Genetic engineering bacteria using key enzymes SrfAD and YbdT to synergistically improve the high production of surfactin by Bacillus

Abstract

The present invention discloses a genetically engineered bacterium that uses the key enzymes SrfAD and YbdT to synergistically improve the high-yield production of surfactin by Bacillus. The genetically engineered bacterium is obtained by transforming the key rate-limiting enzyme genes of SrfAD and / or YbdT into the Bacillus velezensis mutant strain ΔRapF+SerA, which is used as the starting strain. The genetically engineered bacteria ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT constructed in the present invention are used for the high-efficient production of surfactin under fermentation conditions. The constitutive expression of the two key rate-limiting enzyme genes has successfully further increased the surfactin production. Under the optimal medium conditions, about 18.2 g / L and 19.6 g / L of surfactin can be stably produced in a 5 L fermenter for 40-50 h. This genetically engineered bacterium provides raw materials for accelerating the industrialization of surfactin.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a genetically engineered bacterium which utilizes key enzymes SrfAD and YbdT to synergistically improve the high-yield surfactin of Bacillus. Background Art

[0002] Surfactants are cyclic aliphatic heptapeptide lactones produced by Bacillus subtilis and related species. They consist of two acidic amino acids (Glu and Asp) flanked by five nonpolar residues and a 3-hydroxy fatty acid. Due to their outstanding surface activity and antibacterial, antiviral, antitumor, and antiproliferative activities, they have a wide range of biotechnological and therapeutic applications. However, the surfactant yields of native Bacillus strains are typically tens of milligrams per liter, rarely exceeding 1 gram per liter. This makes surfactant production very expensive, thus limiting their practical application. Improving surfactant production through physicochemical mutagenesis and fermentation optimization of native Bacillus strains has been extensively studied. Recently, genetic engineering approaches based on the surfactant biosynthetic pathway and molecular regulatory mechanisms to enhance surfactant production in Bacillus have received significant attention.

[0003] Surfactant biosynthesis is carried out by the SrfAA-D multienzyme complex encoded by the srfA genome, which spans 39 kb and consists of four genomic reading frames. In addition to srfA, the core genome for surfactant synthesis, several other key enzymes unique to Bacillus also play crucial roles in surfactant biosynthesis. The phosphopantetheinyltransferase sfp, which modifies the peptidyl carrier protein domain of peptide synthetase, converts SrfAA-C from the apo form to the holo-synthase. The cytochrome P450 enzyme YbdT catalyzes the conversion of long-chain fatty acids to 3-hydroxy fatty acids. The long-chain fatty acid-CoA ligases LcfA and LcfB then catalyze the conversion of 3-hydroxy fatty acids to fatty acyl CoAs for surfactant biosynthesis. Several enzymes involved in fatty acid, amino acid, and cofactor metabolism are also important for surfactant synthesis, and their functions have been well characterized. In summary, the elucidation of the surfactant biosynthesis pathway lays the foundation for improving its production through metabolic pathway engineering. Summary of the Invention

[0004] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a type of Bacillus velez mutant strain with high surfactin production. The present invention inserts the complete gene fragments of two key enzymes SrfAD and YbdT into pTN-psrfT1T2 and pTC-PlacI-IPTGT1T2 vectors, respectively, and successfully constructs pTN-PsrfT1T2-SrfAD and pTC-PlacI-IPTGT1T2-YbdT. Then, the above-constructed expression vectors are transformed one by one into the Bacillus velez mutant strain ΔRapF+SerA to construct a new Bacillus velez mutant strain with high surfactin production, which can synergistically improve the high surfactin production of Bacillus through the key enzymes SrfAD and YbdT.

[0005] The present invention also provides a construction method and application of the genetically engineered bacteria.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a genetically engineered bacterium that uses the key enzymes SrfAD and YbdT to synergistically improve the high production of surfactin in Bacillus, characterized in that the genetically engineered bacterium uses the Bacillus velez mutant strain ΔRapF+SerA as the starting strain, and transforms the SrfAD and / or YbdT key rate-limiting enzyme genes into the Bacillus velez mutant strain ΔRapF+SerA; the Bacillus velez mutant strain ΔRapF+SerA is obtained by starting with the Bacillus velez HCK2 strain, and knocking out or inactivating the negative regulatory genes RapF and SerA in the strain genome.

