A gene MvLac5 derived from *Lycium verruciformis* and its application

Abstract

The gene derived from *Lysimachia verrucae* provided by this invention MvLac5 This is a gene that encodes laccase. Laccase is one of the key enzymes in lignin degradation, and 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. This invention, through screening the whole genome sequence of *Lactobacillus verrucae*, predicted a gene that may encode laccase and named it... MvLac5 Its nucleotide sequence is shown in SEQ ID No. 1, and the gene was cloned and recombined to obtain Pichia pastoris, yielding GS115-pPICZαB-. MvLac5 The activity of laccase in the fermentation broth was measured, and its activity was 1.645 U / L, proving that the gene... MvLac5 It is the gene that encodes laccase.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the gene MvLac5 derived from *Lactobacillus verrucae* in encoding laccase. Background Technology

[0002] As a major agricultural country, my country boasts abundant crop resources and high yields, but its annual production of agricultural waste (such as straw) ranks first in the world. Crop straw is an important biomass resource, primarily composed of cellulose, hemicellulose, and lignin, but its comprehensive utilization level urgently needs improvement. High-value utilization of lignocellulose raw materials can effectively alleviate the energy crisis, protect the ecological environment, and further promote sustainable agricultural development. Currently, the utilization of lignocellulose raw materials mainly focuses on feed, fertilizer, substrate, materials, and energy. In the process of biomass resource utilization, it has been found that the complex structure of lignin, tightly encapsulating cellulose and hemicellulose, is a key factor affecting the efficient utilization of lignocellulose raw materials. In recent years, scholars have begun to focus on the degradation of lignin in plant biomass. Studies have shown that the effective depolymerization of lignin can break down the physical barrier of lignocellulose, a crucial process for its biological utilization, and 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.

[0003] Microorganisms release specialized extracellular enzymes that act on lignin in plant cell walls, causing its initial degradation. These degradation reactions release highly reactive non-specific free radicals, triggering subsequent cleavage reactions. Complete lignin degradation requires the synergistic action of multiple enzymes. Besides lignin peroxidase (LiP), manganese peroxidase (MnP), multifunctional peroxidase (VP), and laccase, glyoxal oxidase, catalase, aryl alcohol oxidase, glucose oxidase, phenol oxidase, p-coumaroyl esterase, and neoferuloyl esterase are also closely related to lignin degradation. Among these, 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 to bacterial laccase, fungal laccase has a higher redox potential and can oxidize a wider range of substrates.

[0004] Fungal laccases have attracted particular attention due to the stability of their extracellular enzyme components as secretions. Studies have shown that most fungal species, including endophytic fungi, produce at least one laccase. Laccases are found in basidiomycetes, ascomycetes, and deuteromycetes, and are particularly abundant in many white-rot basidiomycetes. Currently, high-laccase-producing fungal strains are mainly concentrated in genera such as *Coriolus*, *Antrodiella*, *Fomes fomentarius*, *Ganoderma*, *Gloeophyllum sepiarium*, *Irpex*, *Lentinusedodes*, *Pleurotus*, *Rigidoporus*, *Schizophyllum commune*, and *Trametes*. Myrothecium verrucaria is a genus belonging to the subphylum Deuteromycetes, class Hyphomycetes, order Myrothecoales, and family Myrothecoalesceae. It is widely distributed in plants and soil.

[0005] 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. Summary of the Invention

[0006] The purpose of this invention is to provide a gene MvLac5 derived from *Lactobacillus verrucae* that encodes a laccase that degrades lignin.

[0007] Another object of the present invention is to provide a pair of primers for amplifying the above-mentioned laccase gene MvLac5.

[0008] Another object of the present invention is to provide the application of the above-mentioned laccase gene MvLac5 in encoding laccase.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A gene MvLac5 derived from *Russula verrucae* has the nucleotide sequence shown in SEQ ID No. 1.

[0011] 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.

[0012] Application of the gene MvLac5 derived from *Lycium verrucosum* in the degradation of lignin.

[0013] Furthermore, this invention provides the application of the above-mentioned gene in encoding laccase, specifically implemented through the following method:

[0014] (1) Cloning of the laccase gene MvLac5

[0015] 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.

[0016] 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.

[0017] (2) Heterologous expression of laccase gene MvLac5 in Pichia pastoris

[0018] 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 overnight at 16℃. 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.

[0019] Successfully transformed single colonies were picked 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 by the ABTS method.

