WO2024239579A1 - 一种共表达芽孢杆菌来源增强因子提高重组蛋白在枯草芽孢杆菌中表达量的方法 - Google Patents

一种共表达芽孢杆菌来源增强因子提高重组蛋白在枯草芽孢杆菌中表达量的方法 Download PDF

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WO2024239579A1
WO2024239579A1 PCT/CN2023/135786 CN2023135786W WO2024239579A1 WO 2024239579 A1 WO2024239579 A1 WO 2024239579A1 CN 2023135786 W CN2023135786 W CN 2023135786W WO 2024239579 A1 WO2024239579 A1 WO 2024239579A1
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pona
bacillus subtilis
protein
recombinant
class
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French (fr)
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张康
吴敬
朱昫飏
罗慧
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Jiangnan University
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Jiangnan University
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • C12N15/09—Recombinant DNA-technology
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    • 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
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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    • C12N9/90—Isomerases (5.)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
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    • C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
    • C12Y504/99011—Isomaltulose synthase (5.4.99.11)

Definitions

  • the invention relates to a method for co-expressing a bacillus-derived enhancing factor to increase the expression amount of a recombinant protein in bacillus subtilis, and belongs to the technical field of enzyme engineering.
  • Bacillus subtilis is a Gram-positive strain of the genus Bacillus Cohn. B. subtilis is listed as a certified strain in the US FDA's GRAS (Generally Recognized as Safe) list because it does not produce toxins and heat-sensitizing proteins. Its easy isolation and culture, high-density fermentation and strong protein secretion make Bacillus subtilis an important industrial strain for the production of a variety of exogenous recombinant proteins and metabolites. Its excellent traits have a high identification value. At the same time, Bacillus subtilis has a clear genetic background and mature molecular biological gene editing methods, which is conducive to genetic modification to enhance the expression ability of Bacillus subtilis as a host for recombinant protein production.
  • the present invention provides a method for increasing the expression of recombinant proteins in Bacillus subtilis, wherein the method is to overexpress Bacillus class A penicillin-binding protein PonA, a truncated form of class A penicillin-binding protein PonA, an ABC pathway transporter OppA or a signal peptide peptidase SppA while expressing the recombinant protein.
  • the PonA is derived from Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium, Class A penicillin-binding proteins from Bacillus megaterium or Geobacillus stearothermophilus.
  • the PonA protein derived from Bacillus subtilis is PonA(Bs), having the amino acid sequence shown in SEQ ID NO.1, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.1.
  • the PonA protein derived from Bacillus licheniformis is PonA(Bl), having the amino acid sequence shown in SEQ ID NO.2, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.2.
  • the PonA protein derived from Bacillus amyloliquefaciens is PonA(Ba), having the amino acid sequence shown in SEQ ID NO.3, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.3.
  • the PonA protein derived from Bacillus megaterium is PonA(Bm), having the amino acid sequence shown in SEQ ID NO.4, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.4.
  • the PonA protein derived from Geobacillus stearothermophilus is PonA(Gs), having the amino acid sequence shown in SEQ ID NO.5, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.5.
  • the PonA truncate is a class A penicillin-binding protein truncate derived from Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus megaterium or Geobacillus stearothermophilus.
  • the truncation of PonA protein derived from Bacillus subtilis is DcPonA(Bs), having the amino acid sequence shown in SEQ ID NO.6, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.6.
  • the PonA protein truncation derived from Bacillus licheniformis is DcPonA(Bl), having the amino acid sequence shown in SEQ ID NO.7, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.7.
  • the PonA protein truncate from Bacillus amyloliquefaciens is DcPonA (Ba), having the amino acid sequence shown in SEQ ID NO.8, or having an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity with SEQ ID NO.8. sequence.
  • the PonA protein truncation derived from Bacillus megaterium is DcPonA(Bm), having the amino acid sequence shown in SEQ ID NO.9, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.9.
  • the truncation of PonA protein derived from Geobacillus stearothermophilus is DcPonA(Gs), having the amino acid sequence shown in SEQ ID NO.10, or having an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.10.
