CN108949784B - Application of sporulation-related gene sigmaF in enzyme production - Google Patents

Abstract

The invention relates to application of a sporulation-related gene sigmaF in enzyme production. The invention discloses the effect of a sporulation related gene sigmaF on enzyme production for the first time, and partial gene fragments of the sigmaF and Cm are subjected to insertion inactivation^rGene fusion, fusion gene sigmaF-Cm^rThe sigmaF gene is inserted into a sequence of the sigmaF gene of the bacillus clausii, so that the sigmaF gene cannot be normally expressed and inactivated, the sporulation rate of the sigmaF gene inactivated strain is reduced to 0.6 percent of that of the original strain, and the fermentation activity of the batch fermentation cellulase is improved by about 2.5 times compared with that of the original strain. The engineering strain constructed by the invention improves the fermentation enzyme activity of the cellulase, and is beneficial to genetic breeding and industrial production of bacillus enzyme preparation production strains.

Description

Application of sigmaF gene related to spore formation in enzyme production

technical field

The invention relates to the application of a spore formation-related gene sigmaF in enzyme production, and belongs to the technical field of molecular biology and the field of enzyme engineering.

Background technique

Bacillus clausii (Bacillus clausii) is a commonly used industrial strain, which can produce protease, amylase, pectinase, lipase, cellulase and other enzymes in the fermentation production process. Among them, cellulase (β-1,4-glucan-4-glucanohydrolase) is a general term for a series of enzymes that can degrade cellulose to generate cellobiose and glucose. Cellulosic material is hydrolyzed to glucose. Cellulase is widely used in food, feed, textile, papermaking and other industries. In recent years, its role in comprehensive utilization of biomass and clean production of bioenergy has attracted more attention. In order to obtain high enzyme activity, different molecular biology methods are applied to the construction of genetically engineered bacteria to further improve enzyme activity and reduce the cost of large-scale production and use of enzymes by means of diversity breeding.

Chinese patent document CN106191013A (application number 201610853941.9) discloses a method for controlling cellulase expression in Bacillus megaterium by temperature and acidity. The invention constructs a homologous recombination plasmid and transforms Bacillus megaterium ATCC14581 to obtain cellulase expression The Bacillus megaterium engineering bacteria can directionally control the expression of the Bacillus megaterium cellulose exonuclease or endonuclease by adjusting the external temperature or acidity. Chinese patent document CN102766615A (application number 201210256710.1) discloses a method for preparing cellulase by using bacillus, which comprises the steps of: taking bacillus as a production strain, and obtaining through slant culture, shaking culture and fermentation culture, wherein the fermentation culture The culture medium includes 0.5-2% of starch, 1-5% of bean cake flour, 8-12% of bean cake flour hydrolyzate, 0.3-0.8% of bran, 1-2% of inorganic salt, 0-0.3% of defoamer, The method obtains cellulase with high activity under neutral pH and alkaline conditions, and is obviously superior to acid cellulase in the application of water-washing enzyme. However, the relationship between spore formation-related genes and cellulase production has not been clearly reported. Some studies have found that spore formation-related genes not only affect spore formation, but also regulate the structure, growth, and metabolite production of strains.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the present invention provides the application of the spore formation related gene sigmaF in enzyme production, selectively knocking out the spore formation related gene sigmaF, and constructing spore deletion bacteria, which can effectively improve the enzymatic activity of strain cellulase.

Summary of the invention:

The invention selectively knocks out sigmaF related gene of spore formation, overlaps and connects Bacillus clausii spore formation related gene sigmaF and Cm ^r fragment, transforms Bacillus clausii after enzyme cutting and concentration, so that sigmaF related to spore formation gene sigmaF Inactivation, the construction of Bacillus clausii spore deletion bacteria, the cell metabolism changes, the cellulase enzyme activity of the strain is significantly improved, and its enzyme activity is about 3.5 times that of the starting strain.

Detailed description of the invention:

The technical scheme of the present invention is as follows:

Application of sigmaF-related gene sigmaF in enzyme production, the nucleotide sequence of said gene sigmaF is shown in SEQ ID NO.1.