[0020] The beneficial effects obtained by this invention are as follows:

[0021] This invention provides the gene MvLac5 derived from *Lactobacillus verrucae*. Extensive research revealed that this gene encodes laccase, which was named MvLac5. Its nucleotide sequence is shown in SEQ ID No. 1. The gene was cloned and recombined into *Pichia pastoris* to obtain GS115-pPICZαB-MvLac5. Laccase activity in the fermentation broth was measured to be 1.645 U / L, confirming that gene MvLac5 encodes laccase. Laccase is a key enzyme in lignin degradation. The high-value utilization of lignocellulose raw materials can effectively alleviate the energy crisis, protect the ecological environment, and further promote sustainable agricultural development. Among these, fungal laccases have attracted particular attention due to the stability of their extracellular enzyme components as secretions. Attached Figure Description

[0022] Figure 1 Electrophoresis image of PCR amplification of the target gene (M: Marker DL5000; Lane 1: PCR amplification of the target gene).

[0023] Figure 2 Electrophoresis image for bacterial culture PCR verification (M: Marker DL5000; lanes 1-6: bacterial culture PCR verification). Figure 3 Electrophoresis image of recombinant plasmid double digestion verification (M: Marker DL5000; Lane 1: pMD-19T-MvLac5 plasmid double digestion verification).

[0024] Figure 4 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).

[0025] Figure 5 Electrophoresis image for bacterial culture PCR verification (M: Marker DL5000; lanes 1-6: bacterial culture PCR verification). Figure 6 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).

[0026] Figure 7 Electrophoresis image of Sac I single enzyme digestion recovery (M: Marker DL5000; Lane 1: pPICZαB-MvLac5 plasmid single enzyme digestion result).

[0027] Figure 8 : Genomic PCR validation electrophoresis image (M: Marker DL5000; lanes 1-3: genomic PCR validation). Specific Implementation

[0028] 1. Research Methods

[0029] 1.1 Cloning and Heterologous Expression of Laccase Gene MvLac5

[0030] 1.1.1 Cloning of the laccase gene MvLac5

[0031] 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.

[0032] 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℃.

[0033] 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 plasmid was then transformed into E. coli DH5α using the heat shock transformation method. The plasmid was sent to a company (Sangon Biotech) for sequencing. The results were compared using DNAman software to analyze whether the cloned target gene had undergone nucleotide site mutations.

[0034] 1.1.2 Heterologous expression of laccase gene MvLac5 in Pichia pastoris

[0035] 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. 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.

[0036] 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.

[0037] 2. Results

[0038] 2.1 Cloning and Heterologous Expression of Lacase Gene MvLac5

[0039] 2.1.1 Construction of the laccase gene cloning vector

[0040] (1) PCR amplification of the target fragment

[0041] 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 1 As shown in the figure, a clear band appears at the target band size, proving that the target fragment was successfully amplified.

[0042] (2) Construction of pMD-19T cloning vector

[0043] 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 2As 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 and cultured. Plasmid was extracted and double-digested with EcoRI and KpnI for verification. The results are as follows... Figure 3 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.

[0044] 2.1.2 Construction of the expression vector for the laccase gene MvLac5

[0045] First, the target fragment and the pPICZαB vector were double-digested using EcoRI and KpnI, respectively. The results are as follows: Figure 4 As shown, the enzyme digestion bands were recovered via gel electrophoresis. The digestion bands were clear and uniform, and corresponded to the length of the target fragment. The recovered enzyme digestion products were ligated and transformed into *E. coli* DH5α. Six single colonies were selected and cultured in LB medium for colony PCR verification. Agarose gel electrophoresis results are shown below. Figure 5 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 6 As shown, lanes 2 and 4 both showed two bands after double enzyme digestion, 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.

[0046] 2.1.3 Screening and Identification of Recombinant Strains

[0047] 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 7 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 8 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.

[0048] 2.1.4 Assay of Laccase Activity of Recombinant Pichia pastoris GS115-pPICZαB-MvLac5

[0049] 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.

Claims

1. A gene derived from *Lysimachia verrucae*. MvLac5 Its features are: Its nucleotide sequence is shown in SEQ ID No.

1.

2. A pair for amplifying the gene as described in claim 1 MvLac5 The primers are characterized by: 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.

3. A gene as described in claim 1 MvLac5 Application in the degradation of lignin.

4. The gene as described in claim 1 MvLac5 Its application in encoding laccase is characterized by: Specifically, this will be implemented through the following methods: (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 overnight at 16°C using T4 ligase. 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 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. Successfully transformed single colonies were picked 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 by the ABTS method.

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