Methods and applications for improving surfactant production using Bacillus subtilis.

Abstract

This invention provides a method and applications for improving the production of surfactants using Bacillus subtilis. [Solution] This method involves transforming the wild-type biotin carboxylase IdeHA of Bacillus subtilis to a specific amino acid sequence, which can effectively improve the production of surfactants. This may further include transforming the wild-type biotin carboxylase accBC, further including transforming the wild-type enoyl reductase fabI, and further including transforming the wild-type malonyltransferase 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, 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, and significantly reduces the surface tension and interfacial tension of liquids, enabling 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 in 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, environment, and medicine. 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. In order 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 ​​​​​​ This involves transforming biotin carboxylase IdeHA, and the transformed biotin The amino acid sequence of carboxylase IdeHA is shown in SEQ ID NO:1. The biotin carboxylase IdeHA after the above transformation is similar to that of wild-type biotin carboxylase. Obtained based on the IdeHA mutation of the wild-type biotin carboxylase IdeHA. The amino acid sequence is shown in SEQ ID NO:5. Specifically, wild-type biotin carboxy The lysine at position 74 of silase IdeHA is mutated to asparagine, and the alanine at position 125 is mutated to The threonine mutation was introduced, the glutamic acid at position 136 was changed to lysine, and the cysteine ​​at position 145 was introduced. By mutating the yin to phenylalanine and the threonine at position 394 to asparagine Then, the transformed biotin carboxylase IdeHA is obtained. Furthermore, this method transforms the wild-type biotin carboxylase accBC of Bacillus subtilis. This further includes the amino acid sequence of biotin carboxylase accBC after transformation. This is indicated by SEQ ID NO:2. The biotin carboxylase accBC obtained after the above transformation is equivalent to wild-type biotin carboxylase. Obtained based on the accBC mutation of wild-type biotin carboxylase accBC. The amino acid sequence is shown in SEQ ID NO:6. Specifically, wild-type biotin carboxy By mutating the serine at position 409 of sylase accBC to glycine, the biotin of the transformed cells was obtained. Obtain carboxylase accBC. Furthermore, this method involves transforming the wild-type enoyl reductase fabI of Bacillus subtilis. It further includes the amino acid sequence SEQ ID of the transformed enoyl reductase fabI. This appears to be in NO:3. The enoyl reductase fabI after the above transformation is similar to wild-type enoyl reductase fab. It is obtained based on the I mutation. The amino acid sequence of wild-type enoyl reductase fabI is SE This is shown in Q ID NO:7. Specifically, the second type of wild-type enoyl reductase fabI. By mutating glycine at position 63 to cysteine, the transformed enoyl reductase fabI To obtain. Furthermore, this method transforms Bacillus subtilis wild-type malonyltransferase fabD. This further includes the amino acid sequence of malonyltransferase fabD after transformation. This is indicated by SEQ ID NO:4. The malonyltransferase fabD after the above transformation is wild-type malonyltransferase Obtained based on the fabD mutation of the wild-type malonyltransferase fabD. The amino acid sequence is shown in SEQ ID NO:8. Specifically, wild-type malonyltrans. By mutating the proline at position 64 of ferase fabD to arginine, the transformed horse chestnut Obtain the transferase fabD. Furthermore, Bacillus subtilis synthesizes β-hydroxy fatty acids (hydrophobic surfactant molecules) via the fatty acid synthesis pathway. Please understand that the acetyl-CoA (sexual part) is produced in the above fatty acid synthesis pathway. The reaction from biotin carboxylase to malonyl-CoA is the rate-limiting step in fatty acid synthesis. IdeHA and biotin carboxylase accBC are key enzymes in this rate-determining step. Malonyltransferase fabD catalyzes malonyl-CoA to malonyl-ACP It generates malonyl-ACP, which is β-ketoacyl-acyl vector protein synthase II. It is catalyzed by I(FabHB) and enters the fatty acid biosynthesis cycle. Enoyl reducter 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 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 solution, 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 transforming the key enzymes involved in the synthesis of surfactants in Bacillus subtilis, but also provides Bacillus subtilis with high production of surfactants. (2) The Bacillus subtilis provided by this invention can produce surfactants highly, 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, the transformed biotin carboxylase accBC, the transformed enoyl reductase fabI and the transformed malonyl transferase fabD provided by this invention are helpful for improving the production of surfactants by Bacillus subtilis. (1) By genetically transforming 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 production of 13.3 g / L, which is conducive to the industrial production and commercial application of surfactants. <00​​​​​​​​​​​​​​​​​​​​​​​​​​​