Preferably according to the present invention, the enzyme producing is cellulase producing, and the enzyme activity of the cellulase is 3-4 times that of the starting strain.

Preferably according to the present invention, the application of the spore formation-related gene sigmaF in enzyme production, the steps are as follows:

(1) extracting the genomic DNA of Bacillus clausii, and using the genomic DNA as a template to carry out PCR amplification to obtain a gene sigmaF related to spore formation of Bacillus clausii, the nucleotide sequence of the gene sigmaF is such as SEQ ID NO.1 shown;

(2) Using pHT01 plasmid as a template, a Cm ^r fragment is obtained through PCR amplification, and the nucleotide sequence of the Cm ^r fragment is shown in SEQID NO.2;

(3) using overlapping PCR technology to fuse the gene sigmaF obtained in step (1) with the Cm ^r fragment obtained in step (2) to obtain a fusion gene sigmaF-Cm ^r , the nucleotides of the fusion gene sigmaF-Cm ^r The sequence is shown in SEQ ID NO.3;

(4) The fusion gene sigmaF-Cm ^r obtained in step (3) is digested with enzyme, concentrated, transformed into competent cells of Bacillus clausii, and positive recombinant bacteria are obtained through screening, which can be used for enzyme production.

Preferably according to the present invention, in the step (1), the nucleotide sequence of the primer amplified by PCR is as follows, and the underline is the BamHI restriction site:

sigmaF-F: CGCGGATCC ATGAGTGCAGAGGTGAAAAACAGCGG,

sigmaF-R: GTGAGCAAAAGGCCAGCAAAATGAGTGGTCGGCAAT;

The PCR amplification system is as follows, with a total volume of 50 μL:

2×HiFi-PCR Master 25 μL, upstream primer sigmaF-F 2.5 μL, downstream primer sigmaF-R 2.5 μL, Bacillus clausii genomic DNA 2.5 μL, ddH ₂ O 17.5 μL;

The PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min 15 s, a total of 30 cycles; continued extension at 72 °C for 10 min.

Preferably according to the present invention, in the step (2), the nucleotide sequence of the primer amplified by PCR is as follows, and the underline is the BamHI restriction site:

Cm ^r -F: CTTGTAGGAACGCTTTTTGCTGGCCTTTTGCTC,

Cm ^r -R: CGCGGATCC TAGTGACTGGCGATGCTGTCGGAATGG;

The PCR amplification system is as follows, with a total volume of 50 μL:

2×HiFi-PCR Master 25 μL, upstream primer Cm ^r -F 2.5 μL, downstream primer Cm ^r -R 2.5 μL, pHT01 plasmid 2.5 μL, ddH ₂ O 17.5 μL;

The PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min 45 s, a total of 30 cycles; continued extension at 72 °C for 10 min.

Preferably according to the present invention, in the step (3), the nucleotide sequence of the amplification primer of the overlapping PCR is as follows, and the underline is the BamHI restriction site:

sigmaF-F: CGCGGATCC ATGAGTGCAGAGGTGAAAAACAGCGG,

Cm ^r -R: CGCGGATCC TAGTGACTGGCGATGCTGTCGGAATGG;

The first round of overlapping PCR amplification system is as follows, with a total volume of 25 μL:

2×HiFi-PCR Master 12.5μL, gene sigmaF 2μL, Cm ^r fragment 2μL, ddH ₂ O 8.5μL;

The first round of overlapping PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min for 40 s, 5 cycles; extension at 72 °C for 10 min.

The second round of overlapping PCR amplification system is supplemented with the following reagents on the basis of the first round of PCR amplification system:

2×HiFi-PCR Master 12.5μL, upstream primer sigmaF-F 1μL, downstream primer Cm ^r -R 1μL, ddH ₂ O 10.5μL;

The second round of overlapping PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 4 min, a total of 30 cycles; extension at 72 °C for 10 min.

Preferably according to the present invention, in the step (4), the reaction system of the enzyme cleavage is as follows, with a total volume of 40 μL:

Overlapping PCR product 20μL, 10×KBuffer 4μL, BamHI endonuclease 2μL, ddH ₂ O 14μL;

The digestion conditions were: 37°C, 1.5h.

