Recombinant bacterium for high-yield surfactin homologue and application of recombinant bacterium

Abstract

The invention relates to a recombinant bacterium for producing surfactin homologues at high yield and application of the recombinant bacterium, and belongs to the technical field of microbial fermentation. According to the application disclosed by the invention, the yield of C14-Surfactin and C15-Surfactin can be obviously improved by knocking out beta-oxidation cycle pathway related genes and amino acid degradation pathway related genes. In addition, according to the invention, a carbon source and a nitrogen source are also optimized, and the production proportions of the C14-Surfactin and the C15-Surfactin are further improved. When the carbon and nitrogen sources are glucose, isobutyric acid and sodium nitrate, the recombinant bacteria without the FadE gene and the glnA gene can produce C14 surfactin at high yield; when the carbon and nitrogen sources are glucose, isovaleric acid and leucine, the recombinant bacteria with the FadD gene and the bkdAB gene knocked out can produce C15 surfactin at high yield. The invention provides a new thought for the production of surfactin homologues, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, in particular to a recombinant bacterium with high yield of surfactin homologues and application thereof. Background Art

[0002] Surfactin is a secondary metabolite produced by Bacillus subtilis. Its structure is a cyclic lipopeptide composed of a heptapeptide chain (L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu) and a C13-C15 β-hydroxy fatty acid chain. Due to the different lengths of the fatty acid chains, surfactin has many homologous components, resulting in different properties such as antibacterial, hemolytic, and emulsifying activities. Studies have shown that C13-surfactin and C14-surfactin have excellent foaming and emulsifying properties and have broader application prospects in daily chemical care. C15-surfactin exhibits extremely strong surface activity, more than 1,000 times higher than the traditional chemical surfactant sodium dodecyl sulfate (SDS), and has higher antibacterial, antiviral, and hemolytic activities, and has broader applications in medicine, environmental remediation, and oil recovery.

[0003] At present, some studies have shown that the proportion of surfactin homologues synthesized can be changed by changing the nutrient components in the culture medium. For example, the existing literature (Zhu Lingyan, Liu Qiang, Liu Yang, et al. Effect of culture medium components on the production of surfactin by Bacillus subtilis [J]. Bioprocessing, 2015, 13(05):8-13) disclosed the optimization of parameters such as pH, carbon source, nitrogen source, carbon-nitrogen ratio, metal ions and amino acids in the fermentation medium of Bacillus subtilis BS-37, which increased the proportion of C15-Surfactin components to 68%; for example, the existing literature (Ding L, Guo W, Chen X. Exogenous addition of alkanoic acids enhanced production of antifungal lipopeptides in Bacillus amyloliquefaciens Pc3. Appl Microbiol Biotechnol. 2019 Jul; 103(13):5367-5377) reported that in Bacillus amyloliquefaciens Adding heptadecanoic acid to the PC3 growth medium can increase the production of surfactin C15 component by 1.83 times, reaching 20% ​​to 30%. In addition, in terms of genetic engineering modification, the existing literature (Dhali D, Coutte F, Arias AA, Auger S, Bidnenko V, Chataigné G, Lalk M, Niehren J, de Sousa J, Versari C, Jacques P. Genetic engineering of the branched fatty acid metabolic pathway of Bacillus subtilis for the overproduction of surfactin C14 isoform. Biotechnol J. 2017 Jul; 12 (7)) disclosed that the knockout of the branched-chain amino acid degradation pathway enzyme lpdV gene increased the proportion of the linear C14-surfactin component to 53%. For example, patent CN202011390286 discloses that heterologous expression of the acyl carrier protein (ACP) thioesterase BTE, which prefers medium-chain C14 acyl groups, from the plant Umbellularia californica and enhanced expression of acetoin dehydrogenase can increase the proportion of nC14-surfactin produced by genetically engineered bacteria to 55%-60%. However, current regulatory strategies have problems such as unclear component synthesis targeting, cumbersome operations, and limited regulatory scope, which are not conducive to industrial production and the needs of different application scenarios. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a method for efficiently synthesizing different surfactin homologues.

