Methanogen lyase having same protein family annotation as PeiR lyase and use thereof

Abstract

The invention discloses methanogen lyase with the same protein family annotation as PeiR lyase and application of the methanogen lyase, and the methanogen lyase is obtained through homologous screening of Pfam protein family database annotation based on known PeiR lyase characteristics aiming at methanogen cell wall peptide bonds. The PeiR 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. According to the invention, by integrating metagenome sequencing data of rumen microorganisms, screening methanogenic bacterium virus protein by using series bioinformatics software, and combining Pfam functional domain annotation to carry out homologous relationship analysis, a series of lyase is finally identified and is successfully expressed in a prokaryotic expression system, and meanwhile, an in-vitro gas production experiment result also shows that the methanogenic bacterium virus protein can be successfully expressed in a prokaryotic expression system. The crude enzyme liquid can significantly reduce the methane generation amount.

Description

Technical Field

[0001] The present invention relates to the field of protein function prediction and genetic engineering, and particularly to a methanogenic archaeal lyase having the same protein family annotation as PeiR lyase and its application. Background Art

[0002] In recent years, in the governance of global climate change, the control of short-cycle greenhouse gases has become a key link in temperature control. As the short-lived greenhouse gas with the strongest warming effect, the greenhouse effect intensity of methane on a 20-year scale can reach 82.5 times that of carbon dioxide. Rumen microorganisms decompose plant fibers through unique cellulose and hemicellulose degradation and metabolism pathways to produce intermediate products such as volatile fatty acids, CO2, and H2, which are then utilized by methanogens to be converted into methane. This process not only intensifies the greenhouse effect but also causes feed energy loss.

[0003] As a special group in the archaeal domain, methanogens have a cell wall structure significantly different from that of bacteria. Research shows that the unique pseudopeptidoglycan structure of Methanobacteriales and Methanopyrales is the key factor for their resistance to conventional lysozymes. The structural characteristics are mainly manifested in: (1) the glycan backbone is composed of N-acetylaminotaropyranuronic acid; (2) the amino sugar modification system linked by β-1,3-glycosidic bonds; (3) due to the lack of D-amino acids between peptide chains, a γ / ε-isopeptide bond cross-linking network is adopted. This unique molecular architecture results in the lack of effective cleavage ability of traditional peptidoglycan hydrolases for them, which poses a major technical challenge for the development of specific cleavage tools.

[0004] In previous studies, although the PeiR enzyme isolated from the genome of rumen methanogen Methanobrevibacter ruminantium M1 has lytic activity (Reference: Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions), its defects such as poor structural stability and short enzyme activity maintenance time have restricted its practical application. To address this technical bottleneck, the present invention innovatively constructs a protein function mining system: first, a large number of peptidase proteins are screened from the rumen microbiome database, and then potential high-efficiency lyase candidate molecules are locked through a homologous alignment strategy based on Pfam annotation. Recombinant proteins are obtained through heterologous expression and their in vitro effects are verified, providing an innovative solution for the construction of a precise rumen methane regulation system. Summary of the Invention

[0005] In view of the problems of high methane emissions and the lack of green and effective methane inhibitors in current animal husbandry, especially in ruminant production, the present invention utilizes the continuously expanding microbial group sequencing data resources and the hot technologies in the fields of bioinformatics and deep learning to screen a series of methanogen lyases Pei036, Pei183, Pei210, Pei325, Pei356, Pei525, Pei750, and Pei945 with the same protein family annotation as PeiR lyase from a series of data, and successfully obtains recombinant enzymes in a prokaryotic expression system, showing a significant inhibitory effect on methane production.

[0006] The object of the present invention is achieved by the following technical solutions: A methanogen lyase having the same protein family annotation as PeiR lyase, and 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 process of screening the lyase is as follows:

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

[0009] Collect the published rumen metagenomic assemblies, metagenome-assembled genomes, and viral genome data, use the virus recognition software geNomad to mine and screen the viral genomes, use CheckV to remove multi-host contaminated and genome-incomplete viral 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 viral operational taxonomic units vOTUs. Use the iPhoP software to predict the potential hosts of vOTUs and retain the viral genomes that infect methanogens in the order Methanobacteriales, and use prodigal-gv to predict the proteins encoded by the viral genomes;

[0010] (2) Screening for proteins homologous to PeiR using the Pfam annotation-based homologous search method:

[0011] Use the Pfam protein family database annotation to perform functional domain alignment on the proteins obtained in step (1), evaluate whether they are homologous to PeiR lyase in terms of protein family annotation dimension, and screen out methanogen lyases that are homologous to PeiR in this dimension.

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

[0013] 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.

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

[0015] Beneficial effects of the present invention: By integrating rumen microbial metagenomic sequencing data, a series of bioinformatics software was used to screen for methanogen virus proteins, and homology analysis was carried out in combination with Pfam functional domain annotation. Finally, a series of lyases were identified and successfully expressed in a prokaryotic expression system. At the same time, the in vitro gas production experiment results also showed that the crude enzyme solution could significantly reduce the methane production. This research has broken through the technical bottleneck of traditional enzyme preparation development and provided an innovative biological solution for greenhouse gas reduction in the livestock industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] 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 use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 is the PCR product of cloning the methanogen lyase;

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

[0019] Figure 3 is the effect of the recombinant protein on the methane production. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0020] The following further details the present invention by combining specific implementation schemes. The following implementation schemes 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 form of change and deformation made to the present invention without departing from the concept of the present invention belongs to the protection scope of the present invention.

