Bacillus producing surfactin and not producing spores and use thereof

Abstract

This invention provides a non-spore-forming Bacillus subtilis that produces surfactants and its applications, relating to the field of synthetic biology. In this invention, spore synthesis-related genes in Bacillus subtilis are knocked out. spoIVB, And by expressing the gene encoding functionally active 4-phosphate pantothenic acid thioethyltransferase. sfpTH For the first time, a high-yield surfactant strain was obtained, with a surfactant production rate of 15.77 g / L and a spore yield of 0% by shake-flask fermentation.

Description

Technical Field

[0001] This application relates to the field of synthetic biology technology, and in particular to a Bacillus that produces surfactants but does not produce spores and its applications. Background Technology

[0002] Surfactin is a cyclic lipopeptide biosurfactant produced by microorganisms such as Bacillus subtilis. Its core advantages lie in its excellent physicochemical properties, biosafety, environmental compatibility, and multiple bioactive functions. Surfactin is one of the most promising biosurfactants currently, with broad application prospects in cosmetics, food, medicine, agriculture, oil extraction, and environmental remediation. However, one of the main bottlenecks to its industrial application is the low fermentation yield and high production cost.

[0003] Currently, methods to increase the fermentation yield of surfactants are no longer limited to optimizing the fermentation process of wild strains and mutagenesis screening. With the continuous deepening of research on surfactant synthesis and the continuous progress of biotechnology, the construction of Bacillus subtilis genetically engineered bacteria has become an important means to increase fermentation yield. Summary of the Invention

[0004] To address the aforementioned problems, this invention aims to provide a recombinant engineered strain that efficiently produces surfactants without spore formation, which can be used for the industrial production of surfactants.

[0005] On the one hand, this application provides a Bacillus that produces surfactants but does not produce spores, said Bacillus comprising any one or two of the following modifications: A1)-A2) A1) Genes regulating spore synthesis spoIVB No expression and / or low expression; A2) Expression and / or overexpression of 4-phosphoubiotylethaminase, wherein the 4-phosphoubiotylethaminase comprises the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 95% identity with SEQ ID No. 3.

[0006] Preferably, the 4-phosphate pantothenicotinyl ethamintransferase comprises the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 3.

[0007] Furthermore, the nucleotide sequence encoding the 4-phosphate pantothenic acid ethaminase comprises the sequence shown in SEQ ID No. 2 or a nucleotide sequence having at least 80% identity with SEQ ID No. 2.

[0008] Preferably, the nucleotide sequence encoding the 4-phosphate pantothenicotinyl ethaminase comprises the sequence shown in SEQ ID No. 2 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 2.

[0009] The gene encoding the 4-phosphate pantothenic acid thioglycol ethanotransferase is sfpTH .

[0010] Furthermore, the overexpression is achieved by expressing 4-phosphate pantothenic acid thioglycol transferase via a strong promoter.

[0011] Preferably, the strong promoter includes a strongly inductive promoter; more preferably, the strongly inductive promoter is a promoter. Pgrac More preferably, the promoter Pgrac The sequence is shown in SEQ ID No. 1.

[0012] Furthermore, the expression and / or overexpression of the 4-phosphate pantothenic acid thioglycotransferase can be either free expression or integrated expression.

[0013] A better approach is to integrate the expression.

[0014] Those skilled in the art can choose the integration site and integration method according to the actual situation, as long as the expression of the target gene can be achieved, and no mandatory restrictions are imposed here.

[0015] More preferably, the gene encoding 4-phosphate pantothenic acid ethanotransferase is inserted into... spoIVB Gene loci, enabling integrated expression.

[0016] More preferably, the integrated expression of 4-phosphate pantothenic acid thioglycolate is achieved using a gene editing plasmid constructed based on the pJOE8999 plasmid.

[0017] Furthermore, the genes regulating spore synthesis... spoIVBMethods of non-expression and / or weak expression include: attenuating or silencing the genes involved in regulating spore synthesis. spoIVB Expression of, and / or introduction of genes that can knock out the aforementioned genes regulating spore synthesis. spoIVB The substance.

