Pseudoxanthomonas strain JC1303 and application thereof

Abstract

The invention relates to the technical field of new strains for degrading cellulose, in particular to a pseudoxanthomonas strain JC1303 and application thereof. Through whole genome sequencing and function annotation, it is systematically revealed for the first time that the strain carries a complete cellulose degrading enzyme system including incision beta-1, 4-glucanase, cellulase and beta-glucosidase, has a plurality of central metabolic pathways supporting efficient degradation and utilization of cellulose, and can be used for degrading cellulose. And specific gene resources are determined through generic genome analysis. The strain has the remarkable technical effects that the strain shows higher cellulase activity after being cultured for 5 days, and cellulose materials such as agricultural wastes and the like can be stably and efficiently degraded in a salt-containing environment by virtue of the unique genetic background and marine source characteristics of the strain.

Description

Technical Field

[0001] This invention relates to the field of novel cellulose-degrading strains, specifically to a *Xanthomonas* strain JC1303 and its applications. Background Technology

[0002] Cellulose is a linear polysaccharide composed of glucose units linked by β-1,4-glycosidic bonds. It is a major component of plant cell walls and is widely recognized as the most abundant organic compound on Earth and an important renewable resource. Cellulose can be used to produce alcohol, gaseous fuels, and biodegradable materials. However, due to its complex chemical structure, efficient utilization presents significant challenges, particularly in agricultural waste treatment—cellulose's resistance to degradation reduces resource utilization and composting efficiency. In China, large quantities of agricultural organic waste are directly incinerated or dumped, resulting in resource waste and environmental pollution. Therefore, developing efficient cellulose resource utilization technologies is of great significance for alleviating resource shortages and promoting environmental protection.

[0003] Microbial cellulases play a central role in cellulose degradation, which is crucial for addressing the waste of cellulose resources. Cellulases mainly include endo-β-1,4-glucanase and β-1,4-glucosidase, which can break down cellulose into glucose monomers. Therefore, screening cellulose-degrading strains is of practical significance for the development and utilization of cellulose resources.

[0004] Marine ecosystems possess abundant microbial resources and highly diverse metabolic capabilities. Microorganisms living in extreme marine environments can produce cellulases with excellent properties such as salt tolerance, acid tolerance, alkali tolerance, and pressure tolerance, which provides an important advantage for the development and application of cellulases. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel marine *Pseudomonas* strain, JC1303, with accession number CCTCC NO: M20252026. Through whole-genome sequencing and functional annotation, this invention systematically reveals for the first time that this strain carries a complete cellulose-degrading enzyme system, including endoglucanase, cellulase, and β-glucosidase, possessing multiple central metabolic pathways supporting efficient cellulose degradation and utilization. Furthermore, its unique gene resources were identified through pan-genome analysis. This strain exhibits high cellulase activity (e.g., FPase activity reaches 0.65±0.11 U / mL) as early as day 5 of culture and can stably and efficiently degrade cellulose materials such as agricultural waste in saline environments.

[0006] The first aspect of this invention provides a strain of Pseudoxanthomonas sp. JC1303, which was deposited at the China Center for Type Culture Collection on September 15, 2025, with accession number CCTCC NO: M 20252026 and classified as Pseudoxanthomonas sp. JC1303.

[0007] After testing, the 16S rRNA gene sequence of Xanthomonas spp. strain JC1303 is shown in SEQ ID NO: 1, and the whole genome sequence is shown in SEQ ID NO: 2.

[0008] A second aspect of the present invention provides a microbial preparation comprising the aforementioned Xanthomonas spp. strain JC1303, and one or more agriculturally acceptable carriers.

[0009] Furthermore, the formulation is in the form of a lyophilized powder or a liquid bacterial agent, and the viable count of the strain JC1303 is not less than 1×10 CFU / g.

[0010] A third aspect of the present invention provides a method for degrading cellulose materials, comprising the following steps: contacting the cellulose material with the above-mentioned Xanthomonas spp. strain JC1303 or the above-mentioned microbial preparation.

[0011] Furthermore, the cellulose material is derived from agricultural waste.

[0012] Furthermore, the cells were cultured for 3-7 days at 25-30°C and 150-200 rpm in a culture medium containing sodium carboxymethyl cellulose or microcrystalline cellulose.

[0013] A fourth aspect of the present invention provides a method for producing reducing sugars, wherein the method described above is used to degrade cellulose material and the resulting reducing sugars containing glucose are collected.

