WO2017215204A1 - Souche recombinante de bacillus subtilis et procédé de préparation correspondant et son application - Google Patents

Souche recombinante de bacillus subtilis et procédé de préparation correspondant et son application Download PDF

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WO2017215204A1
WO2017215204A1 PCT/CN2016/108692 CN2016108692W WO2017215204A1 WO 2017215204 A1 WO2017215204 A1 WO 2017215204A1 CN 2016108692 W CN2016108692 W CN 2016108692W WO 2017215204 A1 WO2017215204 A1 WO 2017215204A1
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seq
bacillus subtilis
upstream
strain
amplifying
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潘力
刘欣
王斌
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华南理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • 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

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to a recombinant strain of Bacillus subtilis, a preparation method and application thereof.
  • Bacillus subtilis belongs to the genus Bacillus, is a Gram-positive bacterium, is not pathogenic, and is recognized as a safe strain (GRAS).
  • B. subtilis has only a single layer of extracellular membrane, which has a fast growth rate, simple nutrient requirements, easy survival, colonization and reproduction. It has a clear heritage background, good protein secretion ability and a series of efficient signal peptides. It has been used for food for a long time. Fermentation and production of important enzyme preparations; ideal hosts for the expression and secretion of foreign proteins in prokaryotic expression systems.
  • B. subtilis has a certain degree of limitation due to factors such as the excessive secretion of its own protease and the instability of the constructed expression plasmid.
  • the transformation of the B. subtilis expression system has focused on knocking out the B. subtilis extracellular protease gene.
  • Protease-deficient strains such as DB104, DB403, WB600, WB700 and WB800 have been obtained by gene knockout technology.
  • WB800 is an eight protease-deficient strain constructed by the experimental team of Wong on the model strain B.subtilis168, knocking out neutral protease (npr), neutral protease B (nprB), alkaline protease (apr), metalloproteinase (mpr), Three serine proteases (bpf, epr, and vpr) and cell wall protease (wprA) [WuSC, YeungJC, WongSL, etal. Functional production and characterization of a fibrin-specific single-chain antibody fragment from Bacillus subtilis: effects of molecular chaperones and a wall-bound Protease on antibody fragment production [J]. Appl. Environ. Microbiol, 2002, 68:3261-269.], enables many genes to achieve stable expression. However, WB800 grows slowly, and residual protease can also effectively degrade highly sensitive foreign proteins, and the host carries a variety of antibiotic genes.
  • the primary object of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a recombinant strain of Bacillus subtilis.
  • Another object of the present invention is to provide a method for preparing the recombinant strain of Bacillus subtilis.
  • a further object of the present invention is to provide the use of the recombinant strain of Bacillus subtilis.
  • the object of the present invention is achieved by the following technical scheme: a recombinant strain of Bacillus subtilis, which is obtained by knocking out or inactivating ten protease genes in Bacillus subtilis, the ten protease genes are: a neutral protease encoding gene ( nprE), neutral protease B encoding gene (nprB), alkaline protease encoding gene (aprE), metalloproteinase (mpr), three serine protease encoding genes (bpf, epr and vpr), cell wall protease encoding gene (wprA) , spore forming factor F coding gene (spollAC) and surfactin synthetase encoding gene (srfAC).
  • nprE neutral protease encoding gene
  • nprB neutral protease B encoding gene
  • aprE alkaline protease en
  • Bacillus subtilis is Bacillus subtilis ATCC6051.
  • the nucleotide sequence of the nprE is shown in Gene ID: 935981; the nucleotide sequence of the nprB is shown in Gene ID: 936384; the nucleotide sequence of the aprE is shown in Gene ID: 939313.
  • the nucleotide sequence of the mpr is as Gene ID: 938430; the nucleotide sequence of the bpf is shown as Gene ID: 939695; the nucleotide sequence of the epr is shown as Gene ID: 937332; the nucleotide sequence of the vpr As shown in Gene ID: 937291; the nucleotide sequence of the wprA is shown as Gene ID: 936350; the nucleotide sequence of the spollAC is shown as Gene ID: 938729; the nucleotide of the srfAC The sequence is shown as Gene ID: 938308.
