Bacillus subtilis engineering bacteria, fermentation product, construction method and application thereof

Abstract

The application belongs to the technical field of synthetic biology, and a bacillus subtilis engineering bacterium is preserved in the China General Microbiological Culture Collection Center on March 11, 2026, with a preservation number of CGMCC No. 37899. The chassis strain bacillus subtilis is preserved in the China General Microbiological Culture Collection Center on March 3, 2026, with a preservation number of CGMCC No. 37834. The modification strategy is to knock out the alpha subunit gene of acetoin dehydrogenase and the phosphotransferase gene from the genome of bacillus subtilis CGMCC No. 37834, and express the acetolactate synthase gene and the alpha-acetolactate decarboxylase gene. The engineering bacterium can be used to prepare fermentation products containing the microbial-crop interaction signal substances acetoin and 2,3-butanediol, and can be used to promote the growth of crop roots, improve the growth and yield of crops.

Description

Technical Field

[0001] This invention relates to the fields of microbial engineering technology and agricultural biotechnology, specifically to a genetically engineered Bacillus subtilis strain, its fermentation preparation method, the obtained fermentation product, and its application in promoting crop growth and improving soil microecology. Background Technology

[0002] Bacillus subtilis, a Gram-positive bacterium, is widely distributed in nature and is characterized by rapid growth, simple nutritional requirements, and mature fermentation technology. It is listed in the "Feed Additive Variety Catalog" by my country's Ministry of Agriculture and Rural Affairs and recognized as a GRAS-rated microorganism by the US FDA. It has been widely used in industrial enzyme preparations, biosurfactants, probiotic preparations, agricultural biological control, and crop growth promotion. However, wild-type or conventionally screened Bacillus subtilis strains have natural metabolic limitations in the targeted synthesis of "microorganism-crop" interaction signaling substances: low yields of target metabolites acetoin and 2,3-butanediol, high accumulation of byproducts, and uncontrollable product synthesis ratios. When directly applied to agricultural production, it suffers from unstable crop growth-promoting effects, limited functionality, and insufficient adaptability to field environments, severely restricting its large-scale application in agricultural settings.

[0003] To enhance the ability of Bacillus subtilis to synthesize target metabolites, metabolic engineering and synthetic biology techniques have been widely used for the genetic modification of this strain. Existing studies have mostly focused on knocking out metabolic competitive pathways and overexpressing key enzymes in the synthetic pathway to increase the yield of substances such as acetoin. However, existing modification strategies still have significant limitations: First, they mostly focus on improving the synthesis efficiency of single products, without taking into account the synergistic synthesis and ratio regulation of acetoin and 2,3-butanediol, two core "microbe-crop" interaction signaling substances, thus failing to achieve functional synergy between the two substances in crop growth-promoting applications. Second, the modified strains are mostly adapted to industrial high-purity monomer synthesis scenarios, lacking strain systems and supporting preparation processes adapted to low-cost, large-scale fermentation in agriculture, making it difficult to achieve stable, low-cost mass production of target products.

[0004] In the field of basic research in applied agriculture, reports have confirmed that volatile organic compounds such as acetoin and 2,3-butanediol can serve as core "microbe-crop" interaction signaling substances, directly promoting plant root growth, increasing crop biomass and yield, and enhancing crop resistance to biotic and abiotic stresses. However, existing research still has significant technological gaps: on the one hand, it only focuses on the basic mechanism of the direct growth-promoting effects of these two signaling substances on crops, without developing industrially applicable technologies for constructing high-yield strains and large-scale preparation; on the other hand, an integrated technical system of "construction of high-yield engineered strains - stable fermentation preparation - efficient field application" has not yet been established, making it impossible to stably obtain fermentation products containing specific concentrations and proportions of signaling substances through engineering methods, and also unable to guarantee the stability and broad-spectrum growth-promoting effects on different crops and under different field conditions.

[0005] In summary, there is an urgent need in this field for a rationally designed and precisely genetically modified Bacillus subtilis strain that can stably and efficiently produce high yields of specific proportions of the "microbe-crop" interaction signaling substances acetoin and 2,3-butanediol. This strain should be coupled with a fermentation preparation process suitable for large-scale agricultural production, enabling its fermentation products to have stable and broad-spectrum effects on promoting crop root growth and increasing yield. This would address the core pain points of existing technologies, such as low target product yield, uncontrollable product proportions, unstable field application effects, and difficulty in industrialization. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides an engineered strain of Bacillus subtilis, a method for constructing the engineered strain, a fermentation preparation method thereof, the resulting fermentation product, and its application in promoting crop growth and yield.

[0007] This application provides a microorganism, which is an engineered Bacillus subtilis strain, and was deposited at the China General Microbiological Culture Collection Center on March 11, 2026, with accession number CGMCC No. 37899.

