Methanogenic lyases structurally homologous to PeiR lyase and their applications

Abstract

This invention discloses a methanogenic lyase homologous to PeiR lyase and its applications. The PeiR lyase protein, targeting the peptide bonds of the methanogenic cell wall, participates in the biological process of hydrolyzing the archaea cell wall, effectively killing methanogens and reducing methane production. Proteins homologous to this lyase possess potential methane-reducing potential. This invention utilizes a series of bioinformatics software to identify protein sequences encoded by methanogenic viruses from a large amount of rumen microbial virome sequencing data, and analyzes the structural homology between these proteins and PeiR. Ultimately, this invention successfully obtained a series of methanogenic lyases homologous to PeiR. To achieve the expression of these lyases, this invention designed multiple primers to synthesize the target sequences and successfully expressed them in a prokaryotic expression system. In vitro gas production experiments with the crude enzyme solution showed that these lyases are effective in reducing methane production.

Description

Technical Field

[0001] This invention relates to the fields of protein function prediction and genetic engineering, specifically to a methanogenic lyase that is structurally homologous to PeiR lyase and its applications. Background Technology

[0002] Methane has a significant impact on global climate change, being the second largest greenhouse gas after carbon dioxide. Ruminants produce large amounts of methane during digestion, making them a major source of methane emissions. The main reason for this is that rumen microorganisms can break down plant cell wall components such as cellulose and hemicellulose, producing intermediate products such as volatile fatty acids, CO2, and hydrogen, which are then utilized by methanogenic bacteria to generate methane.

[0003] Methanogens belong to the archaea, a group of microorganisms distinct from bacteria in terms of evolutionary system, and their cell wall composition differs significantly from that of bacteria. Pseudopeptidoglycan is one of the different cell wall polymers found in archaea, discovered only in the Methanobacteria and Methanogenicales orders. Pseudopeptidoglycan shares a similar overall three-dimensional structure with bacterial cell wall peptidoglycan. The unique characteristics of the methanogenous pseudopeptidoglycan cell wall are mainly reflected in the following aspects: First, the pseudopeptidoglycan cell wall contains archaea-specific sugars, with its glycan backbone being N-acetylamyric acid. Second, it uses β-1,3-glycosidic bonds to link N-acetylglucosamine or N-acetylglucosamine to the glycan backbone. Finally, it lacks D-type amino acids in its peptide chains and uses ε- and γ-isopeptide bonds for cross-linking between peptides. These differences lead to resistance in methanogens containing a pseudopeptidoglycan layer to lysozyme and other bacterial cell wall hydrolases. Therefore, it is necessary to further develop enzymes capable of hydrolyzing methanogenous pseudopeptidoglycan.

[0004] PeiR is a lyase of the rumen-producing methanogen *Methanobrevibacter ruminantium* M1. This enzyme has been shown to reduce methane production in a continuously cultured rumen mimicry system by 15% within 11 days (Reference: Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions). However, the structure of PeiR is relatively unstable, which is not conducive to its subsequent application. Therefore, this invention uses the structural homology software Foldseek to assess the homology between proteins and PeiR, discovering a series of methanogenetic lyases. After obtaining crude enzyme solutions through heterologous expression, in vitro gas production effects were evaluated. The results showed that the crude enzyme solution containing this lyase significantly reduced methane production in rumen fluid, demonstrating the research and application potential of this lyase in the field of methane emission reduction in ruminants. Summary of the Invention

[0005] The purpose of this invention is to address the current situation in livestock farming, especially ruminant production, where methane production is high and there is a lack of green and effective methane inhibitors. Utilizing the growing resources of microbiome sequencing data and cutting-edge technologies in deep learning and computational biology, a series of methanogenic bacterial lysins, Pei444, Pei522, and Pei523, with structures highly similar to PeiR, were obtained through data mining. After expression in a prokaryotic system, the crude enzyme solution of the recombinant protein exhibited good inhibitory effects on methane production.

[0006] The objective of this invention is achieved through the following technical solution: a methanogenic lyase that is structurally homologous to PeiR lyase, wherein the amino acid sequence of the lyase is one of those shown in SEQ ID NO.1 to SEQ ID NO.3.

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

[0008] (1) Rumen metagenomic data collection and archaea virus protein mining:

[0009] Published rumen metagenomic assembly, metagenomic assembly genome, and virome data were collected. Viral genomes were mined using the geNomad virus identification software. CheckV was used to remove multi-host contamination, incomplete viral genomes, and host sequence contamination. Species-level redundancy removal was performed using a threshold of 95% average nucleotide similarity and 85% alignment coverage to obtain viral operational taxonomic units (vOTUs). iPhoP was used to predict potential hosts for vOTUs and to retain viral genomes infecting Methanobacteria archaea. Prodigal-gv was further used to predict encoded proteins. The Merops peptidase database was used to annotate whether the obtained proteins belonged to peptidases, and non-peptidase proteins were removed. To obtain protein structure information, AlphaFold2 software was used to predict protein structures based on sequences.