[0018] The lack of expression may also include the absence of the corresponding gene, and the weak expression or lack of expression may also be achieved by knocking out and / or silencing and / or inhibiting the expression of the protein-coding gene or by introducing a substance capable of knocking out or reducing the expression of the protein-coding gene in the Bacillus genome, as long as it can achieve the lack of protein expression, low expression, inhibition of expression, or inhibition of its activity.

[0019] Preferably, the weak expression or non-expression is achieved by knockout.

[0020] Those skilled in the art can choose the knockout method according to the actual situation, and no specific restrictions are made in this application.

[0021] More preferably, by constructing genes that target spore synthesis. spoIVB The targeting element enables gene knockout.

[0022] Alternatively, the targeting element may include genes related to spore synthesis. spoIVB upstream homologous arms, spore synthesis-related genes spoIVB The downstream homologous arm.

[0023] Alternatively, it may also include genes targeting spore synthesis. spoIVB The sgRNA sequence is shown in P1 and P2.

[0024] More preferably, gene editing systems based on the pJOE8999 plasmid are used to realize genes related to spore synthesis. spoIVB Knockout.

[0025] In a preferred embodiment, genes related to targeted spore synthesis are... spoIVB The sgRNA and targeting element were linked to the pJOE8999 plasmid to construct a knockout plasmid. This knockout plasmid was then transferred into Bacillus subtilis, enabling the expression of genes related to spore synthesis. spoIVB Knockout.

[0026] Furthermore, the genes regulating spore synthesis... spoIVB It contains an amino acid sequence as shown in SEQ ID No. 5 or an amino acid sequence that is at least 95% identical to SEQ ID No. 5.

[0027] Preferably, the gene regulating spore synthesis is... spoIVBThe amino acid sequence comprising the amino acid sequence shown in SEQ ID No. 5 or having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 5.

[0028] Furthermore, the genes regulating spore synthesis... spoIVB It contains a nucleotide sequence as shown in SEQ ID No. 4 or a nucleotide sequence that has at least 85% identity with SEQ ID No. 4.

[0029] Preferably, the gene regulating spore synthesis is... spoIVB Nucleotide sequences containing the nucleotide sequence shown in SEQ ID No. 4 or having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 4.

[0030] Those skilled in the art can use known gene editing tools or gene editing methods to complete the above-mentioned modification operations, and no specific limitations are made in this application.

[0031] In a preferred embodiment, genes related to targeted spore synthesis are... spoIVB The sgRNA, along with the targeting element and the gene encoding 4-phosphate pantothenic acid thioethyltransferase, was ligated into the pJOE8999 plasmid to construct a gene-editing plasmid. This plasmid was then transferred into Bacillus subtilis, enabling the expression of spore-related genes. spoIVB The knockout and simultaneous integration of the 4-phosphate pantothenic acid thioglycol transferase gene.

[0032] More preferably, the gene-editing plasmid contains sgRNA and, in sequence, spore synthesis-related genes. spoIVB upstream homologous arm, promoter Pgrac, Genes encoding 4-phosphopantoylthioethyltransferase and genes related to spore synthesis spoIVB The downstream homologous arm.

[0033] Furthermore, the Bacillus species include any one or more of Bacillus subtilis and Bacillus amyloliquefaciens.

[0034] Preferably, the Bacillus species includes Bacillus subtilis.

[0035] More preferably, the Bacillus subtilis is Bacillus subtilis (B. subtilis) Bacillus subtilis 168.

[0036] This technical solution is suitable for all strains of Bacillus substrate, and no further limitations are made here.

[0037] On the other hand, this application also provides a method for preparing a surfactant, the method comprising: culturing the Bacillus.

[0038] Those skilled in the art can culture Bacillus using conventional methods.

[0039] In a preferred embodiment, the method includes: Step 1: Construct the Bacillus strain; Step 2: Inoculate the Bacillus into the fermentation medium, ferment at 25-40℃ and 100-300 rpm for 1-5 hours, add 0.1-1 mM IPTG (isopropyl-β-D-thiogalactoside) as an inducer, and culture in shake flasks for 24-96 hours.

