WO2020143708A1 - Bacillus licheniformis (b.licheniformis) engineering bacterium capable of restraining autolysis of thalli and construction method and application for b.licheniformis engineering bacterium - Google Patents

Bacillus licheniformis (b.licheniformis) engineering bacterium capable of restraining autolysis of thalli and construction method and application for b.licheniformis engineering bacterium Download PDF

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WO2020143708A1
WO2020143708A1 PCT/CN2020/071158 CN2020071158W WO2020143708A1 WO 2020143708 A1 WO2020143708 A1 WO 2020143708A1 CN 2020071158 W CN2020071158 W CN 2020071158W WO 2020143708 A1 WO2020143708 A1 WO 2020143708A1
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autolysis
medium
pcf
pcr amplification
licheniformis
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PCT/CN2020/071158
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French (fr)
Chinese (zh)
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肖静
李子源
汪俊卿
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齐鲁工业大学
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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
    • 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
    • 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
    • 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
    • C12R2001/10—Bacillus licheniformis

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  • the invention relates to the field of microbial strain transformation, in particular to a Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells and its construction method and application.
  • Bacillus licheniformis is one of the more promising production strains in Bacillus. It can ferment and produce a variety of enzymes such as protease, amylase, lipase, and is widely used in textiles, food, medicine and other industries.
  • the autolysis of the bacteria is mainly related to the external environment and the expression of some specific genes of the bacteria themselves.
  • the regulation of gene expression is a key internal cause of bacterial autolysis.
  • the prophage gene is closely related to the autolysis of Bacillus. It is integrated into the bacterial genome in a latent form, and the gene that normally controls its expression is turned off. State, after induction under specific conditions, it will replicate in large quantities, and finally lyse the bacteria.
  • PBLB is a kind of prophage in Bacillus licheniformis. Under normal circumstances, there will be a small amount of replication. Under the conditions of mitomycin C and other conditions, a large amount of replication will be carried out. Finally, Bacillus licheniformis will be lysed and autolyzed.
  • the object of the present invention is to provide a Bacillus licheniformis engineering bacteria that inhibits autolysis of the microbial cells, and its construction method and application.
  • the present invention provides technical solutions as follows:
  • Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells.
  • the Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells is obtained by knocking out the pcf gene, and the nucleotide sequence of the pcf gene is as SEQ ID NO.1 shown.
  • the structural proteins of PBLB and the enzymes related to the cleavage of the cell wall are controlled by the pcf factor.
  • the pcf factor Through the knockout of the pcf gene and the control of nutritional conditions, the autolysis of the DL-1 cell of B. licheniformis (B. licheniformis) can be inhibited and improved.
  • the accumulation of biomass enhances the production capacity of fermentation products.
  • the method for knocking out the pcf gene is a homologous recombination single exchange method;
  • the culture medium of the Bacillus licheniformis engineering bacteria that inhibits autolysis of the bacterial body is an LB medium supplemented with 0.1% glucose and 2.5% glycerol.
  • the present invention also provides a method for constructing the above-mentioned Bacillus licheniformis engineering bacteria that inhibits autolysis of the bacterial cells, including:
  • Step 1 Extract the genomic DNA of B. licheniformis DL-1, use DNA as a template, design the upstream and downstream primers pcfF and pcfR, and perform PCR amplification to obtain pcf gene fragments.
  • the nucleotide sequence of pcf gene is as SEQ ID NO.1 shown;
  • Step 2 Extract the pHT01 plasmid DNA, use the DNA as a template, design the upstream and downstream primers CmrF and CmrR, and perform PCR amplification to obtain the chloramphenicol-resistant Cmr gene fragment.
  • the nucleotide sequence of the Cmr gene is shown in SEQ ID NO. 2. Show
  • Step 3 The pcf gene fragment prepared in step 1 and the Cmr gene fragment prepared in step 2 are fused by overlapping extension PCR to obtain homologous recombination pcf-Cmr fragments;
  • Step 4 digest the homologous recombination pcf-Cmr fragment obtained in Step 3, digest it, concentrate it, and electroporate it into competent cells of Bacillus licheniformis;
  • Step 5 After resuscitating the competent cells obtained in Step 4, spread on chloramphenicol-containing agar solid medium, select positive recombinant bacteria with chloramphenicol resistance, and prepare a lichen with inhibitory cell autolysis Bacillus engineering bacteria.
  • step 1 the sequences of pcfF and pcfR primers are:
  • the PCR amplification system is: (B. licheniformis) DL-1 genome template 1.5 ⁇ L, upstream primer pcfF 1.5 ⁇ L, downstream primer pcfR 1.5 ⁇ L, 2 ⁇ Phanta Max Master Mix 25 ⁇ L, ddH 2 O 20.5 ⁇ L, total volume 50 ⁇ L;
  • the PCR amplification reaction program was: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 20s, 30 cycles, and final extension at 72°C for 10min.
  • CmrF and CmrR primer sequences in step 2 are:
  • CmrF CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTC-AC
  • CmrR CGCGGATCC TAGTGACTGGCGATGCTGTCGG
  • the PCR amplification system is: pHT01 plasmid template 1.5 ⁇ L, upstream primer CmrF 1.5 ⁇ L, downstream primer CmrR 1.5 ⁇ L, 2 ⁇ Phanta Max Master Mix 25 ⁇ L, ddH 2 O 20.5 ⁇ L, total volume 50 ⁇ L;
  • the PCR amplification reaction program was: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, 30 cycles, and final extension at 72°C for 10min.
  • the primer sequences used in the overlap extension PCR are pcfF and CmrR;
  • Overlap extension PCR amplification is divided into primary PCR amplification and secondary PCR amplification;
  • the system for the first PCR amplification is: pcf gene fragment 2 ⁇ L, Cmr gene fragment 2 ⁇ L, 2 ⁇ Phanta Max Master Mix 12.5 ⁇ L, ddH 2 O 8.5 ⁇ L, total volume 25 ⁇ L;
  • the initial PCR amplification reaction program was: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 40s, 5 cycles, and final extension at 72°C for 5min;
  • the secondary PCR amplification system is as follows: add the following reagents to the primary PCR amplification system, upstream primer pcfF 1.5 ⁇ L, downstream primer CmrR 1.5 ⁇ L, 2 ⁇ Phanta Max Master Mix 12.5 ⁇ L, ddH 2 O 9.5 ⁇ L;
  • the reaction procedure of the second PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
  • step 4 the digestion system and reaction conditions are as follows:
  • the steps of concentration are: add 1/10 volume of 3mol/L sodium acetate solution and 2.5 times volume of absolute ethanol to the digested product, leave at -20°C for 20min, centrifuge at 12000r/min for 5min to obtain DNA precipitate, add 70 Resuspend DNA pellet in% ethanol, centrifuge at 12000r/min for 5min to remove ethanol, air dry at 37°C, add 25 ⁇ L ddH 2 O to resuspend, to obtain pcf-Cmr concentrated recombinant fragment;
  • the steps of electrotransformation are as follows: (B. licheniformis) DL-1 competent cells and pcf-Cmr concentrated recombinant fragments were gently mixed on ice at a ratio of 10:1, and the mixture was transferred into a 2mm electric rotating cup, 2100V, 5ms electric rotating, Then immediately add 1mL RM medium, resuscitate at 37°C, 180r/min for 4h, then spread on LB solid medium containing chloramphenicol (25 ⁇ g/mL) and cultivate.
  • the construction method of the Bacillus licheniformis engineering bacteria for inhibiting autolysis of the bacterial cells further includes step 6: inoculation of the Bacillus licheniformis engineering bacteria prepared for the autolysis of the bacterial cells prepared in step 5 into cultures with different nutritional conditions On the basis, carry out nutrition optimization cultivation.
  • the inoculation amount is a single colony/50ml medium, and the culture conditions are 37°C and 200r/min for 24h; the medium contains 0.5%, 1%, 1.5%, 2%, 2.5%, 3% glycerol in LB medium;
  • LBG medium containing 0.1%, 0.3%, 0.5%, 0.8%, 1% glucose in mass percentage
  • the LB medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH;
  • the LBG medium consists of the following components: peptone 1% (mass percentage), yeast extract 0.5% (mass percentage), sodium chloride 1% (mass percentage), glycerin 1% (volume percentage), using 5mol/ LNaOH adjusts the medium pH to 7.0;
  • the LBP medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH.
  • the medium is an LBP medium containing 2.5% glycerin by mass.
  • the present invention also provides the application of the above-mentioned Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells, which is applied to the production of protease and its preparation.
  • the present invention discloses for the first time a method for controlling the autolysis of bacterial cells by combining the autoclaving related gene pcf knockout of Bacillus licheniformis with nutritional conditions, and improving the biomass of the bacterial cells, and electrically converting the overlapping pcf-Cmr fragments to B. licheniformisDL-1 Competent cells, only one homologous single exchange was used to quickly knock out the pcf gene, and the pcf gene-deleted strain B. licheniformis DL-1 ⁇ pcf was successfully obtained, and the autolysis rate of recombinant bacteria was reduced by 11.2% without optimized nutritional conditions.
  • PCF gene knockout can control the autolysis rate of bacteria;
  • the autolysis rates of the starting strain and the recombinant strain were reduced by 52.6% and 56.3%, respectively.
  • the recombinant bacteria could further increase the biomass and reduce the autolysis rate by autolytic gene knockout combined with nutritional condition control.
  • the autolysis rate was the largest Reduced by 71.2%, knocking out autolytic genes combined with nutritional conditions control can increase the biomass of Bacillus licheniformis, which is beneficial to the acquisition of high-concentration bacteria;
  • Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells provided by the present invention are cultured in an ⁇ -amylase-producing medium, and the enzyme activity is increased by 19.1% compared with the unmodified strain. This method is beneficial for the production of strains of Bacillus licheniformis enzyme preparations Genetic breeding and industrial production.
