Method and use for improving the production of surfactant by Bacillus subtilis

Abstract

Provided are a method and use for improving the production of a surfactant by Bacillus subtilis. 【Solution means】It includes transforming the wild-type biotin carboxylase IdeHA of Bacillus subtilis into a specific amino acid sequence, and can effectively improve the production amount of the surfactant. It may further include transforming the wild-type biotin carboxylase accBC, further include transforming the wild-type enoyl reductase fabI, and further include transforming the wild-type malonyl transferase fabD.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method and use for improving the production of surfactants by Bacillus subtilis.

Background Art

[0002] Surfactin is a kind of natural surfactant mainly synthesized by Bacillus species and widely exists in nature. Surfactin is mainly composed of a cyclic heptapeptide having a hydrophilic group and a hydrophobic group and a β-hydroxy fatty acid chain, which significantly reduces the surface tension and interfacial tension of liquids and enables better mixing and emulsification of water and oil. Compared with conventional chemically synthesized surfactants, biosurfactants have various advantages such as being environmentally friendly and highly safe. For example, they can be used in environmental fields such as the purification of organic pollutants in oil spills and soil, and can also be applied to various fields such as the pharmaceutical and agricultural fields where antibacterial, antiviral, and antitumor effects are exerted. Surfactin shows great potential in applications in industry, the environment, and pharmaceuticals. However, the current production of surfactin mainly depends on microbial fermentation. Since the existing microorganisms have limited production of surfactin, the popularization and application of surfactin are greatly restricted.

Summary of the Invention

[0003] The present invention provides a method and use for improving the production of surfactants by Bacillus subtilis. By transforming Bacillus subtilis, the transformed Bacillus subtilis can improve the production of surfactants. To achieve the above object, the present invention adopts the following technical solutions: A method for improving the production of surfactants by Bacillus subtilis, the method comprising: the wild type of Bacillus subtilis​​​​​​​​​​​​ comprising transforming biotin carboxylase IdeHA, and after transformation, the biotin The amino acid sequence of carboxylase IdeHA is shown in SEQ ID NO: 1. The biotin carboxylase IdeHA after the above transformation is obtained based on the wild-type biotin carboxylase IdeHA mutation. The amino acid sequence of wild-type biotin carboxylase IdeHA is shown in SEQ ID NO: 5. Specifically, lysine at position 74 of wild-type biotin carboxylase IdeHA is mutated to asparagine, alanine at position 125 is mutated to threonine, glutamate at position 136 is mutated to lysine, cysteine at position 145 is mutated to phenylalanine, and threonine at position 394 is mutated to asparagine to obtain the biotin carboxylase IdeHA after transformation. Furthermore, this method further comprises transforming wild-type biotin carboxylase accBC of Bacillus subtilis and the amino acid sequence of biotin carboxylase accBC after transformation is shown in SEQ ID NO: 2. The biotin carboxylase accBC after the above transformation is obtained based on the wild-type biotin carboxylase accBC mutation. The amino acid sequence of wild-type biotin carboxylase accBC is shown in SEQ ID NO: 6. Specifically, serine at position 409 of wild-type biotin carboxylase accBC is mutated to glycine to obtain biotin carboxylase accBC after transformation. Furthermore, this method further comprises transforming wild-type enoyl reductase fabI of Bacillus subtilis and the amino acid sequence of enoyl reductase fabI after transformation is shown in SEQ ID NO: 3. The enoyl reductase fabI after the above transformation is obtained based on the mutation of wild-type enoyl reductase fab I. The amino acid sequence of wild-type enoyl reductase fabI is SE Q shown in ID NO:7. Specifically, glycine at the 2 63rd position of wild-type enoyl reductase fabI is mutated to cysteine to obtain enoyl reductase fabI after the transformation. Furthermore, this method further includes transforming wild-type malonyl transferase fabD of Bacillus subtilis , and the amino acid sequence of malonyl transferase fabD after the transformation is shown in SEQ ID NO:4. The malonyl transferase fabD after the above transformation is obtained based on the mutation of wild-type malonyl transfer lase fabD. The amino acid sequence of wild-type malonyl transferase fabD is shown in SEQ ID NO:8. Specifically, proline at the 64th position of wild-type malonyl transfer lase fabD is mutated to arginine to obtain malonyl transfer lase fabD after the transformation. It should be understood that Bacillus subtilis generates β-hydroxy fatty acids (the hydrophobic part of surfactant molecules) through the fatty acid synthesis pathway. In the above fatty acid synthesis pathway, the reaction from acetyl CoA to malonyl CoA is the rate-limiting step of fatty acid synthesis, and biotin carboxylase IdeHA and biotin carboxylase accBC are key enzymes in this rate-limiting step . Malonyl transferase fabD catalyzes malonyl CoA to generate malonyl ACP . Malonyl-ACP enters the fatty acid biosynthesis cycle under the catalysis of β-ketoacyl-acyl vector protein synthase II I (FabHB). Enoyl reductase ​ZefabI is one of the components of the fatty acid synthase complex and catalyzes the production of acyl-ACP, which is a precursor for the synthesis of fatty acid chains in Bacillus subtilis. By increasing the precursor supply, it is possible to achieve the goal of high production of surfactants in Bacillus subtilis. As can be seen from this, biotin carboxylase IdeHA, biotin carboxylase accBC, enoyl reductase fabI, and malonyl transferase fabD are essential for the synthesis of surfactants. By overexpressing these four enzymes to increase the precursor supply, the production amount of surfactants in Bacillus subtilis can be improved. This invention further provides the use of the method described in any one of the above in the production of surfactants. By adopting the above technical solutions, compared with the prior art, the technical progress achieved by this invention is as follows: (1) This invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis by genetically engineering the key enzymes involved in the synthesis of surfactants in Bacillus subtilis, but also provides Bacillus subtilis that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants and can reach up to 13.3 g / L at most, which is helpful for the realization of industrial production and commercial application of surfactants. (3) The transformed biotin carboxylase IdeHA, transformed biotin carboxylase accBC, transformed enoyl reductase fabI, and transformed malonyl transferase fabD provided by this invention are helpful for improving the production of surfactants by Bacillus subtilis. This invention further provides the use of the method described in any one of the above in the production of surfactants. (1) By genetically engineering the key enzymes involved in surfactant synthesis in Bacillus subtilis, this invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis, but also provides a Bacillus subtilis strain that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants, with a maximum yield of 13.3 g / L, which is beneficial for the industrial production and commercial application of surfactants. (3) The transformed biotin carboxylase IdeHA, transformed biotin carboxylase accBC, transformed enoyl reductase fabI, and transformed malonyl transferase fabD provided by this invention contribute to improving the surfactant production by Bacillus subtilis. By adopting the above technical solutions, compared with the prior art, the technical progress achieved by this invention is as follows: (1) This invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis by genetically engineering the key enzymes involved in surfactant synthesis in Bacillus subtilis, but also provides a Bacillus subtilis strain that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants, with a maximum yield of 13.3 g / L, which is beneficial for the industrial production and commercial application of surfactants. (3) The transformed biotin carboxylase IdeHA, transformed biotin carboxylase accBC, transformed enoyl reductase fabI, and transformed malonyl transferase fabD provided by this invention contribute to improving the surfactant production by Bacillus subtilis. (1) By genetically engineering the key enzymes involved in surfactant synthesis in Bacillus subtilis, this invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis, but also provides a Bacillus subtilis strain that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants, with a maximum yield of 13.3 g / L, which is beneficial for the industrial production and commercial application of surfactants. (3) The transformed biotin carboxylase IdeHA, transformed biotin carboxylase accBC, transformed enoyl reductase fabI, and transformed malonyl transferase fabD provided by this invention contribute to improving the surfactant production by Bacillus subtilis. (1) By genetically engineering the key enzymes involved in surfactant synthesis in Bacillus subtilis, this invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis, but also provides a Bacillus subtilis strain that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants, with a maximum yield of 13.3 g / L, which is beneficial for the industrial production and commercial application of surfactants. (3) The transformed biotin carboxylase IdeHA, transformed biotin carboxylase accBC, transformed enoyl reductase fabI, and transformed malonyl transferase fabD provided by this invention contribute to improving the surfactant production by Bacillus subtilis. (1) By genetically engineering the key enzymes involved in surfactant synthesis in Bacillus subtilis, this invention not only improves the synthesis efficiency of β-hydroxy fatty acids in Bacillus subtilis, but also provides a Bacillus subtilis strain that can highly produce surfactants. (2) The Bacillus subtilis provided by this invention can highly produce surfactants, with a maximum yield of 13.3 g / L, which is beneficial for the industrial production and commercial application of surfactants.