[0040] Preferred, The method includes: Step 1: Construct the Bacillus strain; Step 2: Inoculate the Bacillus into the seed culture medium and culture at 25℃-40℃ and 100-300 rpm for 12-48 h to obtain the seed solution; Step 3: Inoculate the seed culture into the fermentation medium OD 600 The concentration is 0.1-0.5, 25℃-40℃, 100-300rpm, fermentation culture for 1-5 h, add 0.1-1 mM IPTG inducer, and culture in shake flasks for 24-96 h.

[0041] On the other hand, this application also provides the use of the aforementioned Bacillus in the preparation of surfactants and / or in increasing surfactant yield and / or reducing spore yield.

[0042] Preferably, the spore yield is 0, that is, no spores are produced.

[0043] Preferably, the surfactant yield can reach 15.77 g / L.

[0044] On the other hand, this application also provides a method for increasing the yield of Bacillus surfactant and / or decreasing the spore yield and / or increasing the activity of Bacillus, the method comprising modifying Bacillus by any one or two of the following A1)-A2): A1) Genes related to the regulation of spore synthesis spoIVB No expression and / or low expression; A2) Expression and / or overexpression of 4-phosphoubiotylethamintransferase, wherein the 4-phosphoubiotylethamintransferase comprises the sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 95% identity with SEQ ID No. 3.

[0045] Furthermore, the Bacillus species include any one or more of Bacillus subtilis and Bacillus amyloliquefaciens.

[0046] Preferably, the Bacillus species includes Bacillus subtilis.

[0047] More preferably, the Bacillus subtilis is Bacillus subtilis (B. subtilis) Bacillus subtilis 168.

[0048] The present invention has the following beneficial effects: In this invention, spore synthesis-related genes in Bacillus subtilis BS168 are knocked out. spoIVB, And through the strongly inductive promoter P grac Overexpression of the gene encoding functionally active 4-phosphate pantothenic acid thioethyltransferase sfpTH For the first time, a high-yield surfactant strain (S6) was obtained, with a surfactant production rate of up to 15.77 g / L achieved by shake-flask fermentation.

[0049] Furthermore, this invention has discovered that the OD of the modified Bacillus subtilis S6... 600 The higher cell density and bioactivity of Bacillus subtilis S6 compared to the original strain indicate that it has achieved a higher cell density and bioactivity. This provides a new approach for the engineering of surfactant strains and demonstrates that the strain modification and modification methods can be widely applied in industrial production.

[0050] In addition, the modified Bacillus subtilis S6 produced 0% spores during fermentation, which solved the problem of downstream product purification, reduced the difficulty and cost of separation and purification, and lowered production costs. Attached Figure Description

[0051] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1OD of engineered bacteria biomass 600 And a statistical chart of lipopeptide production; Figure 2 This is a graph illustrating bacterial spore formation as verified by the plate count method. Figure 2 A in the diagram is a schematic of the coating of BS168 fermentation broth. Figure 2 B in the diagram is a schematic diagram of the coating of fermentation broth after BS168 heat treatment. Figure 2 C in the diagram represents the coating of the S6 fermentation broth. Figure 2 D is a schematic diagram of the coating of fermentation broth after heat treatment of S6. Detailed Implementation

[0052] Technical terms: Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0053] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0054] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0055] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0056] Recombinant microorganisms (engineered bacteria): bacterial cell lines that use genetic engineering methods to achieve efficient expression of exogenous genes.

[0057] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0058] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and be transmitted independently. They are widely used in genetic engineering and molecular biology research.

[0059] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0060] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0061] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0062] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0063] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0064] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0065] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, analytical chemistry, microbiology, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0066] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0067] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0068] Among them, pJOE8999 plasmid was purchased from Ningbo Taisto Biotechnology Co., Ltd.; Bacillus subtilis ( Bacillus subtilis168 was purchased from Beina Biotechnology (BNCC371816); plasmid PHT08 was purchased from Ningbo Taisto Biotechnology Co., Ltd.