[0008] The Bacillus subtilis engineered strain CGMCC No. 37899 is a genetically engineered bacterium. The first step involved knocking out the α-subunit gene of acetoin dehydrogenase and the phosphoacetyltransferase gene from the genome of Bacillus subtilis CGMCC No. 37834. The second step involved expressing the acetyllactone synthase gene and the α-acetyllactone decarboxylase gene. The amino acid sequence of the α subunit of the acetoin dehydrogenase is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the phosphoacetyltransferase is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4; the amino acid sequence of the acetyllactate synthase is shown in SEQ ID NO.5, and the nucleic acid sequence is shown in SEQ ID NO.6; the amino acid sequence of the α-acetyllactate decarboxylase is shown in SEQ ID NO.7, and the nucleic acid sequence is shown in SEQ ID NO.8.

[0009] This application provides a microbial agent, which includes the engineered Bacillus subtilis strain.

[0010] This application provides a microorganism, namely Bacillus subtilis, which was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37834.

[0011] This application provides a fermentation product obtained by fermentation using the engineered Bacillus subtilis strain.

[0012] According to a preferred embodiment of this application, the fermentation culture medium comprises: a liquid fermentation medium containing a carbon source, a nitrogen source, inorganic salts, and growth factors; wherein the carbon source is selected from at least one of glucose, sucrose, and starch, with a concentration of 10-50 g / L; the nitrogen source is selected from at least one of peptone, yeast extract, and ammonium sulfate, with a concentration of 5-30 g / L; the inorganic salts include phosphates, magnesium salts, and potassium salts, wherein the concentration of potassium dihydrogen phosphate is 1-5 g / L, the concentration of magnesium sulfate heptahydrate is 0.5-2 g / L, and the concentration of potassium chloride is 0.5-2 g / L; the growth factors include vitamins and trace elements, wherein the concentration of vitamin B1 is 0.1-1 mg / L. The pH of the culture medium is adjusted to 6.5-7.5; optionally, The fermentation conditions are as follows: the culture includes an aerobic fermentation process, specifically: inoculum size of 1%-5% (v / v), culture temperature of 30-37°C, stirring speed of 100-300 rpm, aeration rate of 0.5-2.0 vvm, and fermentation time of 24-72 hours. During fermentation, the pH is maintained within the range of 6.5-7.5 by an automatic control system, and a carbon source is added via a feed-and-feed method to maintain metabolic activity. Optionally, the process includes post-processing of the fermentation products, which includes: first, inactivating the strain by maintaining the fermenter pressure at 0.12-0.15 MPa for 20-30 minutes; second, centrifuging the fermentation broth by maintaining a centrifugation speed of ≥5000 rpm / min for ≥30 minutes and collecting the supernatant.

[0013] According to a preferred embodiment of this application, the fermentation product contains a "microbe-crop" interaction signaling substance, which includes acetoin and 2,3-butanediol; wherein the concentration of acetoin in the "microbe-crop" interaction signaling substance is 100 g / L and the concentration of 2,3-butanediol is 10 g / L.

[0014] This application provides the application of the fermentation products in promoting crop root growth and increasing crop yield.

[0015] This application provides a method for constructing the engineered Bacillus subtilis strain, including: Step 1: Using the genomic DNA of Bacillus subtilis CGMCC No.37834 as a template, the upstream and downstream homologous arms of the acetoin dehydrogenase α subunit gene and the phosphoacetyltransferase gene were amplified by PCR. The upstream and downstream genes were ligated with the resistance selection gene by overlapping PCR technology to obtain the linear DNA of the fusion gene of acetoin dehydrogenase α subunit and phosphoacetyltransferase. Step 2: Transform Bacillus subtilis competent cells. Identify the transformed cells by genomic PCR amplification and sequencing. Using the Bacillus subtilis CGMCC No. 37834 genome as a template, amplify the acetolactate synthase gene and α-acetolactate decarboxylase gene by PCR. Ligate the transformed cells to plasmid pMA5 using overlap PCR technology. The promoter is HpaII. The resulting engineered Bacillus subtilis strain CGMCC No. 37899 was obtained.

[0016] This application provides a compound microbial agent, including the above-mentioned Bacillus subtilis engineered bacteria, microbial agent, Bacillus subtilis or fermentation product of Bacillus subtilis engineered bacteria.

[0017] This application provides a bio-fertilizer, comprising the aforementioned engineered Bacillus subtilis bacteria, microbial agents, fermentation products of Bacillus subtilis or engineered Bacillus subtilis bacteria, or the aforementioned compound microbial agents.

[0018] This application is based on the applicant's series of creative discoveries on the mechanism of action and synergistic effect of "microorganism-crop" interaction signaling substances, as well as the targeted regulation technology of Bacillus subtilis metabolic pathways.

[0019] Microbe-crop interaction signaling substances are core small-molecule volatile substances that mediate signaling communication between rhizosphere microorganisms and crops. They can be produced by the metabolism of beneficial microorganisms and can directly regulate the growth and development process of crops, significantly improving crop growth performance and stress adaptability. Among them, acetoin and 2,3-butanediol are two core types of microbe-crop interaction signaling substances, which have complementary and synergistic biological functions in crop growth promotion and stress resistance regulation.

