Bacillus amyloliquefaciens engineering bacteria expressing ice nucleation protein, construction method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to a Bacillus amyloliquefaciens engineering bacterium expressing ice nucleation protein and a construction method and application thereof. The tagV gene, the tagT gene and / or the tagO gene of the Bacillus amyloliquefaciens engineering bacterium are knocked out, and the engineering bacterium carries an expression vector capable of expressing an ice nucleation protein gene. The present application constructs the Bacillus amyloliquefaciens engineering bacterium expressing ice nucleation protein under the condition that the tagV gene, the tagT gene and / or the tagO gene activity is deleted. The tagV gene, the tagT gene and / or the tagO gene belong to the LCP protein family gene, and can affect the synthesis of phosphomucic acid. Deletion of the aforementioned genes can significantly improve the ice nucleation activity and expression amount of the ice nucleation protein. The ice nucleation activity of the obtained engineering bacterium is high, and the Bacillus amyloliquefaciens biosafety strain used can directly utilize the bacterium body expressing the ice nucleation protein to play the ice nucleation role.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein, its construction method, and its application. Background Technology

[0002] When the temperature drops to 0°C, even a very small volume of pure water can remain liquid and not immediately freeze; this phenomenon is called supercooling. Ice-nucleating bacteria can induce and catalyze the formation of ice crystals, thereby raising the freezing temperature of water to allow it to freeze at higher temperatures (-2°C to -7°C). Ice-nucleating bacteria possess ice-nucleating activity because their outer membrane expresses specific ice-nucleating proteins. These proteins act as templates, binding water molecules to form a three-dimensional structure similar to ice-water molecules, thus promoting ice crystal formation. The ability of ice-nucleating bacteria to induce supercooled water to freeze at higher temperatures has been applied in fields such as artificial snowmaking and ice production, seawater desalination, accelerated freezing and insecticide application, and food cold storage, preservation, and freeze-concentration. Ice-nucleating bacteria can increase the ice-nucleation temperature of frozen foods and reduce freezing time. For example, adding ice-nucleating bacteria to food ingredients such as fish, meat, protein, and oil can raise the supercooling temperature to -3 to -4°C for complete freezing, reducing the total cooling time by more than 30%, greatly saving energy and time costs. Moreover, it can solve the problem of excessive loss of flavor and nutrients in frozen foods caused by the formation of large ice crystals due to supercooling, and improve the texture of frozen foods. It has huge application potential and development space in the frozen food industry.

[0003] However, bacteria with ice-nucleation activity mainly belong to species such as *Erwinia herbicola*, *Pseudomonas syringae*, and *Xanthomonas*. The vast majority of these ice-nucleating bacteria are plant pathogens, significantly limiting their application in food processing. To date, only Japan has approved the use of *Xanthomonas camperstris* INXC-1, which possesses ice-nucleation activity, in the food processing industry after high-pressure hydrostatic treatment. While purified ice-nucleation proteins can address the issue of pathogen safety, these proteins are membrane proteins, making them difficult to prepare and purify. Using genetic engineering techniques, biosafety-engineered strains expressing ice-nucleation proteins with high activity hold promise. However, current research on membrane expression of ice-nucleation proteins focuses on *Escherichia coli*, a pathogenic strain, limiting its application in food and other fields. Therefore, the development of safe, non-toxic, and non-pathogenic engineered microbial strains with high ice nucleus activity has a very broad market prospect in the food and other fields, and is of great significance for the large-scale and safe application of ice nucleus proteins. Summary of the Invention

[0004] In view of the problems of pathogenicity and / or poor ice nucleus activity of ice-nucleating bacteria in the prior art, the present invention provides an engineered Bacillus amyloliquefaciens expressing ice nucleus protein, its construction method and application, so as to solve or at least alleviate some of the problems in the prior art.

[0005] This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of the present invention provides an engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein, wherein the tagV gene, tagT gene and / or tagO gene of the engineered Bacillus amyloliquefaciens strain are knocked out, and the strain carries an expression vector capable of expressing ice nucleoprotein genes.

[0007] Furthermore, the tagV gene of the engineered Bacillus amyloliquefaciens strain was knocked out.

[0008] Furthermore, the nucleotide sequence of the tagV gene is shown in SEQ ID NO.1, the nucleotide sequence of the tagT gene is shown in SEQ ID NO.2, and the nucleotide sequence of the tagO gene is shown in SEQ ID NO.3.

[0009] Furthermore, the ice nucleoprotein gene is selected from any one of the inaZ, inaW, inaC, inaE, inaA, inaX, iceE, inaU, inaV and iceE genes derived from *Pseudomonas syringae*, *Pseudomonas fluorescens*, *Erwinia gracilis*, *Erwinia pineapple*, *Erwinia oleifera* wheat pathogenic strain, and *Erwinia uredinioides*.

[0010] Furthermore, the ice nucleoprotein gene is selected from the inaE gene of Erwinia broccoli var. pineapple, and the nucleotide sequence of the inaE gene is shown in SEQ ID NO.4.