[0005] In order to solve the above technical problems, the present invention provides a recombinant bacterium with high production of surfactin homologues and its application. The present invention found that knocking out the relevant genes of the β-oxidation cycle pathway and / or knocking out the relevant genes of the amino acid degradation pathway can significantly increase the production of C14-Surfactin and C15-Surfactin. In addition, the present invention also optimizes the carbon source and nitrogen source to further increase the production ratio of C14-Surfactin and C15-Surfactin. When the carbon and nitrogen sources are glucose, isobutyric acid and sodium nitrate, the recombinant bacteria with only the glnA gene knocked out or the FadE gene and the glnA gene knocked out can produce high C14-Surfactin; when the carbon and nitrogen sources are glucose, isovaleric acid and leucine, the recombinant bacteria with only the bkdAB gene knocked out or the FadD gene and the bkdAB gene knocked out at the same time can produce high C15-Surfactin.

[0006] The first object of the present invention is to provide a recombinant bacterium that produces a high-yield surfactin homolog. The recombinant bacterium is modified in the following manner for Bacillus subtilis EX10: one or more of the following genes are deleted: acyl-CoA synthetase gene, acyl-CoA dehydrogenase gene, acetyl-CoA-carbonyltransferase gene, enoyl-CoA hydratase gene, glutamine synthetase gene, aromatic amino acid aminotransferase gene, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAA protein gene, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAB protein gene, branched-chain α-ketoacid dehydrogenase E2 subunit gene, and branched-chain α-ketoacid dehydrogenase E3 subunit gene.

[0007] Furthermore, the Gene ID of the acyl-CoA synthetase gene is 937336, the Gene ID of the acyl-CoA dehydrogenase gene is 936717, the Gene ID of the acetyl-CoA carbonyltransferase gene is 936729, and the Gene ID of the enoyl-CoA hydratase gene is 937448;

[0008] The Gene ID of the glutamine synthetase gene is 940020, the Gene ID of the aromatic amino acid aminotransferase is 938420, the Gene ID of the branched-chain α-ketoacid dehydrogenase E1 subunit bkdAA protein gene is 938674, the Gene ID of the branched-chain α-ketoacid dehydrogenase E1 subunit bkdAB protein gene is 938672, and the Gene ID of the branched-chain α-ketoacid dehydrogenase E3 subunit gene is 938669.

[0009] Furthermore, the surfactin homologues include surfactins whose β-hydroxy fatty acid chain has a carbon chain length of 13-15.

[0010] Furthermore, the Bacillus subtilis includes Bacillus subtilis EX10, which is disclosed in patent application number 2025102397555.

[0011] The second object of the present invention is to provide a method for producing surfactin homologues, wherein the above-mentioned recombinant bacteria are inoculated into a fermentation medium for fermentation culture to obtain the surfactin homologues.

[0012] Furthermore, the surfactin homologues include surfactins whose β-hydroxy fatty acid chain has a carbon chain length of 13-15.

[0013] Furthermore, the fermentation medium comprises a main carbon source, a supplementary carbon source, a nitrogen source, an inorganic salt and metal ions, and the main carbon source comprises glucose.

[0014] Furthermore, the supplementary carbon source is selected from one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, 2-methylbutyric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, isopentadecanoic acid, stearic acid and arachidic acid.

[0015] Furthermore, the nitrogen source is selected from one or more of sodium nitrate, ammonium sulfate, alanine, glutamic acid, aspartic acid, valine, leucine, glycine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, and histidine.

[0016] Furthermore, the mass ratio of the main carbon source, the supplementary carbon source and the nitrogen source is (40-60): (2-6): (1-30).

[0017] Furthermore, when the supplemental carbon source is isobutyric acid and the nitrogen source is sodium nitrate, and the mass ratio of glucose:isobutyric acid:sodium nitrate is (40-60):(2-6):(10-30), the recombinant bacteria have glnA knocked out alone, FadE and glnA knocked out in combination, or FadE and LpdV knocked out in combination, and the recombinant bacteria produce high C14-Surfactin. Preferably, the mass ratio of glucose:isobutyric acid:sodium nitrate is 40:4:20.

[0018] When the supplemental carbon source is isovaleric acid and the nitrogen source is leucine, and the mass ratio of glucose:isovaleric acid:leucine is (40-60):(2-6):(1-3), the recombinant bacteria with bkdAB knockout or combined knockout of FadD and bkdAB produce high C15-Surfactin. Preferably, the mass ratio of glucose:isovaleric acid:leucine is 60:6:3.

[0019] The third object of the present invention is to provide a use of the above-mentioned recombinant bacteria in the preparation of surfactin homologues.

