Space mutagenesis to obtain high-selenium tolerance and high-selenium cysteine synthesis of bacillus subtilis and its application

Abstract

This invention belongs to the field of microbial technology and provides a space-mutated *Bacillus paralichrysiformis* strain with high synthetic selenocysteine ​​and high sodium selenite tolerance, obtained through space mutagenesis, and its applications. The *Bacillus paralichrysiformis* G1-m strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36525, deposited on November 11, 2025. This strain has significant core advantages: firstly, it possesses extremely high sodium selenite tolerance, able to withstand sodium selenite stress up to 440 mM; secondly, its selenocysteine ​​and nano-selenium synthesis efficiency is significantly improved, not only significantly increasing the strain's resistance threshold to selenium stress but also directionally enhancing the activity of the selenocysteine ​​biosynthetic pathway. Its application potential covers multiple fields, such as selenium-enriched probiotic preparations, nano-drug carriers, functional food additives, and high-efficiency selenium nutritional supplements, laying the foundation for the high-value utilization of selenium resources and the industrial application of functional microorganisms.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a Bacillus paralichrysiformis that has been obtained through space mutagenesis with high synthetic selenocysteine ​​and high sodium selenite tolerance, and its application. Technical Background

[0002] Selenium is an essential trace element for the human body. Selenocysteine, as an important biological form of selenium, participates in the formation of active selenium-containing proteins and is associated with key life processes such as antioxidation and immune regulation. Therefore, the efficient acquisition of selenocysteine ​​has become a research hotspot in the food and pharmaceutical fields.

[0003] Microbial synthesis is an important route for the preparation of selenocysteine ​​due to its mild reaction, environmental friendliness, and ease of scalability. However, in existing studies, few *Bacillus paralichrysiforme* strains have been reported to possess the ability to synthesize selenocysteine. Even when some strains show basic selenium metabolic activity, significant shortcomings exist: firstly, they have low tolerance to sodium selenite, and high concentrations of selenium sources inhibit strain growth, limiting the efficiency of industrial fermentation; secondly, even if the selenium source can be converted at low selenium concentrations, it is difficult to synthesize selenocysteine ​​in a targeted manner, resulting in low selenium source conversion and utilization rates and low synthesis efficiency of the target product, which seriously affects the development of related products.

[0004] Mutagenesis breeding is a common method for improving bacterial strains. Among them, space mutagenesis, relying on the space environment such as microgravity and strong radiation, can efficiently induce gene mutations in microorganisms and is an effective way to select superior strains. However, given the scarcity of reports on Bacillus paralichrysiforme synthesizing selenocysteine, research on the targeted selection of dual-superior strains with "high sodium selenite tolerance + high selenocysteine ​​synthesis efficiency" using space mutagenesis is even more lacking. The industry has long lacked high-quality germplasm resources to support the development of selenocysteine-related products.

[0005] The increasing demand for selenium-enriched probiotics, nanomedicine carriers, and related products has significantly increased the requirements for the selenocysteine ​​synthesis capacity and stress resistance of core strains. Existing microbial resources, due to the aforementioned deficiencies, cannot support product research and development and industrialization, hindering the high-value utilization of selenium resources and creating bottlenecks in the development of the functional microbial industry. Therefore, this invention aims to protect a novel strain of *Bacillus paralichrysogenus* isolated from the root soil of the ultra-selenium-rich plant *Corydalis violaceus* and subsequently obtained through space mutagenesis. This fills the technological gap in *Bacillus paralichrysogenus*'s selenocysteine ​​synthesis and provides core germplasm support for related industries. Summary of the Invention

[0006] In response to the lack of reports on the synthesis and secretion of selenocysteine ​​by *Bacillus paralichrysogenus* in existing technologies, and the fact that existing strains generally suffer from low tolerance to sodium selenite and insufficient selenium form conversion efficiency, this invention aims to provide a new *Bacillus paralichrysogenus* strain isolated from the root soil of the ultra-selenium-rich plant *Corydalis yanhusuo* and bred through space mutagenesis (this strain was carried aboard my country's first reusable recoverable experimental satellite—Shijian-19, launched by a Long March 2D carrier rocket from the Jiuquan Satellite Launch Center on September 27, 2024, and recovered from the Dongfeng Landing Site on October 11 of the same year, operating in orbit for 14 days). This strain possesses both extreme selenium stress resistance and highly efficient selenocysteine ​​conversion ability, filling the technological gap in the synthesis of selenocysteine ​​using *Bacillus paralichrysogenus*, and simultaneously overcoming the technological limitations of microbial methods for producing high-value selenium products.