[0011] Furthermore, the expression vector also includes a promoter located upstream of the ice nucleoprotein gene and a terminator located downstream of the ice nucleoprotein gene, wherein the promoter is selected from the P43 promoter and the terminator is selected from the Tamyl terminator.

[0012] Furthermore, the nucleotide sequence of the P43 promoter is shown in SEQ ID NO.5, and the nucleotide sequence of the Tamyl terminator is shown in SEQ ID NO.6.

[0013] Furthermore, the origin of the engineered Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens BAX-9.

[0014] A second aspect of the present invention provides a method for constructing an engineered Bacillus amyloliquefaciens expressing ice nucleoprotein as described above, comprising the following steps: knocking out the tagV gene, tagT gene and / or tagO gene of Bacillus amyloliquefaciens by homologous recombination technology, and transferring an expression vector expressing an ice nucleoprotein gene into the Bacillus amyloliquefaciens to construct an engineered Bacillus amyloliquefaciens expressing ice nucleoprotein.

[0015] Furthermore, the homologous recombination technology includes the following steps:

[0016] S1. Using the Bacillus amyloliquefaciens genome as a template, the upstream homologous arm sequence A and the downstream homologous arm sequence B are amplified using primers for the upstream and downstream homologous arms of the tagV gene, tagT gene, and tagO gene, respectively; wherein, the nucleotide sequences of the upstream and downstream homologous arm primers of the tagV gene are shown in SEQ ID NO.10-13, the nucleotide sequences of the upstream and downstream homologous arm primers of the tagT gene are shown in SEQ ID NO.14-17, and the nucleotide sequences of the upstream and downstream homologous arm primers of the tagO gene are shown in SEQ ID NO.18-21, respectively;

[0017] S2. The upstream homologous arm sequence A and the downstream homologous arm sequence B are joined together by overlap extension PCR technology to form homologous sequence C; wherein the nucleotide sequences of homologous sequence C of the tagV gene, the tagT gene and the tagO gene are as shown in SEQ ID NO.7-9, respectively;

[0018] S3. Insert the homologous sequence C into the temperature-sensitive knockout plasmid T2(2)-ori to construct the knockout plasmid;

[0019] S4. The knockout plasmid is transferred into the competent cells of Bacillus amyloliquefaciens, and Bacillus amyloliquefaciens mutant strains with the tagV gene, tagT gene and tagO gene knocked out are obtained by single and double crossover screening.

[0020] A third aspect of the invention provides the application of the engineered Bacillus amyloliquefaciens expressing ice nucleoprotein as described above in artificial snowmaking, seawater desalination, insecticide, freeze concentration, or frozen food industries.

[0021] The advantages and positive effects of this invention are as follows:

[0022] 1. This invention obtains an engineered Bacillus amyloliquefaciens strain capable of expressing ice nucleoproteins by knocking out tagV, tagT, and / or tagO of Bacillus amyloliquefaciens and introducing an expression vector capable of expressing ice nucleoprotein genes. The tagV, tagT, and / or tagO genes belong to the LCP protein family and can affect the synthesis of teichoic acid, and further affect the expression activity and level of the membrane protein ice nucleoprotein. Experiments have shown that the deletion of the aforementioned genes can significantly increase the activity and level of ice nucleoproteins. The resulting engineered strain has high ice nucleoactivity and can reach type II ice nucleotemperature.

[0023] 2. The Bacillus amyloliquefaciens strain used in this invention has a biosafety profile and can directly utilize the cells expressing ice nuclei proteins to exert the ice nucleus effect. It is suitable for stable large-scale industrial production, enabling the safe application of ice nuclei bacteria in food, and the application method is more convenient. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a flowchart illustrating the construction process of the LCP protein family gene knockout vectors tagV, tagT, and tagO in an embodiment of the present invention.

[0026] Figure 2 This is a flowchart illustrating the construction of LCP protein family gene deletion mutants through single and double crossovers in an embodiment of the present invention.

[0027] Figure 3 The colony PCR verification diagram of the LCP protein family gene deletion mutant strain constructed in this embodiment of the invention is shown in the diagram. From left to right, lane 1 is the DNA marker, lanes 2-3 are the tagVA-F / tagVB-R amplification fragments, lanes 4-5 are the tagTA-F / tagTB-R amplification fragments, and lanes 6-7 are the tagOA-F / tagVB-R amplification fragments.

[0028] Figure 4 This is a colony PCR verification diagram of a positive transformant carrying an ice nucleoprotein gene expression vector in an embodiment of the present invention. From left to right, lane M is the DNA marker, lanes 1-2 are the promoter P43 amplification fragment, lanes 3-4 are the ice nucleoprotein gene inaE amplification fragment, and lanes 5-6 are the terminator Tamyl amplification fragment.