[0020] Beneficial effects of the present invention:

[0021] The present invention regulates the expression of genes related to the β-oxidation cycle pathway and the expression of genes related to the amino acid degradation pathway, and finds that the related genes can significantly increase the production of C14-Surfactin and C15-Surfactin. In addition, the present invention also optimizes the carbon source and nitrogen source, and finds that different carbon and nitrogen sources can regulate the proportion of C14-Surfactin and C15-Surfactin in the production process of surfactant. Specifically, when the carbon and nitrogen sources are glucose, isobutyric acid and sodium nitrate, recombinant bacteria that only knock out the glnA gene or knock out the FadE gene and glnA gene can produce a high yield of C14-Surfactin, which can reach 85%; when the carbon and nitrogen sources are glucose, isovaleric acid and leucine, recombinant bacteria that only knock out the bkdAB gene or knock out the FadD gene and bkdAB gene at the same time can produce a high yield of C15-Surfactin, which can reach 82%. The present invention provides new ideas for how to regulate the production ratio of C14-Surfactin and C15-Surfactin and how to increase the yield of surfactin, and has broad application prospects. DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0023] The culture medium involved in the following examples is as follows:

[0024] LB medium: peptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g, distilled water 1000 mL, pH 7.0-7.2;

[0025] Fermentation medium: glucose 40 g / L, yeast extract 3 g / L, MgSO4·7H2O 0.15 g / L, FeSO4·7H2O 0.006 g / L, MnSO4·H2O 0.006 g / L, Na2HPO4 23.4 g / L, citric acid 3.4 g / L.

[0026] The growth temperature of E. coli and Bacillus was 37°C and 200 rpm.

[0027] The Bacillus gene editing technology used in the following examples is a homologous recombination scarless knockout technology, including endogenous DNA knockout and heterologous DNA integration technology, see the literature (Gao Ling; Ma W.; Lu Z.; Han J.; Ma Z.; Liu H.; Bie X.; Translocation of subunit PPSE in plipastatin synthase and synthesis of novel lipopeptides[J]. Synthetic and Systems Biotechnology, 2022, 7(4): 1173-1180.).

[0028] The following examples involve the preparation method of Bacillus subtilis competent cells as follows:

[0029] (1) Streak the Bacillus subtilis strain onto an LB plate;

[0030] (2) A single colony was inoculated into 5 mL of GM I medium and cultured overnight at 30°C and 100 rpm.

[0031] (3) Transfer 1 mL of culture medium to 10 mL of GM I medium and incubate at 37°C, 200 rpm, for 3.5 h.

[0032] (4) Transfer 2 mL of culture medium to 18 mL of GMII medium and incubate at 37°C, 100 rpm, for 1.5 h.

[0033] (5) Centrifuge at 5000 g for 10 min at 4°C to collect the cells;

[0034] (6) Gently suspend the bacteria in 2 mL of the original culture supernatant. The suspended bacteria are competent cells and can be directly used for transformation.

[0035] The screening and identification methods of the recombinant strains involved in the following examples are as follows:

[0036] Add 1 μg / mL of the constructed knockout vector to a centrifuge tube containing 200 μL of competent bacterial culture and incubate at 30°C, 100 rpm for 1 hour. Spread the competent strain containing the knockout vector onto a kanamycin-resistant plate and incubate at 30°C for 12-16 hours. Verify transformants by colony PCR. PCR products whose band size matches the theoretical value should be sent for sequencing. Verify transformants by colony PCR. PCR products whose band size matches the theoretical value should be sent for sequencing. Strains that have been sequenced correctly should be expanded and stored at -80°C.

[0037] According to the characteristics of temperature-sensitive plasmid, Bacillus subtilis containing knockout vector was inoculated into LB liquid medium and cultured at 37℃ and 180rpm shaking for 24h. -4 Culture in LB + Kn 50 μg / mL at 37°C overnight to obtain colonies after the first single exchange of the integrated plasmid at the homology arm position; then, pick 1-2 single colonies and inoculate them into LB liquid culture at 30°C and 180 rpm for 48 hours (to induce the second single exchange of the homology arm), dilute and spread on non-resistant plates and culture at 37°C overnight, pick a single colony with a toothpick and gently spot it on the first resistant plate, and gently spot the same toothpick on the second non-resistant plate to screen transformants sensitive to kanamycin, pick sensitive transformants for PCR verification, and finally achieve non-resistance marker knockout.