  • FIG. 1 is an electrophoresis diagram of the pcf gene fragment agarose gel prepared in Example 1 of the present invention; wherein, lane 1 is DNA Maker, and lanes 2 to 4 are pcf gene bands with a size of 418 bp;
  • FIG. 2 is an electrophoresis diagram of the Cmr gene fragment agarose gel prepared in Example 1 of the present invention; wherein, lane 1 is DNA Maker, and lanes 2 to 4 are Cmr gene bands with a size of 1245 bp;
  • FIG. 3 is an agarose gel electrophoresis diagram of the pcf-Cmr gene fragment prepared in Example 1 of the present invention; where lane 1 is DNA Maker, and lanes 2 to 4 are pcf-Cmr gene bands with a size of 1663bp;
  • lane 1 is DNA Maker
  • lanes 2 to 3 are pcf-Cmr gene bands
  • FIG. 5 is a diagram of the relationship between the nutritional conditions of the unmodified B. licheniformis (DL-1) medium and the autolysis of the bacteria in Example 6 of the present invention, where Figure (a) is the unmodified B. licheniformis Growth curve of glucose medium inoculated in LB and added different ratios (0.1%, 0.3%, 0.5%, 0.8%, 1.0%); Figure (b) shows that the unmodified Bacillus licheniformis was inoculated in LB and added different ratios (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) growth curve of glycerol medium; Figure (c) shows that the unmodified Bacillus licheniformis was inoculated in LBG with different proportions (0.1%, 0.3%, 0.5%, 0.8 %, 1.0%) Glucose medium growth curve; Figure (d) shows that unmodified Bacillus licheniformis was inoculated into LBP with different proportions (0.5%, 1.0%, 1.5%, 2.0%
  • FIG. 6 is a graph showing the relationship between the nutrient conditions of the culture medium of B. licheniformis engineering bacteria (B. licheniformis DL-1 ⁇ pcf) prepared by the invention prepared in Example 6 and the autolysis of the microbial cells, in which (a) The growth curve of Bacillus licheniformis engineering bacteria prepared by the present invention for inhibiting autolysis of bacterial cells was inoculated in LB supplemented with different ratios (0.1%, 0.3%, 0.5%, 0.8%, 1.0%) of glucose medium; Figure (b) is The Bacillus licheniformis engineering bacteria prepared by the invention and inhibiting the autolysis of bacterial cells are inoculated in LB and added with different proportions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) of glycerol medium growth curve; Figure (c ) The Bacillus licheniformis engineering bacteria prepared by the present invention, which inhibits autolysis of bacterial cells, is inoculated into LBG and added with different proportions (0.
  • the graph 7 are the growth curves of the unmodified Bacillus licheniformis and the Bacillus licheniformis engineering bacteria prepared by the present invention in different nutrient media in the inhibition of bacterial autolysis;
  • the graph is the growth curve in LB medium,
  • the graph is the growth curve in LBG supplemented with 0.1% glucose medium;
  • the graph is the growth curve in LBP supplemented with 2.5% glycerol medium;
  • FIG. 8 is the measurement results of ⁇ -amylase activity of Bacillus licheniformis engineered bacteria of the unmodified Bacillus licheniformis and the autolysis of bacterial cells prepared in the present invention in Example 7.
  • FIG. 8 is the measurement results of ⁇ -amylase activity of Bacillus licheniformis engineered bacteria of the unmodified Bacillus licheniformis and the autolysis of bacterial cells prepared in the present invention in Example 7.
  • the B. licheniformis (DL-1) used in the present invention was purchased from the American Type Culture Collection Center (ATCC) strain number 27811; the pHT01 plasmid was purchased from Hangzhou Baosai Biotechnology Co., Ltd.
  • the present invention provides a Bacillus licheniformis engineering bacterium that inhibits autolysis of bacteria and its construction method and application, which can effectively improve the autolysis of Bacillus licheniformis bacteria.
  • PCR amplification reaction program pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 20s, 30 cycles, final extension at 72°C for 10min.
  • the PCR product was detected by 1% agarose gel electrophoresis, and a specific band appeared at about 400bp. The result is shown in Figure 1, indicating that the 418bp pcf homologous fragment was successfully obtained.
  • the PCR product homologous fragment was used to recover the reagents using SanPrep column DNA gel The cassette is subjected to gel recovery, and the obtained DNA is directly subjected to the next experiment or stored at -20°C.
  • the nucleotide sequence of the upstream and downstream primers is as follows , Where the underline is the BamH I protection base and the cleavage site:
  • CmrF CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCAC
  • CmrR CGCGGATCC TAGTGACTGGCGATGCTGTCGG
  • PCR amplification reaction program pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 40s, 30 cycles, and final extension at 72°C for 10min.
  • the pcf gene fragment and Cmr gene fragment obtained by gel recovery were fused by overlap extension PCR to obtain pcf-Cmr homologous recombination fragment.
  • the nucleotide sequence of primers used in overlap extension PCR is as follows, in which the underline is BamH I protected base And restriction sites:
  • CmrR CGCGGATCC TAGTGACTGGCGATGCTGTCGG
  • Overlap extension PCR amplification is divided into primary PCR and secondary PCR amplification
  • the initial PCR amplification reaction program was: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 60°C for 30s, extension at 72°C for 40s, 5 cycles, and final extension at 72°C for 5min.
  • the secondary PCR amplification system is as follows: add the following reagents to the primary PCR amplification system, upstream primer pcfF 1.5 ⁇ L, downstream primer CmrR 1.5 ⁇ L, 2 ⁇ Phanta Max Master Mix 12.5 ⁇ L, ddH 2 O 9.5 ⁇ L;
  • the reaction procedure of the second PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
  • the BamH I endonuclease was used to perform single digestion on the pcf-Cmr fusion fragment prepared in step 1, and the digestion system and conditions were as follows:
  • pcf-Cmr homologous recombination fragment 20 ⁇ L, BamH endonuclease 2 ⁇ L, 10 ⁇ FastDigest Buffer 4 ⁇ L, ddH 2 O 4 ⁇ L, total volume 30 ⁇ L; 37°C, digestion for 2h.
  • the digested pcf-Cmr fusion fragment is concentrated, and the concentration steps are as follows:
  • ETM electrotransfer buffer
  • the components of the culture medium involved are as follows:
  • the LB medium is composed of the following components by weight: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0.
  • GM Bacterial Multiplication Medium
  • peptone 1% peptone 1%
  • yeast extract 0.5% yeast extract 0.5%
  • NaCl 1% sorbitol 9.1%
  • pH 7.0 pH 7.0
  • ETM electrotransfer buffer: sorbitol 9.1% (mass percentage), mannitol 9.1% (mass percentage), 10% glycerol (volume percentage).
  • the B. licheniformis DL-1 competent cells prepared in Example 3 and the pcf-Cmr recombinant gene fragments prepared in Example 2 were placed on ice and gently mixed at a ratio of 10:1, and the mixture was transferred into a 2mm electric rotating cup, 2100V, After 5ms electroporation, immediately add 1mL RM medium, revive at 37°C, 180r/min for 4h, then spread on LB solid medium containing chloramphenicol (25 ⁇ g/mL) for cultivation.
  • RM Bacterial Resuscitation Medium
  • peptone 1% peptone 1%
  • yeast extract 0.5% NaCl 1%
  • sorbitol 9.1% mannitol 6.9%
  • its pH is adjusted to 7.0.
  • nucleotide sequences of the upstream and downstream primers involved are as follows, in which the underline is the BamH I protection base and the cleavage site:
  • CmrR CGCGGATCC TAGTGACTGGCGATGCTGTCGG
  • PCR amplification system extract 1.5 ⁇ L of bacterial genome template, upstream primer pcf F1.5 ⁇ L, downstream primer CmrR 1.5 ⁇ L, 2 ⁇ Phanta Max Master Mix 12.5 ⁇ L, ddH 2 O 9.5 ⁇ L;
  • PCR amplification reaction program 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, 72°C final extension for 10 min.
  • the amplified product was verified by 1% agarose gel electrophoresis. The results are shown in Figure 4.
  • the size of the gene amplified fragment is 1663bp, indicating that the Bacillus licheniformis engineering bacteria with the pcf-Cmr recombinant gene fragment has been successfully obtained.
  • B. licheniformis DL-1 Bacillus licheniformis engineering bacteria
  • B. licheniformis DL-1 ⁇ pcf Bacillus licheniformis engineering bacteria
  • composition formula of the medium is shown in the following table:
  • LBG medium peptone 1%, yeast extract 0.5%, sodium chloride 1% (all mass fractions), glycerin 1% (volume fraction), pH 7.0.
  • LBP medium peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1% (all mass fractions).
  • B. licheniformis engineering bacteria B. licheniformis DL-1 ⁇ pcf
  • Bacillus licheniformis prepared by the present invention inhibit the autolysis of bacterial cells.
  • the growth curve of B. licheniformis DL-1 is consistent, and they are most suitable for growth with LBP medium supplemented with 2.5% glycerol.
  • the bacterial autolysis rate of the Bacillus licheniformis engineering bacteria prepared by the present invention is 11.2% lower than that of the unmodified Bacillus licheniformis. It can be seen from Figure 7-(b) that under the culture conditions of LBG supplemented with 0.1% glucose medium, the autolysis rate of the Bacillus licheniformis engineered bacteria prepared by the present invention is higher than that of the unmodified Bacillus licheniformis.
  • the combination of the Bacillus licheniformis engineering bacteria obtained by knocking out the PCF gene knockout and the nutrient conditions of the culture medium has a synergistic superposition effect on the autolysis control of B. licheniformis DL-1 bacteria, and is optimal
  • the nutritional condition is that LB is supplemented with 0.1% glucose and 2.5% glycerol (ie LBP medium + 2.5% glycerol).
  • B.licheniformis DL-1 and recombinant strain B.licheniformis DL-1 ⁇ pcf were inoculated into the fermentation medium at 2% inoculation volume, cultured at 37°C, 200r/min, sampled every 12h, and centrifuged at 4°C, 12000r/min for 8min Qing, the amylase activity was determined according to the method shown in the national standard GB/T24401-2009.
  • the fermentation medium is composed of the following mass percentage components:
  • Soluble starch 2% Soluble starch 2%, peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1%, glycerol 2.5%, pH 7.0.
  • ⁇ -amylase activity 1ml of liquid enzyme, liquefying 1mg of soluble starch in 1min at 70°C and pH6.0, which is 1 unit of enzyme activity, expressed in U/mL.
  • Fig. 8 The results of the measurement of ⁇ -amylase enzyme activity are shown in Fig. 8. From this figure, it can be seen that B. licheniformis DL-1 and B. licheniformis DL-1 ⁇ pcf are both fermented to the maximum enzyme activity at 48h, which are 340U/mL and 405U/mL, respectively. Enzyme activity increased by 19.1%.
  • enzyme activity measurement it can be determined that the PCF gene-deficient Bacillus licheniformis engineering bacteria prepared by the present invention, which inhibits autolysis of bacterial cells, is beneficial to the improvement of ⁇ -amylase enzyme activity.
  • the present invention discloses for the first time a method for controlling the autolysis of bacterial cells by combining the autoclaving related gene pcf knockout of Bacillus licheniformis with nutritional conditions, and improving the biomass of the bacterial cells, and electrically converting the overlapping pcf-Cmr fragments to B. licheniformisDL-1 Competent cells, through a single homologous single exchange to achieve rapid knockout of the pcf gene, successfully obtained pcf gene deletion strain B.licheniformisDL-1 ⁇ pcf.
  • the autolysis rate of the recombinant bacteria was 11.2% lower than that of the starting bacteria.
  • the autolysis rate of the bacterial body can be controlled by the PCF gene knockout; the recombinant bacteria can increase the biomass and reduce the autolysis rate by autolysis gene knockout combined with nutritional control.
  • the maximum reduction rate is 71.2%.
  • Knock-out autolysis genes combined with nutrient control can increase the biomass of Bacillus licheniformis cells, which is beneficial to the acquisition of high-concentration cells; the recombinant bacteria are cultured in ⁇ -amylase-producing medium, and the enzyme activity is increased by 19.1% compared with the starting strain This method is beneficial to the genetic breeding and industrial production of Bacillus licheniformis enzyme preparation production strains.