Brief Description of the Drawings

[0004]

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Modes for Carrying Out the Invention

[0005] Hereinafter, it will be described in more detail in conjunction with the attached drawings and specific examples of the specification of the present invention. Example 1: This example provides a method for improving the production of surfactant by Bacillus subtilis. This method includes transforming the biotin carboxylase IdeHA of Bacillus subtilis, and the amino acid sequence of the transformed biotin carboxylase IdeHA is shown in SEQ ID NO:1, and the above-mentioned Bacillus subtilis is wild-type Bacillus subtilis 168, and its deposit number is ATCC 23857. It should be understood that this wild-type Bacillus subtilis may also be other products of the prior art and is not limited to the Bacillus subtilis corresponding to the above deposit number. The above-mentioned transformed biotin carboxylase IdeHA is the wild-type biotin carboxylase ​The enzyme IdeHA mutation was obtained based on the wild-type biotin carboxylase IdeHA. The amino acid sequence is shown in SEQ ID NO:5. Specifically, the 74th lysine of wild-type biotin carboxylase IdeHA was replaced with asparagine. alanine at position 125 was mutated to threonine, and glutamic acid at position 136 was mutated to 145 cysteine to phenylalanine, and 394 thread The onine was mutated to an asparagine (corresponding to the underlined annotation in SEQ ID NO:1) to obtain the shape After transformation, biotin carboxylase IdeHA was obtained. SEQ ID NO:1 MFTKVLIANRGEIAMRIIRTCSRLGIKTVAVYSEADKDAP HTKAATEAYLIGESRVSESYLNIERIIKTAKKA N ADAIHP GYGLLSENSRFAERCKQENIVFIGPSPDIIAKMGSKIEAR KAME T AGVPVVPGVS K SLGDIEAA F RTASQIGYPVmLKAS AGGGGIGMQRVENEEALKKAYEGNKKRAADFFGDGSMYIE KVIEHARHIEVQLLADQHGHTVHLFERDCSVQRRHQKVIE EAPSPFVDDELRMKIGQTAVKAAKAIGYTNAGTIEFIVDQ KQNFYFLEMNTRLQVEHPVTEEITGLDLVEQQLRIAAGHT LTFSQKDIQRNGHAIEVRIYAEDPKTFFFPSPGTITAFSLP DQKGVRHECAVAKDSTVTPFYDPMIAKMIVKGQ N RTEAIE KLETALRDYRVEGIKTNLPLLIQAAATKAFKEGDVTTDFL KQHL SEQ ID NO:5 MFTKVLIANRGEIAMRIIRTCSRLGIKTVAVYSEADKDAP HTKAATEAYLIGESRVSESYLNIERIIKTAKKAKADAIHP GYGLLSENSRFAERCKQENIVFIGPSPDIIAKMGSKIEAR KAMEAAGVPVVPGVSESLGDIEAACRTASQIGYPVmLKAS AGGGGIGMQRVENEEALKKAYEGNKKRAADFFGDGSMYIE KVIEHARHIEVQLLADQHGHTVHLFERDCSVQRRHQKVIE EAPSPFVDDELRMKIGQTAVKAAKAIGYTNAGTIEFIVDQ KQNFYFLEMNTRLQVEHPVTEEITGLDLVEQQLRIAAGHT LTFSQKDIQRNGHAIEVRIYAEDPKTFFPSPGTITAFSLP DQKGVRHECAVAKDSTVTPFYDPMIAKMIVKGQTRTEAIE KLETALRDYRVEGIKTNLPLLIQAAATKAFKEGDVTTDFL KQHL Next, the above method described in this example will be described in detail,