[0007] The nucleotide sequences of the key rate-limiting enzymes SrfAD and YbdT are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] The method for constructing a genetically engineered bacterium that utilizes the key enzymes SrfAD and YbdT to synergistically increase the surfactin production in Bacillus subtilis comprises the following steps:

[0009] (1) Primers were designed based on the nucleotide sequences of the SrfAD and YbdT genes, and genomic DNA of Bacillus subtilis was used as a template to obtain SrfAD and YbdT gene fragments by PCR amplification;

[0010] (2) Double digesting the SrfAD gene fragment and plasmid with restriction endonucleases to obtain the digested gene fragment and the mitotic plasmid fragment, which were then ligated enzymatically to obtain the recombinant plasmid pTN-PsrfT1T2-SrfAD;

[0011] (3) Double digesting the YbdT gene fragment and plasmid with restriction endonucleases to obtain the digested gene fragment and the mitotic plasmid fragment, which were then ligated to obtain the recombinant plasmid and pTC-PlacI-IPTGT1T2-YbdT;

[0012] (4) The recombinant plasmids pTN-PsrfT1T2-SrfAD and pTC-PlacI-IPTGT1T2-YbdT were transferred into the mutant strain ΔRapF+SerA of Bacillus velez, and the mutant strains ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT of Bacillus velez were constructed.

[0013] Wherein, the primer sequence described in step (1) is as follows:

[0014] SrfADF: 5′-TTTGGATCCAAGGTGAGTGTGAGATGCTT-3′

[0015] SrfADR: 5′-TTTCCATGGGATCTGAAAGAAGGCAGGAA-3′

[0016] YbdTF: 5′-TTTTGCTAGCCGGCGAAGGTGTTTTATGAT-3′

[0017] YbdTR: 5′-TTTACTAGTTAAATCAAGCAGCACCGATG-3′.

[0018] The restriction endonucleases in step (2) are Nhe I and Spe I, and the plasmid is the expression vector pTN-PsrfT1T2; the restriction endonucleases in step (3) are BamH I and Nco I. The plasmid is the expression vector pTC-PlacI-IPTGT1T2.

[0019] In step (2), the recombinant plasmid pTN-PsrfT1T2-SrfAD is transferred into the mutant strain ΔRapF+SerA of Bacillus velez to obtain the mutant strain ΔRapF+SerA+SrfAD of Bacillus velez; and then transferred into the recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT to obtain the mutant strain ΔRapF+SerA+SrfAD+YbdT of Bacillus velez.

[0020] The invention discloses an application of a genetically engineered bacterium that utilizes key enzymes SrfAD and YbdT to synergistically improve the surfactin production of Bacillus sp. in the production of surfactin.

[0021] The production of surfactin is carried out by fermenting genetically engineered bacteria in a fermenter: after activating the bacterial strain, a seed liquid is prepared, which is inoculated into a fresh fermentation medium at a volume ratio of 10-15%, and fermentation is started. During the fermentation process, the pH is controlled to be stable at 6.8 to 7.2, the temperature is maintained at 35-37°C, and the dissolved oxygen is between 25-35%; when the glucose in the culture medium is consumed, a glucose solution is added to maintain the glucose concentration in the fermentation medium at 0.1-5g / L; and the fermentation cycle is 40-50h.

[0022] The fermentation medium is composed of: 10-15 g / L glucose, 5-8 g / L glutamate, 1-3 g / L K2HPO4, 1-3 g / L KH2PO4, 1-2 g / L KCl, 400-500 mg / L MnSO4·7H2O, 4-5 mg / L FeSO4·7H2O, 150-160 mg / L CuSO4·5H2O, and the rest is water, with a pH of 7.0.

[0023] Preferably, the fermentation medium comprises: glucose 20 g / L, glutamic acid 5 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, KCl 1 g / L, MnSO4·7H2O 500 mg / L, FeSO4·7H2O 5 mg / L, and CuSO4·5H2O 160 mg / L.

[0024] The invention discloses an application of the key rate-limiting enzyme genes SrfAD and YbdT in regulating the surfactin production of Bacillus velezinis through cumulative mutation.