Brief Description of the Drawings

[0004] [Figure 1] It is a 3D structure diagram of the transformed biotin carboxylase IdeHA provided by Example 1 of the present application. [Figure 2] It is a 3D structure diagram of the transformed biotin carboxylase accBC provided by Example 2 of the present application. [Figure 3] It is a 3D structure diagram of the transformed enoyl reductase fabI provided by Example 3 of the present application. [Figure 4] It is a 3D structure diagram of the transformed malonyl transferase fabD provided by Example 4 of the present application. [Figure 5] It is a chromatogram of the surfactant in the standard product and NJUXR-CY-4-1 fermentation broth in Example 5 of the present application.

Modes for Carrying Out the Invention

[0005] Hereinafter, it will be further 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, and 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, and it should be understood that this wild-type Bacillus subtilis may 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 ​Based on the IdeHA mutation, the wild-type biotin carboxylase IdeHA The amino acid sequence is shown in SEQ ID NO:5. Specifically, the lysine at position 74 of wild-type biotin carboxylase IdeHA is converted to asparagine. The 125th position alanine is changed to threonine, and the 136th position glutamic acid is changed to It was mutated into gin, the 145th cysteine ​​was mutated into phenylalanine, and the 394th thread By mutating onin to asparagine (corresponding to the underlined note in SEQ ID NO:1), the shape Obtain biotin carboxylase IdeHA after conversion, 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 LTFSQKDIQRNGHAIEVRIYAEDPKTFFFPSPGTITAFSLP DQKGVRHECAVAKDSTVTPFYDPMIAKMIVKGQTRTEAIE KLETALRDYRVEGIKTNLPLLIQAAATKAFKEGDVTTDFL KQHL The above method described in this embodiment will be explained in detail below.

