CN121380144A

CN121380144A - A method for efficiently expressing laccase by regulating the endoplasmic reticulum stress response of Coprinus gracilis.

Info

Publication number: CN121380144A
Application number: CN202511537158.7A
Authority: CN
Inventors: 姚动邦; 李虎; 方泽民; 肖亚中; 刘娟娟; 戚佳茹
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2025-10-27
Filing date: 2025-10-27
Publication date: 2026-01-23

Abstract

This invention discloses a method for efficiently expressing laccase by regulating the endoplasmic reticulum stress response of *Coprinus fusiforme*, belonging to the fields of genetic engineering and fermentation engineering. This invention enhances the recombinant extracellular expression of laccase Lcc5 by regulating the endoplasmic reticulum stress response genes der der 1, Anhac1 Anhac1 , and Cchac Cchac 1 in the filamentous fungus * Coprinus fusiforme*. Specifically, interfering with der der 1, overexpressing Anhac1 , and overexpressing Cchac Cchac 1 increased the extracellular laccase activity of the recombinant bacteria by 18%, 70%, and 26%, respectively, achieving extracellular laccase activities of 31.4, 45.1, and 33.6 U/mL, which are the highest levels reported to date for homologous recombinant expression of laccase Lcc5 in *Coprinus fusiforme*. This invention is the first to demonstrate that regulating the endoplasmic reticulum stress response in *Coprinus fusiforme* can promote efficient laccase expression, and will further promote the development of *Coprinus fusiforme* expression systems.

Description

Method for efficiently expressing laccase by regulating and controlling stress response of endoplasmic reticulum of Coprinus cinereus

Technical Field

The invention relates to a method for efficiently expressing laccase by regulating and controlling stress response of endoplasmic reticulum of Coprinus cinereus, belonging to the fields of genetic engineering and fermentation engineering.

Background

Laccase (lacase, EC 1.10.3.2) is a kind of copper-containing polyphenol oxidase, widely exists in plants, fungi, bacteria, insects and other organisms, can perform oxidation reaction with phenols, carboxylic acid, arylamine compounds, derivatives thereof and the like, has extremely high catalytic efficiency, can catalyze various oxidation reactions in vivo, and has the only byproduct of the reaction of water, so that Laccase is regarded as an environment-friendly enzyme catalyst with huge potential. Thus, laccase enzymes are widely used in a variety of industries, from the environmental field to the cosmetic industry, including the food processing and textile industries (dye biodegradation and synthesis).

Laccase genes are widely distributed in nature and exist in the genomes of plants, insects, bacteria and fungi, and more than 80% of laccase genes found so far come from fungi. The laccase from basidiomycetes has the advantages of wide substrate range, high specific activity, great reduction potential and the like, is the laccase with the most wide application, accounts for more than 90% of the current laccase market share, and has great market demand. Therefore, the laccase from basidiomycete can be efficiently prepared, and has great application prospect.

Laccase from basidiomycetes is often expressed by heterologous recombination based on other hosts such as aspergillus or yeast to increase its production level. However, the enzymatic properties of recombinant laccase obtained based on the above method may vary in terms of protein molecular weight, redox potential, pH stability and thermostability due to its unreasonable post-translational modifications such as incomplete folding and excessive glycosylation compared to the natural laccase, which severely reduces the practical application value of the obtained recombinant laccase.

Coprinus cinereus (Coprinopsis cinerea) is a model strain of the basidiomycete development process. The coprinus cinereus has the advantages of clear genetic background, simple operation, short growth period and the like. Therefore, coprinus cinereus has the potential to be an excellent host for laccase expression. However, compared with aspergillus and yeast expression systems, the coprinus comatus expression system starts later, and the existing lack of an expression strategy capable of effectively improving the recombinant production of proteins makes it difficult to efficiently express basidiomycete laccase based on the coprinus comatus recombination in the early stage, so that the coprinus comatus laccase is difficult to be widely applied in industry.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for efficiently expressing laccase by regulating and controlling endoplasmic reticulum stress reaction, aiming at improving the recombinant expression production level of laccase in Coprinus cinereus. The invention uses genes der1, anhac1 and Cchac1 related to the endoplasmic reticulum stress reaction process as acting genes. Silencing der1 by using an RNA interference technology (RNA INTERFERENCE, RNAI) or over-expressing Anhac and Cchac1 by using a genome over-expression technology, and finally obtaining the recombinant coprinus cinereus, wherein laccase activities in fermentation liquor after 7 days of shaking fermentation are respectively 31.4U/mL, 45.1U/mL and 33.6U/mL, which are respectively 18%, 70% and 26% higher than that of a control.