[0029] Figure 5 This is a plasmid map of the ice nucleus gene expression vector PHY300PLK-INP in an embodiment of the present invention;

[0030] Figure 6 This is a colony PCR verification diagram of the engineered Bacillus amyloliquefaciens expressing ice nucleoprotein in an embodiment of the present invention; wherein, from left to right, lane M is the DNA marker, lane 1 is the B9-INP amplification fragment, lane 2 is the BV9-INP amplification fragment, lane 3 is the BT9-INP amplification fragment, and lane 4 is the BO9-INP amplification fragment.

[0031] Figure 7 This is a graph showing the supercooling point determination of the engineered Bacillus amyloliquefaciens expressing ice nucleoprotein in an embodiment of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0033] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0034] To better understand the invention and not to limit its scope, all figures and other numerical values ​​used in this invention to indicate amounts, percentages, or other quantities should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0035] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0036] The terms “comprising,” “including,” “containing,” “having,” and similar words are non-restrictive and can include other steps and other components that do not affect the result. The term “and / or” should be considered as a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” would be considered to include (i) A, (ii) B, and (iii) A and B.

[0037] The term "engineered bacteria" refers to microorganisms (such as *Bacillus amyloliquefaciens* of this invention) that have been genetically engineered to contain manipulated genes or nucleic acid molecules. The resulting cell lines are generally called "engineered bacteria." Genetic engineering methods include, for example, heterologous gene expression, gene insertion or deletion, gene mutation, alteration of gene expression or inactivation, enzyme engineering, directed evolution, random mutagenesis, gene shuffling, and codon optimization.

[0038] The term "gene" refers to the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. Therefore, gene expression involves transcription and the stable accumulation of coding RNA (mRNA) or functional RNA derived from the gene, and can also refer to the translation of mRNA into a polypeptide or protein. Genes can contain several operatively linked nucleic acid segments, such as the 5' non-coding region, the coding sequence, and the 3' non-coding region containing polyadenylation sites. Non-coding regions generally have regulatory functions in gene expression, such as promoters and terminators.

[0039] "Natural gene" refers to a gene that exists naturally and possesses its own regulatory sequence. In this invention, the LCP protein family gene is a gene that exists naturally in Bacillus amyloliquefaciens. "Heterologous gene" or "exogenous gene" refers to a gene that does not exist naturally in the host organism and is introduced into the host organism through gene transfer. In this invention, the ice nucleoprotein gene is an exogenous gene that does not exist naturally in Bacillus amyloliquefaciens.

[0040] The term "promoter" refers to a DNA sequence that controls the transcription of one or more nucleic acids. It is typically located upstream of the transcription start site (i.e., at the 5' end of the coding sequence). The promoter itself is not transcribed. It is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and may also contain any other DNA sequence, including but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art that directly or indirectly regulates the amount of transcription from the promoter.

[0041] The term "terminator" refers to a DNA sequence that provides a termination signal during transcription. It is located at the 3' end of the coding sequence and functions at the RNA level by forming a stem-loop structure through the transcribed terminator sequence, thereby terminating RNA synthesis.

[0042] The term "operationally ligated" refers to the connection of multiple nucleic acid fragments in a functional relationship. It occurs when one nucleic acid sequence forms a functional relationship with another. For example, if a promoter or transcriptional regulatory sequence affects the transcription of a coding sequence, it is operationally ligated to the coding sequence. Operationally ligated means that the nucleotide sequences being ligated may be contiguous.

[0043] The term "vector" refers to a self-replicating DNA molecule, often in the form of a circular double-stranded DNA molecule, used to transfer foreign genes into a host organism such as *Bacillus amyloliquefaciens* (as described in this invention). Vectors containing foreign genes are called recombinant vectors. Typical vectors include plasmids, viruses, bacteriophages, granules, and minichromosomes. Plasmids are the most common form of vector; therefore, in the context of this invention, plasmids and vectors are used interchangeably.

[0044] The term "expression vector" allows a foreign gene inserted into a vector to be expressed in a host organism, containing regulatory elements (such as promoters) that are expressed in the specified host organism. Expression vectors are used to transform appropriate host organisms, enabling them to express proteins.

[0045] The term "transformation" refers to the transfer of a foreign gene into a host organism, such as a host cell, resulting in stable genetic inheritance. The transformed gene can be in plasmid form retained in the host organism or integrated into the host organism's genome. A host organism containing the transformed gene is referred to as a "transgenic," "recombinant," "transformed," or "engineered" organism. Transformation of host organisms using expression vectors can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. Transformation can also be performed using methods such as microinjection, electroporation, or liposome packaging, if desired.

[0046] The term "knockout" refers to a technique that inactivates or eliminates the function of a specific gene through certain means. In the context of this invention, "knockout," "deletion," or "silencing" have the same meaning. Gene knockout techniques include, but are not limited to, homologous recombination gene knockout technology, CRISPR-Cas9 gene knockout technology, and gene knockout using random mutations.

