A mutant of expansin protein from rumen anaerobic fungus and its coding gene and application

Abstract

The application discloses a mutant of expansin protein from rumen anaerobic fungi, a coding gene and application thereof, and belongs to the technical field of biological genetic engineering. The wild-type expansin protein PFSWO2_1 is subjected to site-directed mutation, and mutants with higher expression, better thermal stability and stronger synergistic effect with cellulase, namely the mutants PFSWO2_1-N1, PFSWO2_1-N2 and PFSWO2_1-N3, are obtained. The mutants are successfully expressed in Pichia pastoris, the hydrogen bond network between cellulose molecules is destroyed, the mutants can significantly enhance the enzymolysis efficiency of cellulase on lignocellulose, the mutants can not only improve the utilization rate of agricultural wastes such as straws in feed, but also can inhibit the generation of methane by regulating the fermentation parameters and microbial community structure in the rumen in vitro fermentation process, and the mutants have important application value in reducing the greenhouse gas emission of the animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a rumen anaerobic fungus-derived expansin protein mutant, its encoding gene, and its applications. Background Technology

[0002] The main obstacle to the resource utilization of straw lies in its complex lignocellulose structure—cellulose, hemicellulose, and lignin are tightly cross-linked through ester and ether bonds, forming a dense three-dimensional network structure, resulting in poor palatability and low digestibility. Improving the feed utilization rate of straw can not only alleviate the conflict between humans and livestock for food but also reduce environmental pollution caused by straw burning.

[0003] Expansins are a class of non-hydrolyzable cell wall relaxants that significantly improve the accessibility of substrates to cellulases by disrupting the hydrogen bond network between cellulose microfibrils, thus loosening the cellulose structure. Microbial expansins have been shown to effectively enhance the enzymatic hydrolysis efficiency of lignocellulose. However, currently commercially available expansins are mainly derived from aerobic microorganisms, and their stability and activity in anaerobic environments such as the rumen are limited.

[0004] The rumen, as the most efficient lignocellulose degradation system in nature, possesses a rich resource of functional genes within its microbial community. Current technologies utilize anaerobic fungi... Neocallimastix sp The source of the expansion hormone gene PFS WO2_1, the protein encoded by this gene (PFSWO2_1), has a typical Swollenin domain, but its expression level is low in heterologous expression systems, which limits its industrial application.

[0005] Furthermore, livestock farming is a significant source of greenhouse gas emissions, especially methane. Methane produced during rumen fermentation in ruminants not only contributes to the greenhouse effect but also leads to a waste of feed energy. Therefore, developing green feed additives that can both improve feed utilization and reduce methane emissions is crucial for achieving carbon neutrality in livestock farming. Summary of the Invention

[0006] Based on the above needs, the purpose of this invention is to provide a rumen anaerobic fungus-derived expansin protein mutant, its encoding gene, and its applications. This invention utilizes the LGM-ZA9 mutant of *Trichophyton mentagrophytes* (…). Neocallimastix sp. By performing site-directed mutagenesis on wild-type lignocellulose protein PFSWO2_1, mutants with higher expression levels, better thermal stability, and stronger synergistic effects with cellulase were obtained, which helps to improve the degradation efficiency of lignocellulose and provides a new technical solution for reducing greenhouse gas emissions from animal husbandry.

[0007] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0008] This invention provides a rumen anaerobic fungus-derived expansin protein mutant, the expansin protein mutant being mutant PFSWO2_1-N1, the amino acid sequence of which is shown in SEQ ID No.3, and is obtained by mutating asparagine at position 119 of the wild-type expansin protein with amino acid sequence SEQ ID No.1 to serine.

[0009] The present invention also provides the encoding gene of the rumen anaerobic fungus-derived expansin protein mutant, the nucleotide sequence of which is shown in SEQ ID No. 4.