The first technical scheme provided by the invention is application of endoplasmic reticulum stress response related genes in the capacity of regulating and controlling laccase expression of Coprinus cinereus, wherein the endoplasmic reticulum stress response related genes comprise genes der1, anhac1 and Cchac.

In certain embodiments, the application is any one of the following:

(1) The ability of Coprinus cinereus to express laccase is improved by interfering with or inhibiting the expression of the Coprinus cinereus gene der1, or over-expressing the gene Anhac or Cchac in Coprinus cinereus;

(2) The expression of Coprinus cinereus laccase is reduced by over-expressing the Coprinus cinereus gene der1, or by interfering with or inhibiting the expression of the gene Cchac in Coprinus cinereus.

In certain embodiments, the nucleotide sequences of the genes der1, anhac1 and Cchac1 are respectively shown in SEQ ID NO. 2-4.

In certain embodiments, the Coprinus cinereus is Coprinus cinereus strain FA2222 or Coprinus cinereus strain Cclcc-13.

In certain embodiments, the Coprinus cinereus Cclcc-13 strain is Coprinus cinereus FA2222 over-expressed laccase, the laccase is laccase Lcc5 gene derived from Coprinus cinereus FA2222, and the nucleotide of the laccase Lcc5 gene is shown in SEQ ID NO. 1.

The second technical scheme provided by the invention is a method for efficiently expressing laccase by using Coprinus cinereus, wherein the method is to perform interference expression on a gene der1 of the Coprinus cinereus or over-express the gene Anhac or Cchac in the Coprinus cinereus.

In certain embodiments, the genes der1 and Cchac1 are derived from the Coprinus cinereus FA2222 genome, and Anhac1 is derived from the Aspergillus niger (Aspergillus niger) genome.

In certain embodiments, the der1 interfering sequence, cchac1 over-expression sequence, and Anhac1 over-expression sequence are set forth in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO. 7.

The third technical scheme provided by the invention is a genetically engineered bacterium which takes Coprinus cinereus as host cells to interfere with a gene der1 of the host cells or over-express the gene Anhac or Cchac1 in the host cells.

In certain embodiments, the host cell is a Coprinus cinereus FA2222 strain or Coprinus cinereus Cclcc-13 strain.

The fourth technical scheme provided by the invention is a method for producing laccase, wherein the method is to use the genetic engineering bacteria of the third technical scheme to ferment and recombine and express to obtain laccase.

In some embodiments, the method comprises inoculating the genetically engineered bacterium of the third technical scheme into a seed culture plate for culturing to obtain seed mycelium blocks, inoculating the seed mycelium blocks into a seed liquid culture medium for culturing to obtain seed mycelium, homogenizing the seed mycelium to obtain seed liquid, and finally inoculating the seed liquid into a fermentation culture medium for culturing.

In some embodiments, the method comprises inoculating the genetically engineered bacterium of the third technical scheme into a seed culture plate, and performing stationary culture for 6-10 d at 37 ℃ to obtain seed mycelium blocks. And then inoculating the seed mycelium blocks into a seed liquid culture medium, and culturing for 3-5 d at 37 ℃ and 100-140 rpm to obtain seed mycelium. And homogenizing the seed mycelium at 3000-4000 rpm for 10-20 s to obtain seed liquid, and finally inoculating the seed liquid into a fermentation medium and culturing for 4-6 d at 37 ℃ and 100-140 rpm.

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

In some embodiments, the seed liquid medium comprises 8-10 g/L malt extract, 2-4 g/L glucose, 2-4 g/L yeast extract.

In some embodiments, 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, 40-50 mg/L magnesium sulfate heptahydrate.

The fifth technical scheme provided by the invention is the method of the second technical scheme, or the genetically engineered bacterium of the third technical scheme, or the application of the method of the fourth technical scheme in preparing laccase or laccase-containing products.

The sixth technical scheme provided by the invention is the method of the second technical scheme, or the genetically engineered bacterium of the third technical scheme, or the application of the method of the fourth technical scheme in the treatment and degradation of oxidized phenols, carboxylic acid, arylamine compounds and derivatives thereof.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, by interfering or overexpressing the endoplasmic reticulum stress response related action genes der1, anhac1 and Cchac of the coprinus cinereus strain, the laccase activity of the obtained recombinant bacteria in the fermentation liquor after shaking is respectively 31.4U/mL, 45.1U/mL and 33.6U/mL, which are respectively 18%, 70% and 26% higher than that of a control.

2. The laccase activity of the recombinant bacterium obtained by over-expressing the endoplasmic reticulum stress response related action gene Anhac of the Coprinus cinereus strain in the invention after shaking the bottle is the highest level reported at present about the homologous recombinant expression of laccase Lcc5 of Coprinus cinereus.

