Methanogen lyase enzymes with the same protein family annotation as pei r lyase and uses thereof

Abstract

This invention discloses methanogenic lyases with the same protein family annotation as PeiR lyases and their applications. These enzymes are obtained through homology screening based on the known characteristics of PeiR lyases targeting peptide bonds in the methanogenic cell wall, using the Pfam protein family database annotation. PeiR lyase proteins participate in the biological process of hydrolyzing archaea cell walls, effectively killing methanogens and reducing methane production. Proteins homologous to this protein have potential methane-reducing potential. This invention integrates rumen microbial metagenomic sequencing data, uses a series of bioinformatics software to screen methanogenic viral proteins, and combines Pfam functional domain annotation for homology analysis, ultimately identifying a series of lyases. These lyases were successfully expressed in a prokaryotic expression system, and in vitro gas production experiments also showed that the crude enzyme solution significantly reduced 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 shares the same protein family annotation as the PeiR lyase and its applications. Background Technology

[0002] In recent years, the management of short-lived greenhouse gases has become a key aspect of global climate change governance. Methane, as the short-lived greenhouse gas with the strongest warming effect, can have a greenhouse effect intensity 82.5 times that of carbon dioxide on a 20-year timescale. Rumen microorganisms decompose plant fibers through a unique cellulose and hemicellulose degradation metabolic pathway, producing intermediate products such as volatile fatty acids, CO2, and H2. These intermediate products are then utilized by methanogens and converted into methane. This process not only exacerbates the greenhouse effect but also causes energy loss in feed.

[0003] Methanogens, as a special group within the Archaea domain, exhibit a cell wall structure significantly different from that of bacteria. Studies have shown that the unique pseudopeptidoglycan structure of Methanobacteria and Methanogenicales is a key factor in their resistance to conventional lysozymes. This structural feature is mainly manifested in: (1) the peptidoglycan backbone is composed of N-acetylamuronic acid; (2) an amino sugar modification system linked by β-1,3-glycosidic bonds; and (3) the interpeptide chains, lacking D-type amino acids, employ a γ / ε-isopeptide bond cross-linking network. This unique molecular architecture results in traditional peptidoglycan hydrolases lacking effective cleavage capabilities, posing a significant technical challenge to the development of specific cleavage tools.

[0004] Previous studies have shown that the PeiR enzyme isolated from the genome of the rumen methanogen *Methanobrevibacter ruminantium* M1 possesses cleavage activity (reference: Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions), but its poor structural stability and short enzyme activity retention time have limited its practical application. To address this technical bottleneck, this invention innovatively constructs a protein function mining system: firstly, a large number of peptidase proteins are screened from the rumen microbiome database; then, a Pfam-based homology alignment strategy is used to identify potential high-efficiency cleavage enzyme candidates. Recombinant proteins are obtained through heterologous expression and their in vitro efficacy is verified, providing an innovative solution for constructing a precise rumen methane regulation system. Summary of the Invention

[0005] This invention addresses the problem of high methane emissions and the lack of green and effective methane inhibitors in current livestock farming, especially ruminant production. Utilizing the ever-expanding microbiome sequencing data resources and cutting-edge technologies in bioinformatics and deep learning, a series of methanogenic lyases—Pei036, Pei183, Pei210, Pei325, Pei356, Pei525, Pei750, and Pei945—with the same protein family annotation as PeiR lyase were screened from a series of data. Recombinant enzymes were successfully obtained in a prokaryotic expression system, demonstrating a significant inhibitory effect on methane production.

[0006] The objective of this invention is achieved through the following technical solution: a methanogenic lyase that has the same protein family annotation as the PeiR lyase, wherein the amino acid sequence of the lyase is one of those shown in SEQ ID NO.1 to SEQ ID NO.8.

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

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

[0009] Published rumen metagenomic assembly, metagenomic assembly, and viral genome data were collected. Viral genomes were mined and screened using the virus identification software geNomad. CheckV was used to remove multi-host contamination and incomplete viral genomes and to remove 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 software was used to predict potential hosts of vOTUs and to retain viral genomes infecting methanogens of the Methanobacterales order. Prodigal-gv was used to predict the proteins encoded by the viral genomes.