[0038] The following examples relate to methods for detecting Surfactin homologues:

[0039] HPLC detection was performed using an Agilent C18 column (4.6 mm × 250 mm × 5 μm). The eluents consisted of 1% trifluoroacetic acid in ultrapure water and 1% trifluoroacetic acid in acetonitrile. The elution gradient was as follows: 0-9 min, 60% (volume) phase B, 40% (volume) phase A; 9-30 min, 93% (volume) phase B, 7% (volume) phase A, at a flow rate of 0.800 mL / min. The injection volume was 20 μL, and the detection wavelength was 210 nm. Phase B consisted of 1% trifluoroacetic acid in acetonitrile, and phase A consisted of 1% trifluoroacetic acid in ultrapure water.

[0040] Example 1: Effects of different carbon sources on the production of surfactin homologues by Bacillus subtilis

[0041] Bacillus subtilis EX10 was inoculated into 30 mL of LB medium, cultured for 12-16 hours, and then transferred to new 50 mL of LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as the seed solution for shake flask fermentation.

[0042] The cells were transferred to 50 mL of fermentation medium at a volume ratio of 3% and shake flask experiments were performed. 4 g / L of supplementary carbon source was added to different fermentation mediums. The shaker temperature was 37°C and the speed was 200 rpm. After 18 h of culture, the cell density OD was measured. 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin, and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Table 1.

[0043] Table 1 Comparison of fermentation with C2-C20 branched and straight chain fatty acids as supplementary carbon sources

[0044]

[0045]

[0046] The results showed that Bacillus subtilis EX10 could synthesize surfactant using C1-C20 saturated fatty acids as supplementary carbon sources, which had different effects on cell growth. 600 =0.3-10, affecting the production of surfactin at 0.2-10 g / L. When using even-chain saturated fatty acids, the proportion of its homologue component C14-Surfactin is relatively high, especially when isobutyric acid and myristic acid are used as supplementary carbon sources, the proportion of C14-Surfactin components is as high as over 50%. When using odd-chain saturated fatty acids, the proportion of its homologue component C15-Surfactin is relatively high, especially when isovaleric acid and 2-methylbutyric acid are used as supplementary carbon sources, the proportion of C15-Surfactin components is as high as over 60%.

[0047] Example 2: Effects of different nitrogen sources on the production of surfactin homologues by Bacillus subtilis

[0048] Bacillus subtilis EX10 was inoculated into 30 mL of LB medium, cultured for 12-16 hours, and then transferred to a new 50 mL of LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as the seed solution for shake flask fermentation.

[0049] Transfer the cells to 50 mL of fermentation medium at a volume ratio of 3% and perform shake flask experiments. The concentration of amino acid nitrogen source is 2 g / L, the concentration of inorganic nitrogen source is 20 g / L, the shaker temperature is 37°C, and the speed is 200 rpm. After 18 h of culture, the cell density OD 600, surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Table 2.

[0050] Table 2 Comparison of fermentation with inorganic carbon sources and 20 amino acids as nitrogen sources

[0051] Serial number Name of supplemental nitrogen source C13-Surfactin (wt%) C14-Surfactin (wt%) C15-Surfactin (wt%) 0 \ 21 40 39 1 sodium nitrate 22 50 28 2 ammonium sulfate 21 34 45 3 Alanine 20 39 41 4 glutamate 20 50 30 5 Aspartic acid 21 31 48 6 Valine 19 55 26 7 Leucine 18 28 54 8 Glycine 21 38 42 9 Isoleucine 16 38 46 10 Methionine 22 41 37 11 Proline 19 45 36 12 Tryptophan 21 36 43 13 Serine 16 48 38 14 Tyrosine 24 40 36 15 Cysteine 22 42 36 16 Phenylalanine 21 40 39 17 Asparagine 19 45 36 18 glutamine 18 44 38 19 Threonine 21 38 41 20 Lysine 21 39 40 21 Arginine 20 41 39 22 Histidine 22 36 42

[0052] The results showed that Bacillus subtilis EX10 could synthesize surfactant by supplementing inorganic nitrogen sources such as sodium nitrate, ammonium sulfate and 20 common amino acids as nitrogen sources, which had different effects on cell growth. 600 =1.2-10, and the surfactin production ranged from 0.8-10g / L. Among them, fermentation using sodium nitrate, valine, and glutamic acid resulted in a relatively high proportion of the homologue component C14-Surfactin, especially when valine was used as the nitrogen source, producing a C14-Surfactin homologue component accounting for up to 55%. Fermentation using ammonium sulfate, leucine, and aspartic acid resulted in a relatively high proportion of the C15-Surfactin homologue component, especially when leucine was used as the nitrogen source, producing a C15-Surfactin homologue component accounting for up to 54%.

