CN119662507A - Bacillus subtilis with high surfactant yield, related enzyme mutant and preparation method thereof - Google Patents

Abstract

The invention discloses a high-yield surfactin Bacillus subtilis, a related enzyme mutant and a preparation method thereof. The Bacillus subtilis is obtained by a genetic modification method and can produce a high yield of surfactin, up to 9.4 g/L. The invention also provides key enzyme mutants involved in the synthesis of surfactin in the Bacillus subtilis, including a biotin carboxylase IdeHA mutant, a biotin carboxylase accBC mutant, an enoyl reductase fabI mutant, a malonyl transferase fabD mutant and a dihydrolipoyl dehydrogenase IPdU mutant. The high-yield surfactin Bacillus subtilis and a preparation method thereof disclosed by the invention are helpful to realize the industrial production and commercial application of surfactin.

Description

Bacillus subtilis with high surfactant yield, related enzyme mutant and preparation method thereof

Technical Field

The invention relates to a microorganism and a preparation method thereof, in particular to bacillus subtilis and a preparation method thereof, and belongs to the field of genetic engineering.

Background

The bacillus subtilis belongs to bacillus, has the advantages of high growth speed, low requirement on nutrition, high-efficiency secretion of a plurality of proteins and metabolites, no toxin generation and the like, is a safe microorganism without pathogenicity, and is widely applied to the fields of aquaculture, plant disease resistance, feed production, medicine and health and the like.

The surfactant is a natural surfactant, is mainly synthesized by bacillus and widely exists in nature. The water-based emulsion is mainly composed of a cyclic heptapeptide and a beta-hydroxy fatty acid chain, has hydrophilic and hydrophobic groups, can obviously reduce the surface and interfacial tension of liquid, and can enable water and oil to be mixed or emulsified better. Compared with the traditional chemical synthesis surfactant, the biosurfactant has the advantages of being ecological, high in safety and the like, is applied to various fields, for example, the environment field, the medicine and agriculture field, and plays roles of resisting bacteria, viruses and tumors by using the biosurfactant to clean leaked petroleum and organic pollutants in soil. The surfactant has great application potential in the fields of industry, environment, medicine and the like. However, the production of the surfactant is mainly dependent on microbial fermentation at present, and the existing yield is limited, so that the popularization and application of the surfactant are greatly limited.

Methods for improving the ability of microorganisms to produce surfactants are numerous, including optimization of culture conditions, genetic engineering, optimization of production processes, and the like. Genetic engineering methods include enhancing the expression of key enzymes, such as by genetically engineering means to regulate the amount of expression of enzymes associated with the production of surfactant. Patent application CN117187286A discloses a method for improving the production of bacillus subtilis surfactant by over-expressing SecA, and the method can obviously improve the yield of the surfactant by over-expressing a proprotein transposase secA gene of a surfactant product secretion pathway in bacillus subtilis genome by a genetic engineering means, so as to strengthen the transmembrane efflux of the surfactant. The yield of the surface active element of the genetically engineered bacterium obtained by the method can reach 9.9g/L, but the wild strain used by the genetically engineered bacterium is limited to a specific strain Bacillus subtilisATCC 21332, and the yield of the original surface active element can reach 6.83g/L. Patent application CN117946952A discloses a high-yield surface active element engineering bacterium, a construction method and application thereof, and uses a genetic engineering means to knock out srfA synthetase negative regulatory factors CodY and PhoP genes in a bacillus subtilis genome, strengthen srfA synthetase expression and can remarkably improve the yield of surface active elements. However, after 48 hours of fermentation, the genetically engineered bacteria obtained by the method have the highest yield of the surfactant of only 4.25g/L. In summary, the current bacillus subtilis capable of producing high-yield surfactant is very limited and has strict preparation conditions.

Disclosure of Invention

The invention aims to provide bacillus subtilis capable of producing surface active substances at high yield, a related enzyme mutant participating in synthesizing the surface active substances in the bacillus subtilis, and a preparation method and application of the bacillus subtilis.