[0007] This invention provides a strain isolated from the root soil of the ultra-selenium-rich plant *Corydalis yanhusuo* and subjected to space mutagenesis, exhibiting high selenocysteine ​​synthesis capacity and high sodium selenite tolerance. This strain is *Bacillus paralicheniformis* (…). Bacillus paralicheniformis G1-m was deposited on November 11, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.36525.

[0008] In addition, the original isolated strain of this strain (i.e., the starting strain G1 before space mutagenesis) naturally possesses the characteristic of "selenium concentration-dependent directional synthesis of selenium forms": it can synthesize nano-selenium in a high concentration sodium selenite environment (10~400 mM) and selenocysteine ​​in a low concentration sodium selenite environment.

[0009] Furthermore, through genome sequencing and transcriptome analysis of the original isolated strain, the key genes and metabolic pathways for the synthesis of selenocysteine ​​and nano-selenium have been identified: among which, cysP , tusA , ssuA It is the core gene responsible for selenite transport; mccA , mccB s ufS , trxA , trxB , trxC It is the core gene for selenocysteine ​​synthesis; cysJ , cysI , cysH , narH , narI , arsC This regulates the reduction process of sodium selenite to nano-selenium.

[0010] In addition, the G1-m strain obtained through space mutagenesis can grow on sodium selenite plates with a maximum growth rate of 440 mM, and its overall growth performance is significantly better than that of the original strain.

[0011] Furthermore, under low-concentration sodium selenite conditions, the G1-m strain achieved a maximum yield of 5.213 μg / g of selenocysteine, which was significantly better than that of the original strain (3.587 μg / g).

[0012] Furthermore, under conditions of higher concentrations of sodium selenite (5 mM), the efficiency of G1-m strain in synthesizing nano-selenium was 93.27%, which was significantly better than that of the original strain (90.19%).

[0013] In addition, both synthetic selenocysteine ​​and nano-selenium are transported and secreted into the extracellular space in large quantities.

[0014] Furthermore, comparing the whole genome sequences of strains G1 and G1-m before and after mutagenesis revealed that the mutagenized strain G1-m had 11 key site mutations, among which... katE Conserved in-frame deletion of genes; cypB The gene is a frameshift mutation (p.Arg600fs), with the deletion of base A causing a reading frame shift; cysJ The gene exhibits a missense mutation (p.Leu507Ser), with a T-base mutation replaced by a C-base substitution. These mutations may significantly enhance the catalytic activity and substrate binding capacity of key enzymes, and are speculated to be the core molecular mechanism underlying the simultaneous increase in selenocysteine ​​synthesis efficiency and sodium selenite tolerance in this strain.

[0015] This invention also discloses the application of Bacillus paralichrysum G1-m in the preparation of animal feed additives for improving the antioxidant status, growth performance or meat and egg quality of animals; in plant biological agents for promoting crop growth or enhancing stress resistance; in soil conditioners for improving the rhizosphere microecological environment, activating soil nutrients and remediating heavy metal pollution; in agricultural product preservatives for improving the storage quality of agricultural products, degrading pesticide residues or inhibiting plant pathogens; in functional foods or health products for antioxidant, anti-inflammatory and immune-enhancing effects through the intestines; and in drugs for intestinal-directed release.

[0016] Strain Preservation Information Bacillus paralichrysogenum G1-m, deposited at: China General Microbiological Culture Collection Center (CGMCC); Postcode: 100101; Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: November 11, 2025; accession number: CGMCC No. 36525; classification and nomenclature: Bacillus paralichrysogenum Bacillus paralicheniformis .

[0017] The advantages of this invention compared to the prior art are: This invention screened and obtained a strain of *Bacillus paralichrysum* G1-m, isolated from the root soil of the ultra-selenium-rich plant *Corydalis yanhusuo* and subjected to space-induced mutagenesis. Its preservation number is CGMCC No. 36525; its classification name is *Bacillus paralichrysum*. Bacillus paralicheniformis This strain has great potential to promote the industrial application of selenium-enriched functional products. 1. Achieve breakthroughs in both the targeted synthesis and high selenium tolerance of selenocysteine. The *Bacillus paralichrysogenus* G1-m provided by this invention not only fills the technological gap in the rare synthesis of selenocysteine ​​by *Bacillus paralichrysogenus*, but also achieves a breakthrough in tolerance to high levels of sodium selenite, growing on sodium selenite plates up to 440 mM. This dual advantage effectively solves the core bottlenecks of existing *Bacillus paralichrysogenus* strains, such as the difficulty in directionally synthesizing selenocysteine ​​and the inhibition of growth at high selenium concentrations, providing strain support for the efficient conversion of selenium sources.