[0020] Acetoin's regulatory effects on crops are mainly reflected in two aspects: growth promotion and enhanced resistance. Acetoin is a key product in the microbial glycolysis metabolic pathway, helping microorganisms avoid excessive environmental acidification during metabolism. It also regulates the redox balance of NAD+ / NADH within microbial cells, enabling targeted carbon source storage. In agricultural ecosystems, acetoin, as a core volatile substance released by rhizosphere growth-promoting bacteria, can directly act on crops, significantly promoting vegetative growth and biomass accumulation. Simultaneously, acetoin can act as a signal inducer, activating systemic disease resistance in crops and helping them resist pathogen infection. Related studies have confirmed its significant inhibitory effect on postharvest rot and spoilage caused by pathogens in citrus. Furthermore, acetoin can be used as an insect pheromone, added to insect traps to target and kill pests, reducing the probability of pest occurrence in the field and ensuring normal crop growth and yield formation.

[0021] 2,3-Butanediol can be used as a crop growth regulator. By regulating the balance of endogenous growth hormones in crops, it can significantly promote the elongation and branching of crop roots and enhance the ability of crop roots to absorb water and nutrients. At the same time, it can improve the crop's resistance to drought, disease and other abiotic and biotic stresses, enhance the photosynthetic efficiency of crop leaves, and thus promote the vegetative growth and reproductive development of crops throughout their entire growth period, achieving simultaneous improvement in crop yield and quality.

[0022] Through extensive experimentation, the inventors unexpectedly discovered that when acetoin and 2,3-butanediol act synergistically in a mass ratio of 9:1 to 11:1, their effects on promoting crop root growth and increasing crop yield are significantly superior to the individual use of either substance, demonstrating a strong synergistic effect. Building upon this, the inventors rationally designed and precisely genetically modified the metabolic network of Bacillus subtilis, establishing a technical solution for the targeted regulation of the synthesis ratio and yield of these two signaling substances. This solution addresses the core technical challenges of low target product yield, high byproduct accumulation, and uncontrollable product synthesis ratio in wild-type Bacillus subtilis, laying a crucial foundation for the large-scale preparation and agricultural industrial application of this type of "microorganism-crop" interaction signaling substance. Attached Figure Description

[0023] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 Phylogenetic tree of Bacillus subtilis SPB1. Detailed Implementation

[0024] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0025] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0026] This application details Definitions and General Terms In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0027] In this document, the terms “optional,” “optional,” “alternatively,” “optional,” or “optional” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0028] In this paper, the term "microbe-crop interaction signaling substance" refers to the chemical signals released during communication between microorganisms and crops. These signaling substances can be small molecule compounds, peptides, proteins, RNA, or combinations thereof, and they play a key role in the interaction between microorganisms and crops, influencing crop growth, development, disease resistance, and environmental adaptability. For example, "microbe-crop interaction signaling substances" are selected from acetoin and 2,3-butanediol.

[0029] In this article, the term "crop" refers to plants that are cultivated and harvested through agricultural activities. They are the primary objects of agricultural production, used to provide food, feed, industrial raw materials, or for ornamental purposes. This includes, but is not limited to: food crops (such as wheat and rice), cash crops (such as cotton and tobacco), feed crops (such as corn), vegetable crops, and fruit trees.

[0030] In a first aspect of this application, a genetically engineered Bacillus subtilis strain CGMCC No. 37899 is proposed. Using Bacillus subtilis CGMCC No. 37834 as a chassis, the α-subunit gene of acetoin dehydrogenase and the phosphoacetyltransferase gene were knocked out from the genome through homologous recombination. Then, acetolactate synthase and α-acetolactate decarboxylase genes were expressed using plasmids to obtain the engineered Bacillus subtilis strain CGMCC No. 37899.

[0031] The amino acid sequence of the α subunit of the acetoin dehydrogenase includes the sequence shown in SEQ ID NO.1.

[0032] SEQ ID NO.1: MELLKREGLSLTEEKALWMYQKMLEIRGFEDKVHELFAQGVLPGFVHLYAGEEAVAVGVCAHLHDGDSITSTHRGHGHCIAKGCDLDGMMAEIFGKATGLCKGKGGSMHIADLDKGMLGANGIVGGGFTLACGSALTAKYKQTKYVSVCFFGDGANNQGTFHEGLNL AAVWNLPVVFVAENNGYGEATPFEYASACDSIADRAAAYNMPGVTVDGKDILAVYQAAEEAIERARNGGGPSLIECMTYRNYGHFEGDAQTYKTKDERVEHLEEKDAIQGFKNYLLKETDANKLSDIEQRVSESIEKAVSFSEDSPYPKDSELLTDVYVSYEKGGM.

[0033] The DNA sequence of the α subunit of acetoin dehydrogenase includes the sequence shown in SEQ ID NO.2.

[0034] SEQ ID NO.2: The amino acid sequence of the phosphoacetyltransferase includes the sequence shown in SEQ ID NO.3.

[0035] SEQ ID NO.3; VADLFSTVQEKVAGKDVKIVFPEGLDERILEAVSKLAGNKVLNPIVIGNENEIQAKAKELNLTLDGVKIYDPHTYEDMEDLVQAFVERRKGKATEEQARKALLDENYFGTMLVYKGLADGLVSGAAHSTADTVRPALQIIKTKEGVKKTSGVFIMARGEEQ YVFADCAINIAPDSQDLAEIAIESANTAKMFDIEPRVAMLSFSTKGSAKSDETEKVADAVKIAKEKAPELTLDGEFQFDAAFVPSVAEKKAPDSEIKGDANVFVFPSLEAGNIGYKIAQRLGNFEAVGPILQGLNMPVNDLSRGCNAEDVYNLALITAAQAL The DNA sequence of the phosphorylated acetyltransferase includes the sequence shown in SEQ ID NO.4.

