Aspergillus niger polysaccharide lytic monooxygenase and use thereof in promoting rumen degradation of straw

Abstract

The application belongs to the technical field of animal feed processing, and particularly relates to an Aspergillus niger polysaccharide-lytic monooxygenase and application thereof in promoting rumen degradation of straw. The amino acid sequence of the Aspergillus niger polysaccharide-lytic monooxygenase is shown in SEQ ID NO. 2. The Aspergillus niger polysaccharide-lytic monooxygenase can change the surface structure of the straw, promote the growth of fiber degradation-related classification bacteria in the rumen, and improve the rumen degradation rate of the straw.

Description

Technical Field

[0001] This invention belongs to the field of animal feed processing technology, specifically relating to a polysaccharide monooxygenase from Aspergillus niger and its application in promoting the rumen degradation of straw. Background Technology

[0002] Straw is a common agricultural byproduct widely distributed around the world, such as rice straw, a byproduct of rice cultivation. The feed value of rice straw is limited by its low rumen degradability. A key reason is the complex cross-linking structure between cellulose, hemicellulose, and lignin, which restricts rumen microorganisms and their hydrolytic enzymes from penetrating the plant fibers. This means that rice straw, once ingested by animals, cannot be effectively converted into meat, milk, or other animal products.

[0003] Existing technologies employ physical and chemical processing methods to improve the rumen digestibility of rice straw, such as steam explosion, alkali and acid treatment. These methods enhance rumen digestibility by disrupting the lignin-carbohydrate complex and cellulose crystallinity in the straw. However, these methods suffer from costly equipment requirements, high energy consumption, and significant risks to animals and the environment (for example, when using alkali to treat straw, residual alkali on the straw surface can be ingested by animals, harming their health; rinsing the straw surface with water can release alkali into the environment, causing pollution). In contrast, the high efficiency, specificity, mild reaction, and environmental friendliness exhibited by bioenzymes in the processing and utilization of agricultural straw have attracted considerable interest from researchers.

[0004] Therefore, it is necessary to find a biological enzyme that can promote the degradation of straw in the rumen. Summary of the Invention

[0005] The purpose of this invention is to discover a polysaccharide monooxygenase (hereinafter also referred to as AnLPMO) from Aspergillus niger that can change the surface structure of straw, promote the growth of fiber-degrading bacteria in the rumen, and improve the rumen degradation rate of straw.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] The present invention provides a polysaccharide monooxygenase lysing Aspergillus niger, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] Another aspect of the present invention provides an isolated nucleic acid molecule that can encode the Aspergillus niger polysaccharide cleavage monooxygenase of the present invention.

[0009] In another aspect, the present invention provides a recombinant expression vector comprising the isolated nucleic acid molecules described in this invention.

[0010] In another aspect, the present invention provides a recombinant engineered cell comprising the isolated nucleic acid molecule or the recombinant expression vector of the present invention.

[0011] In another aspect, the present invention provides the application of the Aspergillus niger polysaccharide monooxygenase of the present invention, or the isolated nucleic acid molecule of the present invention, or the recombinant expression vector of the present invention, or the recombinant engineered cell of the present invention in promoting the rumen degradation of straw.

[0012] The beneficial effects of this invention include at least the following: AnLPMO provided by this invention can change the surface structure of straw, promote the growth of fiber-degrading bacteria in the rumen, and improve the rumen degradation rate of straw. Attached Figure Description

[0013] Figure 1 Electrophoresis results for the AnLPMO gene;

[0014] Figure 2 SDS-PAGE and Western blot analysis of AnLPMO: Lane 1, protein marker; Lane 2, untransformed E. coli BL21(DE3); Lane 3, recombinant AnLPMO cells induced by 0.5 mM IPTG; Lane 4, purified AnLPMO; Lane 5, Western blot analysis of purified AnLPMO.

[0015] Figure 3 The relationship between AnLPMO activity and pH and temperature;

[0016] Figure 4 To illustrate the relationship between the activity of AnLPMO and different substrates;

[0017] Figure 5 The effect of AnLPMO treatment on the FTIR spectrum of rice straw;

[0018] Figure 6 This study examines the effects of AnLPMO on rumen microorganisms during in vitro rumen fermentation of rice straw. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] This invention discovers a polysaccharide-lysing monooxygenase from Aspergillus niger (hereinafter also referred to as AnLPMO), which can alter the surface structure of straw, promote the growth of fiber-degrading bacteria in the rumen, and improve the rumen degradation rate of straw. Specifically:

[0022] This invention provides an Aspergillus niger polysaccharide cleavage monooxygenase, the amino acid sequence of which is shown in SEQ ID NO.2.