[0018] 2. Achieving synergistic performance improvement by relying on key gene mutations The superior performance of strain G1-m of this invention stems from space-induced mutations in key genes specific to the mutation (e.g., cysJ , yumC , ydbD These mutations (site mutations) not only enhance the catalytic activity and substrate binding ability of key enzymes, but also simultaneously improve the efficiency of selenocysteine ​​synthesis and enhance sodium selenite tolerance, forming a synergistic performance advantage, which is different from the limitations of single trait improvement of existing strains.

[0019] 3. By combining unique strain sources with space mutagenesis, we can overcome the limitations of germplasm creation. This invention first isolates a primitive strain with the ability to synthesize selenium speciation from the root soil of the ultra-selenium-rich plant *Corydalis yanhusuo*. Then, by combining space mutagenesis technology (multi-factor synergy including microgravity and strong radiation), it overcomes the dual limitations of traditional mutagenesis ("uncontrollable mutagenesis direction and low beneficial mutation rate") and existing strains ("lack of distinctive selenium metabolic basis"), and successfully creates a breakthrough germplasm that has both the ability to synthesize selenocysteine ​​and high selenium tolerance, which is difficult to achieve with traditional technology.

[0020] 4. Facilitate the improvement of quality, efficiency, and industrialization of selenium product production using microbial methods. The G1-m strain provided by this invention serves as the core germplasm resource, achieving highly efficient synthesis of selenocysteine ​​(maximum yield 5.213 μg / g) under low-concentration sodium selenite conditions, and also synthesizing nano-selenium (conversion rate 93.27%) at high concentrations. Furthermore, both the synthesized selenocysteine ​​and nano-selenium are extensively transported and excreted extracellularly, facilitating concentration and collection. This breakthrough overcomes the bottleneck of "low efficiency and single product" in microbial production of high-value selenium products. Its excellent fermentation adaptability also ensures large-scale green production, powerfully promoting the industrialization of selenium-enriched functional products. Attached Figure Description

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments: Figure 1 Growth characteristics of Bacillus paralicheniformis G1 colonies.

[0022] Figure 2 A phylogenetic tree of Bacillus paralichrysiformis constructed based on 16S rRNA and whole genome sequence.

[0023] Figure 3 Mechanisms of selenite assimilation and dissimilation by Bacillus paralichrysogenus.

[0024] Figure 4 Tolerance of the original strain and the mutant strain on high-concentration sodium selenite plates.

[0025] Figure 5 Tolerance of the original strain and the mutant strain in high-concentration sodium selenite liquid medium. Detailed Implementation

[0026] 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.

[0027] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0028] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to exemplify and further explain and illustrate the content of the present invention, and are not intended to limit the present invention.

[0029] Example 1: Strain Screening and Identification The original strain G1 of *Bacillus paralicheniformis* described in this application was isolated from the root soil of *Cephalotaxus fortunei*. The operation steps are as follows: Select healthy *Cephalotaxus fortunei* plants, and use the five-point sampling method to collect rhizosphere soil samples at a depth of 5-10 cm around the roots. Collect approximately 5 g of soil samples from each plant, mix them, and place them in a sterile sealed bag. Mark the sampling time, location, and host plant information.

[0030] Take 10 g of soil sample and place it in an Erlenmeyer flask containing 90 mL of sterile physiological saline. Add a small amount of sterile glass beads and place the flask on a shaker (150 rpm, 30℃) for 30 min to prepare 10 g of soil sample. -1 Soil suspension was then used; subsequently, a gradient dilution method was employed to sequentially dilute the soil suspension to 10⁻⁶ with sterile physiological saline. -2 10 -3 10 -4 10 -5 10 -6 Concentration gradient. Subsequently, sodium selenite (Na2SeO3) was added to LB medium as a screening pressure and target metabolic substrate to prepare sodium selenite screening medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar. Deionized water was added to bring the volume to 1000 mL, and the pH was adjusted to 7.0-7.2. After autoclaving (121℃, 20 min), when the medium temperature cooled to 50-60℃, sterile sodium selenite stock solution (concentration 1 mol / L) was added in a sterile laminar flow hood to bring the final concentration of sodium selenite in the medium to 100 mM. After thorough mixing, the medium was poured into plates and allowed to solidify for later use.