[0053] Example 3: Effect of Mixed Carbon and Nitrogen Sources on the Production of C14-Surfactin by Bacillus subtilis

[0054] Bacillus subtilis EX10 was inoculated into 30 mL of LB medium, cultured for 12-16 hours, and then transferred to new 50 mL of LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as the seed solution for shake flask fermentation.

[0055] Transfer to 50 mL fermentation medium at a volume ratio of 3% and perform shake flask experiments. The mixed carbon and nitrogen sources are mainly glucose as the main carbon source, 4 g / L of fatty acid carbon source (isobutyric acid, myristic acid) and 20 g / L of inorganic nitrogen source (sodium nitrate) or 2 g / L of amino acid nitrogen source (valine, glutamic acid, serine). The shaker temperature is 37 ° C and the speed is 200 rpm. After 18 hours of culture, the cell density OD is measured. 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin, and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Tables 3-4.

[0056] Table 3 Comparison of fermentation of C14-Surfactin produced by mixed carbon and nitrogen sources

[0057]

[0058] Table 4 Optimization of C14-Surfactin addition concentration in the production of mixed carbon and nitrogen sources

[0059]

[0060] The results showed that when glucose was the main carbon source, the strain growth and product yield were good when additional carbon and nitrogen sources were added. The composition of surfactin homologues was also significantly regulated. Among them, the supplementation of isobutyric acid and sodium nitrate had a significant effect on the composition ratio of surfactin homologues, and the content of C14-Surfactin component increased, reaching a maximum of 72%. The concentration of this group of mixed carbon and nitrogen sources was optimized, and it was found that the best effect was achieved when the ratio of glucose:isobutyric acid:sodium nitrate was 40:4:20.

[0061] Example 4: Effect of Mixed Carbon and Nitrogen Sources on C15-Surfactin Production by Bacillus subtilis

[0062] Bacillus subtilis EX10 was inoculated into 30 mL of LB medium, cultured for 12-16 hours, and then transferred to new 50 mL of LB medium at a volume ratio of 1%, and cultured for another 8-12 hours to serve as the seed solution for shake flask fermentation.

[0063] The mixture was transferred to 50 mL fermentation medium at a volume ratio of 3% and shake flask experiments were performed. The mixed carbon and nitrogen sources were mainly glucose 40 g / L as the main carbon source, fatty acid carbon source (isovaleric acid, 2-methylbutyric acid) 4 g / L and nitrogen source (ammonium sulfate, leucine, aspartic acid, isoleucine) 2 g / L as supplementary carbon and nitrogen sources to obtain the optimal ratio, which was further adjusted by optimizing the added concentration of carbon and nitrogen sources. The shaker temperature was 37 ° C and the speed was 200 rpm. After 18 h of culture, the cell density OD was detected. 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin, and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Tables 5-6.

[0064] Table 5 Comparison of fermentation for producing C15-Surfactin by mixed carbon and nitrogen sources

[0065]

[0066] Table 6 Optimization of C15-Surfactin addition concentration in the production of mixed carbon and nitrogen sources

[0067]

[0068] The results showed that when glucose was the main carbon source, the strain growth and product yield were good when additional carbon and nitrogen sources were added. The composition of surfactin homologues was also significantly regulated. Among them, the supplementation of isovaleric acid and leucine had a significant effect on the composition ratio of surfactin homologues, and the content of C15-Surfactin component increased, reaching a maximum of 67%. The concentration of this group of mixed carbon and nitrogen sources was optimized, and it was found that the best effect was achieved when the ratio of glucose:isovaleric acid:leucine was 60:6:3, and the proportion of C15-Surfactin could reach up to 70%.

[0069] Example 5: Construction of recombinant Bacillus with inactivated β-oxidation cycle genes to produce surfactin

[0070] (1) Construction of recombinant Bacillus with inactivated β-oxidation cycle pathway genes

[0071] Construction of knockout vector:

[0072] Using the genome of Bacillus subtilis 168 as a template, PCR amplification was performed to obtain the gene fragments FadD-L, FadD-R, FadE-L, FadE-R, FadA-L, FadA-R, FadB-L, and FadB-R.