[0006] Step 1: Creation of Bacillus subtilis receptor cells Wild-type Bacillus subtilis was spread onto LB solid medium and incubated at 37°C. Incubate overnight, extract single colonies, inoculate into 4 mL of LB liquid medium, and incubate at 37°C. The cells were cultured at 40 rpm for 6 hours, then washed three times with sterile 10 wt% glycerol solution. Later, the bacterial cells were resuspended in 0.2 mL of 10 wt% glycerol solution, dispensed, and chilled at -80°C. Stored in the warehouse and prepared, Step 2: Production of Bacillus subtilis receptor cells into which the pHT-XCR6 plasmid has been introduced. Here, the pHT-XCR6 plasmid (chloramphenicol resistance in Bacillus subtilis) is C This is a pf1 expression vector, and Cpf1 is regulated by xylose induction, and furthermore, The plasmid further contains NgAgo, improving the efficiency of homologous recombination during the gene editing process. Used for gene editing and expression regulation, the above pHT-XCR6 plasmid is used for gene editing and expression regulation. It is a vector used primarily in the study of Bacillus subtilis. It is used, and the production company is BIO-SCIENCE. Approximately 10 ng of pHT-XCR6 plasmid was added to 0.1 mL of Bacillus subtilis receptor cells, and then placed in an ice bath. The device is placed in a pre-cooled electric shock cup for 5 minutes, and then given one electric shock at 2.0kV. Resuscitate in 1 mL of LB medium for 2 hours, then in LB medium containing 50 ug / mL chloramphenicol. Bacillus subtilis was coated with solid culture medium, incubated overnight at 37°C, and plasmid pHT-XCR6 was introduced. Receptor cells are obtained and prepared by storing them in a refrigerator at -80°C. Step 3: Construction of the PCRF19NM plasmid The pcrF19NM plasmid is a mutation site for wild-type biotin carboxylase IdeHA. It includes information, is inserted between DR2 elements, and contains a crRN with target functionality. The A sequence is shown in Table 1. Recombinant plasmids containing the above mutation site information (also known as pcrF19NM plasmids) The construction process is as follows: (1) Insert a sequence containing site information into the pcrF19NM plasmid. For each mutation site, a pair of primers containing duplicate fragments with a crRNA sequence were designed. These are modified and annealed to form a primer dimer with adhesive ends, PCR The F19NM plasmid was cleaved with Eco31I, and the recovered product was converted into a primer dimer T4. Ligation is performed to obtain the ligation product, (2) Construction of a pcrF19NM plasmid containing the coding gene The above ligation product was transformed into E. coli DH5α and detected by colony PCR. After verification, the plasmid was extracted to obtain the pcrF19NM plasmid containing site information. The above pcrF19NM plasmid is a mutant of wild-type biotin carboxylase IdeHA. It contains positional information and is inserted between two DR2 elements in the pcrF19NM plasmid. The crRNA sequences with target function in the above pcrF19NM plasmid are shown in Table 1. It was shown, Table 1: crRNA sequences for editing the wild-type biotin carboxylase IdeHA gene. JPEG2026113368000001.jpg238168 Step 4: Construction of homologous fragments Homologous fragments are constructed using a complementary primer annealing procedure (also known as annealing PCR). The primer sequences that are built and used are shown in Table 2. Table 2: Primers for constructing homologous fragments for biotin carboxylase IdeHA gene editing array JPEG2026113368000002.jpg238168 Here, "F" represents the upstream primer, "R" represents the downstream primer, and the underlined sequence is unusual. It is a different site and is used for constructing homologous fragments for the biotin carboxylase IdeHA gene editing described above. In the primer sequence, A represents adenine and T represents thymine. C represents cytosine, G represents guanine. N stands for nine, where N represents one of A, T, C, or G, and K (Keto) is T Or it represents G, and M (amino) represents 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 JPEG2026113368000003.jpg238168 Table 4: Annealing PCR procedure JPEG2026113368000004.jpg238168 The PCR product obtained by the above annealing procedure is a homologous fragment. Step 5: Transformation of the PCRF19NM plasmid with homologous fragments. The pcrF19NM plasmid prepared in step 3, and the plasmid prepared in step 4 The homologous fragments are then subjected to plasmid pHT-XCR6 prepared in step 2 above. The cells were transformed into Bacillus subtilis receptor cells and treated with 50 ug / mL chloramphenicol and 50 ug / mL Spread the mixture onto LB solid medium supplemented with L-kanamycin and 3% xylose, and incubate overnight at 30°C. It is possible to perform