[0047] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0048] This invention provides an engineered Bacillus amyloliquefaciens strain that expresses ice nucleoprotein. The tagV gene, tagT gene, and / or tagO gene of this engineered strain are knocked out, and it carries an expression vector capable of expressing ice nucleoprotein genes.

[0049] This invention uses *Bacillus amyloliquefaciens* as the starting bacterium. By knocking out the LCP protein family genes tagV, tagT, and tagO of the starting bacterium and introducing an expression vector capable of expressing ice nucleoprotein genes into them, the aforementioned engineered *Bacillus amyloliquefaciens* strain is obtained. This allows the engineered strain to express ice nucleoproteins even without tagV, tagT, and / or tagO gene activity. *Bacillus amyloliquefaciens* is a Gram-positive bacterium. Teichoic acid is an important surface antigen of Gram-positive bacteria, existing in two forms: wall teichoic acid and membrane teichoic acid. Wall teichoic acid is linked to N-acetylmucolonic acid in peptidoglycan of the cell wall, while membrane teichoic acid, also known as lipteichoic acid, is linked to the cell membrane. Ice nucleoprotein is a membrane protein. The three genes selected for knockout in this invention, tagV, tagT, and / or tagO, belong to the LCP protein family. The six arginine residues in their LCP domains are key enzyme activity sites that can affect the synthesis of teichoic acid, thereby affecting cell membrane synthesis and consequently affecting the expression activity and level of the membrane protein ice nucleoprotein. Experimental verification revealed that introducing expression vectors expressing ice nucleus protein genes into mutant strains and original strains lacking tagV, tagT, and tagO genes yielded Bacillus amyloliquefaciens engineered strains BV9-INP, BT9-INP, BO9-INP, and B9-INP with ice nucleus activity. Under liquid fermentation conditions, all four engineered strains exhibited ice nucleus activity, with BV9-INP, BT9-INP, and BO9-INP showing higher ice nucleus activities than B9-INP. Furthermore, BV9-INP reached type II ice nucleus temperatures (-5°C to -7°C), demonstrating that selective knockout of LCP protein family genes enhances the ice nucleus activity of Bacillus amyloliquefaciens expressing ice nucleus proteins. In addition, the Bacillus amyloliquefaciens strain used in this invention is a generally considered safe (GRAS) strain, and as a probiotic, it can be directly applied through fermentation to exert its ice nucleus effect, ensuring the safety of ice nucleating bacteria in application.

[0050] In this invention, the ice nucleus activities of the engineered bacteria, from highest to lowest, are BV9-INP, BO9-INP, BT9-INP, and B9-INP. Differential scanning calorimetry (DSC) was used to determine the supercooling point of the recombinant bacterial suspension. The supercooling point of the BV9-INP suspension at a concentration of 1 mg / mL was -6.9℃, followed by BO9-INP at -7.2℃. This indicates that BV9-INP has the highest ice nucleus activity, followed by BO9-INP. Therefore, preferably, the tagV and / or tagO genes of the engineered Bacillus amyloliquefaciens are knocked out. More preferably, at least the tagV gene of the engineered Bacillus amyloliquefaciens is knocked out.

[0051] The nucleotide sequence of the tagV gene is shown in SEQ ID NO.1, the nucleotide sequence of the tagT gene is shown in SEQ ID NO.2, and the nucleotide sequence of the tagO gene is shown in SEQ ID NO.3.

[0052] Ice nucleoproteins derived from different ice-nucleating bacteria show high homology and similar primary structures, which can be divided into non-repeating N-terminal and C-terminal domains, and a highly repetitive core domain (R-domain). The regular repetition of the core domain is the reason why water molecules are arranged into an ice-like grid, resulting in ice nucleation. Therefore, different ice nucleoprotein genes can be used to construct the engineered bacteria of this invention. Optionally, the ice nucleoprotein genes include, but are not limited to, the inaZ, inaW, inaC, inaE, inaA, inaX, iceE, inaU, inaV, and iceE genes derived from *Pseudomonas syringae*, *Pseudomonas fluorescens*, *Erwinia herbicola*, *Erwinia ananas*, *Xanthomonas campestris pv. translucens*, and *Erwinia uredovora*.

[0053] In some preferred embodiments, the ice nucleus protein gene is selected from the inaE gene of Erwinia herbicola subsp. ananas. Exemplarily, the nucleotide sequence of the inaE gene is shown in SEQ ID NO. 4.

[0054] To better achieve the expression of the ice nucleoprotein gene, the expression vector may optionally include a promoter located upstream of the ice nucleoprotein gene and a terminator located downstream of the ice nucleoprotein gene. The promoter, the ice nucleoprotein gene, and the terminator are operatively linked, for example, by seamlessly linking the three genes sequentially through homologous recombination and then inserting them into the starting vector to construct the expression vector. The starting bacterium of this invention belongs to the genus Bacillus. Commonly used promoters and terminators in the genus Bacillus can be applied to the construction of the expression vector of this invention. Furthermore, conventional techniques in the art will not be elaborated here. When the aforementioned expression vector is transferred into the starting bacterium and cultured conventionally, the engineered bacteria will express the ice nucleoprotein and produce ice nucleoprotein activity.

