Bacillus subtilis with high yield of specific surfactin and preparation method and application thereof

Abstract

The application discloses a high-yield bacillus subtilis for producing specific surfactin, a preparation method and application of the bacillus subtilis, and relates to the field of genetic engineering, and can be used for improving the yield of surfactin, especially the industrialized production and commercial application of C15 component surfactin.

Description

Technical Field

[0001] This invention relates to a microorganism and its preparation method and application, and more particularly to a Bacillus subtilis and its preparation method and application, belonging to the field of genetic engineering. Background Technology

[0002] Biosurfactants are surface-active substances synthesized by microorganisms, possessing advantages such as low toxicity, biodegradability, and environmental friendliness. Surfactants are a class of biosurfactants with fatty acid and peptide chain structures, widely found in nature, primarily synthesized by Bacillus species, such as Bacillus subtilis. Surfactants consist of a cyclic heptapeptide and a β-hydroxy fatty acid chain (typically 13-15 carbon atoms long). The hydrophilic peptide and hydrophobic fatty acid moieties endow surfactants with both biological and surface activity, thus making them a green alternative to traditional chemical surfactants. Surfactants can be used to enhance oil recovery and are also applied in emulsions, pharmaceuticals, and food; they are particularly suitable for cleaning up oil spills or hazardous compound contamination in soil or water bodies; their antibacterial, antiviral, and antitumor properties also enable their application in the pharmaceutical and agricultural industries. Currently, surfactant production mainly relies on microbial fermentation, but the existing surfactant yield is limited, and the proportion of specific surfactants is low. This low production efficiency significantly restricts the widespread application of surfactants.

[0003] Methods to enhance the ability of microorganisms to produce surfactant include strain screening, genetic modification, and optimization of culture media and fermentation conditions. Genetic modification methods mainly include targeted modification strategies such as promoter engineering, enhanced efflux secretion, modification of NRPS domains, and fatty acid chain synthase systems. The fatty acid chain synthase system includes various enzymes, among which carboxyltransferase (accAD), biotinylate carboxylase (IdeHA and accBC) are components of the acetyl-CoA carboxylase complex, which catalyzes the reaction of acetyl-CoA to malonyl-CoA; fatty acid peroxygenase (cypC) participates in the formation of 3-hydroxy fatty acids, which are used in lipopeptide biosynthesis. All four enzymes are involved in the synthesis of surfactant in Bacillus subtilis. Wang Miaomiao et al. constructed high-surfactant-producing Bacillus subtilis by strengthening key genes in fatty acid biosynthesis and all genes in this pathway. They also optimized conditions such as the types and amounts of amino acids added during fermentation, achieving high-level production of surfactants in a 30L fermenter (Construction and Culture Optimization of High-Surfactant-Producing Recombinant Bacillus subtilis. Chinese Journal of Biotechnology, 2020, 36(11):2377-2386). This demonstrates that genetic modification methods can improve the low yield and low proportion of specific surfactants, thereby promoting the industrial production and commercial application of surfactants. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a Bacillus subtilis strain obtained by genetic modification that can produce high levels of C15 surfactant. This invention also provides an enzyme mutant involved in the production of surfactant by the Bacillus subtilis, as well as a method for preparing and using the Bacillus subtilis strain.

[0005] Technical solution: The present invention provides a Bacillus subtilis strain that produces a high amount of a specific surfactant, comprising carboxyltransferase accAD, biotinylate carboxylase IdeHA, biotinylate carboxylase accBC and fatty acid peroxygenase cypC, the amino acid sequences of which are shown in SEQ ID NO.1-4 in sequence;

[0006] The 16th amino acid threonine in the carboxyltransferase accAD is mutated to serine;

[0007] Alternatively, the 16th amino acid threonine in the carboxyltransferase accAD may be mutated to serine; the 74th amino acid lysine in the biotinylate carboxylase IdeHA may be mutated to asparagine and the 125th amino acid alanine may be mutated to threonine and the 394th amino acid threonine may be mutated to asparagine; and the 409th amino acid serine in the biotinylate carboxylase accBC may be mutated to glycine.