[0014] The fifth aspect of the invention provides the use of the above-described strain JC1303 and / or its metabolites in the preparation of formulations for degrading cellulose, treating agricultural waste, or producing biofuels.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This application provides a novel marine *Pseudomonas* strain, JC1303, with accession number CCTCC NO: M 20252026, and discloses microbial preparations containing this strain, methods for degrading cellulose materials, and methods for producing reducing sugars. Through whole-genome sequencing and functional annotation, this study systematically reveals for the first time that this strain carries a complete cellulose-degrading enzyme system, including endoglucanase, cellulase, and β-glucosidase, possessing multiple central metabolic pathways supporting efficient cellulose degradation and utilization. Furthermore, its unique genetic resources were identified through pan-genome analysis. Because strain JC1303 carries a complete cellulose-degrading enzyme system and its unique genetic resources optimize the central metabolic pathways, this strain exhibits high cellulase activity (e.g., FPase activity reaches 0.65±0.11 U / mL) as early as day 5 of culture without genetic engineering. Furthermore, due to its unique genetic background and marine origin, it can stably and efficiently degrade cellulose materials such as agricultural waste in saline environments. Attached Figure Description

[0016] Figure 1 This is a graph showing the validation data of the cellulose degradation ability of strain JC1303, where... Figure 1 In the diagram, A represents the clear zone of strain JC1303 on a Congo red staining plate. Figure 1 B in the figure represents the cellulase activity of strain JC1303.

[0017] Figure 2 Morphological characteristics of strain JC1303.

[0018] Figure 3 This is a phylogenetic analysis data diagram of strain JC1303; among which... Figure 3 In the figure, A represents the phylogenetic analysis of strain JC1303 constructed based on the 16S rDNA sequence; Figure 3 B in the figure represents the phylogenetic analysis of strain JC1303 based on the genome sequence.

[0019] Figure 4 Heatmap of average nucleotide identity (ANI) analysis for strain JC1303.

[0020] Figure 5 Gene annotation and functional classification of strain JC1303. Figure 5 The 'A' in this context refers to a COG database comment. Figure 5 The 'B' in the text is a GO database comment; Figure 5 The 'C' in this context stands for KEGG database comment. Figure 5 D in the diagram represents the distribution of the carbohydrate active enzyme (CAZy) family.

[0021] Figure 6 Key metabolic pathways reconstructed for strain JC1303.

[0022] Figure 7 For pan-genome analysis of strain JC1303.

[0023] Figure 8 This is a transcriptomic validation result of the expression of cellulose degradation-related genes in strain JC1303 under CMC-Na induction; among them, Figure 8 In the figure, A represents a statistical graph of differentially expressed genes when CMC-Na is used as the carbon source; Figure 8 Figure B in the figure shows the results of verifying the expression of key cellulose degradation and transport-related genes by RT-qPCR.

[0024] Figure 9 The diagram shows the enzymatic properties of the recombinant β-D-glucanase JC1303_2942; among them, Figure 9 A is the graph showing the determination of the optimal reaction temperature. Figure 9 B is the thermal stability measurement diagram. Figure 9 C represents the optimal reaction pH measurement graph. Figure 9 D is the pH stability measurement graph. Figure 9 E is the graph showing the effect of NaCl concentration on enzyme activity. Figure 9 F is a graph showing the effect of different metal ions on enzyme activity.

[0025] Figure 10 The figure shows the substrate specificity assay results for recombinase JC1303_2942. Detailed Implementation

[0026] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0027] The first aspect of this embodiment provides a strain of Pseudoxanthomonas sp. JC1303, which was deposited at the China Center for Type Culture Collection on September 22, 2025, with accession number CCTCC NO: M 20252026 and classified as Pseudoxanthomonas sp. JC1303.

[0028] After testing, the 16S rRNA gene sequence of Xanthomonas spp. strain JC1303 is shown in SEQ ID NO: 1, and the whole genome sequence is shown in SEQ ID NO: 2.

[0029] The second embodiment provides a microbial preparation comprising the aforementioned *Xanthomonas* strain JC1303, and one or more agriculturally acceptable carriers. The agriculturally acceptable carriers include, but are not limited to, peat moss, diatomaceous earth, wheat bran, glycerol, or water.

[0030] In some embodiments, the formulation is a lyophilized powder or a liquid bacterial agent, and the viable count of the strain JC1303 is not less than 1×10 CFU / g.

[0031] The third aspect of this embodiment provides a method for degrading cellulose materials, comprising the following steps: contacting the cellulose material with the above-mentioned Xanthomonas spp. strain JC1303 or the above-mentioned microbial preparation.

[0032] In some embodiments, the cellulose material is derived from agricultural waste.

[0033] In some embodiments, the medium is cultured for 3-7 days at 25°C-30°C and 150-200 rpm in a culture medium containing sodium carboxymethyl cellulose or microcrystalline cellulose.

[0034] The fourth aspect of this embodiment provides a method for producing reducing sugars, wherein the method described above is used to degrade cellulose materials and the resulting reducing sugars containing glucose are collected.

[0035] The fifth aspect of this embodiment provides the use of the above-described strain JC1303 and / or its metabolites in the preparation of formulations for degrading cellulose, treating agricultural waste, or producing biofuels.

[0036] To better understand the technical solutions of the above embodiments, the following more detailed experimental examples are provided to explain some of the embodiments. Example

[0037] 1. Strains Isolation and Culture The JC1303 strain was derived from marine sediment samples.