[0069] The culture media involved in the following examples are as follows: LB medium (1 L): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride (NaCl), and the remainder is water; LB medium plates can be prepared by adding an appropriate amount of agar to adjust the LB medium to a solid state.

[0070] Example 1: Construction of Cas9 Editing Plasmid Construct corresponding editing plasmids based on pJOE8999 plasmid, including spoIVB Knockout sfpTH Integration and use of the strongly inductive promoter P grac overexpression srfAA Related plasmids. spoIVB The nucleotide sequence is shown in SEQ ID No. 4, and the amino acid sequence is shown in SEQ ID No. 5; sfpTH The nucleotide sequence is shown in SEQ ID No. 2, and the amino acid sequence is shown in SEQ ID No. 3; promoter Pgrac As shown in SEQ ID No. 1; srfAA The sequence is shown in SEQ ID No. 6, and the amino acid sequence is shown in SEQ ID No. 7. The specific plasmid construction process is as follows: Construction of pJOE8999-spoIVBg plasmid: In gene sequencing related to spore synthesis spoIVB Specific sgRNA sequences were selected internally, and primers P1 and P2 were designed and synthesized. PCR circularization amplification was performed using pJOE8999 plasmid as a template. After Gibson ligation, the amplified cells were transformed into *E. coli*. Escherichia coli DH5α.

[0071] Construction of pJOE8999-spoIVB plasmid: using Bacillus subtilis ( Bacillus subtilis Using 168 as a template, PCR amplification was performed using primers P3 and P4, and primers P5 and P6, respectively. spoIVB Upstream and downstream fragments were then sequentially spliced ​​together using fusion PCR, and then... SfiI After enzyme digestion and ligation into plasmid pJOE8999-spoIVBg, it was transformed into E. coli. Escherichia coli DH5α.

[0072] Construction of pJOE8999-spoIVB-sfp plasmid: using Bacillus subtilis ( Bacillus subtilisUsing 168 as a template, PCR amplification was performed using primers P3 and P7, and primers P6 and P8 to obtain... spoIVB Upstream and downstream fragments; using plasmid PHT08 as a template, the promoter was obtained by PCR amplification using primers P9 and P10. Pgrac Fragments; artificially synthesized genes sfpTH Using primers P11 and P12 as templates, PCR amplification was performed to obtain... sfpTH Fragments. Then, through two rounds of fusion PCR, the above four PCR fragments were spliced ​​into one PCR fragment, with the first round of fusion PCR... spoIVB Upstream segments and Pgrac promoter fragment splicing, sfpTH Fragments and spoIVB Downstream fragment splicing; the second round of fusion PCR splices the two fragments obtained from the first round of fusion PCR to obtain sequentially ligated fragments. spoIVB Upstream - Pgrac - sfpTH - spoIVB Downstream fragment. Finally, the spliced ​​PCR fragment was digested with SfiI and ligated into plasmid pJOE8999-spoIVBg and transformed into E. coli. Escherichia coli DH5α.

[0073] Construction of pJOE8999-srfAAg plasmid: Based on srfAA A specific sgRNA sequence was designed upstream of the promoter, and primers P13 and P14 were synthesized. PCR circularization amplification was performed using pJOE8999 plasmid as a template, followed by Gibson ligation and transformation into *E. coli*. Escherichia coli DH5α.

[0074] Construction of pJOE8999-srfAA plasmid: using Bacillus subtilis ( Bacillus subtilis Using 168 as a template, PCR amplification was performed using primers P15 and P16, and primers P17 and P18, respectively. srfAA The upstream and downstream fragments of the promoter insertion site; using plasmid PHT08 genome as a template, the promoter was obtained by PCR amplification using primers P19 and P20. Pgrac Fragments. Then, through two rounds of fusion PCR, the above three PCR fragments were spliced ​​into one PCR fragment, wherein the first round of fusion PCR... srfAA Upstream segments and Pgrac Promoter fragment assembly; second-round fusion PCR combines the first-round fusion PCR fragments with... srfAA Downstream segments are spliced ​​together to obtain sequentially connected segments. srfAA Upstream - Pgrac - srfAADownstream fragment. Finally, the spliced ​​PCR fragment was digested with SfiI and ligated into plasmid pJOE8999-srfAAg and transformed into E. coli. Escherichia coli DH5α.