[0055] Specifically, the promoter is selected from the P43 promoter of Bacillus subtilis 168. The terminator is selected from the Tamyl terminator of Bacillus licheniformis WX-02. For example, the nucleotide sequence of the P43 promoter is shown in SEQ ID NO.5, and the nucleotide sequence of the Tamyl terminator is shown in SEQ ID NO.6.

[0056] Optionally, the starting bacterium mentioned above is Bacillus amyloliquefaciens BAX-9.

[0057] Optionally, the starting vector described above is a Bacillus vector, including but not limited to: PHY300 PLK. The P43 promoter from Bacillus subtilis 168, the ice protein gene inaE, and the Tamyl terminator from Bacillus licheniformis WX-02 are seamlessly ligated and inserted into the plasmid PHY300 PLK to construct the expression vector PHY300 PLK-INP.

[0058] Another embodiment of the present invention provides a method for constructing an engineered Bacillus amyloliquefaciens expressing ice nucleoprotein as described above, comprising the following steps: knocking out the tagV gene, tagT gene and / or tagO gene of Bacillus amyloliquefaciens by homologous recombination technology, and transferring an expression vector expressing an ice nucleoprotein gene into the Bacillus amyloliquefaciens to construct an engineered Bacillus amyloliquefaciens expressing ice nucleoprotein.

[0059] The method for constructing the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein has the same advantages over the prior art as described above, and will not be repeated here.

[0060] Specifically, the homologous recombination technology includes the following steps:

[0061] S1. Using the Bacillus amyloliquefaciens genome as a template, the upstream homologous arm sequence A and the downstream homologous arm sequence B are amplified using primers for the upstream and downstream homologous arms of the tagV gene, tagT gene, and tagO gene, respectively; wherein, the nucleotide sequences of the upstream and downstream homologous arm primers of the tagV gene are shown in SEQ ID NO.10-13, the nucleotide sequences of the upstream and downstream homologous arm primers of the tagT gene are shown in SEQ ID NO.14-17, and the nucleotide sequences of the upstream and downstream homologous arm primers of the tagO gene are shown in SEQ ID NO.18-21, respectively;

[0062] S2. The upstream homologous arm sequence A and the downstream homologous arm sequence B are joined together by overlap extension PCR (SOE-PCR) to form homologous sequence C; the nucleotide sequences of homologous sequence C of the tagV gene, the tagT gene and the tagO gene are shown in SEQ ID NO. 7-9, respectively;

[0063] S3. Insert the homologous sequence C into the temperature-sensitive knockout plasmid T2(2)-ori to construct the knockout plasmid;

[0064] S4. The knockout plasmid is transferred into the competent cells of Bacillus amyloliquefaciens, and Bacillus amyloliquefaciens mutant strains with the tagV gene, tagT gene and tagO gene knocked out are obtained by single and double crossover screening.

[0065] The transfer method is an electroconversion method.

[0066] Another embodiment of the present invention provides the application of the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein as described above in artificial snowmaking, seawater desalination, insecticide, freeze concentration, or frozen food industries.

[0067] The advantages of the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein in artificial snowmaking, seawater desalination, insecticide, freeze concentration, or frozen food industry are the same as the advantages of the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein over the prior art, as described above, and will not be repeated here.

[0068] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0069] Example

[0070] 1. Construct Bacillus amyloliquefaciens mutant strains with LCP protein family genes tagV, tagT, and tagO knocked out.

[0071] Using Bacillus amyloliquefaciens BAX-9 as the starting strain, Bacillus amyloliquefaciens BAX-9 is a publicly known biological material that has been reported in the article (Weijie Chen, Lu Li, Changwen Ye, Ziyue Zhao, Kuo Huang, Dian Zou, Xuetuan Wei, Efficient production of extracellular alkaline protease in Bacillus amyloliquefaciens by host strain construction, LWT, Volume 163, 2022, 113620.), and is currently deposited in the Microbial Engineering Laboratory of Huazhong Agricultural University.

[0072] Using all known and putative membrane protein genes of Bacillus amyloliquefaciens listed in the MEROPS database, the corresponding membrane protein genes in the BAX-9 gene sequence of Bacillus amyloliquefaciens were identified by sequence alignment. Combined with the Bacillus amyloliquefaciens membrane protein genes reported in the literature, and using EditSeq to calculate their protein molecular weight, three genes of the LCP protein family, tagV, tagT, and tagO, were finally selected for knockout. The molecular weights of the proteins expressed by tagV, tagT, and tagO genes were 43.7 kDa, 37 kDa, and 33.3 kDa, respectively. The nucleotide sequences are shown in Table 1.

[0073] Table 1. Nucleotide sequences of LCP protein family genes knocked out in Bacillus amyloliquefaciens BAX-9 in this embodiment.