[0010] The present invention also provides a rumen anaerobic fungus-derived expansin protein mutant, wherein the expansin protein mutant is mutant PFSWO2_1-N2, and the amino acid sequence of mutant PFSWO2_1-N2 is shown in SEQ ID No. 5. It is obtained by mutating valine at position 256 of the expansin protein mutant PFSWO2_1-N1 with amino acid sequence SEQ ID No. 3 to leucine.

[0011] The present invention also provides the encoding gene of the rumen anaerobic fungus-derived expansin protein mutant, the nucleotide sequence of which is shown in SEQ ID No. 6.

[0012] The present invention also provides a rumen anaerobic fungus-derived expansin protein mutant, wherein the expansin protein mutant is mutant PFSWO2_1-N3, and the amino acid sequence of mutant PFSWO2_1-N3 is shown in SEQ ID No.7. It is obtained by mutating threonine at position 338 of the expansin protein mutant PFSWO2_1-N2 with amino acid sequence SEQ ID No.5 to alanine.

[0013] The present invention also provides the encoding gene of the rumen anaerobic fungus-derived expansin protein mutant, the nucleotide sequence of which is shown in SEQ ID No. 8.

[0014] The present invention also provides a recombinant expression vector for the above-mentioned encoding gene.

[0015] The present invention also provides a recombinant strain encoding the above-mentioned gene.

[0016] The present invention also provides the aforementioned expansin protein mutant (mutant PFSWO2_1-N1 or mutant PFSWO2_1-N2 or mutant PFSWO2)_ Application of 1-N3 in the preparation of enzyme preparations for degrading lignocellulose.

[0017] Furthermore, the lignocellulose includes at least one of rice straw, wheat straw, corn stalks, and beet pulp.

[0018] The present invention also provides the aforementioned expansin protein mutant (mutant PFSWO2_1-N1 or mutant PFSWO2_1-N2 or mutant PFSWO2) _ Application of 1-N3 in the preparation of feed additives that reduce rumen methane emissions in ruminants.

[0019] Compared with the prior art, the advantages and beneficial technical effects of the present invention are:

[0020] 1. This invention obtains mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 with higher expression levels and better thermostability by site-directed mutagenesis of wild-type expansin PFSWO2_1 derived from anaerobic fungi. This solves the problem of low expression levels of wild-type protein in heterologous systems and lays the foundation for industrial production.

[0021] 2. The mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 provided by this invention work synergistically with cellulase to increase the yield of reducing sugar by 32%, 39%, and 45%, respectively, effectively improving the enzymatic hydrolysis efficiency and feed utilization rate of agricultural wastes such as rice straw and wheat straw.

[0022] 3. This invention is the first to apply expander to the field of greenhouse gas emission reduction. Rumen fermentation experiments show that pretreatment of rice straw with mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 can significantly inhibit methane generation by controlling fermentation parameters (increasing the proportion of propionic acid and decreasing the acetic acid / propionic acid ratio) and microbial community structure, thereby redirecting electron flow from methane generation to propionic acid synthesis and reducing methane emission intensity by 15-20%. Attached Figure Description

[0023] Figure 1 The expression level of the expansion protein mutant in Pichia pastoris.

[0024] Figure 2 The effects of wild-type expansion protein PFSWO2_1 and its mutants on in vitro rumen fermentation and methane production of rice straw. Detailed Implementation

[0025] The technical solutions and beneficial effects of the present invention will be described in detail below with reference to embodiments, so as to fully understand the purpose and features of the present invention. It should be noted that the embodiments described here are merely illustrative and are not intended to limit the entirety of the present invention. Other embodiments obtained by those skilled in the art based on the content of the present invention without creative effort are all within the protection scope of the present invention.

[0026] This invention obtains the swelling hormone gene from *Neisseria gonorrhoeae* LGM-ZA9. PFS WO2_1, *Neopyrobacterium neoformans* LGM-ZA9 is cited from invention patent application number 2023103351263, which describes *Neopyrobacterium neoformans* LGM-ZA9 ( Neocallimastix sp. The accession number of the object is CGMCC No.17592.