  • the ABC pathway transporter OppA derived from Bacillus subtilis has the amino acid sequence shown in SEQ ID NO.21; the signal peptide peptidase SppA derived from Bacillus subtilis has the amino acid sequence shown in SEQ ID NO.22.
  • the recombinant protein includes but is not limited to ultra-high temperature amylase, medium temperature amylase or sucrose isomerase.
  • the ultra-high temperature amylase has an amino acid sequence as shown in SEQ ID NO.23, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.23.
  • the mesophilic amylase has an amino acid sequence as shown in SEQ ID NO.24, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.24.
  • the sucrose isomerase has the amino acid sequence shown in SEQ ID NO.25, or has an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identical to SEQ ID NO.25.
  • pUB110 is used as an expression vector to express ultrahigh temperature amylase, medium temperature amylase or sucrose isomerase.
  • pAD123 is used as an expression vector to express the class A penicillin-binding protein PonA, the class A penicillin-binding protein PonA truncated form, the ABC pathway transporter OppA or the signal peptide peptidase SppA.
  • the method is to express the recombinant protein and the PonA, OppA and SppA in the same host.
  • the host includes but is not limited to Bacillus subtilis SCK6.
  • the recombinant protein is expressed by a constitutive promoter P amyQ' .
  • the nucleotide sequence is shown in SEQ ID NO.31.
  • the class A penicillin-binding protein PonA, the class A penicillin-binding protein PonA truncate, the ABC pathway transporter OppA or the signal peptide peptidase SppA is expressed via the promoter P glv or the promoter PHpaII .
  • nucleotide sequence of the promoter PHpaII is shown as SEQ ID NO.32.
  • nucleotide sequence of the promoter P glv is shown as SEQ ID NO.33.
  • the present invention also provides a recombinant Bacillus subtilis bacterium with increased expression of exogenous proteins.
  • the recombinant Bacillus subtilis bacterium co-expresses the class A penicillin binding protein PonA or DcPonA derived from Bacillus, the ABC pathway transport protein OppA derived from Bacillus subtilis, or the signal peptide peptidase SppA derived from Bacillus subtilis while expressing the exogenous proteins.
  • the exogenous protein refers to an exogenous protein relative to the Bacillus subtilis that expresses and secretes the protein.
  • the exogenous protein can be a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, a protein derived from a virus, or even a protein whose amino acid sequence is artificially designed.
  • the heterologous protein can be a protein derived from humans.
  • the heterologous protein can be a monomeric protein or a multimeric protein.
  • Multimeric protein refers to a protein that can exist as a multimer containing two or more subunits.
  • each subunit can be connected by a covalent bond such as a disulfide bond; can be connected by a non-covalent bond such as a hydrogen bond and a hydrophobic interaction, or can be connected by a combination thereof.
  • a covalent bond such as a disulfide bond
  • a non-covalent bond such as a hydrogen bond and a hydrophobic interaction
  • one or more intermolecular disulfide bonds are contained in the multimer.
  • the multimer can be a homopolymer containing a single type of subunit, or a heteropolymer containing two or more types of subunits. It should be noted that, when the multimeric protein is a heteromultimer, at least one of the subunits constituting the multimer may be a heterologous protein.
  • the heterologous protein may be a naturally secreted protein or a naturally non-secreted protein, preferably a naturally secreted protein.
  • the heterologous protein may be a naturally Tat-dependent secreted protein or a naturally Sec-dependent secreted protein.
  • pUB110 is used as an expression vector to express ultrahigh temperature amylase, medium temperature amylase or sucrose isomerase.
  • pAD123 is used as an expression vector to express the class A penicillin-binding protein PonA, the class A penicillin-binding protein PonA truncated form, the ABC pathway transporter OppA or the signal peptide peptidase SppA.
  • the present invention also provides a method for constructing the recombinant bacillus subtilis, which comprises introducing a gene encoding a recombinant protein and a gene encoding an enhancement factor into the bacillus subtilis.
  • the gene encoding the enhancing factor includes but is not limited to the gene encoding the class A penicillin binding protein PonA, the class A penicillin binding protein PonA truncation, the ABC pathway transporter OppA or the signal peptide peptidase SppA.