[0075] The primer sequences designed above are shown in Table 1.

[0076] Table 1 Primers for PCR amplification

[0077] Example 2: Modification and strain construction of Bacillus subtilis 168 The Cas9 editing plasmid constructed in Example 1 was transformed into Bacillus subtilis using the Spizizen chemical transformation method. Bacillus subtilis In BS168, positive clones were screened by plating the culture onto LB agar plates containing kanamycin (10 mg / L) and mannose (0.02%). After verification by colony PCR and sequencing, positive clones underwent plasmid loss. Plasmid loss was achieved through high-temperature culture utilizing the temperature-sensitive element characteristics of the Cas9-edited plasmid. Specifically, the edited strain was cultured for several generations in LB agar at 45°C and 220 rpm with shaking. Single colonies were obtained by plating the culture onto LB agar plates, and single clones sensitive to kanamycin were screened, indicating successfully plasmid-lost strains. S1 strain construction: The pJOE8999-spoIVB plasmid was transformed into the BS168 strain, and the strain was obtained after selection by antibiotic resistance plates, colony PCR verification, and plasmid loss. spoIVB Knockout strains.

[0078] S2 strain construction: The pJOE8999-srfAA plasmid was transformed into the S1 strain, and the strain was knocked out after screening with antibiotic resistance plates, verification by colony PCR, and plasmid loss. spoIVB, and srfAA The promoter was replaced with grac Strain of the promoter.

[0079] S6 strain construction: The pJOE8999-spoIVB-sfp plasmid was transformed into the BS168 strain. Knockout strains were obtained after antibiotic resistance plate selection, colony PCR verification, and plasmid loss. spoIVB, And integration Pgrac Promoter overexpression sfpTH strains.

[0080] Information on engineered bacteria is shown in Table 2.

[0081] Table 2 Information on engineered bacteria

[0082] Example 3: Shake-flask culture of engineered bacteria and determination of lipopeptide yield The strains BS168, S1, S2, and S6 constructed in Example 2 were inoculated onto LB agar plates and cultured overnight at 37°C. The resulting single clones were then inoculated into LB agar and cultured overnight at 37°C and 220 rpm to obtain seed culture (three replicates for each strain). The seed culture was then transferred to a 500 mL Erlenmeyer flask containing 30 mL of fermentation medium. The fermentation initiation OD was... 600 The concentration was 0.1, and the culture conditions were 37℃ and 220 rpm. After 3 h of fermentation, 0.8 mM IPTG (isopropyl-β-D-thiogalactoside) was added, and the mixture was cultured in shake flasks for 48 h. The biomass OD was then measured. 600 And the lipopeptide content in the fermentation supernatant.

[0083] Fermentation medium formula: 70 g / L brown sugar, 5 g / L yeast powder, 2.5 g / L sodium nitrate, 0.3 g / L potassium dihydrogen phosphate, 1 g / L disodium hydrogen phosphate dodecahydrate, 0.15 g / L magnesium sulfate heptahydrate, 0.0075 g / L calcium chloride, 0.006 g / L ferrous sulfate heptahydrate, 0.006 g / L manganese sulfate heptahydrate, 10 g / L leucine, 6 g / L valine, with the balance being water.

[0084] The concentration of lipopeptides in the fermentation supernatant was determined by high-performance liquid chromatography (HPLC). A Shimadzu C18 column (250 mm × 4.60 mm, 5 μm) was used at a column temperature of 40 °C. The mobile phase for detection was methanol:water = 95:5 (v / v, containing 0.1% trifluoroacetic acid), and the flow rate was 0.8 ml / min. A UV detector was used to detect the lipopeptides at a wavelength of 205 nm. The results are as follows: Figure 1 As shown in Table 3.