[0008] Alternatively, the 16th amino acid threonine in the carboxyltransferase accAD may be mutated to serine; the 74th amino acid lysine in the biotinylate carboxylase IdeHA may be mutated to asparagine, the 125th amino acid alanine may be mutated to threonine, and the 394th amino acid threonine may be mutated to asparagine; the 409th amino acid serine in the biotinylate carboxylase accBC may be mutated to glycine; and the 162nd amino acid serine in the fatty acid peroxygenase cypC may be mutated to proline, the 270th amino acid leucine may be mutated to valine, and the 298th amino acid leucine may be mutated to proline.

[0009] This invention utilizes *Bacillus subtilis* as the chassis cell for gene editing. Site-directed mutagenesis was performed on the amino acid sequences of the following enzymes in the *Bacillus subtilis* genome: carboxyltransferase accAD (SEQ ID No. 1), biotinylate carboxylase IdeHA (SEQ ID No. 2), biotinylate carboxylase accBC (SEQ ID No. 3), and fatty acid peroxygenase cypC (SEQ ID No. 4). Since all four enzymes are involved in the synthesis of surfactants in *Bacillus subtilis*, this invention improves the efficiency of fatty acid synthesis by site-directedly modifying carboxyltransferase accAD, biotinylate carboxylase IdeHA, and accBC. Furthermore, mutations in fatty acid peroxygenase cypC alter its specificity for long-chain fatty acids, thus providing a novel pathway for producing biosurfactants with specific functions and activities.

[0010] This invention involves 3D modeling of the aforementioned carboxyltransferases accAD, biotinylate carboxylases IdeHA and accBC, and fatty acid peroxygenase cypC, and site-directed mutagenesis of key amino acids around the active site or at the protein surface loop position. CRISPR technology is used to edit the genes of these enzymes in *Bacillus subtilis*: a crRNA sequence containing the mutation sites of acetyl-CoA carboxyltransferases accAD, acetyl-CoA carboxylases IdeHA and accBC, and fatty acid peroxygenase cypC is inserted into the pcrF19NM plasmid. The pHT-XCR6 plasmid carrying the Cpf1 gene, the constructed pcrF19NM plasmid, and homologous sequences serving as gene recombination templates are sequentially transformed into competent *Bacillus subtilis* cells. After plasmid removal, fermentation and high-performance liquid chromatography (HPLC) product screening yield *Bacillus subtilis* with increased C15 surfactant production are obtained. Gene sequencing revealed that the 16th amino acid of carboxyltransferase accAD was mutated from threonine to serine; the 74th amino acid of biotinylate carboxylase IdeHA was mutated from lysine to asparagine, and the 125th amino acid of alanine was mutated from threonine, and the 394th amino acid of threonine was mutated from asparagine; the 409th amino acid of biotinylate carboxylase accBC was mutated from serine to glycine; and the 162nd amino acid of fatty acid peroxygenase cypC was mutated from serine to proline, and the 270th amino acid of leucine was mutated from valine, and the 298th amino acid of leucine was mutated from proline.

[0011] The present invention also provides a method for preparing the aforementioned Bacillus subtilis, comprising the following steps:

[0012] Step 1: Prepare competent Bacillus subtilis cells;

[0013] Step 2: Transform the first recombinant plasmid into the competent cells obtained in Step 1 to obtain recombinant cells; the first recombinant plasmid can express a protein with DNA double-strand excision activity;

[0014] Step 3: Construct a second recombinant plasmid; the second recombinant plasmid contains the mutation information;

[0015] Step 4: Construct homologous fragments; the homologous fragments contain primer sequences;

[0016] Step 5: Transform the second recombinant plasmid and the homologous fragment into the recombinant cells obtained in Step 2 to obtain the Bacillus subtilis.

[0017] Preferably, in step 2, the first recombinant plasmid is pHT-XCR6 plasmid.