[0010] (2) Proteins based on structural homology mining and PeiR homology:

[0011] The protein structure files obtained in step (1) were compared with PeiR using Foldseek software. Based on the set TM-Score threshold, proteins that are structurally homologous to the template lysin PeiR were identified, and methanogenic lysins were obtained.

[0012] Furthermore, the specific process for determining whether a protein belongs to a peptidase is as follows: based on the fact that the target substrate is a peptide chain between sugar backbones, the obtained viral protein is annotated by comparing it with the Merops peptidase database using Diamond blastp, and the protein sequence that is a peptidase is retained for subsequent analysis.

[0013] Furthermore, the protein structure was discretized into sequences on the 3Di alphabet, where each letter of 3Di describes the tertiary interaction between an amino acid and its nearest amino acid. A TM-Score ≥ 0.6 was set as the homology screening threshold. Peptidases that met this threshold and were structurally similar to PeiR were classified as PeiR-like proteins and labeled as 1, while the rest were PeiR-non-homologous proteins and labeled as 0.

[0014] Furthermore, based on a recombinant vector and recombinant bacteria containing the methanogenic lyase gene, fermentation-induced expression was performed, and the target protein was obtained through subsequent purification. The effect of the target protein on in vitro microbial methane production was then evaluated.

[0015] On the other hand, the present invention also provides the application of a methanogenic lyase homologous to PeiR lyase in inhibiting methane production.

[0016] The beneficial effects of this invention are as follows: This invention extensively collected a large amount of rumen microbial virome sequencing data, and used a series of bioinformatics software to identify the protein sequences encoded by methanogenic viruses. Based on this, the structural homology between these proteins and PeiR was analyzed. Ultimately, this invention successfully obtained a series of methanogenic lyases structurally homologous to PeiR. To achieve the expression of these lyases, this invention designed multiple primers to synthesize the target sequences and successfully expressed them in a prokaryotic expression system. In vitro gas production experiments with the crude enzyme solution showed that these lyases are effective in reducing methane production. These methanogenic lyases demonstrate good research value and production application potential, laying the foundation for future applications in the field of methane emission reduction. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 It is a PCR product of a cloned methanogenic lysin;

[0019] Figure 2 This is an SDS-PAGE analysis diagram of the crude enzyme solution of recombinant protein;

[0020] Figure 3 This refers to the effect of recombinant proteins on methane production. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that any changes and modifications made to the present invention by those skilled in the art without departing from the concept of the present invention are within the scope of protection of the present invention.

[0022] This invention provides a methanogenic lyase homologous to PeiR lyase, along with its discovery and characterization methods. The invention also analyzes its application effect in inhibiting methane production, including microbiome data mining, sequence homology search, gene synthesis and protein expression, and evaluation of methane inhibition effects. The specific steps are as follows:

[0023] 1. Microbiome data mining

[0024] (1) Data collection and virus identification

[0025] Literature related to the rumen microbiome and virome was collected, and corresponding metagenomic assembly data, metagenomic assembly genome data, and viral genome data were downloaded. Combined with the assembly results of self-tested rumen metagenomic data, the data were input into geNomad software, which can combine gene content and deep neural network information to identify viruses. The viral genomes were then mined and confirmed. CheckV software was used to determine the integrity and contamination of the virus to remove low-quality genomes. At the same time, potential host sequence contamination at both ends of the viral sequence was removed to improve the accuracy of protein coding function identification.

[0026] (2) Database construction and host prediction

[0027] Using 95% average nucleotide similarity and 85% alignment coverage as thresholds, species-level redundancy removal was performed to obtain a ruminant rumen virus genome database. The virus-host relationship prediction tool iPhoP was then used to add 791 non-redundant rumen archaea genomes as candidate hosts to its original reference host genome database to increase specificity. Virus hosts were predicted through pre-virus alignment, CRISPR spacer alignment, and deep learning feature capture of viral genomes, while retaining viral genomes infecting methanogens of the Methanobacterales order.

[0028] (3) Protein screening and structure prediction

[0029] The meta-modality of the prodigal-gv software was used to predict the encoded viral protein set. Based on the target substrate being peptide chains between glycoskeleton components in the archaea cell wall, the obtained viral proteins were annotated using Diamond BlastP alignment against the Merops peptidase database, retaining the peptidase protein sequences for subsequent analysis. To obtain protein structure information, the AlphaFold2 deep learning-based software was used in monomer mode to predict protein structures, yielding five results for each protein. The best structure representative was selected based on the average pLDDT score for further analysis.