[0025] The present invention uses two key rate-limiting enzyme genes, YbdT, to catalyze the biosynthesis gene of 3-hydroxy fatty acids and SrfAD, to activate the fatty acid transfer gene, to achieve constitutive expression and successfully construct a genetically engineered strain. The present invention uses a mutant strain ΔRapF+SerA of Bacillus velezensis as a starting strain, constructs expression vectors of key rate-limiting enzyme genes SrfAD and YbdT, and sequentially transforms them into the mutant strain ΔRapF+SerA to obtain mutant strains ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT. The ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT mutant strains are used to efficiently produce the yield of surfactin under fermentation conditions. The constitutive expression of the two key rate-limiting enzyme genes successfully further increases the yield of surfactin. Under optimal culture medium conditions, about 18.2 g / L and 19.6 g / L of surfactin can be stably produced in a 5L fermenter for 40-50 hours. The high-yield surfactin gene mutant strain and the construction method thereof provide raw materials for accelerating the industrialization of surfactin.

[0026] In the present invention, a constructed SrfAD key rate-limiting enzyme gene expression vector pTN-PsrfT1T2-SrfAD is transferred into a mutant strain ΔRapF+SerA, an exchange occurs at the SrfAD gene site in the genome, and a strain ΔRapF+SerA+SrfAD containing four resistances to kanamycin, spectinomycin, erythromycin and neomycin is screened on a plate with four resistances to kanamycin, spectinomycin, erythromycin and neomycin; and a constructed YbdT key rate-limiting enzyme gene expression vector pTC-PlacI-IPTGT1T2-YbdT is transferred into a mutant strain ΔRapF+SerA+SrfAD, an exchange occurs at the YbdT gene site in the genome, and a strain ΔRapF+SerA+SrfAD+YbdT containing five resistances to kanamycin, spectinomycin, erythromycin, neomycin and chloramphenicol is screened on a plate with five resistances to kanamycin, spectinomycin, erythromycin, neomycin and chloramphenicol.

[0027] The present invention uses the Bacillus velez mutant strain ΔRapF+SerA as a starting strain and, through homologous recombination, inserts mutations in the genes for SrfAD and YbdT, key rate-limiting enzymes that increase surfactin production. The method specifically involves PCR cloning and amplification of partial SrfAD and YbdT sequence DNA, enzyme digestion and ligation, transformation, screening and identification, transfer of the homologous single-crossover recombinant plasmid into the Bacillus velez mutant strain ΔRapF+SerA, and detection and identification. Preliminary studies have shown that the modified strain obtained through this method exhibits enhanced surfactin production, as determined by high-performance liquid chromatography analysis.

[0028] The invention improves the production of surfactin based on the mutant strain ΔRapF+SerA of Bacillus velezinis by accumulating mutations and expressing rate-limiting enzymes, and its effect is better than that of existing Bacillus velezinis and Bacillus subtilis and their engineered bacteria.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0030] By suppressing the expression of the SrfAD and / or YbdT rate-limiting enzyme genes, high-surfactin-producing mutants (ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT) were generated, with surfactin yields of approximately 18.2 g / L and 19.8 g / L, respectively. The ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT mutants increased surfactin production by 25% and 42% compared to the ΔRapF+SerA mutant, and by 505% and 590% compared to Bacillus velezensis HCK2.

[0031] The invention also designs an optimized culture medium, which can increase the surfactin production by about 50% compared to conventional LB culture medium. The invention's Bacillus velezensis mutant strain, its construction method, and fermentation application provide raw materials for accelerating the industrialization of surfactin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A. Construction of the recombinant plasmid pTN-PsrfT1T2-SrfAD. BM, 15000 bp DNA marker; 1. Expression vector pTN-PsrfT1T2 single enzyme digest (6193 bp). CM, 2000 bp DNA marker; 1, 2, SrfAD PCR fragment (376 bp). DM, 15000 bp DNA marker; 1, 3, recombinant plasmid pTN-PsrfT1T2-SrfAD double enzyme digest (6193 bp and 376 bp); 2, 4, recombinant plasmid pTN-PsrfT1T2-SrfAD single enzyme digest (6569 bp).

[0033] Figure 2 A. Construction of the recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT; BM, DNA 15000bp marker; 1. Expression vector pTC-PlacI-IPTGT1T2 single enzyme digest (7795bp). CM, DNA 5000bp marker; 1, 2. YbdT PCR fragment (794bp). DM, DNA 10000bp marker; 1. Recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT single enzyme digest (8577bp); 2. Recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT double enzyme digest (7783bp and 794bp).

