Methanogen lyase homologous with PeiP lyase structure and application of methanogen lyase

Abstract

The invention discloses methanogen lyase homologous with a PeiP lyase structure and application of the methanogen lyase, and the methanogen lyase is screened from rumen microbiome sequencing data through a structure comparison method based on known PeiP lyase protein characteristics. The PeiP lyase protein can participate in the biological process of hydrolyzing archaea cell walls, methanogens are effectively killed, methane production is reduced, and the protein homologous with the protein has potential methane reduction potential. Protein sequences coded by methanogens viruses are extracted from rumen microbiome sequencing data, the structural similarity between the proteins and PeiP proteins is analyzed by using a bioinformatics tool, and finally two lyases are successfully identified. In order to realize the expression, multiple segments of primers are designed and synthesized to construct a target sequence, and high-efficiency expression is realized in a prokaryotic expression system. In-vitro gas production experiments show that the crude enzyme liquid can significantly reduce methane production.

Description

Technical Field

[0001] The present invention relates to the field of protein function prediction and genetic engineering, and particularly to a methanogen lyase homologous to the structure of PeiP lyase and its application. Background Art

[0002] Methane has a particularly significant impact on global climate change. A large amount of methane is produced during the digestion of ruminants. Rumen microorganisms can decompose plant cell wall components such as cellulose and hemicellulose to generate intermediate products such as volatile fatty acids, CO2, and hydrogen, and methanogens use these substrates to produce methane.

[0003] Methanogens belong to archaea, a unique class of microorganisms with a distinct phylogenetic evolution different from bacteria, and their cell wall components are also different from bacteria. Among them, pseudopeptidoglycan is a cell wall polymer unique to some archaea, only found in Methanobacteriales and Methanopyrales. Pseudopeptidoglycan is similar to bacterial peptidoglycan in the overall three-dimensional structure, but has the following significant characteristics: First, its glycan backbone is composed of N-acetyltalosaminuronic acid. Second, N-acetylglucosamine or N-acetylgalactosamine is linked to the glycan backbone through β-1,3-glycosidic bonds. Finally, the peptide chain lacks D-amino acids and uses ε- and γ-isopeptide bonds in peptide chain cross-linking. These structural characteristics make the pseudopeptidoglycan layer of methanogens resistant to lysozyme and most bacterial cell wall hydrolases. Therefore, it is of great significance to develop enzymes that can specifically hydrolyze methanogen pseudopeptidoglycan.

[0004] Previous studies have found the methanogen lyase PeiP from the prophage genome of methanogen Methanothermobacter wolfeii ΨM100 (Reference: Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains). However, PeiP has low activity at 37°C, requires metal ion activation, and has poor stability, which is not conducive to practical applications. The present invention collects rumen microbiome data from the literature and self-tests, screens potential proteases, and evaluates their similarity to PeiP through structure homology search. Finally, two novel methanogen lyases are discovered. Their functions are characterized by heterologous expression, and their methane reduction effects are evaluated in in vitro experiments. The discovery of this novel lyase is beneficial to reducing methane emissions in the livestock industry and improving the feed conversion efficiency of animals by reducing methane production. Summary of the Invention

[0005] The present invention aims to solve the problems of high methane emissions and the lack of green and efficient methane inhibitors in the livestock industry, especially in the production process of ruminants. By utilizing the growing microbial group sequencing data resources and combining deep learning with computational biology techniques, the present invention screens out two methanogen lyases, Pei429 and Pei513, which are structurally homologous to PeiP, through data mining. After prokaryotic system expression, the crude enzyme solution of the recombinant protein exhibits good methane inhibition effect.

[0006] The object of the present invention is achieved by the following technical solutions: A methanogen lyase structurally homologous to the PeiP lyase, and the amino acid sequence of the lyase is one of those shown in SEQ ID NO.1 to SEQ ID NO.2.

