test tube one day in advance, and culture overnight at 37 ℃ on a shaking table, and collect the bacterial cells by centrifugation at low temperature at the maximum speed.

[0116] (2) Add 0.2 mL of pre-cooled lysis buffer I to the collected bacterial pellet, flick the tube, and mix the resuspended bacteria.

[0117] (3) Add 0.4 mL of lysis buffer II, invert the tube 3-5 times, and mix the contents.

[0118] (4) Add 0.3 mL of lysis buffer III, invert the tube 5-6 times, and mix the contents. Place the tube on ice for 5 min.

[0119] (5) Centrifuge the chilled sample at 12000 x g for 5 min. Transfer the supernatant to a new sterile tube.

[0120] (6) Add an equal volume of DNA extraction solution to the tube, shake vigorously for 15 min, and repeat three times to mix the organic and aqueous phases. Centrifuge at maximum speed for 3 min in a low-temperature centrifuge, carefully remove the tube, and transfer the supernatant to a new 1.5 mL sterile tube.

[0121] (7) Add 600 μL of isopropanol solution, shake vigorously for 40 s, mix the contents, and let stand at room temperature for 3 min.

[0122] (8) Centrifuge at 12000 x g for 5 min at room temperature, gently pipette the liquid from the tube, invert the EP tube to dry the liquid, and collect the white precipitate.

[0123] (9) Add 1000 μL of 70% ethanol solution (freshly prepared) to the white precipitate, shake vigorously to resuspend the precipitate, and wash the precipitate. Let stand at room temperature for 10 min, and centrifuge at maximum speed for 5 min in a low-temperature centrifuge.

[0124] (10) Carefully remove the ethanol solution from the tube, leaving the white precipitate at the bottom, and invert the tube on a clean paper towel to evaporate the ethanol and remove water droplets from the walls.

[0125] (11) Add 40 μL of pre-sterilized pure water to dissolve the recombinant plasmid pYSK-anti- der 1, pYSK- Anhac 1, and pYSK- Cchac 1, and store at -20 °C for future use.

[0126] 3, Transfer of recombinant vector into Cclcc 5-13 The components shown in Table 12 below were transferred into 2.0 mL EP tubes pre-chilled Table 12 Fungal transformation system

[0127] The mixture in the EP tube was placed on ice for 20 min, 500 μL sterile PEG solution was added and mixed carefully, and the reaction was carried out at room temperature for 5 min. 1 mL sterilized STC buffer was added, and 350 μL of the mixture was spread on a regeneration medium plate and cultured in a constant temperature incubator at 37 °C for 24 h.

[0128] 4. PCR amplification verification of Coprinus cinereus recombinant strains Part of the mycelium growing on the YMG plate was carefully scraped with a sterile spatula and placed in a 2 mL centrifuge tube. 150 μL of Mighty Prep reagent for DNA was added, and the solution and mycelium were thoroughly mixed by using a vortex shaker and a gun head blowing and sucking. The solution was boiled in 95 °C hot water for 10 min, and the centrifuge tube was taken out every 2 min and mixed by inverting. After centrifugation at 12000xg, 4 °C for 2 min, 50 μL of supernatant was aspirated and transferred to a sterile centrifuge tube. The supernatant was used as a template for PCR with Fj- der 1 and Rj- der 1, Fj- Anhac 1 and Rj- Anhac 1, Fj- Cchac 1 and Rj- Cchac 1 as primers. The PCR system is shown in Table 13.

[0129] Table 13 Genomic verification system

[0130] The results are shown in Table 14. Figure 3 Figure 3 A is a positive transformant of the 1 interference strain; der B is a positive transformant of the 1 overexpression strain; Figure 3 C is a positive transformant of the 1 overexpression strain. Anhac Figure 3 The 1 interference recombinant strain Anti- Cchac 1,The 1 overexpression recombinant strain OE- 1 andThe 1 overexpression recombinant strain OE-

[0131] 1. der der Anhac Anhac Cchac Cchac

[0132] ​​​​​Example 5: qRT-PCR of recombinant strains of *Coprinus gracilistylus* The specific steps are as follows: (1) Pick up the mycelial block of the recombinant strain from the glycerol tube of the preserved strain with an inoculation loop, inoculate it onto a seed culture plate, and culture at 37°C for about 6 to 10 days.

[0133] (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.

[0134] (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℃, collect the mycelium, and store it at -80℃ for later use.

[0135] Following the method in Example 1, the obtained mycelium was lysed and total RNA was extracted. Genomic DNA was removed and then reverse transcribed into cDNA for qRT-PCR detection. The primers for qRT-PCR are shown in Table 14.

[0136] Table 14 qRT-PCR primer sequences

[0137] The specific reaction system for qRT-PCR is shown in Table 15.

[0138] Table 15 qRT-PCR reaction system

[0139] qRT-PCR reaction conditions: 95℃, 30 s (x35) and 95℃, 5 s (x35); 60℃, 30 s.