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Abstract

Provided are a B.licheniformis engineering bacterium capable of restraining autolysis of thalli and a construction method and application for the B.licheniformis engineering bacterium. The B.licheniformis engineering bacterium capable of restraining the autolysis of thalli is obtained by knocking out a pcf gene, and the nucleotide sequence of the pcf gene is as shown in SEQ ID NO.1. According to the B.licheniformis engineering bacterium capable of restraining the autolysis of thalli, the biomass is significantly increased; compared with the unmodified B.licheniformis, the autolysis rate is reduced by 11.2%, and fermentation production of α-amylase can be improved.

Description

抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用Bacillus licheniformis engineering bacteria for inhibiting bacterial autolysis, construction method and application thereof 技术领域Technical field
本发明涉及微生物菌种改造领域，具体涉及一种抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用。The invention relates to the field of microbial strain transformation, in particular to a Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells and its construction method and application.
背景技术Background technique
地衣芽孢杆菌是芽孢杆菌中较具应用潜力的生产菌种之一，能够发酵生产蛋白酶、淀粉酶、脂肪酶等多种酶，在纺织、食品、医药等行业有广泛应用。Bacillus licheniformis is one of the more promising production strains in Bacillus. It can ferment and produce a variety of enzymes such as protease, amylase, lipase, and is widely used in textiles, food, medicine and other industries.
酶高效生产最重要的条件之一就是高浓度的菌体，而本发明所涉及的地衣芽孢杆菌(B.licheniformis)DL-1合成酶能力强，但菌体易自溶，在进行目标酶高效表达的基因工程改造后，菌体自溶现象更加明显，菌体生长的稳定期及有效产酶时间缩短。为了保证生产菌株的连续高密度发酵，提高发酵生产效率，需要采取有效措施对菌体自溶进行控制。One of the most important conditions for the efficient production of enzymes is a high concentration of bacteria, and the B. licheniformis DL-1 synthetase involved in the present invention has strong ability, but the bacteria are easy to dissolve and the target enzyme is highly efficient. After the genetic engineering of the expression, the autolysis of the bacteria is more obvious, and the stable period of bacterial growth and the effective enzyme production time are shortened. In order to ensure the continuous high-density fermentation of production strains and improve the efficiency of fermentation production, effective measures need to be taken to control the autolysis of the bacterial cells.
菌体自溶主要和外部环境以及菌体自身的某些特定的基因表达有关。基因表达调控是菌体自溶的一个关键性内因，其中，原噬菌体基因与芽孢杆菌菌体自溶关系密切，它以一种潜伏的形式整合在细菌基因组上，通常控制其表达的基因处于关闭状态，特定条件诱导后，它就会大量复制，最后将细菌裂解。PBLB是地衣芽孢杆菌中的一种原噬菌体，在正常情况下也会有少量复制，在丝裂霉素C等条件诱导下会进行大量复制，最终使地衣芽孢杆菌裂解自溶。The autolysis of the bacteria is mainly related to the external environment and the expression of some specific genes of the bacteria themselves. The regulation of gene expression is a key internal cause of bacterial autolysis. Among them, the prophage gene is closely related to the autolysis of Bacillus. It is integrated into the bacterial genome in a latent form, and the gene that normally controls its expression is turned off. State, after induction under specific conditions, it will replicate in large quantities, and finally lyse the bacteria. PBLB is a kind of prophage in Bacillus licheniformis. Under normal circumstances, there will be a small amount of replication. Under the conditions of mitomycin C and other conditions, a large amount of replication will be carried out. Finally, Bacillus licheniformis will be lysed and autolyzed.
发明内容Summary of the invention
为解决地衣芽孢杆菌菌体存在自溶不足，本发明的目的在于提供一种抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用。In order to solve the lack of autolysis of Bacillus licheniformis, the object of the present invention is to provide a Bacillus licheniformis engineering bacteria that inhibits autolysis of the microbial cells, and its construction method and application.
为解决上述技术问题，本发明提供技术方案如下：To solve the above technical problems, the present invention provides technical solutions as follows:
本发明，一方面提供一种抑制菌体自溶的地衣芽孢杆菌工程菌，所述抑制菌体自溶的地衣芽孢杆菌工程菌采用敲除pcf基因获得，所述pcf基因核苷酸序 列如SEQ ID NO.1所示。According to one aspect of the present invention, there is provided a Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells. The Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells is obtained by knocking out the pcf gene, and the nucleotide sequence of the pcf gene is as SEQ ID NO.1 shown.
PBLB的结构蛋白以及与裂解细胞壁相关的酶是由pcf因子控制表达的，通过对pcf基因进行敲除，结合营养条件控制可以抑制地衣芽孢杆菌(B.licheniformis)DL-1菌体自溶，提高生物量的积累，提升发酵产物的生产能力。The structural proteins of PBLB and the enzymes related to the cleavage of the cell wall are controlled by the pcf factor. Through the knockout of the pcf gene and the control of nutritional conditions, the autolysis of the DL-1 cell of B. licheniformis (B. licheniformis) can be inhibited and improved. The accumulation of biomass enhances the production capacity of fermentation products.
进一步的，所述敲除pcf基因的方法为同源重组单交换方法；所述抑制菌体自溶的地衣芽孢杆菌工程菌的培养基为添加0.1％葡萄糖、2.5％甘油的LB培养基。Further, the method for knocking out the pcf gene is a homologous recombination single exchange method; the culture medium of the Bacillus licheniformis engineering bacteria that inhibits autolysis of the bacterial body is an LB medium supplemented with 0.1% glucose and 2.5% glycerol.
另一方面，本发明还提供上述抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，包括：On the other hand, the present invention also provides a method for constructing the above-mentioned Bacillus licheniformis engineering bacteria that inhibits autolysis of the bacterial cells, including:
步骤1：提取地衣芽孢杆菌(B.licheniformis)DL-1基因组DNA，以DNA为模板，设计上下游引物pcfF和pcfR，进行PCR扩增，获得pcf基因片段，pcf基因核苷酸序列如SEQ ID NO.1所示；Step 1: Extract the genomic DNA of B. licheniformis DL-1, use DNA as a template, design the upstream and downstream primers pcfF and pcfR, and perform PCR amplification to obtain pcf gene fragments. The nucleotide sequence of pcf gene is as SEQ ID NO.1 shown;
步骤2：提取pHT01质粒DNA，以该DNA为模板，设计上下游引物CmrF和CmrR，进行PCR扩增，获得氯霉素抗性Cmr基因片段，Cmr基因核苷酸序列如SEQ ID NO.2所示；Step 2: Extract the pHT01 plasmid DNA, use the DNA as a template, design the upstream and downstream primers CmrF and CmrR, and perform PCR amplification to obtain the chloramphenicol-resistant Cmr gene fragment. The nucleotide sequence of the Cmr gene is shown in SEQ ID NO. 2. Show
步骤3：将步骤1制备得到的pcf基因片段与步骤2制备得到的Cmr基因片段，采用重叠延伸PCR进行融合，获得同源重组pcf-Cmr片段；Step 3: The pcf gene fragment prepared in step 1 and the Cmr gene fragment prepared in step 2 are fused by overlapping extension PCR to obtain homologous recombination pcf-Cmr fragments;
步骤4：将步骤3获得的同源重组pcf-Cmr片段，酶切，浓缩后，电转化至地衣芽孢杆菌感受态细胞；Step 4: digest the homologous recombination pcf-Cmr fragment obtained in Step 3, digest it, concentrate it, and electroporate it into competent cells of Bacillus licheniformis;
步骤5：将步骤4获得的感受态细胞，复苏后，涂布含氯霉素的琼脂固体培养基上，筛选具有氯霉素抗性的阳性重组菌，制得具有抑制菌体自溶的地衣芽孢杆菌工程菌。Step 5: After resuscitating the competent cells obtained in Step 4, spread on chloramphenicol-containing agar solid medium, select positive recombinant bacteria with chloramphenicol resistance, and prepare a lichen with inhibitory cell autolysis Bacillus engineering bacteria.
其中，所述步骤1中，pcfF和pcfR引物序列为：Among them, in step 1, the sequences of pcfF and pcfR primers are:
pcfF： CGCGGATCCTGTATAAGCCCTATCAAGATG pcfF: CGCGGATCC TGTATAAGCCCTATCAAGATG
pcfR：TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAA-CAGpcfR: TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAA-CAG
PCR扩增体系为：(B.licheniformis)DL-1基因组模板1.5μL，上游引物pcfF  1.5μL，下游引物pcfR 1.5μL，2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； The PCR amplification system is: (B. licheniformis) DL-1 genome template 1.5 μL, upstream primer pcfF 1.5 μL, downstream primer pcfR 1.5 μL, 2×Phanta Max Master Mix 25 μL, ddH 2 O 20.5 μL, total volume 50 μL;
PCR扩增反应程序为：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸20s，30循环，72℃终延伸10min。The PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 20s, 30 cycles, and final extension at 72℃ for 10min.
其中，所述步骤2中CmrF和CmrR引物序列为：Wherein, the CmrF and CmrR primer sequences in step 2 are:
CmrF：CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTC-ACCmrF: CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTC-AC
CmrR： CGCGGATCCTAGTGACTGGCGATGCTGTCGG CmrR: CGCGGATCC TAGTGACTGGCGATGCTGTCGG
PCR扩增体系为：pHT01质粒模板1.5μL，上游引物CmrF 1.5μL，下游引物CmrR 1.5μL，2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； The PCR amplification system is: pHT01 plasmid template 1.5 μL, upstream primer CmrF 1.5 μL, downstream primer CmrR 1.5 μL, 2×Phanta Max Master Mix 25 μL, ddH 2 O 20.5 μL, total volume 50 μL;
PCR扩增反应程序为：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸40s，30循环，72℃终延伸10min。The PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 40s, 30 cycles, and final extension at 72℃ for 10min.
