Methanogen lyase homologous with PeiR lyase sequence and application of methanogen lyase

Abstract

The invention discloses methanogen lyase homologous with a PeiR lyase sequence and application of the methanogen lyase, and aims to solve the problem that PeiR lyase protein of a methanogen cell wall peptide bond can participate in a biological process of hydrolyzing archaea cell walls, effectively kill methanogen and reduce methane production, and protein homologous with the PeiR lyase protein has potential methane reduction potential. According to the invention, a series of biological information software is utilized to identify protein sequences coded by methanogens virus from rumen microbiome sequencing data, and sequence homology between the proteins and PeiR protein is further analyzed to obtain a series of methanogens lyases. In order to realize the expression of the lyase, multiple segments of primers are designed to synthesize a target sequence, and the target sequence is successfully expressed in a prokaryotic expression system. Through the detection of crude enzyme liquid, the effectiveness of the lyase in the aspect of reducing the methane yield is verified in an in-vitro gas production test.

Description

Technical Field

[0001] The present invention relates to the fields of protein function prediction and genetic engineering, and particularly relates to a methanogenic archaeal lyase homologous to the PeiR lyase sequence and its application. Background Art

[0002] Methane has a significant impact on global climate change. Its global warming potential over a 20-year scale is 82.5 times that of CO2. Ruminants produce a large amount of methane during digestion and are an important source of methane emissions. Rumen microorganisms can decompose plant cell wall components such as cellulose and hemicellulose to produce intermediate products such as volatile fatty acids, CO2, and hydrogen, which are then utilized by methanogens to generate methane.

[0003] Methanogens belong to archaea, a unique group of microorganisms with a distinct phylogenetic evolution different from bacteria, and there are significant differences in the composition of their cell walls from those of bacteria. Pseudopeptidoglycan is one of the different cell wall polymers present in archaea and is only found in the orders Methanobacteriales and Methanopyrales. Pseudopeptidoglycan has a similar overall three-dimensional structure to bacterial cell wall peptidoglycan. The particularity of the methanogen pseudopeptidoglycan cell wall is mainly reflected in the following aspects. Firstly, the pseudopeptidoglycan cell wall contains archaeal-specific sugars, and its glycan backbone is N-acetyltalosaminuronic acid. Secondly, β-1,3-glycosidic bonds are used to link N-acetylglucosamine or N-acetylgalactosamine to the glycan backbone. Finally, D-amino acids are lacking in the peptide chain, and ε- and γ-isopeptide bonds are used in the peptide and peptide cross-linking. These differences result in methanogens containing a pseudopeptidoglycan layer being resistant to lysozyme and other bacterial cell wall hydrolases. Therefore, it is necessary to conduct in-depth research and development on enzymes capable of hydrolyzing methanogen pseudopeptidoglycan.

[0004] Previous studies have found a methanogenic archaeal lyase PeiR from the genome of the rumen methanogen Methanobrevibacter ruminantium M1 (Reference: Tailored Nanoparticles With the Potential to Reduce Ruminant Methane Emissions). However, the structure of PeiR is relatively unstable, which is not conducive to subsequent applications. Therefore, the present invention uses sequence homology search to evaluate the homology of proteins with PeiR, and finally discovers a series of methanogenic archaeal lyases, and characterizes their characteristics through heterologous expression and evaluates their in vitro effects, which has important significance and application value for practical production aspects such as reducing methane production in animal husbandry and improving feed conversion efficiency. Summary of the Invention

[0005] The object of the present invention is to address the current situation in the livestock industry, especially in ruminant production, where methane production is high and there is a lack of green and effective methane inhibitors. By utilizing the growing microbial group sequencing data resources and hot technologies in the field of computational biology, a series of methanogen lyases Pei008, Pei099, Pei137, Pei185, Pei231, Pei274, and Pei284 homologous to PeiR are obtained through a series of data mining. After prokaryotic system expression, the crude enzyme solution of the recombinant protein has a good effect on inhibiting methane production.

[0006] The object of the present invention is achieved through the following technical solutions: A methanogen lyase homologous to the PeiR lyase sequence, and the amino acid sequence of the lyase is one of those shown in SEQ ID NO.1 to SEQ ID NO.7.

[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 the published rumen metagenomic assembly data, metagenome-assembled genomes, and virus genome data. Use geNomad to mine and screen the virus genomes, use CheckV to remove multi-host contaminated and genome-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 methanogenic archaea in the order Methanobacteriales, and 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 candidate proteins.

[0010] (2) Mining of proteins homologous to PeiR based on sequence homology:

[0011] Use the sequence alignment software Blastp and the sequence-level homology software HH-suite3 based on hidden Markov models to perform alignment analysis on the candidate proteins screened in step (1) to determine whether they are homologous to the known PeiR lyase at the sequence level; through the above sequence homology analysis, screen out the methanogen lyases homologous to PeiR in the sequence dimension.

[0012] Furthermore, the specific process of determining whether a protein belongs to a peptidase is as follows: Based on the target substrate being a 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] Further, proteins that meet the following threshold conditions are considered to be homologous to PeiR at the sequence level: the E-value of the Blastp alignment result ≤ 1e-5 and the E-value of the HH-suite3 alignment result ≤ 1e-5.

[0014] Further, 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.

