A homologous overexpression engineered bacterium of the laccase gene MvLac5 and its application

Abstract

The present invention provides the gene MvLac5 from *Varicospora verrucae*, which is a gene encoding laccase, and its nucleotide sequence is shown in SEQ ID No. 1. The gene was cloned and recombined to obtain *Pichia pastoris* GS115-pPICZαB-. MvLac5 The laccase activity in the fermentation broth was measured, and its activity was 1.645 U / L. The laccase gene provided by this invention... MvLac5 The method and techniques for homologous overexpression of engineered bacteria overcome the difficulties encountered when introducing exogenous gene fragments into filamentous fungi. The wild-type strain of *M. verrucaria* of this invention achieved a maximum laccase activity of 1676.97 U / L after 108 h. Recombinant strains... MvLac5‑2 It increased by 1.13 times compared to the wild type; MvLac5‑7 It increased by 1.19 times; MvLac5‑9 It increased by 2.47 times. The strain with the highest laccase activity was selected from the overexpressing strain. MvLac5‑9 Analysis of lignin degradation rate showed that the wild-type strain had the highest degradation rate of alkali lignin at 36.73%, while the engineered strain overexpressing the gene had a maximum degradation rate as high as 77.60%, which was 2.11 times that of the wild-type strain. This indicates that... M.verrucaria Overexpression of laccase gene Mv Lake5 It can effectively improve the lignin degradation ability of the strain.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an engineered bacterium that homologously overexpresses the laccase gene MvLac5 and its application in lignin degradation. Background Technology

[0002] Crop straw is an important biomass resource, mainly composed of cellulose, hemicellulose, and lignin, but its comprehensive utilization level urgently needs improvement. Lignin degradation can effectively improve the utilization efficiency of biomass resources in feed, fertilizer, materials, and bioconversion. Compared with physical and chemical methods, microbial methods have advantages such as environmental safety, green friendliness, and high specificity. Laccase is one of the key enzymes in lignin degradation. Laccase catalyzes the oxidation of various phenolic compounds, lignin-related compounds, and environmental pollutants through a free radical catalytic reaction mechanism. Compared with bacterial laccase, fungal laccase has a higher redox potential and can oxidize a variety of substrates.

[0003] Studies have shown that laccases typically exist as a non-allelic polygenous family in the genomes of various microorganisms, insects, and plants, with multiple isoenzymes in most species. With the rapid development of sequencing technology, whole-genome identification has become increasingly common, revealing significant differences in laccases from different species and sources. Currently, most genomic data from brown-rot fungi, animal and plant pathogens, ectomycorrhizal fungi, white-rot fungi, and saprophytic organisms are publicly available, revealing the presence of multiple laccase genes in fungi. Furthermore, members of the laccase gene family have been identified in plants such as flax, sweet sorghum, and rice. However, the laccase gene sequence in *M. verrucaria* has not been reported.

[0004] Fungal laccases have attracted particular attention due to the stability of their extracellular enzyme components as secretions. However, compared to single-celled microorganisms such as *Escherichia coli* and *Saccharomyces cerevisiae*, filamentous fungi have a more complex genetic background, specific morphology, and thicker cell walls, making the introduction of exogenous gene fragments more difficult. Filamentous fungi also possess physiological characteristics such as apical growth, heteronuclear nature, low homologous recombination efficiency, and a lack of genetic markers, posing challenges to the mature genetic modification of these microorganisms.

[0005] In recent years, various genetic transformation methods have been developed for the genetic modification of filamentous fungi, including: CaCl2 / polyethylene glycol (PEG)-mediated protoplast transformation (PMT), Agrobacterium tumefaciens-mediated transformation (ATMT), electroporation transformation (EP), and biolistic transformation (BT). Due to the complexity and high equipment requirements of BT, the first three methods are currently the most commonly used in laboratories.

[0006] Agrobacterium-mediated transformation (ATMT) was first used for genetic transformation of plants and, after modification, for filamentous fungi, such as *Aspergillus awamori*, *Aspergillus fumigatus*, *M. thermophila*, and *Metarhizium anisopliae*. No reports have been found regarding the use of ATMT for the genetic transformation of *Lactobacillus verrucosum*. The ATMT method primarily utilizes *Agrobacterium tumefaciens* to infect budding spores of filamentous fungi, transferring exogenous T-DNA into host cells. Compared to the protoplast-mediated transformation (PMT) method, the ATMT method has several advantages, such as not requiring protoplast preparation, using filamentous fungal spores as the transformation target, resulting in fewer false positive transformants; lower copy number of the introduced exogenous gene, making it easier to obtain transformants with single-copy exogenous gene insertion; and the ability to mediate the introduction of large DNA fragments. It has been reported that in the genetic transformation of *Aspergillus awamori*, the transformation efficiency of the ATMT method is 600 times that of the PMT method. However, the ATMT method is time-consuming, requires the preparation of Agrobacterium rhizogenes containing the target T-DNA, and can only introduce a single target DNA fragment each time. In addition, some filamentous fungi are sensitive to acetylsuccinone used in the ATMT method, resulting in low transformation efficiency.

[0007] Overexpression technology involves linking an overexpression vector to a target gene and then genetically transforming the strain to increase the expression level of the target gene. By studying the phenotypic changes and physiological and biochemical characteristics of the overexpressing strain, the function of the target gene can be further investigated. The target gene is typically inserted into an overexpression vector containing a fungal constitutive or inducible promoter to regulate the gene and enhance its expression level.