其中，所述步骤3中，重叠延伸PCR所使用的引物序列为pcfF和CmrR；Among them, in step 3, the primer sequences used in the overlap extension PCR are pcfF and CmrR;
重叠延伸PCR扩增分为初次PCR扩增和二次PCR扩增；Overlap extension PCR amplification is divided into primary PCR amplification and secondary PCR amplification;
所述初次PCR扩增的体系为：pcf基因片段2μL，Cmr基因片段2μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 8.5μL，总体积25μL； The system for the first PCR amplification is: pcf gene fragment 2 μL, Cmr gene fragment 2 μL, 2×Phanta Max Master Mix 12.5 μL, ddH 2 O 8.5 μL, total volume 25 μL;
初次PCR扩增反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸40s，5循环，72℃终延伸5min；The initial PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 40s, 5 cycles, and final extension at 72℃ for 5min;
所述二次PCR扩增的体系为：在初次PCR扩增体系中补加如下试剂，上游引物pcfF 1.5μL，下游引物CmrR 1.5μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 9.5μL； The secondary PCR amplification system is as follows: add the following reagents to the primary PCR amplification system, upstream primer pcfF 1.5 μL, downstream primer CmrR 1.5 μL, 2×Phanta Max Master Mix 12.5 μL, ddH 2 O 9.5 μL;
所述二次PCR扩增的反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸1min，30循环，72℃终延伸10min。The reaction procedure of the second PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
其中，步骤4中，酶切的体系及反应条件如下：Among them, in step 4, the digestion system and reaction conditions are as follows:
pcf-Cmr同源重组片段20μL，BamH Ⅰ内切酶2μL，10×FastDigest Buffer 4μL，ddH 2O 4μL，总体积30μL；37℃，酶切2h，获得酶切后产物； pcf-Cmr homologous recombination fragment 20μL, BamH endonuclease 2μL, 10×FastDigest Buffer 4μL, ddH 2 O 4μL, total volume 30μL; 37°C, digestion for 2h to obtain the digested product;
浓缩的步骤为：向酶切后的产物中，加入1/10体积3mol/L醋酸钠溶液和2.5倍体积的无水乙醇，-20℃放置20min，12000r/min离心5min得DNA沉淀， 加入70％乙醇重悬DNA沉淀，12000r/min离心5min除去乙醇，37℃风干，加入25μL ddH 2O重悬，得到pcf-Cmr浓缩重组片段； The steps of concentration are: add 1/10 volume of 3mol/L sodium acetate solution and 2.5 times volume of absolute ethanol to the digested product, leave at -20℃ for 20min, centrifuge at 12000r/min for 5min to obtain DNA precipitate, add 70 Resuspend DNA pellet in% ethanol, centrifuge at 12000r/min for 5min to remove ethanol, air dry at 37°C, add 25μL ddH 2 O to resuspend, to obtain pcf-Cmr concentrated recombinant fragment;
电转化的步骤为：将(B.licheniformis)DL-1感受态细胞与pcf-Cmr浓缩重组片段于冰上以10:1比例轻轻混匀，将混合物移入2mm电转杯，2100V、5ms电转，然后立即加入1mL RM培养基，37℃，180r/min复苏4h后涂布于含氯霉素(25μg/mL)的LB固体培养基中培养。The steps of electrotransformation are as follows: (B. licheniformis) DL-1 competent cells and pcf-Cmr concentrated recombinant fragments were gently mixed on ice at a ratio of 10:1, and the mixture was transferred into a 2mm electric rotating cup, 2100V, 5ms electric rotating, Then immediately add 1mL RM medium, resuscitate at 37℃, 180r/min for 4h, then spread on LB solid medium containing chloramphenicol (25μg/mL) and cultivate.
进一步的，所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，还包括步骤6：将步骤5制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种到营养条件不同的培养基上，进行营养优化培养。Further, the construction method of the Bacillus licheniformis engineering bacteria for inhibiting autolysis of the bacterial cells further includes step 6: inoculation of the Bacillus licheniformis engineering bacteria prepared for the autolysis of the bacterial cells prepared in step 5 into cultures with different nutritional conditions On the basis, carry out nutrition optimization cultivation.
进一步的，接种量为单菌落/50ml培养基，培养条件为37℃，200r/min培养24h；所述培养基分别为含质量百分比为0.5％、1％、1.5％、2％、2.5％、3％甘油的LB培养基；Further, the inoculation amount is a single colony/50ml medium, and the culture conditions are 37°C and 200r/min for 24h; the medium contains 0.5%, 1%, 1.5%, 2%, 2.5%, 3% glycerol in LB medium;
含质量百分比为0.1％、0.3％、0.5％、0.8％、1％葡萄糖的LB培养基；LB medium containing 0.1%, 0.3%, 0.5%, 0.8%, 1% glucose in mass percentage;
含质量百分比为0.1％、0.3％、0.5％、0.8％、1％葡萄糖的LBG培养基；LBG medium containing 0.1%, 0.3%, 0.5%, 0.8%, 1% glucose in mass percentage;
含质量百分比为0.5％、1％、1.5％、2％、2.5％、3％甘油的LBP培养基；LBP medium containing 0.5%, 1%, 1.5%, 2%, 2.5% and 3% glycerol in mass percentage;
所述LB培养基由以下质量百分比组分组成：蛋白胨1％、酵母浸粉0.5％、氯化钠1％；用5mol/LNaOH调节培养基pH为7.0；The LB medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH;
所述LBG培养基由以下组分组成：蛋白胨1％(质量百分比)、酵母浸粉0.5％(质量百分比)、氯化钠1％(质量百分比)、甘油1％(体积百分比)，用5mol/LNaOH调节培养基pH为7.0；The LBG medium consists of the following components: peptone 1% (mass percentage), yeast extract 0.5% (mass percentage), sodium chloride 1% (mass percentage), glycerin 1% (volume percentage), using 5mol/ LNaOH adjusts the medium pH to 7.0;
所述LBP培养基由以下质量百分比组分组成：蛋白胨1％、酵母浸粉0.5％、氯化钠1％、葡萄糖0.1％；用5mol/LNaOH调节培养基pH为7.0。The LBP medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH.
优选的，所述培养基为含质量百分比为2.5％甘油的LBP培养基。Preferably, the medium is an LBP medium containing 2.5% glycerin by mass.
再一方面，本发明还提供上述抑制菌体自溶的地衣芽孢杆菌工程菌的应用，应用于蛋白酶及其制剂的生产。In still another aspect, the present invention also provides the application of the above-mentioned Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells, which is applied to the production of protease and its preparation.
本发明具有以下有益效果：The invention has the following beneficial effects:
本发明首次公开了一种地衣芽孢杆菌自溶相关基因pcf敲除与营养条件结合控制菌体自溶，提高菌体生物量的方法，将重叠的pcf-Cmr片段电转化至 B.licheniformisDL-1感受态细胞，仅通过一次同源单交换实现对pcf基因快速敲除，成功获得pcf基因缺失菌株B.licheniformis DL-1Δpcf，在无优化营养条件下重组菌自溶率降低了11.2％，证实通过pcf基因敲除可以控制菌体自溶率；The present invention discloses for the first time a method for controlling the autolysis of bacterial cells by combining the autoclaving related gene pcf knockout of Bacillus licheniformis with nutritional conditions, and improving the biomass of the bacterial cells, and electrically converting the overlapping pcf-Cmr fragments to B. licheniformisDL-1 Competent cells, only one homologous single exchange was used to quickly knock out the pcf gene, and the pcf gene-deleted strain B. licheniformis DL-1Δpcf was successfully obtained, and the autolysis rate of recombinant bacteria was reduced by 11.2% without optimized nutritional conditions. PCF gene knockout can control the autolysis rate of bacteria;
通过优化营养条件，出发菌株与重组菌株菌体自溶率分别降低52.6％与56.3％，重组菌通过自溶基因敲除结合营养条件控制可以进一步提高生物量，降低自溶率，自溶率最大降低71.2％，敲除自溶基因结合营养条件控制可使地衣芽孢杆菌菌体生物量得到提高，有利于高浓度菌体获得；By optimizing the nutritional conditions, the autolysis rates of the starting strain and the recombinant strain were reduced by 52.6% and 56.3%, respectively. The recombinant bacteria could further increase the biomass and reduce the autolysis rate by autolytic gene knockout combined with nutritional condition control. The autolysis rate was the largest Reduced by 71.2%, knocking out autolytic genes combined with nutritional conditions control can increase the biomass of Bacillus licheniformis, which is beneficial to the acquisition of high-concentration bacteria;
本发明提供的抑制菌体自溶的地衣芽孢杆菌工程菌在产α-淀粉酶培养基中培养，酶活较未改造菌株提高了19.1％，该方法有益于地衣芽孢杆菌类酶制剂生产菌株的遗传育种及工业化生产。The Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells provided by the present invention are cultured in an α-amylase-producing medium, and the enzyme activity is increased by 19.1% compared with the unmodified strain. This method is beneficial for the production of strains of Bacillus licheniformis enzyme preparations Genetic breeding and industrial production.