3. The invention indicates that the recombinant expression level of laccase in Coprinus cinereus can be effectively improved by regulating and controlling the endoplasmic reticulum stress reaction of Coprinus cinereus for the first time, which not only accelerates the industrial application process of laccase, but also promotes the development of the Coprinus cinereus expression system.

Drawings

FIG. 1 is a graph showing qRT-PCR results of endoplasmic reticulum stress response genes. Wherein panels A-D are the expression levels of genes der1, hrd1, pdi1 and bip1, respectively.

FIG. 2 shows the present invention schematic diagram of vector construction.

FIG. 3 is a PCR-based verification electrophoresis chart of a recombinant strain positive transformant of the present invention. Wherein M is DNA MAEKER, FIG. A is the selection of der1 interference strain positive transformants, FIG. B is the selection of Anhac1 over-expression strain positive transformants, and FIG. C is the selection of Cchac1 over-expression strain positive transformants.

FIG. 4 is a graph showing the results of screening for hygromycin resistant concentrations of Coprinus cinereus of the present invention.

FIG. 5 is a graph showing the results of qRT-PCR verification of recombinant strains of the present invention. FIG. A shows the expression level of the Anti-der1-2 gene of the interference strain, FIG. B shows the expression level of the OE-Anhac1-8 gene of the over-expression strain, and FIG. C shows the expression level of the OE-Cchac1-8 gene of the over-expression strain.

FIG. 6 shows the fermentation curve of laccase production by recombinant strains of the invention.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for better illustration of the invention, and should not be construed as limiting the invention.

The testing method comprises the following steps:

the method for measuring the total enzyme activity of laccase comprises the following steps:

mu.L Sodium tartrate of solution (100 mmol/L; pH 4.0), 33. Mu.L ABTS (15 mmol/L) and 17. Mu.L of enzyme solution were taken and the system was added to a 2m sterile EP tube using a pipette. Vortex vibration 10s, put in water bath kettle at 30 ℃ to react 3 min, then rapidly take out, ice bath on ice 30 s, then immediately measure the value of the absorbance value OD420, count enzyme activity according to absorbance value (absorbance value OD420 needs to be zero by using a solution system without enzyme liquid before measuring absorbance of laccase enzyme activity, when ABTS is taken as substrate by using the system, the calculation formula of enzyme activity is E (U/L) =OD 420× 555.56.

Materials used in the examples:

The strain Coprinus cinereus FA2222 (C.cinereaFA2222) referred to in the examples below is disclosed in Zhang Jingna, zhou Gang, liu Juanjuan, fang Zemin, shore. Homologous overexpression of laccase Lcc9 in Coprinus cinereus [ J ]. J.biol., 2022,39 (06): 47-51.

The Coprinus cinereus laccase overexpressing strain 5-13 (Cclcc-13) referred to in the examples below is disclosed in ：Yao, Dongbang, et al. "Enhanced extracellular production of laccase inCoprinopsis cinereaby silencing chitinase gene."Applied Microbiology and Biotechnology108.1 (2024): 324.

Expression vectors pYSK referred to in the examples below are disclosed in Zhang Jingna, zhou Gang, liu Juanjuan, fang Zemin, shore. Homologous overexpression of laccase Lcc9 in Coprinus cinereus [ J ]. J.Biol.2022, 39 (06): 47-51.

The molecular biology experimental methods not specifically described in the following examples were carried out with reference to the specific methods listed in the "guidelines for molecular cloning experiments" (third edition) j.

The reagents, media and buffers involved in the following experimental examples were as follows:

1. The biochemical reagent is Green Premix Pro TAQ HS QPCR KIT purchased from Hunan Ai Kerui bioengineering Co., ltd., and the other reagents are domestic reagents (all can be purchased from common biochemical reagent company).

2. Culture medium:

(1) The strain culture basal medium YMG comprises 8-10 g/L malt extract, 2-4 g/L glucose and 2-4 g/L yeast extract, and is subjected to sterilization treatment for 30min at 115 ℃ in 1L deionized water.

(2) E.coli culture medium LB, 8-10 g/L sodium chloride, 8-10 g/L tryptone and 1-2 g/L yeast extract, and sterilizing with pure water to 1000 mL at 121 ℃ for 20 min.

(3) The enzyme-producing culture medium mKjalke 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, and the enzyme-producing culture medium is subjected to sterilization treatment in 1-L deionized water at 115 ℃ for 30-min.

3. Buffer solution:

MM Buffer (25 mL) the 12.5: 12.5 mL sterile 1. 1M mannitol solution, 6.25 mL 0.2M sterile maleate Buffer, 12.5: 12.5 mL sterile water were pipetted and mixed thoroughly.