[0031] In a sterile laminar flow hood, take the above 10 -4 10 -5 10 -6 Three concentration gradients of soil dilution, 100 μL each, were evenly spread onto sodium selenite selection medium plates, with three replicates for each concentration gradient. Sterile physiological saline plates were used as blank controls. The spread plates were inverted and placed in a constant temperature incubator at 30℃ for 24–48 h. During this period, colony growth and morphological characteristics were observed, with particular attention paid to the presence of colonies exhibiting "nano-selenium characteristics."

[0032] Single colonies exhibiting red colony characteristics were selected from the screening plates and inoculated onto fresh sodium selenite screening medium plates. Purification was performed using the streak plating method, and the culture was maintained at 37℃ for 24 h. This streak purification was repeated 2-3 times until a homogeneous, uncontaminated pure culture strain was obtained. After preliminary screening, a strain G1 with the strongest sodium selenite reducing ability was obtained (Figure 1). To further investigate the selenium conversion characteristics of strain G1, it was cultured under different sodium selenite concentrations, and the product components were analyzed by atomic fluorescence spectrometry. The results showed that strain G1 could convert sodium selenite to selenocysteine ​​(maximum yield 3.587 μg / g) in a low-concentration sodium selenite environment; while under higher selenite conditions (5 mM), it mainly converted sodium selenite to nano-selenium (conversion rate of 90.19% after 48 h). Centrifugation and ceramic membrane concentration separation of the bacterial cells and fermentation broth revealed that both synthesized selenocysteine ​​and nano-selenium were extensively transported and secreted extracellularly. In summary, strain G1 is a high-performance selenium-transforming strain: it can synthesize nano-selenium under high-concentration sodium selenite conditions and can directionally convert to organic selenium under low-concentration sodium selenite conditions. It possesses diversified selenium metabolism capabilities, providing high-quality strain material for subsequent research on efficient utilization of selenium resources and related applications.

[0033] To further identify strain G1, purified bacterial genomic DNA was used as a template for PCR amplification using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the bacterial 16S rRNA gene. The amplified products were sequenced, and the obtained sequences were compared for homology using BLAST to screen for the known strain sequence with the highest similarity. Finally, a neighbor-joining phylogenetic tree was constructed using MEGA12 software to analyze the evolutionary relationship between the candidate strain and known strains (Figure 2). It was determined that strain G1 clustered with the standard strain of *Bacillus paralichrysogenus* on the phylogenetic tree. Combined with the homology comparison results (similarity ≥ 99%), strain G1 was identified as *Bacillus paralichrysogenus*. The 16S rRNA gene sequence is shown in SEQ ID No:1.

[0034] Example 2: Gene Prediction and Mechanism Analysis To investigate the characteristics and regulatory mechanisms of selenium metabolism-related genes in Bacillus paralichrysiforme G1 under different sodium selenite concentrations and to clarify the molecular basis of its diversified selenium metabolism capabilities, this study explored potential functional genes through third-generation genome sequencing and analyzed differential expression patterns using transcriptome sequencing.

[0035] Using *Bacillus paralichrysogenus* G1 purified in Example 1 as the research object, three treatment groups were set up for culture: the control group used LB liquid medium without sodium selenite; the low concentration group used LB liquid medium with sodium selenite added to a final concentration of 1 mg / kg; and the high concentration group used LB liquid medium with sodium selenite added to a final concentration of 400 mg / kg. Three biological replicates were set up for each group. After inoculating strain G1 into the corresponding medium, the culture was placed on a shaker (150 rpm, 37℃) for 24 h, centrifuged (8000 rpm, 4℃, 10 min) to remove the supernatant, washed twice with sterile physiological saline, and stored at -80℃ for subsequent genome and transcriptome sequencing.

[0036] After genome sequencing, structural analysis of the genome was performed, focusing on genes related to the "selenium metabolism pathway" (map00450) and "cysteine ​​and methionine metabolism pathway" (map00270) in the KEGG database. At the same time, combined with selenium reductase genes and nano-selenium synthesis-related genes reported in the Nr database, potential selenium metabolism functional genes in the genome of strain G1 were screened.