附图说明BRIEF DESCRIPTION
图1为本发明实施例1中制备得到的pcf基因片段琼脂糖凝胶电泳图；其中，泳道1为DNA Maker，泳道2～4为pcf基因条带，大小418bp；FIG. 1 is an electrophoresis diagram of the pcf gene fragment agarose gel prepared in Example 1 of the present invention; wherein, lane 1 is DNA Maker, and lanes 2 to 4 are pcf gene bands with a size of 418 bp;
图2为本发明实施例1制备得到的Cmr基因片段琼脂糖凝胶电泳图；其中，泳道1为DNA Maker，泳道2～4为Cmr基因条带，大小1245bp；FIG. 2 is an electrophoresis diagram of the Cmr gene fragment agarose gel prepared in Example 1 of the present invention; wherein, lane 1 is DNA Maker, and lanes 2 to 4 are Cmr gene bands with a size of 1245 bp;
图3为本发明实施例1制备得到的pcf-Cmr基因片段琼脂糖凝胶电泳图；其中，泳道1为DNA Maker，泳道2～4为pcf-Cmr基因条带，大小1663bp；FIG. 3 is an agarose gel electrophoresis diagram of the pcf-Cmr gene fragment prepared in Example 1 of the present invention; where lane 1 is DNA Maker, and lanes 2 to 4 are pcf-Cmr gene bands with a size of 1663bp;
图4为实施例5鉴定抑制菌体自溶的地衣芽孢杆菌工程菌pcf-Cmr基因片段琼脂糖凝胶电泳图；其中，泳道1为DNA Maker，泳道2～3为pcf-Cmr基因条带；4 is an agarose gel electrophoresis diagram of the pcf-Cmr gene fragment of the Bacillus licheniformis engineering strain identified in Example 5 to inhibit autolysis of the bacterial cell; wherein, lane 1 is DNA Maker, and lanes 2 to 3 are pcf-Cmr gene bands;
图5为本发明实施例6中未经改造的地衣芽孢杆菌(B.licheniformis DL-1)培养基营养条件与菌体自溶的关系图，其中图(a)为未经改造的地衣芽孢杆菌接种于LB添加不同比例(0.1％、0.3％、0.5％、0.8％、1.0％)葡萄糖培养基生长曲线；图(b)为未经改造的地衣芽孢杆菌接种于LB添加不同比例(0.5％、1.0％、1.5％、2.0％、2.5％、3.0％)甘油培养基生长曲线；图(c)为未经改造的地衣芽孢杆菌接种于LBG添加不同比例(0.1％、0.3％、0.5％、0.8％、1.0％)葡萄糖培养基生长曲线；图(d)为未经改造的地衣芽孢杆菌接种于LBP添加不同 比例(0.5％、1.0％、1.5％、2.0％、2.5％、3.0％)甘油培养基长曲线；5 is a diagram of the relationship between the nutritional conditions of the unmodified B. licheniformis (DL-1) medium and the autolysis of the bacteria in Example 6 of the present invention, where Figure (a) is the unmodified B. licheniformis Growth curve of glucose medium inoculated in LB and added different ratios (0.1%, 0.3%, 0.5%, 0.8%, 1.0%); Figure (b) shows that the unmodified Bacillus licheniformis was inoculated in LB and added different ratios (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) growth curve of glycerol medium; Figure (c) shows that the unmodified Bacillus licheniformis was inoculated in LBG with different proportions (0.1%, 0.3%, 0.5%, 0.8 %, 1.0%) Glucose medium growth curve; Figure (d) shows that unmodified Bacillus licheniformis was inoculated into LBP with different proportions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) glycerol culture Base length curve
图6为实施例6中本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌(B.licheniformis DL-1Δpcf)的培养基营养条件与菌体自溶的关系图，其中图(a)为本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种于LB添加不同比例(0.1％、0.3％、0.5％、0.8％、1.0％)葡萄糖培养基生长曲线；图(b)为本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种于LB添加不同比例(0.5％、1.0％、1.5％、2.0％、2.5％、3.0％)甘油培养基生长曲线；图(c)为本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种于LBG添加不同比例(0.1％、0.3％、0.5％、0.8％、1.0％)葡萄糖培养基生长曲线；图(d)为本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种于LBP添加不同比例(0.5％、1.0％、1.5％、2.0％、2.5％、3.0％)甘油培养基长曲线；6 is a graph showing the relationship between the nutrient conditions of the culture medium of B. licheniformis engineering bacteria (B. licheniformis DL-1Δpcf) prepared by the invention prepared in Example 6 and the autolysis of the microbial cells, in which (a) The growth curve of Bacillus licheniformis engineering bacteria prepared by the present invention for inhibiting autolysis of bacterial cells was inoculated in LB supplemented with different ratios (0.1%, 0.3%, 0.5%, 0.8%, 1.0%) of glucose medium; Figure (b) is The Bacillus licheniformis engineering bacteria prepared by the invention and inhibiting the autolysis of bacterial cells are inoculated in LB and added with different proportions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) of glycerol medium growth curve; Figure (c ) The Bacillus licheniformis engineering bacteria prepared by the present invention, which inhibits autolysis of bacterial cells, is inoculated into LBG and added with different proportions (0.1%, 0.3%, 0.5%, 0.8%, 1.0%) of glucose medium growth curve; Figure (d) Long-curve glycerol medium with different proportions (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%) added to the LBP engineering bacteria prepared by the invention for inhibiting the autolysis of bacterial cells and inoculated with LBP;
图7中(a)、(b)、(c)为未经改造的地衣芽孢杆菌与本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌在不同营养培养基中的生长曲线；(a)图为在LB培养基中的生长曲线，(b)图为在LBG添加0.1％葡萄糖培养基中的生长曲线；(c)图为在LBP添加2.5％甘油培养基中的生长曲线；7 (a), (b), (c) are the growth curves of the unmodified Bacillus licheniformis and the Bacillus licheniformis engineering bacteria prepared by the present invention in different nutrient media in the inhibition of bacterial autolysis; ( a) The graph is the growth curve in LB medium, (b) the graph is the growth curve in LBG supplemented with 0.1% glucose medium; (c) the graph is the growth curve in LBP supplemented with 2.5% glycerol medium;
图8为实施例7中对未经改造的地衣芽孢杆菌与本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌α-淀粉酶酶活测定结果。FIG. 8 is the measurement results of α-amylase activity of Bacillus licheniformis engineered bacteria of the unmodified Bacillus licheniformis and the autolysis of bacterial cells prepared in the present invention in Example 7. FIG.
具体实施方式detailed description
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合附图及具体实施例进行详细描述。但本发明绝非限于这些例子。以下所述仅为本发明较好的实施例，仅仅用以解释本发明，并不能因此而理解为对本发明专利范围的限制。应当指出的是，凡在本发明的精神和原则之内所做的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。因此，本发明专利的保护范围应以所附权利要求为准。In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will describe in detail with reference to the accompanying drawings and specific embodiments. However, the present invention is by no means limited to these examples. The following are only preferred embodiments of the present invention, which are only used to explain the present invention and should not be construed as limiting the patent scope of the present invention. It should be pointed out that any modification, equivalent replacement and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the invention patent shall be subject to the appended claims.
除特殊说明，本发明所用组分均为市售产品。Unless otherwise specified, the components used in the present invention are all commercially available products.
本发明中采用的地衣芽孢杆菌(B.licheniformis DL-1)购自美国模式菌种收集中心(ATCC)菌种编号27811；pHT01质粒购买于杭州宝赛生物科技有限公 司。The B. licheniformis (DL-1) used in the present invention was purchased from the American Type Culture Collection Center (ATCC) strain number 27811; the pHT01 plasmid was purchased from Hangzhou Baosai Biotechnology Co., Ltd.
本发明提供一种抑制菌体自溶的地衣芽孢杆菌工程菌及其构建方法和应用，能够有效改善地衣芽孢杆菌菌体自溶现象。The present invention provides a Bacillus licheniformis engineering bacterium that inhibits autolysis of bacteria and its construction method and application, which can effectively improve the autolysis of Bacillus licheniformis bacteria.
实施例1Example 1
同源重组pcf-Cmr基因片段的制备Preparation of homologous recombination pcf-Cmr gene fragment
采用DNA提取试剂盒提取B.licheniformis DL-1基因组，根据pcf基因核苷酸序列设计上、下游引物，以B.licheniformisDL-1基因组为模板，进行pcf基因PCR扩增，上下游引物核苷酸序列如下，其中下划线为BamH Ⅰ保护碱基及酶切位点：Use DNA extraction kit to extract B.licheniformis DL-1 genome, design the upstream and downstream primers according to the nucleotide sequence of pcf gene, and use B.licheniformisDL-1 genome as a template for PCR amplification of pcf gene, upstream and downstream primer nucleotides The sequence is as follows, where the underline is the BamH I protection base and the cleavage site:
pcfF： CGCGGATCCTGTATAAGCCCTATCAAGATG pcfF: CGCGGATCC TGTATAAGCCCTATCAAGATG
pcfR：TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAACAGpcfR: TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAACAG
PCR扩增体系：PCR amplification system:
B.licheniformis DL-1基因组模板1.5μL，上游引物pcfF 1.5μL，下游引物pcfR 1.5μL，2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； B. licheniformis DL-1 genome template 1.5μL, upstream primer pcfF 1.5μL, downstream primer pcfR 1.5μL, 2×Phanta Max Master Mix 25μL, ddH 2 O 20.5μL, total volume 50μL;
PCR扩增反应程序：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸20s，30循环，72℃终延伸10min。PCR amplification reaction program: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 20s, 30 cycles, final extension at 72℃ for 10min.
1％琼脂糖凝胶电泳检测PCR产物，400bp左右出现特异性条带，结果见图1，表明成功获得418bp的pcf同源片段，将PCR产物pcf同源片段，使用SanPrep柱式DNA胶回收试剂盒进行胶回收，所得DNA直接进行下一步实验或-20℃保存。The PCR product was detected by 1% agarose gel electrophoresis, and a specific band appeared at about 400bp. The result is shown in Figure 1, indicating that the 418bp pcf homologous fragment was successfully obtained. The PCR product homologous fragment was used to recover the reagents using SanPrep column DNA gel The cassette is subjected to gel recovery, and the obtained DNA is directly subjected to the next experiment or stored at -20°C.
采用DNA提取试剂盒提取含氯霉素抗性pHT01质粒DNA，根据Cmr基因核苷酸序列设计上、下游引物，以pHT01质粒为模板，进行Cmr基因PCR扩增，上下游引物核苷酸序列如下，其中下划线为BamH Ⅰ保护碱基及酶切位点：Using a DNA extraction kit to extract the pHT01 plasmid DNA containing chloramphenicol, design the upstream and downstream primers according to the nucleotide sequence of the Cmr gene, and use the pHT01 plasmid as a template to perform PCR amplification of the Cmr gene. The nucleotide sequence of the upstream and downstream primers is as follows , Where the underline is the BamH I protection base and the cleavage site:
CmrF：CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCACCmrF: CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTCAC
CmrR： CGCGGATCCTAGTGACTGGCGATGCTGTCGG CmrR: CGCGGATCC TAGTGACTGGCGATGCTGTCGG
PCR扩增体系：PCR amplification system:
pHT01质粒DNA模板1.5μL，上游引物CmrF 1.5μL，下游引物CmrR 1.5μL， 2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； pHT01 plasmid DNA template 1.5μL, upstream primer CmrF 1.5μL, downstream primer CmrR 1.5μL, 2×Phanta Max Master Mix 25μL, ddH 2 O 20.5μL, total volume 50μL;
PCR扩增反应程序：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸40s，30循环，72℃终延伸10min。PCR amplification reaction program: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 40s, 30 cycles, and final extension at 72℃ for 10min.
1％琼脂糖凝胶电泳检测，1200bp左右出现特异性条带，结果见图2，表明成功获得1245bp的Cmr片段，使用SanPrep柱式DNA胶回收试剂盒进行胶回收，所得DNA直接进行下一步实验或-20℃保存。Detection by 1% agarose gel electrophoresis showed a specific band around 1200bp. The result is shown in Figure 2. It shows that the Cmr fragment of 1245bp was successfully obtained. The SanPrep column-type DNA gel recovery kit was used for gel recovery. The resulting DNA was directly used in the next experiment. Or store at -20℃.
将胶回收获得的pcf基因片段、Cmr基因片段，通过重叠延伸PCR进行融合，获得pcf-Cmr同源重组片段，重叠延伸PCR所使用的引物核苷酸序列如下，其中下划线为BamH Ⅰ保护碱基及酶切位点：The pcf gene fragment and Cmr gene fragment obtained by gel recovery were fused by overlap extension PCR to obtain pcf-Cmr homologous recombination fragment. The nucleotide sequence of primers used in overlap extension PCR is as follows, in which the underline is BamH I protected base And restriction sites:
pcfF： CGCGGATCCTGTATAAGCCCTATCAAGATG pcfF: CGCGGATCC TGTATAAGCCCTATCAAGATG
CmrR： CGCGGATCCTAGTGACTGGCGATGCTGTCGG CmrR: CGCGGATCC TAGTGACTGGCGATGCTGTCGG
重叠延伸PCR扩增共分初次PCR和二次PCR扩增；Overlap extension PCR amplification is divided into primary PCR and secondary PCR amplification;
初次PCR扩增体系：Initial PCR amplification system:
pcf片段2μL，Cmr片段2μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 8.5μL，总体积25μL； pcf fragment 2μL, Cmr fragment 2μL, 2×Phanta Max Master Mix 12.5μL, ddH 2 O 8.5μL, total volume 25μL;
初次PCR扩增反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸40s，5循环，72℃终延伸5min。The initial PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 40s, 5 cycles, and final extension at 72℃ for 5min.