[0008] In summary, the physiological characteristics of filamentous fungi, such as apical growth, heteronuclear nature, low homologous recombination efficiency, and lack of genetic markers, remain major challenges for homologous overexpression. Summary of the Invention

[0009] The purpose of this invention is to provide a laccase gene MvLac5 derived from *Lactobacillus verrucae*.

[0010] Another object of the present invention is to provide a pair of primers for amplifying the above-mentioned laccase gene MvLac5.

[0011] Another object of the present invention is to provide an engineered bacterium that homologously overexpresses the above-mentioned laccase gene MvLac5.

[0012] Furthermore, another objective of this invention is to provide a method for constructing a homologous overexpression engineered bacterium of the above-mentioned laccase gene MvLac5.

[0013] Furthermore, the present invention also provides the application of the above-mentioned engineered bacteria that homologously overexpress the laccase gene MvLac5 in the efficient degradation of lignin.

[0014] The objective of this invention is achieved through the following technical solution:

[0015] A gene encoding laccase, MvLac5, derived from *Lactobacillus verruciformis*, has the nucleotide sequence shown in SEQ ID No. 1.

[0016] A pair of primers for amplifying the laccase gene MvLac5, the upstream primer nucleotide sequence is shown in SEQ ID No. 2, and the downstream primer nucleotide sequence is shown in SEQ ID No. 3.

[0017] An engineered bacterium that overexpresses the aforementioned laccase gene MvLac5.

[0018] An expression vector, characterized in that: the expression vector is formed by linking the above-mentioned MvLac5 gene with pBARGPE1-hygro to form a pBARGPE1-hygro-MvLac5 expression vector. This expression system, when transformed into Agrobacterium EHA105, successfully achieved homologous overexpression of the MvLac5 gene in *Varicospora verrucae*.

[0019] This invention also provides a method for constructing an engineered bacterium that homologously overexpresses the above-mentioned laccase gene MvLac5: the specific method is as follows:

[0020] (1) Cloning of the laccase gene MvLac5

[0021] After culturing the strain for 3 days, total RNA was extracted and reverse transcribed to obtain cDNA; PCR amplification was performed using primers MvLac5-F / R. The base sequence of MvLac5-F is shown in SEQ ID No. 2, and the base sequence of MvLac5-R is shown in SEQ ID No. 3.

[0022] Using pMD-19T as the cloning vector, the vector was digested with EcoRI and KpnI and then ligated with the target fragment overnight at 16°C using T4 ligase. The vector was then transformed into E. coli DH5α using the heat shock transformation method. The plasmid was sent to the company for sequencing (Sangon Biotech). The results were compared using DNAman software to analyze whether the cloned target gene had undergone nucleotide site mutations.

[0023] (2) Heterologous expression of laccase gene MvLac5 in Pichia pastoris

[0024] After digesting the pMD-19T cloning vector and pPICZαB plasmid with EcoRI and KpnI, respectively, they were verified by agarose gel electrophoresis and recovered from the gel. The recovered product MvLac5 and the expression vector pPICZαB were ligated with T4 ligase overnight at 16°C. The ligation product was transformed into Escherichia coli DH5α by heat shock transformation. The successfully verified plasmid was linearized and transformed into Pichia pastoris GS115 competent cells by electroporation transformation.

[0025] Successfully transformed single colonies were picked and activated in YPD medium, then inoculated into BMGY medium. After culturing at 28℃ and 200 r / min for 24 h, the cells were collected by centrifugation and transferred to BMMY medium for induced expression. The laccase activity in the fermentation broth was determined by ABTS method.

[0026] (3) Homologous overexpression of the laccase gene MvLac5

[0027] a. Construction of overexpression plasmids

[0028] The MvLac5 fragment digested with EcoRI and KpnI and the expression vector pBARGPE1-hygro were ligated overnight at 16°C using T4 ligase. The ligation product was then transformed into E. coli DH5α for storage.

[0029] b. Overexpression vector freeze-thaw transformation of Agrobacterium tumefaciens EHA105

[0030] The bacterial strain containing the pBARGPE1-hygro-MvLac5 expression vector was expanded and plasmids were extracted. The strain was then transformed into Agrobacterium EHA105 using the freeze-thaw method.

[0031] c. Agrobacterium tumefaciens EHA105-mediated transformation of Lactospora verrucosum.

[0032] M. verrucaria was cultured on PDA plates at 28°C until fresh spores appeared. The spores were washed away with sterile water and prepared into 1×10⁻⁶ spores. 5 concentration per mL;

[0033] Agrobacterium tumefaciens EHA105 cells successfully transformed into the pBARGPE1-hygro-MvLac5 expression vector were inoculated into YEB liquid medium containing 25 μg / mL rifampin and 100 μg / mL ampicillin and cultured. After centrifugation and discarding the supernatant, the cells were transferred to IM liquid medium and cultured at 28°C and 160 rpm until OD200. 600 =0.5-0.8;

[0034] Take 100 μL of the above-mentioned fresh M. verrucaria spores and the above-mentioned positive transformant solution, mix them well, spread them on IM medium (containing AS 0.2 mM) lined with cellophane, and culture them in the dark at 22.5℃ for 72 h. Then transfer the cellophane to MM medium containing hygromycin resistance (200 μg / mL) and cefotaxime (0.2 mmol / L) and culture at 30℃.