The invention provides bacillus subtilis with high yield of surface active elements, which comprises biotin carboxylase IdeHA, biotin carboxylase accBC, enoyl reductase fabI, malonyl transferase fabD and dihydrolipoyl dehydrogenase IPdU, wherein the amino acid sequences of the bacillus subtilis are shown as SEQ ID NO.1-5 in sequence;

the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, and the 394 th threonine is mutated to asparagine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, and the 409 th serine of the biotin carboxylase accBC is mutated to glycine;

or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, the 409 th serine of the biotin carboxylase accBC is mutated to glycine, and the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, and the 409 th serine of the biotin carboxylase accBC is mutated to glycine, and the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine, and the 64 th proline of the malonyl transferase fabD is mutated to arginine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, the 409 th serine of the biotin carboxylase accBC is mutated to glycine, the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine, the 64 th proline of the malonyl transferase fabD is mutated to arginine, the 163 th valine of the dihydrolipoyl dehydrogenase IPdU is mutated to alanine, and the 287 th glycine is mutated to serine.

According to the invention, bacillus subtilis (Bacillus subtilis) is taken as a chassis cell, 3D modeling is carried out on 5 enzymes involved in surfactant synthesis, key amino acids around an active center or at loop positions on the surface of protein are selected for site-directed mutagenesis, and finally 5 strains with high yield of surfactant are selected from the bacillus subtilis transformed by hundreds of strains. The 5 enzymes are respectively biotin carboxylase IdeHA with the sequence of SEQ ID No.1, biotin carboxylase accBC with the sequence of SEQ ID No.2, enoyl reductase fabI with the sequence of SEQ ID No.3, malonyl transferase fabD with the sequence of SEQ ID No.4 and dihydrolipoyl dehydrogenase IPdU with the sequence of SEQ ID No. 5. The invention improves the efficiency of fatty acid synthesis by genetically engineering the key enzyme participating in the synthesis of the surfactant in the bacillus subtilis, and provides the bacillus subtilis with high yield of the surfactant.

The invention also provides a preparation method of the bacillus subtilis, which comprises the following steps:

step 1, preparing bacillus subtilis competent cells;

Step 2, transforming a first recombinant plasmid into the competent cells obtained in the step 1 to obtain recombinant cells, wherein the first recombinant plasmid can express a protein with DNA double-strand excision activity;

Step3, constructing a second recombinant plasmid, wherein the second recombinant plasmid contains the information of the mutation in claim 1;

step 4, constructing a homologous fragment, wherein the homologous fragment contains a primer sequence;

And 5, transforming the second recombinant plasmid and the homologous fragment into the recombinant cell obtained in the step 2 to obtain the bacillus subtilis of claim 1.

The present invention uses CRISPR technology to carry out gene editing on the enzyme in bacillus subtilis, wherein a crRNA sequence is inserted into pcrF NM plasmid, and the sequence contains mutation site information of the enzyme. The pHT-XCR6 plasmid with Cpf1 gene and the pcrF NM plasmid constructed as well as the homologous sequence as the template of gene recombination are transformed into competent cells of Bacillus subtilis in sequence. After eliminating the plasmid, fermenting and detecting the surfactant, and finally screening out the bacillus subtilis with high yield of the surfactant.

Preferably, in step 2, the first recombinant plasmid is a pHT-XCR6 plasmid.

Preferably, in step 3, the second recombinant plasmid is pcrF NM plasmid.

Further, in step 2, the protein having a double strand cleavage activity of DNA is Cpf1. In step3, the second recombinant plasmid comprises a crRNA sequence.

