WO2020113365A1 - 一种高产脂肽的基因工程菌及其应用 - Google Patents

一种高产脂肽的基因工程菌及其应用 Download PDF

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WO2020113365A1
WO2020113365A1 PCT/CN2018/118893 CN2018118893W WO2020113365A1 WO 2020113365 A1 WO2020113365 A1 WO 2020113365A1 CN 2018118893 W CN2018118893 W CN 2018118893W WO 2020113365 A1 WO2020113365 A1 WO 2020113365A1
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genetically engineered
thy
gene
engineered bacterium
lipopeptide
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于慧敏
王苗苗
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清华大学
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12P21/00—Preparation of peptides or proteins
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a genetically engineered bacterium with high lipopeptide production and its application.
  • Lipopeptide biosurfactants are a class of amphoteric substances composed of a hydrophilic cyclic oligopeptide and a hydrophobic fatty acid chain connected by a lactone bond, and are mainly synthesized by microorganisms such as Bacillus and Streptomyces. Bacillus lipopeptides can be divided into surfactants, fenin, and subtilisin. Among them, surfactants that form a unique "saddle" conformation at the gas/liquid interface have excellent surface activity, biodegradability, and antibacterial activity. It has broad application prospects in the fields of petroleum exploitation, biological control, medicine and daily chemical industry.
  • methods to improve the fermentation level of microbial lipopeptide include optimization of culture conditions, mutation breeding, enhanced surfactant transmembrane transport, enhanced expression of surfactant synthase, etc.
  • Patent documents CN 101892176A, CN 101775427A, WO 2002026961A), etc. have optimized the culture medium and culture conditions during fermentation of Bacillus subtilis to produce surfactants, which has increased the yield of surfactants in the fermentation broth.
  • CN101928677A discloses the treatment of Streptomyces roseosporus by ultraviolet mutagenesis
  • US05227294 discloses the treatment of Bacillus subtilis with nitrosomethyl urethane.
  • Chinese patent document CN103898038A discloses that the transmembrane protein YcxA is transported from intracellular to extracellular by enhancing lipopeptide, the yield of surfactant produced by Bacillus subtilis fermentation is increased by 97%, and Chinese patent document CN1554747A discloses the expression of comA in Bacillus subtilis Gene, lipopeptide production increased by 50%.
  • Chinese patent document CN105400784A By replacing the promoter of the lipopeptide synthase gene cluster with the inducible strong promoter Pg3, the yield of surfactant was increased by 17.7 times.
  • Literature (Dhali D, Coutte F, Arias A, et al. Biotechnology journal, 2017, 12 (7): 1600574.) discloses the lpdV gene in the BKD operon that knocks out the production of fatty acids from branched chain amino acids in Bacillus subtilis The yield of surfactant increased by 1.4 times.
  • a spore-free strain By constructing its defective strains, a spore-free strain can be successfully obtained, but the transcriptional expression of the intracellular maltose operon is affected (Yu Zhiqiang, Construction of the integrated expression vector of the Bacillus subtilis maltose promoter P ⁇ glvA and a preliminary discussion on the promoter function [D], 2004), acetoin fermentation yield decreased (Li Xin. Bacillus subtilis sporulation blocking and carbon flow regulation Effect on the synthesis of acetoin [M], 2017). Therefore, an inappropriate spore gene knockout program will not only increase the target product yield, but will reduce the target product yield. No studies have been reported on which spore synthesis genes have been knocked out to construct spore-free engineering bacteria to increase the yield of surfactant and at the same time reduce the potential safety risks of Bacillus subtilis application.
  • the purpose of the present invention is to provide a genetically engineered bacterium with high lipopeptide production in order to overcome the problems in the prior art. No spores are produced during the fermentation process and the production of lipopeptide can be improved.
  • a genetically engineered bacterium with high lipopeptide production according to the present invention is constructed by inactivating at least one of the genes regulating bud cell wall formation in the original lipopeptide-producing strain.
  • the genes regulating bud cell wall formation include spoIVA, spoIVB, spoIVC, spoIVF, spoVA, spoVB, spoVD and spoVE.
  • the genes that regulate the formation of bud cell walls have different gene numbers in different strains.
  • For the sequence take the number in the selection of B. subtilis model bacteria B.subtilis168 as an example, and their GeneIDs are:
  • spoIVC including spiIVCA (GeneID: 937799) and spoIVCB (GeneID: 937803));
  • spoIVF including spoIVFB (GeneID: 937505) and spoIVFA (GeneID: 937501));
  • spoVA including spoVAA (GeneID: 938734), spoVAB (GeneID: 938933), spoVAC (GeneID: 938733), spoVAD (GeneID: 938932), spoVAEA (GeneID: 8303022), spoVAEB (GeneID: 8303020), and spoVAF (GeneID: 938936) ));
  • the inactivation means that the gene is not expressed or is not completely expressed.
  • the method of inactivation is gene knockout or gene mutation.
  • the original strain is Bacillus subtilis, Bacillus cereus or Pseudomonas.
  • the surfactant synthase in the original strain is controlled by an inducible strong promoter, and the inducible strong promoter is Pg3.
  • the original strain is Bacillus subtilis THY-7.
  • the Bacillus subtilis THY-7 was deposited on March 11, 2014 in the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the deposit registration number is CGMCC No. 8906, and is disclosed in Chinese patent document CN105400784A.
  • the original strain is Bacillus subtilis THY-7/Pg3-srfA.
  • the Bacillus subtilis THY-7/Pg3-srfA refers to the addition of an inducible strong promoter Pg3 to Bacillus subtilis THY-7, so that the strong promoter Pg3 controls the expression of surfactant synthase srfA.
  • the inducible strong promoter Pg3 Bacillus subtilis THY-7/Pg3-srfA and its specific preparation method are disclosed in Chinese patent document CN105400784A.
  • the above genetically engineered bacteria can be constructed as follows, including:
  • the plasmid containing the cre recombinase gene is transformed into a genetically engineered bacterium containing a resistance selection marker, and the genetically engineered bacterium with a high lipoprotein-producing peptide from which the resistance gene is removed is cultured and selected.
  • the antibiotic resistance gene may be selected from the group consisting of erythromycin resistance gene, bleomycin resistance gene, kanamycin resistance gene, phenocycin resistance gene and neomycin resistance gene Sex genes, etc.
  • a bleomycin resistance gene is used.
  • the above genetic engineering bacteria construction method is:
  • the specific construction method of the left-antibiotic resistance gene-right fragment is:
  • left-F and left-R as upstream and downstream primers, using the original strain genome as a template for polymerase chain reaction to amplify and obtain the homologous arm left gene fragment located upstream of the target gene in the genome.
  • the three gene fragments obtained above are recovered and mixed as a template, and left-F and right-R are used as upstream and downstream primers to perform PCR amplification to obtain the linear gene fragment left-antibiotic resistance for the target gene knockout. Sex gene-right.