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

[0016] Advantages of the present invention: The present invention measured and widely collected a large amount of rumen microbiome sequencing data, used a series of bioinformatics software to identify the protein sequences encoded by methanogenic archaea viruses therefrom, and further analyzed the sequence homology between these proteins and PeiR proteins. Finally, the present invention successfully obtained a series of methanogen lyases. In order to achieve the expression of these lyases, the present invention designed multiple primers to synthesize the target sequence and successfully expressed it in a prokaryotic expression system. Through the detection of the crude enzyme solution, the present invention verified the effectiveness of these lyases in reducing methane production in in vitro gas production experiments. This series of methanogen lyases demonstrated good research value and production application potential, laying a foundation for future applications in the field of methane reduction. Description of the Drawings

[0017] 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, other drawings can be obtained based on these drawings without creative efforts.

[0018] Figure 1 is the PCR product of cloning methanogen lyase;

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

[0020] Figure 3 is the effect of the recombinant protein on methane production. Detailed Embodiments

[0021] The present invention will be further described in detail below 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 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.

[0022] The present invention provides a methanogenic archaeal lyase homologous to the PeiR lyase sequence, and methods for its mining 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:

[0023] 1. Microbiome data mining

[0024] 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 therein. Use the CheckV software to judge the integrity and contamination degree of the viruses to remove low-quality genomes, and at the same time trim the potential host sequence contamination at both ends of the viral sequences to improve the accuracy of protein-coding function identification. Using 95% average nucleotide similarity and 85% alignment coverage as thresholds, perform species-level dereplication to obtain the rumen virus genome database vOTUs of ruminants. Further use the 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 the 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. Based on the target substrate being the peptide chain between sugar skeletons, annotate the obtained viral proteins by Diamond blastp alignment against the Merops peptidase database, and retain the protein sequences that are peptidases for subsequent analysis.

[0025] 2. Sequence homology search

[0026] Sequence homology search integrates two sequence-level homology analysis tools, including Blastp (2.15.0) based on sequence alignment and HH-suite3 (3.3.0) based on hidden Markov models. Since protein sequence data is easily accessible, Blastp is widely used for preliminary homology screening, and in this invention, pairs with an Evalue less than or equal to 1e-5 are selected as reliable results. However, Blastp has limitations in detecting distant homology relationships, so the HMM model of HH-suite3 is further adopted to judge homology by calculating the probability of amino acids at each position, and pairs with an Evalue less than or equal to 1e-5 are also selected as reliable results. Through the double screening of this step, this invention can more comprehensively and accurately identify candidate proteins homologous to PeiR at the sequence dimension. The finally obtained 7 methanogen lyases are homologous to PeiR in both of the above two sequence homology dimensions, as shown in the following table:

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

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

[0032] The high-purity and high-concentration recombinant plasmid Pei_pET-30a(+) was extracted using a plasmid miniprep kit and introduced into Escherichia coli BL21(DE3) competent cells by heat shock method. After recovery growth, positive clones were obtained through kanamycin screening. As Figure 1 shown, after identifying the gene sequence correctness by colony PCR and verifying it by Sanger sequencing, the engineered 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 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 induction 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 are 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 amount 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 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 at time t; P t is the pressure (mPa) read at time t; 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 at each time period.

[0039] Methane production: The methane content of the collected gas was measured by a gas chromatograph. Methane production = gas production × methane content. The results show ( Figure 3 ): Proteins Pei008, Pei099, Pei137, Pei185, Pei231, Pei274, and Pei284 can significantly reduce the methane production of rumen microorganisms.

[0040] The above embodiments are used to explain the present invention, rather than limiting 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 sequence homology to 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.

7.

2. A methanogen lyase having a sequence homologous to PeiR 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: Published rumen metagenomic assembly data, metagenomic assembly genomes and viral genome data were collected, geNomad was used to mine and screen viral genomes, CheckV was used to remove multi-host contamination and incomplete viral genomes and to cut out host sequence contamination, and vOTUs were obtained by species-level de-redundancy with an average nucleotide similarity of 95% and an alignment coverage of 85% as thresholds. iPhoP was used to predict potential hosts of vOTUs and retain viral genomes that infect methanogenic archaea of ​​the Methanobacteriales order, and prodigal-gv was used to predict the encoded proteins. The Merops peptidase database was used to annotate whether the obtained proteins belonged to peptidases and remove the non-peptidase parts to obtain candidate proteins. (2) Proteins based on sequence homology mining and PeiR homology: The candidate proteins screened in step (1) were compared and analyzed using sequence alignment software Blastp and sequence-level homology software HH-suite3 based on the hidden Markov model to determine whether they have homology with the known PeiR lyase at the sequence level; through the above sequence homology analysis, methanogenic lyases that are homologous to PeiR in the sequence dimension were screened out.

3. A methanogen lyase homologous to the PeiR lyase sequence 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 homologous to the PeiR lyase sequence according to claim 2, characterized in that: Proteins that met the following threshold conditions were considered to have homology with PeiR at the sequence level: E-value ≤ 1e-5 for Blastp alignment results and E-value ≤ 1e-5 for HH-suite3 alignment results.

5. A methanogen lyase having homology to the PeiR lyase sequence 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.

6. Use of a methanogenic bacterial lyase with homology to the PeiR lyase sequence in inhibiting methane production.

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