  • the inactivation is that the gene is not correctly transcribed or translated.
  • the preparation method of the recombinant strain of Bacillus subtilis is obtained by a technique for inhibiting transcription, correct transcription or translation of the ten protease genes.
  • Such techniques include gene knockout techniques, frameshift mutation techniques, and antisense nucleotide interference techniques.
  • the gene knockout technique is preferably a homologous recombination method.
  • the homologous recombination method preferably includes the following steps:
  • nprE neutral protease B encoding gene
  • aprE alkaline protease encoding gene
  • mpr metalloproteinase
  • bpf, epr and vpr cell wall protease encoding gene
  • wprA cell wall protease encoding gene
  • spollAC sporulation factor F coding gene
  • srfAC surfactin synthase encoding gene
  • protease genes were cloned into the temperature sensitive plasmid pKS2, respectively, to obtain ten knockout vectors of protease genes: pKS2-nprE, pKS2-nprB, pKS2-aprE, pKS2-mpr, pKS2-bpf, pKS2-epr, pKS2-vpr, pKS2-wprA, pKS2-spollAC, pKS2-srfAC;
  • the upstream homology arm of the nprE is obtained by amplifying the primer DNA as shown in SEQ ID NO. 12 and SEQ ID NO. 13 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the nprE is obtained by amplifying the primer DNA as shown in SEQ ID NO. 14 and SEQ ID NO. 15 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the nprB is obtained by amplifying the primer DNA as shown in SEQ ID NO. 16 and SEQ ID NO. 17 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of nprB is obtained by amplifying the primer DNA as shown in SEQ ID NO. 18 and SEQ ID NO. 19 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the aprE is obtained by amplifying the primer DNA as shown in SEQ ID NO. 20 and SEQ ID NO. 21 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the aprE is obtained by amplifying the primer DNA as shown in SEQ ID NO. 22 and SEQ ID NO. 23 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the mpr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 24 and SEQ ID NO.
  • the downstream homology arm of the mpr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 26 and SEQ ID NO. 27 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the bpf is obtained by amplifying the primer DNA as shown in SEQ ID NO. 28 and SEQ ID NO.
  • the downstream homology arm of the bpf is obtained by amplifying the primer DNA as shown in SEQ ID NO. 30 and SEQ ID NO.
  • the upstream homology arm of the epr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 32 and SEQ ID NO. 33 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the epr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 34 and SEQ ID NO. 35 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the vpr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 36 and SEQ ID NO. 37 with the B. subtilis strain DNA as a template.
  • the downstream homology arm of the vpr is obtained by amplifying the primer DNA as shown in SEQ ID NO. 38 and SEQ ID NO. 39 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the wprA is obtained by amplifying the primer DNA as shown in SEQ ID NO. 40 and SEQ ID NO. 41 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the wprA is obtained by amplifying the primer DNA as shown in SEQ ID NO. 42 and SEQ ID NO.
  • the upstream homology arm of the spollAC is obtained by amplifying the primer DNA as shown in SEQ ID NO. 44 and SEQ ID NO. 45 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the spollAC is obtained by amplifying the primer DNA as shown in SEQ ID NO. 46 and SEQ ID NO. 47 using the B. subtilis strain DNA as a template.
  • the upstream homology arm of the srfAC is obtained by amplifying the primer DNA as shown in SEQ ID NO. 48 and SEQ ID NO. 49 using the B. subtilis strain DNA as a template.
  • the downstream homology arm of the srfAC is obtained by amplifying the primer DNA as shown in SEQ ID NO. 50 and SEQ ID NO. 51 using the B. subtilis strain DNA as a template.
  • the step of merging is preferably as follows: using the upstream homologous arm and the downstream homology arm as a template to amplify the upstream primer of the upstream homologous arm and the downstream primer of the downstream homologous arm as a primer pair to obtain the upstream and downstream Source arm.
  • sequence of the upstream and downstream homology arms of the wprA is set forth in SEQ ID NO.
  • sequence of the upstream and downstream homology arms of the srfAC is shown in SEQ ID NO.