  • the specific construction method of the pHK-cre plasmid is:
  • the ligation product was transformed into E. coli TOP10 competent cells, spread on kanamycin LB plates, and placed in a 37°C incubator for overnight cultivation.
  • the resistant clones grown on the plate were picked and cultured, and the extracted plasmid was sequenced and verified to obtain an expression plasmid containing the recombinase cre gene sequence.
  • the specific construction method of the shuttle plasmid pHK is:
  • Kana gene and shuttle plasmid pHT08 were subjected to NheI and AflII double digestion, respectively, to purify the digested product, and the two digested products were ligated using T4 DNA ligase to obtain the ligated product.
  • the ligation product was transformed into E. coli TOP10 competent cells, spread on kanamycin LB plates, and placed in a 37°C incubator for overnight cultivation. The resistant clones grown on the plate were picked and cultured to obtain the shuttle plasmid pHK containing the kanamycin resistance gene.
  • primer sequence is:
  • NheI-Kana-F GCTAGCAGATCCTTTGATCTTTTCTACGGGG
  • AflII-Kana-R CTTAAGTTAGAAAAACTCATCGAGCATCAAA
  • the plasmid pET28a was purchased from Merck KGa, and the shuttle plasmid pHT08 was purchased from MoBiTec.
  • the method of deleting the antibiotic resistance gene is:
  • the object of the present invention is also to provide the application of the above genetically engineered bacteria in preparing lipopeptides.
  • a method for preparing lipopeptide includes the following steps:
  • the method for expanding the culture is: culturing for 10-20 hours under the conditions of 35-40° C. and a shaker rotation speed of 150-200 rpm.
  • the fermentation culture method is: cultivating for 1.5-4 hours under the conditions of 35-40° C. and a shaker rotation speed of 150-200 rpm, and adding the inducer to continue culturing for 40-60 hours.
  • the composition of the fermentation medium may be selected as: sugar 30-100g/L, inorganic nitrogen source 10-50g/L, organic nitrogen source 0.5-3g/L, KH 2 PO 4 0.1-1g/L , Na 2 HPO 4 ⁇ 12H 2 O 0.5-0.3g/L, CaCl 2 0.002-0.01g/L, MnSO 4 ⁇ H 2 O 0.002-0.01g/L, FeSO 4 ⁇ 7H 2 O 0.002-0.01g/L , pH 6.5-7.5.
  • the invention adopts genetic engineering technology to construct genetically engineered bacteria, knocks out the relevant genes of the fourth and fifth stages of spore synthesis in the genome of lipopeptide strains, blocks the synthesis of spores, and thus obtains no spores and significantly increases the lipopeptide content Genetic engineering.
  • the genetically engineered bacteria obtained by the present invention can be kept intact under the environment of high concentration of surfactant compared with the genetically engineered bacteria whose key response regulator protein gene spo0A or spoIIIE gene of the third stage of spore formation is knocked out from the vegetative form of the vegetative form. Compared with the starting strain, no spores were produced during the fermentation process and the lipopeptide production was significantly increased.
  • the surfactant yield reached up to 9.9g/L, which was 25% higher than the starting strain.
  • the spoIVB genetically engineered bacteria was knocked out. After 10 hours of secondary inoculation culture, the number of cells increased by a maximum of 3.6 times compared to the starting strain of the same condition.
  • Figure 1 is a schematic diagram of the construction of spoIVB-deficient Bacillus subtilis.
  • Figure 2 is a PCR electrophoresis diagram of left, right, bleo, and left-bleo-right gene fragments; lane 1 is the DNA molecular weight standard; lane 2 is the left gene, 800bp; lane 3 is the right gene, 800bp; lane 4 is the bleo gene, 650bp ; Lane 5 is a left-bleo-right fragment, 2.2 kb.
  • Figure 3 shows the verification results of spoIVB knockout bacteria.
  • Lane 1 is the DNA molecular weight standard;
  • lane 2 is the result of PCR amplification of the original strain THY-7/Pg3-srfA with left-F and right-R, and a 2.9 kb band can be obtained;
  • lane 3 is the genetically engineered strain THY- 7/Pg3-srfA ⁇ spoIVB PCR amplification using left-F and right-R results in a 2.2 kb band.
  • Fig. 4 is the spore diagram observed by the original strain THY-7/Pg3-srfA and the genetically engineered strain THY-7/Pg3-srfA ⁇ spo0A cell morphology microscope; wherein, 1 is the original strain cell morphology diagram, and the arrow indicates the spore; 2 It is the cell morphology diagram of spore-deficient genetically engineered bacteria THY-7/Pg3-srfA ⁇ spo0A.
  • Fig. 5 is a chromatogram of the detection of surfactant in the fermentation broth of the original strain THY-7/Pg3-srfA and the genetically engineered strain THY-7/Pg3-srfA ⁇ spo0A.
  • Figure 6 is the cell morphology observation diagram of the original strain THY-7/Pg3-srfA and the genetically engineered bacteria THY-7/Pg3-srfA ⁇ spoIIIE microscope and transmission electron microscope; where, 1 is the cell morphology observation diagram of the original strain, indicated by arrows in the figure For spores; 2 for morphology of the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIIIE; 3 for the morphology of the original strain cell transmission electron microscopy; 4 for the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIIIE transmission electron microscopy Cell morphology map.
  • Figure 7 is the cell morphology observation diagram of the original strain THY-7/Pg3-srfA and the genetically engineered bacteria THY-7/Pg3-srfA ⁇ spoIVB microscope and transmission electron microscope; wherein, 1 is the cell morphology observation diagram of the original strain, indicated by arrows in the figure For spores; 2 for morphology of spore-deficient genetically engineered bacteria THY-7/Pg3-srfA ⁇ spoIVB; 3 for morphology of the original strain cell transmission electron microscope observation; 4 for spore-deficient genetically engineered bacteria THY-7/Pg3-srfA ⁇ spoIVB transmission electron microscope observation Cell morphology map.
  • Fig. 8 shows the concentration of surfactant in the fermentation product of the original strain THY-7/Pg3-srfA and the genetically engineered strain THY-7/Pg3-srfA ⁇ spoIVB.
  • Figure 9 is the count result of the number of cells in the secondary culture of the original strain THY-7/Pg3-srfA and the genetically engineered strain THY-7/Pg3-srfA ⁇ spoIVB.
  • Figure 10 is the cell morphology observation diagram of the original strain THY-7/Pg3-srfA and the genetically engineered bacteria THY-7/Pg3-srfA ⁇ spoVD microscope and transmission electron microscope; wherein, 1 is the cell morphology observation diagram of the original strain, indicated by arrows in the figure It is a spore; 2 is the morphology of the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoVD.
  • biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means in the books of those skilled in the art.
  • the bacterial genome extraction kit from Omega was used to extract the B. subtilisTHY-7 genome.
  • PCR amplification was performed using the upstream primer left-F and the downstream primer left-R to obtain the homology arm left fragment upstream of the spoIVB gene in the genome.