[0021] The present invention provides a series of methanogen lyases with the same protein family annotation as PeiR lyase, and methods for their discovery, characterization, and analysis of their application effects in inhibiting methane production, including microbiome data mining, sequence homology search, gene synthesis, protein expression, and methane inhibition effect evaluation. The specific steps are as follows:

[0022] 1. Microbiome data screening

[0023] Collect relevant literature on rumen microbiome and virome, download the corresponding metagenomic assembly data, metagenome-assembled genome data, and viral genome data, and add the assembly results of self-tested rumen metagenomic data. Input them into the software geNomad that combines information from gene content and deep neural networks for virus judgment to mine and confirm the viral genomes. Use the CheckV software to judge the integrity and contamination of the viruses to remove low-quality genomes, and at the same time trim potential host sequence contamination at both ends of the viral sequences to facilitate accurate identification of the functions encoded by the proteins. Perform dereplication at the species level with an average nucleotide similarity of 95% and an alignment coverage of 85% as the threshold to obtain a rumen viral genome database. Further use the integrated virus-host relationship prediction tool iPhoP, and add 791 non-redundant rumen archaeal genomes to its original library as candidate hosts to increase specificity. Predict virus hosts through means such as provirus alignment, CRISPR spacer alignment, and deep learning feature capture of viral genomes, and retain viral genomes that infect methanogenic archaea of the order Methanobacteriales. Further use the meta mode of the prodigal-gv software to predict the encoded viral protein set.

[0024] 2. Homology search based on Pfam annotation

[0025] The homology search based on Pfam annotation performs functional domain annotation on the obtained protein sequences through the Pfam protein family database to determine whether they contain functional domains related to PeiR lyase. The Pfam database collects a large number of protein families formed by classifying protein sequence data in the UniProtKB database using multiple sequence alignment and hidden Markov models, and is widely used to infer the domain arrangement form 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 with annotation results belonging to the above families were considered to be homologous to PeiR. Through this method, the present invention successfully screened out 8 methanogen lyases homologous to PeiR in terms of Pfam annotation, as shown in the following table:

[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 multiple segments were assembled into a complete target sequence by Overlap PCR. The pET-30a(+) vector was double-digested and linearized with XhoⅠ and NdeⅠ restriction endonucleases. The target DNA fragment was ligated to the linearized vector using seamless cloning technology, and then transformed into Escherichia coli DH5α competent cells. Positive clones were obtained by screening with kanamycin, and after amplification culture, they were preserved.

[0031] (2) Transformation of recombinant plasmid and construction of expression strain

[0032] A plasmid miniprep kit was used to extract high-purity and high-concentration recombinant plasmid Pei_pET-30a(+), which was introduced into Escherichia coli BL21(DE3) competent cells by heat shock method. After recovery growth, positive clones were obtained by screening with kanamycin. As Figure 1 shown, after verifying the correctness of the gene sequence by colony PCR identification and Sanger sequencing, the engineering strain was preserved.

[0033] (3) Induced expression of recombinant protein

[0034] The verified engineering strain was inoculated into LB medium containing kanamycin for expansion culture. When the OD of the bacterial liquid 600 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.

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

[0036] 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 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. When culturing in a 39 °C incubator for 12 h, 24 h, and 48 h, the pressure in the gas production bottle was read with a pressure sensor, and the gas was collected.

[0037] Gas production: calculation formula

[0038] 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 volume of the bottle; 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

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

[0040] The above embodiments are used to explain the present invention, rather than 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 with the same protein family annotation as PeiR 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.

8.

2. The methanogen lyase having the same protein family annotation as PeiR lyase according to claim 2, characterized in that, The screening process of the lyase is as follows: (1) Integration of rumen metagenomic data and mining of archaeal virus proteins: Collect published rumen metagenomic assemblies, metagenome-assembled genomes, and virus genome data. Use the virus recognition software geNomad to mine and screen virus genomes. Use CheckV to remove multi-host contaminated and genome-incomplete virus genomes and trim host sequence contamination. Perform species-level dereplication with an average nucleotide similarity of 95% and an alignment coverage of 85% as the threshold to obtain virus operational taxonomic units (vOTUs). Use the iPhoP software to predict the potential hosts of vOTUs and retain virus genomes that infect methanogens in the order Methanobacteriales. Use prodigal-gv to predict the proteins encoded by the virus genomes. (2) Screening for proteins homologous to PeiR based on Pfam annotation homology search method: Use the Pfam protein family database annotation to perform functional domain alignment on the proteins obtained in step (1), and evaluate whether they are homologous to the PeiR lyase in terms of protein family annotation dimension, and screen out methanogen lyases homologous to PeiR in this dimension.

3. A methanogen lyase having the same protein family annotation as PeiR lyase according to claim 2, characterized in that If one of the Pfam family domains of PF03412, PF13529, or PF09373 is included in the protein annotation result, it is considered homologous to PeiR in the Pfam annotation dimension; otherwise, it is recorded as non-homologous.

4. A methanogen lyase having the same protein family annotation as PeiR lyase according to claim 1, characterized in that, Based on the recombinant vector and recombinant bacteria containing the methanogen lyase gene, perform fermentation-induced expression, and obtain the target protein through subsequent purification, and evaluate the effect of the target protein on in vitro microbial methane production.

5. Application of a methanogen lyase with the same protein family annotation as the PeiR lyase in inhibiting methane production.

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