  • the upstream and downstream homology arms of the ten protease genes were separately cloned into the temperature sensitive plasmid pKS2, and the upstream and downstream homologous arms of the ten protease genes were cloned into the EcoRV cleavage site of the temperature sensitive plasmid pKS2. point.
  • the Bacillus subtilis starting strain is preferably Bacillus subtilis ATCC6051.
  • the recombinant bacteria 1 is preferably Bacillus subtilis lacking the nprE gene.
  • the recombinant bacteria 2 is preferably Bacillus subtilis which lacks the nprE and nprB genes.
  • the recombinant bacteria 3 is preferably Bacillus subtilis which lacks the nprE, nprB, and aprE genes.
  • the recombinant bacteria 4 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, and mpr genes.
  • the recombinant bacteria 5 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, mpr, bpf genes.
  • the recombinant bacteria 6 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, mpr, bpf, epr genes.
  • the recombinant bacteria 7 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, mpr, bpf, epr, vpr genes.
  • the recombinant bacteria 8 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA genes.
  • the recombinant bacterium 9 is preferably Bacillus subtilis lacking the nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, and spollAC genes.
  • the screening is carried out by heating at 37 ° C and cooling at 30 ° C, and then screening by PCR and sequencing verification; preferably including the following steps:
  • the knockout vector of the protease gene was transferred into Bacillus subtilis to obtain a bacterial solution coated on an LB plate containing 5 ⁇ g/mL erythromycin (erm) resistance, and cultured at 30 ° C to obtain a positive clone positively confirmed;
  • step LB plate containing 5 ⁇ g/mL erythromycin resistance inoculate in 10 mL LB liquid medium, incubate at 30 ° C, 200 rpm for 12 h; dilute the culture solution by 10 5 times, and take 100 ⁇ L of bacterial solution Buried on LB solid plate, cultured at 37 ° C for 12 h;
  • the foreign protein is preferably a microbial transglutaminase (MTG).
  • MTG microbial transglutaminase
  • the present invention has the following advantages and effects: in the transformation of Bacillus subtilis, due to the change of the expression of Bacillus subtilis self-protein, although the degree of degradation of foreign proteins is reduced, it often affects the grass The growth and reproduction rate of Bacillus.
  • the present invention inactivates ten extracellular secretory protease genes of Bacillus subtilis, a recombinant strain having a fast growth and a high expression level of a foreign protein is obtained.
  • the strain provided by the present invention can be used as a host for efficient expression of a foreign protein.
  • Figure 1 is a schematic diagram showing the construction of a gene knockout plasmid.
  • Fig. 2 is a diagram showing the results of PCR identification of Bacillus subtilis ATCC6051 knockout mutant; wherein lanes 1-10 are B. subtilis ATCC6051, lane 11 is recombinant bacteria 9, lane 12 is recombinant bacteria 1, and lane 13 is recombinant Bacterium 3, Lane 14 is recombinant bacteria 10, Lane 15 is recombinant bacteria 2, Lane 16 is recombinant bacteria 6, Lane 17 is recombinant bacteria 4, Lane 18 is recombinant bacteria 7, Lane 19 is recombinant bacteria 8, Lane 20 is recombinant bacteria 5. Lane M is a DNA marker.
  • Figure 3 is a graph showing growth curves of B. subtilis ATCC 6051, recombinant Bacillus subtilis ATCC6051 ⁇ 10, Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 ⁇ 10 (pBEp43-proMTG).
  • Figure 4 is a graph showing the secretion and expression of transglutaminase in the supernatant of recombinant fermentation broth by SDS-PAGE protein electrophoresis; lane 1 is Bacillus subtilis ATCC6051, lane 2 is Bacillus subtilis ATCC6051 ⁇ 10, lane 3 is band Bacillus subtilis ATCC6051 with pBEp43-proMTG plasmid and lane 4 with Bacillus subtilis ATCC6051 ⁇ 10 with pBEp43-proMTG plasmid.
  • the primers in Table 1 were used to amplify FXL, RXL and FXR, and RXR (X is the gene to be knocked, the same below).