[0085] Table 3

[0086] The lipopeptide (surfactant) production results of Bacillus subtilis showed that BS168, S1, and S2 produced 0 lipopeptide, while strain S6 achieved the highest lipopeptide production at 24 h, reaching 15.77 g / L. Furthermore, the OD of the modified Bacillus subtilis S6... 600 The higher density than the original strain indicates that Bacillus subtilis S6 has achieved a higher cell density and greater biological activity.

[0087] Example 4: Determination of spore yield of engineered strain S6 Based on the characteristic that spores are heat-resistant while vegetative cells are not, this embodiment uses the heat resistance plate counting method to functionally verify the spore generation efficiency of the S6 strain constructed in Example 2.

[0088] After inoculating BS168 or S6 strains onto LB agar plates and culturing overnight at 37°C, single clones of BS168 and S6 were inoculated into LB agar plates and cultured at 37°C and 220 rpm for 48 h to obtain fermentation broth. The fermentation broth and the heat-treated broth (80°C water bath for 15 min) were serially diluted and plated onto LB agar plates, incubated at 37°C for 12 h, and clone counts (CFU) were performed. Spore count (%) = (Number of surviving spores after heat treatment / Total number of viable cells before heat treatment) × 100%.

[0089] Experimental results are as follows Figure 2 As shown, strain BS168, without heat treatment, produced 327 single colonies on the diluted plate, while after heat treatment, it produced 276 single colonies. Strain S6, without heat treatment, produced 212 single colonies on the diluted plate, while after heat treatment, it produced 0 single colonies. The final calculation showed that strain BS168 had a spore rate of 74.2%, while strain S6 had a spore rate of 0%.

[0090] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A Bacillus strain for producing surfactants, characterized in that, The Bacillus species includes any one or two of the following modifications: A1)-A2): A1) Genes regulating spore synthesis spoIVB No expression and / or low expression; A2) Expression and / or overexpression of 4-phosphoubiotylethaminase, wherein the 4-phosphoubiotylethaminase comprises the amino acid sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 95% identity with SEQ ID No.

3.

2. The Bacillus according to claim 1, characterized in that, The nucleotide sequence encoding the 4-phosphate pantothenic acid ethamine transferase comprises the sequence shown in SEQ ID No. 2 or a nucleotide sequence having at least 80% identity with SEQ ID No.

2.

3. The Bacillus according to claim 1, characterized in that, The genes related to regulating spore synthesis spoIVB Methods of non-expression and / or weak expression include: attenuating or silencing the genes involved in regulating spore synthesis. spoIVB Expression of, and / or introduction of genes that can knock out the aforementioned genes regulating spore synthesis. spoIVB The substance.

4. The Bacillus according to claim 1 or 3, characterized in that, The genes related to regulating spore synthesis spoIVB It contains an amino acid sequence as shown in SEQ ID No. 5 or an amino acid sequence that is at least 95% identical to SEQ ID No.

5.

5. The Bacillus according to claim 4, characterized in that, The genes related to regulating spore synthesis spoIVB It contains a nucleotide sequence as shown in SEQ ID No. 4 or a nucleotide sequence that has at least 85% identity with SEQ ID No.

4.

6. The Bacillus species according to any one of claims 1-5, characterized in that, The Bacillus species mentioned include any one or more of Bacillus subtilis and Bacillus amyloliquefaciens.

7. A method for preparing a surfactant, characterized in that, The preparation method includes: culturing Bacillus as described in any one of claims 1-6.

8. The use of Bacillus as described in any one of claims 1-6 in increasing surfactant yield and / or reducing spore yield.

9. A method for increasing the yield of Bacillus surfactant and / or decreasing spore yield and / or increasing Bacillus activity, characterized in that, The method includes modifying Bacillus spp. with one or both of the following A1)-A2): A1) Genes regulating spore synthesis spoIVB No expression and / or low expression; A2) Expression and / or overexpression of 4-phosphoubiotylethamintransferase, wherein the 4-phosphoubiotylethamintransferase comprises the sequence shown in SEQ ID No. 3 or an amino acid sequence having at least 95% identity with SEQ ID No.

3.

10. The method according to claim 9, characterized in that, The Bacillus species mentioned include any one or more of Bacillus subtilis and Bacillus amyloliquefaciens.

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