[0006] Step 1, Preparation of Bacillus subtilis receptor cells Wild-type Bacillus subtilis was spread on an LB solid medium and cultured overnight at 37 °C Single colonies were picked out, inoculated into 4 mL of LB liquid medium, and cultured at 37 °C, 1 40 rpm for 6 h. The cells were washed three times with a sterilized 10 wt% glycerol solution, and finally The cells were resuspended in 0.2 mL of 10 wt% glycerol solution, dispensed, and stored in a -80 °C freezer for preparation, Step 2, Preparation of Bacillus subtilis receptor cells transfected with the pHT-XCR6 plasmid Here, the pHT-XCR6 plasmid (chloramphenicol resistance in Bacillus subtilis) is C a pf1 expression vector, where Cpf1 is controlled by the induction of xylose. Furthermore, this plasmid further contains NgAgo and is used to improve the efficiency of homologous recombination during gene editing. The above pHT-XCR6 plasmid is a vector for gene editing and expression control, mainly used in the research of Bacillus subtilis. Its production company is BIO-SCIENCE. Approximately 10 ng of the pHT-XCR6 plasmid is added to 0.1 mL of Bacillus subtilis receptor cells, placed in an ice bath in a pre-cooled electric shock cup for 5 minutes, and then given an electric shock at 2.0 kV once. It is then resuscitated and cultured in 1 mL of LB medium for 2 hours, spread on LB solid medium containing 50 μg / mL chloramphenicol, and cultured overnight at 37°C to obtain Bacillus subtilis receptor cells transfected with the plasmid pHT-XCR6. These cells are then stored in a -80°C refrigerator for later use. Step 3, Construction of the pcrF19NM plasmid The pcrF19NM plasmid contains the mutation site information of wild-type biotin carboxylase IdeHA and is inserted between the DR2 elements. Among them, the crRNA sequence with target function is shown in Table 1. The construction process of the recombinant plasmid with the above mutation site information (also called the pcrF19NM plasmid) is as follows: (1) Insert the sequence with site information into the pcrF19NM plasmid For each mutation site, a pair of primers containing overlapping fragments with the crRNA sequence are designed, denatured and annealed to form sticky-ended primer dimers, and then used for pcr to amplify the target fragment. The amplified fragment is then digested with restriction enzymes and ligated into the pcrF19NM plasmid. The resulting plasmid is then transformed into competent cells and screened on plates containing antibiotics to obtain the recombinant plasmid pcrF19NM. The construction process of the plasmid with the above mutation site information (also called the pcrF19NM plasmid) is as follows: (1) Insert the sequence with site information into the pcrF19NM plasmid For each mutation site, a pair of primers containing overlapping fragments with the crRNA sequence are designed, denatured and annealed to form sticky-ended primer dimers, and then used for pcr The F19NM plasmid was digested with Eco31I, and the product after recovery was ligated with primer dimer T4 to obtain a ligation product, (2) Construction of the pcrF19NM plasmid carrying the coding gene The above ligation product was transformed into E. coli DH5α, verified by colony PCR, and then the plasmid was extracted to obtain the pcrF19NM plasmid with site information, The above pcrF19NM plasmid contains the mutation site information of wild-type biotin carboxylase IdeHA, is inserted between two DR2 elements in the pcrF19NM plasmid, The crRNA sequence with target function in the above pcrF19NM plasmid is shown in Table 1, Table 1: crRNA sequences for editing the wild-type biotin carboxylase IdeHA gene JPEG0007710127000001.jpg238168 Step 4, construction of homologous fragments The homologous fragments were constructed using the complementary primer annealing procedure (also called annealing PCR), and the primer sequences used are shown in Table 2, JPEG0007710127000002.jpg238168 Table 2: Primer sequences for constructing homologous fragments for editing the biotin carboxylase IdeHA gene Here, "F" represents the upstream primer, "R" represents the downstream primer, the underlined sequences are variable sites, and in the primer sequences for constructing homologous fragments for editing the above biotin carboxylase IdeHA gene, A represents adenine, T represents thymine, C represents cytosine, G represents guanine, N represents any one of A, T, C, G, and K (keto) represents T JPEG0007710127000002.jpg238168 Table 2: Primer sequences for constructing homologous fragments for editing the biotin carboxylase IdeHA gene Sequence JPEG0007710127000002.jpg238168 Here, "F" represents the upstream primer, "R" represents the downstream primer, the underlined sequences are variable sites, and in the primer sequences for constructing homologous fragments for editing the above biotin carboxylase IdeHA gene, A represents adenine, T represents thymine, C represents cytosine, G represents guanine, N represents any one of A, T, C, G, and K (keto) represents T is the different site, and in the primer sequences for constructing homologous fragments for editing the above biotin carboxylase IdeHA gene, A represents adenine, T represents thymine, C represents cytosine, G represents guanine, N represents any one of A, T, C, G, and K (keto) represents T In the primer sequences for constructing homologous fragments for editing the above biotin carboxylase IdeHA gene, A represents adenine, T represents thymine, C represents cytosine, G represents guanine, N represents any one of A, T, C, G, and K (keto) represents T ymine, C represents cytosine, G represents guanine, N represents any one of A, T, C, G, and K (keto) represents T nine, N represents any one of A, T, C, G, and K (keto) represents T represents either G or M (amino, Amino) represents either A or C, The annealing PCR system is shown in Table 3, and the annealing PCR procedure is shown in Table 4. Table 3: Annealing PCR System JPEG0007710127000003.jpg238168 Table 4: Annealing PCR Procedure JPEG0007710127000004.jpg238168 The PCR product obtained by the above annealing procedure is a homologous fragment. Step 5, Transformation of pcrF19NM plasmid and homologous fragment The pcrF19NM plasmid prepared in Step 3 and the homologous fragment prepared in Step 4 were introduced into the Bacillus subtilis receptor cells prepared in Step 2 with the plasmid pHT-XCR6, transformed, and plated on an LB solid medium supplemented with 50 μg / mL chloramphenicol, 50 μg / m L kanamycin, and 3% xylose, and cultured overnight at 30 °C. It is possible to perform liquid culture and fermentation verification using the grown single colonies. And Step 6, Production of surfactant by Bacillus subtilis culture and fermentation Forty-eight single colonies of Bacillus subtilis were picked from the plate (i.e., LB solid medium), and each was inoculated into a 10 mL test tube containing 3 mL of LB liquid medium and cultured overnight at 37 °C, 200 rpm, on a shaker floor to obtain a seed culture solution. 