[0018] Furthermore, in step 2, the protein with DNA double-strand excision activity is Cpf1.

[0019] Preferably, in step 3, the second recombinant plasmid is pcrF19NM plasmid.

[0020] Furthermore, in step 3, the second recombinant plasmid contains a crRNA sequence.

[0021] Furthermore, the crRNA sequence is as follows:

[0022] accAD-T16 GGCGATTTTGGTTTGCAGTTCAATC IdeHA-K74 CGCGTCGGCTTTTGCTTTTTTC IdeHA-A125 GACACCTGCAGCCTCCATTGC IdeHA-T394 CTGTTCTGGTTTGGCCTTTG accBC-S409 CGAATTCGCTCAATGCGCGC cypC-S162 GAAAACGACTTCCGGAACAGCGCTG cypC-L270 GCTTGATTTATATGGAACGAAC cypC-L298 GGAAGAAAATCTGTTTGATATG

[0023] Furthermore, in step 4, the primer sequence is:

[0024]

[0025]

[0026] The present invention also provides a carboxyltransferase accAD mutant of Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO.5.

[0027] The present invention also provides a biotin carboxylase IdeHA mutant of Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO.6.

[0028] The present invention also provides a biotin carboxylase accBC mutant of Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO.7.

[0029] The present invention also provides a fatty acid peroxygenase cypC mutant of Bacillus subtilis, the amino acid sequence of which is shown in SEQ ID NO.8.

[0030] The present invention also provides an application of the aforementioned Bacillus subtilis in the production of surfactants.

[0031] Preferably, the surfactant is a C15 component surfactant.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention provides a higher yield of surfactant from Bacillus subtilis, and the proportion of C15 surfactant is significantly increased; 2. The present invention screens out the carboxyltransferase accAD, biotin carboxylase IdeHA and accBC mutants, as well as the fatty acid peroxygenase cypC mutant, which assist Bacillus subtilis in high surfactant production by site-directed mutagenesis involving key enzymes in surfactant synthesis; 3. The present invention also provides a method for preparing the Bacillus subtilis and its application, which helps to realize the industrial production and commercial application of surfactant. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the protein structure of the carboxyltransferase accAD;

[0034] Figure 2 This is a schematic diagram of the protein structure of biotin carboxylase IdeHA.

[0035] Figure 3 This is a schematic diagram of the protein structure of biotin carboxylase accBC.

[0036] Figure 4 This is a schematic diagram of the protein structure of fatty acid peroxygenase cypC;

[0037] Figure 5 Chromatogram of surfactant in wild-type Bacillus subtilis fermentation broth;

[0038] Figure 6 This is a chromatogram of surfactant in the fermentation broth of Bacillus subtilis ZT-3, which produces a high amount of specific surfactants, as provided in Example 3 of the present invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0040] Example 1: A Bacillus subtilis strain that produces a high level of a specific surfactant

[0041] In this embodiment, Bacillus subtilis with the carboxyltransferase accAD mutant was first constructed through steps 1-5, and then strains with high production of specific surfactants were screened through steps 6-8.

[0042] Step 1. Prepare Bacillus subtilis competent cells

[0043] Wild-type Bacillus subtilis was spread on LB solid medium and incubated overnight at 37°C. Single colonies were picked and inoculated into 4 mL of LB liquid medium and incubated at 37°C and 140 rpm for 6 h. The cells were washed three times with sterile 10% glycerol solution, and finally resuspended in 0.2 mL of 10% glycerol and aliquoted for storage at -80°C for later use.

[0044] Step 2. Prepare Bacillus subtilis competent cells with plasmid pHT-XCR6

[0045] Approximately 10 ng of plasmid pHT-XCR6 was added to 0.1 mL of Bacillus subtilis competent cells, and the cells were incubated on ice for 5 min in a pre-chilled electroporation cuvette. Electroporation was performed once at 2.0 kV, followed by recovery and culture in 1 mL of LB medium for 2 h. The cells were then spread onto LB solid medium containing 50 μg / mL chloramphenicol and cultured overnight at 37°C. Competent cells carrying plasmid pHT-XCR6 were prepared using this method and stored at -80°C for later use.