  • THY-7 genome was used as a template, PCR amplification was performed using the upstream primer right-F and the downstream primer right-R to obtain a homology arm right fragment located downstream of the spoIVB gene in the genome, 800 bp (as shown in FIG. 2).
  • primer sequence is as follows:
  • the primer was synthesized by Platinum Biotechnology (Shanghai) Co., Ltd., dissolved in sterile water and diluted to 10 ⁇ M for use.
  • the polymerase, buffer and restriction enzyme used for PCR amplification were purchased from TaKaRa.
  • the PCR amplification reaction system is:
  • spoIVA-right-R CGGTAGACCTCTTTATAGAATGGGA
  • spoIVC-left-R CCTGTGTGAAATTGTTATCCTCGTGATGAAATAGGGAATAGGTTG
  • spoIVC-right-R ACCCCCCTTTTGTAATTACAATCTC
  • spoVA-left-R CCTGTGTGAAATTGTTATCCTTTGGAGATTGAAGCTGAGGATGTT
  • the left-bleo-right fragment used in spoIVB gene knockout constructed in Example 1 was transformed into competent cells of Bacillus subtilis THY-7 and THY-7/Pg3-srfA by electroporation, respectively, to obtain spoIVB-deficient type Genetically engineered bacteria THY-7/spoIVB:bleo, THY-7/Pg3-srfA/spoIVB:bleo.
  • the preparation and electrotransformation of Bacillus subtilis THY-7/Pg3-srfA competent cells adopt the method in Chinese patent document CN105400784A.
  • the plasmid pHK-cre containing cre recombinase was introduced into B. subtilis THY-7 and THY-7/Pg3-srfA/spoIVB:bleo, and the positive clones were picked and inoculated into LB medium (containing 1mM inducer IPTG) for cultivation 8-10h, dilute the antibiotic-free LB plate, invert it in a 37°C incubator for overnight cultivation, pick a single colony and select B. subtilis THY-7 (pHK-cre) and B. THY-7/Pg3-srfA ⁇ spoIVB (pHK-cre).
  • the sifting process in the farming mode is as follows: single colonies on the plate are picked and mixed in 10uL of sterile physiological saline, and 2uL of bacterial suspensions are respectively taken on LB plates with and without bleomycin and placed upside down Strains grown overnight in a 37°C incubator without antibiotic plates but without bleomycin plates are engineered bacteria that have lost bleomycin resistance genes.
  • B. subtilis THY-7 (pHK-cre) and THY-7/Pg3-srfA ⁇ spoIVB (pHK-cre) strains were selected and inoculated in LB liquid medium, subcultured at 37°C, 200rpm, and diluted with antibiotic-free LB Plate, farming method screening finally obtained spoIVB-deficient genetically engineered bacteria
  • B. subtilis THY-7 ⁇ spoIVB and THY-7/Pg3-srfA ⁇ spoIVB were selected and inoculated in LB liquid medium, subcultured at 37°C, 200rpm, and diluted with antibiotic-free LB Plate, farming method screening finally obtained spoIVB-deficient genetically engineered bacteria B. subtilis THY-7 ⁇ spoIVB and THY-7/Pg3-srfA ⁇ spoIVB.
  • the above-mentioned genetically engineered bacteria were used for lipopeptide production through expansion culture and fermentation culture.
  • the respective yields of lipopeptides produced by spoIVB-deficient genetically engineered bacteria B. subtilis THY-7 ⁇ spoIVB and THY-7/Pg3-srfA ⁇ spoIVB were better than their original strains.
  • THY-7/Pg3-srfA ⁇ spoIVB's lipopeptide production growth factor is better than B. subtilis THY-7 ⁇ spoIVB, so B. subtilis THY-7/Pg3-srfA was selected as the original strain for the next experiment.
  • the genetically engineered bacterium B. subtilis THY-7/Pg3-srfA ⁇ spo0A deficient in the spo0A gene of the first stage of spore synthesis and the genetically engineered bacterium B. subtilis THY-7/Pg3-srfA ⁇ spoIIIE lacking the gene spoIIIE of the third stage spore synthesis, Genetically engineered bacteria B. subtilis THY-7/Pg3-srfA ⁇ spoIVA, B. subtilis THY-7/Pg3-srfA ⁇ spoIVC, B. subtilis THY-7/Pg3-srfA ⁇ spoIVF and spores Synthesis of genetically engineered bacteria B.
  • subtilis THY-7/Pg3-srfA ⁇ spoVA B. subtilis THY-7/Pg3-srfA ⁇ spoVB
  • B. subtilis THY-7/Pg3-srfA ⁇ spoVD and spoVA B.subtilisTHY-7/Pg3-srfA ⁇ spoVE.
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spo0A obtained in Example 2 was inoculated in LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • composition of the fermentation medium used is: sugar 30-100g/L, inorganic nitrogen source 10-50g/L, organic nitrogen source 0.5-3g/L, KH 2 PO 4 0.1-1g/L, Na 2 HPO 4 ⁇ 12H 2 O 0.5-0.3g / L, CaCl 2 0.002-0.01g / L, MnSO 4 ⁇ H 2 O 0.002-0.01g / L, FeSO 4 ⁇ 7H 2 O 0.002-0.01g / L, pH 6.5-7.5 .
  • the detection of surfactant in fermentation broth adopts the method in Chinese patent document CN105400784A.
  • the chromatogram of surfactant in the fermentation broth of genetic engineering THY-7/Pg3-srfA ⁇ spo0A and starting strain THY-7/Pg3-srfA is shown in Figure 5. After knocking out the spo0A gene in the spore synthesis pathway, no surface was detected in the fermentation broth Activin.
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIIIE obtained in Example 2 was inoculated in LB liquid medium and cultured at 37°C and 200 rpm for 16 hours to obtain a genetically engineered bacterial solution;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIVA obtained in Example 2 was inoculated in LB liquid medium and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIVB obtained in Example 2 was inoculated in LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain a genetically engineered bacterial solution;
  • the fermentation broth cultivated for 24 hours was inoculated into fresh fermentation medium at 37° C. and 200 rpm for secondary culture.
  • the inoculation time and the secondary culture for 10 hours were used to dilute the bacterial solution and counted on a plate.
  • the detection of surfactant in fermentation broth adopts the method in Chinese patent document CN105400784A.
  • the statistical results of the concentration of surfactant in the fermentation broth of genetic engineering THY-7/Pg3-srfA ⁇ spoIVB and the starting strain THY-7/Pg3-srfA are shown in Figure 9:
  • the yield of surfactant of THY-7/Pg3-srfA ⁇ spoIVB is 9.9g/
  • L is 25% higher than the starting bacteria (7.9g/L).
  • the cells were fermented for 24h, and the cell count results at 0 and 10 hours of inoculation are shown in Figure 10: After equal inoculation (the initial cfu is 1 ⁇ 10 8 ), after 10 hours, the number of THY-7/Pg3-srfA ⁇ spoIVB cells It is 4.6 times that of the starting strain, reaching 5.15 ⁇ 10 9 /mL (shown in FIG. 9).