1 mL of the seed culture solution 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 cultured at 37 °C, 200 rpm, on a shaker floor for 48 h to obtain a Bacillus subtilis fermentation broth. Step 7, Calculation of the content of the surfactant produced by Bacillus subtilis fermentation Centrifuge the Bacillus subtilis fermentation broth, take 1 mL of the supernatant, and centrifuge it at 12,000 rpm for 3 min. Take 200 μL of the supernatant, add methanol to make it 3 - 10 times the volume, shake well, and then centrifuge at 12 00 rpm for 10 min. Pass the supernatant through a membrane to obtain the test sample, and use Agilen 1260 high-performance liquid chromatography as the detection tool. The column used is A methyst C18-H column (5μm, 250×4.6 mM). The mobile phase used is 90% methanol and 10% water. In addition, 0.05% trifluoroacetic acid is added. The flow rate is 0.8 mL / min, the injection volume is 20 μL, and the detection wavelength is 214 nm. Prepare the surfactant standard product with gradient concentrations, create a standard curve, calculate the content of the surfactant in the fermentation broth sample, and select the 3 strains of bacteria with the most increased surfactant production from the 200 strains of Bacillus subtilis isolated in step 6, which are respectively designated as NJUXR-CY-1-1, CY-1-2, and CY-1-3. Step 8, Genomic sequencing of dominant strains Using a Gram-positive bacteria genomic DNA extraction kit (Beijing Solarbio), extract the genomes of NJUXR-CY- 1-1, CY-1-2, and CY-1-3 respectively. Sequence the biotin carboxylase IdeHA gene on the NJUXR-CY-1-1 genome, CY-1-2 genome, and CY-1-3 genome. Here, the sequence of the biotin carboxylase IdeHA gene on the NJUXR-CY-1-1 genome is shown in SEQ ID NO:1, mutate lysine at position 74 to aspartic acid, mutate alanine at position 125 to threonine, mutate glutamic acid at position 136 to lysine, mutate cysteine at position 145 to phenylalanine, and at position 39 ​​​Mutate the 4 - threonine to asparagine, and the position of the transformed biotin carboxylase Id eHA in the 3D structure corresponding to positions 74, 125, 1 36, 145, and 394 is shown in Figure 1, Based on this, the example of this application preserved the above - mentioned Bacillus subtilis NJUXR - CY - 1 - 1. The above Bacillus subtilis NJUXR - CY - 1 - 1 was preserved in the China Center for Type Culture Collection. The preservation location is Wuhan University, Wuhan, China. The preservation date is January 13, 2025, and the preservation number assigned by the preservation unit is CCTCC NO:M 2025097. The taxonomic name of the above - mentioned Bacillus subtilis NJU XR - CY - 1 - 1 (also called biological material) is Bacillus subtilis NJUXR - CY - 1 - 1, and the Latin name of this taxonomic name is Bacillus subtilis N JUXR - CY - 1 - 1. Example 2: This example is different from Example 1 in the following points. It further includes transforming the wild - type biotin carboxylase accBC of Bacillus subtilis. The amino acid sequence of the transformed biotin carboxylase accBC is shown in SEQ ID NO:2, The above - mentioned transformed biotin carboxylase accBC is obtained based on the mutation of the wild - type biotin carboxylase accBC. The amino acid sequence of the wild - type biotin carboxylase accBC is shown in SEQ ID NO:6. Specifically, serine at position 409 of the wild - type biotin carboxylase accBC is mutated to glycine (corresponding to the underlined annotation in SEQ ID NO:2 ) to obtain the transformed biotin carboxylase accBC, SEQ ID NO:2 MIKKLLIANRGEIAVRIIRACRELGIETVAVYSEADKDAL MIKKLLIANRGEIAVRIIRACRELGIETVAVYSEADKDAL HVQMADEAFCIGPKASKDSYLNVTNIVSVAKLTGTDAIHP GYGFLAENADFAELCEEVNVTFVGPSADAISKMGTKDVAR ETMKQAGVPIVPGSQGIIENVEEAVSLANEIGYPVIIKAT AGGGGKGIRVARTEEELINGIKITQQEAATAFGNPGVYIE KYIEDFRHVEIQVLADNYGNTIHLGERDCSIQRRLQKLLE ESPSPALDSEIREQMGDAAVKAAKAVGYTGAGTVEFIYDY NEQRYYFMEMNTRIQVEHPVTEMVTGTDLIKEQIKVASGM ELSLKQEDVEFEGWAIECRINAENPSKNFMPSPGEIKMYL PPGGLGVRVDSAAYPGYSIPPYYDSMIAKVITYGKTRDEA IARMKRAL G EFVIEGIETTIPFHLKLLEHETFVSGEFNTK FLETYDVMGS SEQ ID NO:6 MIKKLLIANRGEIAVRIIRACRELGIETVAVYSEADKDAL HVQMADEAFCIGPKASKDSYLNVTNIVSVAKLTGTDAIHP GYGFLAENADFAELCEEVNVTFVGPSADAISKMGTKDVAR ETMKQAGVPIVPGSQGIIENVEEAVSLANEIGYPVIIKAT AGGGGKGIRVARTEEELINGIKITQQEAATAFGNPGVYIE KYIEDFRHVEIQVLADNYGNTIHLGERDCSIQRRLQKLLE ESPSPALDSEIREQMGDAAVKAAKAVGYTGAGTVEFIYDY NEQRYYFMEMNTRIQVEHPVTEMVTGTDLIKEQIKVASGM ELSLKQEDVEFEGWAIECRINAENPSKNFMPSPGEIKMYL PPGGLGVRVDSAAYPGYSIPPYYDSMIAKVITYGKTRDEA IARMKRALSEFVIEGIETTIPFHLKLLEHETFVSGEFNTK FLETYDVMGS The above method described in this example will be described in detail below, Step 1: Preparation of Bacillus subtilis recipient cells This step is basically the same as Step 1 of Example 1, except for the following points. In Example 1, the wild-type Bacillus subtilis used is replaced with the high-yield strain NJUXR- CY-1-1 screened in Example 1, Step 2: Preparation of Bacillus subtilis recipient cells introduced with plasmid pHT-XCR6 This step is the same as Step 2 of Example 1, Step 3: Construction of pcrF19NM plasmid This step is basically the same as Step 3 of Example 1, except for the following points. The crR NA sequence is replaced with the one shown in Table 5, Table 5: crRNA sequences for editing the biotin carboxylase accBC gene JPEG0007710127000005.jpg238168 Step 4: Construction of homologous fragments This step is basically the same as Step 4 of Example 1, except for the following points. The homologous fragment construction primer sequences are replaced with the ones shown in Table 6, Table 6: Primer sequences for constructing homologous fragments for editing the biotin carboxylase accBC gene sequences JPEG0007710127000006.jpg238168 Step 5: Transformation of pcrF19NM plasmid and homologous fragments This step is the same as Step 5 of Example 1, Step 6, Production of surfactant by culturing and fermenting Bacillus subtilis This step is the same as step 6 of Example 1, Step 7, Calculation of the content of the surfactant produced by Bacillus subtilis fermentation This step is the same as step 7 of Example 1. Specifically, from the 2 00 strains of Bacillus subtilis isolated in step 6, the 3 strains of strains with the most increased production of surfactant were designated as NJUXR-CY-2- 1, CY-2-2, and CY-2-3, Step 8, Sequencing of the dominant strain genome This