  • the gene encoding the class A penicillin-binding protein PonA has a nucleotide sequence shown in any one of SEQ ID NO.11 to SEQ ID NO.15.
  • the gene encoding the class A penicillin-binding protein PonA truncate has a nucleotide sequence shown in any one of SEQ ID NO.16 to SEQ ID NO.20.
  • the gene encoding the recombinant protein includes but is not limited to a gene encoding an ultrahigh temperature amylase, a medium temperature amylase or a sucrose isomerase.
  • the gene encoding the ultra-high temperature amylase has a nucleotide sequence shown in SEQ ID NO.28.
  • the encoding gene of the mesophilic amylase has a nucleotide sequence shown in SEQ ID NO.29.
  • the gene encoding the sucrose isomerase has a nucleotide sequence shown in SEQ ID NO.30.
  • the present invention also provides the use of the recombinant Bacillus subtilis in producing ultrahigh temperature amylase, medium temperature amylase or sucrose isomerase.
  • the application is to culture the recombinant bacteria in a fermentation medium for a period of time and collect the ultrahigh temperature amylase, medium temperature amylase or sucrose isomerase.
  • the use is fermentation at 30-40°C, or 30-37°C, or 35-40°C, or 37-40°C, or 35-37°C.
  • the fermentation is fermented for at least 24 hours, or at least 36 hours, or at least 42 hours, or at least 48 hours, or at least 60 hours.
  • the fermentation medium comprises yeast powder, soy peptone, ammonium citrate and metal ions.
  • the fermentation medium contains 9.0 g/L yeast powder, 18 g/L soy peptone, 1.0 g /L ammonium citrate, 14.66 g/ L K2HPO4 ⁇ 3H2O, 2.68 g/L (NH4)2SO4 , 2.0 g / L Na2SO3 , 3.49 g/L NaH2PO4, 0.49 g/L MgSO4, and 0.3% v/v TES metal ion solution.
  • the TES metal ion solution contains: CaCl 2 0.5 g/L, ZnSO 4 ⁇ 7H 2 O 0.18 g/L, MnSO 4 ⁇ H 2 O 0.1 g/L, FeCl 3 8.35 g/L, CuSO 4 ⁇ 5H 2 O 0.16 g/L, CoCl 2 ⁇ 6H 2 O 0.18 g/L, and Na 2 ⁇ EDTA 10.5 g/L.
  • feeding is also performed during the fermentation process.
  • the feed is a feed medium containing glucose.
  • the feed medium contains glucose, soy peptone, yeast powder and metal ions.
  • the feed medium contains 250.0 g/L glucose, 66.66 g/L soy peptone, 33.33 g/L yeast powder, 0.39 g/L MgSO 4 , and 4.0% v/v TES metal ion solution.
  • the present invention also provides the application of the recombinant Bacillus subtilis bacteria or the method in producing products in the fields of food, chemical industry, sugar making, feed, etc.
  • the present invention significantly increases the expression level of the recombinant protein in Bacillus subtilis by overexpressing the enhancing factors PonA, OppA or SppA derived from Bacillus, and can increase the activity of ultrahigh temperature amylase to 2.03 times, 1.42 times and 1.92 times that of the wild type.
  • the present invention also screened the enhancing factor PonA protein from different sources, which can increase the expression level of the co-expressed ultra-high temperature amylase to 1.71 to 3.96 times compared with the control strain.
  • the present invention truncated the FN3 domain of PonA protein from different sources, and the obtained truncated DcPonA was co-expressed with ultra-high temperature amylase, which could increase the expression level of ultra-high temperature amylase by 2.02 to 3.35 times compared with the control strain.
  • FIG1 is a schematic diagram showing the effect of recombinant bacteria co-expressing PonA, OppA or SppA from Bacillus subtilis on the enzymatic activities of ultra-high temperature amylase, mesophilic amylase and sucrose isomerase.
  • FIG. 2 is a schematic diagram of the shake flask bacterial cell concentration and enzyme activity of the recombinant strain co-expressing Bacillus-derived PonA or its genetically engineered truncation DcPonA and ultra-high temperature amylase.