[0007] Furthermore, the process of screening the lyase is as follows:

[0008] (1) Collection of rumen metagenomic data and mining of archaeal virus proteins

[0009] Collect publicly published rumen metagenomic assemblies, metagenome-assembled genomes and virome data, use the virus recognition software geNomad to mine virus genomes, use CheckV to remove multi-host contaminated and genomically incomplete virus genomes and trim host sequence contamination, and perform species-level dereplication with an average nucleotide similarity of 95% and an alignment coverage of 85% as the threshold to obtain vOTUs. Use iPhoP to predict the potential hosts of vOTUs and retain the virus genomes that infect archaea of the order Methanobacteriales, and further use prodigal-gv to predict the encoded proteins; use the Merops peptidase database to annotate whether the obtained proteins belong to peptidases and remove the non-peptidase parts; to obtain protein structure information, use the AlphaFold2 software to predict the protein structure based on the sequence.

[0010] (2) Screening by structural homology alignment

[0011] Use the Foldseek software to perform a structural alignment of the protein structure file set obtained in step (1) and PeiP, and determine the proteins that are structurally homologous to the template lyase PeiP according to the set TM-Score threshold to obtain novel methanogen lyases.

[0012] Furthermore, the specific process of determining whether a protein belongs to a peptidase is as follows: Based on the target substrate being the peptide chain between sugar backbones, align the obtained virus proteins with the Merops peptidase database by Diamond blastp for annotation, and retain the protein sequences that are peptidases for subsequent analysis.

[0013] Furthermore, the ProtParam module in the Biopython SeqUtils package was used to predict the stability of the obtained protein.

[0014] Furthermore, the protein structure was discretized into a sequence on the 3Di alphabet, where each letter of 3Di describes the tertiary structure interaction between an amino acid and its nearest amino acid; a TM-Score ≥ 0.6 was set as the homology screening threshold, and proteins that met this threshold in comparison with PeiP were classified as PeiP homologous proteins.

[0015] Furthermore, based on the recombinant vector and recombinant bacteria containing the methanogen lyase gene, fermentation induction expression was carried out, and the target protein was obtained through subsequent purification, and the effect of the target protein on in vitro microbial methane production was evaluated.

[0016] On the other hand, the present invention also provides an application of a methanogen lyase homologous to the PeiP lyase structure in inhibiting methane production.

[0017] Advantages of the present invention: The present invention extracted the protein sequences encoded by methanogen viruses from rumen microbiome sequencing data, and used bioinformatics tools to analyze the structural similarity of these proteins with PeiP proteins, and finally successfully identified two lyases. To achieve their expression, the present invention designed and synthesized multiple primers to construct the target sequence, and achieved high-efficiency expression in a prokaryotic expression system. In vitro gas production experiments showed that the crude enzyme solution could significantly reduce methane production. These methanogen lyases have important research significance and application prospects, providing important support for the development and application of future methane reduction technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings without creative efforts based on these drawings.

[0019] Figure 1 is the PCR product of cloning the methanogen lyase;

[0020] Figure 2 is the SDS-PAGE analysis diagram of the crude enzyme solution of the recombinant protein;

[0021] Figure 3 is the effect of the recombinant protein on methane production. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention will be further described in detail below by combining specific implementation embodiments. The following implementation embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, any formal changes and deformations made to the present invention without departing from the concept of the present invention fall within the protection scope of the present invention.

[0023] The present invention provides a methanogen lyase homologous to the PeiP lyase structure, and methods for its discovery and characterization, and analyzes its application effect in inhibiting methane production, including the processes of microbiome data mining, sequence homology search, gene synthesis, protein expression, and methane inhibition effect evaluation. The specific steps are as follows:

[0024] 1. Microbiome data mining

[0025] By collecting relevant literature on rumen microbiome and virome, obtaining corresponding metagenomic assembly data, metagenome-assembled genome data, and viral genome data, and combining the self-tested rumen metagenomic assembly results, the geNomad software is used to mine and confirm viral genomes. geNomad can integrate gene content and deep neural network information for virus identification. Subsequently, the CheckV software is used to evaluate the integrity and contamination of viral genomes, remove low-quality genomes, and trim potential host sequence contamination at both ends of the viral sequences to improve the accuracy of protein-coding function identification. And species-level redundancy removal is performed with an average nucleotide similarity of 95% and an alignment coverage of 85% as the threshold to construct a ruminant rumen viral genome database. Further, the virus-host relationship prediction tool iPhoP is used to add 791 non-redundant rumen archaeal genomes as candidate hosts to its original reference host genome library. The virus hosts are predicted by methods such as provirus alignment, CRISPR spacer alignment, and deep learning feature capture of viral genomes, and the viral genomes that infect methanogenic archaea of the order Methanobacteriales are screened out. The prodigal-gv software in meta mode is used to predict the protein set encoded by the virus. Based on the target substrate being the peptide chain between the glycan skeletons of the archaeal cell wall, the viral proteins are aligned with the Merops peptidase database by Diamond blastp, and the peptidase protein sequences are screened out for subsequent analysis. To obtain protein structure information, the monomer mode of the AlphaFold2 software is used to predict the protein structure, and 5 results are generated for each protein. Based on the average pLDDT score, the structure with the highest confidence is selected for subsequent analysis.

[0026] 2. Protein structure homology search

[0027] Although homology search based on sequence similarity can identify a certain number of homologous proteins, detecting distant evolutionary relationships still poses challenges. Since the divergence rate of protein structures is slower than that of sequences, detecting protein similarity through three-dimensional structures can provide higher sensitivity, thereby discovering proteins that cannot be identified by sequence homology methods. The structure homology search uses Foldseek (9.427df8a) for structure alignment, discretizes the protein structure into 3Di letter sequences, and uses a pre-trained 3Di substitution matrix, combined with the k-mer and gapless alignment pre-filter of MMseqs2 for 3Di sequence search. By default, the Smith-Waterman local alignment that combines 3Di and amino acid substitution scores is used to screen out high-score matching results. The TM-Score ≥ 0.6 is set as the homology screening threshold, and proteins that meet this condition are classified as PeiP homologous proteins. These proteins have no homology with PeiP at the sequence level, highlighting the necessity and innovation of structure homology search. The stability of PeiP homologous proteins is predicted using the ProtParam module in the Biopython SeqUtils package. If the instability_index ≤ 40, the protein is considered to be able to exist stably.

[0028] The two novel methanogen lyases obtained in this invention are homologous to PeiP in terms of structure dimension and can exist stably, as shown in the following table:

[0029]

[0030] 3. Heterologous expression of lyase

[0031] (1) Synthesis of lyase gene and construction of recombinant plasmid

[0032] The gene sequence of the target protein is designed in segments, and multiple segments are assembled into a complete target sequence through Overlap PCR. The pET-30a(+) vector is digested linearly with XhoⅠ and NdeⅠ restriction endonucleases, and the target DNA fragment is ligated to the linearized vector using seamless cloning technology, then transformed into Escherichia coli DH5α competent cells. Positive clones are obtained through kanamycin screening, and after amplification culture, they are preserved.

[0033] (2) Transformation of recombinant plasmid and construction of expression strain

[0034] A high-quality recombinant plasmid Pei_pET-30a(+) is extracted using a plasmid mini-prep kit and introduced into Escherichia coli BL21(DE3) competent cells by heat shock method. After recovery growth, positive clones are obtained through kanamycin screening. As Figure 1 shown, after verifying the correctness of the gene sequence by colony PCR identification and Sanger sequencing, the engineered strain is preserved.

[0035] (3) Induced expression of recombinant protein

[0036] The verified engineered strain was inoculated into LB medium containing kanamycin for expansion culture. When the OD600 of the bacterial solution reached 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.25 mM was added, and induced expression was carried out at 16 °C for 18 hours. After centrifuging to collect the bacterial cells, lysis buffer was added to resuspend and remove cell debris to obtain crude enzyme solution. Protein expression was verified by SDS-PAGE electrophoresis combined with Coomassie Brilliant Blue staining, and the results were as Figure 2 shown.