step is basically the same as step 8 of Example 1, except for the following points. The sequence content is the biotin carboxylase accBC gene of the NJUXR-CY-2-1 genome, the CY-2-2 genome, and the CY-2-3 genome. Here, the mutation method of the biotin carboxylase accBC gene of the NJUXR-CY-2- 1 genome is to mutate serine at position 409 to glycine, and its sequence is shown in SEQ ID NO:2. The position in the 3D structure of the transformed biotin carboxylase accBC corresponding to the above mutation site (corresponding to position 4 09) is shown in Figure 2. Based on this, the example of the present application preserved the above-mentioned Bacillus subtilis NJUXR-CY-2-1. The above-mentioned Bacillus subtilis NJUXR-CY-2-1 is preserved in the China Center for Type Culture Collection. The preservation location is Wuhan University, Wuhan, China, and the preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025099. The taxonomic name of the above-mentioned Bacillus subtilis NJU XR-CY-2-1 (also called biological material) is Bacillus subtilis NJUXR-CY-2- 1, and the Latin name of this taxonomic name is Bacillus subtilis N JUXR-CY-2-1. The position is shown in Figure 2. Based on this, the example of the present application preserved the above-mentioned Bacillus subtilis NJUXR-CY-2-1. The above-mentioned Bacillus subtilis NJUXR-CY-2-1 is preserved in the China Center for Type Culture Collection. The preservation location is Wuhan University, Wuhan, China, and the preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025099. The taxonomic name of the above-mentioned Bacillus subtilis NJU XR-CY-2-1 (also called biological material) is Bacillus subtilis NJUXR-CY-2- 1, and the Latin name of this taxonomic name is Bacillus subtilis N JUXR-CY-2-1. The preservation location is Wuhan University, Wuhan, China, and the preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025099. The taxonomic name of the above-mentioned Bacillus subtilis NJU XR-CY-2-1 (also called biological material) is Bacillus subtilis NJUXR-CY-2- 1, and the Latin name of this taxonomic name is Bacillus subtilis N JUXR-CY-2-1. The preservation location is Wuhan University, Wuhan, China, and the preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025099. The taxonomic name of the above-mentioned Bacillus subtilis NJU XR-CY-2-1 (also called biological material) is Bacillus subtilis NJUXR-CY-2- Example 3: This example is different from Example 2 in the following aspect. It further includes transforming the wild-type enoyl reductase of Bacillus subtilis, fabI. After transformation, the amino acid sequence of enoyl reductase fab I is shown in SEQ ID NO:3. The enoyl reductase fabI after the above transformation is obtained based on the mutation of wild-type enoyl reductase fab I. The amino acid sequence of wild-type enoyl reductase fabI is SEQ ID NO:7. Specifically, glycine at position 263 of wild-type enoyl reductase fabI is mutated to cysteine (corresponding to the underlined annotation in SEQ ID NO:3) to obtain the enoyl reductase fabI after transformation. SEQ ID NO:3 MSLLNIGGFIHMNFSLEGRNIVVMGVANKRSIAWGIARSL HEAGARLIFTYAGERLEKSVHELAGTLDRNDSIILPCDVT NDAEIETCFASIKEQVGVIHGIAHCIAFANKEELVGEYLN TNRDGFLLAHNISSYSLTAVVKAARPMMTEGGSIVTLTYL GGELVMPNYNVMGVAKASLDASVKYLAADLGKENIRVNSI SAGPIRTLSAKGISDFNSILKDIEERAPLRRTTTPEEVGD TAAFLFSDMSRGITGENLHVDS FHITAR C FHITAR SEQ ID NO:7 MSLLNIGGFIHMNFSLEGRNIVVMGVANKRSIAWGIARSL HEAGARLIFTYAGERLEKSVHELAGTLDRNDSIILPCDVT NDAEIETCFASIKEQVGVIHGIAHCIAFANKEELVGEYLN TNRDGFLLAHNISSYSLTAVVKAARPMMTEGGSIVTLTYL GGELVMPNYNVMGVAKASLDASVKYLAADLGKENIRVNSI SAGPIRTLSAKGISDFNSILKDIEERAPLRRTTTPEEVGD TAAFLFSDMSRGITGENLHVDSGFHITAR The method described in this example is different from the method described in Example 2 in the following aspects: In step 1, the high-yield strain NJUXR-CY-2-1 is used as the Bacillus subtilis recipient cell, In step 3, the crRNA sequence on the pcrF19NM plasmid is shown in Table 7, , Table 7 crRNA sequences for enoyl reductase fabI gene editing JPEG0007710127000007.jpg238168 In step 4, the primer sequences of the constructed homologous fragment are shown in Table 8, Table 8 Primer sequences for constructing homologous fragments for enoyl reductase fabI gene editing JPEG0007710127000008.jpg238168 In the primer sequences for constructing homologous fragments for enoyl reductase fabI gene editing above, , N represents any one of A, T, C, and G, In step 5, the extracted single colonies are replaced from 200 to 100, In step 6, one Bacillus subtilis strain is extracted from each of the 100 single colonies, and 1 00 Bacillus subtilis strains are obtained, In step 7, from the 100 Bacillus subtilis strains extracted in step 6, the 3 strains with the most increased surfactant production are designated as NJUXR-CY-3-1, CY-3-2, and CY-3-3, In step 8, the sequence content is the NJUXR-CY-3-1 genome, CY-3-2 ge In step 8, the sequence content is the NJUXR-CY-3-1 genome, CY-3-2 ge The enoyl reductase fabI gene of Nome and CY-3-3 genome, where N The mutation method of enoyl reductase fabI of JUXR-CY-3-1 is that glycine at position 263 is mutated to cysteine, and the sequence is shown in SEQ ID NO:3. The position of the enoyl reductase fabI after transformation at this mutation site (corresponding to position 2 63) in the 3D structure is , as shown in Figure 3 Based on this, the examples of this application preserved the above-mentioned Bacillus subtilis NJUXR-CY-3-1. The above mentioned Bacillus subtilis NJUXR-CY-3-1 was preserved in the China Center for Type Culture Collection. The preservation location is Wuhan University, Wuhan, China. The preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025103. The taxonomic name of the above-mentioned Bacillus subtilis NJ UXR-CY-3-1 (also called biological material) is Bacillus subtilis NJUXR-CY 3-1, and the Latin name of this taxonomic name is Bacillus subtilis NJUXR-CY-3-1. Example 4: This example is different from Example 3 in the following points: It further includes transforming the wild-type malonyltransferase fabD of the above-mentioned Bacillus subtilis, and the amino acid sequence of the transformed malonyltransferase fabD is shown in SEQ ID NO:4 . The above-mentioned transformed malonyltransferase fabD is obtained based on the mutation of wild-type malonyltransferase fabD. The amino acid sequence of wild-type malonyltransferase fabD is shown in SEQ ID NO:8. Specifically, proline at position 64 of wild-type malonyltransferase fabD is mutated to arginine (corresponding to the underlined annotation in SEQ ID NO:4 ) to obtain the transformed malonyltransferase fabD . The above-mentioned transformed malonyltransferase fabD is