Further, the crRNA sequence is:

CrRNA name	Sequence (5 '-3')
		IdeHA-K74	GCGAAAAAAGCAAAAGCCGACGCG
IdeHA-A125	GCAATGGAGGCTGCAGGTGTC
		IdeHA-E136	GGCGTTTCTGAATCCCTCGGA
IdeHA-C145	GAGGCAGCCTGCCGCACCGCA
		IdeHA-T394	AAAGGCCAAACCAGAACAGAA
accBC-S409	CGCGCATTGAGCGAATTCGTC
		fabI-G263	GTTGATTCTGGTTTCCATATC
fabD-P64	AATGCGCAGCCTGCTTTGCTT
		IPdU-V163	GGTCTTGAAGTGGACGGTAAG
IPdU-G287	AATATCGAAGGCATCGGCCTA

Further, in step 4, the primer sequence is:

the invention also provides a biotinylase IdeHA mutant of the bacillus subtilis, and the amino acid sequence of the biotinylase IdeHA mutant is shown as SEQ ID NO. 6.

The invention also provides a biotinylase accBC mutant of the bacillus subtilis, and the amino acid sequence of the mutant is shown as SEQ ID NO. 7.

The invention also provides an enoyl reductase fabI mutant of the bacillus subtilis, and the amino acid sequence of the enoyl reductase fabI mutant is shown as SEQ ID NO. 8.

The invention also provides a malonyl transferase fabD mutant of the bacillus subtilis, and the amino acid sequence of the malonyl transferase fabD mutant is shown as SEQ ID NO. 9.

The invention also provides a dihydrolipoyl dehydrogenase IPdU mutant of the bacillus subtilis, and the amino acid sequence of the dihydrolipoyl dehydrogenase IPdU mutant is shown as SEQ ID NO. 10.

The invention also provides application of the bacillus subtilis in production of the surfactant.

Compared with the prior art, the bacillus subtilis provided by the invention has the remarkable advantages that 1, the bacillus subtilis can produce the surfactant with high yield of 9.4g/L at most, the preparation method and the application of the bacillus subtilis are beneficial to realizing industrial production and commercial application of the surfactant, and 2, the mutant of the biotin carboxylase IdeHA, the mutant of the biotin carboxylase accBC, the mutant of the enoyl reductase fabI, the fabD mutant of the malonyl transferase and the IPdU mutant of the dihydrolipoyl dehydrogenase are beneficial to the high yield of the surfactant.

Drawings

FIG. 1 is a schematic diagram of the protein structure of biotin carboxylase IdeHA;

FIG. 2 is a schematic representation of the protein structure of the biotinylase accBC;

FIG. 3 is a schematic diagram of the protein structure of enoyl reductase fabI;

FIG. 4 is a schematic diagram showing the protein structure of malonyl transferase fabD;

FIG. 5 is a schematic diagram showing the protein structure of dihydrolipoyl dehydrogenase IPdU;

FIG. 6 is a chromatogram of a surfactant in a fermentation broth of Bacillus subtilis CY-5 provided in example 5 of the invention.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

The wild type Bacillus subtilis, plasmid pHT-XCR6 and pcrF NM plasmids used in the examples were all stored in the laboratory. Other materials are commercially available unless specified.

Example 1 Bacillus subtilis for high production of surfactant

In the embodiment, bacillus subtilis with biotin carboxylase IdeHA mutant is constructed by the preparation method in the step 1-5, and then the strain with high-yield surfactant is screened out in the step 6-8.

Step 1, preparing bacillus subtilis competent cells

Wild-type bacillus subtilis was spread in LB solid medium and cultured overnight at 37 ℃. Single colonies were picked and inoculated in 4mL LB liquid medium, cultured at 37℃and 140rpm for 6h, the cells were washed 3 times with sterile 10% glycerol solution, and finally the cells were resuspended with 0.2mL 10% glycerol and sub-packaged for storage in a-80℃refrigerator for use.

Step2, preparing bacillus subtilis competent cells with plasmid pHT-XCR6

About 10ng of plasmid pHT-XCR6 was added to 0.1mL of competent cells of Bacillus subtilis, and the mixture was subjected to ice-bath for 5min in a precooled electric cuvette and 1 time of 2.0kv electric shock, and resuscitated and cultured in 1mL of LB medium for 2 hours, spread on LB solid medium containing 50ug/mL of chloramphenicol, and cultured overnight at 37 ℃. The bacillus subtilis competent cells with plasmid pHT-XCR6 were obtained and stored in a-80℃refrigerator for use.