Preferably according to the present invention, in the step (4), the concentration of the concentrated fusion gene sigmaF-Cm ^r is 300-500 ng/μL.

Preferably according to the present invention, in the step (4), the preparation method of the competent cells of Bacillus reuteri is as follows:

Pick a fresh single colony of Bacillus clausii, cultivate at 37°C, 220 r/min until the bacterial concentration OD ₆₀₀ = 0.9-1.0, cool on ice, centrifuge after cooling, and then wash the bacteria with pre-cooled electrotransfer buffer The cells were resuspended in electroporation buffer for 3 to 5 times, and then dispensed into pre-cooled sterile EP tubes to obtain Bacillus clausii competent cells.

Further preferably, the components of the electrotransfer buffer are as follows:

The mass percentage is 9.1% sorbitol, the mass percentage is 9.1% mannitol, the volume percentage is 10% glycerol, and the balance is water.

Preferably according to the present invention, in the step (4), the competent cells of Bacillus clausii are transformed, and the steps are as follows:

The fusion gene sigmaF-Cm ^r after digestion and concentration was electro-transformed into Bacillus clausii competent cells, the voltage of electro-transformation was 1500-1800V, and the electric shock time was 4-5ms, and then the cells were recovered and cultured in liquid at 37°C. Incubate in the medium for 3 to 4 hours.

Further preferably, the components of the liquid resuscitation medium are as follows, all in percentage by weight:

1% peptone, 0.5% yeast extract, 1% sodium chloride, 9% sorbitol, 7% mannitol, pH=7.0-7.4.

Preferably according to the present invention, in the described step (4), the screening step is as follows:

The transformed Bacillus clausii competent cells were coated on an LB plate containing chloramphenicol, cultured at 37°C for 12-24 hours, and then the transformants were identified by colony PCR, and positive recombinant bacteria were obtained by screening.

Further preferably, the LB plate containing chloramphenicol is an LB solid medium with a chloramphenicol concentration of 25 μmol/mL.

Beneficial effects:

1. The present invention discloses for the first time the role of sigmaF, a gene related to spore formation, in enzyme production. By means of insertional inactivation, part of the sigmaF gene fragment is fused with the ^Cmr gene, and the fusion gene sigmaF- ^Cmr is inserted into Bacillus clausii. In the sigmaF gene sequence, the sigmaF gene cannot be expressed normally and inactivated, the spore formation rate of the sigmaF gene inactivated strain is reduced to 0.6% of the starting strain, and the fermentation activity of batch fermentation cellulase is about 2.5 times higher than that of the starting strain.

2. The engineering strain constructed by the present invention improves the fermentative enzyme activity of cellulase, which is beneficial to the genetic breeding and industrial production of Bacillus-like enzyme preparation production strains.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of Bacillus clausii sigmaF gene fragment of the present invention;

In the figure, lane M is the DNA molecular weight marker (DNA marker), and lanes 1-2 are the sigmaF gene fragment bands with a size of 579 bp.

Fig. 2 is the agarose gel electrophoresis figure of ^Cmr gene fragment of the present invention;

Swimming lane M in the figure is a DNA molecular weight marker (DNA marker), and lanes 1-2 are Cm ^r gene fragment bands with a size of 1260 bp.

Fig. 3 is the agarose gel electrophoresis figure that the Bacillus clausii sigmaF gene inactivation transformant of the present invention is verified;

In the figure, lane M is a DNA molecular weight marker (DNA marker), and lanes 1-2 are transformant bands with a size of 1803 bp.

Detailed ways

The technical solutions of the present invention will be further described below with reference to the embodiments, but the protection scope of the present invention is not limited thereto.

Terms used in this method, unless otherwise specified, generally have the meanings commonly understood by one of ordinary skill in the art.

In the following examples, various procedures and methods that are not described in detail and are conventional methods well known in the art. The sources, trade names of the reagents used, and those necessary to list their components are indicated when they appear for the first time, and the same reagents used thereafter will be the same as those indicated for the first time unless otherwise specified.