  • the amplified product XL (the homologous arm upstream of the gene to be knocked out) and XR (the homologous arm downstream of the gene to be knocked out) were obtained.
  • the X-L fragment and the X-R fragment were used as templates, and F-X-L and R-X-R were used as primers to carry out fusion PCR, and the corresponding ten fusion products (L+R fragments) were obtained. Ten fusion products were cloned into the pMD18-T vector and sequenced.
  • the ten fusion fragments are the nucleotides shown in sequence 1 of the sequence table (DNA molecules of the nprE coding gene homology arm), and the sequence 2
  • the nucleotide shown (nprB encodes the DNA molecule of the gene homology arm), the nucleotide shown in the sequence 3 (the DNA molecule of the aprE coding gene homology arm), and the nucleotide shown in the sequence 4 (the mpr coding gene is the same) a DNA molecule of the source arm, a nucleotide represented by the sequence 5 (a DNA molecule encoding the homologous arm of the bpf gene), a nucleotide represented by the sequence 6 (a DNA molecule encoding the homologous arm of the epr gene), and a sequence of 7 Nucleotide (DNA molecule of vpr-encoding gene homology arm), nucleotide represented by SEQ ID NO:8 (DNA molecule encoding wprA-
  • the EcoRV cleavage site of SEQ ID NO:11) gave the recombinant plasmid pKS2-X(L+R) (see Figure 1), ie the knockout vectors of ten genes: pKS2-nprE, pKS2-nprB, pKS2-aprE, pKS2- Mpr, pKS2-bpf, pKS2-epr, pKS2-vpr, pKS2-wprA, pKS2-spollAC, pKS2-srfAC.
  • Table 1 shows the relevant primer sequences for gene knockout vector construction as follows:
  • the PCR reaction system is as follows:
  • the PCR program was 98 ° C for 5 min; 35 cycles: 98 ° C for 30 s, 54 ° C for 30 s, 72 ° C for 40 s; 72 ° C for 10 min.
  • the recombinant plasmid pKS2-nprE was first transferred into Escherichia coli TG1 (purchased from Bio-Engineering (Dalian) Co., Ltd.), and the plasmid was extracted. The other recombinant plasmids were also subjected to the same operation. The plasmid pKS2-nprE was then transferred into Bacillus subtilis ATCC6051 by electroporation (see Natalia P, Zakataeva, Oksana V et al. A simple method to introduce marker-free genetic modification into chromosome of naturally nontransformable Bacillus). Amyloliquefaciens strains [J]. Appl Microbiol Biotechnol. 2010, 85: 1201-1209.), inverted culture at 30 ° C, and positive clones were screened with 5 ⁇ g/mL erythromycin (erm)-resistant LB plates.
  • Step 4 The single colonies grown on the anti-plate (Step 4) were transferred to the LB anti-plate and the LB-resistant plate (5 ⁇ g/mL erm), and the phenotype was expressed on the LB anti-plate, while The suspected clones were not long on the LB-resistant plates, and were further verified by PCR using the primers F-nprE-L (SEQ ID NO. 12) and R-nprE-R (SEQ ID NO. 15). 2; After sequencing, recombinant bacteria 1 (Bacillus subtilis ATCC6051 ⁇ nprE) was obtained.
  • the method of operation is basically the same as step (1), except that the starting strain is the recombinant strain 1 obtained in the above step, and pKS2- is used.