Step 3 construction pcrF NM plasmid

PcrF19NM plasmid contains the mutation site information of biotin carboxylase IdeHA and is inserted between DR2 elements. Wherein the crRNA sequence with targeting function is shown in Table 1.

TABLE 1 crRNA sequence for the Gene editing of Biotin carboxylase IdeHA

CrRNA name	Sequence (5 '-3')
		IdeHA-K74	GCGAAAAAAGCAAAAGCCGACGCG
IdeHA-A125	GCAATGGAGGCTGCAGGTGTC
		IdeHA-E136	GGCGTTTCTGAATCCCTCGGA
IdeHA-C145	GAGGCAGCCTGCCGCACCGCA
		IdeHA-T394	AAAGGCCAAACCAGAACAGAA

Step 4, constructing homologous fragments

Construction of homologous fragments was constructed using a complementary primer annealing procedure, with the primer sequences shown in Table 2.

TABLE 2 primer sequences constructed from homologous fragments edited by the Gene of Biotin carboxylase IdeHA

Primer name	Sequence (5 '-3')
		IdeHA-K74-F	AAAAAAGCAAACGCCGACGCGATCCACCCGGGATATG
IdeHA-K74-R	CGCGTCGGCGTTTGCTTTTTTCGCCGTCTTTATG
		IdeHA-A125-F	GCAATGGAGACGGCAGGTGTCCCTGTGGTGCCGGGC
IdeHA-A125-R	GACACCTGCCGTCTCCATTGCTTTTCGCGCTTCAATTTTG
		IdeHA-E136-F	GGCGTTTCTNNKTCCCTCGGAGATATAGAGGCAGCC
IdeHA-E136-R	TCCGAGGGAMNNAGAAACGCCCGGCACCACAGGGAC
		IdeHA-C145-F	GAGGCAGCCNNKCGCACCGCAAGTCAAATCGGCTATC
IdeHA-C145-R	TGCGGTGCGMNNGGCTGCCTCTATATCTCCGAGGG
		IdeHA-T394-F	AAAGGCCAANNKAGAACAGAAGCAATTGAAAAACTAG
IdeHA-T394-R	TTCTGTTCTMNNTTGGCCTTTGACAATCATCTTAG

Wherein "F" represents the upstream primer and "R" represents the downstream primer, and the underlined sequence is the mutation site.

The system of annealing PCR is shown in Table 3, and the procedure is shown in Table 4.

TABLE 3 annealing PCR System

Composition of components	Volume of
		10 Pmol/. Mu.L of upstream primer	5μL
10 Pmol/. Mu.L downstream primer	5μL

TABLE 4 annealing PCR procedure

The PCR product is the homologous fragment.

Step 5. Transformation pcrF of the NM plasmid and homologous fragment

The pcrF NM plasmid and homologous fragments were transformed into competent cells of Bacillus subtilis with plasmid pHT-XCR6 according to the second experimental procedure described above and plated on LB solid medium supplemented with 50ug/mL chloramphenicol, 50ug/mL kanamycin and 3% xylose, and cultured overnight at 30 ℃. The single colony can be used for liquid culture and fermentation verification.

Step 6, culturing and fermenting bacillus subtilis to produce the surfactant

From the plate, 48 single colonies of Bacillus subtilis were picked, inoculated into 10mL test tubes containing 3mL LB liquid medium, and cultured overnight at 37℃with a shaker at 200rpm, respectively, as primary seed solutions. 1mL of the primary seed solution was added to a 250mL Erlenmeyer flask containing 30mL of LB liquid medium, and cultured in a shaker at 37℃and 200rpm for 16 hours, as a secondary seed solution. 5mL of the secondary seed solution was added to a 2L conical flask containing 1L of a fermentation medium, and the mixture was cultured in a shaking table at 37℃and 200rpm for 48 hours, wherein the 1L fermentation medium had the components of 40g of soluble starch, 7g of sodium nitrate, L g g of monopotassium phosphate, 1g of yeast extract, 0.5g of magnesium sulfate, 0.5g of potassium chloride, 0.28g of ferrous sulfate heptahydrate, 1.7mg of manganese sulfate and 0.16mg of copper sulfate pentahydrate.