liquid culture and fermentation verification using a single colony that has been nurtured and grown. the law of nature, Step 6: Production of surfactants by Bacillus subtilis culture and fermentation 48 single colonies of Bacillus subtilis were extracted from the plate (i.e., LB solid medium), and Each sample was inoculated into a 10 mL test tube containing 3 mL of LB liquid medium, and then incubated at 37°C at 200 rpm with shaking. Cultivate overnight in a potting bed to obtain a starter culture solution. Then, add 30 mL of fermentation medium (20 g / L gluco) to 1 mL of starter culture solution. - 3g / L tryptone, 3g / L K2HPO4, 10g / L NaH2PO4, 0 In addition to 0.02g / L mgSO4 (1g / L L-leucine), 37℃, 200rpm, vibration After culturing in a bed for 48 hours, a Bacillus subtilis fermentation solution is obtained. Step 7: Calculation of the surfactant content produced by Bacillus subtilis fermentation. The Bacillus subtilis fermentation liquid is centrifuged, 1 mL of the supernatant is taken, and centrifuged at 12000 rpm for 3 minutes. Take 200uL of supernatant, dilute it 3 to 10 times with methanol, shake well, and then 120 Centrifuged at 00 rpm for 10 minutes, passed the supernatant through a membrane to obtain the test sample, and then Agilen The t1260 high-performance liquid chromatography is used as the detection tool, and the column used is A It is a methyst C18-H column (5 μm, 250 × 4.6 mM), The mobile phase used is 90% methanol and 10% water, in addition to 0.05% Add lefluoroacetic acid, flow rate 0.8 mL / min, injection volume 20 μl, detection wavelength 214 The values ​​are nm, surfactant standards are prepared as gradient concentrations, a standard curve is created, and the fermented liquid is used. The surfactant content in the pull was calculated, and from the 200 strains of Bacillus subtilis extracted in step 6, the K Three strains were selected that showed the greatest increase in surfactant production, and each was designated as NJUXR-CY-1-1. These are denoted as CY-1-2, CY-1-3, Step 8: Genome sequencing of dominant strains Using the Gram-positive bacterial genome DNA extraction kit (Beijing Suolaibao), NJUXR-CY- The genomes of 1-1, CY-1-2, and CY-1-3 were extracted, and the NJUXR-CY-1-1 genome was extracted. Biotin carboxylase IdeHA gene on CY-1-2 and CY-1-3 genomes The offspring were sequenced, and here, biotin carboxylase on the NJUXR-CY-1-1 genome was identified. The sequence of the -se IdeHA gene is shown in SEQ ID NO:1, and lysine at position 74 is as The paragine was mutated, the alanine at position 125 was mutated to threonine, and the glutamic acid at position 136 was mutated. The cysteine ​​at position 145 was mutated to lysine, and the cysteine ​​at position 145 was mutated to phenylalanine, and position 39 The threonine at position 4 is mutated to asparagine, and the above mutation sites (positions 74, 125, and 1) Biotin carboxylase Id after transformation (corresponding to positions 36, 145, and 394) The position of eHA in the 3D structure is shown in Figure 1. Based on this, the embodiment of this application preserved the above-mentioned Bacillus subtilis NJUXR-CY-1-1. The Bacillus subtilis NJUXR-CY-1-1 is preserved at the China Center for the Preservation of Typical Cultures, and the storage location is China. It is located in Wuhan, Wuhan University, and was preserved on January 13, 2025, from the preservation unit. The assigned storage number is CCTCC NO:M 2025097, and the above Bacillus subtilis NJU The taxonomic name for XR-CY-1-1 (also called biomaterial) is Bacillus subtilis NJUXR-CY-1- 1, and the Latin name for this taxonomic name is Bacillus subtilis N It is JUXR-CY-1-1. Example 2: This example differs from Example 1 in the following respects, and the wild-type biotin carboxy of Bacillus subtilis is used. This further includes transforming sylase accBC and the transformed biotin carboxylase. The amino acid sequence of las accBC is shown in SEQ ID NO:2. The biotin carboxylase accBC obtained after the above transformation is equivalent to wild-type biotin carboxylase. Obtained based on the accBC mutation, and is an a mutation of wild-type biotin carboxylase accBC. The amino acid sequence is shown in SEQ ID NO:6, specifically wild-type biotin carboxyla - See the underlined note in SEQ ID NO:2 for serine at position 409 of -zeaccBC. (The corresponding mutation was introduced to obtain the transformed biotin carboxylase accBC.) SEQ ID NO:2 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 embodiment will be explained in detail below. Step 1: Creation of Bacillus subtilis receptor cells This step is basically the same as step 1 of Example 1, except for the following points, and Example The wild-type Bacillus subtilis used in 1 is the high-yielding strain NJUXR- screened in Example 1. Replace