[0037] After transcriptome sequencing, a cDNA library was constructed. Once the library passed quality control, it was sequenced using a next-generation high-throughput sequencing platform. Differentially expressed genes (DEGs) were screened and their functional enrichment was analyzed to identify key pathways and core genes related to selenium metabolism.

[0038] Genome sequencing and selenium metabolism-related gene prediction results showed that the Bacillus paralichrysum G1 genome consists of a single complete circular chromosome (4,380,176 bp in length, GC content 45.87%), without plasmid sequences. Genome structure analysis revealed that repetitive sequences accounted for approximately 0.39% of the genome, and four gene island regions and five CRISPR sites were predicted. Genome annotation results predicted a total of 4293 CDS genes, 82 tRNA genes, and 24 rRNA genes (8 5S rRNAs, 8 16S rRNAs, and 8 23S rRNAs). Annotation using general and proprietary databases further clarified the specific functions of 4289 CDS genes, among which 45 potential selenium metabolism-related genes were predicted, including selenite transport-related genes. tusA, cysP, thiS, ssuC, ssuA Etc.; Genes related to organoselenium synthesis include mccA, mccB, metK, mtaD Etc.; genes related to nano-selenium synthesis include narJ, narI, narH, cysJ, cysI etc.; selenium tolerance-related genes include ahpC, sodA, dsbE wait.

[0039] Transcriptome sequencing and differential expression analysis revealed 150 DEGs in the low-concentration group vs. the control group, including 60 upregulated genes and 90 downregulated genes. KEGG enrichment analysis showed that DEGs were significantly enriched in "selenium compound metabolism" (map00450), "sulfur metabolism" (map00920), and "amino acid metabolism" (map00250, map00260, map00280) (Table 1). This indicates that under low-concentration sodium selenite induction, *Bacillus paralichrysiformis* G1 promotes the conversion of sodium selenite to selenocysteine ​​by significantly regulating the expression of genes related to organoselenium synthesis, consistent with the phenotypic results of "directed generation of organoselenium at low concentrations" in Example 1.

[0040] Table 1. Differential gene enrichment pathways in the low concentration group vs. the control group.

[0041] DEG analysis between the high-concentration group and the control group identified 1624 DEGs, including 751 upregulated genes and 873 downregulated genes. KEGG enrichment analysis showed that DEGs were significantly enriched in "glutathione metabolism" (map00480), "sulfur metabolism" (map00920), "ascorbic acid and aldehyde metabolism" (map00053), and "purine and pyrimidine metabolism" (map00230, map00240) (Table 2). Changes in the expression of selenium metabolism-related genes showed... trxA , trxC , trxB , narI , cysI The gene expression levels were 3.8, 33.6, 3, 5.1, and 52.7 times that of the control group, respectively; at the same time, the expression level of thiosulfate-transferase activity TusA was 649.2 times that of the control group. This indicates that under high concentration sodium selenite pressure, Bacillus paralichrysum G1 promotes the synthesis of nano-selenium by upregulating thioredoxin-related genes and selenium reduction-related genes (nitrate reductase, sulfite reductase, etc.) to catalyze the reduction of sodium selenite. At the same time, it reduces the antioxidant damage caused by selenite by upregulating superoxide dismutase and enhances its own tolerance, which is consistent with the phenotypic results of "synthesis of nano-selenium under high concentration" in Example 1.

[0042] Table 2. Differential gene enrichment pathways in the high-concentration group vs. the control group.

[0043] Based on the gene localization information from the genome sequencing and the differential expression analysis results from the transcriptome sequencing, it can be clearly seen that the mechanism of selenite reduction by Bacillus paralichrysogenus G1 exhibits concentration-dependent regulatory characteristics (Figure 3). Under low concentrations of sodium selenite, the strain utilizes selenite transport-related genes (such as...)cysAWPT , ssuAC (etc.) take selenite into cells, and then through the thioredoxin system ( trxABC It is reduced to selenide (H2Se); H2Se combines with O-acetylserine, which is produced from serine by serine acetyltransferase, and synthesizes selenocysteine ​​under the action of cysteine ​​synthase; on the other hand, it combines with O-acetylhomoserine under the catalysis of cystathionine β-synthiazide, cystathionine γ-lyase, and S-adenosylmethionine synthase to synthesize selenocysteine. Under high concentrations of sodium selenite, the strain mainly synthesizes nano-selenium through three pathways: one is the thioredoxin system ( trxABC The redox regulation mediated by (etc.) is twofold: first, the assimilation sulfite reduction pathway (etc.) cysHCJI The reducing effect of nitrite and arsenate, and the genes related to nitrite and arsenate (etc.) narJ / I / H , arsC The reduction process, involving substances such as sodium selenite, ultimately converts high-concentration sodium selenite into nano-selenium.