Source of biological material:

Bacillus clausii (Bacillus clausii) in the embodiment was purchased from Beijing Beina Chuanglian Institute of Biotechnology, strain number: BNCC160124, which is a common commercially available strain;

Plasmid pHT01 was purchased from Hangzhou Baosai Biological Co., Ltd.

The components of LB solid medium are as follows, all in mass percentage:

1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar, pH=7.0～7.4

The components of LB medium are as follows, all in mass percentage:

Peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, pH=7.0～7.4

Example 1: Construction of target fragments

(i) Extracting the genomic DNA of Bacillus clausii (according to the instructions of the Ezup column bacterial genomic DNA extraction kit).

The primers were designed according to the nucleotide sequence of the sigmaF gene, the BamHI restriction site was introduced into the upstream primer, and the 21 bases at the 5' end of the Cm ^r fragment were added to the downstream primer. synthesis. The sigmaF gene was amplified by using the 2×HiFi-PCR Master polymerase from Bao Bioengineering Co., Ltd. with the extracted genomic DNA of Bacillus clausii as a template.

Wherein, the primer nucleotide sequence is as follows, wherein the underline is the BamHI restriction site.

sigmaF-F: CGCGGATCC ATGAGTGCAGAGGTGAAAAACAGCGGG

sigmaF-R: GTGAGCAAAAGGCCAGCAAAATGAGTGGTCGGCAAT;

The PCR amplification system is as follows, with a total volume of 50 μL:

2×HiFi-PCR Master 25 μL, upstream primer sigmaF-F 2.5 μL, downstream primer sigmaF-R 2.5 μL, Bacillus clausii genomic DNA 2.5 μL, ddH ₂ O 17.5 μL;

The PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, extension at 72 °C for 1 min 15 s, a total of 30 cycles; continued extension at 72 °C for 10 min.

The PCR product was checked by agarose gel electrophoresis. The results are shown in Figure 1. The length of the target gene sigmaF is 579bp (SEQ ID NO. 1). Recover, and store the obtained DNA solution at -20°C for later use.

(ii) Acquisition of Cm ^r Fragments

Using the pHT01 plasmid as a template, the Cm ^r fragment was obtained by PCR amplification.

Among them, the nucleotide sequence of the PCR amplification primer is as follows, the BamHI restriction site is introduced into the downstream primer, and the upstream primer adds 15 bases at the 3' end of the gene sigmaF, wherein the underline is the BamHI restriction site:

Cm ^r -F: ATTGCCGACCACTCATTTTGCTGGCCTTTTGCTCAC

Cm ^r -R: CGCGGATCC TAGTGACTGGCGATGCTGTCGGAATGG;

The PCR amplification system is as follows, with a total volume of 50 μL:

2×HiFi-PCR Master 25 μL, upstream primer Cm ^r -F 2.5 μL, downstream primer Cm ^r -R 2.5 μL, pHT01 plasmid 2.5 μL, ddH ₂ O 17.5 μL;

The PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min 45 s, a total of 30 cycles; continued extension at 72 °C for 10 min.

The PCR product was checked by agarose gel electrophoresis. The results are shown in Figure 2. The length of the Cm ^r fragment is 1260 bp (SEQ ID NO. 2). The amplified PCR product Cm ^r was gelatinized using the SanPrep column DNA gel recovery kit. Recover, and store the obtained DNA solution at -20°C for later use.

(iii) The gene sigmaF obtained in step (i) and the Cm ^r fragment obtained in step (ii) are fused by overlapping PCR to obtain the fusion gene sigmaF-Cm ^r .

Among them, the nucleotide sequences of the primers for overlapping PCR are as follows, and the BamHI restriction site is underlined:

sigmaF-F: CGCGGATCC ATGAGTGCAGAGGTGAAAAACAGCGGG

Cm ^r -R: CGCGGATCC TAGTGACTGGCGATGCTGTCGGAATGG

The first round of overlapping PCR amplification system is as follows, with a total volume of 25 μL:

2×HiFi-PCR Master 12.5μL, gene sigmaF 2μL, Cm ^r fragment 2μL, ddH ₂ O 8.5μL;

The first round of overlapping PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min for 40 s, 5 cycles; extension at 72 °C for 10 min.