[0046] Step 3. Construct pcrF19NM plasmid

[0047] The pcrF19NM plasmid contains mutation site information for the carboxyltransferase accAD and is inserted between DR2 elements. The crRNA sequences with targeting functions are shown in Table 1.

[0048] Table 1. crRNA sequences edited by the carboxyltransferase accAD gene.

[0049] accAD-T16 GGCGATTTTGGTTTGCAGTTCAATC

[0050] Step 4. Construct homologous fragments

[0051] Homologous fragments were constructed using a complementary primer annealing procedure, and the primer sequences used are shown in Table 2.

[0052] Table 2 Primer sequences for constructing homologous fragments of the carboxyltransferase accAD gene editing.

[0053]

[0054] In this context, "F" represents the upstream primer, "R" represents the downstream primer, and the underlined sequence represents the mutation site.

[0055] The annealing PCR system is shown in Table 3, and the procedure is shown in Table 4.

[0056] Table 3 Annealing PCR System

[0057] 10 pmol / μL upstream primer 5μL 10 pmol / μL downstream primer 5μL

[0058] Table 4 Annealing PCR Procedure

[0059]

[0060] The resulting PCR product is the homologous fragment.

[0061] Step 5. Transform pcrF19NM plasmid and homologous fragment

[0062] Following the experimental procedure in step two above, the pcrF19NM plasmid and homologous fragment were transformed into competent Bacillus subtilis cells carrying plasmid pHT-XCR6, and plated on LB solid medium supplemented with 50 μg / mL chloramphenicol, 50 μg / mL kanamycin, and 3% xylose, and incubated overnight at 30°C. Single colonies that grow can then be used for liquid culture and fermentation verification.

[0063] Step 6. Bacillus subtilis culture and fermentation to produce surfactants

[0064] Forty-eight single colonies of Bacillus subtilis were picked from the plate and inoculated into 10 mL test tubes containing 3 mL of LB liquid medium. The culture was incubated overnight at 37°C and 200 rpm to obtain the seed culture. One mL of the seed culture was added to 30 mL of fermentation medium (20 g / L glucose, 3 g / L tryptone, 3 g / L K₂HPO₄, 10 g / L NaH₂PO₄, 0.02 g / L MgSO₄, 1 g / L L-leucine) and incubated at 37°C and 200 rpm for 48 h.

[0065] Step 7. Detection of the content and composition of surfactants produced by Bacillus subtilis fermentation.

[0066] Centrifuge the Bacillus subtilis fermentation broth, collect 1 ml of supernatant, and centrifuge at 12000 rpm for 3 min. Take 200 μL of the supernatant, add 3-10 times the appropriate amount of methanol, shake thoroughly, and centrifuge at 12000 rpm for 10 min. Filter the supernatant through a membrane; this is the sample to be tested. Use an Agilent 1260 high-performance liquid chromatograph as the detection tool, with an Amethyst C18-H column (5 μm, 250 × 4.6 mm). The mobile phase is 90% methanol and 10% water, with 0.05% trifluoroacetic acid added. The flow rate is 0.8 mL / min. The injection volume is 20 μL. The detection wavelength is 214 nm. Surfactant standards were prepared in gradient concentrations, and a standard curve was constructed to calculate the surfactant content and composition ratio in the fermentation broth sample. Among the 48 Bacillus subtilis strains selected in step 6, the strain with the highest increase in surfactant production in the C15 fraction was designated ZT-1. Figure 5 The chromatogram of surfactant in the fermentation broth of wild-type Bacillus subtilis is shown in Table 5. Table 5 shows the surfactant content and component ratio in the fermentation broth of wild-type Bacillus subtilis and ZT-1.

[0067] Table 5. Surfactant content and component ratio in wild-type Bacillus subtilis and ZT-1 fermentation broth.