[0036] SEQ ID NO.4: GTGGCAGATTTATTTTCAACAGTGCAAGAAAAAGTAGCTGGAAAAGACGTTAAAATTGTATTTCCTGAAGGCTTAGACGAGCGTATTTTAGAAGCGGTCAGCAAGCTTGCGGGAAACAAAGTGCTGAATCCGATTGTGATCGGCAATGAAAATGAGATCCAAGCAAAAGCAAAAGAATTGAACCTTACGCTGGACGGCGTTAAGATTTATGATCCTCATACATATGAAGACATGGAAGACCTTGTACAAGCATTCGTAGAACGCCGCAAAGGCAAAGCGACAGAAGAACAGGCTCGCAAAGCGTTATTAGACGAGAACTACTTCGGTACAATGCTGGTGTATAAAGGCCTTGCAGACGGACTCGTAAGCGGAGCTGCTCACTCGACAGCTGACACTGTCCGCCCGGCTCTTCAAATCATCAAAACAAAAGAAGGCGTGAAAAAGACTTCAGGCGTGTTCATCATGGCTCGCGGAGAAGAGCAATACGTATTCGCAGATTGCGCGATCAACATTGCGCCTGACAGCCAAGATCTTGCCGAGATTGCGATCGAAAGTGCCAATACGGCAAAAATGTTTGACATTGAGCCTCGCGTGGCAATGCTCAGCTTCTCTACAAAAGGCTCAGCAAAATCTGATGAAACAGAAAAAGTAGCGGATGCAGTGAAAATCGCGAAAGAAAAAGCGCCTGAACTGACACTTGACGGCGAATTCCAATTTGATGCTGCATTTGTTCCATCTGTAGCTGAGAAAAAAGCGCCGGATTCCGAGATCAAAGGGGACGCTAACGTATTCGTATTCCCAAGCCTTGAAGCAGGAAACATCGGCTATAAAATTGCTCAGCGTTTGGGCAACTTTGAAGCGGTAGGACCAATCCTGCAAGGTTTAAATATGCCTGTAAACGACCTTTCAAGAGGATGTAACGCTGAAGATGTTTACAATCTCGCATTAATTACAGCGGCGCAAGCACTGTAA The amino acid sequence of the acetolactate synthase includes the sequence shown in SEQ ID NO.5.

[0037] SEQ ID NO.5: .

[0038] The nucleic acid sequence of the acetolactate synthase includes the sequence shown in SEQ ID NO.6.

[0039] SEQ ID NO.6:

[0040] The amino acid sequence of the α-acetolactate decarboxylase includes the sequence shown in SEQ ID NO.7.

[0041] SEQ ID NO.7: MKRESNIQVLSRGQKDQPVSQIYQVSTMTSLLDGVYDGDFELSEIPKYGDFGIGTFNKLDGELIGFDGEFYRLRSDGTATPVQKGDRSPFCSFTFFTPDMTHKIDVKMTREDFEKEINSMLPSRNLF YAIRIDGLFKKVQTRTVELQEKPYVPMVEAVKTQPIFNFDNVRGTIVGFLTPAYANGIAVSGYHLHFIDEGRNSGGHVFDYVLEDCTVTISQKMNMNLRLPNTADFFNANLDNPDFAKDIETTEGSPE The nucleic acid sequence of the α-acetolactate decarboxylase includes the sequence shown in SEQ ID NO.8.

[0042] SEQ ID NO.8: .

[0043] The second aspect of this application discloses a fermentation technology using engineered Bacillus subtilis strains for the efficient preparation of fermentation products containing "microbe-crop" interaction signaling substances (acetoin and 2,3-butanediol). This technology is based on metabolic engineering and fermentation process optimization to ensure stable and high-yield production of the target metabolites by the engineered strains.

[0044] Seed culture preparation: Inoculate the engineered Bacillus subtilis strain with preservation number CGMCC No.37899 into a solid activation medium (such as LB agar medium) and culture at 30-37°C for 12-24 hours; pick a single colony and inoculate it into a liquid seed medium (composition similar to the main fermentation medium, but concentration halved), and culture under the same conditions with shaking (100-200 rpm) for 12-16 hours until the OD600 reaches 1.0-2.0 to obtain the first-grade seed culture.