MMC Buffer (25 mL) the pipettor aspirates 12.5 mL of the prepared sterile 1M mannitol solution, 6.25 mL maleate Buffer (0.2M), 0.625 mL CaCl ₂ solution (1M), 5.625 mL sterile water, and mix thoroughly for use.

Alkaline lysate I (80 mL): 0.72 g glucose, 0.24 g Tris,0.232 g EDTA.

Alkaline lysate II (40 mL) 0.32: 0.32 g sodium hydroxide, 0.4: 0.4 g SDS.

Alkaline lysate III (80 mL) 23.52 g potassium acetate, 9.2 mL glacial acetic acid.

1M mannitol solution (80 mL) mannitol 14.5728 g.

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

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

1M CaCl ₂ solution (50 mL) 7.35. 7.35 g calcium chloride.

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

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

The muramidase solution (10 mL) is prepared by weighing 400 mg cellulase and 1 mg chitinase, adding MM Buffer solution, dissolving and fixing volume to 10mL, centrifuging at 4deg.C and 3000 Xg for 30min, vacuum filtering in low temperature environment, and packaging in 2 mL sterile centrifuge tube.

Stock solution A (80 mL) was weighed 2.3 g KH ₂PO₄,7.2 g Na₂HPO₄,0.928 g Na₂SO₃, 1.6. 1.6 g ammonium tartrate. The mixture was dissolved in pure water and the volume was adjusted to 80 mL, and 400. Mu.L of chloroform was added.

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

Stock solution C (50 mL) 1.25: 1.25 g MgSO ₄·7H₂ O was weighed, dissolved in pure water and set to a volume of 50. 50 mL, and 200. Mu.L of chloroform was added.

100A solution of sodium tartrate (pH 4.0, 3L) was prepared by weighing 90 g L-tartaric acid, dissolving in pure water, adjusting pH to 4.0 with sodium hydroxide, and fixing volume to 3L.

15 MM ABTS (100 mL) 0.822: 0.822 g ABTS is weighed, the absolute ethanol 10: 10mL is weighed by a dosage cylinder, dissolved and then added with water and the volume is fixed to 100: 100 mL.

The regenerated solid culture medium (1L) is prepared by weighing the prepared stock solution A25 mL, stock solution B1 mL, stock solution C10 mL,172 g of sucrose, 2g soluble starch, 2g asparagine and 10 g agar powder, and then using pure water to fix the volume to 1L. Separately, 5g glucose was weighed and dissolved in 10mL pure water, and sterilized at high pressure of 115℃for 30 min. Mixing the above two materials in an ultra-clean workbench, and pouring into a flat plate.

EXAMPLE 1 excavation of the critical action genes for stress response in the endoplasmic reticulum of Coprinus cinereus

The method comprises the following specific steps:

1. cclcc5-13 mycelium collection

(1) And picking Cclcc-13 mycelium blocks from a glycerol tube for preserving strains by using an inoculating loop, inoculating the mycelium blocks to a seed culture plate, and culturing for about 6-10 d at 37 ℃.

(2) The mycelia on the plate were divided into mycelia with a diameter of about 1 cm a by an inoculating loop, and 4 mycelia were selected and placed in a seed liquid medium, and subjected to shaking culture at 37℃under 120 rpm to obtain 4 pieces d.

(3) After 15: 15 s was homogenized at 3500 rpm with a homogenizer, the medium was inoculated with 5% (v/v) of the seed liquid medium and placed in shaking culture at 37℃and 120 rpm. Samples were taken every 12 h, centrifuged at 4℃and 12000 Xg for 25: 25 min, and mycelia were collected and stored at-80℃for later use.

2. Total RNA extraction of Cclcc-13

(1) Mycelium frozen at different fermentation moments in a-80 ℃ refrigerator was thawed and added to 1 mL RNAiso Plus (available from baori doctor materials technology (beijing) inc.).

(2) After 6 min on ice, the RNASE FREE centrifuge tube was sealed tightly, and the supernatant was transferred to a new RNASE FREE centrifuge tube after grinding in a fully automatic sample freeze grinder (12000×g,5 min).

(3) Adding 300 mu L of chloroform, shaking and mixing uniformly by a vortex shaker, and standing on ice for 5 min hours after the chloroform and the vortex shaker are mixed uniformly.

(4) After centrifugation at 15min at 12000 Xg at 4 ℃, the supernatant was transferred to a new RNASE FREE centrifuge tube.

(5) 800 Μl of isopropanol was added again and 10 min were placed on ice.

(6) Centrifuging 10min the supernatant at 12000 Xg at 4deg.C, adding 1 mL of 75% ethanol

(7) The supernatant was centrifuged at 5min Xg at 4℃and 12000 Xg, and after leaving about 2 min, the pellet was dissolved in 30. Mu.L DEPC water and stored in a-80℃refrigerator for further use.