[0037] 4. Evaluation of the effect of recombinant protein on microbial methane production in vitro

[0038] The in vitro gas production technique was used to evaluate the effect of recombinant protein on microbial methane production. The rumen fluid of three dairy cows was extracted by a vacuum pump, mixed, filtered through four layers of gauze, and then injected into a gas production bottle together with artificial saliva at an addition ratio of 5 mL:45 mL as the culture substrate; the fermentation substrate was TMR diet (collected from the pasture), and 500 mg of dry matter feed was added to each gas production bottle. 1 mL of crude enzyme solution was added to the treatment group, and the treatment was repeated 10 times; the control group (CON) did not add recombinant protein, and 1 mL of pure water was used as the negative control. At 12 h, 24 h, and 48 h of incubation in a 39 °C incubator, the pressure in the gas production bottle was read using a pressure sensor, and the gas was collected.

[0039] Gas production: calculation formula

[0040] GP t is the gas production (mL) of the sample in the t time period; P t is the pressure (mPa) read in the t time period; V0 is the bottle volume; 101.3 is the standard atmospheric pressure (mPa); W is the dry matter weight of the sample. The total cumulative gas production during the gas production process is the sum of the gas production in each time period.

[0041] Methane production: The methane content of the collected gas was measured using a gas chromatograph. Methane production = gas production × methane content. The results showed ( Figure 3 ) that proteins Pei429 and Pei513 could significantly reduce the methane production of rumen microorganisms.

[0042] The above embodiments are used to explain and illustrate the present invention, rather than to limit the present invention. Any modifications and changes made within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention.

Claims

1. A methanogen lyase having structural homology to PeiP lyase, characterized in that: The amino acid sequence of the lyase is one of those shown in SEQ ID NO.1 to SEQ ID NO.

2.

2. A methanogen lyase having a structural homology to PeiP lyase according to claim 1, characterized in that: The lytic enzyme screening process is as follows: (1) Rumen metagenomic data collection and archaeal viral protein mining Publicly published rumen metagenomic assemblies, metagenomic assembly genomes, and virome data were collected. The virus identification software geNomad was used to mine viral genomes. CheckV was used to remove multi-host contamination and incomplete viral genomes and to trim host sequence contamination. 95% average nucleotide similarity and 85% alignment coverage were used as thresholds for species-level de-redundancy to obtain vOTUs. iPhoP was used to predict potential hosts of vOTUs and viral genomes that infect Methanobacteriales archaea were retained. Prodigal-gv was further used to predict the encoded proteins. The Merops peptidase database was used to annotate whether the obtained proteins belonged to peptidases and to remove the non-peptidase parts. To obtain protein structure information, AlphaFold2 software was used to predict the protein structure based on sequence. (2) Structural homology comparison and screening The protein structure file set obtained in step (1) was compared with PeiP using Foldseek software, and proteins with structural homology to the template lyase PeiP were determined based on the set TM-Score threshold, thereby obtaining a novel methanogenic lyase.

3. A methanogen lyase having a structural homology to PeiP lyase according to claim 2, characterized in that: The specific process of determining whether a protein is a peptidase is as follows: based on the target substrate being a peptide chain between sugar backbones, the obtained viral protein is compared with the Merops peptidase database annotation by Diamond blastp, and the protein sequence that is a peptidase is retained for subsequent analysis.

4. A methanogen lyase having a structural homology to PeiP lyase according to claim 2, characterized in that: The ProtParam module in the BiopythonSeq Utils package was used to predict the stability of the resulting proteins.

5. A methanogen lyase having a structural homology to PeiP lyase according to claim 2, characterized in that: The protein structure was discretized into a sequence on the 3Di alphabet, where each letter of 3Di describes the interaction of the tertiary structure between an amino acid and its nearest amino acid; TM-Score ≥ 0.6 was set as the homology screening threshold, and proteins that met this threshold in the comparison with PeiP were classified as PeiP homologous proteins.

6. The methanogen lyase having a structural homology to PeiP lyase according to claim 1, characterized in that: Based on the recombinant vector and recombinant bacteria containing the methanogen lyase gene, fermentation-induced expression was carried out, and the target protein was obtained through subsequent purification, and the effect of the target protein on in vitro microbial methane production was evaluated.

7. Application of a methanogenic bacteria lyase with structural homology to PeiP lyase in inhibiting methane production.

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