[0034] Figure 3 Middle is a schematic diagram of the HPLC analysis of surfactin production of Bacillus velez mutant strains ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT;

[0035] Figure 4 The hemolytic activity of Bacillus velez strain HCK2 and Bacillus velez mutants ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT;

[0036] Figure 5Schematic diagram of the inhibitory activity of Bacillus veleboldii mutant strains ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT against Staphylococcus aureus. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and examples.

[0038] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are generally performed under conventional conditions or the conditions recommended by the manufacturer.

[0039] The starting strain Bacillus velez mutant ΔRapF+SerA used in the present invention is ΔRapF+SerA in Bacillus velez HCK2. The Bacillus velez mutant ΔRapF+SerA has a specific construction process in Chinese patent 202111677296.7, wherein Bacillus velez HCK2: Deposition number CCTCC NO: M2019396; all are provided by Huaiyin Institute of Technology.

[0040] The expression vectors pTC-PlacI-IPTGT1T2 and pTN-PsrfT1T2 of the present invention have specific construction processes in Chinese Patent 202111680804.7, and the nucleotide sequences are shown in sequences 5 and 6 in Patent 202111680804.7, respectively.

[0041] Bacillus subtilis 168 (CCTCC AB 130001) was purchased from China Center for Type Culture Collection.

[0042] Preparation of competent cells of Bacillus velezensis mutant strains: GCHE liquid medium: 200 ml as an example (glucose 100 g, potassium L-glutamate 0.4 g, trisodium citrate 0.18 g, ammonium ferric citrate 0.022 g, L-tryptophan 0.101 g, casein compound 20 g, K2HPO4 2.2822 g, KH2PO4 1.3609 g);

[0043] GE liquid culture medium: Taking 200 ml as an example (glucose 100 g, potassium L-glutamate 0.4 g, trisodium citrate 0.18 g, ammonium ferric citrate 0.022 g, L-tryptophan 0.101 g, K2HPO4 2.2822 g, KH2PO4 1.3609 g).

[0044] Example 1

[0045] Construction of recombinant plasmid vector

[0046] (1) Primer design

[0047] Primers were designed based on the gene nucleotide sequence of Bacillus subtilis 168, and genomic DNA of 168 was used as a template to amplify the SrfAD and YbdT gene fragments by PCR (the SrfAD gene fragment is 376 bp, and the YbdT gene fragment is 794 bp). The primer sequences are as follows:

[0048] SrfADF: 5′-TTTGGATCCAAGGTGAGTGTGAGATGCTT-3′

[0049] SrfADR: 5′-TTTCCATGGGATCTGAAAGAAGGCAGGAA-3′

[0050] YbdTF: 5′-TTTTGCTAGCCGGCGAAGGTGTTTTATGAT-3′

[0051] YbdTR: 5′-TTTACTAGTTAAATCAAGCAGCACCGATG-3′

[0052] The PCR system is as shown in Table 1:

[0053] Table 1

[0054]

[0055] The PCR reaction program was as follows: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 53°C annealing for 45 s, 72°C extension for 30 s (SrfAD) / 1 min (YbdT), 33 cycles, and 72°C extension for 7 min.

[0056] (2) Enzyme digestion and ligation

[0057] The PCR amplification product and plasmids (pTN-PsrfT1T2, pTC-PlacI-IPTGT1T2) were double-digested using BamH I / Nco I and Nhe I / Spe I restriction endonucleases. The 80 μL double-digestion system was designed as follows (Table 2, Table 3). The digestion was carried out at 37°C for 2 hours:

[0058] Table 2

[0059]

[0060]

[0061] Table 3

[0062]

[0063] The digested product was detected by agarose gel electrophoresis and recovered. The resulting digested gene fragment and the linearized plasmid fragment were ligated using T4 DNA ligase. The 12 μL linearized plasmid and DNA fragment ligation system was as follows (Table 4):

[0064] Table 4

[0065]

[0066] Ligation was performed at 16°C for 3 h, wherein the pTN-PsrfT1T2 plasmid was ligated to the purified SrfAD DNA fragment, and the pTC-PlacI-IPTGT1T2 plasmid was ligated to the purified YbdT DNA fragment.

[0067] (3) Transformation, screening and identification

[0068] The ligation product obtained in the previous step was transformed into Escherichia coli DH5α by the calcium chloride transformation method and spread on LB solid medium (10 g / L tryptone, 5 g / L yeast extract powder, 5 g / L NaCl) containing 100 μL / ml ampicillin to screen positive clones and extract plasmids.