3. Cclcc5-13 removal of genomic DNA

The reaction conditions and reaction system were set and adjusted to remove genomic DNA according to the instructions of Evo M-MLV reverse transcription kit (purchased from Ai Kerui Bio Inc.), wherein the total RNA sample concentration was uniformly adjusted to 800 ng. The experimental reaction system is shown in table 1.

TABLE 1 genomic DNA removal reaction System

The reaction conditions for removing the genomic DNA were 42℃for 2 min and 4 ℃.

4. Cclcc5-13 cDNA Generation

The reaction solution was prepared according to the contents of the following table, and the reverse transcription reaction was performed.

TABLE 2 RNA reverse transcription reaction system

The reaction conditions were 37℃for 15 min, 85℃for 5s.

5. QRT-PCR reaction for determining endoplasmic reticulum stress related gene

The gene sequences of der1 (CC 1G-14444), hrd1 (CC 1G-02491), pdi1 (CC 1G-00344) and bip1 (CC 1G-06790) in Coprinus cinereus FA2222 were first found using NCBI, and then aligned using SNAPEGENE, a gene sequence fragment spanning two or more introns was selected for primer design, and the primer sequences are shown in Table 3 below.

TABLE 3 qRT primer sequences for PCR

The reaction system and conditions for qRT-PCR were set according to the instructions of SYBR cube Green Premix Pro TAQ HS QPCR kit (available from Ai Kerui Bio-engineering Co., ltd.). The experiment was performed using a fluorescence quantification apparatus (PCR LIGHTCYCLER.cndot.96 real-TIME PCR SYSTEM) and the transcript levels were calculated according to the 2-. DELTA.CT formula. The specific reaction system of qRT-PCR is shown in Table 4.

TABLE 4 qRT-PCR reaction System

The reaction conditions of qRT-PCR are 95 ℃, 30s (x 35) and 95 ℃, 5 s (x 35), 60 ℃ and 30 s. Wherein, the calculation method of the 2-delta CT formula is 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.

The relative transcriptional level results for each gene are shown in FIG. 1:

As shown in FIG. 1A, the relative transcription levels of der1 at 4, 8 and 12 h were 0, 0.121, 0.195, and that of Cclcc5-13 at 4, 8 and 12 h were 0.112, 0.165, 0.332.

As shown in FIG. 1B, the hrd1 relative transcript levels at 4, 8 and 12h were 0.008, 0.006, 6.526, and the Cclcc5-13 at 4, 8 and 12h were 0.008, 0.328, 9.211.

As shown in FIG. 1C, the relative transcript levels of pdi1 at 4, 8 and 12 h were 0, 0.003, 5..291 and that of Cclcc5-13 at 4, 8 and 12 h were 0.004, 0.0352 and 3.4.

As shown in FIG. 1D, the relative transcription level of bip1 at 4, 8 and 12h for Coprinus cinereus FA2222 was 0.051, 0.003, 53.321, and the relative transcription level of bip1 at 4, 8 and 12h for Cclcc5-13 was 0.001, 0.379, 23.001.

6. Digging hac gene in Coprinus cinereus Cclcc-13 and homologous hac1 gene in Aspergillus niger

The invention selects hac (Cchac 1) endogenous to Coprinus cinereus and Aspergillus niger hac (Anhac 1) which are also filamentous fungi, and the nucleotide sequences of the genes are SEQ ID NO.3 and SEQ ID NO.4.

EXAMPLE 2 construction of interference vectors and overexpression vectors

The specific steps are as follows (fig. 2):

1. gene fragment amplification

The amplification primers of designed der1 interference sequences are Fi-der1 and Ri-der1, anhac and the amplification primers of the overexpression sequences of Fe-Anhac1 and Re-Anhac1, cchac1 are Fe-Cchac1 and Re-Cchac1 to extract total RNA of Cclcc-13 and Aspergillus niger, then reverse transcription is carried out to cDNA, and the interference sequences and the overexpression sequences are obtained by PCR amplification with the cDNA as a template. The primer sequences for gene amplification are shown in Table 5.

TABLE 5 primer sequences

The PCR system is shown in Table 6.

TABLE 6 PCR reaction system

PCR conditions were 94℃for 4 min, 98℃for 10 s,55℃for 5 s,72℃for 1 min for 3,30 cycles, and the PCR products were gel recovered.

2. Vector amplification

PYSK vectors are obtained by amplification using primers L22 and L24, and after linearization of the vectors, the PCR amplified products are subjected to nucleic acid gel electrophoresis and then recovered for ligation.

TABLE 7 PCR amplification of vector fragment primers

The PCR system is shown in Table 8.