The second round of overlapping PCR amplification system is supplemented with the following reagents on the basis of the first round of PCR amplification system:

2×HiFi-PCR Master 12.5μL, upstream primer sigmaF-F 1μL, downstream primer Cm ^r -R 1μL, ddH ₂ O 10.5μL;

The second round of overlapping PCR amplification procedure is as follows:

Pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 4 min, a total of 30 cycles; extension at 72 °C for 10 min.

Similarly, the PCR product was checked by agarose gel electrophoresis, and the length of the target gene sigmaF-Cm ^r was detected to be 1803 bp (SEQ ID NO. 3). The amplified PCR product sigmaF-Cm ^r was recovered using the SanPrep column DNA gel recovery reagent. The cassette was used for gel recovery, and the resulting DNA solution was stored at -20°C for later use.

Example 2: Preparation of Bacillus clausii competent cells

(i) Picking a single colony of Bacillus clausii on the surface of fresh LB solid medium, inoculating it in 10 mL LB medium, culturing overnight at 37° C., 220 r/min;

(ii) Transfer 2.0 mL of bacterial liquid to 50 mL of GM medium, at 37° C., 220 r/min, and cultivate for 4 h until OD ₆₀₀ =1.0;

(iii) Transfer the bacterial liquid to a 50 mL centrifuge tube, and take an ice bath for 10 minutes to stop the growth of the bacterial cells;

(IV) Centrifuge at 4°C and 5000 r/min for 5 min after ice bath, and collect bacterial cells;

(V) The cells after centrifugation were washed 3 times with pre-cooled electrotransfer buffer (ETM);

(Ⅵ) After washing, resuspend the cells with 1000 μL electroporation buffer;

(VII) Dispense the prepared competent cells into 100 μL/tube and store at -80°C for later use.

Among them, GM medium: LB medium + 0.5mol/L sorbitol

Electrotransfer buffer (ETM): 9.1% sorbitol by mass, 9.1% mannitol by mass, 10% glycerol by volume, and balance water.

Example 3: Electrotransformation of Bacillus clausii competent cells with fusion gene sigmaF-Cm ^r

(i) digest the fusion gene sigmaF- ^Cmr with the restriction enzyme BamHI;

The digestion system (40 μL) is as follows:

10×K Buffer 4 μL, BamHI endonuclease 2 μL, overlapping PCR product 20 μL, ddH ₂ O 14 μL;

The digestion conditions were: 37°C, 1.5h;

(ii) Concentrate and purify the digested product

(1) Take the enzyme digestion product, add 1/10 volume of 3mol/L sodium acetate and 2.5 times volume of absolute ethanol, and place it in a -20°C refrigerator for 20min;

(2) 12000r/min, centrifuge for 5min to get sediment;

(3) 300 μL of 75% (volume percent) absolute ethanol solution was added to the precipitation, and the precipitation was resuspended;

(4) 12000r/min, centrifuged for 5min, air-dried at 37°C for 30min to remove ethanol;

(5) Add 20 μL of ddH ₂ O to resuspend the DNA, and store at -20°C for later use.

(iii) Electroconversion

First, the concentration of the concentrated fusion gene sigmaF-Cm ^r was measured with a nucleic acid ultra-micro spectrophotometer. After the concentration reached 500 ng/μL, the competent cells and the concentrated product were added to the electroporation cup, and the electroconversion was carried out after ice bathing for 5 minutes. , electroporated for 5 ms, the cells after electroporation were recovered and cultured in liquid recovery medium RM at 37 °C for 3 to 4 h, centrifuged at 4000 r/min for 5 min, 100 μL supernatant was suspended, and 25 μmol/mL chloramphenicol LB plate was coated. Incubate at 37°C for 12-24 hours, and screen transformants with chloramphenicol resistance;

The liquid resuscitation medium RM components are as follows, all in percentage by weight:

1% peptone, 0.5% yeast extract, 1% sodium chloride, 9% sorbitol, 7% mannitol, pH=7.0.