  • Figure 3 is a graph showing the bacterial concentration and enzyme activity of recombinant strains co-expressing PonA (Bs) and ultra-high temperature amylase in a 3L bioreactor; (a) SCK6/pfa-ck, (b) SCK6/pfa-ponA (Bs).
  • FIG. 4 is an electrophoretic diagram of SDS-PAGE analysis of a fermentation sample of a recombinant strain co-expressing PonA (Bs) and ultra-high temperature amylase in a 3 L bioreactor.
  • FIG. 5 is a schematic diagram of the shake flask cell concentration and enzyme activity of the recombinant strain co-expressing PonA or its homologous protein teichoic acid synthase LtaS from Bacillus subtilis and ultra-high temperature amylase.
  • PonA class A penicillin-binding protein
  • OppA ABC pathway transporter
  • SppA signal peptide peptidase
  • ponA(Bs) Class A penicillin-binding protein from Bacillus subtilis 168;
  • ponA(Bl) Class A penicillin-binding protein from Bacillus licheniformis DSM13;
  • ponA(Ba) Class A penicillin-binding protein from Bacillus amyloliquefaciens DSM7;
  • ponA(Bm) Class A penicillin-binding protein from Bacillus megaterium DSM319;
  • ponA(Gs) Class A penicillin-binding protein from Geobacillus stearothermophilus ATCC 7953;
  • DcPonA(Bs) a truncated form of the class A penicillin-binding protein from Bacillus subtilis 168;
  • DcPonA(Bl) a truncated form of the class A penicillin-binding protein from Bacillus licheniformis DSM13;
  • DcPonA(Ba) a truncated form of the class A penicillin-binding protein from Bacillus amyloliquefaciens DSM7;
  • DcPonA(Bm) a truncated form of the class A penicillin-binding protein from Bacillus megaterium DSM319;
  • DcPonA(Gs) A truncated form of the class A penicillin-binding protein from Geobacillus stearothermophilus ATCC 7953;
  • LB Luria-Bertani medium, used for the culture of Bacillus subtilis.
  • the bacterial concentration (OD 600 ) is used to express the sample. After sampling, the sample is diluted with deionized water to an appropriate multiple and placed in a spectrophotometer to measure the absorbance at 600 nm.
  • the culture medium was prepared with ddH 2 O and sterilized at 121°C for 20 min.
  • LB liquid culture medium yeast powder 5.0 g/L, trypsin 10.0 g/L, NaCl 10.0 g/L.
  • TB liquid culture medium yeast powder 24.0 g/L, tryptone 12.0 g/L, glycerol 5.0 g/L, K 2 HPO 4 ⁇ 3H 2 O 16.43 g/L, KH 2 PO 4 2.31 g/L.
  • 3L fermenter basic medium yeast powder 9.0g/L, soy peptone 18g/L, ammonium citrate 1.0g/L, K2HPO4 ⁇ 3H2O 14.66g/L, ( NH4 )2SO4 2.68g/L , Na2SO3 2.0g/L, NaH2PO4 3.49g / L , MgSO4 0.49g /L, TES metal ion solution 0.3% v /v.
  • 3L fermenter feed medium glucose 250.0 g/L, soy peptone 66.66 g/L, yeast powder 33.33 g/L, MgSO 4 0.39 g/L, TES metal ion solution 4.0% v/v.
  • TES metal ion solution CaCl 2 0.5 g/L, ZnSO 4 ⁇ 7H 2 O 0.18 g/L, MnSO 4 ⁇ H 2 O 0.1 g/L, FeCl 3 8.35 g/L, CuSO 4 ⁇ 5H 2 O 0.16 g/L, CoCl 2 ⁇ 6H 2 O 0.18 g/L, Na 2 ⁇ EDTA 10.5 g/L.
  • Example 1 Construction of recombinant bacteria expressing ultra-high temperature amylase, medium temperature amylase or sucrose isomerase
  • the nucleotide sequences of gene fragments pfa, amyS and si were amplified from ultra-high temperature amylase, medium temperature amylase and sucrose isomerase expression strains by PCR method, respectively. After gel recovery, it was connected to the pUB110 vector by the poe-pcr method, and the expression of the recombinant protein was regulated by the constitutive promoter P amyQ' shown in SEQ ID NO.31.