[0044] Example 3: Space-induced mutagenesis and screening To investigate the mutagenic effects of the combined space environment (high vacuum, microgravity, high-energy cosmic ion radiation, etc.) on Bacillus paralichrysiforme G1 and to screen for mutant strains with superior selenium conversion capabilities, a space-borne experiment was conducted. The specific procedures are as follows: Bacillus paralichrysogenus G1 was cultured to the logarithmic growth phase (37℃, 150 rpm, LB medium), and the bacterial cells were collected by centrifugation (8000 rpm, 4℃, 10 min). The cells were washed three times with sterile 0.1 M phosphate buffer (pH 7.0) and the precipitate was collected. A lyophilization protectant (10% skim milk powder) was added to the bacterial cells, and the mixture was freeze-dried to prepare a lyophilized bacterial powder. The lyophilized powder was sealed in preservation tubes and launched into space aboard the "Shijian-19" satellite. It operated for 13 days in an orbit with a perigee of 175–200 km and an apogee of 300–500 km, during which time the strain was continuously exposed to the complex space environment (including high vacuum, microgravity, and mutagenic factors such as high-energy cosmic ion radiation). After the satellite returned to Earth, a portion of the lyophilized bacterial powder was immediately revived and cultured to restore damaged cells and proliferate the mutant population.

[0045] After resuscitation and culture, the bacterial suspension was appropriately diluted and spread onto solid screening plates containing gradient concentrations of sodium selenite. The selenium concentration gradients were set at 240, 280, 320, 360, 400, and 440 mM. The plates were incubated at 37°C for 3 days, and the growth of colonies on each concentration plate was observed and recorded periodically. Single colonies that grew well and had normal morphology on plates with concentrations significantly higher than the tolerance of the original strain were selected as the initial screening selenium-tolerant mutant strains. The mutant strains obtained from the initial screening were numbered and preserved in glycerol tubes. Among them, the mutant strain numbered G1-m showed the highest tolerance to sodium selenite and was used as the subject of subsequent research.

[0046] Example 4: Comparison of reduction characteristics between original and mutant strains To comprehensively evaluate the tolerance and transformation characteristics of the space-mutated selenite-resistant mutant strains obtained in the initial screening of Example 3, and to compare them with the corresponding ability of the original strain G1, this example conducts a comprehensive analysis from the following two aspects: Selenite tolerance assessment: Fresh bacterial suspensions of the original strain and the mutant strain G1-m (logarithmic growth phase OD) were prepared. 600 (≈0.8) The microbial concentration was inoculated onto solid LB agar plates containing gradient sodium selenite concentrations (240, 280, 320, 360, 400, 440 mM) using the dilution plating method. The plates were incubated at 37℃ for 4 days. Colonies of each strain on plates with different selenium concentrations were observed and recorded (observation indicators: size, morphology, and growth abundance). Particular attention was paid to whether colonies could form and their growth status at high selenium concentrations. For liquid culture, liquid LB medium containing gradient sodium selenite concentrations of 240, 280, 320, 360, 400, and 440 mM was prepared. Fresh logarithmic-phase bacterial suspensions of the original strain and mutant strains were inoculated into the corresponding medium at a 1% inoculation rate and incubated at 37℃ and 150 rpm for 24 h. The color of the bacterial solution was observed, paying attention to the characteristic red color of nano-selenium and its intensity.

[0047] Selenocysteine ​​synthesis capacity determination: Fresh bacterial suspensions of the original strain and the mutant strain were inoculated into liquid LB medium containing low concentrations of sodium selenite, respectively. The inoculated medium was then placed in a shaker (150 rpm, 37℃) for 24 h. After incubation, the bacterial suspensions were collected, and the amount of selenocysteine ​​produced in the culture medium was detected by atomic fluorescence spectrometry. The selenocysteine ​​conversion rate of the two strains under different concentrations of sodium selenite was calculated. The differences in the ability of the original strain and the mutant strain to synthesize selenocysteine ​​in a low-concentration selenite environment were compared and analyzed, with a focus on the conversion efficiency and synthesis capacity of the two strains at the optimal concentration.