[0023] It should be noted that polysaccharide lysing monooxygenases (LPMOs) are a class of copper-dependent oxidases. Their most unique and attractive feature is their ability to attack polysaccharides with stubborn structures, such as crystalline cellulose and cellulose-hemicellulose complexes, and they exhibit a high degree of synergistic effect with cellulases. It is well known that the rumen contains a large number of microorganisms such as bacteria, protozoa, and fungi, which collectively produce a variety of cellulases, meaning that the rumen provides a natural enzymatic environment for LMPOs. However, current research on LPMOs has largely focused on the industrial utilization of fiber resources, such as the production of biofuels and chemical products, and their application in animal-related fields has not yet been observed. This invention clones a polysaccharide lysing monooxygenase from Aspergillus niger, named AnLPMO, and introduces it into the field of ruminant roughage utilization, providing a new approach to improving straw utilization.

[0024] Another embodiment of the present invention provides an isolated nucleic acid molecule that encodes the Aspergillus niger polysaccharide cleavage monooxygenase of the present invention. In some specific embodiments, the sequence of the isolated nucleic acid molecule is shown in SEQ ID NO.1.

[0025] It should be noted that the isolated nucleic acid molecule in this invention can be any nucleic acid molecule that can encode the Aspergillus niger lysing polysaccharide monooxygenase in this invention, such as the sequence shown in SEQ ID NO.1, or the promoter optimized sequence shown in SEQ ID NO.1, etc.

[0026] Another embodiment of the present invention provides a recombinant expression vector comprising the isolated nucleic acid molecule described in this invention. It should be noted that the isolated nucleic acid molecule described in this invention can be ligated with an expression vector to form a recombinant expression vector for the expression of the AnLPMO gene; the expression vector can be any type known in the art.

[0027] Another embodiment of the present invention provides a recombinant engineered cell, which includes the isolated nucleic acid molecule or the recombinant expression vector of the present invention. It should be noted that the isolated nucleic acid molecule or the recombinant expression vector of the present invention can be transferred into engineered cells (such as Escherichia coli) for expression.

[0028] Another embodiment of the present invention provides the application of the Aspergillus niger polysaccharide monooxygenase of the present invention, or the isolated nucleic acid molecule of the present invention, or the recombinant expression vector of the present invention, or the recombinant engineered cell of the present invention in promoting the rumen degradation of straw.

[0029] It should be noted that the optimal environment for the activity of Aspergillus niger polysaccharide monooxygenase in straw is pH 6.5 and 40℃, and the rumen environment is just right for Aspergillus niger polysaccharide monooxygenase.

[0030] In some specific embodiments, the above applications include: the application of the Aspergillus niger polysaccharide monooxygenase of the present invention, the isolated nucleic acid molecules of the present invention, the recombinant expression vector of the present invention, or the recombinant engineered cells of the present invention in degrading cellulose and hemicellulose on the surface of straw and increasing the lignin content of the surface.

[0031] In some specific embodiments, the above applications include: the Aspergillus niger lysing polysaccharide monooxygenase of the present invention, or the isolated nucleic acid molecules of the present invention, or the recombinant expression vector of the present invention, or the recombinant engineered cells of the present invention, in increasing the content of Proteobacteria, Tenerigutia, Fusobacteria, and Spirochetes in the rumen, and decreasing the content of Firmicutes in the rumen. In some specific embodiments, at the genus level, AnLPMO increased 9 taxa, among which Vibrio succinate, Rikenellaceae_RC9_gut_group, and Oribacteria were dominant bacteria; and decreased 8 taxa, among which Streptococcus, Prevotella_1, and Anaerovibrio were dominant bacteria. The growth of Vibrio succinate, Rikenellaceae_RC9_gut_group, and Oribacteria all benefit the degradation of fibrous material in the rumen.

[0032] In some specific embodiments, the amount of Aspergillus niger polysaccharide monooxygenase added in the above applications is 1.3U-3.3U / g straw. It should be noted that while the amount of Aspergillus niger polysaccharide monooxygenase added in this invention is 1.3U-3.3U / g straw, if the Aspergillus niger polysaccharide monooxygenase is obtained by expressing isolated nucleic acid molecules, recombinant expression vectors, or recombinant engineered cells as described in this invention, the amount of isolated nucleic acid molecules, recombinant expression vectors, or recombinant engineered cells can be obtained by adjusting the amount of Aspergillus niger polysaccharide monooxygenase added.