with CY-1-1, Step 2: Creation of Bacillus subtilis receptor cells into which plasmid pHT-XCR6 has been introduced. This step is the same as step 2 of Example 1, Step 3: Construction of the PCRF19NM plasmid This step is basically the same as step 3 of Example 1, except for the following: crR Replace the NA sequence with the one shown in Table 5. Table 5: crRNA sequences for biotin carboxylase accBC gene editing JPEG2026113368000005.jpg238168 Step 4: Construction of homologous fragments This step is basically the same as step 4 of Example 1, except for the following points, and is homologous. Replace the primer sequences for fragment construction with those shown in Table 6. Table 6: Primer configuration for constructing homologous fragments for biotin carboxylase accBC gene editing column JPEG2026113368000006.jpg238168 Step 5: Transformation of the PCRF19NM plasmid and homologous fragments This step is the same as step 5 of Example 1, Step 6: Production of surfactants by Bacillus subtilis culture and fermentation This step is the same as step 6 of Example 1, Step 7: Calculation of the surfactant content produced by Bacillus subtilis fermentation. This step is the same as step 7 of Example 1, specifically the two extracted in step 6. From 00 strains of Bacillus subtilis, the three strains that showed the greatest increase in surfactant production were selected as NJUXR-CY-2- 1, CY-2-2, CY-2-3 are denoted as follows: Step 8: Sequencing of the dominant strain genome. This step is basically the same as step 8 of Example 1, except for the following: The quench content includes the NJUXR-CY-2-1 genome, the CY-2-2 genome, and the CY-2-3 genome. This is the biotin carboxylase accBC gene, where NJUXR-CY-2- The mutation pattern of the biotin carboxylase accBC gene in one genome is serine at position 409. The mutated portion was changed to glycine, and its sequence is shown in SEQ ID NO:2, with the above mutation site (4 In the 3D structure of biotin carboxylase accBC after transformation (corresponding to rank 9) The location is shown in Figure 2. Based on this, the embodiment of this application preserved the above-mentioned Bacillus subtilis NJUXR-CY-2-1. The Bacillus subtilis NJUXR-CY-2-1 is preserved at the China Center for the Preservation of Typical Cultures, and the storage location is China. It is located in Wuhan, Wuhan University, and was preserved on January 13, 2025, from the preservation unit. The assigned storage number is CCTCC NO:M 2025099, and the above Bacillus subtilis NJU The taxonomic name for XR-CY-2-1 (also called biomaterial) is Bacillus subtilis NJUXR-CY-2- 1, and the Latin name for this taxonomic name is Bacillus subtilis N It is JUXR-CY-2-1. Example 3: This example differs from Example 2 in the following respects, and involves the wild-type enoyl reducter of Bacillus subtilis. This further includes transforming zefabI, and the transformed enoyl reductase fab The amino acid sequence of I is shown in SEQ ID NO:3. The enoyl reductase fabI after the above transformation is similar to wild-type enoyl reductase fab. Based on the I mutation, the amino acid sequence of wild-type enoyl reductase fabI is SEQ ID NO:7 is shown, specifically, of wild-type enoyl reductase fabI, number 263. By mutating glycine at position 3 to cysteine ​​(corresponding to the underlined note in SEQ ID NO:3), Obtain enoyl reductase fabI after transformation, SEQ ID NO:3 MSLLNIGGFIHMNFSLEGRNIVVMGVANKRSIAWGIARSL HEAGARLIFTYAGERLEKSVHELAGTLDRNDSIILPCDVT NDAEIETCFASIKEQVGVIHGIAHCIAFANKEELVGEYLN TNRDGFLLAHNISSYSLTAVVKAARPMMTEGGSIVTLTYL GGELVMPNYNVMGVAKASLDASVKYLAADLGKENIRVNSI SAGPIRTLSAKGISDFNSILKDIEERAPLRRTTTPEEVGD TAAFLFSDMSRGITGENLHVDS C FHITAR SEQ ID NO:7 MSLLNIGGFIHMNFSLEGRNIVVMGVANKRSIAWGIARSL HEAGARLIFTYAGERLEKSVHELAGTLDRNDSIILPCDVT NDAEIETCFASIKEQVGVIHGIAHCIAFANKEELVGEYLN TNRDGFLLAHNISSYSLTAVVKAARPMMTEGGSIVTLTYL GGELVMPNYNVMGVAKASLDASVKYLAADLGKENIRVNSI SAGPIRTLSAKGISDFNSILKDIEERAPLRRTTTPEEVGD TAAFLFSDMSRGITGENLHVDSGFHITAR The method described in this embodiment differs from the method described in Example 2 in the following respects: In Step 1, the high-yielding strain NJUXR-CY-2-1 is used as the Bacillus subtilis receptor 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. JPEG2026113368000007.jpg238168 In Step 4, the primer sequences of the constructed homologous fragments are shown in Table 8. Table 8. Homologous fragment construction primer sequences for enoyl reductase fabI gene editing. JPEG2026113368000008.jpg238168 In the homologous fragment construction primer sequence for enoyl reductase fabI gene editing described above... N represents one of A, T, C, or G. In step 5, replace the extracted single colonies from 200 to 100. In step 6, one strain of Bacillus subtilis was extracted from each of the 100 single colonies, and 