[0035] d. Screening and validation of transformants

[0036] Agrobacterium EHA105 and M. verrucaria were co-cultured at 30℃ for 4 days. Colonies with hygromycin B resistance growing on the surface of MM medium were observed. Transformants were randomly selected and screened again in resistant medium. Genomic DNA of the transformants was extracted using a fungal genomic DNA extraction kit for PCR verification. Wild-type M. verrucaria genomic DNA was used as a negative control. Strains with correct PCR verification results were selected and frozen in 50% glycerol.

[0037] e. Real-time quantitative PCR detection of laccase gene expression levels

[0038] Genomic DNA contaminating the total RNA was removed using a reverse transcription kit, and the resulting cDNA was reverse transcribed. Using the cDNA as a template, the expression level of the laccase gene was quantitatively detected in real time using a quantitative fluorescence kit. After the reaction, 2... -ΔΔCt The relative expression levels of different target genes were calculated, and expression level analysis was performed; 18S RNA was used as an internal control for gene expression levels.

[0039] (4) Lacase activity assay and lignin degradation of MvLac5 overexpressing engineered bacteria

[0040] a. Laccase activity assay

[0041] Laccase activity was determined using the 2,2'-adiazono-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay.

[0042] b. Determination of lignin degradation rate

[0043] The absorbance of alkali lignin in the fermentation broth of each strain was measured at 230 nm using a multifunctional microplate reader. The content of alkali lignin in the culture medium was calculated according to the alkali lignin standard curve, and the removal rate of alkali lignin was calculated according to the formula: alkali lignin removal rate = (C1-C2) / C1*100%.

[0044] Furthermore, the present invention also provides the application of the above-mentioned engineered bacteria that homologously overexpress the laccase gene MvLac5 in lignin degradation.

[0045] The beneficial effects obtained by this invention are:

[0046] Laccase is one of the key enzymes in lignin degradation. The high-value utilization of lignocellulose raw materials can effectively alleviate the energy crisis, protect the ecological environment, and further promote the sustainable development of agriculture. Among them, fungal laccases have attracted special attention due to the stability of their extracellular enzyme components as secretions.

[0047] The present invention provides the gene MvLac5, derived from *Lactobacillus verrucosum*, which is a gene encoding laccase. The present invention obtained a laccase-encoding gene by screening the whole genome sequence of *Lactobacillus verrucosum*, and named it MvLac5, whose nucleotide sequence is shown in SEQ ID No. 1. The gene was cloned and recombined to obtain *Pichia pastoris* GS115-pPICZαB-MvLac5. The laccase activity in the fermentation broth was measured, and the activity was 1.645 U / L, proving that the gene MvLac5 is indeed a laccase-encoding gene.

[0048] The present invention provides an engineered bacterium and method for homologous overexpression of the laccase gene MvLac5, overcoming the difficulties in introducing exogenous gene fragments into filamentous fungi, and is more efficient and convenient than previous methods. First, filamentous fungi grow through the division of apical hyphae, making it difficult for exogenous plasmids to be evenly distributed among the dividing hyphae. Cytoplasmic components and even organelles of the hyphae can enter adjacent cells through septal pores, causing false positives in resistance selection. Second, some filamentous fungi have two different nuclei in their somatic cells. If a transformant containing the resistance selection marker does not integrate into both nuclei simultaneously, subsequent transformants will no longer contain the resistance gene due to dilution during division. Third, in most filamentous fungi, the repair mechanism of non-homologous terminal tuberculosis dominates at low temperatures, making precise gene editing based on homologous recombination difficult. These factors pose significant challenges to the genetic modification of filamentous fungi. Our previous attempts to construct this overexpression engineered bacterium using protoplast fusion and other methods failed. Finally, we successfully achieved homologous overexpression of the target gene in *Lactobacillus verrucae* using the *Agrobacterium*-mediated method described in this invention.

[0049] This invention integrates the target fragment into the overexpression vector pBARGPE1-Hygro with a strong promoter, transforms it into Agrobacterium tumefaciens EHA105 via freeze-thaw transformation, and then infects the mycelium into the host cell *Varicospora verrucosum*. Based on the determination of the minimum inhibitory concentration of hygromycin B against *Varicospora verrucosum*, positive recombinants are screened, thereby achieving homologous overexpression of the target gene. In this process, the success of the target gene transformation, the integration site of the target gene, and the hygromycin B screening system all affect the subsequent acquisition of positive recombinants.

[0050] This invention successfully constructed an engineered bacterium that homologously overexpresses the laccase gene MvLac5 of *M. verrucaria*. Activity assays showed that the wild-type *M. verrucaria* strain reached its maximum laccase activity of 1676.97 U / L after 108 hours. The recombinant strains MvLac5-2, MvLac5-7, and MvLac5-9 showed improved laccase activity compared to the wild-type *M. verrucaria*, with MvLac5-2 showing a 1.13-fold increase, MvLac5-7 a 1.19-fold increase, and MvLac5-9 a 2.47-fold increase. The lignin degradation rate of the overexpressing strain MvLac5-9, which had the highest laccase activity, was analyzed using the wild-type strain as a control. The results showed that the wild-type strain had the highest degradation rate of alkali lignin at 36.73%, while the engineered strain with the overexpressed gene had a maximum degradation rate of 77.60%, which was 2.11 times that of the wild-type strain. This indicates that overexpression of the laccase gene Lac5 in M. verrucaria can effectively improve the lignin degradation ability of the strain. Attached Figure Description

[0051] Figure 1 : Standard curve of alkali lignin.