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIVC obtained in Example 2 was inoculated in LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoIVF obtained in Example 2 was inoculated in LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoVA obtained in Example 2 was inoculated in LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoVB obtained in Example 2 was inoculated in LB liquid medium and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoVD obtained in Example 2 was inoculated into LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;
  • the spore-deficient genetically engineered bacterium THY-7/Pg3-srfA ⁇ spoVE obtained in Example 2 was inoculated in LB liquid medium and cultured at 37°C and 200 rpm for 16 hours to obtain genetically engineered bacterial liquid;

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Abstract

一种高产脂肽的基因工程菌及其应用。所述高产脂肽的基因工程菌，其是使原始菌株中芽孢合成第四阶段和第五阶段中的至少一个基因失活构建而成，其在高浓度表面活性素环境下能够保持完整的细胞形态；与出发菌株相比，发酵过程中无芽孢产生且显著提高脂肽产量，摇瓶中表面活性素产量最高达到9.9g/L，比出发菌株提高了25％，细胞活性也得到增强。

Description

一种高产脂肽的基因工程菌及其应用 技术领域
本发明属于基因工程技术领域，具体涉及一种高产脂肽的基因工程菌及其应用。
背景技术
脂肽(lipopeptide)类生物表面活性剂是一类由亲水的环状寡肽与疏水的脂肪酸链以内酯键连接而成的两性物质，主要由芽孢杆菌、链霉菌等微生物合成。芽孢杆菌脂肽可分为表面活性素、芬芥素、伊枯草菌素等，其中在气/液界面形成独特“马鞍”构象的表面活性素具有优良的表面活性、生物降解性及抗菌活性，在石油开采、生物防治、医药及日化等领域都有广阔的应用前景。但由于芽孢杆菌发酵生产表面活性素产率低限制了表面活性素的工业化生产和应用，同时芽孢杆菌发酵中后期产生大量芽孢休眠体，占用了部分营养源且芽孢在发酵生产结束后灭菌排放时也会有环境安全隐患，这些进一步制约了表面活性素的工业化生产和应用。
目前，提高微生物脂肽发酵水平的方法包括培养条件优化、诱变育种、强化表面活性素跨膜运输、强化表面活性素合成酶表达等。专利文献(CN 101892176A、CN 101775427A、WO 2002026961A)等通过对枯草芽孢杆菌发酵生产表面活性素发酵过程中培养基及培养条件进行优化，提高了发酵液中表面活性素的产量。CN101928677A公开了通过紫外诱变处理玫瑰孢链霉菌，US05227294公开了用亚硝基甲替尿烷处理枯草芽孢杆菌，上述文献中的微生物在诱变处理后，其表面活性素合成量都有增加。中国专利文献CN103898038A公开了通过强化脂肽由胞内向胞外运输的跨膜蛋白YcxA，枯草芽孢杆菌发酵生产的表面活性素产量提高了97％，中国专利文献CN1554747A公开了在枯草芽孢杆菌中表达comA基因，脂肽产量提高了50％。中国专利文献CN105400784A 通过将脂肽合成酶基因簇启动子替换成诱导型强启动子Pg3，表面活性素产量提高了17.7倍。文献(Dhali D,Coutte F,Arias A A,et al.Biotechnology journal,2017,12(7):1600574.)公开了敲除枯草芽孢杆菌中催化支链氨基酸生成脂肪酸的BKD操纵子中的lpdV基因，表面活性素产量提高了1.4倍。
另外，枯草芽孢杆菌中芽孢生成过程已有部分基础研究，芽孢合成的整个过程受多个调控蛋白调控，而这些调控蛋白又与参与胞内的其它代谢途径，如spo0A与枯草芽孢杆菌胞内500多个基因表达相关(Virginie Molle,et al.Molecular Microbiology，2003,50(5):1683-1701)，通过构建其缺陷型菌株能成功获得无芽孢菌株，但胞内麦芽糖操纵子转录表达受到影响(余志强，枯草芽孢杆菌麦芽糖启动子PΔglvA整合表达载体的构建及启动子功能的初步探讨[D],2004)，乙偶姻发酵产量减少(李欣.枯草芽孢杆菌芽孢形成阻断及碳流调控对乙偶姻合成的影响[M]，2017)。因此，不合适的芽孢基因敲除方案，不仅不会提高目标产物产量，反而会降低目标产物的产量。敲除何种芽孢合成基因构建无芽孢工程菌来提高表面活性素产量、同时降低枯草芽孢杆菌应用安全隐患的研究未见报道。
发明内容
本发明的目的在于为了克服现有技术中的问题，提供一种高产脂肽的基因工程菌，其发酵过程中无芽孢产生并且可以提高脂肽的产量。
本发明的一种高产脂肽的基因工程菌，其是使产脂肽的原始菌株中调控芽胞壁形成的基因中的至少一个失活构建而成，所述调控芽胞壁形成的基因包括spoIVA、spoIVB、spoIVC、spoIVF、spoVA、spoVB、spoVD和spoVE。
上述调控芽胞壁形成的基因，在不同的菌株中的基因编号不同。其序列，以选择枯草芽孢杆菌模式菌B.subtilis 168中的编号为例，其GeneID分别为：
spoIVA(GeneID:938991)；
spoIVB(GeneID:938654)；
spoIVC(包含spiIVCA(GeneID:937799)和spoIVCB(GeneID:937803))；
spoIVF(包括spoIVFB(GeneID:937505)和spoIVFA(GeneID:937501))；
spoVA(包含spoVAA(GeneID:938734)、spoVAB(GeneID:938933)、spoVAC (GeneID:938733)、spoVAD(GeneID:938932)、spoVAEA(GeneID:8303022)、spoVAEB(GeneID:8303020)和spoVAF(GeneID:938936))；
spoVB(GeneID:938022)；
spoVD(GeneID:936661)；
和spoVE(GeneID:936953)。
所述失活是指基因不表达或者不完全表达。
所述失活的方法为基因敲除或基因变异。
所述原始菌株为枯草芽孢杆菌、蜡状芽孢杆菌或假单胞菌。
在一实施例中，所述原始菌株中的表面活性素合成酶由诱导型强启动子控制，所述诱导型强启动子为可以选择Pg3。
在一实施例中，所述原始菌株为枯草芽孢杆菌Bacillus subtilis THY-7。
所述枯草芽孢杆菌Bacillus subtilis THY-7，于2014年3月11日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏登记号为CGMCC No.8906，并在中国专利文献CN105400784A中公开。
在一实施例中，所述原始菌株为枯草芽孢杆菌Bacillus subtilis THY-7/Pg3-srfA。
所述枯草芽孢杆菌Bacillus subtilis THY-7/Pg3-srfA，是指在枯草芽孢杆菌Bacillus subtilis THY-7中加入诱导型强启动子Pg3，使强启动子Pg3控制表面活性素合成酶srfA的表达。关于诱导型强启动子Pg3、枯草芽孢杆菌Bacillus subtilis THY-7/Pg3-srfA及其具体的制备方法在中国专利文献CN105400784A中公开。
上述基因工程菌的可以按下述方法构建，包括：
(1)扩增原始菌株基因组中需要敲除的目的基因的左、右同源臂和抗生素抗性基因片段，通过搭桥PCR得到用于敲除目的基因且含有抗性基因的线性片段；
(2)将上述线性片段导入原始菌株，筛选得到抗性基因原位替换目的基因的工程菌，得到含有抗性筛选标记的基因工程菌；