[0010] (2) Screening proteins homologous to PeiR based on the Pfam annotation homology search method:

[0011] The proteins obtained in step (1) were annotated using the Pfam protein family database to perform functional domain alignment and assess whether they were homologous to PeiR lyase in the protein family annotation dimension. Methanogenic lyases that were homologous to PeiR in this dimension were then screened out.

[0012] Furthermore, if the protein annotation result contains one of the Pfam family domains of PF03412, PF13529, or PF09373, it is considered to be homologous to PeiR in the Pfam annotation dimension; otherwise, it is recorded as non-homologous.

[0013] 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.

[0014] On the other hand, the present invention also provides the application of a series of methanogenic lyases with the same protein family annotation as PeiR lyase in inhibiting methane production.

[0015] The beneficial effects of this invention are as follows: By integrating rumen microbial metagenomic sequencing data, this invention utilizes a series of bioinformatics software to screen methanogenic viral proteins, and combines Pfam functional domain annotation for homology analysis, ultimately identifying a series of lysins. These lysins were successfully expressed in a prokaryotic expression system. Simultaneously, in vitro gas production experiments showed that the crude enzyme solution significantly reduced methane production. This research breaks through the technical bottlenecks of traditional enzyme preparation development and provides an innovative biological solution for greenhouse gas emission reduction in animal husbandry. Attached Figure Description

[0016] 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.

[0017] Figure 1 It is a PCR product of a cloned methanogenic lysin;

[0018] Figure 2 This is an SDS-PAGE analysis diagram of the crude enzyme solution of recombinant protein;

[0019] Figure 3 This refers to the effect of recombinant proteins on methane production. Detailed Implementation

[0020] 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.

[0021] This invention provides a series of methanogenic lyases annotated with the same protein family as PeiR lyases, along with their mining and characterization methods. It also analyzes their application effects 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:

[0022] 1. Microbiome data screening

[0023] 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. The assembly results of self-tested rumen metagenomic data were added, and the data were input into geNomad software, which combines information from gene content and deep neural networks for virus identification, to mine and confirm viral genomes. CheckV software was used to assess viral integrity and contamination levels to remove low-quality genomes and to remove potential host sequence contamination at both ends of the viral sequences, facilitating accurate identification of protein-coding functions. Species-level redundancy removal was performed using a threshold of 95% average nucleotide similarity and 85% alignment coverage to obtain a rumen viral genome database. The virus-host relationship integrated prediction tool iPhoP was further used to add 791 non-redundant rumen archaea genomes as candidate hosts to increase specificity. Viral hosts were predicted using pre-virus alignment, CRISPR spacer alignment, and deep learning feature capture of viral genomes, while retaining viral genomes infecting methanogenic archaea of ​​the Methanobacterales order. The meta-model of prodigal-gv software was then used to predict the encoded viral protein set.

[0024] 2. Homologous search based on Pfam annotations

[0025] Based on Pfam annotation homology search, the obtained protein sequences were functionally annotated using the Pfam protein family database to determine whether they contained functional domains related to PeiR lyases. The Pfam database collects a large number of protein families formed by classifying protein sequence data from the UniProtKB database using multiple sequence alignment and hidden Markov models. It is widely used to infer the domain arrangement and function of proteins through sequence alignment. To screen for target proteins homologous to PeiR, this study set the Pfam annotation targets as PF03412, PF13529, and PF09373. Peptidases whose annotation results belong to these families were considered homologous to PeiR. Using this method, this invention successfully screened eight methanogenic lyases homologous to PeiR along the Pfam annotation dimension, as shown in the table below:

[0026]

[0027]

[0028] 3. Heterologous expression of lyase

[0029] (1) Synthesis of lyase gene and construction of recombinant plasmid

[0030] 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.

[0031] (2) Transformation of recombinant plasmids and construction of expression strains

[0032] 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.

[0033] (3) Recombinant protein induced expression

[0034] 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.

[0035] 4. Evaluation of the effect of recombinant protein on in vitro microbial methane production

[0036] 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.

[0037] Gas production: Calculation formula

[0038] 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 at each time interval.

[0039] Methane production: The methane content of the collected gas was determined using a gas chromatograph. Methane production = gas production rate × methane content. The results show ( Figure 3 Proteins Pei036, Pei183, Pei210, Pei325, Pei356, Pei525, Pei750, and Pei945 can significantly reduce methane production in rumen microbes.

[0040] 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 annotated with the same protein family as 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 protein family annotation of the PeiR lyase described in claim 1, in inhibiting methane production.

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