[0052] Figure 2 Electrophoresis image of PCR amplification of the target gene (M: Marker DL5000; Lane 1: PCR amplification of the target gene).

[0053] Figure 3 Electrophoresis image for bacterial culture PCR verification (M: Marker DL5000; lanes 1-6: bacterial culture PCR verification). Figure 4 Electrophoresis image of recombinant plasmid double digestion verification (M: Marker DL5000; Lane 1: pMD-19T-MvLac5 plasmid double digestion verification).

[0054] Figure 5 Electrophoresis images of the target fragment and pPICZαB plasmid double digestion recovery (M: Marker DL5000; lanes 1 and 2: pPICZαB plasmid double digestion recovery; lanes 3 and 4: target fragment double digestion recovery).

[0055] Figure 6 Electrophoresis image for bacterial culture PCR verification (M: Marker DL5000; lanes 1-6: bacterial culture PCR verification). Figure 7 Electrophoresis image of pPICZαB-MvLac5 plasmid double digestion verification (M: Marker DL5000; lanes 1, 3: pPICZαB-MvLac5 plasmid; lanes 2, 4: pPICZαB-MvLac5 plasmid double digestion results). Figure 8 Electrophoresis image of Sac I single enzyme digestion recovery (M: Marker DL5000; Lane 1: pPICZαB-MvLac5 plasmid single enzyme digestion result).

[0056] Figure 9 Genomic PCR validation electrophoresis image (M: Marker DL5000; lanes 1-3: genomic PCR validation).

[0057] Figure 10 Electrophoresis images of the target fragment and pBARGPE1-Hygro plasmid after double digestion (M: Marker DL15000; lanes 1 and 2: target fragment after double digestion; lanes 3 and 4: pBARGPE1-Hygro plasmid after double digestion).

[0058] Figure 11 Electrophoresis image of recombinant plasmid PCR verification (M: Marker DL15000; Lane 1: Negative control with water as template; Lanes 2-8: PCR verification results of bacterial culture).

[0059] Figure 12 Electrophoresis image of recombinant plasmid double digestion verification (M: Marker DL15000; lanes 1-7: pBARGPE1-Hygro-MvLac5 plasmid double digestion).

[0060] Figure 13 Transformant PCR validation (M: Marker DL5000; Lane 1: Negative control using wild-type as template; Lanes 2-11: Transformant genomic PCR validation).

[0061] Figure 14 qRT-PCR of recombinant strain laccase gene.

[0062] Figure 15 : Enzyme production curve of recombinant strain.

[0063] Figure 16 Alkali lignin removal rate of wild-type strains and engineered strains. Detailed Implementation

[0064] 1. Research Methods

[0065] 1.1 Cloning and Heterologous Expression of Laccase Gene MvLac5

[0066] 1.1.1 Cloning of the laccase gene MvLac5

[0067] A gene, MvLac5, was predicted to encode laccase from the whole genome data obtained in the early stage of the experiment. Primers were designed based on the laccase gene information to amplify the relevant gene fragment in M. verrucaria. The primer sequences are as follows: MvLac5-F base sequence is shown in SEQ ID No.2, and MvLac5-R base sequence is shown in SEQ ID No.3.

[0068] Total RNA was extracted from the strain after culturing for 3 days and quality control was performed by agarose gel electrophoresis. Then, the total RNA was immediately reverse transcribed and amplified using a reverse transcription kit to obtain cDNA. The cDNA was then amplified by PCR to obtain the target fragment, identified by gel electrophoresis, and the target fragment was recovered using a gel recovery kit and stored at -20℃.

[0069] Using pMD-19T as the cloning vector, the vector was digested with EcoRI and KpnI restriction enzymes and then ligated with the target fragment using T4 ligase overnight at 16°C. The vector was then transformed into E. coli DH5α using the heat shock transformation method. The plasmid was sent to the company for sequencing (Sangon Biotech). The results were compared using DNAman software to analyze whether the cloned target gene had undergone nucleotide site mutations.

[0070] 1.1.2 Heterologous expression of laccase gene MvLac5 in Pichia pastoris

[0071] Plasmids were extracted using the SanPrep column-based plasmid DNA mini-extraction kit. The pMD-19T cloning vector and pPICZαB plasmid were digested with EcoRI and KpnI, respectively, and then verified by agarose gel electrophoresis and gel recovery. The recovered product MvLac5 and the expression vector pPICZαB were ligated overnight at 16°C using T4 ligase. The ligation product was transformed into *E. coli* DH5α, and single colonies on low-salt LB agar plates were selected for colony PCR verification. Positive transformants were inoculated into low-salt LB medium containing 30 μg / mL bleomycin and cultured for 12 h. After preservation, plasmids were extracted and verified by double digestion with EcoRI and KpnI restriction enzymes. The successfully validated plasmids were sent to Sangon Biotech for sequencing. Plasmids from strains with correct sequencing results were extracted, linearized, and transformed into Pichia pastoris GS115 competent cells using electroporation. The transformed cells were plated on YPD solid medium containing 100 μg / mL bleomycin and incubated at 30°C for 2–4 days. Single colonies were inoculated into YPD liquid medium containing 100 μg / mL bleomycin for amplification. Genomic DNA was extracted using a yeast DNA extraction kit and used as a template for PCR verification to confirm the presence of the target gene in the transformants.

[0072] Successfully validated single colonies were selected and activated in YPD medium, then inoculated into BMGY medium. After culturing at 28°C and 200 r / min for 24 h, the cells were collected by centrifugation and transferred to BMMY medium for induced expression. The laccase activity in the fermentation broth was determined using the ABTS method.