[0073] The upstream and downstream gene fragments of genes FadD, FadE, FadA and FadB were homologously recombined into the temperature-sensitive vector pKS2 plasmid between the restriction sites KpnI and SalI by enzyme ligation (enzyme ligation sites KpnI and SalI), and knockout vectors pKS2-FadD, pKS2-FadE, pKS2-FadA and pKS2-FadB were constructed.

[0074] According to the above method, the knockout plasmids pKS2-FadD, pKS2-FadE, pKS2-FadA, and pKS2-FadB were introduced into Bacillus subtilis EX10 competent cells, respectively. According to the above screening and identification method of recombinant strains, strains with inactivated genes FadD, FadE, FadA, and FadB were constructed, namely: BS-ΔFadD, BS-ΔFadE, BS-ΔFadA, and BS-ΔFadB, respectively.

[0075] (2) Verification of surfactin production by recombinant Bacillus

[0076] The recombinant strains BS-ΔFadD, BS-ΔFadE, BS-ΔFadA, and BS-ΔFadB constructed above were inoculated into 30 mL of LB medium, respectively. After culturing for 12-16 h, they were transferred to new 50 mL of LB medium at a volume ratio of 1% and cultured for another 8-12 h to serve as seed liquid for shake flask fermentation.

[0077] Transfer the cells to 50 mL of fermentation medium at a volume ratio of 3% and perform a shake flask experiment with a shaker temperature of 37°C and a speed of 200 rpm. After culturing for 18 h, the cell density OD 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Table 7.

[0078] Table 7 Comparison of glucose fermentation by different recombinant Bacillus

[0079] Strain name C13-Surfactin (wt%) C14-Surfactin (wt%) C15-Surfactin (wt%) EX10 21 40 39 BS-ΔFadD 19 45 46 BS-ΔFadE 18 51 41 BS-ΔFadA 17 43 40 BS-ΔFadB 20 43 37

[0080] The results showed that inactivation of β-oxidation cycle pathway related genes FadD, FadE, FadA, and FadB could significantly promote the synthesis of Surfactin and improve the growth and production of the strain. 600 =6-10, the production of surfactin is 6-10 g / L, especially the knockout of FadE, which increases the proportion of the homologue C14-Surfactin component to more than 50%, an increase of 27% compared with the starting strain.

[0081] Example 6: Construction of recombinant Bacillus with inactivated amino acid degradation pathway genes to produce surfactin

[0082] (1) Construction of recombinant Bacillus expressing key proteins in the amino acid degradation pathway

[0083] Construction of knockout vectors: glutamine synthetase glnA protein, aromatic amino acid aminotransferase ilvE protein, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAA protein, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAB protein, branched-chain α-ketoacid dehydrogenase E2 subunit bkdB protein, branched-chain α-ketoacid dehydrogenase E3 subunit LpdV protein;

[0084] Using the genome of Bacillus subtilis 168 as a template, PCR amplification was performed to obtain the gene fragments glnA-L, glnA-R, ilvE-L, ilvE-R, bkdAA-L, bkdAA-R, bkdAB-L, bkdAB-R, bkdB-L, bkdB-R, LpdV-L, and LpdV-R.

[0085] The upstream and downstream gene fragments of genes glnA, ilvE, bkdAA, bkdAB, bkdB, and LpdV were homologously recombined between the restriction sites KpnI and SalI of the temperature-sensitive vector pKS2 plasmid by enzyme ligation (enzyme ligation sites are KpnI and SalI), and knockout vectors pKS2-glnA, pKS2-ilvE, pKS2-bkdAA, pKS2-bkdAB, and pKS2-LpdV were constructed.

[0086] According to the above method, the knockout plasmids pKS2-glnA, pKS2-ilvE, pKS2-bkdAA, pKS2-bkdAB, and pKS2-LpdV were introduced into Bacillus subtilis EX10 competent cells, respectively. According to the above screening and identification method of recombinant strains, strains with inactivated genes glnA, ilvE, bkdAA, bkdAB, bkdB, and LpdV were constructed, namely: BS-ΔglnA, BS-ΔilvE, BS-ΔbkdAA, BS-ΔbkdAB, and BS-ΔLpdV.

[0087] (2) Verification of surfactin production by recombinant Bacillus

[0088] The recombinant strains BS-ΔglnA, BS-ΔilvE, BS-ΔbkdAA, BS-ΔbkdAB, and BS-ΔLpdV constructed above were inoculated into 30 mL of LB medium, cultured for 12-16 h, and then transferred to new 50 mL of LB medium at a volume ratio of 1% and cultured for another 8-12 h to serve as seed liquid for shake flask fermentation.