[0069] After double enzyme digestion of the recombinant plasmid pTN-PsrfT1T2-SrfAD, 1% agarose gel electrophoresis confirmed that two bands of 6193 bp and 376 bp were obtained, which was consistent with the expected result; after single enzyme digestion of the recombinant plasmid pTN-PsrfT1T2-SrfAD, 1% agarose gel electrophoresis confirmed that a band of 6569 bp was obtained, which was consistent with the expected result (see Figure 1 ), the sequence of which is shown in SEQ ID NO.3.

[0070] After double digestion of the recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT, 1% agarose gel electrophoresis confirmed that two bands of 7783 bp and 794 bp were obtained, which was consistent with the expected result. After single digestion of the recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT, 1% agarose gel electrophoresis confirmed that a band of 8577 bp was obtained, which was consistent with the expected result (see Figure 2 ), the sequence of which is shown in SEQ ID NO.4.

[0071] Example 2

[0072] Construction of mutant strains

[0073] The recombinant expression vector was transferred into the mutant strain ΔRapF+SerA of Bacillus velezensis, and screened at 37°C using a culture medium containing resistance. The screened transformants were randomly inserted into the recombinant plasmid to verify the correct mutant strain.

[0074] The specific method for constructing the ΔRapF+SerA+SrfAD mutant is as follows:

[0075] A single colony on the plate containing the mutant strain ΔRapF + SerALB was picked and inoculated into 5 mL of GCHE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL), and erythromycin (1 μg / mL) and cultured at 37°C with shaking at 160 rpm / min for 12 h;

[0076] Take 1 mL of bacterial suspension and add it to 24 mL of fresh GCHE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL) and erythromycin (1 μg / mL), and culture on a shaker at 28°C and 180 rpm / min.

[0077] At 2 h, 2.5 h, 3 h, 3.5 h, and 4 h, 1 mL of culture medium was taken and added to 1 mL of GE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL), and erythromycin (1 μg / mL), and cultured at 37 °C and 200 rpm / min for 1 h.

[0078] After culturing for 1 hour in each of the above time periods, the cells were centrifuged at 4000 rpm for 10 minutes and concentrated to 400 uL to obtain competent cells. The corresponding recombinant plasmid pTN-PsrfT1T2-SrfAD (prepared in Example 1, about 10 μg) was added and cultured at 37°C, 180 rpm / min for 90 minutes, followed by centrifugation at 3000 rpm for 5 minutes. 300 μL of supernatant was discarded, and the precipitated bacteria were mixed with the remaining supernatant to obtain 100 μL of bacterial suspension, which was applied to four LB resistance plates containing kanamycin, spectinomycin, erythromycin, and neomycin (including kanamycin 10 μg / mL, spectinomycin 50 μg / mL, erythromycin 1 μg / mL, and neomycin 10 μg / mL) and cultured at 37°C for 24 h.

[0079] A single colony was picked and inoculated onto LB liquid medium containing kanamycin, spectinomycin, erythromycin, and neomycin antibiotics, and cultured overnight at 37°C, 180 rpm / min. The strain was then streaked onto solid LB plates containing the four antibiotics (20 μg / mL kanamycin, 100 μg / mL spectinomycin, 10 μg / mL erythromycin, and 20 μg / mL neomycin) using an inoculating loop and cultured at 37°C for 24 hours. PCR verification was performed to obtain the correct mutant strain, ΔRapF+SerA+SrfAD.

[0080] The specific construction of the ΔRapF+SerA+SrfAD+YbdT mutant strain is the same as above:

[0081] The constructed recombinant plasmid vector pTC-PlacI-IPTGT1T2-YbdT was transformed into competent cells of the ΔRapF+SerA+SrfAD mutant strain of Bacillus velezensis (the preparation of competent cells and the introduction of plasmids were carried out according to the above steps), and exchange occurred at the genomic YbdT gene site. Resistant strains were screened on resistant plates containing 10 μg / mL of kanamycin, 50 μg / mL of spectinomycin, 1 μg / mL of erythromycin, 10 μg / mL of neomycin, and 12.5 μg / mL of chloramphenicol, and PCR verification was performed to obtain the correct mutant strain ΔRapF+SerA+SrfAD+YbdT. The above mutant strain can be used in the production of the lipopeptide antibiotic Surfactin to produce Bacillus velezensis antimicrobial peptide products.