Example 4: Cultivation and identification of positive recombinant bacteria

Pick the above transformants with chloramphenicol resistance, inoculate them into liquid LB medium containing chloramphenicol resistance, cultivate at 37°C for 12 hours, freeze and thaw the bacterial liquid repeatedly for 3 to 4 times, and use the bacterial liquid as a template. sigmaF-F and Cm ^r -R were used as primers for colony PCR amplification, and the amplified products were verified by agarose gel electrophoresis.

The PCR primer sequences are as follows, and the underline is the BamHI restriction site:

sigmaF-F: CGCGGATCC CCTTAAAAGCGGAGCCAAAAC

Cmr-R: CGCGGATCC TAGTGACTGGCGATGCTG

The PCR amplification system is 25 μL:

2×HiFi-PCR Master 12.5μL, bacterial solution 2μL, upstream primer sigmaF-F 1μL, downstream primer Cm ^r -R 1μL, ddH ₂ O 8.5μL;

The PCR amplification procedure is as follows:

Pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 4min25s, 30 cycles; extension at 72°C for 10min, PCR products were checked by agarose gel electrophoresis, and the verification results are shown in Figure 3.

Example 5: Determination of cellulase enzymatic activity

(1) The strains identified as positive recombinant bacteria in Example 4 were repeatedly activated twice in LB solid medium containing 25 μmol/mL chloramphenicol, and cultured at 37°C for 20 h; single colonies were placed in 10 mL liquid LB medium, Culture at 37°C, 200r/min for 12h, inoculate 3% of the inoculum in 100mL liquid LB medium containing 25μmol/mL chloramphenicol, culture at 37°C, 200r/min, take the fermentation broth of different incubation times, remove by centrifugation Bacteria get the enzyme solution to be tested;

(2) The specific method for the determination of cellulase enzymatic activity is:

i. Plot the standard curve

According to the amount specified in Table 1, draw the standard glucose solution, buffer solution and DNS reagent into each standard tube, each group of 3 parallel samples, and mix with a vortex mixer; put the standard tube in a boiling water bath at the same time, After 10 min of reaction, it was rapidly cooled to room temperature, the volume was adjusted to 25 mL, and the mixture was mixed; absorbance was measured at 540 nm, and a standard curve was drawn.

Table 1 Glucose standard curve

ⅱ. Sample determination

Take four test tubes, add 2 mL of CMC-Na (sodium carboxymethyl cellulose) solution prepared by buffer solution to them respectively; add 0.5 mL of the diluted enzyme solution to be tested into the three sample tubes, and mix with a vortex mixer. After incubating the four test tubes at 50°C for 30 min at the same time, add 3 mL of DNS reagent to each tube, add 0.5 mL of diluted enzyme solution to be tested to the blank tube, and shake well. Put the four tubes into a boiling water bath at the same time, heat them for 10 min, then quickly cool to room temperature, and set the volume to 25 mL; the blank tube is set to zero for the control group, and the absorbance value is measured under a 540 nm spectrophotometer.

Definition of cellulase activity unit: 1mL of liquid enzyme, at pH 4.8 and 50℃, hydrolyzes sodium carboxymethylcellulose substrate to produce 1umol reducing sugar per minute, which is one enzyme activity unit, expressed in U/mL.

The unit of amylase activity was converted to U/g by measuring the dry weight of the bacterial cells.

It was determined that the spore formation rate of the Bacillus clausii sigmaF deletion strain was 0.6% of that of the starting strain, and the cellulase enzyme activity of the Bacillus clausii sigmaF deletion strain reached 23.2×10 ³ U/g after 96 hours of fermentation, which is about the starting strain. 3.5 times. The insertion inactivation of sigmaF, a gene related to sporulation, can effectively improve the enzymatic activity of Bacillus clausii cellulase.