[0045] Main fermentation process: The primary seed culture is transferred to the fermenter at an inoculum rate of 1%-5%, and the fermenter is filled with culture medium. A fed-batch strategy is employed during fermentation: In the initial fermentation stage (0-12 hours), the temperature is controlled at 30-37°C, pH at 6.5-7.5, the stirring speed at 100-200 rpm, and the aeration rate at 1.0-1.5 vvm to promote cell growth; after entering the product synthesis phase (12-72 hours), a residual sugar concentration of 5-10 g / L is maintained by feeding a carbon source (such as glucose solution), and the dissolved oxygen level is optimized (20%-40%) to induce the accumulation of acetoin and 2,3-butanediol. The fermentation cycle is typically 48-72 hours. The culture medium is a liquid fermentation medium containing a carbon source, a nitrogen source, inorganic salts, and growth factors. The carbon source is selected from at least one of glucose, sucrose, and starch, with a concentration of 10-50 g / L. The nitrogen source is selected from at least one of peptone, yeast extract, and ammonium sulfate, with a concentration of 5-30 g / L. The inorganic salts include phosphates, magnesium salts, and potassium salts, with potassium dihydrogen phosphate at a concentration of 1-5 g / L, magnesium sulfate heptahydrate at a concentration of 0.5-2 g / L, and potassium chloride at a concentration of 0.5-2 g / L. The growth factors include vitamins and trace elements, with vitamin B1 at a concentration of 0.1-1 mg / L. The pH of the culture medium is adjusted to 6.5-7.5.

[0046] Technical advantages: This fermentation technology maximizes the metabolic flux of engineered bacteria by precisely controlling nutrient conditions and environmental parameters, avoiding the accumulation of by-products, and is suitable for large-scale production.

[0047] The third aspect of this application discloses a post-processing procedure for fermentation products of engineered Bacillus subtilis. The first step involves strain inactivation, maintaining the fermenter temperature at 121℃ (approximately 0.12-0.15 MPa pressure) for 20-30 minutes. The second step involves centrifuging the fermentation broth at a speed ≥5000 rpm for ≥30 minutes, collecting the supernatant. The supernatant contains small-molecule "microorganism-crop" interaction signaling substances acetoin and 2,3-butanediol.

[0048] The fourth aspect of this application discloses the application of a fermentation product containing acetoin and 2,3-butanediol, small-molecule "microbe-crop" interaction signaling substances. In crop cultivation, such as wheat, corn, tomatoes, and peppers, applying the fermentation product containing acetoin and 2,3-butanediol can promote root growth and increase crop yield.

[0049] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application.

[0050] Example 1: Identification of Bacillus subtilis strain in chassis cells The basal cell strain *Bacillus subtilis* SPB1 was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37834. The 16S rRNA gene sequence (1472 bp) of this strain SPB1 is shown below, and the phylogenetic tree is as follows. Figure 1 .

[0051] The above results indicate that the strain is Bacillus subtilis.

[0052] Example 2: Construction of engineered Bacillus subtilis strain Using the genomic DNA of *Bacillus subtilis* CGMCC No. 37834 as a template, the upstream and downstream homologous arms of the acetoin dehydrogenase α-subunit gene and the phosphoacetyltransferase gene were amplified by PCR. The upstream and downstream genes were ligated to an antibiotic resistance selection gene using overlap PCR to obtain linear DNA of the acetoin dehydrogenase α-subunit and phosphoacetyltransferase fusion gene. This DNA was then transformed into competent *Bacillus subtilis* cells, and the transformants were identified by genomic PCR amplification and sequencing. Using the *Bacillus subtilis* CGMCC No. 37834 genome as a template, the acetolactate synthase gene and α-acetolactate decarboxylase gene were amplified by PCR and ligated to plasmid pMA5 using overlap PCR with the HpaII promoter. Through these steps, engineered *Bacillus subtilis* strain CGMCC No. 37899 was obtained.

[0053] Example 3: Preparation of fermentation products containing small molecule "microorganism-crop" interaction signaling substances This embodiment details the specific process of preparing fermentation products using engineered Bacillus subtilis strain (preservation number CGMCC No. 37899). The final product contains 100 g / L of acetoin and 10 g / L of 2,3-butanediol.

[0054] Strain activation: The engineered bacteria were taken from the -80°C glycerol storage tube and streaked onto LB solid medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, pH 7.0) and incubated in a 37°C incubator for 24 hours to obtain single colonies.

[0055] Seed culture: Pick a single colony and inoculate it into 100 mL of liquid seed culture medium (composition: glucose 15 g / L, peptone 7.5 g / L, yeast extract 5 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 0.75 g / L, potassium chloride 0.5 g / L, microbial B1 0.25 mg / L, pH 7.0) in a 500 mL shake flask. Incubate at 37°C and 200 rpm for 12 hours until the OD600 is about 1.5. This is the first-stage seed culture.

[0056] Primary fermentation: The primary seed culture was inoculated into a 5 L fermenter (working volume 3 L) at a 2% inoculation rate. The culture medium composition in the tank was as follows: glucose 30 g / L, peptone 15 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, potassium chloride 1 g / L, microbial B1 0.5 mg / L, and trace element solution (containing FeSO4, MnSO4, ZnSO4) 2 mL / L. The pH was adjusted to 7.0. Fermentation conditions were controlled as follows: temperature 37°C, stirring speed 150 rpm, aeration rate 1.2 vvm, and pH maintained at 7.0 ± 0.2 by automatic addition of acid (1 M HCl) or alkali (1 M NaOH). During fermentation, samples were taken every 12 hours to detect OD600, residual sugar concentration (using DNS method), and metabolites (analyzed by HPLC). After 24 hours of fermentation, glucose solution (500 g / L) was added to maintain the residual sugar concentration at 5-8 g / L.