[0140] The results are as follows Figure 5 As shown, Figure 5 A is relative to Cclcc 5. Interfering strain Anti- der1 -2 der1 The level of expression decreased by half; Figure 5 B is relative to Cclcc 5. Overexpression strain OE- Anhac 1-8 Anhac 1. Expression levels increased by 5.7 times; Figure 5 C relative to Cclcc 5. Overexpression strain OE- Cchac 1-8 Cchac 1. Expression level increased by 11.06 times.

[0141] Example 6: Shake flask fermentation of grey Ganoderma recombinant strains 1. Shake flask fermentation culture (1) Take the recombinant bacteria blocks stored in glycerol tubes, inoculate them on YMG solid culture medium, and place them in a 37℃ constant temperature water bath incubator for culture for 6-8 days; (2) Select the plates with good growth state, use sterile inoculation ring operation, divide the bacteria of the plates into blocks with a diameter of about 1 cm, and select 8 blocks into YMG liquid culture medium; (3) Set three groups of parallel for each experiment, and place them in a 37℃ shaker with a rotation speed of 150 r / min for constant culture; (4) After the strains are cultured in the shaker for 4 days, use a high-speed homogenizer at 3500 r / min for 15 s, inoculate them into freshly prepared fermentation culture medium at a 5% (v / v) inoculation amount, and place them in a 150 r / min, 37℃ shaker for continuous constant culture; (5) Take samples every 24 h, and use the supernatant after centrifugation for detection of laccase enzyme activity.

[0142] 2. Fermentation results During the fermentation, samples were taken once a day at a fixed time, and the laccase activity of the shake flask fermentation supernatant was determined. The fermentation results are shown in Table 1. Figure 6 As shown in Table 1, most of the transformed positive strains can overexpress laccase Lcc5, but the laccase enzyme activities of different strains differ greatly. The enzyme activity of the control group Cclcc 5-13 reached 26.4 U / mL, and the enzyme activities of the transformants Anti- der 1, OE- Anhac 1 and OE- Cchac 1 were 31.4, 45.1 and 33.61 U / mL, respectively, which were increased by 18%, 70% and 26% compared with the enzyme activity of the control group Cclcc 5-13.

[0143] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. Use of endoplasmic reticulum stress-related genes in regulating the ability of Coprinus cinereus to express laccase, wherein the endoplasmic reticulum stress-related genes comprise genes der 1, Anhac 1, Cchac 1, the nucleotide sequences of the genes der 1, Anhac 1, Cchac 1 are shown in SEQ ID NO. 2-4, respectively.

2. Use according to claim 1, characterized in that, The application is any one of the following: (1) by interfering with or suppressing the expression of a Coprinus cinereus gene der 1, or overexpressing a gene Anhac 1 or Cchac 1 in Coprinus cinereus, to increase the ability of Coprinus cinereus to express laccase; (2) Reducing the expression of laccase in Coprinus cinereus by overexpressing a gene in Coprinus cinereus der 1, or interfering with or inhibiting the expression of a gene in Coprinus cinereus Cchac 1, to reduce the expression of laccase in Coprinus cinereus.

3. Use according to claim 1 or 2, characterized in that, The Coprinus cinereus is a Coprinus cinereus FA2222 strain or a Coprinus cinereus Cclcc 5-13 strain.

4. A method for high-level expression of laccase from Coprinus cinereus, characterized by, The method described is for the genes of the gray-capped coprinus. der 1. To interfere with gene expression, or to overexpress the gene in *Coprinus comatus*. Anhac 1 or Cchac 1. The gene der 1. Anhac 1. Cchac The nucleotide sequences of 1 are shown in SEQ ID NO.2~4 respectively.

5. The method of claim 4, wherein, The genes der 1 Interfering sequences, genes Cchac 1 Overexpression sequences and genes Anhac 1 Overexpression sequences are shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO.

7.

6. The method of claim 4, wherein, The Coprinus cinereus is a Coprinus cinereus FA2222 strain or a Coprinus cinereus Cclcc 5-13 strain.

7. A genetically engineered bacterium, characterized by, The genetically engineered bacteria are the Coprinus cinereus FA2222 strain or the Coprinus cinereus Cclcc 5-13 strain as a host cell, interfering with the genes of the host cell der 1; or overexpressing genes in the host cell Anhac 1 or Cchac 1, the genes der 1, Anhac 1, Cchac 1, the nucleotide sequences of which are shown in SEQ ID NO. 2~4, respectively.

8. A method for producing laccase, characterized by, The method is to obtain the laccase by fermentation of the genetically engineered bacteria of claim 7.

9. Use of the method of any one of claims 4-6, or the genetically engineered bacteria of claim 7, or the method of claim 8 in the preparation of laccase or a product containing laccase.

10. Use of the method of any one of claims 4-6, or the genetically engineered bacteria of claim 7, or the method of claim 8 in the treatment or degradation of phenolic, carboxylic acid and arylamine compounds and derivatives thereof.

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