SEQUENCE LISTING

<110> Qilu University of Technology

<120> Application of sigmaF gene related to spore formation in enzyme production

<160> 3

<170> PatentIn version 3.5

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caaattgccg accactcatt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 600

cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 660

cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcatg 720

cttaagttat tggtatgact ggttttaagc gcaaaaaaag ttgctttttc gtacctatta 780

atgtatcgtt ttagaaaacc gactgtaaaa agtacagtcg gcattatctc atattataaa 840

agccagtcat taggcctatc tgacaattcc tgaatagagt tcataaacaa tcctgcatga 900

taaccatcac aaacagaatg atgtacctgt aaagatagcg gtaaatatat tgaattacct 960

ttattaatga attttcctgc tgtaataatg ggtagaaggt aattactatt attattgata 1020

tttaagttaa acccagtaaa tgaagtccat ggaataatag aaagagaaaa agcattttca 1080

ggtataggtg ttttgggaaa caatttcccc gaaccattat atttctctac atcagaaagg 1140

tataaatcat aaaactcttt gaagtcattc tttacaggag tccaaatacc agagaatgtt 1200

ttagatacac catcaaaaat tgtataaagt ggctctaact tatcccaata acctaactct 1260

ccgtcgctat tgtaaccagt tctaaaagct gtatttgagt ttatcaccct tgtcactaag 1320

aaaataaatg cagggtaaaa tttatatcct tcttgtttta tgtttcggta taaaacacta 1380

atatcaattt ctgtggttat actaaaagtc gtttgttggt tcaaataatg attaaatatc 1440

tcttttctct tccaattgtc taaatcaatt ttattaaagt tcatttgata tgcctcctaa 1500

atttttatct aaagtgaatt taggaggctt acttgtctgc tttcttcatt agaatcaatc 1560

ctttttttaaa agtcaatatt actgtaacat aaatatatat tttaaaaata tcccacttta 1620

tccaattttc gtttgttgaa ctaatgggtg ctttagttga agaataaaag accacattaa 1680

aaaatgtggt cttttgtgtt tttttaaagg atttgagcgt agcgaaaaat ccttttcttt 1740

cttatcttga taataagggt aactattgcc gatcgtccat tccgacagca tcgccagtca 1800

cta 1803

Claims (8)

1. The application of the sporulation related gene sigmaF in enzyme production is characterized by comprising the following steps:

(1) extracting genome DNA of the Bacillus clausii, and carrying out PCR amplification by taking the genome DNA as a template to obtain a Bacillus clausii spore formation related gene sigmaF, wherein the nucleotide sequence of the gene sigmaF is shown as SEQ ID NO. 1;

(2) using pHT01 plasmid as template, obtaining Cm by PCR amplification^rFragment of Cm^rThe nucleotide sequence of the fragment is shown as SEQ ID NO. 2;

(3) adopting an overlapping PCR technology to combine the gene sigmaF obtained in the step (1) with the Cm obtained in the step (2)^rFusing the fragments to obtain a fused gene sigmaF-Cm^rThe fusion gene sigmaF-Cm^rThe nucleotide sequence of (A) is shown as SEQ ID NO. 3;

(4) the fusion gene sigmaF-Cm prepared in the step (3) is used for^rAfter enzyme digestion, concentrating, transforming the competent cells of the Bacillus clausii, and screening to obtain positive recombinant bacteria which can be applied to enzyme production; the concentrated fusion gene sigmaF-Cm^rThe concentration of (a) is 300-500 ng/muL;

the enzyme production is cellulase, and the enzyme activity of the cellulase is 3-4 times of that of the original strain.

2. The use according to claim 1, wherein in step (1), the nucleotide sequence of the primers amplified by PCR is as follows, and the BamHI cleavage sites are underlined:

sigmaF-F：CGCGGATCCATGAGTGCAGAGGTGAAAAACAGCGG，

sigmaF-R：GTGAGCAAAAGGCCAGCAAAATGAGTGGTCGGCAAT；

the PCR amplification system was as follows, with a total volume of 50. mu.L:

2 XHiFi-PCR Master 25. mu.L, upstream primer sigmaF-F2.5. mu.L, downstream primer sigmaF-R2.5. mu.L, Bacillus clausii genomic DNA 2.5. mu.L, ddH₂O 17.5μL；

The PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 1min for 15s for 30 cycles; extension was continued for 10min at 72 ℃.