[0033] It should be noted that the straw mentioned above is an agricultural by-product known in this field, such as rice straw.

[0034] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0035] I. Cloning of the AnLPMO gene and construction of its expression vector

[0036] The *Aspergillus niger* used in this invention was purchased from the China General Microbiological Culture Collection Center (CGMCC 3.17612). Total RNA was extracted from *Aspergillus niger*, and cDNA was synthesized using a kit (AE311-02, TransGen Biotech). The *AnLPMO* gene was amplified using primers AnLPMO-F (AAGAAGGAGATATACGAATTCATGAAGACTACCACCTACAGTT) and AnLPMO-R (GTGGTGGTGGTGGTGGTGGTGCTCGAGCTGAGACGCAACGCAC). The amplified *AnLPMO* gene was detected by electrophoresis, and the results are as follows: Figure 1 As shown, the results indicate that there is a bright and clear band around 1200bp, consistent with the predicted size.

[0037] In addition, the amplified AnLPMO gene was ligated into the pET-28a vector, named pET-AnLPMO, and introduced into E. coli DH5α cells by heat shock. The pET-AnLPMO plasmid was extracted, confirmed by PCR amplification, and the AnLPMO gene sequence on the vector was analyzed. The results are as follows:

[0038] AnLPMO gene sequence (SEQ ID NO.1):

[0039]

[0040] AnLPMO amino acid sequence (SEQ ID NO.2):

[0041] MKTTTYSLLALAAASKLASAHTTVQAVWINGEDQGLGNSADGYIRSPPSNSPVTDVTSTDMTCNVNGDQAASKTLSVKAGDVVTFEWHHSDRSDSDDIIASSHKGPVQVYMAPTAKGSNGNNWVKIAEDGYHKSSDEWATDILIANKGKHNITVPDVPAGNYLFRPEIIALHEGNREGGAQFYMECVQFKVTSDGSSELPSGV SIPGVYTATDPGILFDIYNSFDSYPIPGPDVWDGSSSGSSSGSSSSAAAAATTSAVAATTPATQAAVAVSSSSAAAVVESTSSAAAATTEAAAPVVSQQATSAVTSQAQAPTTFATSSKSSKTACKNKTKSKSKVAASSTEAVVAPAPTSSVVPAVSASASASAGGVAKKYERCGGINHTGPTTCESGSVCKKWNPYYYQCVASQ.

[0042] II. Expression and Purification of AnLPMO

[0043] The recombinant plasmid pET-AnLPMO was transformed into competent *E. coli* BL21(DE3) cells. The transformed cells were plated on LB agar plates containing kanamycin and incubated overnight at 37°C. Positive clones were selected and inoculated into 2 mL of liquid LB medium. Simultaneously, untransformed *E. coli* BL21(DE3) cells were cultured as a control. 250 μL of the overnight culture was inoculated into 25 mL of LB medium and cultured at 37°C with shaking until the OD600 value reached 0.8. Recombinant protein expression was then induced with isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0–0.8 mM. The induction process was carried out at 20°C for 20 hours. Subsequently, the bacterial culture was collected and centrifuged at 8000 rpm for 15 minutes at 4°C. The resulting precipitate was then resuspended in phosphate-buffered saline (PBS) and sonicated in an ice-water bath. After centrifugation, the supernatant containing AnLPMO was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.

[0044] SDS-PAGE results are as follows Figure 2As shown, the results indicated that, compared to the control group without the AnLPMO gene, a thicker band with a molecular weight of approximately 51 kDa appeared in the cells transformed with the AnLPMO gene, the supernatant after induced cell lysis, and the purified sample. Western blot analysis confirmed that this band was indeed AnLPMO (… Figure 2 Lane 5 indicates that AnLPMO has been successfully expressed in Escherichia coli.

[0045] AnLPMO was then purified using a Ni column. The purification process was as follows: the lysed supernatant crude enzyme solution was appropriately diluted with binding buffer (containing 0.05 M sodium dihydrogen phosphate and 0.3 M sodium chloride, pH 8.0). The diluted solution was loaded onto a 5 mL Ni-affinity chromatography column using a low-pressure chromatography system (Biologic LP, Bio-Rad, Hercules, CA, USA) at a flow rate of 1.0 mL / min. After loading, the column was first washed with binding buffer containing 0.02 M imidazole, and finally eluted with binding buffer containing 0.25 M imidazole to obtain purified AnLPMO. The total protein concentration of the purified solution was determined using a Bradford protein assay kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China), and the purity of AnLPMO protein was determined by SDS-PAGE (purity 78.1%).