Step 7, the content of the surfactant produced by fermenting the bacillus subtilis

The Bacillus subtilis broth was centrifuged, 1mL of supernatant was taken, and centrifuged at 12000rpm for 3min. Taking 200uL of supernatant, using methanol for proper 3-10 times, fully oscillating, centrifuging at 12000rpm for 10min, and obtaining the sample to be detected after the supernatant passes through a membrane. Agilent 1260 high performance liquid chromatography was used as detection tool, using a column of AMETHYST C-Hcolumn (5 μm, 250X 4.6 mm). The mobile phase used was 90% methanol and 10% water, and 0.05% trifluoroacetic acid was additionally added at a flow rate of 0.8mL/min. The sample injection amount is 20ul. The detection wavelength was 214nm. The surfactant standard was formulated as a gradient concentration and a standard curve was made to calculate the surfactant content in the broth sample. Among the 48 Bacillus subtilis strains selected in the step 6, the strain with the most improved surfactant yield was designated CY-1, and the surfactant yields are shown in Table 13.

Step 8, dominant strain genome sequencing

The genome DNA of CY-1 is extracted by using a gram positive bacterium genome DNA extraction kit (Beijing Soy Bao), the gene sequencing of biotin carboxylase IdeHA on the genome is carried out, the sequence of a mutant of biotin carboxylase IdeHA of CY-1 is shown as SEQ ID No.6, the mutation of lysine at 74 is asparagine, the mutation of alanine at 125 is threonine, the mutation of glutamic acid at 136 is lysine, the mutation of cysteine at 145 is phenylalanine, and the mutation of threonine at 394 is asparagine. The position of the mutation site in the 3D structure of the enzyme is shown in FIG. 1.

Example 2 Bacillus subtilis for high production of surfactant

Similar to example 1, in this example, bacillus subtilis with biotin carboxylase IdeHA mutant and biotin carboxylase accBC mutant was first constructed by the preparation method of steps 1-5, and then strains with high-yield surfactant were screened by steps 6-8.

Step 1, preparing bacillus subtilis competent cells

This procedure is substantially the same as in step 1 of example 1, except that the wild-type Bacillus subtilis used in example 1 is changed to the high-yielding strain CY-1 selected in example 1.

Step2, preparing bacillus subtilis competent cells with plasmid pHT-XCR6

This step is the same as step 2 of example 1.

Step 3 construction pcrF NM plasmid

This procedure is essentially the same as in step 3 of example 1, except that the crRNA sequence is changed to that shown in Table 5.

TABLE 5 crRNA sequence edited by the biotinylase accBC Gene

CrRNA name	Sequence (5 '-3')
		accBC-S409	CGCGCATTGAGCGAATTCGTC

Step 4, constructing homologous fragments

This procedure was substantially the same as in step 4 of example 1, except that the homologous fragment constructing primer sequences were changed as shown in Table 6.

TABLE 6 construction of primer sequences for homologous fragments edited by the ACcBC Gene of biotinylase

Primer name	Sequence (5 '-3')
		accBC-S409-F	CGCGCATTGGGCGAATTCGTCATCGAAGGCATTGAG
accBC-S409-R	GACGAATTCGCCCAATGCGCGCTTCATGCGGGCAATC

Step 5. Transformation pcrF of the NM plasmid and homologous fragment

This procedure was essentially the same as in step 5 of example 1, except that the selection of 48 single colonies was changed to 192 single colonies.

Step 6, culturing and fermenting bacillus subtilis to produce the surfactant

This step is the same as step 6 of example 1.

Step 7, the content of the surfactant produced by fermenting the bacillus subtilis

This step is the same as step 7 of example 1. Among 192 Bacillus subtilis strains selected in the step 6, the strain with the most improved surfactant yield was designated CY-2, and the surfactant yield is shown in Table 13.