[0068] C13 component 35.2％ 28.2％ C14 component 20.8％ 20.5％ C15 component 44.0％ 51.3％ Total output 0.6g / L 1.1g / L

[0069] Step 8. Genome sequencing of the dominant strain

[0070] Genomic DNA of ZT-1 was extracted using a Gram-positive bacteria genomic DNA extraction kit (Beijing Solarbio). The carboxyltransferase accAD gene on the genome was sequenced. The mutant sequence of the ZT-1 carboxyltransferase accAD is shown in SEQ ID No. 5, with the 16th amino acid, threonine, mutated to serine. The location of this mutation site in the 3D structure of the protein is shown in [image missing]. Figure 1 As shown.

[0071] Example 2: A Bacillus subtilis strain that produces a high level of a specific surfactant

[0072] Similar to Example 1, this example first constructs Bacillus subtilis with carboxyltransferase accAD mutant and biotin carboxylase IdeHA and accBC mutant through steps 1-5, and then screens strains that produce high levels of specific surfactants through steps 6-8.

[0073] Step 1. Prepare Bacillus subtilis competent cells

[0074] This step is the same as step 1 of Example 1, except that the wild-type Bacillus subtilis is replaced with the strain ZT-1, which was selected in Example 1 and showed the highest increase in surfactant production from the C15 component.

[0075] Step 2. Prepare Bacillus subtilis competent cells with plasmid pHT-XCR6

[0076] This step is the same as step 2 in Example 1.

[0077] Step 3. Construct pcrF19NM plasmid

[0078] This step is the same as step 3 in Example 1, except that the crRNA sequence is changed to that shown in Table 6.

[0079] Table 6. crRNA sequences edited by biotinylate carboxylase IdeHA and accBC genes.

[0080] IdeHA-K74 CGCGTCGGCTTTTGCTTTTTTC IdeHA-A125 GACACCTGCAGCCTCCATTGC IdeHA-T394 CTGTTCTGGTTTGGCCTTTG accBC-S409 CGAATTCGCTCAATGCGCGC

[0081] Step 4. Construct homologous fragments

[0082] This step is the same as step 4 in Example 1, except that the primer sequences for constructing homologous fragments are changed as shown in Table 7.

[0083] Table 7 Primer sequences for constructing homologous fragments of biotinylate carboxylase IdeHA and accBC gene editing.

[0084]

[0085] Step 5. Transform pcrF19NM plasmid and homologous fragment

[0086] This step is the same as step 5 in Example 1, except that 48 single colonies are picked instead of 192 single colonies.

[0087] Step 6. Bacillus subtilis culture and fermentation to produce surfactants

[0088] This step is the same as step 6 in Example 1.

[0089] Step 7. Detection of the content and composition of surfactants produced by Bacillus subtilis fermentation.

[0090] This step is the same as step 7 in Example 1. Among the 192 Bacillus subtilis strains selected in step 6, the strain with the highest increase in surfactant production in the C15 component was designated as ZT-2. Table 8 shows the surfactant content and component ratio in the fermentation broth of ZT-2.

[0091] Table 8. Surfactant content and component ratio in ZT-2 fermentation broth

[0092] C13 component 19.8％ C14 component 11.3％ C15 component 68.9％ Total output 1.2g / L

[0093] Step 8. Genome sequencing of the dominant strain

[0094] This step is the same as step 8 of Example 1, except that the sequenced genes are the biotinylate carboxylase IdeHA and accBC genes of the ZT-2 genome. The mutation mode of the ZT-2 biotinylate carboxylase IdeHA is K74N / A125T / T394N, as shown in SEQ ID No. 6, and the mutation mode of the biotinylate carboxylase accBC is S409G, as shown in SEQ ID No. 7. The positions of the mutation sites in the 3D structure of the protein are as follows... Figure 2 and 3 As shown.

[0095] Example 3: A Bacillus subtilis strain that produces a high level of a specific surfactant

[0096] Similar to Example 1, this example first constructs Bacillus subtilis with carboxyltransferase accAD mutant, biotin carboxylase IdeHA and accBC mutant and fatty acid peroxygenase cypC mutant through steps 1-5, and then screens strains that produce high levels of specific surfactants through steps 6-8.