(3)将含有cre重组酶基因的质粒转化至含有抗性筛选标记的基因工程菌中，培养筛选得到去除抗性基因的高产脂肽的基因工程菌。
上述基因工程菌的构建方法中，抗生素抗性基因可以选择红霉素抗性基因、博来霉素抗性基因、卡那霉素抗性基因、状观霉素抗性基因和新霉素抗性基因等。优选的，使用博来霉素抗性基因。
在一实施例中，上述基因工程菌构建方法为：
1、合成两端带有lox位点的抗生素抗性基因。
2、利用需要敲除的目的基因左、右同源臂上下游引物，以原始菌株基因组为模板进行聚合酶链式反应，扩增并获得基因组上位于目的基因上下游的同源臂left和right片段。
3、搭桥PCR获得left-抗生素抗性基因-right片段并转入原始菌株中，涂布于含有抗生素的LB平板上，挑取阳性克隆进行培养，用left-F和right-R引物进行PCR验证，得到抗生素抗性基因原位替换目的基因的目的基因缺陷菌株。
4、合成识别lox位点的cre重组酶基因，构建含有重组酶的pHK-cre质粒。
5、将质粒pHK-cre导入抗生素抗性基因原位替换目的基因的目的基因缺陷菌株，得到含有cre重组酶基因的目的基因缺陷菌株。
6、挑取上述含有cre重组酶基因的目的基因缺陷菌株接种至LB(含1mM IPTG)培养基中，培养并筛选得到抗生素基因缺失的目的基因缺陷型菌株。
7、将上述抗生素基因缺失的目的基因缺陷型菌株传代培养丢掉质粒pHK-cre得到无芽孢的高产脂肽的基因工程菌。
上述基因工程菌的制备方法中，left-抗生素抗性基因-right片段的具体构建方法为：
1、以left-F和left-R为上下游引物，以原始菌株基因组为模板进行聚合酶链式反应，扩增并获得位于基因组中目的基因上游的同源臂left基因片段。
2、以right-F和right-R为上下游引物，以原始菌株基因组为模板进行聚合酶链式反应，扩增并获得位于基因组中目的基因下游的同源臂right基因片段。
3、按照公开数据库NCBI上公布的p7z6质粒序列(NCBI：EU541492.1)合成两端带有lox识别位点的抗生素抗性基因片段bleo。
4、将上述得到的3个基因片段回收后混合做为模板，以left-F和right-R做为上、下游引物进行PCR扩增得到用于目的基因敲除的线性基因片段left-抗生素抗性基因-right。
上述基因工程菌的构建方法中，pHK-cre质粒的具体构建方法为：
1、按照公开数据库NCBI上公布的p06-PgrapA-cre质粒序列(GenBank:MG014197.1)合成编码重组酶的cre基因片段，合成时基因cre上下游分别加上XbaI和XmaI酶切位点。
2、将合成的cre基因以及穿梭质粒pHK分别进行XbaI和XmaI双酶切，纯化酶切产物，使用T4 DNA连接酶将两种酶切产物进行连接，得到连接产物。
3、将连接产物转化大肠杆菌E.coli TOP10感受态细胞，涂布于卡纳霉素的LB平板，倒置于37℃培养箱中过夜培养。挑取平板上长出来的抗性克隆进行培养，提取质粒测序验证，得到含有重组酶cre基因序列的表达质粒。
上述基因工程菌的制备方法中，穿梭质粒pHK的具体构建方法为：
1、以pET28a质粒为模板，以NheI-Kana-F和AflII-Kana-R为引物扩增得到卡那霉素抗性基因Kana
2、将Kana基因及穿梭质粒pHT08分别进行NheI和AflII双酶切，纯化酶切产物，使用T4DNA连接酶将两种酶切产物进行连接，得到连接产物。
3、将连接产物转化大肠杆菌E.coli TOP10感受态细胞，涂布于卡纳霉素的LB平板，倒置于37℃培养箱中过夜培养。挑取平板上长出来的抗性克隆进行培养，得到含有卡纳霉素抗性基因的穿梭质粒pHK。
其中，所述引物序列为：
NheI-Kana-F:GCTAGCAGATCCTTTGATCTTTTCTACGGGG
AflII-Kana-R:CTTAAGTTAGAAAAACTCATCGAGCATCAAA
所述质粒pET28a购自于Merck KGa公司，穿梭质粒pHT08购自于MoBiTec公司。
上述基因工程菌的制备方法中，抗生素抗性基因缺失的方法为：
1、将pHK-cre质粒导入基因组中目的基因被原位替换成抗性基因的工程菌株中，涂布于含卡那霉素的LB平板，倒置于37℃培养箱中过夜培养。
2、挑取平板上的阳性克隆接种于10ml含有IPTG诱导剂的LB培养基中培养，14～20h后取菌液稀释涂布于不含抗生素的LB平板，倒置于37℃培养箱中过夜培养。
3、挑取平板上的单菌落接于10uL的无菌生理盐水中混匀制成菌悬液，吸 取2uL分别滴在含抗生素的LB平板和无抗性素平板，倒置于37℃培养箱中过夜培养。
4、挑取无抗平板上生长而卡那霉素平板上不生长的菌株即为抗生素基因缺失的工程菌。
本发明的目的也在于提供上述基因工程菌在制备脂肽中的应用。
一种脂肽的制备方法，包括步骤如下：
将上述基因工程菌进行扩大培养后，再进行发酵培养，含有脂肽的发酵液。
所述扩大培养的方法为：在35-40℃、摇床转速为150-200rpm的条件下培养10-20h。
所述发酵培养的方法为：在35-40℃、摇床转速为150-200rpm的条件下培养1.5-4h，加入诱导剂后继续培养40-60h。
上述方法中，所述发酵培养基的组成可以选择为：糖类30-100g/L，无机氮源10-50g/L，有机氮源0.5-3g/L，KH 2PO 4 0.1-1g/L,Na 2HPO 4·12H 2O 0.5-0.3g/L,CaCl 2 0.002-0.01g/L,MnSO 4·H 2O 0.002-0.01g/L,FeSO 4·7H 2O 0.002-0.01g/L,pH 6.5-7.5。
本发明的优点和有益效果：
本发明采用基因工程技术构建基因工程菌，敲除了产脂肽菌株基因组中芽孢合成第四阶段和第五阶段的相关基因，阻断了芽孢的合成，从而得到了无芽孢并且显著提高脂肽含量的基因工程。本发明所得基因工程菌与敲除细胞自营养型进入芽孢合成阶段的关键应答调节蛋白基因spo0A或芽孢形成第三阶段基因spoIIIE的基因工程菌相比，在高浓度表面活性素环境下能够保持完整的细胞形态；与出发菌株相比，发酵过程中无芽孢产生且显著提高脂肽产量，表面活性素产量最高达到9.9g/L，比出发菌株提高了25％；其中敲除spoIVB基因工程菌，二次接种培养10小时后，细胞数比同等条件的出发菌株最多增长了3.6倍。
附图说明
图1为用于spoIVB缺陷型枯草芽孢杆菌构建示意图。
图2为left、right、bleo和left-bleo-right基因片段PCR电泳图；泳道1为DNA分子量标准；泳道2为left基因，800bp；泳道3为right基因，800bp；泳道4为bleo基因，650bp；泳道5为left-bleo-right片段，2.2kb。
图3为spoIVB敲除菌验证结果。泳道1为DNA分子量标准；泳道2为原始菌株THY-7/Pg3-srfA用left-F和right-R进行PCR扩增的结果，可得到2.9kb的条带；泳道3为基因工程菌THY-7/Pg3-srfAΔspoIVB用left-F和right-R进行PCR扩增的结果，可得到2.2kb的条带。
图4为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspo0A细胞形态显微镜观测的芽孢图；其中，1为原始菌株细胞形态图，图中箭头所指为芽孢；2为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspo0A细胞形态图。
图5为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspo0A的发酵液检测表面活性素色谱图。
图6为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspoIIIE显微镜及透射电镜的细胞形态观测图；其中，1为原始菌株细胞形态显微镜观测图，图中箭头所指为芽孢；2为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspoIIIE细胞形态图；3为原始菌株细胞形态透射电镜观测；4为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspoIIIE透射电镜观测细胞形态图。