[0038] Weigh 5g of the sediment and add it to 50mL of sterile PBS buffer. Shake at 28℃ and 180rpm for 1 hour. Add 10... -3 -10 -7 100 μL of the serially diluted solution was spread onto a carboxymethyl cellulose sodium (CMC-Na) selective medium. The medium consisted of: CMC-Na 10 g / L, KH₂PO₄ 1 g / L, (NH₄)₂SO₄ 2 g / L, MgSO₄·7H₂O 0.5 g / L, NaCl 10 g / L, yeast extract 1 g / L, agar 20 g / L, pH 7.0. The medium was incubated at 28°C for 3 days. High-yielding cellulase-producing bacteria were screened using Congo red staining: the isolated strains were inoculated onto CMC-Na solid medium and incubated at 28°C for 3 days; subsequently, they were stained with 1 g / L Congo red solution at room temperature for 1 hour, and then destained with 1 mol / L NaCl solution for 30 minutes. Colonies forming a clear halo were selected for further research. Figure 1As shown.

[0039] 2. Morphological observation The re-screened strain JC1303 was streaked onto 2216E solid medium and incubated at 28℃ for 48 h. The color and morphological characteristics of the strain were observed. A single colony was taken and placed in the center of a glass slide, mixed with a small amount of 0.85% physiological saline, spread into a thin film, and fixed by drying at 37℃. Gram staining was performed according to the standard procedure: primary staining with crystal violet for 1 min, mordanting with iodine solution for 1 min, destaining with destaining solution (95% absolute ethanol + 5% acetone) for 30 s, and counterstaining with safranin for 1 min. The cells were slowly rinsed with distilled water between each step. After staining, the bacterial staining reaction (Gram-positive bacteria appear purple, Gram-negative bacteria appear red), morphology, and size were observed and photographed under an oil immersion microscope (100×). Strain JC1303 was inoculated into 2216E liquid medium and cultured for 24 h. The bacterial culture was collected by centrifugation at 8000 rpm for 10 min, washed three times with PBS buffer (pH 7.4), and fixed with electron microscopy fixative (Wuhan Saiweier, G1102) at room temperature for 2 h, then transferred to 4°C for storage. The fixed samples were rinsed three times (15 min each) with 0.1 mol / L phosphate buffer (PB, pH 7.4), then fixed with 1% osmium tetroxide solution at room temperature in the dark for 1-2 h, followed by three rinses with PB (15 min each). The samples were dehydrated stepwise with a gradient of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%, 100%) for 15 min each step, followed by 15 min of isoamyl acetate replacement. After critical drying and gold sputtering, the samples were observed and photographed under a scanning electron microscope (SEM). (See attached image for bacterial morphology). Figure 2 ).

[0040] 3. Cellulase activity assay JC1303 was inoculated into enzyme-producing liquid medium and cultured at 28℃ and 180 rpm for 7 days. The medium formulation was: CMC-Na 10 g / L, KH2PO4 1 g / L, (NH4)2SO4 2 g / L, MgSO4·7H2O 0.5 g / L, NaCl 10 g / L, yeast extract 1 g / L, and tryptone 5 g / L. Samples were taken every 24 hours, centrifuged at 8000 rpm for 10 minutes at 4℃, and the supernatant was used to determine enzyme activity. Cellulase activity was determined based on the 3,5-dinitrosalicylic acid (DNS) method, using CMC-Na (for CMCase activity), microcrystalline cellulose (for CXase), salicin (for β-Gase), and quantitative filter paper (for FPase) as substrates. Specific method: Mix 0.5 mL of enzyme solution with 0.5 mL of substrate in a 5 mL centrifuge tube and react at 50 °C for 30 minutes; add 1 mL of DNS solution, heat in a 100 °C water bath for 5 minutes, cool, and measure the absorbance at 540 nm. Calculate the reducing sugar concentration using a glucose standard curve. An inactivated enzyme solution serves as a control. Unit enzyme activity is defined as the amount of enzyme required to release 1 μg of glucose per milliliter of enzyme solution per minute under specified conditions. Furthermore, this example evaluated the tolerance characteristics of cellulase from strain JC1303 by testing its adaptability to temperature, pH, and salt concentration using crude fermented enzyme solution. For temperature adaptability testing, the crude enzyme solution was reacted with CMC-Na substrate at temperatures ranging from 20–70 °C for 30 minutes, and the relative CMCase enzyme activity was measured. pH adaptability test: The pH of the reaction system was adjusted using a 50mM citrate-disodium hydrogen phosphate buffer system (pH 4.0-8.0) and a 50mM glycine-sodium hydroxide buffer system (pH 8.0-10.0) to determine enzyme activity, with the optimal pH value representing 100% enzyme activity. Salt tolerance test: Different concentrations of NaCl (0-5%, w / v) were added to the standard reaction system, and the reaction was carried out under optimal conditions for 30 minutes. CMCase activity was then measured, with the salt-free condition value representing 100% enzyme activity.