[0074]

[0075] 1.1 Construction of LCP protein family gene knockout vectors

[0076] Taking the tagV gene as an example, the construction process is as follows: Figure 1As shown, primers for the two homologous arms of the tagV gene were designed based on the approximately 500 bp upstream and downstream sequences of the tagV gene in the whole genome sequence of Bacillus amyloliquefaciens BAX-9. The upstream and downstream homologous arm primers were named tagV-AF / AR and tagV-BF / BR, respectively. Using the gene of Bacillus amyloliquefaciens BAX-9 as a template, the upstream homologous arm sequence A and the downstream homologous arm sequence B of the tagV gene were amplified. After purification and recovery of the PCR product, the upstream and downstream homologous arms A and B were ligated by SOE-PCR to obtain the homologous sequence fragment tagV(A+B), as shown in SEQ ID NO.7. After purification and recovery of the product, tagV(A+B) was double-digested with restriction endonucleases BamHI and XbaI. The digested tagV(A+B) and the temperature-sensitive knockout plasmid T2(2)-ori, which had been treated with the same double enzyme digestion, were ligated with T4 ligase at 25℃ for 2 h. The ligation product was transformed into E. coli DH5α competent cells. Colony PCR was performed on single colonies using T2-F / R primers. After preliminary confirmation as a positive transformant, the plasmid was extracted for double digestion and DNA sequencing verification. After verification, the tagV gene knockout vector was obtained and named: T2ΔtagV.

[0077] Except for the different upstream and downstream homologous arm primers, T2ΔtagT and T2ΔtagO were constructed using the same method. The upstream and downstream homologous arm primers for the tagT gene were named tagT-AF / AR and tagT-BF / BR, respectively. The sequence of the fragment tagT(A+B) connecting the upstream homologous arm sequence A and the downstream homologous arm sequence B is shown in SEQ ID NO.8. The upstream and downstream homologous arm primers for the tagO gene were named tagO-AF / AR and tagO-BF / BR, respectively. The sequence of the fragment tagO(A+B) connecting the upstream homologous arm sequence A and the downstream homologous arm sequence B is shown in SEQ ID NO.9.

[0078] Table 2. Nucleotide sequences of tagV(A+B), tagT(A+B), and tagO(A+B) in this embodiment.

[0079]

[0080] 1.2 Constructing LCP protein family gene deletion mutant strains

[0081] The constructed knockout plasmid T2ΔtagV was electroporated into Bacillus amyloliquefaciens BAX-9 competent cells, plated on kanamycin-resistant plates, and incubated at 37°C for 16–20 h. Transformants were verified by colony PCR using primers T2-F / tagVB-R and T2-R / tagVA-F, and the products were detected by agarose gel electrophoresis. Correct positive transformants underwent single and double crossover; the procedure is described in [link to procedure]. Figure 2 Specifically, positive transformants were cultured in LB medium containing 20 μg / mL of Kan at 45°C for a single crossover process for 10-12 hours. Further, strains that had completed the single crossover were picked and placed in PA bottles containing 5 mL of LB medium (without Kan) for homologous recombination double crossover at 37°C in a shaker (the culture time and number of transductions were fine-tuned according to the actual crossover results). The double crossover culture was then diluted 10-fold. -3 -10 -6 Dilute 10 -3 The culture was continued to be incubated in shake flasks at 37°C, and the 10-fold dilution was... -6 The culture was spread on LB plates without Kan, and the single colonies that grew were spotted onto plates containing 20 μg / mL Kan and plates without Kan, respectively. The single colonies that grew on plates without Kan but not on plates containing Kan were verified by PCR using primers tagVA-F / tagVB-R. Double-exchange strains were screened to obtain the Bacillus amyloliquefaciens BAX-9 mutant strain with the tagV gene deleted, which was named B9△tagV.

[0082] Except for the different primers used for colony PCR, B9△tagT and B9△tagO were constructed using the same method.

[0083] The nucleotide sequences of the primers used above are shown in Table 3.

[0084] Table 3. Nucleotide sequences of primers used to construct the knockout vector and deletion mutant in this embodiment.

[0085]

[0086]

[0087] Figure 3 The image shows PCR validation diagrams of B9△tagV, B9△tagT, and B9△tagO colonies. Lane 1 contains a DNA marker; lanes 2-3 represent the amplified fragments of tagVA-F / tagVB-R; lanes 4-5 represent the amplified fragments of tagTA-F / tagTB-R; and lanes 6-7 represent the amplified fragments of tagOA-F / tagVB-R. The diagram shows that double crossovers occurred in B9△tagV, B9△tagT, and B9△tagO, resulting in amplified bands of varying sizes.

[0088] 2. Construction of ice nucleoprotein gene expression vector

[0089] Based on the genomic sequences and homologous recombination requirements of Bacillus subtilis 168, the ice nucleoprotein gene inaE of Erwinia bromelain var. pineapple, and the terminator Tamyl of Bacillus licheniformis WX-02 published by NCBI, the promoter P43, the ice nucleoprotein gene inaE, and the terminator Tamyl sequence were amplified by PCR. The nucleotide sequences of the three gene fragments and their amplification primers are shown in Tables 4-5.