[0082] A single colony from the LB plate containing the mutant strain ΔRapF+SerA+SrfAD was picked and inoculated into 5 mL of GCHE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL), erythromycin (1 μg / mL), and neomycin (10 μg / mL) and cultured at 37°C with shaking at 160 rpm / min for 12 h;

[0083] Take 1 mL of bacterial suspension and add it to 24 mL of fresh GCHE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL), erythromycin (1 μg / mL), and neomycin (10 μg / mL), and culture on a shaker at 28°C and 180 rpm / min.

[0084] At 2 h, 2.5 h, 3 h, 3.5 h, and 4 h, 1 mL of culture medium was taken and added to 1 mL of GE liquid medium containing kanamycin (10 μg / mL), spectinomycin (50 μg / mL), erythromycin (1 μg / mL), and neomycin (10 μg / mL), and cultured at 37 °C and 200 rpm / min for 1 h.

[0085] After culturing for 1 hour in each time period, the cells were centrifuged at 4000 rpm for 10 minutes and concentrated to 400 μL to obtain competent cells. The corresponding recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT (prepared in Example 1, about 10 μg) was added and cultured at 37°C, 180 rpm / min for 90 minutes, followed by centrifugation at 3000 rpm for 5 minutes. 300 μL of supernatant was discarded, and the precipitated cells were mixed with the remaining supernatant to obtain a 100 μL bacterial suspension, which was applied to five LB resistance plates containing kanamycin, spectinomycin, erythromycin, neomycin, and chloramphenicol (wherein kanamycin 10 μg / mL, spectinomycin 50 μg / mL, erythromycin 1 μg / mL, neomycin 10 μg / mL, chloramphenicol 12.5 μg / mL) and cultured at 37°C for 24 hours.

[0086] A single colony was picked out and inoculated into LB liquid culture medium containing kanamycin, spectinomycin, erythromycin, neomycin and chloramphenicol antibiotics, and cultured overnight at 37°C and 180rpm / min. Then, an inoculation loop was used to streak culture on an LB solid plate containing the above five double antibiotics (20μg / mL of kanamycin, 100μg / mL of spectinomycin, 10μg / mL of erythromycin, 20μg / mL of neomycin, and 25μg / mL of chloramphenicol) at 37°C for 24 hours, and PCR verification was performed to obtain the correct mutant strain ΔRapF+SerA+SrfAD+YbdT. The mutant strain described above can be used in the production of the lipopeptide antibiotic Surfactin to produce antimicrobial peptide products of Bacillus velezensis.

[0087] Example 3

[0088] Fermentation extraction method of surfactant

[0089] Qualitative and quantitative analysis of the surfactant production by fermentation of the Bacillus velez mutant strain constructed in Example 2.

[0090] Single colonies of different mutant strains of Bacillus velezensis constructed in Example 2 were picked from the plates and transferred to 5 mL of LB liquid containing the corresponding resistance medium (the ΔRapF+SerA+SrfAD mutant was a resistance medium containing kanamycin 10 μg / mL, spectinomycin 50 μg / mL, erythromycin 1 μg / mL, and neomycin 10 μg / mL; the ΔRapF+SerA+SrfAD+YbdT mutant was a resistance medium containing kanamycin 10 μg / mL, spectinomycin 50 μg / mL, erythromycin 1 μg / mL, neomycin 10 μg / mL, and chloramphenicol 12.5 μg / mL) at 37°C and 180 rpm for 16 h, and then transferred to 50 mL The cells were cultured in LB liquid medium at 37 °C and 180 rpm for 24 h to prepare seed solution, which was inoculated into a 5 L fermentor at a volume ratio of 10%, fresh LB fermentation medium (10 g / L tryptone, 5 g / L yeast extract powder, and 5 g / L NaCl) and optimized medium (glucose 20 g / L, glutamic acid 5 g / L, K2HPO4 1 g / L, KH2PO4 1 g / L, KCl 1 g / L, MnSO4·7H2O 500 mg / L, FeSO4·7H2O 5 mg / L, CuSO4·5H2O 5 mg / L) were added. Fermentation was initiated with a 160 mg / L fermentation medium. During fermentation, the pH was maintained at 7.0, the temperature was maintained at 37°C, and the dissolved oxygen content was approximately 30%. The reducing sugar content in the fermentation broth was determined by the DNS method. After the glucose in the culture medium was consumed, glucose solution was fed to maintain the glucose concentration in the fermentation medium at 3 g / L to facilitate subsequent product accumulation. Fermentation was continued for 48 hours. The fermentation broth was centrifuged at 8000 rpm for 10 minutes to obtain a fermentation supernatant. The supernatant was adjusted to pH 2.5 with 6 mol concentrated hydrochloric acid and refrigerated overnight at 4°C. The supernatant was centrifuged at 12000 rpm for 20 minutes to obtain a precipitate. The crude fermentation extract was dissolved in 100% methanol solution, refrigerated overnight, and centrifuged at 8000 rpm for 10 minutes. The extract was then filtered through a 0.22 μm organic filter to remove large particulate impurities to prepare a preliminary extract of surfactin. Bacillus velez HCK2 and the Bacillus velez mutant strain ΔRapF+SerA were used as controls under the same conditions as above.