Step 8, dominant strain genome sequencing

This step is essentially the same as step 8 of example 1, except that the biotin carboxylase accBC gene of the CY-2 genome is sequenced. The mutation mode of the biotin carboxylase accBC of CY-2 is that serine at 409 th site is mutated into glycine, and the sequence is shown as SEQ ID No. 7. The position of the mutation site in the 3D structure of the protein is shown in fig. 2.

Example 3 Bacillus subtilis for high production of surfactant

Similar to example 1, in this example, bacillus subtilis with biotin carboxylase IdeHA, accBC mutant and enoyl reductase fabI mutant is constructed by the preparation method of steps 1-5, and then the strain with high-yield surfactant is screened by steps 6-8.

Step 1, preparing bacillus subtilis competent cells

This procedure is substantially the same as in step 1 of example 1, except that the wild-type Bacillus subtilis is changed to the high-yielding strain CY-2 selected in example 2.

Step2, preparing bacillus subtilis competent cells with plasmid pHT-XCR6

This step is the same as step 2 of example 1.

Step 3 construction pcrF NM plasmid

This step is essentially the same as step 3 of example 1, except that the crRNA sequence is changed to that shown in table 7.

TABLE 7 Enyl reductase fabI Gene edited crRNA sequences

CrRNA name	Sequence (5 '-3')
		fabI-G263	GTTGATTCTGGTTTCCATATC

Step 4, constructing homologous fragments

This procedure was substantially the same as in step 4 of example 1, except that the homologous fragment constructing primer sequences were changed as shown in Table 8.

TABLE 8 construction of primer sequences for homologous fragments for the gene editing of enoyl reductase fabI

Primer name	Sequence (5 '-3')
		fabI-G263-F	GTTGATTCTNNKTTCCATATCACTGCCCGCTAAG
fabI-G263-R	GATATGGAAMNNAGAATCAACGTGAAGATTTTCACC

Step 5. Transformation pcrF of the NM plasmid and homologous fragment

This procedure was essentially the same as in step 5 of example 1, except that the selection of 48 single colonies was changed to 96 single colonies.

Step 6, culturing and fermenting bacillus subtilis to produce the surfactant

This step is the same as step 6 of example 1.

Step 7, the content of the surfactant produced by fermenting the bacillus subtilis

This step is the same as step 7 of example 1. Among 96 Bacillus subtilis strains selected in the step 6, the strain with the most improved surfactant yield was designated CY-3, and the surfactant yield is shown in Table 13.

Step 8, dominant strain genome sequencing

This procedure is essentially the same as in step 8 of example 1, except that the gene for the enoyl reductase fabI of the CY-2 genome is sequenced. The mutation mode of the CY-3 enoyl reductase fabI is that the 263 rd glycine is mutated into cysteine, and the sequence is shown as SEQ ID No. 8. The position of the mutation site in the 3D structure of the protein is shown in fig. 3.

Example 4 Bacillus subtilis for high production of surfactant

Similar to example 1, in this example, bacillus subtilis with biotin carboxylase IdeHA, accBC mutant, enoyl reductase fabI mutant and malonyl transferase fabD is constructed by the preparation method of steps 1-5, and then the strain with high yield of surfactant is screened by steps 6-8.

Step 1, preparing bacillus subtilis competent cells

This procedure is substantially the same as in step 1 of example 1, except that the wild-type Bacillus subtilis is changed to example 3 to screen out the strain CY-3 having the most improved production of surfactant.

Step2, preparing bacillus subtilis competent cells with plasmid pHT-XCR6

This step is the same as step 2 of example 1.

Step 3 construction pcrF NM plasmid

This step is essentially the same as step 3 of example 1, except that the crRNA sequence is changed to that shown in table 9.

TABLE 9 crRNA sequence for malonyl transferase fabD Gene editing

CrRNA name	Sequence (5 '-3')
		fabD-P64	AATGCGCAGCCTGCTTTGCTT

Step 4, constructing homologous fragments

This procedure was substantially the same as in step 4 of example 1, except that the homologous fragment constructing primer sequences were changed as shown in Table 10.