[0027] Example 1: Wild-type expansion hormone gene PFS Acquisition and Sequence Analysis of WO2_1

[0028] This invention relates to the novel *Echinococcus neoformans* LGM-ZA9 ( Neocallimastix sp. The expansion hormone gene was obtained from ) PFS WO2_1, with a nucleotide sequence shown in SEQ ID No. 2, is 1656 bp in length and encodes 552 amino acids, the amino acid sequence of which is shown in SEQ ID No. 1. This protein has a theoretical molecular weight of 62.01 kDa, a theoretical isoelectric point (pI) of 4.3, and an instability coefficient of 39.38, classifying it as a stable protein. Domain analysis indicates that this protein belongs to the Swollenin subclass, containing a DPBB_1 domain (PF03330) and an allergen-1 domain (PF01357), as well as a C-terminal CBM10 carbohydrate-binding module.

[0029] Example 2: Rational design of mutation sites and construction of mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3

[0030] Using the wild-type expansin protein PFSWO2_1 shown in SEQ ID No.1 as the parent protein, its amino acid sequence was subjected to physicochemical property analysis and structural prediction analysis, including: amino acid hydrophobicity distribution analysis, surface accessibility analysis, secondary structure prediction and potential interface region identification.

[0031] Comprehensive analysis revealed that asparagine (Asn) at position 119 is located on the protein surface and is a potential N-glycosylation site. Mutating it to serine (Ser) can reduce glycosylation modification and increase its expression level in Pichia pastoris. Valine (Val) at position 256 is located in the hydrophobic core region inside the domain. Mutating it to leucine (Leu) can enhance hydrophobic interactions and improve protein stability. Threonine (Thr) at position 338 is located in the relatively exposed region on the surface of the connection region between the CBM10 domain and the catalytic domain of the protein and is not in the highly conserved core region. Mutating it to alanine (Ala) can enhance the rigidity of the local structure, reduce the exposure of polar groups, and thus improve the overall thermal stability of the protein.

[0032] Site-directed mutagenesis was performed using overlap extension PCR. A plasmid containing the wild-type PFSWO2_1 gene was used as a template, and three rounds of overlap extension PCR were conducted using specific mutation primers. Reaction conditions: 94℃ for 5 min; 94℃ for 20 s, 56℃ for 20 s, 72℃ for 3 min, 30 cycles; 72℃ for 10 min. The PCR products were transformed into *E. coli* DH5α, and single clones were selected for sequencing verification.

[0033] The specific mutations include the following:

[0034] The asparagine (Asn) at position 119 of the wild-type expansin protein PFSWO2_1 (SEQ ID No. 1) was mutated to serine (Ser) to obtain the fungal expansin protein mutant PFSWO2_1-N1. The primer sequences for the site-directed mutagenesis of the above-mentioned fungal expansin protein PFSWO2_1 are as follows:

[0035] N119S-F: 5'-CTATGAGAACAATGATTGG TCT GGAATAATAGC-3' (SEQ ID No. 9);

[0036] N119S-R: 5'-GCTATTATTCC AGA CCAATCATTGTTCTCATAG-3' (SEQ ID No. 10);

[0037] The amino acid sequence of the obtained mutant PFSWO2_1-N1 is shown in SEQ ID No. 3, and its nucleotide sequence is shown in SEQ ID No. 4.

[0038] The valine (Val) at position 256 of the expansin protein mutant PFSWO2_1-N1 (SEQ ID No. 3) was mutated to leucine (Leu) to obtain the fungal expansin protein mutant PFSWO2_1-N2. The primer sequences for site-directed mutagenesis of the above-mentioned fungal expansin protein mutant PFSWO2_1-N2 are as follows:

[0039] V256L-F: 5'-GCTTGCGGCAAGAAC CTG CGCTACGGCGGAGAT-3' (SEQ ID No. 11);

[0040] V256L-R: 5'-ATCTCCGCCGTAGCG CAG GTTCTTGCCGCAAGC-3' (SEQ ID No. 12);

[0041] The amino acid sequence of the obtained mutant PFSWO2_1-N2 is shown in SEQ ID No. 5, and its nucleotide sequence is shown in SEQ ID No. 6.