[0041] 4. Transcriptomics Validation of Cellulose Degradation-Related Gene Expression: Objective: To verify whether cellulose degradation-related genes in the genome annotation are expressed under cellulose-induced conditions.

[0042] Materials Methods To verify the expression of the cellulase gene, strain JC1303 was inoculated into liquid medium with CMC-Na (10 g / L) or glucose (10 g / L) as the sole carbon source. The medium composition was as follows: KH₂PO₄ 1 g / L, (NH₄)₂SO₄ 2 g / L, MgSO₄·7H₂O 0.5 g / L, NaCl 10 g / L, and pH adjusted to 7.0. After culturing at 28℃ and 180 rpm with shaking for 5 days, the bacterial cells were collected by centrifugation at 12,000 rpm and 4℃ for 10 min, immediately followed by flash freezing in liquid nitrogen and storage at 80℃. Total RNA was extracted using a bacterial RNA extraction kit (Omega, USA) according to the kit instructions. The integrity and purity of the RNA were detected using an Agilent Bioanalyzer 2100 system and a NanoDrop 2000 spectrophotometer, respectively. Qualified RNA samples were sequenced using paired-end transcriptome sequencing by OneMore-Tech Co., Ltd. on the Illumina NovaSeq 6000 platform. After quality control and adapter sequence removal of the raw sequencing data, the obtained high-quality clean reads were aligned to the JC1303 reference genome using HISAT2 software. Gene expression levels were quantified using FPKM (Fragments Per Kilobase Million) values. Differentially expressed genes were identified using the DESeq2 software package, with selection criteria of |log(fold change)| ≥ 1 and adjusted p-value < 0.05. To verify the reliability of the RNA-seq results, genes related to cellulose degradation were selected for RT-qPCR analysis. Specific primer sequences are shown in Table S3, with the gapdh gene used as an internal reference gene.

[0043] To verify the functional relevance of cellulose degradation-related genes obtained from genome annotation in the cellulose degradation process, we conducted a comparative transcriptome analysis. The results showed that under CMC-Na induction conditions, a total of 1,465 genes exhibited significant differential expression, of which 719 were upregulated and 746 were downregulated. Figure 8(A) Among cellulose degradation-related genes, the endoglucanase gene JC1303_01352 was significantly upregulated, suggesting it may act as a key enzyme in the initial attack on cellulose polymers. Furthermore, we found that gene JC1303_02942 is annotated in the cellulose degradation pathway and is significantly upregulated. Notably, several transporter-related genes (including JC1303_03320, JC1303_00515, JC1303_00516, and JC1303_00517) were significantly upregulated, which facilitates efficient uptake of the products cellobiose and cellodextrins, and maintains intracellular metabolism. RT-qPCR analysis showed that the expression trends of all validation genes were consistent with the RNA-seq data, thus confirming the reliability of this transcriptome dataset. Figure 8 (B in the middle).

[0044] 5. Heterologous expression and enzymatic properties analysis of gene JC1303_02942 Gene cloning, plasmid construction and protein expression The target gene JC1303_2942 was amplified by PCR using 2×Phanta Max Master Mix (Nanjing Novizan Biotechnology Co., Ltd., China). After gel purification, the amplified gene fragment was ligated into the linearized pET28a vector via homologous recombination using the ClonExpress UltraOne Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd., China). The recombinant plasmid (named pET28a-2942) was transformed into *E. coli* DH5α chemocompetent cells. Transformants were screened on LB agar plates containing 50 μg / mL kanamycin. Positive clones were initially screened by colony PCR, followed by Sanger sequencing to confirm correct gene insertion and sequence accuracy. The sequence-verified recombinant plasmid was then transformed into *E. coli* BL21(DE3) competent cells for heterologous protein expression.

[0045] Recombinant *E. coli* BL21(DE3) cells were cultured in LB medium containing 50 μg / mL kanamycin at 37°C with shaking at 200 rpm until the OD reached 0.6–0.8. Protein expression was induced by the addition of 0.2 mM isopropyl-β-D-thiogalactoside (IPTG), followed by incubation at 16°C for 20 h. Cells were collected by centrifugation at 10,000 rpm for 10 min at 4°C. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.1% Triton X-100, 0.2 mM PMSF) and sonicated. The lysis buffer was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was filtered through a 0.22 μm filter.