1 We obtained 00 strains of Bacillus subtilis, In step 7, the amount of surfactant produced from the 100 strains of Bacillus subtilis extracted in step 6 was The three strains that showed the greatest increase were designated NJUXR-CY-3-1, CY-3-2, and CY-3-3. In step 8, the sequence content is NJUXR-CY-3-1 genome, CY-3-2 genome This is the enoyl reductase fabI gene of the nom and CY-3-3 genome, where N The mutation pattern of enoyl reductase fabI in JUXR-CY-3-1 is glycine at position 263. The mutation was performed on cysteine, and the sequence is shown in SEQ ID NO:3, and this mutation site (2 The position of the transformed enoyl reductase fabI (corresponding to rank 63) in the 3D structure is As shown in Figure 3, Based on this, the embodiment of this application preserved the above-mentioned Bacillus subtilis NJUXR-CY-3-1. The Bacillus subtilis species NJUXR-CY-3-1 is preserved at the China Center for the Preservation of Typical Cultures, and the storage location is It is located at Wuhan University in Wuhan, China, and the preservation date is January 13, 2025. The assigned storage number is CCTCC NO:M 2025103, and the above Bacillus subtilis NJ The taxonomic name of UXR-CY-3-1 (also called biomaterial) is Bacillus subtilis NJUXR-CY- 3-1, and the Latin name for this taxonomic name is Bacillus subtilis It is NJUXR-CY-3-1. Example 4: This example differs from Example 3 in the following respects: the wild-type maloniltran of Bacillus subtilis. The process further includes transforming with spherase fabD, and the post-transformation malonyltransfer The amino acid sequence of ferase fabD is shown in SEQ ID NO:4. The malonyltransferase fabD after the above transformation is wild-type malonyltransferase Based on the fabD mutation, the wild-type malonyltransferase fabD The malonyltransferase sequence is shown in SEQ ID NO:8, specifically wild-type malonyltransferase. The 64th position of the enzyme fabD is proline, which is replaced by arginine (underlined note in SEQ ID NO: 4). (corresponding to) the mutated form is used 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 method described in this embodiment differs from the method described in Example 3 in the following respects: In Step 1, the high-yielding strain CY-3-1 is used as the Bacillus subtilis receptor cell. In step 3, the crRNA sequence on the pcrF19NM plasmid is shown in Table 9. , Table 9: crRNA sequences for malonyltransferase fabD gene editing JPEG2026113368000009.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 malonyltransferase fabD gene editing array JPEG2026113368000010.jpg238168 Primer sequences for constructing homologous fragments for malonyltransferase fabD gene editing. In this, N represents one of A, T, C, or G. In Step 7, the amount of surfactant produced was determined from the 96 strains of Bacillus subtilis extracted in Step 6. The three strains that also increased were designated NJUXR-CY-4-1, CY-4-2, and CY-4-3. In step 8, the sequence content is NJUXR-CY-4-1 genome, CY-4-2 genome This is the malonyltransferase fabD gene of the nom and CY-4-3 genome, and here So, the mutation pattern of malonyltransferase fabD in the NJUXR-CY-4-1 genome is The 64th amino acid, proline, was mutated to arginine, and the sequence is shown in SEQ ID NO:4. Therefore, the malonyltransferase fa after transformation of the aforementioned mutation site (corresponding to position 64) The position of bD in the 3D structure is shown in Figure 4. This example uses a surfactant standard (99% purity, Shanghai McLin) and NJUXR-CY-4- 1. Detect the surfactant in the fermentation liquid and compare it with the standard (i.e., the surfactant) and NJUXR-C. The chromatogram of surfactants in the Y-4-1 fermentation liquid is shown in Figure 5, and as can be seen from Figure 5... Compared to the wild-type strain, the amount of surfactant produced by the constructed NJUXR-CY-4-1 strain was The production of NJUXR-CY-4-1 increased significantly, and its production was 14.7% higher compared to wild-type Bacillus subtilis. The gene mutation method described in the present invention doubled, and effectively improved the production volume of surfactants. Reflecting this, Based on this, the above Bacillus subtilis NJUXR-CY-4-1 was preserved, and this Bacillus subtilis NJUXR-C Y-4-1 was placed in the China Center for the Preservation of Typical Cultures on January 13, 2025, and The reference number is CCTCC M 2025098. Based on this, the embodiments of this application preserve the above-mentioned Bacillus subtilis NJUXR-CY-4-1. Bacillus subtilis NJUXR-CY-4-1 is preserved at the China Center for the Preservation of Typical Cultures, located in China. It is located in Wuhan, Wuhan University, and the preservation date is January 13, 2025, and it is attached to the preservation unit. The