[0057] Fermentation endpoint and product harvest: When fermentation reaches 60 hours, the concentration of acetoin reaches 100 g / L, the concentration of 2,3-butanediol reaches 10 g / L, and the cell OD600 is stable at 15-20, which is judged as the fermentation endpoint.

[0058] The fermentation broth underwent post-treatment: First, the temperature in the fermenter was raised to 121°C (tank pressure 0.12-0.15 MPa) and maintained for 20 minutes to inactivate the bacterial strain. Then, the fermentation broth was centrifuged at 5000 rpm for 30 minutes at 4°C, and the supernatant was collected, which is the fermentation product containing the signal substance. This product is colorless and transparent, with a pH of approximately 6.5, and can be used directly in agricultural applications or further concentrated.

[0059] Validation analysis: The concentrations of acetoin and 2,3-butanediol in the product were verified by high-performance liquid chromatography (HPLC), and the purity of the substances was confirmed by gas chromatography-mass spectrometry (GC-MS). This example demonstrates that the fermentation technology can stably produce high yields of the target signaling substance, providing a foundation for subsequent applications.

[0060] (In the following Examples 4-7, Sample 1 refers to the same thing, and Sample 2 refers to the same thing.) Example 4: Rapeseed Plot Experiment This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested vegetable was Shanghai Bok Choy. The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. The plot area was 15㎡ (3m×5m). Specific treatments are as follows: CK: Control group, rapeseed was treated with an equal amount of water at the three-leaf stage.

[0061] T1: Sample 1, dosage 1L / mu, diluted 1000 times (i.e., diluted with 1000L / mu of water), applied by fertigation at the three-leaf stage of rapeseed (approximately 15-20 days after sowing). Sample 1 contains 100g / L of acetoin and 10g / L of 2,3-butanediol. Preparation method: Dissolve 100g of pure acetoin and 10g of pure 2,3-butanediol in 1L of ultrapure water. The pure acetoin was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number A109410, with a purity of 99%; the pure 2,3-butanediol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number B424414, with a purity of 99%.

[0062] T2: Sample 2, with the same dosage and application method as T1. Sample 2 is the fermentation product from Example 2, containing 100 g / L of acetoin and 10 g / L of 2,3-butanediol.

[0063] The previous crop at the experimental site was melons, and the soil was fertile loam. Before sowing, the soil was deeply tilled, and 30 kg of compound fertilizer per mu (approximately 0.067 hectares) was applied as basal fertilizer. The fertilizer was purchased from Sinochem Shandong Fertilizer Co., Ltd., with a specification of N-P2O5-K2O=15-15-15. Sowing was carried out on March 10, 2024, using a row sowing method with a row spacing of 15 cm. After sowing, a thin layer of soil was applied and the soil was thoroughly watered. After emergence, seedlings were thinned to three leaves, and the plant spacing was adjusted to 8-10 cm to ensure uniform growth. Unified water, fertilizer, and pest and disease management was implemented throughout the entire growth period.

[0064] During the rapeseed harvest period (approximately 40 days after sowing), 20 representative plants were collected from each plot for measurement. Root biomass: The complete root system was carefully dug up, washed, blanched at 105℃ for 30 minutes, and dried at 80℃ to constant weight. The dry weight (g / plant) was then measured.

[0065] Yield determination: Collect all plants within a 2㎡ area in the center of the plot, weigh their fresh weight, and calculate the yield per mu (kg / mu).

[0066] The results are as follows:

[0067] The results show that both T1 and T2 can increase rapeseed root biomass, with T1 and T2 increasing by 28.0% and 64.0% respectively compared to CK, and T2 increasing by 28.1% compared to T1. Both T1 and T2 can also increase rapeseed yield, with T1 and T2 increasing by 10.4% and 21.6% respectively compared to CK, and T2 increasing by 10.1% compared to T1. This example confirms that applying either Sample 1 or Sample 2 (1 L / acre, diluted 1000 times) during rapeseed cultivation can effectively promote rapeseed root growth and increase yield.

[0068] Example 5: Wheat Field Trial This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested wheat variety was Jimai 22 (a semi-winter variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. Each plot was 20 m² (4 m × 5 m). Specific treatments are as follows: CK: Control group, which received the same amount of water during the wheat's greening stage.

[0069] T1: Sample 1, dosage 2L / mu, diluted 1000 times (2000L / mu of water), applied by irrigation during the wheat greening stage (before jointing).

[0070] T2: Sample 2, dosage and application method are the same as T1.

[0071] The previous crop in the experimental field was summer maize, and the soil type was brown soil with medium fertility. Deep plowing was carried out before sowing, and 50 kg of compound fertilizer (N-P2O5-K2O=15-15-15) was applied per mu as basal fertilizer. Wheat was sown mechanically in rows on October 12, 2023, with a row spacing of 25 cm, and the basic seedling density was controlled at 1.8 million plants per mu. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0072] Representative plant samples were collected from each plot at the wheat maturity date (June 5, 2024) for testing: Root biomass: Dig up roots from the 0–20 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh them (g / plant).