[0046] His-tagged proteins were purified using Ni-NTA agarose resin (Shanghai Sangon Biotech Co., Ltd., China) and eluted with elution buffers containing different concentrations of imidazole (10 mM, 20 mM, 250 mM dissolved in 50 mM Tris-HCl, pH 8.0, 300 mM NaCl). The eluted fractions were analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining with Coomassie Brilliant Blue G-250 ultrafast staining solution (Shanghai Sangon Biotech Co., Ltd., China) to assess protein purity. The purified protein was concentrated using 10 kDa ultrafiltration tubes (Shanghai Sangon Biotech Co., Ltd., China). Protein concentration was determined using the Bradford Protein Assay Kit (Beyotime Biotechnology Co., Ltd., China). The purified protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0047] Enzyme activity assay and biochemical characterization β-D-glucanase activity was determined using the DNS method: The reaction system contained 50 μL of diluted enzyme solution and 150 μL of 5 mg / mL β-D-glucan solution (50 mM NaHPO4-citrate buffer, pH 5.0). The reaction was carried out at 40 °C for 10 min, then 200 μL of DNS reagent was added to terminate the reaction. The mixture was then boiled in a water bath for 5 min for color development, cooled to room temperature, and the absorbance was measured at 540 nm. One unit of enzyme activity was defined as the amount of enzyme required to release 1 μg of reducing sugar per minute under the above conditions.

[0048] The optimal temperature was determined by measuring enzyme activity at different temperatures (4, 10, 20, 30, 40, 50, 60, 70, 80 °C) under pH 5.0 conditions. Thermal stability was assessed by pre-incubating the enzyme at each temperature for 1 hour, followed by measuring residual activity under standard assay conditions.

[0049] The optimal pH was determined by measuring enzyme activity in different pH buffers (pH 3.0–10.0) at the optimal temperature (NaHPO4-citric acid buffer for pH 3.0–6.0, Tris-HCl buffer for pH 7.0–8.0, and glycine-NaOH buffer for pH 9.0–10.0). pH stability was determined by incubating the enzyme in different pH buffers at 4°C for 1 hour, followed by measuring residual activity at the optimal temperature and pH.

[0050] The effect of NaCl on enzyme activity was studied by measuring the enzyme activity at different NaCl concentrations (0, 171, 342, 684, 1026, 1368, 1710, 2565, 3420 mM) under optimal temperature and pH conditions.

[0051] By adding various additives to the reaction system at final concentrations of 1 mM and 10 mM, respectively, the study of metal ions (Mg) was conducted. 2 Ca 2 Mn 2 Cu 2 Zn 2 Co 2 Ba 2 Ni 2 Fe 2 Fe 3 Al 3 The effect of any added substance on enzyme activity. Activity without any added substance is defined as 100% (control).

[0052] The maximum enzyme activity observed in all characterization experiments was defined as 100% relative activity, and all assays were repeated three times.

[0053] Substrate specificity and enzyme kinetics To evaluate the substrate specificity of enzyme JC1303_2942, β-D-glucan, sodium alginate, CMC-Na, guar gum, locust bean gum, and laminarin (all at a concentration of 5 mg / mL) were used as substrates. The reactions were carried out for 10 minutes at optimal temperature and pH. The optimal substrate activity was set as 100% relative activity.

[0054] result Biochemical characteristics of β-D-glucanase The optimal reaction temperature for JC1303_2942 is 40℃. Figure 9 It is worth noting that the enzyme activity of JC1303_2942 is less than 50% above 40℃. Thermostability tests show that the JC1303_2942 enzyme exhibits maximum activity after incubation at 4℃ for 1 hour; stability gradually decreases with increasing temperature, eventually tending towards complete inactivation. Figure 9B). After incubation at 40°C for 1 hour, JC1303_2942 retained only 23.4% of its initial activity, indicating that JC1303_2942 is a psychrophilic enzyme. Figure 9 B).

[0055] pH activity profiles show that the optimal pH for JC1303_2942 is 5.0. Figure 9 C). JC1303_2942 retained 74.1% of its activity at pH 4.0, while it had 33.0% of its activity at pH 7.0. Figure 9 C). pH stability tests showed that after treatment with pH 5.0-7.0 buffer at 4°C for 1 hour, the JC1303_2942 enzyme retained more than 50% of its activity, with JC1303_2942 still retaining a significant 94.6% activity at pH 5.0. Figure 9 D). In summary, JC1303_2942 is characterized by its resistance to weak acids.

[0056] NaCl promotes the catalytic activity of JC1303_2942 and exhibits a biphasic effect, meaning that the activity first increases and then decreases with increasing salt concentration. Figure 9 E). The maximum enzyme activity of JC1303_2942 occurred at a NaCl concentration of 2565 mM (E). Figure 9 E), which indicates that the JC1303_2942 enzyme has strong salt tolerance.

[0057] The effect of metal ions on the enzyme activity of JC1303_2942 showed that at a concentration of 1 mM, Ca 2 Zn 2 Cu 2 Mg 2 Ba 2 and Co 2 It promotes enzyme activity; conversely, Mn 2 Al 3 Fe 2 Ni 2 Fe 3 It significantly inhibits enzyme activity, and all metal ions show inhibitory effects at a concentration of 10 mM. Figure 9 F).

[0058] Substrate specificity and enzyme kinetic parameters Substrate specificity assays revealed significant differences in preference among the JC1303_2942 enzymes. JC1303_2942 exhibited the highest activity towards β-D-glucan, followed by laminarin, with an activity 22.1% of that of β-D-glucan. Figure 10 It has no activity against CMC-Na and sodium alginate. Figure 10 ).