TABLE 8 PCR amplification reaction System

The PCR reaction procedure was 94℃for 4 min, 98℃for 10 s,55℃for 5s, 72℃for 10min, 30 cycles, and the gel recovery of the PCR product.

3. Ligation of fragments to vectors

In vitro ligation was performed using an in vitro homologous recombination method, and ligation products were transferred into E.coli (ESCHERICHIA COLI) DH 5. Alpha. Competence for amplification, constructing pYSK-anti-der1, pYSK-Anhac1 and pYSK-Cchac1 vectors. The recovered three segments of der1, anhac1 and Cchac1 with homology arms were mixed with pYSK vector segments according to the required molar ratio of insert and vector of 2:1, and the ligation products were transferred into DH 5. Alpha. Competent cells after ligation using the following ligation system, which was shown in Table 9 after plating LB solid plates overnight at 37 ℃.

TABLE 9 homologous recombination ligation reaction System

The connection reaction condition is that the water bath heating is carried out at 37 ℃ and the temperature is 30 min.

4. PCR validation of positive monoclonal

And (3) picking out the positive monoclonal obtained by overnight culture into sterile water, and performing PCR amplification verification by taking the positive monoclonal as a template to avoid false positive.

The PCR amplification primers are shown in Table 10.

TABLE 10 monoclonal PCR amplification primers

The PCR amplification system is shown in Table 11:

TABLE 11 monoclonal PCR System

PCR conditions were 94℃for 10 min, 98℃for 10 s,55℃for 5 s,72℃for 1 min and 30 cycles, and the PCR products were detected by agarose gel electrophoresis.

Recombinant strains E. coliDH. Alpha./pYSK-anti-der 1, E. coliDH. Alpha./pYSK-Anhac 1 and E. coliDH. Alpha./pYSK-Cchac 1 were obtained.

EXAMPLE 3 hygromycin resistance Activity test

The method comprises the following specific steps:

(1) Protoplasts of preparation strains Cclcc-13 were plated on regeneration medium plates.

(2) Culturing in a constant temperature incubator at 37 ℃ for 24 h.

(3) A certain amount of hygromycin B is added into a culture plate of the strain under aseptic condition, namely, a regeneration culture medium containing hygromycin B is added onto the surface of the plate. Slightly coating, and setting different hygromycin B gradients (0-80 mug/mL).

(4) The plates covered with hygromycin B were placed again in a 37℃incubator for 3-4 days, and the growth of the protoplasts was observed.

As a result, as shown in FIG. 4, hygromycin B at a concentration of 80. Mu.g/mL can completely inhibit the growth of Cclcc-13 protoplasts.

EXAMPLE 4 construction and screening of recombinant Coprinus cinereus strains

The method comprises the following specific steps:

1. protoplast preparation

(1) Cclcc5-13 spore collection:

Selecting a laccase over-expression strain culture dish with good growth state, adding 5 mL sterile water into the culture dish in an ultra-clean workbench, slightly scraping hyphae by using a medicine spoon burnt and sterilized by an alcohol lamp, and filtering by a spore filter to obtain filtrate. Centrifugation at 2600 Xg, centrifugation at 10 min at 4℃and removal of supernatant under aseptic conditions, addition of 8 mL MM buffer to resuspend spores, centrifugation at 2600 Xg again at the above conditions and removal of supernatant.

(2) Cclcc5-13 protoplast preparation:

To the collected spore suspension, 1 mL cell lysate was added to resuspend spores and placed in a 37 ℃ incubator for incubation 3.5-4 h. Sterile sampling at intervals of 15 min and observing spore lysis effect under microscope, and adding 5mL freshly prepared MMC buffer when spore lysis effect reaches above 60%. The enzymatic hydrolysis reaction is terminated. 640 Xg, centrifugation at 10min at 4℃and removal of supernatant under sterile conditions. Proper amount of MMC buffer solution is added to adjust the concentration of protoplast.

2. Extraction of plasmid by alkaline lysis

(1) Recombinant strains E. coliDH5 alpha/pYSK-anti-der 1, E.collDH5alpha/pYSK-Anhac 1 and E. coliDH 5alpha/pYSK-Cchac of example 2 were inoculated into liquid LB tubes of the corresponding resistances one day in advance, cultured overnight at 37℃with shaking, centrifuged at maximum rotation speed at low temperature, and the cells were collected.

(2) Adding 0.2mL of pre-cooled alkaline lysis solution I into the collected bacterial strain precipitate, flicking a centrifuge tube, and uniformly mixing and re-suspending the bacterial cells.

(3) Adding 0.4 mL alkali lysate II, repeatedly reversing for 3-5 times, and mixing the contents.

(4) The addition of 0.3 mL alkaline lysates III was continued and reversed 5-6 times. The mixture was mixed well and the tube was placed on ice 5 min.