[0073] 2.2 Homologous overexpression of the laccase gene MvLac5

[0074] 2.2.1 Construction of overexpression plasmids

[0075] After activating the previously constructed MvLac5 cloning vector, the plasmid was extracted. The target gene MvLac5 and the overexpression vector pBARGPE1-hygro were digested with restriction endonucleases EcoRI and KpnI. The digestion products were then recovered by agarose gel electrophoresis. The MvLac5 fragment digested with EcoRI and KpnI and the expression vector pBARGPE1-hygro were then ligated with T4 ligase at 16°C overnight. The ligation product was transformed into E. coli DH5α and stored.

[0076] 2.2.2 Transformation of Agrobacterium tumefaciens EHA105 using the freeze-thaw method with overexpression vector

[0077] The bacterial strain containing the pBARGPE1-hygro-MvLac5 expression vector was expanded and plasmids were extracted. The plasmids were then transformed into Agrobacterium EHA105 using the freeze-thaw method.

[0078] After the competent Agrobacterium cells taken out at -80℃ were partially thawed, they were inserted into ice.

[0079] Add the target plasmid and mix well. Let stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0080] Add 700 μL of antibiotic-free YEB liquid medium and incubate at 28°C and 200 rpm for 2–3 h.

[0081] Spread the resuscitation solution onto YEB plates containing the corresponding antibiotics and incubate upside down at 28°C for 2–3 days.

[0082] After the transformants have grown into single bacteria, they are verified by PCR in the bacterial culture.

[0083] 2.2.3 Agrobacterium tumefaciens EHA105-mediated transformation of *Epipremnum aureum*

[0084] (1) M. verrucaria was cultured on a PDA plate at 28℃ until fresh spores appeared. The spores were washed away with sterile water and prepared into 1×10⁻⁶ spores. 5 concentration per mL;

[0085] (2) The positive transformants obtained in the previous stage were inoculated into YEB liquid medium containing 25 μg / mL rifampin and 100 μg / mL ampicillin and cultured. After centrifugation and discarding the supernatant, the bacterial cells were transferred to IM liquid medium and cultured at 28℃ and 160 rpm until OD. 600 =0.5-0.8.

[0086] (3) Take 100 μL of the solutions from step 1 and step 2 respectively, mix them, spread them on IM medium (containing AS 0.2 mM) lined with cellophane, and culture them in the dark at 22.5℃ for 72 h. Then transfer the cellophane to MM medium containing hygromycin resistance (200 μg / mL) and cefotaxime (0.2 mmol / L) and culture at 30℃.

[0087] 2.2.4 Screening and Validation of Transformants

[0088] Agrobacterium EHA105 and M. verrucaria were co-cultured at 30℃ for 4 days, and colonies with hygromycin B resistance growing on the surface of MM medium were observed. Transformants were randomly selected and screened again in resistant medium, and genomic DNA was extracted from the transformants using a fungal genomic DNA extraction kit for PCR verification. Wild-type M. verrucaria genomic DNA was used as a negative control. Strains with correct PCR verification results were selected and frozen in 50% glycerol.

[0089] 2.2.5 Real-time quantitative PCR detection of laccase gene expression levels

[0090] Genomic DNA contaminating the total RNA was removed using a reverse transcription kit, and the resulting cDNA was reverse transcribed. Using the cDNA as a template, the expression level of the laccase gene was detected in real-time using a quantitative fluorescence assay kit. After the reaction, 2... -ΔΔCt The relative expression levels of different target genes were calculated, and expression level analysis was performed. 18S RNA was used as an internal control for gene expression levels.

[0091] 2.3 Lacase activity and lignin degradation by MvLac5-overexpressing engineered bacteria

[0092] 2.3.1 Laccase Activity Assay

[0093] Laccase activity was determined using the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay.

[0094] Laccase activity was determined at room temperature with an ABTS substrate concentration of 0.5 mmol / L and a sodium acetate buffer concentration of 0.1 mol / L (pH 4.8). An inactivated enzyme solution served as the control. Enzyme activity was defined as the amount of enzyme required to convert 1 μmol / L ABTS per minute. The calculation formula is as follows:

[0095]

[0096] ΔOD: Absorbance change over reaction time; Vtotal: Total volume of the reaction system; N: Dilution factor; Δt: Reaction time; Venzyme: Volume of enzyme solution added; ε: ABTS molar extinction coefficient (ε0) 420 =3.6×10 4 mol / L·cm -1 ); d: thickness of the absorbent layer of the reaction solution.

[0097] 2.3.2 Determination of Lignin Degradation Rate

[0098] (1) Standard curve of alkali lignin

[0099] A 1 g / L alkali lignin solution was prepared, and a full-band scan was performed in the wavelength range of 200 nm-400 nm to determine that the maximum absorption wavelength of alkali lignin was 230 nm. Standard solutions of alkali lignin with concentrations of 20, 40, 60, 80, and 100 mg / L were prepared by dilution. The absorbance A of the standard solutions at 230 nm was measured. Based on the measured values, the concentration C (mg / L) was regressed against A to obtain the regression equation for alkali lignin: y = 0.00534x + 0.03353(R²). 2 =0.9972). See the standard curve. Figure 1 .