所述二次PCR扩增的体系为：在初次PCR扩增体系中补加如下试剂，上游引物pcfF 1.5μL，下游引物CmrR 1.5μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 9.5μL； The secondary PCR amplification system is as follows: add the following reagents to the primary PCR amplification system, upstream primer pcfF 1.5 μL, downstream primer CmrR 1.5 μL, 2×Phanta Max Master Mix 12.5 μL, ddH 2 O 9.5 μL;
所述二次PCR扩增的反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸1min，30循环，72℃终延伸10min。The reaction procedure of the second PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
1％琼脂糖凝胶电泳检测，1600bp左右出现特异性条带结果见图3，表明成功获得1663bp同源重组片段pcf-Cmr，使用SanPrep柱式DNA胶回收试剂盒进行胶回收，所得DNA直接进行下一步实验或-20℃保存。1% agarose gel electrophoresis detection, specific bands around 1600bp results shown in Figure 3, indicating that the 1663bp homologous recombination fragment pcf-Cmr was successfully obtained, using SanPrep column DNA gel recovery kit for gel recovery, the resulting DNA was directly carried out Next experiment or store at -20℃.
实施例2Example 2
同源重组pcf-Cmr基因片段的酶切及浓缩Digestion and concentration of homologous recombination pcf-Cmr gene fragment
利用BamH Ⅰ内切酶对步骤1制备得到的胶回收pcf-Cmr融合片段进行单酶 切，其酶切体系及条件如下：The BamH I endonuclease was used to perform single digestion on the pcf-Cmr fusion fragment prepared in step 1, and the digestion system and conditions were as follows:
pcf-Cmr同源重组片段20μL，BamH Ⅰ内切酶2μL，10×FastDigest Buffer 4μL，ddH 2O 4μL，总体积30μL；37℃，酶切2h。 pcf-Cmr homologous recombination fragment 20μL, BamH endonuclease 2μL, 10×FastDigest Buffer 4μL, ddH 2 O 4μL, total volume 30μL; 37℃, digestion for 2h.
对酶切后的pcf-Cmr融合片段，进行浓缩，浓缩步骤如下：The digested pcf-Cmr fusion fragment is concentrated, and the concentration steps are as follows:
向酶切后的产物中，加入1/10体积3mol/L醋酸钠溶液和2.5倍体积的无水乙醇，-20℃放置20min，12000r/min离心5min得DNA沉淀；加入70％乙醇重悬DNA沉淀，12000r/min离心5min除去乙醇，37℃风干，加入25μLddH 2O重悬，制得300ng/μL的pcf-Cmr浓缩DNA溶液，直接进行下一步实验或或-20℃保存。 To the digested product, add 1/10 volume of 3mol/L sodium acetate solution and 2.5 volumes of absolute ethanol, stand at -20℃ for 20min, and centrifuge at 12000r/min for 5min to obtain DNA precipitate; add 70% ethanol to resuspend the DNA Precipitate, centrifuge at 12000r/min for 5min to remove ethanol, air dry at 37℃, add 25μL ddH 2 O to resuspend, prepare 300ng/μL pcf-Cmr concentrated DNA solution, and proceed directly to the next experiment or store at -20℃.
实施例3Example 3
B.licheniformis DL-1感受态细胞的制备B. Preparation of Licheniformis DL-1 competent cells
将B.licheniformis DL-1划线于LB固体平板培养基上，37℃培养24h，挑取单菌落接种50mL LB培养基，37℃、200r/min培养18h传代两次；取二代活化的菌液2mL转接50mLGM(菌体增殖培养基)中，使初始菌液OD600＝0.15，培养至OD600＝0.90；将菌液倒入预冷的50mL离心管中，10min，4℃，5000r/mim离心10min，收集菌体；用预冷的20mL ETM(电转缓冲液)电转缓冲液洗涤3次后，ETM重悬、分装，即为制备好的感受态细胞。Streak B.licheniformis DL-1 on LB solid plate medium, incubate at 37°C for 24h, pick single colonies and inoculate 50mL of LB medium, and incubate at 37°C and 200r/min for 18h for two passages; take the second-generation activated bacteria Transfer 2mL of solution to 50mL of GM (cell proliferation medium), make the initial bacterial solution OD600 = 0.15, cultivate to OD600 = 0.90; pour the bacterial solution into a pre-cooled 50mL centrifuge tube, 10min, 4 ℃, 5000r/mim centrifugation After 10 min, collect the cells; wash them with pre-cooled 20 mL ETM (electrotransfer buffer) electrotransfer buffer three times, and then resuspend and aliquot the ETM to prepare the competent cells.
其中，涉及到的培养基，其组分如下：Among them, the components of the culture medium involved are as follows:
LB培养基由以下重量百分比组分组成：蛋白胨1％，酵母浸粉0.5％，NaCl1％，pH7.0。The LB medium is composed of the following components by weight: peptone 1%, yeast extract 0.5%, NaCl 1%, pH 7.0.
GM(菌体增殖培养基)由以下质量百分比组分组成：蛋白胨1％，酵母浸粉0.5％，NaCl 1％，山梨醇9.1％，pH7.0。GM (Bacterial Multiplication Medium) is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, NaCl 1%, sorbitol 9.1%, pH 7.0.
ETM(电转缓冲液)：山梨醇9.1％(质量百分比)，甘露醇9.1％(质量百分比)，10％甘油(体积百分比)。ETM (electrotransfer buffer): sorbitol 9.1% (mass percentage), mannitol 9.1% (mass percentage), 10% glycerol (volume percentage).
实施例4Example 4
pcf-Cmr同源重组片段的电转化Electrotransformation of pcf-Cmr homologous recombination fragment
将实施例3制备的B.licheniformisDL-1感受态细胞与实施例2制备得到的pcf-Cmr重组基因片段置于冰上以10:1比例轻轻混匀，将混合物移入2mm电转杯，2100V、5ms电转，然后立即加入1mL RM培养基，37℃，180r/min复苏 4h后涂布于含氯霉素(25μg/mL)的LB固体培养基中培养。The B. licheniformis DL-1 competent cells prepared in Example 3 and the pcf-Cmr recombinant gene fragments prepared in Example 2 were placed on ice and gently mixed at a ratio of 10:1, and the mixture was transferred into a 2mm electric rotating cup, 2100V, After 5ms electroporation, immediately add 1mL RM medium, revive at 37℃, 180r/min for 4h, then spread on LB solid medium containing chloramphenicol (25μg/mL) for cultivation.
RM(菌体复苏培养基)由以下质量百分比组分组成：蛋白胨1％，酵母浸粉0.5％，NaCl 1％，山梨醇9.1％，甘露醇6.9％，调整其pH为7.0。RM (Bacterial Resuscitation Medium) is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, NaCl 1%, sorbitol 9.1%, mannitol 6.9%, and its pH is adjusted to 7.0.
实施例5Example 5
抑制菌体自溶的地衣芽孢杆菌工程菌(B.licheniformis DL-1Δpcf)的阳性筛选与鉴定Positive screening and identification of B. licheniformis engineering bacteria (DL-1Δpcf) inhibiting autolysis of bacteria
挑取实施例4制备得到的LB固体培养基单菌落，接种到含氯霉素抗性的LB液体培养基，37℃，200r/min培养24h，采用DNA提取试剂盒提取液体培养基中的菌体DNA，以以提取的基因组为模板，pcfF与CmrR为引物，进行PCR扩增。Single colonies of LB solid medium prepared in Example 4 were picked, inoculated into LB liquid medium containing chloramphenicol resistance, cultured at 37°C, 200r/min for 24h, and the bacteria in the liquid medium were extracted using DNA extraction kit DNA was amplified by PCR using the extracted genome as a template and pcfF and CmrR as primers.
所述涉及的上下游引物核苷酸序列如下，其中下划线为BamH Ⅰ保护碱基及酶切位点：The nucleotide sequences of the upstream and downstream primers involved are as follows, in which the underline is the BamH I protection base and the cleavage site:
pcfF： CGCGGATCCTGTATAAGCCCTATCAAGATG pcfF: CGCGGATCC TGTATAAGCCCTATCAAGATG
CmrR： CGCGGATCCTAGTGACTGGCGATGCTGTCGG CmrR: CGCGGATCC TAGTGACTGGCGATGCTGTCGG
PCR扩增体系：提取菌体基因组模板1.5μL，上游引物pcf F1.5μL，下游引物CmrR 1.5μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 9.5μL； PCR amplification system: extract 1.5 μL of bacterial genome template, upstream primer pcf F1.5 μL, downstream primer CmrR 1.5 μL, 2×Phanta Max Master Mix 12.5 μL, ddH 2 O 9.5 μL;
PCR扩增反应程序：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸1min，30循环，72℃终延伸10min。PCR amplification reaction program: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, 72°C final extension for 10 min.
扩增产物用1％琼脂糖凝胶电泳进行验证，结果见图4，基因扩增片段大小为1663bp，表明已成功获得具有pcf-Cmr重组基因片段的地衣芽孢杆菌工程菌。The amplified product was verified by 1% agarose gel electrophoresis. The results are shown in Figure 4. The size of the gene amplified fragment is 1663bp, indicating that the Bacillus licheniformis engineering bacteria with the pcf-Cmr recombinant gene fragment has been successfully obtained.
实施例6Example 6
营养条件与菌体自溶率的关系探索Exploring the relationship between nutritional conditions and bacterial autolysis rate
将未经改造的地衣芽孢杆菌(B.licheniformis DL-1)与本发明制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌(B.licheniformis DL-1Δpcf)分别接种到不同营养组分的100ml培养基中，37℃，200r/min培养，每隔4～6h分别取样测定菌液OD600，以菌体浓度来衡量B.licheniformis DL-1菌体自溶情况。Inoculate unmodified Bacillus licheniformis (B. licheniformis DL-1) and Bacillus licheniformis engineering bacteria (B. licheniformis DL-1Δpcf) prepared by the present invention into 100 ml of different nutritional components The medium was cultured at 37°C and 200r/min, and the bacterial solution OD600 was sampled every 4 to 6 hours, and the autolysis of B. licheniformis DL-1 cells was measured by the cell concentration.
培养基的组分配方如下表所示：The composition formula of the medium is shown in the following table:
Figure PCTCN2020071158-appb-000001
Figure PCTCN2020071158-appb-000001
注：√表示添加，/表示未添加。Note: √ means added, / means not added.
其中，LBG培养基：蛋白胨1％、酵母浸粉0.5％、氯化钠1％(均为质量分数)、甘油1％(体积分数)，pH 7.0。Among them, LBG medium: peptone 1%, yeast extract 0.5%, sodium chloride 1% (all mass fractions), glycerin 1% (volume fraction), pH 7.0.
LBP培养基：蛋白胨1％、酵母浸粉0.5％、氯化钠1％、葡萄糖0.1％(均为质量分数)。LBP medium: peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1% (all mass fractions).