(5) The ice cooled sample was centrifuged at 12000 Xg at low temperature for 5min. Transfer the supernatant to a new sterile centrifuge tube.

(6) Adding the DNA extracting solution with equal volume into the centrifuge tube, and shaking vigorously for 15 min times, repeating for three times, so that the organic phase and the water phase are uniformly mixed. Centrifuge 3min in a cryocentrifuge at maximum speed, slowly remove the centrifuge tube, carefully transfer the upper aqueous phase to a new 1.5 mL sterile centrifuge tube.

(7) 600 Μl of isopropyl alcohol solution was added, and 40 s was vigorously shaken to mix the contents uniformly, and allowed to stand at room temperature for 3: 3 min.

(8) Centrifuging at room temperature at 12000 Xg for 5 min min, slightly sucking with a pipette to remove the liquid in the tube, inverting the EP tube, allowing the liquid in the tube to drain, and collecting the white precipitate.

(9) To the white precipitate was added 1000. Mu.L of 70% ethanol solution (which was ready for use). Shaking vigorously to make white nucleus become electric suspension, washing precipitate. And standing at room temperature for 10 min. In the cryocentrifuge, 5 min was centrifuged at maximum speed.

(10) Taking out the centrifuge tube, carefully removing the ethanol solution, keeping the white nucleic acid sediment at the bottom, inverting the centrifuge tube on a clean paper towel, fully volatilizing ethanol in the centrifuge tube, and removing water drops on the inner wall.

(11) 40. Mu.L of pure water sterilized in advance is added, and recombinant plasmids pYSK-anti-der1, pYSK-Anhac1 and pYSK-Cchac1 are dissolved and frozen at-20 ℃ for later use.

3. Recombinant vector transfer Cclcc-13

The components shown in Table 12 below were transferred into a 2.0 mL EP tube pre-chilled in advance

TABLE 12 fungal transformation systems

The mixture in the EP tube was placed on ice for 20 min g, 500. Mu.L of sterile PEG solution was added and carefully mixed and reacted at room temperature for 5g min g. After the sterilization, STC buffer 1 mL was added, 350. Mu.L of the mixed solution was spread on a regeneration medium plate, and the mixture was cultured in a constant temperature incubator at 37℃for 24: 24 h.

4. PCR amplification verification of Coprinus cinereus recombinant strain

Part of the cells grown on YMG plates was carefully scraped with a sterile drug spoon into a 2 mL centrifuge tube. 150 mu L MIGHTY PREP REAGENT for DNA was added, and the solution was thoroughly mixed with the cells by vortex shaking and gun tip sucking. 95. Boiling 10min in hot water at the temperature of C, taking out the centrifuge tube every 2: 2 min, and mixing. 12000 Xg, 4 ℃,2 min, after centrifugation, 50. Mu.L of supernatant was aspirated and transferred to a sterile centrifuge tube. PCR was performed using the supernatant as a template and Fj-der1 and Rj-der1, fj-Anhac1 and Rj-Anhac1, fj-Cchac1 and Rj-Cchac1 as primers, and the PCR system was shown in Table 13.

TABLE 13 genome verification System

The results are shown in FIG. 3, FIG. 3A shows the positive transformant of the der1 interference strain, FIG. 3B shows the positive transformant of Anhac A over-expression strain, and FIG. 3C shows the positive transformant of Cchac A over-expression strain.

Obtaining the der1 interference recombinant strains Anti-der1, anhac1 over-expression recombinant strain OE-Anhac1 and Cchac1 over-expression recombinant strain OE-Cchac1.

EXAMPLE 5 qRT-PCR of recombinant Coprinus cinereus strain

The method comprises the following specific steps:

(1) The mycelium blocks of the recombinant strain are picked up from the glycerol tube of the preserved strain by an inoculating loop, inoculated on a seed culture plate and cultured for about 6-10 d at 37 ℃.

The obtained mycelia were disrupted, total RNA was extracted, genomic DNA was removed and reverse transcribed into cDNA, and qRT-PCR was performed according to the method of example 1, and primers of qRT-PCR are shown in Table 14.

TABLE 14 qRT-PCR primer sequences

The specific reaction system of qRT-PCR is shown in Table 15.

TABLE 15 qRT-PCR reaction System

The reaction conditions of qRT-PCR were 95℃and 30 s (x 35) and 95℃and 5 s (x 35), 60℃and 30 s.

As a result, as shown in FIG. 5, the expression level of der1 in the interference strain Anti-der1-2 was reduced by 1-fold in FIG. 5A relative to Cclcc, the expression level of Anhac1 in the over-expression strain OE-Anhac1-8 was increased by 5.7-fold in FIG. 5B relative to Cclcc5, and the expression level of Cchac1 in the over-expression strain OE-Cchac1-8 was increased by 11.06-fold in FIG. 5C relative to Cclcc 5.