obtained based on the mutation of wild-type malonyltransferase fabD. The amino acid sequence of wild-type malonyltransferase fabD is shown in SEQ ID NO:8. Specifically, proline at position 64 of wild-type malonyltransferase fabD is mutated to arginine (corresponding to the underlined annotation in SEQ ID NO:4 ) to obtain the transformed malonyltransferase fabD. The amino acid sequence of wild-type malonyltransferase fabD is shown in SEQ ID NO:8. Specifically, proline at position 64 of wild-type malonyltransferase fabD is mutated to arginine (corresponding to the underlined annotation in SEQ ID NO:4 ) to obtain the transformed malonyltransferase fabD. The amino acid sequence of wild-type malonyltransferase fabD is shown in SEQ ID NO:8. Specifically, proline at position 64 of wild-type malonyltransferase fabD is mutated to arginine (corresponding to the underlined annotation in SEQ ID NO:4 ) to obtain the transformed malonyltransferase fabD. The amino acid sequence of wild-type malonyltransferase fabD is shown in SEQ ID NO:8. Specifically, proline at position 64 of wild-type malonyltransferase fabD is mutated to arginine (corresponding to the underlined annotation in SEQ ID NO:4 ) to obtain the transformed malonyltransferase fabD SEQ ID NO:4 MSKIAFLFPGQGSQFIGMGKELYEQVPAAKRLFDEADETL ETKLSSLIFEGDAEELTLTYNAQ R ALLTTSIAVLEKFKES GITPDFTAGHSLGEYSALVAAGALSFKDAVYTVRKRGEFM NEAVPAGEGAMAAILGMDAEALKQVTDKVTEEGNLVQLAN LNCPGQIVISGTAKGVELASELAKENGAKRAIPLEVSGPF HSELMKPAAEKLKEVLDACDIKDADVPVISNVSADVMTEK ADIKEKLIEQLYSPVRFEESINKLIAEGVTTFIEIGPGKV LSGLVKKVNRRLKTIAVSDPETIELAIQTLKEENDNA SEQ ID NO:8 MSKIAFLFPGQGSQFIGMGKELYEQVPAAKRLFDEADETL ETKLSSLIFEGDAEELTLTYNAQPALLTTSIAVLEKFKES GITPDFTAGHSLGEYSALVAAGALSFKDAVYTVRKRGEFM NEAVPAGEGAMAAILGMDAEALKQVTDKVTEEGNLVQLAN LNCPGQIVISGTAKGVELASELAKENGAKRAIPLEVSGPF HSELMKPAAEKLKEVLDACDIKDADVPVISNVSADVMTEK ADIKEKLIEQLYSPVRFEESINKLIAEGVTTFIEIGPGKV LSGLVKKVNRRLKTIAVSDPETIELAIQTLKEENDNA The above method described in this example is different from the method described in Example 3 in the following aspects: In step 1, the high-yield strain CY-3-1 is used as the Bacillus subtilis recipient cell, In step 3, the crRNA sequence on the pcrF19NM plasmid is shown in Table 9 , Table 9: crRNA sequences for editing the malonyltransferase fabD gene JPEG0007710127000009.jpg238168 In step 4, the primer sequences of the constructed homologous fragments are shown in Table 10, Table 10: Primers for constructing homologous fragments for editing the malonyltransferase fabD gene Sequence JPEG0007710127000010.jpg238168 For the primer sequences for constructing homologous fragments for editing the above malonyltransferase fabD gene , N represents any one of A, T, C, and G, In step 7, from the 96 Bacillus subtilis strains isolated in step 6, the 3 strains with the highest increase in surfactant production are designated as NJUXR-CY-4-1, CY-4-2, and CY-4-3, In step 8, the sequence content is the malonyltransferase fabD gene of the NJUXR-CY-4-1 genome, CY-4-2 genome, and CY-4-3 genome. Here, the mutation mode of malonyltransferase fabD in the NJUXR-CY-4-1 genome is to mutate proline at the 64th amino acid to arginine, and the sequence is shown in SEQ ID NO:4, The position of the transformed malonyltransferase fabD in the 3D structure at the mutation site (corresponding to the 64th position) is shown in Figure 4, This example detected the surfactant in the surfactant standard product (purity 99%, Shanghai Macklin) and the fermentation broth of NJUXR-CY-4- 1. The chromatograms of the surfactant in the standard product (i.e., the surfactant) and the fermentation broth of NJUXR-C Y-4-1 are shown in Figure 5. As can be seen from Figure 5 ​​​, Compared with the wild-type strain, the production amount of surfactant of the constructed NJUXR-CY-4-1 strain significantly increased. Compared with wild-type Bacillus subtilis, the production amount of NJUXR-CY-4-1 increased by 14.7 times. The gene mutation method described in the present invention effectively improved the production amount of surfactant, reflecting this, Based on this, the above-mentioned Bacillus subtilis NJUXR-CY-4-1 was preserved. This Bacillus subtilis NJUXR-C Y-4-1 was preserved in the China Center for Type Culture Collection on January 13, 2025, and its preservation number is CCTCC M 2025098. Based on this, the examples of the present application preserve the above-mentioned Bacillus subtilis NJUXR-CY-4-1. The above-mentioned Bacillus subtilis NJUXR-CY-4-1 was preserved in the China Center for Type Culture Collection. The preservation location is Wuhan, China. Wuhan University, and the preservation date is January 13, 2025. The preservation number assigned by the preservation unit is CCTCC NO:M 2025098. The taxonomic name of the above-mentioned Bacillus subtilis NJUX R-CY-4-1 (also called biological material) is Bacillus subtilis NJUXR-CY-4-1 and the Latin name of this taxonomic name is Bacillus subtilis NJ UXR-CY-4-1. Example 5: This example provides the use of the methods described in Examples 1 to 4 in the production of surfactant, specifically, wild-type Bacillus subtilis, NJUXR-CY-1-1, CY-1-2, CY-1-3, NJUX R-CY-2-1, CY-2-2, CY-2-3, NJUXR-CY-3-1, CY-3-2, CY- 3-3, NJUXR-CY-4-1, CY-4-2 and CY-4-3 strains were scribed on LB solid plates ( i.e., LB solid medium), cultured overnight at 37 °C, and single colonies were picked out and Inoculate 10 mL test tubes each containing 3 mL of LB liquid medium, and culture overnight at 37 °C and 200 r pm on a shaking table to obtain a seed culture solution. Add 1 mL of the seed culture solution to 30 mL of fermentation medium (20 g / L glucose, 3 g / L tryptone, 3 g / L K2HPO4, 10 g / L NaH2P O4, 0.02 g / L mgSO4, 1 g / L L-leucine), and culture at 37 °C and 200 r pm on a shaking table for 48 h to obtain fermentation broths of wild-type Bacillus subtilis, NJUXR-CY-1- 1, fermentation broth of CY-1-2, fermentation broth of CY-1-3, fermentation broth of NJUXR-CY-2-1, fermentation broth of CY-2-2, fermentation broth of CY-2-3, fermentation broth of NJUXR-CY-3-1, CY- fermentation broth of 3-2, fermentation broth of CY-3-3, fermentation broth of NJUXR-CY-4-1, CY-4-2 fermentation broth and fermentation broth of CY-4-3 were centrifuged, 1 mL of the supernatant was taken, centrifuged at 12000 rpm for 3 min, 200 uL of the supernatant was taken, diluted with methanol to 3 - 10 times the volume, shaken well, and then centrifuged at 12000 rpm for 10 min. After passing the supernatant through a membrane, it was detected by high-performance liquid chromatography. The production amounts of surfactants in the above 13 fermentation broths finally obtained are shown in Table 11. The production