[0089] Transfer the cells to 50 mL of fermentation medium at a volume ratio of 3% and perform a shake flask experiment with a shaker temperature of 37°C and a speed of 200 rpm. After culturing for 18 h, the cell density OD 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Table 8.

[0090] Table 8 Comparison of glucose fermentation by different recombinant Bacillus

[0091] Strain name C13-Surfactin (wt%) C14-Surfactin (wt%) C15-Surfactin (wt%) EX10 21 40 39 BS-ΔglnA 19 50 31 BS-ΔilvE 22 42 36 BS-ΔbkdAA 24 34 42 BS-ΔbkdAB 16 33 51 BS-ΔLpdV 20 48 32

[0092] The results showed that inactivation of key amino acid degradation pathway protein-related genes glnA, ilvE, bkdAA, bkdAB, bkdB, and LpdV could significantly promote the synthesis of Surfactin and improve the growth and production of the strain. 600 =5-10, the impact on the production of surfactin was 5-10 g / L, especially the knockout of bkdAB and glnA. The knockout of bkdAB increased the proportion of the homologue C15-Surfactin component to more than 50%, an increase of 30.8% compared with the starting strain. The knockout of glnA increased the proportion of the homologue C14-Surfactin component to more than 50%, an increase of 25% compared with the starting strain.

[0093] Example 7: Production of surfactin homologue C14-Surfactin by recombinant Bacillus using mixed carbon sources

[0094] The above experiments confirmed that knockout of the FadE, glnA, and LpdV genes significantly improved the production of surfactin C14. The gene knockout methods described in Examples 5-6 were used to knock out two genes in combination, yielding recombinant strains BS-ΔFadE-ΔglnA and BS-ΔFadE-ΔLpdV.

[0095] The recombinant strains BS-ΔFadE, BS-ΔglnA, BS-ΔFadE-ΔglnA and BS-ΔFadE-ΔLpdV obtained above were inoculated into 30 mL of LB medium, respectively. After culturing for 12-16 h, they were transferred to new 50 mL of LB medium at a volume ratio of 1% and continued to be cultured for 8-12 h as the seed liquid for shake flask fermentation.

[0096] Transfer the cells to 50 mL of fermentation medium at a volume ratio of 3% and perform shake flask experiments. Glucose: isobutyric acid: sodium nitrate ratio of 40:4:20 is selected as the main components of the culture medium. The shaker temperature is 37°C and the speed is 200 rpm. After 18 hours of culture, the cell density OD 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin and C15-Surfactin components. Three parallel experiments were set up for each group of experiments, and the results were averaged. The results are shown in Table 9.

[0097] Table 9 Comparison of C14-Surfactin production by different recombinant Bacillus using mixed carbon and nitrogen sources

[0098] Serial number C13-Surfactin (wt%) C14-Surfactin (wt%) C15-Surfactin (wt%) EX10 12 72 16 BS-ΔFadE 13 70 17 BS-ΔglnA 8 79 13 BS-ΔLpdV 13 71 16 BS-ΔFadE-ΔglnA 6 85 9 BS-ΔFadE-ΔLpdV 10 78 12

[0099] The results showed that glucose, isobutyric acid and sodium nitrate were used as combined carbon and nitrogen sources, and the density was 600 =6.5-10, the production of surfactin can be significantly improved in the range of 7-10 g / L by inactivated recombinant bacteria to the homologue C14-Surfactin component, especially the recombinant strain BS-ΔFadE-ΔglnA. While maintaining the same total surfactin production, the proportion of C14-Surfactin component can reach up to 85%.

[0100] Example 8: Production of surfactin homologue C15-Surfactin by recombinant Bacillus using mixed carbon sources

[0101] Knockout of the FadD, bkdAB, and LpdV genes significantly improved the production of surfactin C15. The three genes were knocked out using the gene knockout methods described in Examples 5-6 to obtain recombinant strains BS-ΔFadD-ΔbkdAB and BS-ΔFadD-ΔLpdV.

[0102] The recombinant strains BS-ΔFadD, BS-ΔbkdAB, BS-ΔFadD-ΔbkdAB, and BS-ΔFadD-ΔLpdV obtained above were inoculated into 30 mL of LB medium, respectively. After culturing for 12-16 h, they were transferred to new 50 mL of LB medium at a volume ratio of 1% and continued to be cultured for 8-12 h as seed liquid for shake flask fermentation.