[0073] Yield determination: The dry weight of the grains was measured after threshing the middle three rows of plants in the actual harvest plot, and the yield per mu (kg / mu) was calculated. As shown in the table below, treatments T1 and T2 were significantly better than CK in terms of root biomass, yield, and soil enzyme activity, with T2 showing the best results.

[0074]

[0075] The root biomass of treatments T1 and T2 increased by 27.1% and 55.3% respectively compared to the control (CK), and the yield increased by 6.9% and 15.3% respectively. Compared with T1, T2 showed an increase of 22.2% in root biomass and 7.8% in yield per acre.

[0076] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the wheat greening stage can effectively promote wheat root growth and increase yield. Sample 2 (T2) showed the most significant effect, providing a reliable basis for expanding the application of this technology in grain crops.

[0077] Example 6: Field Trial of Maize This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested maize variety was Zhengdan 958 (a compact, high-density tolerant variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. The plot area was 30 m² (5 m × 6 m). Specific treatments are as follows: CK: Control group, which received the same amount of water during the corn jointing stage.

[0078] T1: Sample 1, dosage 2L / mu, diluted 1000 times (2000L / mu of water), applied by irrigation during the corn jointing stage.

[0079] T2: Sample 2, dosage and application method are the same as T1.

[0080] The previous crop at the experimental site was winter wheat, and the soil type was alluvial brown soil with moderate fertility. Deep plowing (25 cm depth) was carried out before sowing, and 50 kg of compound fertilizer (N-P2O5-K2O=15-15-15) was applied. Corn was precision-planted mechanically on June 15, 2024, with a row spacing of 60 cm, a plant spacing of 28 cm, and a planting density of 4000 plants / mu. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0081] Representative plant samples were collected from each plot at the corn maturity date (October 10, 2024) for testing. Root biomass: Dig up roots from the 0–30 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh them (g / plant).

[0082] Yield determination: The dry weight of the grains in the middle 4 rows of plants in the actual harvest plot was measured after threshing, and the yield per mu (kg / mu) was calculated.

[0083] As shown in the table below, treatments T1 and T2 were significantly better than the control (CK) in terms of root biomass, yield, and soil enzyme activity, with T2 showing the best results.

[0084] The root biomass of treatments T1 and T2 increased by 25.4% and 54.6% respectively compared to the control (CK), and the yield increased by 11.8% and 22.1% respectively. The root biomass and yield of treatment T2 increased by 23.3% and 9.2% compared to T1.

[0085] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the jointing stage of maize can effectively promote maize root growth, increase yield, and enhance the activation capacity of rhizosphere soil phosphorus, with Sample 2 (T2) showing the most significant effect. These results further validate the application potential of the tested samples on major food crops and provide a reliable basis for expanding the application of this technology in high-yield maize cultivation.

[0086] Example 7: Tomato Field Trial This experiment was conducted at the open-field plot experimental base of Sinochem Agriculture (Linyi) R&D Center Co., Ltd. The tested tomato variety was Provence (a large-fruited pink variety). The experiment consisted of three treatment groups, arranged in a randomized block design with three replicates. Each plot was 25 m² (5 m × 5 m). Specific treatments are as follows: CK: Control group, which was irrigated with the same amount of water during the tomato flowering period.

[0087] T1: Sample 1, dosage 2L / mu, diluted 1000 times (1000L / mu of water), applied by drip irrigation during the tomato flowering period (when the first cluster of flowers opens).

[0088] T2: Sample 2, dosage and application method are the same as T1.

[0089] The previous crop in the experimental field was leafy vegetables, and the soil type was light loam. Deep tillage was carried out before sowing, and 3000 kg of well-rotted cow manure and 40 kg of compound fertilizer (N-P2O5-K2O=15-15-15) were applied per acre as basal fertilizer. Tomatoes were raised in plug trays on February 20, 2024, and transplanted on March 25, with a row spacing of 60 cm and a plant spacing of 35 cm, resulting in a planting density of approximately 3200 plants per acre. Uniform water and fertilizer management and pest and disease control were implemented throughout the entire growth period to ensure consistent environmental conditions across all treatments.

[0090] During the peak tomato fruiting period (June 15, 2024), representative plant samples were collected from each plot for testing. Root biomass: Dig up roots from the 0–25 cm soil layer, wash them, blanch them at 105℃ for 30 minutes, dry them at 80℃ to constant weight, and weigh them (g / plant).

[0091] Yield determination: All fruits in the plot were harvested in three batches, and the total fresh weight was measured and converted into yield per mu (kg / mu).

[0092] As shown in the table below, treatments T1 and T2 were significantly better than CK in terms of root biomass, yield, soil enzyme activity and quality indicators, with T2 showing the best results.

[0093]

[0094] The root biomass of treatments T1 and T2 increased by 26.4% and 55.2% respectively compared to the control (CK), and the yield increased by 15.5% and 26.7% respectively. Compared with T1, T2 showed a 22.8% increase in root biomass and a 9.7% increase in yield per acre.