[0091] Surfactin detection method

[0092] The detection conditions for surfactin by HPLC were as follows: Agilent 1200 series HPLC and C18 column (5 μm, 4 mm × 250 mm; Merck, Frankfurt, Germany) with mobile phase (acetonitrile: water: trifluoroacetic acid (20:80:0.05 (V / V)), detection wavelength was 210 nm, flow rate was 0.8 mL / min, column temperature was 30°C, and the amount of surfactin produced by fermentation of the above-mentioned Bacillus velezensis mutant strain was shown in Table 1. Figure 3 and Table 5.

[0093] Table 5 Surfactant production by fermentation of Bacillus velezensis mutants

[0094]

[0095] Depend on Figure 3 As shown in Table 5, the mutant strains ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT constructed in the present invention produced large amounts of surfactin after fermentation, significantly outperforming the Bacillus velez mutant strain ΔRapF+SerA and Bacillus velez HCK2. Furthermore, using the optimized culture medium of the present invention further increased surfactin production.

[0096] Example 4

[0097] Determination of hemolytic activity

[0098] The hemolytic activity of the Velez Bacillus mutant strains was determined using a hemolytic plate. The specific method is as follows: Use an inoculation loop to pick single colonies of the wild-type Velez Bacillus strain HCK2 and the Velez Bacillus mutant strains ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT and streak them onto an agar medium containing 5% fresh sheep red blood cells. Incubate at 37°C for 48 hours, observe the hemolytic transparent zone around the colony, and determine the hemolytic activity of the wild-type Velez Bacillus HCK2 and its mutant strains ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT. The results are as follows Figure 4The HCK2 strain and its mutants, ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT, showed differences in their ability to form hemolytic rings on blood agar plates. At 8 hours of culture, the mutants ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT began to form hemolytic rings, with the ΔRapF+SerA+SrfAD+YbdT mutant forming the largest ring. At 12 hours of culture, the hemolytic rings of the mutants became larger, and HCK2 also exhibited a hemolytic ring. At 24 hours of culture, both strains formed distinct hemolytic rings, but the rings of the mutants were more pronounced. The hemolytic rings reached their maximum at 48 hours of culture, indicating that knockout of the mutants enhanced the hemolytic ability of surfactin on blood agar plates.

[0099] Example 5

[0100] Inhibition zone test

[0101] The Oxford cup method was used to determine the antibacterial activity of Bacillus velezensis mutants ΔRapF+SerA, ΔRapF+SerA+SrfAD, and ΔRapF+SerA+SrfAD+YbdT against Staphylococcus aureus. The specific method was as follows: a single colony of Staphylococcus aureus was picked and inoculated into 5 mL of LB liquid culture medium and cultured at 37°C and 180 rpm / min for 12 h. The Staphylococcus aureus liquid was inoculated into 100 mL of LB solid culture medium cooled to 40°C at a volume ratio of 2%, mixed and poured into a sterile plate, the plate was divided equally and marked, and the Oxford cup was clamped and placed vertically on the sterile plate under sterile operation. 200 uL of the fermentation supernatant of ΔRapF+SerA, ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT Velez Bacillus mutant strains (prepared according to the method of Example 3) were added to the Oxford cup for experimental comparison. The cells were placed flat in a 37°C incubator and cultured for 24 hours. The inhibition zone around the Oxford cup was observed. The diameter of the inhibition zone (mm) = the total measured diameter (mm) - the diameter of the Oxford cup (mm). The results are as follows. Figure 5 It showed that the antibacterial effect of the fermentation supernatant of mutant strains ΔRapF+SerA+SrfAD and ΔRapF+SerA+SrfAD+YbdT under optimized culture medium was significantly better than that of mutant strain ΔRapF+SerA.