[0100] (2) Calculation of alkali lignin removal rate

[0101] The absorbance of alkali lignin in the fermentation broth of each strain was measured at 230 nm using a multifunctional microplate reader. The content of alkali lignin in the culture medium was calculated based on the alkali lignin standard curve, and the removal rate of alkali lignin was calculated using the following formula:

[0102] Alkali lignin removal rate = (C1-C2) / C1*100%

[0103] Among them: the content of alkali lignin in the C1-uninoculated culture medium;

[0104] The content of alkali lignin in the culture medium after degradation by C2- strain.

[0105] 3. Results

[0106] 3.1 Cloning and Heterologous Expression of Laccase Gene MvLac5

[0107] 3.1.1 Construction of the laccase gene cloning vector

[0108] (1) PCR amplification of the target fragment

[0109] Using M. verrucaria cDNA as a template, MvLac5-F / R primers were designed for PCR amplification of the target fragment. The results were analyzed by gel electrophoresis. Figure 2 As shown in the figure, a clear band appears at the target band size, proving that the target fragment was successfully amplified.

[0110] (2) Construction of pMD-19T cloning vector

[0111] After transformation by ligation of the pMD-19T cloning vector with the target fragment, single colonies of newly formed transformants were picked from LB solid medium containing ampicillin resistance. Six single colonies were then cultured in LB medium for colony PCR verification. The results are as follows: Figure 3 As shown, lanes 1 and 3 showed no band at the target band size, indicating unsuccessful transformation; the remaining four transformants were positive. Subsequently, one successfully transformed strain was randomly selected for culture, and plasmid was extracted for EcoRI and KpnI double digestion verification. The results are as follows... Figure 4 As shown, the obtained bands are the same size as the expected target bands, proving that the recombinant plasmid was successfully constructed and then stored.

[0112] 3.1.2 Construction of the expression vector for the laccase gene MvLac5

[0113] First, the target fragment and the pPICZαB vector were double-digested with EcoRI and KpnI, respectively, and then recovered using a gel. The results are as follows: Figure 5As shown. The restriction enzyme bands were clear and single, and corresponded to the length of the target fragment. The recovered restriction enzyme products were ligated and transformed into *E. coli* DH5α. Six single colonies were selected and cultured in LB medium for colony PCR verification. The agarose gel electrophoresis results are shown below. Figure 6 As shown, all six transformants exhibited clear bands at the expected band size, and the length matched that of the target gene band, thus preliminarily identifying them as positive transformants. Plasmids were extracted from two positive transformants and subjected to enzyme digestion for verification. The verification results are shown below. Figure 7 As shown, lanes 2 and 4 both showed two bands after double digestion with EcoRI and KpnI, and the band sizes were consistent with the expected results, proving that the expression vector was successfully constructed. The two successfully validated strains were preserved.

[0114] 3.1.3 Screening and Identification of Recombinant Strains

[0115] The recombinant plasmid pPICZαB-MvLac5 was digested with Sac I, and the product was recovered and subjected to agarose gel electrophoresis. The results are as follows: Figure 8 As shown, the recombinant plasmid pPICZαB-MvLac5 exhibited a single and clear band, suitable for subsequent experiments. The linearized plasmid was transformed into GS115 competent cells and cultured on antibiotic plates. Three single colonies were randomly selected, cultured, and their genomes were extracted for PCR verification. The results are as follows: Figure 9 As shown, lane 3 contains a band that matches the target gene fragment. This demonstrates the successful construction of recombinant Pichia pastoris GS115-pPICZαB-MvLac5.

[0116] 3.1.3 Assay of Laccase Activity in Recombinant Pichia pastoris GS115-pPICZαB-MvLac5

[0117] The activity of laccase in the fermentation broth of recombinant Pichia pastoris GS115-pPICZαB-MvLac5 was measured after shake-flask culture. The activity was 1.645 U / L, proving that the gene MvLac5 is the gene encoding laccase.

[0118] 3.2 Homologous overexpression of the laccase gene MvLac5

[0119] 3.2.1 Construction of the overexpression vector pBARGPE1-Hygro-MvLac5

[0120] The overexpression vectors pBARGPE1-Hygro plasmid and pMD-19T-MvLac5 plasmid were digested with EcoRI and KpnI, respectively, and then recovered. The results are as follows: Figure 10 As shown, a clear band was obtained at the expected band size, proving that the enzyme digestion result was correct and can be used for subsequent ligation.

[0121] The recombinant pBARGPE1-Hygro-MvLac5 overexpression plasmid was transformed into competent E. coli DH5α cells, and the transformed strain was confirmed to be positive by PCR after preservation of the bacterial culture. Results are as follows: Figure 11 As shown, clear bands appeared in all lanes, and the bands were the same size as the target bands. Plasmids were extracted from the seven positive transformants and verified by double enzyme digestion. The results are as follows. Figure 12 As shown, after double digestion with EcoRI and KpnI, two clear bands appeared at 1794bp and 5992bp, consistent with the expected results. The successfully validated plasmid was sent to Sangon Biotech for sequencing, and DNAman analysis showed no mutation sites, confirming the successful construction of the overexpression vector pBARGPE1-Hygro-MvLac5.