EXAMPLE 6 shake flask fermentation of Coprinus cinereus recombinant strains

1. Shaking flask fermentation culture

(1) Taking out recombinant fungus blocks stored in the glycerol pipe, inoculating the recombinant fungus blocks on YMG solid culture medium, and placing the YMG solid culture medium in a 37 ℃ constant temperature water bath incubator for culturing for 6-8 days;

(2) Selecting a flat plate with good growth state, performing aseptic inoculating loop operation, dividing thallus of the flat plate into bacterial blocks with the diameter of about 1cm, and selecting 8 bacterial blocks into YMG liquid culture medium;

(3) Three groups of parallel experiments are simultaneously arranged in each group of experiments and are placed in a shaking table at 37 ℃ with the rotating speed of 150r/min, and the continuous culture is carried out at constant temperature;

(4) After the strain is cultured in a shaking table for 4 days, a high-speed homogenizing refiner 3500r/min 15s is used, the strain is inoculated in a freshly prepared fermentation culture medium with an inoculum size of 5% (v/v), and the strain is placed in the shaking table at 150r/min and 37 ℃ for continuous constant-temperature culture;

(5) Samples were taken every 24h and the supernatant after centrifugation was used to detect laccase enzyme activity.

2. Fermentation results

Samples were taken at fixed times daily during fermentation and shake flask fermentation supernatants were assayed for laccase activity. As shown in FIG. 6, most of the transformed positive strains can overexpress laccase Lcc5, but the enzyme activities of laccase between different strains are relatively large, the enzyme activities of control group Cclcc-13 reach 26.4U/mL, the extracellular laccase activities of transformant Anti-der1, OE-Anhac1 and OE-Cchac1 are 31.4, 45.1 and 33.61U/mL respectively, and the enzyme activities are improved by 18%,70% and 26% respectively compared with the enzyme activities of control group Cclcc 5-13.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of endoplasmic reticulum stress response-related genes in regulating the expression of laccase by Coprinus comatus, characterized in that the endoplasmic reticulum stress response-related genes include genes der1 , Anhac1 , and Cchac1 , and the nucleotide sequences of genes der1 , Anhac1 , and Cchac1 are shown in SEQ ID NO.2~4, respectively.

2. The application according to claim 1, wherein the application is any one of the following:

(1) By interfering with or inhibiting the expression of the der 1 gene in Coprinus gracilistylus, or by overexpressing the gene Anhac 1 or Cchac 1 in Coprinus gracilistylus, the ability of Coprinus gracilistylus to express laccase can be improved.

(2) By overexpressing the gene der 1 in Coprinus fusiforme, or by interfering with or inhibiting the expression of gene Cchac 1 in Coprinus fusiforme, the expression of Coprinus fusiforme laccase can be reduced.

3. The application according to claim 1 or 2, wherein the *Coprinus comatus* strain is *Coprinus comatus* FA2222 or *Coprinus comatus * Cclcc 5-13.

4. A method for efficient expression of laccase in Coprinus gravidus, characterized in that the method involves interfering with the expression of the gene der1 of Coprinus gravidus, or overexpressing the gene Anhac1 or Cchac1 in Coprinus gravidus, wherein the nucleotide sequences of the genes der1 , Anhac1 , and Cchac1 are shown in SEQ ID NO. 2~4, respectively.

5. The method according to claim 4, wherein the gene der 1 interference sequence, gene Cchac 1 overexpression sequence and gene Anhac 1 overexpression sequence are as shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

6. The method according to claim 4, wherein the *Coprinus comatus* strain is *Coprinus comatus* FA2222 or *Coprinus comatus * Cclcc 5-13.

7. A genetically engineered bacterium, characterized in that the genetically engineered bacterium uses *Coprinus fusiforme* strain FA2222 or *Coprinus fusiforme* strain Cclcc 5-13 as the host cell, and interferes with the host cell's gene der 1; or overexpresses the gene Anhac 1 or Cchac 1 in the host cell, wherein the nucleotide sequences of the genes der 1, Anhac 1, and Cchac 1 are as shown in SEQ ID NO. 2-4, respectively.

8. A method for producing laccase, characterized in that the method involves fermentation and recombinant expression of laccase using the genetically engineered bacteria described in claim 7.

9. The method according to any one of claims 4 to 6, or the genetically engineered bacteria according to claim 7, or the method according to claim 8, in the preparation of laccase or laccase-containing products.

10. The application of the method according to any one of claims 4 to 6, or the genetically engineered bacteria according to claim 7, or the method according to claim 8 in the treatment and degradation of oxidized phenols, carboxylic acids and aromatic amines and their derivatives.