[0046] III. Characteristic Analysis of AnLPMO

[0047] In this embodiment of the invention, the mass-volume ratio refers to the "proportion of solid substances in liquid". For example, a 1% mass-volume ratio means that 100 ml of liquid contains 1 g of substrate. In addition, the liquid in the reaction system is not water, but a citrate-disodium hydrogen phosphate buffer solution with different pH values.

[0048] (I) pH-dependent analysis

[0049] At 37°C, using 1% (w / v) microcrystalline cellulose (Avicel pH-101, Merck Sigma Biotech) as the reaction substrate, the degradation of microcrystalline cellulose by AnLPMO was determined within a pH range of 3.0–8.0 (citric acid-disodium hydrogen phosphate buffer, containing 50 mmol / L citric acid and disodium hydrogen phosphate; i.e., first prepare 50 mmol / L citric acid solution and 50 mmol / L disodium hydrogen phosphate solution, mix them, and observe pH changes while mixing until all desired pH values ​​are achieved). The reaction time was 6 h, and the release of reducing sugars was detected using the DNS method. The pH with the highest activity was defined as the optimum pH, and its activity was defined as 100%.

[0050] The results are as follows Figure 3As shown, the results indicate that AnLPMO exhibits maximum activity at pH 6.5 and retains approximately 80% of its maximum activity at pH 7.0-8.0. However, when the pH drops below 6.0, the activity of AnLPMO decreases significantly, resulting in its maximum activity being less than 40%. Therefore, the optimal reaction pH for AnLPMO is 6.5.

[0051] (II) Temperature Dependence Analysis

[0052] Using 1% microcrystalline cellulose as the reaction substrate, the degradation of microcrystalline cellulose by AnLPMO at 30℃-55℃ and pH 6.5 was detected, with a reaction time of 6 h. The amount of reducing sugar released was determined using the DNS method. The temperature at which the highest activity was defined as the optimum temperature, and its activity was defined as 100%.

[0053] The results are as follows Figure 3 As shown, AnLPMO exhibits maximum activity at 40°C and retains over 60% of its maximum activity between 30-50°C. Therefore, the optimal reaction temperature for AnLPMO is 40°C.

[0054] (III) Substrate Specificity

[0055] The substrate specificity of AnLPMO was determined at pH 6.5 and 40°C using 1% rice straw xylan, sodium carboxymethyl cellulose, microcrystalline cellulose, chitosan, and rice straw as substrates, respectively.

[0056] The results are as follows Figure 4 As shown, AnLPMO exhibits the highest activity towards rice straw xylan, followed by microcrystalline cellulose and rice straw, and the weakest activity towards sodium carboxymethyl cellulose and chitosan.

[0057] IV. Effects of AnLPMO on the surface structure of rice straw

[0058] 0.3 U AnLPMO and 50 mg of rice straw were placed in 5 mL of sodium citrate-phosphate buffer (0.05 M, pH 6.5) and incubated with shaking at 40 °C for 6 hours. The control group, which did not contain AnLPMO, was incubated under the same conditions. After incubation, the reaction mixture was centrifuged at 12,000 rpm for 2 minutes and the supernatant was discarded. The precipitate was dried at 65 °C for 48 hours, and the structure of the rice straw was analyzed by Fourier transform infrared spectroscopy (FTIR).

[0059] The results are as follows Figure 5 As shown, compared with the control group, the AnLPMO treatment group reached approximately 1161 cm. -1 1246cm -1 1371cm -1 1429cm -1 1455cm -1and 3407cm -1 The peak intensity at these locations decreased significantly, while it decreased at 1516, 1545, 1628, and 1648 cm⁻¹. -1 The peak intensity near 3407 cm⁻¹ increases significantly. -1 1371cm -1 1429cm -1 1455cm -1 The peak at 1246 cm⁻¹ is a characteristic peak of cellulose. -1 The peak at 1516 cm⁻¹ is a characteristic peak of hemicellulose. -1 and 1648cm -1 The peak at that location is a characteristic peak of lignin. The above data indicates that AnLPMO alters the surface structure of rice straw, degrading some of the cellulose and hemicellulose on the straw surface, thereby increasing the lignin content of the straw surface.

[0060] V. Effects of AnLPMO on in vitro rumen fermentation of rice straw

[0061] Rumen fluid was collected from beef cattle via a rumen fistula, filtered through four layers of coarse cotton cloth, and mixed with buffer at a ratio of 1:2 (v / v). 600 mg of rice straw, 0-2.0 U AnLPMO, and 60 mL of the mixed rumen fluid were placed in a 120 mL glass bottle and anaerobically fermented in a 39 °C water bath for 48 h. After the experiment, the fermentation bottle was placed in ice to cool and terminate the fermentation. The fermentation broth was filtered using a crucible to calculate the in vitro digestibility (IVDMD) of the rice straw. In addition, the pH, volatile fatty acids (VFA), and ammonia nitrogen concentration of the filtrate were measured, and the results are shown in Table 1 below.