assigned storage number is CCTCC NO:M 2025098, and it corresponds to the above-mentioned Bacillus subtilis NJUX. The taxonomic name for R-CY-4-1 (also called biological material) is Bacillus subtilis NJUXR-CY-4-1 The Latin name for this taxonomic name is Bacillus subtilis NJ It is UXR-CY-4-1. Example 5: This example demonstrates the application of the surfactant described in Examples 1-4 in the production of surfactants. To serve, 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- Strains 3-3, NJUXR-CY-4-1, CY-4-2, and CY-4-3 were placed on LB solid plates. (i.e., scribe onto LB solid medium), incubate overnight at 37°C, and extract single colonies.) Then, each sample was inoculated into a 10 mL test tube containing 3 mL of LB liquid medium, and incubated at 37°C for 200°C. In the afternoon, culture overnight on a shaking bed to obtain the starter culture solution, and then add 1 mL of starter culture solution to 30 mL of fermentation medium (20 g / L-glucose, 3g / L tryptone, 3g / L K2HPO4, 10g / L NaH2P In addition to O4, 0.02g / L mgSO4, 1g / L L-leucine), 37℃, 200r The fermentation broth of wild-type Bacillus subtilis and NJUXR-CY-1- were cultured for 48 hours in a shaking bed, respectively. Fermentation liquid of 1, fermentation liquid of CY-1-2, fermentation liquid of CY-1-3, fermentation of NJUXR-CY-2-1 Liquid, fermented liquid of CY-2-2, fermented liquid of CY-2-3, fermented liquid of NJUXR-CY-3-1, CY- Fermentation liquid of 3-2, fermentation liquid of CY-3-3, fermentation liquid of NJUXR-CY-4-1, fermentation liquid of CY-4-2 The fermentation liquid and the fermentation liquid of CY-4-3 were centrifuged, 1 mL of the supernatant was taken, and it was divided at 12000 rpm for 3 minutes. Centrifuge for 1 minute, take 200 uL of supernatant, add methanol to 3 to 10 times the volume, and shake well. After that, the mixture was centrifuged at 12000 rpm for 10 minutes, and the supernatant was passed through a membrane before being subjected to high-performance liquid chromatography. It was detected using matrixing. The final production amounts of surfactants in the 13 types of fermentation liquids obtained are shown in Table 11. Table 11: Production volume of surfactants from different bacterial strains JPEG2026113368000011.jpg238168 NJUXR-CY-1-1, CY-1-2, and CY-1-3 listed in Table 11 above are biotincarb It is a xylase IdeHA mutant strain, and among them, the surfactant NJUXR-CY-1-1 The production volume is the highest at 1.5g / L, and it is NJUXR-CY-1-1 biotin carboxylate. The lysine at position 74 of zeIdeHA is mutated to asparagine, and the alanine at position 125 is threo By mutating to nin, the glutamic acid at position 136 is mutated to lysine, and the cysteine ​​at position 145 is The phenylalanine was mutated, and the threonine at position 394 was mutated to asparagine, as shown in the table. The listed NJUXR-CY-2-1, CY-2-2, and CY-2-3 are biotin carboxylase ac It is a cBC mutant strain, and among them, the surfactant production volume of NJUXR-CY-2-1 is 5 It has the highest concentration at 0.4g / L, and is NJUXR-CY-2-1 biotin carboxylase accBC. The serine at position 409 is mutated to glycine, resulting in NJUXR-CY-3-1, CY-3-2, CY- 3-3 is a mutant strain of enoyl reductase fabI, and among them, NJUXR-CY- The production volume of surfactant 3-1 was the highest at 9.5g / L, and NJUXR-CY-3-1 Enoi By mutating the glycine at position 263 of lureductase fabI to cysteine, NJUXR-C Y-4-1, CY-4-2, and CY-4-3 are mutant strains of malonyltransferase fabD. Of these, the production volume of the surfactant NJUXR-CY-4-1 was the highest at 13.3 g / L. Highly, the 64th amino acid of malonyltransferase fabD of NJUXR-CY-4-1 The proline acid is mutated into arginine. As can be seen from Table 13, Examples 1 to 4 improved the production of surfactants by Bacillus subtilis. This is the Bacillus subtilis bacterium that produced the largest amount of surfactant by the method, and its surfactant activity The production volume of the agent was superior to that of wild-type Bacillus subtilis in all cases, as shown in Examples 1 to 4. The obtained strains NJUXR-CY-1-1~CY-4-3, compared to the original wild-type Bacillus subtilis, This demonstrated an improvement in the production capacity of surfactants. As can be seen from the analysis of Table 13, regarding the production volume of surfactants, NJUXR-C Y-4-1>NJUXR-CY-3-1>NJUXR-CY-2-1>NJUXR-CY-1-1> This is a wild-type Bacillus subtilis, and as can be seen from this, biotin carboxy in wild-type Bacillus subtilis IdeHA enzyme, biotin carboxylase accBC, enoyl reductase fab After transformation with I and malonyltransferase fabD, the resulting Bacillus subtilis is at the interface The production of surfactants increased most dramatically, and the amount of surfactant produced was far greater