图7为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspoIVB显微镜及透射电镜的细胞形态观测图；其中，1为原始菌株细胞形态显微镜观测图，图中箭头所指为芽孢；2为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspoIVB细胞形态图；3为原始菌株细胞形态透射电镜观测；4为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspoIVB透射电镜观测细胞形态图。
图8为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspoIVB的发酵产物中表面活性素的浓度。
图9为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspoIVB二次培养细胞数计数结果。
图10为原始菌株THY-7/Pg3-srfA与基因工程菌THY-7/Pg3-srfAΔspoVD显微镜及透射电镜的细胞形态观测图；其中，1为原始菌株细胞形态显微镜观测图，图中箭头所指为芽孢；2为芽孢缺陷型基因工程菌THY-7/Pg3-srfAΔspoVD细胞形态图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。如未特别指明，实施例中所用的生化试剂均为市售试剂，实施例中所用的技术手段为本领域技术人员书中的常规手段。
实施例1用于敲除芽孢基因的线性片段left-bleo-right的构建
构建流程如图1所示，以spoIVB基因、博来霉素抗性基因为例。
按照数据库NCBI上公布的p7z6质粒序列(NCBI：EU541492.1)合成两端带有lox识别位点的博来霉素抗性基因片段bleo，650bp。
使用Omega公司的细菌基因组提取试剂盒提取枯草芽孢杆菌B.subtilisTHY-7基因组。以得到的基因组为模板，使用上游引物left-F和下游引物left-R进行PCR扩增得到基因组中位于spoIVB基因上游的同源臂left片段。以THY-7基因组为模板，使用上游引物right-F和下游引物right-R进行PCR扩增得到基因组中位于spoIVB基因下游的同源臂right片段，800bp(如图2所示)。
将上述得到left、bleo和right 3个基因片段混合做为模板，以left-F和right-R做为上、下游引物进行PCR扩增left-bleo-right片段，2.2kb(如图2所示)。
其中，所述引物序列如下：
left-F:GAAAAACATGGGCGAAAAAGCT
left-R:CCTGTGTGAAATTGTTATCCTTCACTACTTCACTCTCCTCGCTCC
right-F:TCGTGACTGGGAAAACCCTGGCGACTGCCGGAGTTTCCGGCAGTTT
right-R:ATAGAGATAGCCTTTAATATGGGCT
引物由铂尚生物技术(上海)有限公司合成，用无菌水溶解并稀释至10μM备用。PCR扩增所用聚合酶、缓冲液和限制性内切酶购自TaKaRa公司。PCR扩增反应体系为：
Figure PCTCN2018118893-appb-000001
热循环条件为
Figure PCTCN2018118893-appb-000002
同样的，利用上述方法，得到用于敲除spo0A、spoIIIE、spoIVA、spoIVC、spoIVF、spoVA、spoVB、spoVD、spoVE基因的含博来霉素抗性基因的线性片段，其中用到的引物序列为：
spo0A-left-F:ATGGACACAAAGAAACCAATTGTAGTGGAGA
spo0A-left-R:CCTGTGTGAAATTGTTATCCGCTCACGCTTGCTTTTTCT
spo0A-right-F:TCGTGACTGGGAAAACCCTGGCGACATGAGCTTATTAAGTGGT
spo0A-right-R:CTGCCGAAACGATTCGGCAGTCTTTTTTCCC
spoIIIE-left-F:AATGTTTTCAAAAGGAAACTTAACA
spoIIIE-left-R:CCTGTGTGAAATTGTTATCCCACCTTACTGCGTTAAAAAATGTAA
spoIIIE-right-F:TCGTGACTGGGAAAACCCTGGCGTGAAGGGAGTTCCGCTTTCTATAGTTGTCAA
spoIIIE-right-R:CTCTTCGTATCATCTTCAGACGGCA
spoIVA-left-F:GTGATCCCCTCCCGGACTTCCTATC
spoIVA-left-R:CTGTGTGAAATTGTTATCCCTTACAAGGATGTGCTATCTCGTGA
spoIVA-right-F:TCGTGACTGGGAAAACCCTGGCGTAAGTGCATCTAAGATCGTATCAAA
spoIVA-right-R:CGGTAGACCTCTTTATAGAATGGGA
spoIVC-left-F:TATTAAATAATGATAGCAATCGTTA
spoIVC-left-R:CCTGTGTGAAATTGTTATCCTCGTGATGAAATAGGGAATAGGTTG
spoIVC-right-F:TCGTGACTGGGAAAACCCTGGCGTGCTGCTTACCAAAGCCGGACTCCC
spoIVC-right-R:ACCCCCCTTTTGTAATTACAATCTC
spoIVF-left-F:GAAAATAGAATTATTTACGATCTGG
spoIVF-left-R:CCTGTGTGAAATTGTTATCCTTGCCATCATCCTTTGCATTCGT
spoIVF-right-F:TCGTGACTGGGAAAACCCTGGCGAAACTGATTGACAAACGCCTTGTATT
spoIVF-right-R:CCTCCTATATTATGTTTGTGGTCAC
spoVA-left-F:TCATTGATCACCATCTTTCGGTGGT
spoVA-left-R:CCTGTGTGAAATTGTTATCCTTTGGAGATTGAAGCTGAGGATGTT
spoVA-right-F:TCGTGACTGGGAAAACCCTGGCGGATTTCATAGAAATTATCCACCACA
spoVA-right-R:AAACTGACTGAAGAGTATGATAATG
spoVB-left-F:TCAATAGAACGAAAAGGAAAAACGC
spoVB-left-R:CCTGTGTGAAATTGTTATCCTTTACCCCCTGCCTTCTCAAACTAC
spoVB-right-F:TCGTGACTGGGAAAACCCTGGCGTGGTCACGTGCGGTGCCCATCTTTT
spoVB-right-R:GTAGACGATCATCTATTTCATACCA
spoVD-left-F:CTGACTCAGATAAGGAAGAAACAAA
spoVD-left-R:CTGTGTGAAATTGTTATCCAGAGACCGTTCACTCCTTATTTAGG
spoVD-right-F:TCGTGACTGGGAAAACCCTGGCGTTTTATTGCAGAAAAAATGCTGATA
spoVD-right-R:CTTCGTCGGCTGTCCGTAAAACGCA
spoVE-left-F:TTTACTGGTGTGAAGCACCGCCTCC
spoVE-left-R:CCTGTGTGAAATTGTTATCCACACCCCAAGCTTAAAGTCATTAGG
spoVE-right-F:TCGTGACTGGGAAAACCCTGGCGTCCAAGCCTCCTGTCTAACATGAAG
spoVE-right-R:AAACTTATTCGTGATGCCCGGCAGG
实施例2 spoIVB缺陷型基因工程菌的构建
将实施例1中构建的用于spoIVB基因敲除的left-bleo-right片段以电穿孔法分别转化枯草芽孢杆菌THY-7和THY-7/Pg3-srfA的感受态细胞，可得到spoIVB缺陷型基因工程菌THY-7/spoIVB:bleo、THY-7/Pg3-srfA/spoIVB:bleo。其中，枯草芽孢杆菌THY-7/Pg3-srfA感受态细胞的制备及电转化采用中国专利文献CN105400784A中的方法。
复苏后取100uL菌液涂布于含10-30μg/mL博来霉素的LB固体培养基上，倒置于37℃培养箱中过夜培养，挑取单菌落，使用上下游引物left-F和right-R进行PCR验证，验证时同时以原始菌株基因组为模板以上述相同引物PCR做为对照，原始菌株可扩增出约2.9kb条带，spoIVB敲除菌可扩增出约2.2kb条带，验证结果如图3所示，即获得了芽孢合成相关基因spoIVB缺陷型基因工程菌B.subtilis THY-7/spoIVB:bleo和B.subtilis THY-7/Pg3-srfA/spoIVB:bleo。