[0062] Table 1 Effects of AnLPMO on in vitro fermentation of rice straw

[0063]

[0064] Note: In the table, different lowercase letters in the same row indicate significant differences (P<0.05).

[0065] As shown in Table 1 above, compared with the control group, the addition of 0.8U-2.0U of AnLPMO significantly increased the in vitro dry matter digestibility of rice straw (P<0.01), with the 1.4U group showing the best effect, increasing by 9.59 percentage points; AnLPMO supplementation increased the total gas production (P<0.01), with the 2.0U group showing an increase of 19.3 mL; when the AnLPMO supply reached 1.1U-2.0U, the concentrations of total VFA and acetic acid significantly increased (P<0.01); the addition of AnLPMO increased the propionic acid concentration (P<0.01); the supplementation of AnLPMO decreased the butyric acid concentration (P<0.01); with the increase of AnLPMO addition, the ammonia nitrogen concentration gradually increased (P<0.01), especially after the addition of 2U of AnLPMO, the ammonia nitrogen concentration increased by 17.2 mM.

[0066] VI. Effects of AnLPMO on Rumen Microorganisms during In Vitro Fermentation of Rice Straw

[0067] Rumen fluid was collected from beef cattle via a rumen fistula, filtered through four layers of coarse cotton cloth, and mixed with buffer at a 1:2 ratio (v / v). 600 mg of straw, 1.4 U AnLPMO or 1.4 U inactivated AnLPMO (control group), and 60 mL of the mixed rumen fluid were placed in a 120 mL glass bottle and anaerobically fermented in a 39°C water bath for 48 h. After the experiment, the fermentation bottle was placed on ice to cool and terminate the fermentation. The filtrate was collected using crucible filtration and the microbial community was determined using 16S rRNA high-throughput sequencing technology.

[0068] The results are as follows Figure 6 As shown, at the phylum level, AnLPMO increased the content of Proteobacteria, Teneriguts, Fusobacteria, and Spirochetes, while decreasing the content of Firmicutes. At the genus level, AnLPMO increased 9 taxa, with *Vibrio succinate*, *Rikenellaceae_RC9_gut_group*, and *Oribacteria* being the dominant bacteria; and decreased 8 taxa, with *Streptococcus*, *Prevotella_1*, and *Anaerovibrio* being the dominant bacteria. The growth of *Vibrio succinate*, *Rikenellaceae_RC9_gut_group*, and *Oribacteria* all promoted the degradation of fibrous material in the rumen.

[0069] The above results indicate that AnLPMO can alter the surface structure of rice straw, promote the growth of fiber-degrading bacteria in the rumen, and improve the rumen degradation rate of rice straw. The optimal amount of AnLPMO added is 1.3U-3.3U / g rice straw.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of Aspergillus niger lysing polysaccharide monooxygenase, or isolated nucleic acid molecules, or recombinant expression vectors, or recombinant engineered cells in promoting rumen degradation of straw; the amino acid sequence of the Aspergillus niger lysing polysaccharide monooxygenase is shown in SEQ ID NO.2; the isolated nucleic acid molecule encodes the Aspergillus niger lysing polysaccharide monooxygenase; the recombinant expression vector includes the isolated nucleic acid molecule; the recombinant engineered cells include the isolated nucleic acid molecule or the recombinant expression vector.

2. The application according to claim 1, characterized in that, The sequence of the isolated nucleic acid molecule is shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, Applications include: degrading cellulose and hemicellulose on the surface of straw and increasing the lignin content on the surface.

4. The application according to claim 1 or 2, characterized in that, Applications include: increasing the content of Proteobacteria, Tenerigutia, Fusobacteria, and Spirochetes in the rumen, and decreasing the content of Firmicutes in the rumen.

5. The application according to claim 1 or 2, characterized in that, Applications include: increasing *Vibrio succinate*, *Rikenellaceae_RC9_gut_group*, and *Oribacteria*, and decreasing *Streptococcus*, *Prevotella_1*, and *Anaerovibrio*.

6. The application according to claim 1 or 2, characterized in that, The addition amount of Aspergillus niger polysaccharide monooxygenase was 1.3U-3.3U / g straw.

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