Claims

1. A method for synergistically improving the surfactin production and hemolytic ability of genetically engineered Bacillus velezinis using the key enzymes SrfAD and YbdT. The genetically engineered bacteria is obtained by using the Velez subtilis mutant strain ΔRapF+SerA as the starting strain, and transforming the genes encoding the key enzymes SrfAD and YbdT into the Velez subtilis mutant strain ΔRapF+SerA; the Velez subtilis mutant strain ΔRapF+SerA is obtained by using the Velez subtilis HCK2 as the starting strain, and knocking out or inactivating the negative regulatory genes RapF and SerA in the strain genome; the deposit number of the Velez subtilis HCK2 is CCTCC NO:M2019396; the nucleotide sequences encoding the key enzymes SrfAD and YbdT are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

2. The use according to claim 1, characterized in that The method for constructing the genetically engineered bacteria comprises the following steps: (1) Primers were designed based on the nucleotide sequences of the SrfAD and YbdT genes, and genomic DNA of Bacillus subtilis was used as a template to obtain SrfAD and YbdT gene fragments by PCR amplification; (2) Double digestion of the SrfAD gene fragment and plasmid with restriction endonucleases to obtain the digested gene fragment and the mitochondrial plasmid fragment, which were then ligated by enzymes to obtain the recombinant plasmid pTN-PsrfT1T2-SrfAD; (3) Double digestion of the YbdT gene fragment and plasmid with restriction endonucleases to obtain the digested gene fragment and the mitotic plasmid fragment, which were then ligated to obtain the recombinant plasmid and pTC-PlacI-IPTGT1T2-YbdT; (4) The recombinant plasmids pTN-PsrfT1T2-SrfAD and pTC-PlacI-IPTGT1T2-YbdT were transferred into the mutant strain ΔRapF+SerA of Bacillus velezini to construct the genetically engineered strain ΔRapF+SerA+SrfAD+YbdT of Bacillus velezini; Wherein, the primer sequence described in step (1) is as follows: SrfADF: 5′-TTTGGATCCAAGGTGAGTGTGAGATGCTT-3′ SrfADR: 5′-TTTCCATGGGATCTGAAAGAAGGCAGGAA-3′ YbdTF: 5′-TTTTGCTAGCCGGCGAAGGTGTTTTATGAT-3′ YbdTR: 5′-TTTACTAGTTAAATCAAGCAGCACCGATG-3′.

3. The use according to claim 2, characterized in that The restriction endonucleases in step (2) are Nhe I and Spe I, and the plasmid is the expression vector pTN-PsrfT1T2; the restriction endonucleases in step (3) are BamH I and Nco I; and the plasmid is the expression vector pTC-PlacI-IPTGT1T2.

4. The use according to claim 2, characterized in that The recombinant plasmid pTN-PsrfT1T2-SrfAD in step (2) is transferred into the mutant strain ΔRapF+SerA of Bacillus velez to construct the mutant strain ΔRapF+SerA+SrfAD of Bacillus velez; and then transferred into the recombinant plasmid pTC-PlacI-IPTGT1T2-YbdT to obtain the genetically engineered strain ΔRapF+SerA+SrfAD+YbdT of Bacillus velez.

5. The use according to claim 1, characterized in that The production of surfactant is carried out by fermentation in a fermenter using genetically engineered bacteria: after activating the bacterial strain, a seed solution is prepared, which is inoculated into a fresh fermentation medium at a volume ratio of 10-15%, and fermentation is started. During the fermentation process, the pH is controlled to be stable at 6.8 to 7.2, the temperature is maintained at 35-37°C, and the dissolved oxygen is between 25-35%. When the glucose in the culture medium is consumed, a glucose solution is added to maintain the glucose concentration in the fermentation medium at 0.1-5g / L. The fermentation cycle is 40-50 hours.

6. The use according to claim 5, characterized in that The fermentation medium comprises: 10-15 g / L glucose, 5-8 g / L glutamic acid, 1-3 g / L K2HPO4, 1-3 g / L KH2PO4, 1-2 g / L KCl, 400-500 mg / L MnSO4•7H2O, 4-5 mg / L FeSO4•7H2O, and 150-160 mg / L CuSO4•5H2O.

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