将含有cre重组酶的质粒pHK-cre分别导入B.subtilis THY-7和THY-7/Pg3-srfA/spoIVB:bleo中，挑取阳性克隆接种至LB培养基中(含1mM诱导剂IPTG)培养8-10h，稀释涂不含抗生素的LB平板，倒置于37℃培养箱中过夜培养，挑取单菌落采取种田方式筛选丢失博来霉素基因的B.subtilis THY-7(pHK-cre)和THY-7/Pg3-srfAΔspoIVB(pHK-cre)。种田方式筛菌过程为：挑取平板上的单菌落于10uL无菌生理盐水中混匀，分别取2uL菌悬液滴于含博来霉素和不含博来霉素的LB平板，倒置于37℃培养箱中过夜培养，无抗生素平板生长而博来霉素平板不生长的菌株，即为丢失博来霉素抗性基因的工程菌。
分别挑取B.subtilis THY-7(pHK-cre)和THY-7/Pg3-srfAΔspoIVB(pHK-cre)菌株接种于LB液体培养基中，37℃，200rpm传代培养，稀释涂不含抗生素的LB平板，种田法筛选最终得到spoIVB缺陷型基因工程菌B.subtilis THY-7ΔspoIVB 和THY-7/Pg3-srfAΔspoIVB。
将上述基因工程菌，经过扩大培养和发酵培养进行脂肽生产，spoIVB缺陷型基因工程菌B.subtilis THY-7ΔspoIVB和THY-7/Pg3-srfAΔspoIVB所产脂肽的分别产量优于其原始菌株，但是THY-7/Pg3-srfAΔspoIVB的脂肽生产增长倍数优于B.subtilis THY-7ΔspoIVB，故选用B.subtilis THY-7/Pg3-srfA作为原始菌株进行下一步实验。
同样的，得到芽孢合成第一阶段基因spo0A缺陷的基因工程菌B.subtilis THY-7/Pg3-srfAΔspo0A，芽孢合成第三阶段基因spoIIIE缺失的基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIIIE，芽孢合成第四阶段基因spoIVA、spoIVC、spoIVF缺失的基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIVA、B.subtilis THY-7/Pg3-srfAΔspoIVC、B.subtilis THY-7/Pg3-srfAΔspoIVF以及芽孢合成第五阶段基因spoVA、spoVB、spoVD、spoVE缺失的基因工程菌B.subtilis THY-7/Pg3-srfAΔspoVA、B.subtilis THY-7/Pg3-srfAΔspoVB、B.subtilis THY-7/Pg3-srfAΔspoVD和B.subtilis THY-7/Pg3-srfAΔspoVE。
实施例3使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspo0A生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspo0A接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
所使用的发酵培养基的组成为：糖类30-100g/L，无机氮源10-50g/L，有机氮源0.5-3g/L，KH 2PO 4 0.1-1g/L，Na 2HPO 4·12H 2O 0.5-0.3g/L,CaCl 20.002-0.01g/L,MnSO 4·H 2O 0.002-0.01g/L,FeSO 4·7H 2O 0.002-0.01g/L,pH 6.5-7.5。
发酵过程中细胞芽孢形态显微镜观测采用文献Bacteriology Proceedings,1959,38-39中的方法。基因工程THY-7/Pg3-srfAΔspo0A与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图4所示，敲除芽孢合成途径中spo0A基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基 因工程THY-7/Pg3-srfAΔspo0A与出发菌株THY-7/Pg3-srfA发酵液中表面活性素色谱图如图5所示，敲除芽孢合成途径中spo0A基因后，发酵液中未检测出表面活性素。
实施例4使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIIIE生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoIIIE接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
基因工程THY-7/Pg3-srfAΔspoIIIE与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图6所示，敲除芽孢合成途径中spoIIIE基因后，菌体细胞中无芽孢产生。
发酵过程中细胞芽孢形态透射电镜观测采用文献Microscopy Research and Technique 2005,66:307-311中的方法。基因工程THY-7/Pg3-srfAΔspoIIIE与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图6所示：敲除芽孢合成途径中spoIIIE基因后，菌体在高浓度表面活性素(8.4g/L)条件下不能维持正常的细胞形态。
实施例5使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIVA生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoIVA接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoIVA基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoIVA表面活性素产量为8.9g/，L比出发菌(7.9g/L)提高了12.7％。
实施例6使用基因工程菌THY-7/Pg3-srfAΔspoIVB生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoIVB接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，即得到含脂肽的发酵液。
将培养至24h的发酵液接种至新鲜发酵培养基中于37℃、200rpm条件下二次培养，取接种时刻和二次培养10h菌液稀释涂平板计数。
基因工程THY-7/Pg3-srfAΔspoIVB与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图7所示，敲除芽孢合成途径中spoIVB基因后，菌体细胞中无芽孢产生。
基因工程THY-7/Pg3-srfAΔspoIVB与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图8所示，敲除芽孢合成途径中spoIVB基因后，菌体在高浓度表面活性素(9.9g/L)条件下仍然能维持比较完整的细胞形态。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoIVB与出发菌株THY-7/Pg3-srfA发酵液中表面活性素浓度的统计结果如图9所示：THY-7/Pg3-srfAΔspoIVB表面活性素产量为9.9g/，L比出发菌(7.9g/L)提高了25％。
将发酵24h的细胞二次接种，接种0小时及10小时时细胞计数结果如图10所示：等量接种(初始cfu为1×10 8)10小时后，THY-7/Pg3-srfAΔspoIVB细胞数是出发菌株的4.6倍，达到5.15×10 9/mL(图9所示)。
实施例7使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIVC生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoIVC接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoIVC基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoIVC表面活性素产量为8.8g/，L比出发菌(7.9g/L)提高了11.4％。