[0059] Enzymatic characterization of recombinant β-D-glucanase JC1303_2942 revealed significant salt tolerance, but its substrate specificity indicated that it primarily acts on β-D-glucan, rather than CMC-Na. Combined with genome-wide annotation... Figure 5 D) and transcriptome data analysis Figure 8 This study demonstrates that the highly efficient cellulose degradation ability of strain JC1303 is the result of the synergistic action of its multiple endoglucanases (such as the GH5 and GH9 families), β-glucosidases, and other related enzyme systems. JC1303_2942, as a salt-tolerant β-D-glucanase, may play an auxiliary role in the further hydrolysis of specific glucan intermediates under saline conditions, thus contributing to the overall salt-tolerant degradation phenotype of the strain.

[0060] 6. Molecular identification and phylogenetic analysis of strain JC1303 16S rRNA sequencing Genomic DNA was extracted from strain JC1303 using the Qiagen Bacterial Genomic DNA Extraction Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. DNA quality was assessed by 1% agarose gel electrophoresis and Nanodrop 2000 (Thermo Scientific, USA), and concentration was determined using a Qubit 3.0 quantitative PCR instrument. Primer 27F (5′-GAGTTTGATCMTGGCTCAG-3) was used to extract the DNA. ′ The 16S rRNA gene was amplified using 1492R (5′-GGTTACCTTGTTACGACTT-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The total volume of the PCR system was 25 μL, including 12.5 μL of Taq enzyme, 1 μL of template DNA, 1 μL of forward primer, 1 μL of reverse primer, and 9.5 μL of nucleic acid-free water. The amplification program was as follows: pre-denaturation at 94℃ for 4 min; followed by 30 cycles (denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1.5 min); and a final extension at 72℃ for 10 min. The amplified products were sequenced by Shanghai Sangon Biotech Co., Ltd.

[0061] The 16S rRNA gene sequence is shown in SEQ ID NO: 1.

[0062]

[0063] 7. Whole genome sequencing and annotation of strain JC1303: DNA extraction, library construction and sequencing The genome sequencing of strain JC1303 was performed by Wuhan Wanmo Biotechnology Co., Ltd. Quality-controlled DNA samples underwent damage repair, end repair, and magnetic bead purification sequentially; after ligating barcode tags, they were purified again with magnetic beads and then ligated with sequencing adapters. The constructed DNA libraries were sequenced on Illumina and Nanopore platforms, respectively. After filtering low-quality reads, they were mixed and assembled using Unicycler software (version 0.4.9). Error correction was performed using Pilon (version 1.23) and Bowtie2 (version 2.4.1) to obtain a high-precision genome sequence. The complete genome sequence is shown in SEQ ID NO: 2.

[0064] Genome annotation Protein-coding genes were predicted using Prodigal software (version 2.6.2); tRNA genes were predicted using the tRNAscan-SE tool (version 2.0); and rRNA genes were predicted using Barrnap software (version 0.7). A circular genome map was constructed using the "circlize" program in R software. Protein sequences were BLAST-aligned with Nr, Swiss-Prot, GO, eggNOG / COG, KOG, KEGG, and Pfam databases to obtain functional annotation information. Genes encoding carbohydrate-active enzymes were identified using the dbCAN online annotation platform of the CAZy database.

[0065] Phylogenetic analysis Fourteen 16S rRNA sequences were selected from the EZbioCloud database. A phylogenetic tree was constructed using the neighbor-joining method with MEGA software (version 11.0), and 1000 bootstrapping tests were performed. *Vulcaniibacterium tengchongense* was considered an outgroup. Whole-genome phylogenetic analysis was performed using Type Strain Genome Server (TYGS) based on the GBDP (Genome BLAST Distance Phylogeny) method. The "trimming" algorithm was used for accurate intergenomic distance inference, and a minimal phylogenetic tree was constructed using FASTME (version 2.1.6.1) combined with SPR post-processing. Branch support was calculated using 100 pseudo-bootstrapping replicates. The average nucleotide identity (ANI) was calculated using the JSpecies online platform, and the digital DNA-DNA hybridization (dDDH) value was calculated using the TYGS platform. (See [link to JSpecies online platform]). Figure 3 A in the middle.

[0066] pan-genome analysis The whole genome sequences of 22 strains of *Pseudoxanthomonas* were downloaded from the NCBI database (Table S2). Pan-genome and core genome analyses were performed using the integrated prokaryotic genome and pan-genome analysis platform IPGA (version 1.09) (Liu et al., 2022) with default parameters. Genes within the genome were predicted and identified using Roary software, analyzing core genes (genes common to all strains) and unique genes (genes contained only in a single strain). All genes were annotated based on the COG database.