[0122] 3.2.2 Construction and Screening Identification of Strains Homologously Overexpressing the Lacase Gene MvLac5

[0123] The pBARGPE1-Hygro-MvLac5 overexpression plasmid was transformed into Agrobacterium tumefaciens EHA105. After successful verification, wild-type *M. verrucaria* was transformed using *Agrobacterium tumefaciens* EHA105 containing pBARGPE1-Hygro-MvLac5. Hygromycin resistance genes, used as fungal selection markers, are present in the expression vector pBARGPE1-Hygro. Cefotaxime inhibits the growth of *Agrobacterium tumefaciens*, therefore transformants were screened on MM plates containing hygromycin B and cefotaxime. Ten transformant strains were obtained, and these ten colonies were transferred to PDA plates for growth, followed by PD liquid culture to extract genomic DNA from the transformants. PCR verification was performed using the corresponding primers. Primers were designed using a portion of the overexpression vector fragment and a portion of the laccase gene MvLac5 sequence, totaling approximately 2000 bp. The primer sequences are shown in SEQ ID No. 4 for LF-S-1 and SEQ ID No. 5 for LR-S-1. The results are as follows. Figure 13As shown. Since the amplified fragment is not the original sequence in the wild-type genome, the corresponding amplified sequence can only be obtained in positive transformants. When amplified using this primer, lane 7 did not show a band at the target band size, indicating that the overexpression vector had not integrated into the *M. verrucaria* genome. The remaining lanes all showed clear bands at the expected target band size, proving the transformation result was correct. The positive transformants were sent to Sangon Biotech for sequencing, and the sequencing results matched the original sequence, proving that the transformation result could be used for subsequent experiments. A total of 9 recombinant strains were obtained, named MvLac5-1, MvLac5-2, MvLac5-3, MvLac5-4, MvLac5-5, MvLac5-6, MvLac5-7, MvLac5-8, and MvLac5-9, and their fresh mature spores were preserved.

[0124] 3.2.3 Quantitative fluorescence analysis of laccase gene overexpression levels in positive transformants

[0125] The transcriptional level of MvLac5 laccase in recombinant strains was analyzed using RT-qPCR. RNA was extracted from positive and wild-type M. verrucaria strains, and the stably expressed 18S RNA was selected as an internal reference gene for quantitative laccase gene analysis. Each sample was tested in triplicate, using 2... -ΔΔCt The method is used to process the data, and the results are as follows: Figure 14 As shown, compared with wild-type M. verrucaria, the laccase transcription levels of the five recombinant strains MvLac5-2, MvLac5-4, MvLac5-6, MvLac5-7, and MvLac5-9 were increased. Specifically, the laccase transcription level in MvLac5-2 was 2.07 times higher than that in wild-type M. verrucaria; the laccase transcription level in MvLac5-4 was 1.45 times higher; the laccase transcription level in MvLac5-6 was 1.58 times higher; the laccase transcription level in MvLac5-7 was 2.16 times higher; and the laccase transcription level in MvLac5-9 was 3.44 times higher.

[0126] 3.3 Lacase activity and lignin degradation by MvLac5 overexpressing engineered bacteria

[0127] 3.3.1 Laccase Activity Assay

[0128] Wild-type and positively transformed strains of *M. verrucaria*, MvLac5-1, MvLac5-2, MvLac5-3, MvLac5-4, MvLac5-5, MvLac5-6, MvLac5-7, MvLac5-8, and MvLac5-9, were subjected to liquid fermentation in basal fermentation medium. After three parallel fermentations, the enzyme production curves of the positive transformants are shown below. Figure 15 As shown in the figure, the recombinant strains and wild-type strains exhibit similar enzyme production characteristics. With increasing time, laccase activity gradually stabilizes, showing a slight decreasing trend. The wild-type strain reaches its maximum laccase activity of 1676.97 U / L at 108 h. The recombinant strains MvLac5-2, MvLac5-7, and MvLac5-9 show increased laccase activity compared to the wild-type *M. verrucaria*, with MvLac5-2 showing a 1.13-fold increase, MvLac5-7 a 1.19-fold increase, and MvLac5-9 a 2.47-fold increase. The recombinant strains MvLac5-3, MvLac5-5, and MvLac5-8 show decreased laccase activity compared to the wild-type, while the recombinant strains MvLac5-1, MvLac5-4, and MvLac5-6 show no significant change in laccase activity compared to the wild-type. The results are largely consistent with the results of laccase relative overexpression transcription level, indicating that the transcription level is basically consistent with the protein level expression.

[0129] 3.3.2 Determination of Alkali Lignin Degradation Rate

[0130] The lignin degradation rate of the overexpressing strain MvLac5-9, which exhibited the highest laccase activity, was analyzed, with the wild-type strain serving as a control. The results are as follows: Figure 16 As shown. By Figure 16 It can be seen that under alkali lignin culture conditions, the removal of alkali lignin by both the wild-type strain and the engineered strain showed a rapid increasing trend within 0-84 hours, reaching its maximum at 84 hours and 96 hours respectively, and then stabilizing. With further extension of culture time, cell autolysis occurred, and the alkali lignin removal rate no longer increased. The wild-type strain exhibited the highest alkali lignin degradation rate of 36.73%, while the engineered strain with overexpressed gene achieved a maximum degradation rate as high as 77.60%, 2.11 times that of the wild-type strain. This indicates that overexpression of the laccase gene Lac5 in M. verrucaria effectively enhances the lignin degradation capacity of the strain.

Claims

1. A fungus containing *Russula verrucae* ( Myrothecium verrucaria laccase gene MvLac5 The engineered bacteria are characterized by: Gene MvLac5 The nucleotide sequence is shown in SEQ ID No.

1.

2. The engineered bacteria as described in claim 1, characterized in that: It is the aforementioned MvLac5 The gene is linked with pBARGPE1-hygro to form pBARGPE1-hygro- MvLac5 The expression vector was transformed into Agrobacterium EHA105 to realize the gene expression. MvLac5 Homologous overexpression in *Epipremnum aureum*.