TABLE 10 construction of primer sequences for homologous fragments edited by malonyl transferase fabD Gene

Primer name	Sequence (5 '-3')
		fabD-P64-F	AATGCGCAGNNKGCTTTGCTTACGACAAGCATCGCTG
fabD-P64-R	AAGCAAAGCMNNCTGCGCATTGTATGTAAGTGTTAATTC

Step 5. Transformation pcrF of the NM plasmid and homologous fragment

This procedure was essentially the same as in step 5 of example 1, except that the selection of 48 single colonies was changed to 96 single colonies.

Step 6, culturing and fermenting bacillus subtilis to produce the surfactant

This step is the same as step 6 of example 1.

Step 7, the content of the surfactant produced by fermenting the bacillus subtilis

This step is the same as step 7 of example 1. Among 96 Bacillus subtilis strains selected in the step 6, the strain with the most improved surfactant yield was designated CY-4, and the surfactant yield is shown in Table 13.

Step 8, dominant strain genome sequencing

This procedure is essentially the same as in step 8 of example 1, except that the malonyl transferase fabD gene of the CY-2 genome is sequenced. The mutation mode of the malonyl transferase fabD of CY-4 is that the 64 th amino acid proline is mutated into arginine, and the sequence is shown as SEQ ID No. 9. The position of the mutation site in the 3D structure of the protein is shown in fig. 4.

Example 5 Bacillus subtilis for high production of surfactant

Similar to example 1, in this example, bacillus subtilis with biotin carboxylase IdeHA, accBC mutant, enoyl reductase fabI mutant, malonyl transferase fabD and dihydrolipoyl dehydrogenase IPdU mutant is constructed by the preparation method of steps 1-5, and then the strain with high yield of surfactant is screened by steps 6-8.

Step 1, preparing bacillus subtilis competent cells

This procedure is substantially the same as in step 1 of example 1, except that the wild-type Bacillus subtilis is changed to example 4 to screen out the strain CY-4 having the most improved production of surfactant.

Step2, preparing bacillus subtilis competent cells with plasmid pHT-XCR6

This step is the same as step 2 of example 1.

Step 3 construction pcrF NM plasmid

This step is essentially the same as step 3 of example 1, except that the crRNA sequence is changed to that shown in table 11.

TABLE 11 crRNA sequence edited by the dihydrolipoyl dehydrogenase IPdU Gene

CrRNA name	Sequence (5 '-3')
		IPdU-V163	GGTCTTGAAGTGGACGGTAAG
IPdU-G287	AATATCGAAGGCATCGGCCTA

Step 4, constructing homologous fragments

This procedure was substantially the same as in step 4 of example 1, except that the homologous fragment constructing primer sequences were changed as shown in Table 12.

TABLE 12 construction of primer sequences for homologous fragments edited by the dihydrolipoyl dehydrogenase IPdU Gene

Primer name	Sequence (5 '-3')
		IPdU-V163-F	GGTCTTGAANNKGACGGTAAGTCTGTACTGACTTCAGATG
IPdU-V163-R	CTTACCGTCMNNTTCAAGACCCGGAAGCATTCTCGGTCTTG
		IPdU-G287-F	AATATCGAANNKATCGGCCTAGAGAACACCGATATTG
IPdU-G287-R	TAGGCCGATMNNTTCGATATTTGCCTGTCTGCCGATG

Step 5. Transformation pcrF of the NM plasmid and homologous fragment

This procedure was essentially the same as in step 5 of example 1, except that the selection of 48 single colonies was changed to 96 single colonies.

Step 6, culturing and fermenting bacillus subtilis to produce the surfactant

This step is the same as step 6 of example 1.

Step 7, the content of the surfactant produced by fermenting the bacillus subtilis

This step is the same as step 7 of example 1. Among 96 Bacillus subtilis strains selected in the step 6, the strain with the most improved surfactant yield was designated CY-5, and the surfactant yield is shown in Table 13. FIG. 6 is a chromatogram of a surfactant in a standard and CY-5 broth.