经过上述不同营养组分培养基的摸索，由图5、图6可知，本发明制备出的抑制菌体自溶的地衣芽孢杆菌工程菌(B.licheniformis DL-1Δpcf)与未经改造的地衣芽孢杆菌(B.licheniformis DL-1)生长曲线一致，均最适于添加有2.5％甘油的LBP培养基生长，与常规LB培养基培养相比，出发菌株与重组菌株菌体自溶率分别降低52.6％与56.3％；由图6-(d)可知重组菌通过自溶基因敲除结合营养条件控制可以进一步提高生物量，降低自溶率，自溶率最大降低71.2％(计算公式＝(OD600max1-OD600max2)/OD600max2，其中，OD600max1为B.licheniformis DL-1Δpcf在最适营养条件下600nm的最大吸光度值；OD600max2为B.licheniformis DL-1在LB培养基条件下600nm的最大吸光度值)After exploring the medium of different nutrient components, it can be seen from FIG. 5 and FIG. 6 that B. licheniformis engineering bacteria (B. licheniformis DL-1Δpcf) and unmodified Bacillus licheniformis prepared by the present invention inhibit the autolysis of bacterial cells. The growth curve of B. licheniformis DL-1 is consistent, and they are most suitable for growth with LBP medium supplemented with 2.5% glycerol. Compared with conventional LB medium culture, the autolysis rate of the starting strain and the recombinant strain is reduced by 52.6 respectively % And 56.3%; it can be seen from Figure 6-(d) that recombinant bacteria can further increase biomass and reduce autolysis rate by autolytic gene knockout combined with nutritional condition control, and the autolysis rate can be reduced by a maximum of 71.2% (calculation formula = (OD600max1- OD600max2)/OD600max2, where OD600max1 is the maximum absorbance value of B.licheniformis DL-1Δpcf at 600nm under optimal nutritional conditions; OD600max2 is the maximum absorbance value of B.licheniformis DL-1 at 600nm under LB medium conditions)
由图7-(a)可知，在普通LB培养基培养条件下，本发明制备的抑制菌体自溶的地衣芽孢杆菌工程菌比未改造的地衣芽孢杆菌的菌体自溶率降低了11.2％；由图7-(b)可知，在LBG添加0.1％葡萄糖培养基培养条件下，本发明制备的抑制菌体自溶的地衣芽孢杆菌工程菌比未改造的地衣芽孢杆菌的菌体自溶率降低了12.8％；由图7-(c)可知，在LBP添加2.5％甘油培养基培养条件下，本发明制备的抑制菌体自溶的地衣芽孢杆菌工程菌比未改造的地衣芽孢杆菌的 菌体自溶率降低了13.3％。As can be seen from FIG. 7-(a), under the general LB medium culture conditions, the bacterial autolysis rate of the Bacillus licheniformis engineering bacteria prepared by the present invention is 11.2% lower than that of the unmodified Bacillus licheniformis. It can be seen from Figure 7-(b) that under the culture conditions of LBG supplemented with 0.1% glucose medium, the autolysis rate of the Bacillus licheniformis engineered bacteria prepared by the present invention is higher than that of the unmodified Bacillus licheniformis. Reduced by 12.8%; as can be seen from Figure 7-(c), under the culture conditions of LBP supplemented with 2.5% glycerol medium, the Bacillus licheniformis engineered bacteria prepared by the present invention inhibited autolysis of the bacterial cells compared to the unmodified Bacillus licheniformis bacteria The body autolysis rate decreased by 13.3%.
由上述数据可知，通过pcf基因敲除获得抑制菌体自溶的地衣芽孢杆菌工程菌与培养基营养条件相结合，对B.licheniformis DL-1菌体自溶控制具有协同叠加效应，且最佳的营养条件为LB添加0.1％葡萄糖、2.5％甘油(即LBP培养基+2.5％甘油)。From the above data, it can be seen that the combination of the Bacillus licheniformis engineering bacteria obtained by knocking out the PCF gene knockout and the nutrient conditions of the culture medium has a synergistic superposition effect on the autolysis control of B. licheniformis DL-1 bacteria, and is optimal The nutritional condition is that LB is supplemented with 0.1% glucose and 2.5% glycerol (ie LBP medium + 2.5% glycerol).
实施例7Example 7
B.licheniformis DL-1与本发明制备的B.licheniformis DL-1Δpcfα-淀粉酶酶活测定B.licheniformis DL-1 and B.licheniformis DL-1Δpcfα-amylase prepared by the invention
将B.licheniformis DL-1和重组菌株B.licheniformis DL-1Δpcf以2％接种量接种于发酵培养基，37℃、200r/min培养，每隔12h取样，4℃、12000r/min离心8min取上清，按照国标GB/T24401-2009所示方法测定淀粉酶酶活。B.licheniformis DL-1 and recombinant strain B.licheniformis DL-1Δpcf were inoculated into the fermentation medium at 2% inoculation volume, cultured at 37℃, 200r/min, sampled every 12h, and centrifuged at 4℃, 12000r/min for 8min Qing, the amylase activity was determined according to the method shown in the national standard GB/T24401-2009.
所述发酵培养基由以下质量百分比组分组成：The fermentation medium is composed of the following mass percentage components:
可溶性淀粉2％、蛋白胨1％、酵母浸粉0.5％、氯化钠1％、葡萄糖0.1％、甘油2.5％，pH 7.0。 Soluble starch 2%, peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1%, glycerol 2.5%, pH 7.0.
α-淀粉酶活力定义：1ml液体酶，于70℃、pH6.0条件下，1min液化1mg可溶性淀粉，即为1个酶活力单位，以U/mL表示。Definition of α-amylase activity: 1ml of liquid enzyme, liquefying 1mg of soluble starch in 1min at 70℃ and pH6.0, which is 1 unit of enzyme activity, expressed in U/mL.
α-淀粉酶酶活测定结果见图8，由该图可知B.licheniformis DL-1与B.licheniformis DL-1Δpcf均在发酵至48h酶活达到最大，分别为340U/mL和405U/mL，最大酶活提高了19.1％。通过酶活测定可以确定本发明制备的pcf基因缺失的抑制菌体自溶的地衣芽孢杆菌工程菌，有利于α-淀粉酶酶活的提高。The results of the measurement of α-amylase enzyme activity are shown in Fig. 8. From this figure, it can be seen that B. licheniformis DL-1 and B. licheniformis DL-1Δpcf are both fermented to the maximum enzyme activity at 48h, which are 340U/mL and 405U/mL, respectively. Enzyme activity increased by 19.1%. Through enzyme activity measurement, it can be determined that the PCF gene-deficient Bacillus licheniformis engineering bacteria prepared by the present invention, which inhibits autolysis of bacterial cells, is beneficial to the improvement of α-amylase enzyme activity.
本发明首次公开了一种地衣芽孢杆菌自溶相关基因pcf敲除与营养条件结合控制菌体自溶，提高菌体生物量的方法，将重叠的pcf-Cmr片段电转化至B.licheniformisDL-1感受态细胞，仅通过一次同源单交换实现对pcf基因快速敲除，成功获得pcf基因缺失菌株B.licheniformisDL-1Δpcf。重组菌自溶率较出发菌降低11.2％，证实通过pcf基因敲除可以控制菌体自溶率；重组菌通过自溶基因敲除结合营养条件控制可以提高生物量，降低自溶率，自溶率最大降低71.2％。敲除自溶基因结合营养条件控制可使地衣芽孢杆菌菌体生物量得到提高，有利于高浓度菌体获得；重组菌在产α-淀粉酶培养基中培养，酶活较出发菌株提高 19.1％，该方法有益于地衣芽孢杆菌类酶制剂生产菌株的遗传育种及工业化生产。The present invention discloses for the first time a method for controlling the autolysis of bacterial cells by combining the autoclaving related gene pcf knockout of Bacillus licheniformis with nutritional conditions, and improving the biomass of the bacterial cells, and electrically converting the overlapping pcf-Cmr fragments to B. licheniformisDL-1 Competent cells, through a single homologous single exchange to achieve rapid knockout of the pcf gene, successfully obtained pcf gene deletion strain B.licheniformisDL-1Δpcf. The autolysis rate of the recombinant bacteria was 11.2% lower than that of the starting bacteria. It was confirmed that the autolysis rate of the bacterial body can be controlled by the PCF gene knockout; the recombinant bacteria can increase the biomass and reduce the autolysis rate by autolysis gene knockout combined with nutritional control. The maximum reduction rate is 71.2%. Knock-out autolysis genes combined with nutrient control can increase the biomass of Bacillus licheniformis cells, which is beneficial to the acquisition of high-concentration cells; the recombinant bacteria are cultured in α-amylase-producing medium, and the enzyme activity is increased by 19.1% compared with the starting strain This method is beneficial to the genetic breeding and industrial production of Bacillus licheniformis enzyme preparation production strains.
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is a preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims (10)

  1. 一种抑制菌体自溶的地衣芽孢杆菌工程菌，其特征在于，所述抑制菌体自溶的地衣芽孢杆菌工程菌采用敲除pcf基因获得，所述pcf基因核苷酸序列如SEQ ID NO.1所示。A Bacillus licheniformis engineering bacterium that inhibits autolysis of bacterial cells, characterized in that the Bacillus licheniformis engineering bacterium that inhibits autolysis of cells is obtained by knocking out the pcf gene, and the nucleotide sequence of the pcf gene is as SEQ ID NO .1 shown.
  2. 根据权利要求1所述的抑制菌体自溶的地衣芽孢杆菌工程菌，其特征在于，所述敲除pcf基因的方法为同源重组单交换方法；所述抑制菌体自溶的地衣芽孢杆菌工程菌的培养基为添加0.1％葡萄糖、2.5％甘油的LB培养基。The Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 1, wherein the method of knocking out the pcf gene is a homologous recombination single exchange method; The medium for engineering bacteria is LB medium supplemented with 0.1% glucose and 2.5% glycerol.
  3. 根据权利要求1或2所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，包括：The method for constructing Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 1 or 2, characterized in that it comprises:
    步骤1：提取地衣芽孢杆菌(B.licheniformis)DL-1基因组DNA，以DNA为模板，设计上下游引物pcfF和pcfR，进行PCR扩增，获得pcf基因片段，pcf基因核苷酸序列如SEQ ID NO.1所示；Step 1: Extract the genomic DNA of B. licheniformis DL-1, use DNA as a template, design the upstream and downstream primers pcfF and pcfR, and perform PCR amplification to obtain pcf gene fragments. The nucleotide sequence of pcf gene is as SEQ ID NO.1 shown;
    步骤2：提取pHT01质粒DNA，以该DNA为模板，设计上下游引物CmrF和CmrR，进行PCR扩增，获得氯霉素抗性Cmr基因片段，Cmr基因核苷酸序列如SEQ ID NO.2所示；Step 2: Extract the pHT01 plasmid DNA, use the DNA as a template, design the upstream and downstream primers CmrF and CmrR, and perform PCR amplification to obtain the chloramphenicol-resistant Cmr gene fragment. The nucleotide sequence of the Cmr gene is shown in SEQ ID NO. 2. Show
    步骤3：将步骤1制备得到的pcf基因片段与步骤2制备得到的Cmr基因片段，采用重叠延伸PCR进行融合，获得同源重组pcf-Cmr片段；Step 3: The pcf gene fragment prepared in step 1 and the Cmr gene fragment prepared in step 2 are fused by overlapping extension PCR to obtain homologous recombination pcf-Cmr fragments;
    步骤4：将步骤3获得的同源重组pcf-Cmr片段，酶切，浓缩后，电转化至地衣芽孢杆菌感受态细胞；Step 4: digest the homologous recombination pcf-Cmr fragment obtained in Step 3, digest it, concentrate it, and electroporate it into competent cells of Bacillus licheniformis;
    步骤5：将步骤4获得的感受态细胞，复苏后，涂布含氯霉素的琼脂固体培养基上，筛选具有氯霉素抗性的阳性重组菌，制得具有抑制菌体自溶的地衣芽孢杆菌工程菌。Step 5: After resuscitating the competent cells obtained in Step 4, spread on chloramphenicol-containing agar solid medium, select positive recombinant bacteria with chloramphenicol resistance, and prepare a lichen with inhibitory cell autolysis Bacillus engineering bacteria.