[0095] This embodiment confirms that applying either Sample 1 or Sample 2 (2 L / acre, diluted 1000 times) during the tomato flowering period can effectively promote tomato root growth, increase yield, improve fruit quality, and enhance rhizosphere soil phosphorus activation capacity, with Sample 2 (T2) showing the most significant effect. These results further validate the application potential of the tested samples in fruit and vegetable crops, providing a reliable basis for expanding the application of this technology in high-quality, high-yield cultivation of greenhouse tomatoes.

[0096] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0097] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A microorganism, characterized in that, The microorganism in question is an engineered Bacillus subtilis strain, which was deposited on March 11, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37899.

2. The microorganism according to claim 1, characterized in that, The Bacillus subtilis engineered strain CGMCC No. 37899 is a genetically engineered bacterium. The first step involved knocking out the α-subunit gene of acetoin dehydrogenase and the phosphoacetyltransferase gene from the genome of Bacillus subtilis CGMCC No. 37834. The second step involved expressing the acetyllactone synthase gene and the α-acetyllactone decarboxylase gene. The amino acid sequence of the α subunit of the acetoin dehydrogenase is shown in SEQ ID NO.1, and the nucleic acid sequence is shown in SEQ ID NO.2; the amino acid sequence of the phosphoacetyltransferase is shown in SEQ ID NO.3, and the nucleic acid sequence is shown in SEQ ID NO.4; the amino acid sequence of the acetolactate synthase is shown in SEQ ID NO.5, and the nucleic acid sequence is shown in SEQ ID NO.6; the amino acid sequence of the α-acetolactate decarboxylase is shown in SEQ ID NO.7, and the nucleic acid sequence is shown in SEQ ID NO.

8.

3. A microorganism, characterized in that, The microorganism in question is Bacillus subtilis, which was deposited on March 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37834.

4. A fermentation product, characterized in that, The fermentation product is obtained by fermentation using the engineered Bacillus subtilis strain as described in claim 1 or 2.

5. The fermentation product according to claim 4, characterized in that, The fermentation medium comprises: a liquid fermentation medium containing a carbon source, a nitrogen source, inorganic salts, and growth factors; wherein the carbon source is selected from at least one of glucose, sucrose, and starch, with a concentration of 10-50 g / L; the nitrogen source is selected from at least one of peptone, yeast extract, and ammonium sulfate, with a concentration of 5-30 g / L; the inorganic salts include phosphates, magnesium salts, and potassium salts, wherein the concentration of potassium dihydrogen phosphate is 1-5 g / L, the concentration of magnesium sulfate heptahydrate is 0.5-2 g / L, and the concentration of potassium chloride is 0.5-2 g / L; the growth factors include vitamins and trace elements, wherein the concentration of vitamin B1 is 0.1-1 mg / L. The pH of the medium is adjusted to 6.5-7.5; optionally, The fermentation conditions are as follows: the culture includes an aerobic fermentation process, specifically: inoculum size of 1%-5% (v / v), culture temperature of 30-37°C, stirring speed of 100-300 rpm, aeration rate of 0.5-2.0 vvm, and fermentation time of 24-72 hours. During fermentation, the pH is maintained within the range of 6.5-7.5 by an automatic control system, and a carbon source is added via a feed-and-feed method to maintain metabolic activity. Optionally, the process includes post-processing of the fermentation products, which includes: first, inactivating the strain by maintaining the fermenter pressure at 0.12-0.15 MPa for 20-30 minutes; second, centrifuging the fermentation broth by maintaining a centrifugation speed of ≥5000 rpm / min for ≥30 minutes and collecting the supernatant.

6. The fermentation product according to any one of claims 4 to 5, characterized in that, The fermentation product contains a "microbe-crop" interaction signaling substance, which includes acetoin and 2,3-butanediol; wherein the concentration of acetoin is 100 g / L and the concentration of 2,3-butanediol is 10 g / L.

7. The use of the fermentation product according to any one of claims 4 to 6 in promoting crop root growth and increasing crop yield.

8. The method for constructing the microorganisms according to claims 1 and 3, characterized in that, include: Step 1: Using the genomic DNA of Bacillus subtilis CGMCC No.37834 as a template, the upstream and downstream homologous arms of the acetoin dehydrogenase α subunit gene and the phosphoacetyltransferase gene were amplified by PCR. The upstream and downstream genes were ligated with the resistance selection gene by overlapping PCR technology to obtain the linear DNA of the fusion gene of acetoin dehydrogenase α subunit and phosphoacetyltransferase. Step 2: Transform Bacillus subtilis competent cells. Identify the transformed cells by genomic PCR amplification and sequencing. Using the Bacillus subtilis CGMCC No. 37834 genome as a template, amplify the acetolactate synthase gene and α-acetolactate decarboxylase gene by PCR. Ligate the transformed cells to plasmid pMA5 using overlap PCR technology with HpaII promoter to obtain engineered Bacillus subtilis strain CGMCC No. 37899.

9. A compound microbial agent, characterized in that, It includes the microorganisms described in any one of claims 1 to 3 or the fermentation products described in any one of claims 4 to 6.

10. A bio-fertilizer, characterized in that, It includes the microorganisms described in any one of claims 1 to 3, the fermentation products described in any one of claims 4 to 6, or the compound microbial agent described in claim 9.

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