[0030] 2. Protein structure homology search

[0031] While sequence similarity-based homology searches can uncover a certain number of homologous proteins, detecting distant evolutionary relationships from sequences remains challenging. Protein structures differentiate and mutate more slowly than sequences; therefore, detecting similarity between protein structures using 3D structures offers higher sensitivity and can uncover proteins that sequence homology methods cannot detect. The structural homology search involves using the structural homology software Foldseek (9.427df8a) for structure alignment, discretizing protein structures into sequences on a 3Di alphabet, using a pre-trained 3Di substitution matrix, and applying k-mer and gap-free alignment pre-filters from MMseqs2 to the 3Di sequence search. By default, Smith-Waterman local comparisons combining 3Di and amino acid substitution scores are used to obtain high-scoring matches. A TM-Score ≥ 0.6 is set as the homology screening threshold; proteins meeting this threshold are classified as PeiR homologs. These three proteins do not share sequence homology with PeiR, demonstrating the necessity and novelty of structural homology searches.

[0032] The three methanogenic lysins obtained in this invention are structurally highly similar to PeiR, as shown in the table below:

[0033]

[0034] 3. Heterologous expression of lyase

[0035] (1) Synthesis of lyase gene and construction of recombinant plasmid

[0036] The gene sequence of the target protein was designed in segments, and the multiple segments were assembled into a complete target sequence using overlap PCR. The pET-30a(+) vector was linearized by double digestion with XhoI and NdeI restriction endonucleases. The target DNA fragment was ligated to the linearized vector using seamless cloning technology, transformed into E. coli DH5α competent cells, and positive clones were obtained by kanamycin selection. After expansion, the clones were preserved.

[0037] (2) Transformation of recombinant plasmids and construction of expression strains

[0038] High-purity and high-concentration recombinant plasmid Pei_pET-30a(+) was extracted using a plasmid miniprep kit and introduced into *E. coli* BL21(DE3) competent cells via heat shock. After growth recovery, positive clones were obtained by kanamycin selection. Figure 1 As shown, the engineered strain was preserved after colony PCR identification and Sanger sequencing verification of the gene sequence correctness.

[0039] (3) Recombinant protein induced expression

[0040] The validated engineered strain was inoculated into LB medium containing kanamycin for expansion culture. When the bacterial culture OD... 600 When the concentration reached 0.6, isopropyl β-D-1-thiogalactoside (IPTG) was added to a final concentration of 0.25 mM, and expression was induced at 16°C for 18 hours. After centrifugation to collect the bacterial cells, lysis buffer was added for resuspending and cell debris was removed to obtain the crude enzyme solution. Protein expression was verified by SDS-PAGE electrophoresis combined with Coomassie brilliant blue staining, and the results are as follows. Figure 2 As shown.

[0041] 4. Evaluation of the effect of recombinant protein on in vitro microbial methane production

[0042] The effect of recombinant protein on microbial methane production was evaluated using in vitro gas generation technology. Rumen fluid from three dairy cows was extracted using a vacuum pump, mixed, filtered through four layers of gauze, and then added to gas-generating bottles at a ratio of 5 mL:45 mL with artificial saliva as the culture substrate. The fermentation substrate was a TMR diet (collected from the farm), with 500 mg of dry matter added to each gas-generating bottle. The treatment group received 1 mL of crude enzyme solution, and the treatment was repeated 10 times. The control group (CON) received no recombinant protein, and 1 mL of pure water served as a negative control. After incubation at 39℃ for 12 h, 24 h, and 48 h, the pressure inside the gas-generating bottles was read using a pressure sensor, and the gas was collected.

[0043] Gas production: Calculation formula

[0044] GP t P represents the gas production (mL) of the sample during time period t; t Vt represents the pressure (mPa) read during time interval t; V0 represents the bottle volume; 101.3 represents the standard atmospheric pressure (mPa); and W represents the dry weight of the sample. The total accumulated gas production during the gas production process is the sum of the gas production amounts in each time interval.

[0045] Methane production: The methane content of the collected gas was determined using a gas chromatograph. Methane production = gas production × methane content. The results show ( Figure 3Proteins Pei444, Pei522, and Pei523 can significantly reduce methane production by rumen microorganisms.

[0046] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A methanogenic lyase structurally homologous to PeiR lyase, characterized in that, The amino acid sequence of this lyase is shown in SEQ ID NO.

1.

2. The application of a methanogenic lyase based on the structure of PeiR lyase as described in claim 1 in inhibiting methane production.

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