[0097] Step 1. Prepare Bacillus subtilis competent cells

[0098] This step is the same as step 1 of Example 1, except that the wild-type Bacillus subtilis is replaced with the strain ZT-2, which was selected in Example 2 and showed the highest increase in surfactant production from the C15 component.

[0099] Step 2. Prepare Bacillus subtilis competent cells with plasmid pHT-XCR6

[0100] This step is the same as step 2 in Example 1.

[0101] Step 3. Construct pcrF19NM plasmid

[0102] This step is the same as step 3 in Example 1, except that the crRNA sequence is changed to that shown in Table 9.

[0103] Table 9. crRNA sequence of fatty acid peroxygenase cypC gene editing

[0104]

[0105]

[0106] Step 4. Construct homologous fragments

[0107] This step is the same as step 4 in Example 1, except that the primer sequences for constructing homologous fragments are changed as shown in Table 10.

[0108] Table 10 Primer sequences for constructing homologous fragments of fatty acid peroxygenase cypC gene editing.

[0109]

[0110] Step 5. Transform pcrF19NM plasmid and homologous fragment

[0111] This step is the same as step 5 in Example 1, except that 48 single colonies are picked instead of 96 single colonies.

[0112] Step 6. Bacillus subtilis culture and fermentation to produce surfactants

[0113] This step is the same as step 6 in Example 1.

[0114] Step 7. Detection of the content and composition of surfactants produced by Bacillus subtilis fermentation.

[0115] This step is the same as step 7 in Example 1. Among the 96 Bacillus subtilis strains selected in step 6, the strain with the highest increase in surfactant production of the C15 component is designated as ZT-3. Figure 6The chromatogram of surfactant in ZT-3 fermentation broth is shown in Table 11. Table 11 shows the surfactant content and component ratio in wild-type Bacillus subtilis and ZT-3 fermentation broth.

[0116] Table 11 Surfactant content and component ratio in ZT-3 fermentation broth

[0117]

[0118]

[0119] Step 8. Genome sequencing of the dominant strain

[0120] This step is the same as step 8 in Example 1, except that the sequenced gene is the fatty acid peroxygenase cypC gene of the ZT-3 genome. The ZT-3 fatty acid peroxygenase cypC mutation mode is S162P / L270V / L298P, and the sequence is shown in SEQ ID No. 8. The location of the mutation site in the 3D structure of the protein is as follows: Figure 4 As shown.

[0121] As shown in Tables 5, 8, and 11, the surfactant production of the ZT-1, ZT-2, and ZT-3 strains constructed in Examples 1-3 was significantly increased compared to that of wild-type Bacillus subtilis, with the highest increase being 3.5 times. The surfactant proportion of the C15 component increased from 44.0% to 72.5%, becoming the main product, demonstrating that the Bacillus subtilis strain provided by this invention effectively increased the surfactant production of the C15 component.

Claims

1. A Bacillus subtilis strain comprising carboxyltransferase accAD, biotinylate carboxylase IdeHA, biotinylate carboxylase accBC, and fatty acid peroxygenase cypC, the amino acid sequences of which are shown in SEQ ID NO. 1-4 in sequence; characterized in that: The 16th threonine in the carboxyltransferase accAD is mutated to serine; the 74th lysine in the biotinylate carboxylase IdeHA is mutated to asparagine, the 125th alanine is mutated to threonine, and the 394th threonine is mutated to asparagine; the 409th serine in the biotinylate carboxylase accBC is mutated to glycine; and the 162nd serine in the fatty acid peroxygenase cypC is mutated to proline, the 270th leucine is mutated to valine, and the 298th leucine is mutated to proline.

2. The application of Bacillus subtilis as described in claim 1 in the production of surfactants.

3. Use according to claim 2, characterized in that, The surfactant is a C15 component surfactant.

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