[0067] result Isolation and enzyme activity determination of cellulose-degrading bacteria The JC1303 strain isolated from the sediments exhibited cellulose degradation activity. Figure 1 (A) To evaluate the cellulose degradation ability of JC1303, the activities of its CMCase, CXase, β-Gase, and FPase were measured. The activities of all four enzymes showed a trend of first increasing and then decreasing. Figure 1 The concentrations of B in the culture reached their peak on day 5: CMCase 1.66±0.07 U / mL, CXase 2.11±0.17 U / mL, β-Gase 1.54±0.15 U / mL, and FPase 0.65±0.11 U / mL. Figure 1(B in the text). Strain JC1303 forms light-colored, irregularly shaped colonies on 2216E plates, with a moist and slightly rough surface. Gram staining shows that the bacterium is Gram-negative, usually arranged singly or in short chains. Scanning electron microscopy reveals that the cells are slender rod-shaped (…). Figure 2 This strain can grow at temperatures of 15-35℃, salinity of 0-5%, and pH of 5-9. The crude enzyme activity of strain JC1303 retains 20% CMCase activity at 50℃ and 70% at pH 9. In a reaction system with a sodium chloride concentration of 5%, the crude enzyme activity can still be maintained at 57%.

[0068] Phylogenetic analysis The phylogenetic tree constructed based on the 16S rDNA sequence is shown below. Figure 3 In the phylogenetic tree, A, JC1303 clustered with species of the genus *Pseudoxanthomonas*, confirming its taxonomic position. JC1303 is most closely related to *Pseudoxanthomonasspadix* DSM 18855. (A phylogenetic tree constructed based on the whole genome is shown.) Figure 3 Figure B) shows that JC1303 clusters with P. spadix DSM 18855 and Pseudoxanthomonas winnipegensis NML 130738. The ANI heatmap results indicate that the ANI values ​​of JC1303 with P. winnipegensis NML 130738 and P. spadix DSM 18855 are 82.23% and 81.18%, respectively, both well below the 95% species classification threshold (see Figure B). Figure 4 The dDDH value was also significantly lower than the 70% species definition standard, with the highest being 40% of P. winnipegensis NML 130738 (Table 1), suggesting that JC1303 may be a new species of the genus Pseudoxanthomonas.

[0069] Table 1. DDH values ​​of strain JC1303 compared to other strains. strain name DDH value Pseudoxanthomonas winnipegensis NML130738 40 Pseudoxanthomonas spadix DSM 18855 32.3 Pseudoxanthomonas composti GSS15 27.4 Xanthomonas youngii AmX2 21.7 Xanthomonas sontii CFBP 8688 20.6 Xanthomonas surreyensis Sa3BUA13 21 Xanthomonas rydalmerensis DAR 34855 20.8 Xanthomonas chitinilytica H13-6 21.4 Xanthomonas bonasiae CFBP 8703 21.2 Xanthomonas hawaiiensis A6251 21.3 Pseudoxanthomonas putridarboris WD12 19.2 Xanthomonas indica PPL560 21.6 Xanthomonas sacchari CFBP 4641 21.4 Xanthomonas protegens PPL118 21.6 Pseudoxanthomonas helianthi 110414 18.4

[0070] Genomic characteristics The JC1303 genome has a coverage of 392×, is a single complete chromosome, and is 4,365,879 bp in length with a G+C content of 67.41% (Table 2). Gene prediction results show a total of 3,871 genes, including 3,803 protein-coding genes, 4 rRNA genes, and 61 tRNA genes (Table 2). COG annotation covers a total of 2,592 genes (see Table 2). Figure 5In the A category, the most numerous are those with unknown functions (483 genes), followed by amino acid transport and metabolism (216 genes), cell wall / membrane / capsule biosynthesis (196 genes), energy production and conversion (179 genes), translation and ribosome structure and biosynthesis (170 genes), and carbohydrate transport and metabolism (164 genes). GO annotations cover a total of 2,303 genes, encompassing three main categories: biological processes, cellular composition, and molecular function (see [link to GO annotation]). Figure 5 The classification is divided into 52 subclasses, with cellular processes (1,416) and metabolic processes (1,234) being the most abundant in biological processes; among the cellular composition subclasses, cells (1,433) and cellular components (1,425) are dominant; and among the molecular functions subclasses, catalytic activity (1,552) and binding (1,332) are the most abundant. Figure 5 B). KEGG annotations included 1,988 genes, with metabolism being the most numerous (1,523) across 6 major categories. Among these, carbohydrate metabolism (324), global and overview metabolism (276), and amino acid metabolism (257) were the most significant (see [link to KEGG annotation]). Figure 5 The C in the text is missing. Annotation of carbohydrate-active enzymes (CAZy) shows that JC1303 contains 319 related genes, including 97 glycoside hydrolases (GH), 82 glycosyltransferases (GT), 74 carbohydrate esterases (CE), 48 accessory activity (AA) proteins, 10 carbohydrate binding modules (CBM), and 8 polysaccharide lyases (PL) (see...). Figure 5 Among them, GH3 and GH5 families are associated with cellulose degradation, while GH10 and GH43 are involved in xylan degradation, etc.