  4. 根据权利要求3所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，所述步骤1中，pcfF和pcfR引物序列为：The method for constructing Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 3, characterized in that, in step 1, the sequences of pcfF and pcfR primers are:
    pcfF： CGCGGATCCTGTATAAGCCCTATCAAGATG pcfF: CGCGGATCC TGTATAAGCCCTATCAAGATG
    pcfR：TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAA-CAGpcfR: TGTGAGCAAAAGGCCAGCAAAAGTTTCACATTGGTTTGAA-CAG
    PCR扩增体系为：(B.licheniformis)DL-1基因组模板1.5μL，上游引物pcfF 1.5μL，下游引物pcfR1.5μL，2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； The PCR amplification system is: (B. licheniformis) DL-1 genome template 1.5 μL, upstream primer pcfF 1.5 μL, downstream primer pcfR 1.5 μL, 2×Phanta Max Master Mix 25 μL, ddH 2 O 20.5 μL, total volume 50 μL;
    PCR扩增反应程序为：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸20s，30循环，72℃终延伸10min；The PCR amplification reaction program is: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 20s, 30 cycles, and final extension at 72℃ for 10min;
    所述步骤2中CmrF和CmrR引物序列为：The CmrF and CmrR primer sequences in the step 2 are:
    CmrF：CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTC-ACCmrF: CTACTGTTCAAACCAATGTGAAACTTTTGCTGGCCTTTTGCTC-AC
    CmrR： CGCGGATCCTAGTGACTGGCGATGCTGTCGG CmrR: CGCGGATCC TAGTGACTGGCGATGCTGTCGG
    PCR扩增体系为：pHT01质粒模板1.5μL，上游引物CmrF 1.5μL，下游引物CmrR 1.5μL，2×Phanta Max Master Mix 25μL，ddH 2O 20.5μL，总体积50μL； The PCR amplification system is: pHT01 plasmid template 1.5 μL, upstream primer CmrF 1.5 μL, downstream primer CmrR 1.5 μL, 2×Phanta Max Master Mix 25 μL, ddH 2 O 20.5 μL, total volume 50 μL;
    PCR扩增反应程序为：95℃预变性5min，95℃变性30s，58℃退火30s，72℃延伸40s，30循环，72℃终延伸10min。The PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 40s, 30 cycles, and final extension at 72℃ for 10min.
  5. 根据权利要求3所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，所述步骤3中，重叠延伸PCR所使用的引物序列为pcfF和CmrR；The method for constructing the Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 3, wherein in step 3, the primer sequences used in the overlap extension PCR are pcfF and CmrR;
    重叠延伸PCR扩增分为初次PCR扩增和二次PCR扩增；Overlap extension PCR amplification is divided into primary PCR amplification and secondary PCR amplification;
    所述初次PCR扩增的体系为：pcf基因片段2μL，Cmr基因片段2μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 8.5μL，总体积25μL； The system for the first PCR amplification is: pcf gene fragment 2 μL, Cmr gene fragment 2 μL, 2×Phanta Max Master Mix 12.5 μL, ddH 2 O 8.5 μL, total volume 25 μL;
    初次PCR扩增反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸40s，5循环，72℃终延伸5min；The initial PCR amplification reaction program was: pre-denaturation at 95℃ for 5min, denaturation at 95℃ for 30s, annealing at 60℃ for 30s, extension at 72℃ for 40s, 5 cycles, and final extension at 72℃ for 5min;
    所述二次PCR扩增的体系为：在初次PCR扩增体系中补加如下试剂，上游引物pcfF1.5μL，下游引物CmrR1.5μL，2×Phanta Max Master Mix 12.5μL，ddH 2O 9.5μL； The secondary PCR amplification system is as follows: add the following reagents to the primary PCR amplification system, upstream primer pcfF1.5μL, downstream primer CmrR1.5μL, 2×Phanta Max Master Mix 12.5μL, ddH 2 O 9.5μL;
    所述二次PCR扩增的反应程序为：95℃预变性5min，95℃变性30s，60℃退火30s，72℃延伸1min，30循环，72℃终延伸10min。The reaction procedure of the second PCR amplification is: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, 30 cycles, and final extension at 72°C for 10 minutes.
  6. 根据权利要求3所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，所述步骤4中，酶切的体系及反应条件如下：The method for constructing Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 3, characterized in that in step 4, the enzyme-cut system and reaction conditions are as follows:
    pcf-Cmr同源重组片段20μL，BamHⅠ内切酶2μL，10×FastDigest Buffer 4μL，ddH 2O 4μL，总体积30μL；37℃，酶切2h，获得酶切后产物； pcf-Cmr homologous recombination fragment 20μL, BamHI endonuclease 2μL, 10×FastDigest Buffer 4μL, ddH 2 O 4μL, total volume 30μL; 37°C, digestion for 2h to obtain the digested product;
    浓缩的步骤为：向酶切后的产物中，加入1/10体积3mol/L醋酸钠溶液和2.5倍体积的无水乙醇，-20℃放置20min，12000r/min离心5min得DNA沉淀，加入70％乙醇重悬DNA沉淀，12000r/min离心5min除去乙醇，37℃风干，加入25μL ddH 2O重悬，得到pcf-Cmr浓缩重组片段； The concentration step is: add 1/10 volume of 3mol/L sodium acetate solution and 2.5 times volume of absolute ethanol to the digested product, leave at -20℃ for 20min, centrifuge at 12000r/min for 5min to obtain DNA precipitate, add 70 Resuspend DNA pellet in% ethanol, centrifuge at 12000r/min for 5min to remove ethanol, air dry at 37°C, add 25μL ddH 2 O to resuspend, to obtain pcf-Cmr concentrated recombinant fragment;
    电转化的步骤为：将(B.licheniformis)DL-1感受态细胞与pcf-Cmr浓缩重组片段于冰上以10:1比例轻轻混匀，将混合物移入2mm电转杯，2100V、5ms电转，然后立即加入1mL RM培养基，37℃，180r/min复苏4h后涂布于含氯霉素(25μg/mL)的LB固体培养基中培养。The steps of electrotransformation are as follows: (B. licheniformis) DL-1 competent cells and pcf-Cmr concentrated recombinant fragments were gently mixed on ice at a ratio of 10:1, and the mixture was transferred into a 2mm electric rotating cup, 2100V, 5ms electric rotating, Then immediately add 1mL RM medium, resuscitate at 37℃, 180r/min for 4h, then spread on LB solid medium containing chloramphenicol (25μg/mL) and cultivate.
  7. 根据权利要求3-6任一所述的所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，还包括步骤6：将步骤5制备得到的抑制菌体自溶的地衣芽孢杆菌工程菌接种到营养条件不同的培养基上，进行营养优化培养。The method for constructing B. licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to any one of claims 3 to 6, further comprising step 6: autolysis of the bacterial suppression bacteria prepared in step 5 The engineering bacteria of Bacillus licheniformis were inoculated on the medium with different nutritional conditions to carry out nutrition optimization culture.
  8. 根据权利要求7所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，接种量为单菌落/50ml培养基，培养条件为37℃，200r/min培养24h；所述培养基分别为含质量百分比为0.5％、1％、1.5％、2％、2.5％、3％甘油的LB培养基；The method for constructing Bacillus licheniformis engineering bacteria for inhibiting bacterial autolysis according to claim 7, characterized in that the inoculation amount is a single colony/50ml medium, and the culture conditions are 37°C and 200r/min for 24h; The medium is LB medium containing 0.5%, 1%, 1.5%, 2%, 2.5%, and 3% glycerin;
    含质量百分比为0.1％、0.3％、0.5％、0.8％、1％葡萄糖的LB培养基；LB medium containing 0.1%, 0.3%, 0.5%, 0.8%, 1% glucose in mass percentage;
    含质量百分比为0.1％、0.3％、0.5％、0.8％、1％葡萄糖的LBG培养基；LBG medium containing 0.1%, 0.3%, 0.5%, 0.8%, 1% glucose in mass percentage;
    含质量百分比为0.5％、1％、1.5％、2％、2.5％、3％甘油的LBP培养基；LBP medium containing 0.5%, 1%, 1.5%, 2%, 2.5% and 3% glycerol in mass percentage;
    所述LB培养基由以下质量百分比组分组成：蛋白胨1％、酵母浸粉0.5％、氯化钠1％；用5mol/LNaOH调节培养基pH为7.0；The LB medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH;
    所述LBG培养基由以下组分组成：蛋白胨1％(质量百分比)、酵母浸粉0.5％(质量百分比)、氯化钠1％(质量百分比)、甘油1％(体积百分比)，用5mol/LNaOH调节培养基pH为7.0；The LBG medium consists of the following components: peptone 1% (mass percentage), yeast extract 0.5% (mass percentage), sodium chloride 1% (mass percentage), glycerin 1% (volume percentage), using 5mol/ LNaOH adjusts the medium pH to 7.0;
    所述LBP培养基由以下质量百分比组分组成：蛋白胨1％、酵母浸粉0.5％、氯化钠1％、葡萄糖0.1％；用5mol/LNaOH调节培养基pH为7.0。The LBP medium is composed of the following mass percentage components: peptone 1%, yeast extract 0.5%, sodium chloride 1%, glucose 0.1%; the pH of the medium is adjusted to 7.0 with 5mol/LNaOH.
  9. 根据权利要求8所述的抑制菌体自溶的地衣芽孢杆菌工程菌的构建方法，其特征在于，所述培养基为含体积百分比为2.5％甘油的LBP培养基。The method for constructing Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 8, wherein the culture medium is an LBP medium containing 2.5% glycerol by volume.
  10. 根据权利要求1或2所述的抑制菌体自溶的地衣芽孢杆菌工程菌的应 用，其特征在于，应用于淀粉酶及其制剂的生产。The application of the Bacillus licheniformis engineering bacteria for inhibiting autolysis of bacterial cells according to claim 1 or 2, characterized in that it is applied to the production of amylase and its preparation.
PCT/CN2020/071158 2019-01-11 2020-01-09 Bacillus licheniformis (b.licheniformis) engineering bacterium capable of restraining autolysis of thalli and construction method and application for b.licheniformis engineering bacterium WO2020143708A1 (en)

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