  • the poe-pcr product was then transformed into Bacillus subtilis SCK6, ice bathed for 20 minutes, 37°C water bathed for 20 minutes, 37°C, 200rpm resuscitation for 3 hours, coated on LB plates containing kanamycin resistance (40 ⁇ g/mL), and recombinant bacteria were obtained by screening and verification after overnight culture.
  • the recombinant bacteria were named BSP, BSA and BSS respectively.
  • the recombinant bacteria used to obtain the pfa, amyS and si genes were constructed by the inventor team in the early stage (published in: Tan Ruiting, Zhang Kang, Wu Jing. Secretory expression of hyperthermogenic ⁇ -amylase from thermophilic archaea in B.
  • subtilis [J]. Genomics and Applied Biology; Yao D, Zhang K, Zhu X, et al. Enhanced extracellular ⁇ -amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment [J]. Journal of Industrial Microbiology and Biotechnology, 2021(1): 1.; Liu Juntong, Wu Jing, Chen Sheng. Expression and fermentation optimization of dispersed Pantoea sucrose isomerase in Escherichia coli [J]. Journal of Biotechnology, 2016, 032(008): 1070-1080).
  • the ponA gene fragment shown in SEQ ID NO.11 was amplified from the Bacillus subtilis genome by PCR. After gel recovery, it was connected to the pAD123 vector by a one-step cloning kit (ClonExpress II One Step Cloning Kit), and the expression was regulated by the maltose-inducible promoter P glv shown in SEQ ID NO.33, and then transformed into Escherichia coli JM109, ice bathed for 30 minutes, 42°C water bathed for 90 seconds, ice bathed for 3 minutes, 37°C, 200rpm resuscitation for 1 hour, and then coated on LB plates containing ampicillin resistance (100 ⁇ g/mL) and cultured overnight. The recombinant plasmid was obtained after screening and verification.
  • the nucleotide sequence of oppA gene fragment shown in SEQ ID NO.26 was amplified from the genome of Bacillus subtilis by PCR method, and its sequence was connected to pAD123 vector by ClonExpress II One Step Cloning Kit after gel recovery, and the expression was regulated by maltose inducible promoter P glv shown in SEQ ID NO.33, and then transformed into Escherichia coli JM109, ice bathed for 30 minutes, 42°C water bathed for 90 seconds, ice bathed for 3 minutes, 37°C, 200rpm resuscitation for 1 hour, and then coated on LB plate containing ampicillin resistance (100 ⁇ g/mL), and cultured overnight. The recombinant plasmid was obtained after screening and verification.
  • the sppA gene fragment was amplified from the genome of B. subtilis 168 by PCR, and its sequence was shown in SEQ ID NO.22. After gel recovery, it was connected to the pAD123 vector by a one-step cloning kit (ClonExpress II One Step Cloning Kit), and the expression was regulated by the maltose-inducible promoter P glv shown in SEQ ID NO.33, and then transformed into Escherichia coli JM109, ice bathed for 30 minutes, 42°C water bathed for 90 seconds, ice bathed for 3 minutes, 37°C, 200rpm resuscitation for 1 hour, and then coated on LB plates containing ampicillin resistance (100 ⁇ g/mL) and cultured overnight. The recombinant plasmid was obtained after screening and verification.
  • Example 3 Construction of co-expression recombinant bacteria producing ultra-high temperature amylase, medium temperature amylase or sucrose isomerase
  • the recombinant plasmids pAD123-PonA, pAD123-OppA and pAD123-SppA constructed correctly in Example 2 were transformed into the competent cells of the recombinant bacteria BSP, BSA and BSS constructed in Example 1 respectively. Ice bath for 20 min, 37 ° C water bath for 20 min, 37 ° C, 200 rpm recovery for 3 h, coated on LB plates containing kanamycin resistance (40 ⁇ g / mL) and chloramphenicol resistance (10 ⁇ g / mL), cultured overnight, and recombinant bacteria expressing recombinant proteins and enhancing factors were obtained.