实施例8使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoIVF生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoIVF接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoIVF基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoIVA表面活性素产量为9.0g/，L比出发菌(7.9g/L)提高了13.9％。
实施例9使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoVA生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoVA接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoVA基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoVA表面活性素产量为9.1g/，L比出发菌(7.9g/L)提高了15.2％。
实施例10使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoVB生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoVB接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoVB基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoVA表面活性素产量为9.0g/，L比出发菌(7.9g/L) 提高了13.9％。
实施例11使用基因工程菌THY-7/Pg3-srfAΔspoVD生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoVD接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，即得到含脂肽的发酵液。
基因工程THY-7/Pg3-srfAΔspoVD与出发菌株THY-7/Pg3-srfA发酵液中菌体细胞形态显微镜观测结果如图10所示:敲除芽孢合成途径中spoVD基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoVD表面活性素产量为9.2g/，L比出发菌(7.9g/L)提高了16％。
实施例12使用基因工程菌B.subtilis THY-7/Pg3-srfAΔspoVE生产脂肽类表面活性剂—表面活性素
将实施例2所得到的芽孢缺陷型的基因工程菌THY-7/Pg3-srfAΔspoVE接种于LB液体培养基中，37℃、200rpm条件下培养16h，得到基因工程菌菌液；
以5％的比例接入装有100mL发酵培养基的摇瓶中，37℃、200rpm条件下培养2-6h时加入1mM的IPTG，继续培养至2d，发酵结束。
敲除芽孢合成途径中spoVE基因后，菌体细胞中无芽孢产生。
发酵液中表面活性素检测采用中国专利文献CN105400784A中的方法。基因工程THY-7/Pg3-srfAΔspoVE表面活性素产量为8.6g/，L比出发菌(7.9g/L)提高了8.9％。

Claims (11)

  1. 一种高产脂肽的基因工程菌，其是使产脂肽的原始菌株中调控芽胞壁形成的基因中的至少一个失活构建而成，所述调控芽胞壁形成的基因包括spoIVA、spoIVB、spoIVC、spoIVF、spoVA、spoVB、spoVD和spoVE。
  2. 根据权利要求1所述的基因工程菌，其特征在于，所述失活的方法为基因敲除或基因变异。
  3. 根据权利要求1所述的基因工程菌，其特征在于，所述原始菌株中的表面活性素合成酶由诱导型强启动子控制。
  4. 根据权利要求3所述的基因工程菌，其特征在于，所述诱导型强启动子为Pg3。
  5. 根据权利要求1所述的基因工程菌，其特征在于，所述原始菌株为枯草芽孢杆菌、蜡状芽孢杆菌或假单胞菌。
  6. 根据权利要求5所述的基因工程菌，其特征在于，所述原始菌株为枯草芽孢杆菌Bacillus subtilis THY-7。
  7. 根据权利要求5所述的基因工程菌，其特征在于，所述原始菌株为枯草芽孢杆菌Bacillus subtilis THY-7/Pg3-srfA。
  8. 权利要求1-7任一项所述基因工程菌在生产脂肽中的应用。
  9. 一种脂肽的制备方法，其特征在于，包括步骤如下：
    权利要求1-7任一项所述基因工程菌进行扩大培养后，再进行发酵培养，含有脂肽的发酵液。
  10. 根据权利要求9所述的方法，其特征在于，所述扩大培养的方法为：在35-40℃、摇床转速为150-200rpm的条件下培养10-20h。
  11. 根据权利要求9所述的方法，其特征在于，所述发酵培养的方法为：在35-40℃、摇床转速为150-200rpm的条件下培养1.5-4h，加入诱导剂后继续培养40-60h。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1160765A (zh) * 1995-08-30 1997-10-01 山道士有限公司 苏云金杆菌芽孢形成基因
CN1190434A (zh) * 1995-07-07 1998-08-12 诺沃挪第克公司 用不能形成芽孢的芽孢杆菌生产蛋白质的方法
CN104471066A (zh) * 2012-01-31 2015-03-25 巴斯夫欧洲公司 表达方法
CN105400784A (zh) * 2015-10-10 2016-03-16 清华大学 一种枯草芽孢杆菌诱导性强启动子及其应用
WO2017123946A1 (en) * 2016-01-15 2017-07-20 University Of Massachussetts Anthelmintic compositions and methods
WO2018217807A1 (en) * 2017-05-23 2018-11-29 University Of Massachusetts Purified anthelmintic compositions and related methods

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1190434A (zh) * 1995-07-07 1998-08-12 诺沃挪第克公司 用不能形成芽孢的芽孢杆菌生产蛋白质的方法
CN1160765A (zh) * 1995-08-30 1997-10-01 山道士有限公司 苏云金杆菌芽孢形成基因
CN104471066A (zh) * 2012-01-31 2015-03-25 巴斯夫欧洲公司 表达方法
CN105400784A (zh) * 2015-10-10 2016-03-16 清华大学 一种枯草芽孢杆菌诱导性强启动子及其应用
WO2017123946A1 (en) * 2016-01-15 2017-07-20 University Of Massachussetts Anthelmintic compositions and methods
WO2018217807A1 (en) * 2017-05-23 2018-11-29 University Of Massachusetts Purified anthelmintic compositions and related methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHI, YAN: "Mechanism of MicroRNA-203 Inhibiting Proliferation and Migration of Osteosarcoma Cells", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE; ENGINEERING SCIENCE AND TECHNOLOGY I, no. 4, 15 April 2018 (2018-04-15), ISSN: 1674-022x, DOI: 20190723165539Y *

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