[0042] The threonine (Thr) at position 338 of the expansin protein mutant PFSWO2_1-N2 (SEQ ID No. 5) was mutated to alanine (Ala) to obtain the fungal expansin protein mutant PFSWO2_1-N3. The primer sequences for site-directed mutagenesis of the above-mentioned fungal expansin protein mutant PFSWO2_1-N3 are as follows:

[0043] T338A-F: 5'-GAAGGTTACACTCCCCCTG GCC CTTTTATCCGTGAT-3' (SEQ ID No. 13);

[0044] T338A-R: 5'-ATCACGGATAAAAGG GCC AGGGGGAGTGTAACCTTC-3' (SEQ ID No. 14).

[0045] The amino acid sequence of the obtained mutant PFSWO2_1-N3 is shown in SEQ ID No. 7, and its nucleotide sequence is shown in SEQ ID No. 8.

[0046] Example 3: Expression and purification of mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 in Pichia pastoris

[0047] The gene fragments PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 were inserted into the pPICZαA expression vector to construct the recombinant plasmids pPICZαA-PFSWO2_1-N1, N2, and N3. The linearized recombinant plasmids were electroporated into Pichia pastoris GS115 competent cells, and positive transformants were screened using YPD plates containing bleomycin.

[0048] Positive single colonies were picked and inoculated into BMGY medium for amplification. After centrifugation, the bacterial cells were collected and resuspended in BMGY medium. Expression was induced by adding 1% methanol every 24 h for 120 h. The fermentation supernatant was collected by centrifugation, purified by Ni-NTA affinity chromatography, and detected by SDS-PAGE electrophoresis.

[0049] like Figure 1 The results showed that the mutant expression levels reached 73.18 mg / L, 75.50 mg / L, and 77.34 mg / L at 120 h, which were 2.42, 2.50, and 2.56 times that of the wild type, respectively. This indicates that the mutation effectively reduced glycosylation sites and improved the secretory expression efficiency in Pichia pastoris. The purified protein had a molecular weight of approximately 78 kDa, slightly higher than the theoretical value, which was attributed to residual glycosylation modification.

[0050] Example 4: Enzyme activity assay and synergistic effect analysis of mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3

[0051] Using sodium carboxymethyl cellulose (CMC-Na), microcrystalline cellulose (Avicel), and xylan as substrates, the reducing sugar release was determined by the DNS method. Table 1 shows that there was no significant difference in reducing sugar release between the PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 treatment groups and the blank control group (BSA). P > 0.05), confirming that the mutant does not possess hydrolytic enzyme activity, and its role in promoting cellulose degradation originates from a non-hydrolytic mechanism.

[0052] Table 1. Reducing sugar release (mg / mL) of different substrates

[0053]

[0054] Using rice straw as a substrate, a control group (with cellulase added alone) and a mutant pretreatment group (pretreated with PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3 at 50℃ and pH 4.8 for 24 h, respectively, before adding cellulase) were set up. Table 2 shows that the reducing sugar yield in the pretreatment group was 45% higher than that in the control group. P< 0.05). This result indicates that the mutant increases the accessibility of cellulase to cellulose by disrupting the substrate structure, thereby producing a significant synergistic effect.

[0055] Using xylan as a substrate, the synergistic effect between the mutant and xylanase was detected using the same method. Table 2 shows that the synergistic effect of PFSWO2_1-N1 on xylanase was only 18%, significantly lower than its synergistic effect on cellulase (…). P <0.05). This result indicates that the mutant mainly acts on the cellulose backbone, rather than the hemicellulose side chains, and its mechanism of action is substrate-selective.

[0056] Table 2 Degradation effects of different substrates N1

[0057]

[0058] Example 5: Effects of mutant PFSWO2_1-N1 on in vitro rumen fermentation and methane production of rice straw

[0059] Using rice straw as the substrate, four groups were established: a control group (BSA treatment), a wild-type group (PFSWO2_1 treatment), and mutant groups 1 (PFSWO2_1-N1 pretreatment for 24 h), 2 (PFSWO2_1-N2 pretreatment for 24 h), and 3 (PFSWO2_1-N3 pretreatment for 24 h). The pretreated substrate was mixed with rumen fluid and fermented at 39°C for 72 h. Gas production, fermentation parameters, and microbial community structure were measured.