[0071] Table 2. Genomic characteristics of strain JC1303 feature numerical values Genome size (bp) 4,365,879 N50 4,365,879 N90 4,365,879 GC content (%) 67.41％ Number of genes 3,871 Number of protein-coding genes 3,803 tRNA count 61 rRNA quantity 4

[0072] 8. Metabolic pathways and pangenome analysis of strain JC1303 Metabolic pathways The metabolic pathway of JC1303 was analyzed based on KEGG annotation (see...). Figure 6 The study found that strain JC1303 contained three endoglucanase genes, two cellulase genes, and four β-glucosidase genes, but no exoglucanase genes were detected. Unlike typical cellulose-degrading microorganisms, we found that the genome of strain JC1303 lacks exoglucanase. It is speculated that strain JC1303 may possess novel exoglucanases, or that unannotated exoglucanases are present. Another possibility is that strain JC1303 does not require exoglucanases, but instead degrades cellulose into cellobiose through the synergistic action of endoglucanases and β-glucosidases, which are then further converted into glucose. The absence of exoglucanases has been reported in the literature for some cellulose-degrading bacteria.

[0073] This bacterium possesses a complete metabolic pathway encompassing glycolysis / gluconeogenesis, the tricarboxylic acid cycle, pentose phosphate, and pyruvate, including key enzymes such as hexokinase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and pyruvate kinase, and multiple amino acid synthesis pathways. Its genome encodes a two-component system and ABC transporters, enabling it to sense cellulose and transport extracellular sugars. For example, gene 3320 is involved in the transport of glucose / mannose, cellobiose, chitobiose, and α-glucosides, while genes 513, 515, 516, and 517 are responsible for the transport of sorbitol / mannitol. Furthermore, JC1303 contains a complete respiratory chain composed of a type IV complex.

[0074] pan-genome analysis Pan-genome analysis of core and specific genes from 23 strains of *Xanthomonas* using IPGA yielded 26,278 pan-gene clusters, of which only 847 were core gene clusters, accounting for 3%. Figure 7 In the A), the number of unique genes of different strains is 387-1,372 (in the A), and the number of unique genes of different strains is 387-1,372 (in the A). Figure 7 (B in the original text). JC1303 possesses 936 unique genes, sharing 228 genes with its closely related species, *P. winnipegensis* NML130738. Core gene COG annotations show 345 metabolic genes, 261 information storage and processing genes, and 217 cellular process and signaling genes; among the JC1303-specific genes, 163 are metabolic, 68 are information storage and processing, and 138 are cellular process and signaling genes. Figure 7 (C in the middle).

[0075] In summary, this application provides a novel marine *Pseudomonas* strain JC1303 with accession number CCTCC NO: M 20252026, and discloses microbial preparations containing this strain, methods for degrading cellulose materials, and methods for producing reducing sugars. Through whole-genome sequencing and functional annotation, this application systematically reveals for the first time that this strain carries a complete cellulose-degrading enzyme system, including endoglucanase, cellulase, and β-glucosidase, possessing multiple central metabolic pathways supporting efficient cellulose degradation and utilization. Furthermore, its unique genetic resources were identified through pan-genome analysis. The above technical solutions achieve significant technical effects: this strain exhibits high cellulase activity (e.g., FPase activity reaches 0.65±0.11 U / mL) as early as day 5 of culture, and thanks to its unique genetic background and marine origin, it can stably and efficiently degrade cellulose materials such as agricultural waste in saline environments.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of *Xanthomonas* JC1303, characterized in that, It was deposited at the China Center for Type Culture Collection on September 15, 2025, with accession number CCTCC NO: M 20252026 and classification name Pseudoxanthomonas sp.JC1303.

2. A microbial preparation, characterized in that, It comprises the Xanthomonas strain JC1303 as described in claim 1, and one or more agriculturally acceptable vectors.

3. The microbial preparation according to claim 2, characterized in that, The formulation is in the form of lyophilized powder or liquid bacterial agent, and the viable count of the strain JC1303 is not less than 1×10 CFU / g.

4. A method for degrading cellulose materials, characterized in that, The method includes the following steps: contacting the Xanthomonas genus strain JC1303 of claim 1, or the microbial preparation of claim 2 or 3, with a cellulose material.

5. The method according to claim 4, characterized in that, The cellulose material is derived from agricultural waste.

6. The method according to claim 4, characterized in that, Incubate in a culture medium containing sodium carboxymethyl cellulose or microcrystalline cellulose at 25℃-30℃ and 150-200 rpm for 3-7 days.

7. A method for producing reducing sugars, characterized in that, Cellulose materials are degraded using the method described in any one of claims 4-6, and the resulting reducing sugars containing glucose are collected.

8. Use of strain JC1303 of claim 1 and / or its metabolites in the preparation of formulations for degrading cellulose, treating agricultural waste or producing biofuels.

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