  • the nprB plasmid was obtained to obtain recombinant bacteria 2. Further verification by PCR using primers F-nprB-L (SEQ ID NO. 16) and R-nprB-R (SEQ ID NO. 19), the results of PCR identification are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 2 obtained in the above step, and the recombinant strain 3 is obtained by using the pKS2-aprE plasmid. Further verification was carried out by PCR using primers F-aprE-L (SEQ ID NO: 20) and R-aprE-R (SEQ ID NO: 23), and the results of PCR identification are shown in Fig. 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 3 obtained in the above step, and the recombinant strain 4 is obtained by using the pKS2-mpr plasmid. Further verification was carried out by PCR using primers F-mpr-L (SEQ ID NO. 24) and R-mpr-R (SEQ ID NO. 27), and the results of PCR identification are shown in Fig. 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 4 obtained in the above step, and the recombinant strain 5 is obtained by using the pKS2-bpf plasmid. Further verification by PCR using primers F-bpf-L (SEQ ID NO. 28) and R-bpf-R (SEQ ID NO. 31), the results of PCR identification are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 5 obtained in the above step, and the recombinant strain 6 is obtained by using the pKS2-epr plasmid. Further verification by PCR using primers F-epr-L (SEQ ID NO. 32) and R-epr-R (SEQ ID NO. 35), the results of PCR identification are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 6 obtained in the above step, and the recombinant strain 7 is obtained by using the pKS2-vpr plasmid. Further verification by PCR using primers F-vpr-L (SEQ ID NO. 36) and R-vpr-R (SEQ ID NO. 39), the results of PCR identification are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 7 obtained in the above step, and the recombinant strain 8 is obtained by using the pKS2-wprA plasmid. Further verification by PCR using primers F-wprA-L (SEQ ID NO. 40) and R-wprA-R (SEQ ID NO. 43), the results of PCR identification are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant bacteria 8 obtained in the above step, and the recombinant strain 9 is obtained by using the pKS2-spollAC plasmid. Further verification by PCR using the primers F-spollAC-L (SEQ ID NO. 44) and R-spollAC-R (SEQ ID NO. 47), the PCR identification results are shown in Figure 2.
  • the operation method is basically the same as the step (1), except that the starting strain is the recombinant strain 9 obtained in the above step, and the pKS2-aprE plasmid is used to obtain the target strain Bacillus subtilis ATCC6051 ⁇ 10 (nprE, nprB, aprE, mpr, bpf, epr, Vpr, wprA, spollAC, srfAC). Further verification by PCR using primers F-srfAC-L (SEQ ID NO. 48) and R-srfAC-R (SEQ ID NO. 51), the results of PCR identification are shown in Figure 2.
  • the plasmid pBEp43-proMTG (according to CN201210052578.2, a recombinant Bacillus subtilis and its method for producing transglutaminase) was used as an expression plasmid, and was transformed into Bacillus subtilis ATCC6051 and Bacillus subtilis ATCC6051 ⁇ 10 by electroporation. Two transformed strains Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 ⁇ 10 (pBEp43-proMTG).
  • Single colonies of Bacillus subtilis ATCC6051 (pBEp43-proMTG) and Bacillus subtilis ATCC6051 ⁇ 10 (pBEp43-proMTG) were inoculated separately in 10 mL of LB medium (kanamycin 20 ⁇ g/mL) for activation at 37 ° C, 200 rpm for 12 h, and activated.
  • the seed solution was inoculated into 50 mL of LB medium (Kanamycin 20 ⁇ g/mL), the inoculum amount was 1% (volume ratio), and fermentation was carried out at 37 ° C, 200 rpm for 48 h.
  • the starting strains Bacillus subtilis ATCC6051 and Bacillus subtilis ATCC6051 ⁇ 10 were used as controls.
  • the OD 600 values were measured at six time points, 3h, 6h, 12h, 24h, 36h and 48h.
  • the growth rate of the recombinant Bacillus subtilis ATCC6051 ⁇ 10 was higher than that of the Bacillus subtilis ATCC6051 and Bacillus subtilis ATCC6051 ⁇ 10 (pBEp43-proMTG).
  • the speed is also faster than Bacillus subtilis ATCC6051 (pBEp43-proMTG) and reaches its highest value at 48h (see Figure 3).

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Abstract

La présente invention concerne une souche recombinante de bacillus subtilis et un procédé de préparation correspondant et son application. La souche recombinante est obtenue par l'invalidation ou l'inactivation de dix gènes de la protéase à partir de Bacillus subtilis, et les dix gènes de la protéase comprennent nprE, nprB, aprE, mpr, bpf, epr, vpr, wprA, spollAC et srfAC.
PCT/CN2016/108692 2016-06-15 2016-12-06 Souche recombinante de bacillus subtilis et procédé de préparation correspondant et son application WO2017215204A1 (fr)

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