amounts of surfactants in the above 13 fermentation broths finally obtained are shown in Table 11. Table 11: Production amounts of surfactants of different strains JPEG0007710127000011.jpg238168 NJUXR-CY-1-1, CY-1-2, and CY-1-3 described in Table 11 above are biotin carboxylase xylase IdeHA mutant strains. Among them, the production amount of surfactant of NJUXR-CY-1-1 is the highest at 1.5 g / L. The 74th lysine of biotin carboxylase IdeHA of NJUXR-CY-1-1 was mutated to asparagine, and the 125th alanine was mutated to threonine. ​​Mutate it into Nin, mutate glutamic acid at position 136 into lysine, and cysteine at position 145 into phenylalanine, mutate threonine at position 394 into asparagine, and as shown in the table NJUXR-CY-2-1, CY-2-2, and CY-2-3 listed are mutant strains of biotin carboxylase ac cBC, among which the production amount of surfactant of NJUXR-CY-2-1 is 5 .4 g / L, which is the highest, mutate serine at position 409 of biotin carboxylase accBC of NJUXR-CY-2-1 into glycine, and NJUXR-CY-3-1, CY-3-2, CY- 3-3 are mutant strains of enoyl reductase fabI, among which the production amount of surfactant of NJUXR-CY- 3-1 is the highest at 9.5 g / L, mutate glycine at position 263 of enoyl reductase fabI of NJUXR-CY-3-1 into cysteine, and NJUXR-C Y-4-1, CY-4-2, and CY-4-3 are mutant strains of malonyl transferase fabD, among which the production amount of surfactant of NJUXR-CY-4-1 is the highest at 13.3 g / L, and mutate proline at position 64 of malonyl transferase fabD of NJUXR-CY-4-1 into arginine. As can be seen from Table 13, Examples 1 to 4 are Bacillus subtilis with the highest production amount of surfactant obtained by a method for improving the production of surfactant by Bacillus subtilis, and the production amount of its surfactant is better than that of wild-type Bacillus subtilis in all cases. This indicates that the strains NJUXR-CY-1-1 to CY-4-3 obtained in Examples 1 to 4 have improved surfactant production ability compared with the original wild-type Bacillus subtilis. Continuing the analysis of Table 13, as can be seen, regarding the production amount of surfactant, NJUXR-C As can be seen from Table 13, Examples 1 to 4 are Bacillus subtilis in which the production amount of surfactant obtained by the method of improving the production of surfactant by Bacillus subtilis has increased the most, and the production amount of its surfactant is better than that of wild-type Bacillus subtilis in all cases. This indicates that the strains NJUXR-CY-1-1 to CY-4-3 obtained in Examples 1 to 4 have improved surfactant production ability compared with the original wild-type Bacillus subtilis. As can be seen by continuing the analysis of Table 13, regarding the production amount of surfactant, NJUXR-C Y-4-1 > NJUXR-CY-3-1 > NJUXR-CY-2-1 > NJUXR-CY-1-1 > is a wild-type Bacillus subtilis, and as can be seen from this, biotin carboxylase IdeHA, biotin carboxylase accBC, enoyl reductase fab I and malonyl transferase fabD were transformed, and the resulting Bacillus subtilis had the highest increase in the production amount of surfactant, and its surfactant production amount was much higher than that of the wild-type Bacillus subtilis. <Copy of the deposit certificate of the microorganism> JPEG0007710127000012.jpg 238169 JPEG0007710127000013.jpg 229166 JPEG0007710127000014.jpg 231166 JPEG0007710127000015.jpg 238169 [Sequence Listing] <st26sequencelisting originalfreetextlanguagecode="ja" dtdversion="V1_3" filenam e="枯草菌による界面活性剤の生産を向上させる方法および用途.xml" softwarename="WIP O Sequence" softwareversion="2.3.0" productiondate="2025-03-31"> <applicationidentification> <ipofficecode>JP< / 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<INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q12"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MIKKLLIANRGEIAVRIIRACRELGIETVAVYSEADKDALHVQMADEAFCIGPKASKDSYLNVTNIVSVAKLTGTDAIHP GYGFLAENADFAELCEEVNVTFVGPSADAISKMGTKDVARETMKQAGVPIVPGSQGIIENVEEAVSLANEIGYPVIIKAT AGGGGKGIRVARTEEELINGIKITQQEAATAFGNPGVYIEKYIEDFRHVEIQVLADNYGNTIHLGERDCSIQRRLQKLLE ESPSPALDSEIREQMGDAAVKAAKAVGYTGAGTVEFIYDYNEQRYYFMEMNTRIQVEHPVTEMVTGTDLIKEQIKVASGM ELSLKQEDVEFEGWAIECRINAENPSKNFMPSPGEIKMYLPPGGLGVRVDSAAYPGYSIPPYYDSMIAKVITYGKTRDEA IARMKRALSEFVIEGIETTIPFHLKLLEHETFVSGEFNTKFLETYDVMGS < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="7"> <insdseq> <INSDSeq_length>269< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..269< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q14"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MSLLNIGGFIHMNFSLEGRNIVVMGVANKRSIAWGIARSLHEAGARLIFTYAGERLEKSVHELAGTLDRNDSIILPCDVT NDAEIETCFASIKEQVGVIHGIAHCIAFANKEELVGEYLNTNRDGFLLAHNISSYSLTAVVKAARPMMTEGGSIVTLTYL GGELVMPNYNVMGVAKASLDASVKYLAADLGKENIRVNSISAGPIRTLSAKGISDFNSILKDIEERAPLRRTTTPEEVGD TAAFLFSDMSRGITGENLHVDSGFHITAR < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="8"> <insdseq> <INSDSeq_length>317< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..317< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q16"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MSKIAFLFPGQGSQFIGMGKELYEQVPAAKRLFDEADETLETKLSSLIFEGDAEELTLTYNAQPALLTTSIAVLEKFKES GITPDFTAGHSLGEYSALVAAGALSFKDAVYTVRKRGEFMNEAVPAGEGAMAAILGMDAEALKQVTDKVTEEGNLVQLAN LNCPGQIVISGTAKGVELASELAKENGAKRAIPLEVSGPFHSELMKPAAEKLKEVLDACDIKDADVPVISNVSADVMTEK ADIKEKLIEQLYSPVRFEESINKLIAEGVTTFIEIGPGKVLSGLVKKVNRRLKTIAVSDPETIELAIQTLKEENDNA < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="9"> <insdseq> <INSDSeq_length>24< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..24< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q18"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcgaaaaaagcaaaagccgacgcg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="10"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q20"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcaatggaggctgcaggtgtc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="11"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q22"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggcgtttctgaatccctcgga< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="12"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q24"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gaggcagcctgccgcaccgca< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="13"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q26"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aaaggccaaaccagaacagaa< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="14"> <insdseq> <INSDSeq_length>37< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..37< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q28"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