[0090] The PCR amplification products were purified by gel electrophoresis and their concentration was detected. Using the laboratory-preserved expression vector PHY300 PLK as the initial starting vector, a linearized cloning vector was prepared by double digestion of the PHY300 PLK plasmid with BamHI and XbaI. This vector was then purified by gel electrophoresis and its concentration was detected. Next, the P43 promoter, the ice nucleoprotein gene inaE, and the Tamyl terminator fragment were mixed with the prepared linearized cloning vector. The vector volume was 10-100 ng, the molar ratio of vector to insert fragment was 1:1-1:10, and the molar ratio among the three fragments was 1:1. A one-step ligation system was used, and the reaction was carried out at 50℃ for 30 min. The ligation product was immediately transformed into E. coli DH5α. Transformants were selected for colony PCR verification. Positive transformants that passed PCR verification were inoculated into PA bottles containing 5 mL of LB medium (20 μg / mL tetracycline). Plasmids were extracted and sequenced. The plasmid with correct sequencing was the ice nucleoprotein gene expression vector, named PHY300PLK-INP.

[0091] Table 4. Nucleotide sequences of promoter, ice nucleoprotein gene and terminator in this embodiment.

[0092]

[0093]

[0094] Table 5 shows the nucleotide sequences of the primers used to construct the ice nucleus gene expression vector in this embodiment.

[0095]

[0096] Figure 4The diagram shows the colony PCR verification of positive transformants carrying the ice nucleus protein gene expression vector. From left to right, lane M is the DNA marker, lanes 1-2 are the amplified fragment of promoter P43 (305 bp), lanes 3-4 are the amplified fragment of the ice nucleus gene inaE (3777 bp), and lanes 5-6 are the amplified fragment of the terminator Tamyl (502 bp). As can be seen from the diagram, all inserted genes of this invention can be cloned, indicating that promoter P43, the ice nucleus gene inaE, and the terminator Tamyl were successfully inserted into the starting vector, successfully constructing the ice nucleus gene expression vector PHY300PLK-INP (plasmid map shown). Figure 5 (As shown).

[0097] 3. Constructing an engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein.

[0098] The constructed recombinant plasmid PHY300PLK-INP was electroporated into Bacillus amyloliquefaciens native strain BAX-9, mutant strains B9△tagV, B9△tagT, and B9△tagO competent cells. The electroporation cuvettes were pre-chilled on ice for 15 min after drying. Then, 100 μL of Bacillus amyloliquefaciens competent cells were mixed with 10 μL of the recombinant vector and added to the cuvette. After pre-chilling on ice for 3–5 min, electroporation was performed at 2.4 kV for 4.8–5.2 ms. Immediately afterwards, 800 μL of recovery medium was added and transferred to a 1.5 mL EP tube. After incubation at 37℃ and 100rpm for 3h, the bacteria were plated on LB plates containing 20μg / mL tetracycline resistance and incubated at 37℃ for 48h. After transformants grew, colony PCR and plasmid extraction were performed using primers pHY300-F / R (see Table 6). After successful verification, the bacterial strains were preserved to obtain recombinant bacteria B9-INP, BV9-INP, BT9-INP, and BO9-INP.

[0099] Table 6. Nucleotide sequences of primers used in the engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein in this embodiment.

[0100]

[0101] Figure 6The image shows colony PCR verification diagrams of engineered Bacillus amyloliquefaciens strains B9-INP, BT9-INP, BO9-INP, and BV9-INP expressing ice nucleus proteins. From left to right, lane M is the DNA marker, lane 1 is the amplified fragment of B9-INP, lane 2 is the amplified fragment of BT9-INP, lane 3 is the amplified fragment of BO9-INP, and lane 4 is the amplified fragment of BV9-INP. The image shows that amplification using the vector-specific primers pHY300-F / R yielded clear bands, indicating that the ice nucleus gene expression vector PHY300PLK-INP was successfully transformed into Bacillus amyloliquefaciens BAX-9, and that BAX-9 stably carries this expression vector. Therefore, the engineered strains were successfully constructed.

[0102] 4. Freezing point determination of engineered Bacillus amyloliquefaciens

[0103] Fermentation culture of engineered Bacillus amyloliquefaciens BAX-9, including the following culture medium and conditions:

[0104] a) Seed culture medium (pH 7.0-7.2): 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 μg / mL tetracycline; dispensed into PA bottles at 5 mL per bottle, seed culture time is 48 h, used to activate cells.

[0105] b) Fermentation medium (pH 7.0-7.2): 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride; dispensed into 250 mL Erlenmeyer flasks at a volume of 100 mL each, with an inoculum size of 1%, a fermentation cycle of 20 h, and a shaking speed of 150 rpm, for large-scale culture.