Step 8, dominant strain genome sequencing

This procedure is essentially the same as in step 8 of example 1, except that the dihydrolipoyl dehydrogenase IPdU gene of the CY-2 genome is sequenced. The mutation mode of the dihydrolipoyl dehydrogenase IPdU of CY-5 is that the amino acid valine at 163 is mutated into alanine, the amino acid glycine at 287 is mutated into serine, the sequence is shown as SEQ ID No.10, and the position of the mutation site in the 3D structure of the protein is shown as figure 5.

The surfactant content of wild type Bacillus subtilis and CY-1, CY-2, CY-3, CY-4, CY-5 strains are shown in Table 13. Compared with wild type bacillus subtilis, the 5 bacillus subtilis has obviously improved surfactant yield which is improved by 15.7 times at most.

TABLE 13 production of surface active substances by different strains of Bacillus subtilis

Claims (10)

1. The bacillus subtilis comprises biotin carboxylase IdeHA, biotin carboxylase accBC, enoyl reductase fabI, malonyl transferase fabD and dihydrolipoyl dehydrogenase IPdU, and the amino acid sequences of the bacillus subtilis are shown as SEQ ID NO.1-5 in sequence, and the bacillus subtilis is characterized in that:

the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, and the 394 th threonine is mutated to asparagine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, and the 409 th serine of the biotin carboxylase accBC is mutated to glycine;

or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, the 409 th serine of the biotin carboxylase accBC is mutated to glycine, and the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, and the 409 th serine of the biotin carboxylase accBC is mutated to glycine, and the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine, and the 64 th proline of the malonyl transferase fabD is mutated to arginine;

Or the 74 th lysine of the biotin carboxylase IdeHA is mutated to asparagine, the 125 th alanine is mutated to threonine, the 136 th glutamic acid is mutated to lysine, the 145 th cysteine is mutated to phenylalanine, the 394 th threonine is mutated to asparagine, the 409 th serine of the biotin carboxylase accBC is mutated to glycine, the 263 rd glycine of the enoyl reductase fabI is mutated to cysteine, the 64 th proline of the malonyl transferase fabD is mutated to arginine, the 163 th valine of the dihydrolipoyl dehydrogenase IPdU is mutated to alanine, and the 287 th glycine is mutated to serine.

2. A method for producing bacillus subtilis according to claim 1, comprising the steps of:

step 1, preparing bacillus subtilis competent cells;

Step 2, transforming a first recombinant plasmid into the competent cells obtained in the step 1 to obtain recombinant cells, wherein the first recombinant plasmid can express a protein with DNA double-strand excision activity;

Step3, constructing a second recombinant plasmid, wherein the second recombinant plasmid contains the information of the mutation in claim 1;

step 4, constructing a homologous fragment, wherein the homologous fragment contains a primer sequence;

And 5, transforming the second recombinant plasmid and the homologous fragment into the recombinant cell obtained in the step 2 to obtain the bacillus subtilis of claim 1.

3. The method according to claim 2, wherein in step 2, the first recombinant plasmid is a pHT-XCR6 plasmid.

4. The method of claim 2, wherein in step 3, the second recombinant plasmid is pcrF NM plasmid.

5. A mutant biotinylase IdeHA of bacillus subtilis according to claim 1, characterized in that it has the amino acid sequence shown in SEQ ID No. 6.

6. A mutant of the biotinylase accBC of bacillus subtilis according to claim 1, wherein the amino acid sequence is shown in SEQ ID No. 7.

7. A mutant of the enoyl reductase fabI of bacillus subtilis according to claim 1, characterized in that the amino acid sequence is shown in SEQ ID No. 8.

8. A malonyl transferase fabD mutant of bacillus subtilis according to claim 1, characterized in that the amino acid sequence is shown in SEQ ID No. 9.

9. A mutant of the dihydrolipoyl dehydrogenase IPdU of bacillus subtilis according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID No. 10.

10. Use of the bacillus subtilis of claim 1 for the production of a surfactant.