[0060] Figure 2 The results showed that the total gas production in all mutant groups was significantly higher than that in the wild-type group and the control group. （P <0.05). Furthermore, mutant group 3 was significantly higher than all treatment groups. （P <0.05). This result indicates that mutant pretreatment makes the substrate more readily available to rumen microorganisms and improves fermentation efficiency, with PFSWO2_1-N3 showing the best effect.

[0061] Volatile fatty acid (VFA) analysis showed that the mutant group had significantly higher levels of total VFAs, propionic acid, and butyric acid. P <0.05), while the acetic acid / propionic acid ratio was further reduced compared to the wild-type group. The propionic acid generation pathway is a competitive utilization pathway for hydrogen ions. The increased propionic acid ratio means that hydrogen originally used for methane generation is diverted to propionic acid synthesis, thereby inhibiting methane generation.

[0062] This invention utilizes a mutation strategy to target the rumen-derived *Lithocarpus neonatorum* LGM-ZA9 (… Neocallimastix sp. Inflatin-like protein from ) PFSFunctionally targeted modification of WO2_1 yielded mutants PFSWO2_1-N1, PFSWO2_1-N2, and PFSWO2_1-N3. These mutants, while maintaining their non-hydrolyzable properties, significantly enhanced synergistic effects with cellulase, improving the degradation efficiency of lignocellulose. Furthermore, by regulating the rumen fermentation pathway (increasing propionic acid ratio and decreasing the acetic acid / propionic acid ratio) and microbial community structure, they successfully induced the ability to inhibit rumen methanogenic archaea, reducing methane emission intensity by 15-20%. This invention provides a new technical solution for developing green feed additives that simultaneously improve feed utilization and reduce greenhouse gas emissions.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A mutant of the expansin protein derived from rumen anaerobic fungi, characterized in that, The expansin mutant is mutant PFSWO2_1-N1, and the amino acid sequence of mutant PFSWO2_1-N1 is shown in SEQ ID No.

3. It is obtained by mutating asparagine at position 119 of wild-type expansin protein with amino acid sequence SEQ ID No.1 to serine.

2. The encoding gene of the rumen anaerobic fungus-derived expansin protein mutant according to claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

4.

3. A mutant of the expansin protein derived from rumen anaerobic fungi, characterized in that, The expansin mutant is mutant PFSWO2_1-N2, and the amino acid sequence of mutant PFSWO2_1-N2 is shown in SEQ ID No.

5. It is obtained by mutating valine at position 256 of expansin mutant PFSWO2_1-N1 (amino acid sequence of SEQ ID No. 3) to leucine.

4. The encoding gene of the rumen anaerobic fungus-derived expansin protein mutant according to claim 3, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

6.

5. A mutant of the expansin protein derived from rumen anaerobic fungi, characterized in that, The expansin mutant is mutant PFSWO2_1-N3, and the amino acid sequence of mutant PFSWO2_1-N3 is shown in SEQ ID No.

7. It is obtained by mutating threonine at position 338 of the expansin mutant PFSWO2_1-N2 with amino acid sequence SEQ ID No.5 to alanine.

6. The encoding gene of the rumen anaerobic fungus-derived expansin protein mutant according to claim 5, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID No.

8.

7. A recombinant expression vector comprising the coding gene of claim 2, 4, or 6.

8. A recombinant strain comprising the encoding gene of claim 2, 4, or 6.

9. The use of the expansion protein mutant according to claim 1, 3 or 5 in the preparation of formulations that reduce methane emissions and improve cellulase hydrolysis efficiency.

10. The use of the expansion protein mutant according to claim 1, 3 or 5 in the preparation of feed additives that reduce rumen methane emissions in ruminants.

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