3. An expression vector for constructing the engineered bacteria as described in claim 1, characterized in that: The expression vector is used to deliver the... MvLac5 The gene is linked to pBARGPE1-hygro to form pBARGPE1-hygro- MvLac5 Expressive vehicle.

4. The laccase gene containing *Varistomium verrucosum* as described in claim 1 or 2 MvLac5 Application of engineered bacteria in lignin degradation.

5. The method for constructing the engineered bacterium containing the laccase gene MvLac5 of *Lactobacillus verrucae* as described in claim 1 or 2, characterized in that: The specific construction method is as follows: (1) Laccase gene MvLac5 Cloning After culturing the strain for 3 days, total RNA was extracted and reverse transcribed to obtain cDNA; PCR amplification was performed using primers MvLac5-F / R. The base sequence of MvLac5-F is shown in SEQ ID No. 2, and the base sequence of MvLac5-R is shown in SEQ ID No.

3. Using pMD-19T as the cloning vector, the vector was utilized... EcoR I and Kpn I After double digestion, the target fragment was ligated with T4 ligase overnight at 16°C. The plasmid was then transformed into E. coli DH5α using the heat shock transformation method. The plasmid was sent to the company for sequencing. The results were compared using DNAman software to analyze whether the cloned target gene had undergone nucleotide site mutations. (2) Laccase gene MvLac5 Heterologous expression in Pichia pastoris use EcoR I and Kpn I After digesting the pMD-19T cloning vector and pPICZαB plasmid with enzymes, the results were verified by agarose gel electrophoresis and the gel was recovered. MvLac5 The expression vector pPICZαB was ligated with T4 ligase overnight at 16°C. The ligation product was transformed into E. coli DH5α by heat shock transformation. The successfully verified plasmid was linearized and transformed into Pichia pastoris GS115 competent cells by electroporation transformation. Successfully transformed single colonies were picked and activated in YPD medium, then inoculated into BMGY medium. After culturing at 28℃ and 200 r / min for 24 h, the cells were collected by centrifugation and transferred to BMMY medium for induced expression. The laccase activity in the fermentation broth was determined by ABTS method. (3) Laccase gene MvLac5 Homologous overexpression a. Construction of overexpression plasmids will EcoR I and Kpn I Double enzyme digestion MvLac5 The fragment and expression vector pBARGPE1-hygro were ligated overnight at 16°C using T4 ligase, and the ligation product was transformed into E. coli DH5α for storage. b. Overexpression vector freeze-thaw transformation of Agrobacterium tumefaciens EHA105 For those containing pBARGPE1-hygro- MvLac5 The bacteria expressing the vector were cultured and plasmids were extracted. The plasmids were then transformed into Agrobacterium EHA105 using the freeze-thaw method. c. Agrobacterium tumefaciens EHA105-mediated transformation of Lactospora verrucosum. Incubate at 28°C on a PDA plate. Myrothecium verrucaria Once fresh spores have grown, wash them off with sterile water and prepare a 1×10⁻⁶ solution. 5 concentration per mL; Successfully converted to pBARGPE1-hygro- MvLac5 Agrobacterium tumefaciens EHA105 expressing the vector was inoculated into YEB liquid medium containing 25 μg / mL rifampin and 100 μg / mL ampicillin and cultured. After centrifugation and discarding the supernatant, the bacterial cells were transferred to IM liquid medium and cultured at 28°C and 160 rpm until OD200. 600 =0.5-0.8 to obtain the positive transformant solution; Take 100 μL of the above Myrothecium verrucaria Fresh spores and the above positive transformant solution were mixed and spread on IM medium containing 0.2 mM AS on cellophane. After co-culturing at 22.5℃ in the dark for 72 h, the cellophane was transferred to MM medium containing 200 μg / mL hygromycin and 0.2 mmol / L cefotaxime and cultured at 30℃. d. Screening and validation of transformants Agrobacterium EHA105 and Myrothecium verrucaria The bacteria were co-cultured at 30℃ for 4 days, and colonies with hygromycin B resistance growing on the surface of MM medium were observed. Transformants were randomly selected and screened again in resistant medium, and genomic DNA of the transformants was extracted using a fungal genomic DNA extraction kit for PCR verification. Wild-type... Myrothecium verrucaria Genomic DNA was used as a negative control; strains with correct PCR verification results were selected and frozen in 50% glycerol. e. Real-time quantitative PCR detection of laccase gene expression levels Genomic DNA contaminating the total RNA was removed using a reverse transcription kit, and the resulting cDNA was reverse transcribed. Using the cDNA as a template, the expression level of the laccase gene was quantitatively detected in real time using a quantitative fluorescence kit. After the reaction, 2... - Ct The relative expression levels of different target genes were calculated, and expression level analysis was performed; 18S RNA was used as an internal control for gene expression levels. (4) Overexpression MvLac5 Laccase activity assay and lignin degradation by engineered bacteria a. Laccase activity assay Laccase activity was determined using the 2,2'-adiazono-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. b. Determination of lignin degradation rate The absorbance of alkali lignin in the fermentation broth of each strain was measured at 230 nm using a multifunctional microplate reader. The content of alkali lignin in the culture medium was calculated according to the alkali lignin standard curve, and the removal rate of alkali lignin was calculated according to the formula: Alkali lignin removal rate = (C1 - C2) / C1 100%.

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