[0106] The fermentation broth from the expanded culture was centrifuged at 6000 rpm for 15 min, and the bacterial cells were collected, dissolved in sterile deionized water to prepare a bacterial suspension, and then lyophilized. The lyophilized bacterial cells were serially diluted to 1 mg / mL, and the supercooling point was determined using differential scanning calorimetry (DSC). The supercooling point of the bacterial suspension was measured by DSC using a thermal analyzer (NETZS 204F1, Germany) equipped with liquid nitrogen and compressed nitrogen. 10 μL of sample was transferred to a covered aluminum crucible and placed in the apparatus. The temperature ramp of the DSC unit was set to a freezing rate of 1 °C / min, from 4 °C to -25 °C, using an empty crucible as a reference. The temperature point showing the observed maximum heat flux was recorded as the supercooling point. The results are shown in [Figure number missing]. Figure 7 .

[0107] As can be seen from the figure, the engineered Bacillus amyloliquefaciens strains B9-INP, BV9-INP, BT9-INP, and BO9-INP all exhibit significant ice nucleus activity. The supercooling points of B9-INP, BV9-INP, BO9-INP, and BT9-INP are -9℃, -6.9℃, -7.2℃, and -8℃, respectively. BV9-INP has the highest supercooling point and the strongest ice nucleus activity.

[0108] 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. An engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein, characterized in that, The tagV gene, tagT gene, or tagO gene of the engineered Bacillus amyloliquefaciens strain is knocked out, and it carries an expression vector capable of expressing the ice nucleoprotein gene. The nucleotide sequence of the tagV gene is shown in SEQ ID NO.1, the nucleotide sequence of the tagT gene is shown in SEQ ID NO.2, and the nucleotide sequence of the tagO gene is shown in SEQ ID NO.

3. The ice nucleoprotein gene is selected from the inaE gene of Erwinia broccoli var. pineapple, and the nucleotide sequence of the inaE gene is shown in SEQ ID NO.

4.

2. The engineered Bacillus amyloliquefaciens expressing ice nucleoprotein according to claim 1, characterized in that, The tagV gene of the engineered Bacillus amyloliquefaciens strain was knocked out.

3. The engineered Bacillus amyloliquefaciens expressing ice nucleoprotein according to claim 1, characterized in that, The expression vector further includes a promoter located upstream of the ice nucleoprotein gene and a terminator located downstream of the ice nucleoprotein gene, wherein the promoter is selected from the P43 promoter and the terminator is selected from the Tamyl terminator. The nucleotide sequence of the P43 promoter is shown in SEQ ID NO.5, and the nucleotide sequence of the Tamyl terminator is shown in SEQ ID NO.

6.

4. The engineered Bacillus amyloliquefaciens expressing ice nucleoprotein according to claim 1, characterized in that, The starting strain of the engineered Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens BAX-9.

5. A method for constructing an engineered Bacillus amyloliquefaciens strain expressing ice nucleoprotein, characterized in that, The method for constructing the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein as described in any one of claims 1-4 includes the following steps: By knocking out the tagV, tagT, or tagO genes of Bacillus amyloliquefaciens using homologous recombination technology, and then transferring an expression vector expressing the ice nucleoprotein gene into the Bacillus amyloliquefaciens, an engineered Bacillus amyloliquefaciens strain expressing the ice nucleoprotein was constructed.

6. The method for constructing an engineered Bacillus amyloliquefaciens expressing ice nucleoprotein according to claim 5, characterized in that, The homologous recombination technology includes the following steps: S1. Using the genome of Bacillus amyloliquefaciens as a template, amplify the upstream homologous arm sequence A and the downstream homologous arm sequence B respectively using primers for the upstream and downstream homologous arms of the tagV gene, tagT gene, or tagO gene. The nucleotide sequences of the upstream and downstream homologous arm primers of the tagV gene are shown in SEQ ID NO.10-13, the nucleotide sequences of the upstream and downstream homologous arm primers of the tagT gene are shown in SEQ ID NO.14-17, and the nucleotide sequences of the upstream and downstream homologous arm primers of the tagO gene are shown in SEQ ID NO.18-21. S2. The upstream homologous arm sequence A and the downstream homologous arm sequence B are linked together using overlap extension PCR technology to form homologous sequence C; The nucleotide sequences of the homologous sequence C of the tagV gene, the tagT gene, and the tagO gene are shown in SEQ ID NO.7-9, respectively. S3. Insert the homologous sequence C into the temperature-sensitive knockout plasmid T2(2)-ori to construct the knockout plasmid; S4. The knockout plasmid is transferred into the competent cells of Bacillus amyloliquefaciens, and Bacillus amyloliquefaciens mutant strains with the tagV gene, tagT gene and tagO gene knocked out are obtained by single and double crossover screening.

7. The use of the Bacillus amyloliquefaciens engineered strain expressing ice nucleoprotein as described in any one of claims 1-4 in artificial snowmaking, seawater desalination, insecticide, freeze concentration, or frozen food industries.

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