[0103] Transfer the cells to 50 mL of fermentation medium at a volume ratio of 3% and perform shake flask experiments. Glucose and isovaleric acid:leucine ratio of 60:6:3 were selected as the main components of the culture medium. The shaker temperature was 37°C and the speed was 200 rpm. After 18 hours of culture, the cell density OD was measured. 600 , surfactin content and the proportion of homologues C13-Surfactin, C14-Surfactin and C15-Surfactin components. Each group of experiments had three parallels and the results were averaged. The results are shown in Table 10.

[0104] Table 10 Comparison of C15-Surfactin production by different recombinant Bacillus using mixed carbon and nitrogen sources

[0105] Serial number C13-Surfactin (wt%) C14-Surfactin (wt%) C15-Surfactin (wt%) EX10 12 18 70 BS-ΔFadD 14 20 66 BS-ΔbkdAB 9 15 76 BS-ΔFadD-ΔbkdAB 8 10 82 BS-ΔFadD-ΔLpdV 10 12 78

[0106] The results showed that when glucose, isovaleric acid and leucine were used as combined carbon sources, the density was 600=4-10, the impact on the production of surfactin at 6-10 g / L can significantly improve the conversion rate of the inactivated recombinant bacteria to the homologue C15-Surfactin component, especially the recombinant strain BS-ΔFadD-bkdAB. While maintaining the same total surfactin production, the proportion of C15-Surfactin component can reach up to 82%.

[0107] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A recombinant bacterium that produces a high yield of surfactin homologues, characterized in that: The recombinant bacteria are modified in the following ways on Bacillus subtilis: one or more of the following genes are deleted: acyl-CoA synthetase gene, acyl-CoA dehydrogenase gene, acetyl-CoA-carbonyl transferase gene, enoyl-CoA hydratase gene, glutamine synthetase gene, aromatic amino acid aminotransferase gene, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAA protein gene, branched-chain α-ketoacid dehydrogenase E1 subunit bkdAB protein gene, branched-chain α-ketoacid dehydrogenase E2 subunit gene, and branched-chain α-ketoacid dehydrogenase E3 subunit gene.

2. The recombinant bacterium according to claim 1, characterized in that The Gene ID of the acyl-CoA synthetase gene is 937336, the Gene ID of the acyl-CoA dehydrogenase gene is 936717, the Gene ID of the acetyl-CoA carbonyltransferase gene is 936729, and the Gene ID of the enoyl-CoA hydratase gene is 937448; The Gene ID of the glutamine synthetase gene is 940020, the Gene ID of the aromatic amino acid aminotransferase is 938420, the Gene ID of the branched-chain α-ketoacid dehydrogenase E1 subunit bkdAA protein gene is 938674, the Gene ID of the branched-chain α-ketoacid dehydrogenase E1 subunit bkdAB protein gene is 938672, and the Gene ID of the branched-chain α-ketoacid dehydrogenase E3 subunit gene is 938669.

3. The recombinant bacterium according to claim 1, characterized in that The Bacillus subtilis includes Bacillus subtilis EX10, which is disclosed in patent application number 2025102397555.

4. A method for producing a surfactin homologue, characterized in that: The production method comprises: inoculating the recombinant bacteria according to any one of claims 1 to 3 into a fermentation medium for fermentation and culturing to obtain the surfactin homologue.

5. The production method according to claim 4, characterized in that The surfactin homologues include surfactins whose β-hydroxy fatty acid chain has a carbon chain length of 13-15.

6. The production method according to claim 4, characterized in that The fermentation medium comprises a main carbon source, a supplementary carbon source, a nitrogen source, inorganic salts and metal ions, wherein the main carbon source comprises glucose.

7. The production method according to claim 6, characterized in that The supplementary carbon source is selected from one or more of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, 2-methylbutyric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, capric acid, lauric acid, myristic acid, isopentadecanoic acid, stearic acid, oleic acid, linoleic acid, and arachidic acid.

8. The production method according to claim 6, characterized in that The nitrogen source is selected from one or more of sodium nitrate, ammonium sulfate, alanine, glutamic acid, aspartic acid, valine, leucine, glycine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, lysine, arginine, and histidine.

9. The production method according to claim 6, characterized in that The mass ratio of the main carbon source, the supplementary carbon source and the nitrogen source is (40-60): (2-6): (1-30).

10. Use of the recombinant bacterium according to any one of claims 1 to 3 in the preparation of surfactin homologues.

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