[0048] Observation of plate images revealed significant differences in the growth performance of the original strain and the mutant strain G1-m on high-concentration sodium selenite plates (Figure 4): Under high-concentration sodium selenite (260~400 mM), both strains could form colonies, but the mutant strain G1-m had slightly more colonies than the original strain, and the colonies were fuller and more abundant. As the sodium selenite concentration increased to 440 mM, the original strain showed almost no growth, while the mutant strain maintained a high colony count and stable growth. The same phenomenon was observed in LB liquid medium with the same sodium selenite concentration (Figure 5), indicating that the selenium tolerance of the mutant strain was significantly better than that of the original strain.

[0049] Atomic fluorescence spectrometry analysis showed that both the original strain and the mutant strain G1-m could synthesize selenocysteine ​​in a low-concentration sodium selenite environment, but their synthetic abilities differed significantly. The mutant strain G1-m exhibited higher selenocysteine ​​production and conversion rates than the original strain, with the highest production reaching 5.213 μg / g. Furthermore, in a higher concentration of sodium selenite (5 mM), the mutant strain's efficiency in synthesizing nano-selenium after 48 h of culture was also superior to that of the original strain (original strain: 90.19% vs. mutant strain: 93.27%). In summary, the mutant strain G1-m demonstrates significantly better selenite tolerance than the original strain, specifically exhibiting a higher colony count at the same sodium selenite concentration and the ability to survive and grow in higher selenium concentrations; its selenocysteine ​​and nano-selenium synthesis capabilities are also significantly stronger than those of the original strain.

[0050] Example 5: Molecular Mechanism Analysis of Mutagenic Strains To reveal the molecular basis for the significant enhancements in selenite tolerance and selenocysteine ​​synthesis capacity of the space-mutated mutant strain G1-m compared to the original strain G1, and to clarify the mutational differences at the genomic level, this embodiment uses genome sequencing and SNP analysis to locate key functional mutation sites. The specific operations and results are as follows: Take 5 mL of fresh bacterial culture from both the original strain and the mutant strain, both in the logarithmic growth phase, and place them in sterile centrifuge tubes. Centrifuge at 8000 rpm for 10 min at 4°C and collect the bacterial pellet. Extract DNA from both strains using a bacterial genomic DNA extraction kit. After passing quality control, the DNA samples are sent to Meiji Biotechnology Co., Ltd. for sequencing. Subsequently, under the Linux CentOS 7.9 operating system environment, Snippy (version 4.6.0) was used to perform SNP detection on the genomes of the two strains. The steps are as follows: Reference genome construction: The high-quality clean reads of the original strain G1 were assembled into a genome using SPAdes software (version 3.15.5) to obtain a complete genome sequence with a contig N50 ≥ 100kb, and annotated using prokka. The gbk annotation file was used as the reference genome for subsequent SNP analysis. Sequence alignment and variant detection: Snippy commands were called to align the gene sequence file of the mutant strain G1-m with the reference genome of the original strain G1, automatically completing read length localization, base mismatch identification, and preliminary screening of SNP sites. SNP filtering: Snippy's built-in filtering module was used to remove suspected SNP sites with a coverage depth < 10× and a mutation frequency < 90%, retaining high-confidence SNPs.

[0051] Analysis using the Snippy tool revealed multiple high-confidence genomic variant sites in the mutagenic strain's genome (Table 3), covering several contig regions including BEJOFAJN_1, BEJOFAJN_3, BEJOFAJN_5, and BEJOFAJN_9. Missense and nonsense mutations were the predominant types. Further analysis, combined with the strain's core functional phenotypes of enhanced selenite tolerance and selenocysteine ​​synthesis, identified 11 key genes potentially directly related to functional changes after mutagenesis. katE, cypB, wprA, nagC (etc.). Its specific variation information and functional association analysis are as follows: katE The gene undergoes a conserved in-frame deletion (p.Glu439_Ile443del), which results in the loss of 5 amino acids due to the deletion of 15 bases without disrupting the protein framework. The spatial conformation of the catalase HPII it encodes may be altered, which may enhance the ability to scavenge reactive oxygen species induced by high selenium and increase the antioxidant threshold of the strain. cypB The gene is a frameshift mutation (p.Arg600fs), with the deletion of base A leading to a reading frame shift. The encoded bifunctional cytochrome P450 reductase can enhance the catalytic conversion efficiency of selenite and regulate cellular redox homeostasis, thereby reducing high selenium toxicity. wprAThe gene is a missense mutation (p.Glu363Gly), with a base A mutation to G causing an amino acid substitution, which may enhance the activity of the encoded cell wall-associated protease, strengthen the cell wall barrier function, repair selenium ion damage, and provide a stable microenvironment for the synthesis of organic selenium. All three factors work together to enhance the strain's resistance to oxidative stress and its ability to detoxify high selenium levels.