3. The use according to claim 1, wherein in step (2), the nucleotide sequence of the primers amplified by PCR is as follows, and the BamHI cleavage sites are underlined:

Cm^r-F：CTTGTAGGAACGCTTTTTGCTGGCCTTTTGCTC，

Cm^r-R：CGCGGATCCTAGTGACTGGCGATGCTGTCGGAATGG；

the PCR amplification system was as follows, with a total volume of 50. mu.L:

2 × HiFi-PCR Master 25 μ L, upstream primer Cm^r2.5. mu.L of-F, the downstream primer Cm^r2.5. mu.L of R, 2.5. mu.L of pHT01 plasmid, ddH₂O 17.5μL；

The PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 2min for 45s for 30 cycles; extension was continued for 10min at 72 ℃.

4. The use according to claim 1, wherein in step (3), the nucleotide sequences of the amplification primers of the overlapping PCR are as follows, with the BamHI cleavage sites underlined:

sigmaF-F：CGCGGATCCATGAGTGCAGAGGTGAAAAACAGCGG，

Cm^r-R：CGCGGATCCTAGTGACTGGCGATGCTGTCGGAATGG；

the first round of overlap PCR amplification system was as follows, with a total volume of 25. mu.L:

2 × HiFi-PCR Master 12.5 μ L, Gene sigmaF 2 μ L, Cm^rFragment 2. mu.L, ddH₂O 8.5μL；

The first round of overlap PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min for 40s, 5 cycles; continuing to extend for 10min at 72 ℃;

the second round of overlapping PCR amplification system is that the following reagents are added on the basis of the first round of PCR amplification system:

2 XHiFi-PCR Master 12.5. mu.L, upstream primer sigmaF-F1. mu.L, downstream primer Cm^r-R 1μL，ddH₂O10.5μL；

The second round of overlap PCR amplification procedure was as follows:

pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 4min for 30 cycles; extension was continued for 10min at 72 ℃.

5. The use of claim 1, wherein in step (4), the reaction system of the enzyme digestion is as follows, and the total volume is 40 μ L:

overlapping PCR products 20. mu.L, 10 XK Buffer 4. mu.L, BamHI endonuclease 2. mu.L, ddH₂O 14μL；

The enzyme digestion conditions are as follows: 37 ℃ for 1.5 h.

6. The use of claim 1, wherein in step (4), said bacillus thioninus competent cells are prepared by:

selecting fresh Bacillus clausii single colony, culturing at 37 deg.C and 220r/min to thallus concentration OD₆₀₀Putting the bacillus clausii strain;

wherein the components of the electrotransfer buffer solution are as follows:

sorbitol with the mass percent of 9.1 percent, mannitol with the mass percent of 9.1 percent, glycerol with the volume percent of 10 percent and the balance of water.

7. The use of claim 1, wherein in step (4), said transformed c.

The fused gene sigmaF-Cm after enzyme digestion and concentration is used^rElectrically transforming the bacillus clausii competent cells, wherein the voltage of the electric transformation is 1500-1800V, the electric shock time is 4-5 ms, and then culturing in a liquid recovery culture medium at 37 ℃ for 3-4 h to obtain the bacillus clausii competent cells;

the liquid recovery culture medium comprises the following components in percentage by weight:

peptone 1%, yeast extract powder 0.5%, sodium chloride 1%, sorbitol 9%, mannitol 7%, and pH 7.0-7.4.

8. The use of claim 1, wherein in step (4), the screening step is as follows:

coating the transformed bacillus clausii competent cells on an LB (Luo-Beauveria-biosciences) flat plate containing chloramphenicol, culturing at 37 ℃ for 12-24 h, then carrying out transformant identification through colony PCR (polymerase chain reaction), and screening to obtain positive recombinant bacteria;

wherein the LB plate containing chloramphenicol is LB solid medium with chloramphenicol concentration of 25 mu mol/mL.

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