aaaaaagcaaacgccgacgcgatccacccgggatatg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="15"> <insdseq> <INSDSeq_length>34< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..34< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q30"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>cgcgtcggcgtttgcttttttcgccgtctttatg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="16"> <insdseq> <INSDSeq_length>36< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..36< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q32"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gcaatggagacggcaggtgtccctgtggtgccgggc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="17"> <insdseq> <INSDSeq_length>40< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..40< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q34"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gacacctgccgtctccattgcttttcgcgcttcaattttg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="18"> <insdseq> <INSDSeq_length>36< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..36< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q37"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ggcgtttctnnktccctcggagatatagaggcagcc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="19"> <insdseq> <INSDSeq_length>36< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..36< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q39"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>tccgagggamnnagaaacgcccggcaccacagggac< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="20"> <insdseq> <INSDSeq_length>37< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..37< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q41"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gaggcagccnnkcgcaccgcaagtcaaatcggctatc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="21"> <insdseq> <INSDSeq_length>35< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..35< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q43"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>tgcggtgcgmnnggctgcctctatatctccgaggg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="22"> <insdseq> <INSDSeq_length>37< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..37< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q45"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aaaggccaannkagaacagaagcaattgaaaaactag< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="23"> <insdseq> <INSDSeq_length>35< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..35< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q47"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>ttctgttctmnnttggcctttgacaatcatcttag< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="24"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q49"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>cgcgcattgagcgaattcgtc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="25"> <insdseq> <INSDSeq_length>36< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..36< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q51"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>cgcgcattgggcgaattcgtcatcgaaggcattgag< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="26"> <insdseq> <INSDSeq_length>37< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..37< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q53"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gacgaattcgcccaatgcgcgcttcatgcgggcaatc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="27"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q55"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gttgattctggtttccatatc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="28"> <insdseq> <INSDSeq_length>34< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..34< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q57"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gttgattctnnkttccatatcactgcccgctaag< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="29"> <insdseq> <INSDSeq_length>36< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..36< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q59"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>gatatggaamnnagaatcaacgtgaagattttcacc< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="30"> <insdseq> <INSDSeq_length>21< / INSDSeq_length> <INSDSeq_moltype>RNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..21< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other RNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q61"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aatgcgcagcctgctttgctt< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="31"> <insdseq> <INSDSeq_length>37< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..37< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q63"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aatgcgcagnnkgctttgcttacgacaagcatcgctg< / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="32"> <insdseq> <INSDSeq_length>39< / INSDSeq_length> <INSDSeq_moltype>DNA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..39< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>other DNA< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q65"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence>aagcaaagcmnnctgcgcattgtatgtaagtgttaattc< / INSDSeq_sequence> < / insdseq> < / sequencedata> < / st26sequencelisting>

[0007]

Claims

**Claim 1** A method for improving the production of a surfactant by Bacillus subtilis, comprising transforming the wild-type biotin carboxylase IdeHA of Bacillus subtilis, wherein the amino acid sequence of the transformed biotin carboxylase IdeHA is shown in SEQ ID NO : 1, A method for improving the production of a surfactant by Bacillus subtilis, characterized by the above. **Claim 2** Further comprising transforming the wild-type biotin carboxylase accBC of said Bacillus subtilis, wherein the amino acid sequence of the transformed biotin carboxylase accBC is shown in SEQ ID NO:

2. The method according to claim 1, characterized by the above. **Claim 3** Further comprising transforming the wild-type enoyl reductase fabI of said Bacillus subtilis, and the form wherein the amino acid sequence of the transformed enoyl reductase fabI is shown in SEQ ID NO: 3 The method according to claim 2, characterized by the above. **Claim 4** Further comprising transforming the wild-type malonyl transferase fabD of said Bacillus subtilis, wherein the amino acid sequence of the transformed malonyl transferase fabD is shown in SEQ ID NO:

4. The method according to claim 3, characterized by the above.

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