[0052] cysJ A missense mutation (p.Leu507Ser) occurred in the gene, with a base T mutation to C-induced amino acid substitution. The encoded sulfite reductase [NADPH] flavoprotein subunit has cross-catalytic activity for selenite due to its selenium-sulfur homology. The mutation enhances the enzyme's substrate affinity for selenite, accelerates the conversion of selenite to selenide, and the conversion product can be used as a precursor to synthesize selenocysteine, achieving a highly efficient "reduction-synthesis" linkage. This is the key molecular basis for the strain's enhanced selenocysteine ​​synthesis capacity.

[0053] yumC The gene is a missense mutation (p.Asn121Asp), where the base A mutation is replaced by G, resulting in an amino acid substitution that enhances the electron transfer efficiency of the encoded ferroredoxin-NADP reductase, providing energy for selenium reduction-related enzymes and regulating redox balance. ytbE The gene is a missense mutation (p.Asn13Asp), with a base A mutation replaced by G, resulting in an amino acid substitution. This enhances the membrane localization ability of the encoded oxidoreductase, which can catalyze the reduction of extracellular selenite concentration and stabilize organic selenium. bshC The gene is a missense mutation (p.Ile139Val), with a base A mutation to G-induced amino acid substitution, which enhances the activity of the encoded cysteine ​​ligase, promoting glutathione synthesis for detoxification and sulfur supply; ydbD The gene is a missense mutation (p.Cys17Tyr), with a base G mutation to A leading to an amino acid substitution. This encodes manganese catalase, which has enhanced manganese ion binding capacity, improves hydrogen peroxide scavenging efficiency, and assists in the activity of selenium metabolic enzymes. These four factors provide auxiliary support for optimizing the selenium metabolic function of the strain from aspects such as electron transport, substrate catalysis, and substance synthesis.

[0054] Based on the experimental data on selenite tolerance and selenocysteine ​​synthesis capacity in the aforementioned examples, it can be inferred that the superior performance of the mutant strain G1-m originates from genome-specific mutations caused by space mutagenesis—missense mutations in multiple key genes such as oxidative stress detoxification, sulfur metabolism association, and stress assist. By enhancing selenite tolerance and increasing synthase activity, the strain's selenium metabolism performance is improved, providing a clear molecular mechanism to support the application of the G1-m strain.

[0055] Table 3. Genomic SNP sites of the mutant strain and the original strain

Claims

1. A strain of Bacillus paralichrysogenus with high synthetic selenocysteine ​​and high sodium selenite tolerance obtained by space mutagenesis, wherein the Bacillus paralichrysogenus is named G1-m and is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36525 and deposit date of November 11, 2025.

2. The use of Bacillus paralichrysogenum as described in claim 1 in the preparation of animal feed additives for improving the antioxidant status, growth performance or meat and egg quality of animals.

3. The use of Bacillus paralichrysogenum according to claim 1 in the preparation of plant biological agents for promoting crop growth or enhancing stress resistance.

4. The application of Bacillus paralichrysum as described in claim 1 in the preparation of soil conditioners for improving the rhizosphere microecological environment, activating soil nutrients, and remediating heavy metal pollution.

5. The application of Bacillus paralichrysogenum according to claim 1 in the preparation of agricultural product preservatives for improving the storage quality of agricultural products, degrading pesticide residues or inhibiting plant pathogens.

6. The use of Bacillus paralichrysum as described in claim 1 in the preparation of functional foods or health products for intestinal antioxidant, anti-inflammatory and immune-enhancing effects.

7. The use of Bacillus paralichrysiformis as a carrier in the preparation of intestinal-directed release drugs according to claim 1.

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