than that of wild-type Bacillus subtilis. It's expensive. <Copy of the deposit certificate for the deposit of microorganisms> JPEG2026113368000012.jpg238169 JPEG2026113368000013.jpg229166 JPEG2026113368000014.jpg231166 JPEG2026113368000015.jpg238169 [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< / ipofficecode> <applicationnumbertext / > <filingdate / > < / applicationidentification> <applicantfilereference> 10284< / applicantfilereference> <earliestpriorityapplicationidentification> <ipofficecode> CN< / ipofficecode> <applicationnumbertext> 202411924439.3< / applicationnumbertext> <filingdate> 2024-12-25< / filingdate> < / earliestpriorityapplicationidentification> <applicantname languagecode="ja"> Nanjing University< / applicantname> <applicantnamelatin> Nanjing University< / applicantnamelatin> <inventiontitle languagecode="ja">A method to improve the production of surfactants by Bacillus subtilis. 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INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q8"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MSKIAFLFPGQGSQFIGMGKELYEQVPAAKRLFDEADETLETKLSSLIFEGDAEELTLTYNAQRALLTTSIAVLEKFKES GITPDFTAGHSLGEYSALVAAGALSFKDAVYTVRKRGEFMNEAVPAGEGAMAAILGMDAEALKQVTDKVTEEGNLVQLAN LNCPGQIVISGTAKGVELASELAKENGAKRAIPLEVSGPFHSELMKPAAEKLKEVLDACDIKDADVPVISNVSADVMTEK ADIKEKLIEQLYSPVRFEESINKLIAEGVTTFIEIGPGKVLSGLVKKVNRRLKTIAVSDPETIELAIQTLKEENDNA < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="5"> <insdseq> <INSDSeq_length>444< / INSDSeq_length> <INSDSeq_moltype>AA< / INSDSeq_moltype> <INSDSeq_division>PAT< / INSDSeq_division> <INSDSeq_feature-table> <insdfeature> <INSDFeature_key>source< / INSDFeature_key> <INSDFeature_location>1..444< / INSDFeature_location> <INSDFeature_quals> <insdqualifier> <INSDQualifier_name>mol_type< / INSDQualifier_name> <INSDQualifier_value>protein< / INSDQualifier_value> < / insdqualifier> <insdqualifier id="q10"> <INSDQualifier_name>organism< / INSDQualifier_name> <INSDQualifier_value>synthetic construct< / INSDQualifier_value> < / insdqualifier> < / INSDFeature_quals> < / insdfeature> < / INSDSeq_feature-table> <INSDSeq_sequence> MFTKVLIANRGEIAMRIIRTCSRLGIKTVAVYSEADKDAPHTKAATEAYLIGESRVSESYLNIERIIKTAKKAKADAIHP GYGLLSENSRFAERCKQENIVFIGPSPDIIAKMGSKIEARKAMEAAGVPVVPGVSESLGDIEAACRTASQIGYPVMLKAS AGGGGIGMQRVENEEALKKAYEGNKKRAADFFGDGSMYIEKVIEHARHIEVQLLADQHGHTVHLFERDCSVQRRHQKVIE EAPSPFVDDELRMKIGQTAVKAAKAIGYTNAGTIEFIVDQKQNFYFLEMNTRLQVEHPVTEEITGLDLVEQQLRIAAGHT LTFSQKDIQRNGHAIEVRIYAEDPKTFFPSPGTITAFSLPDQKGVRHECAVAKDSTVTPFYDPMIAKMIVKGQTRTEAIE KLETALRDYRVEGIKTNLPLLIQAAATKAFKEGDVTTDFLKQHL < / INSDSeq_sequence> < / insdseq> < / sequencedata> <sequencedata sequenceidnumber="6"> <insdseq> <INSDSeq_length>450< / 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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> aaaggccaaaccagaacaga< / 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> aaaaaagcaacgccgacgcgatccacccgggatg< / 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> gcaatggagacggcaggtgtccctgtgggcggc< / 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> ggcgtttctnnctccctcggagatatagaggcagcc< / 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> gaggcagccnncgcaccgcaagtcaaatcggctatc< / 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> tgcggtgcgmngctgcctctatatctccgaggg< / 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> gacgaattcgcccaatgcgcgcttgcgggcaatc< / 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

1. A method for improving the production of surfactants by Bacillus subtilis, This involves transforming the wild-type biotin carboxylase IdeHA of Bacillus subtilis, The amino acid sequence of biotin carboxylase IdeHA after transformation is SEQ ID NO. : As shown in 1, A method for improving the production of surfactants by Bacillus subtilis, characterized by the features described above.

2. The process further includes transforming the wild-type biotin carboxylase accBC of Bacillus subtilis. The amino acid sequence of biotin carboxylase accBC after transformation is SEQ ID The method according to claim 1, characterized by being shown in NO:

2.

3. The method further includes transforming the wild-type enoyl reductase fabI of Bacillus subtilis, The amino acid sequence of enoyl reductase fabI after conversion is shown in SEQ ID NO:

3. The method according to the second method, characterized in that it is performed.

4. The process further includes transforming the Bacillus subtilis with wild-type malonyltransferase fabD. The amino acid sequence of malonyltransferase fabD after transformation is SEQ ID The method according to the present invention as shown in NO:

4.

5. Applications in the production of surfactants according to the method described in any one of claims 1 to 4.

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