  • kanamycin resistance 40 ⁇ g / mL
  • chloramphenicol resistance 10 ⁇ g / mL
  • the correct recombinant bacteria were inoculated in 10 mL of LB medium containing kanamycin resistance (40 ⁇ g / mL) and chloramphenicol resistance (10 ⁇ g / mL) with an inoculum size of 2 ⁇ , and cultured at 37 ° C, 200 rpm for 10 h as fermentation seed liquid. Then, the inoculum was transferred to 50 mL of TB medium containing the same concentration of antibiotics at a 5% inoculum, and 5 g/L maltose was added to induce expression. The culture was first cultured at 37 °C for 2 h and then the temperature was adjusted to 33 °C until the end of the fermentation. The ultra-high temperature amylase expression strain was cultured for 60 h, and the medium temperature amylase and sucrose isomerase expression strains were cultured for 48 h, and the fermentation supernatant was taken and appropriately diluted to determine the enzyme activity.
  • the activities of the three recombinant proteases in the fermentation supernatant are shown in Figure 1.
  • the model enzyme is ultra-high temperature amylase
  • the co-expression of PonA, OppA and SppA increases the enzyme activity to 2.03 times, 1.42 times and 1.92 times of the wild type, respectively, and there is no significant difference in the bacterial OD.
  • the model enzyme is mesophilic amylase
  • the co-expression of PonA and OppA increases the enzyme activity to 1.49 times and 1.38 times of the wild type, respectively, and there is no significant difference in the bacterial OD during the fermentation of the PonA and OppA overexpression strains.
  • the co-expression of PonA and SppA increases the enzyme activity to 1.52 times and 2.26 times of the wild type, respectively.
  • the overexpression of PonA significantly reduces the bacterial OD, while the overexpression of SppA significantly increases the bacterial concentration.
  • the ponA(Bs), ponA(Bl), ponA(Ba), ponA(Bm) and ponA(Gs) gene fragments were amplified from the genomes of B.subtilis 168, Bacillus licheniformis DSM13, Bacillus amyloliquefaciens DSM7, Bacillus megaterium DSM319 and Geobacillus stearothermophilus ATCC7953 by PCR, and their nucleotide sequences were shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively.
  • the C-terminal FN3 domain of PonA was truncated by homologous recombination using the PCR method to obtain DcPonA(Bs), DcPonA(Bl), DcPonA(Ba), DcPonA(Bm), and DcPonA(Gs) fragments as shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, and SEQ ID NO.20, respectively; the PCR products were recovered by gel according to the same strategy as in Example 4 and ligated to the pAD123 vector via a ClonExpress II One Step Cloning Kit, and then the template was digested with dpnI and transformed into Escherichia coli JM109, incubated on ice for 30 min, in a water bath at 42°C for 90 s, in an ice bath for 3 min, and revived at 37°C, 200 rpm for 1 h, then spread on an LB plate containing amp
  • Example 6 Shake flask fermentation of PonA or DcPonA co-expressing recombinant bacteria to synthesize ultra-high temperature amylase
  • Example 4 and Example 5 The recombinant plasmids constructed correctly in Example 4 and Example 5 were transformed into the recombinant bacteria BSP constructed in Example 1, ice bathed for 20 minutes, 37°C water bathed for 20 minutes, 37°C, 200rpm resuscitation for 3 hours, coated on LB plates containing kanamycin resistance (40 ⁇ g/mL) and chloramphenicol resistance (10 ⁇ g/mL), and cultured overnight to obtain recombinant bacteria expressing ultra-high temperature amylase Pfa and PonA proteins at the same time, or recombinant bacteria expressing ultra-high temperature amylase Pfa and DcPonA proteins at the same time.
  • kanamycin resistance 40 ⁇ g/mL
  • chloramphenicol resistance 10 ⁇ g/mL
  • the verified recombinant bacteria were inoculated in 10mL of LB medium containing kanamycin resistance (40 ⁇ g/mL) and chloramphenicol resistance (10 ⁇ g/mL) at an inoculum size of 2 ⁇ , and cultured at 37°C, 200rpm for 10 hours as fermentation seed liquid. Then, the inoculum was transferred to 50 mL of TB medium containing the same concentration of antibiotics at a 5% inoculum, and 5 g/L maltose was added to induce expression. The culture was first cultured at 37 ° C for 2 h, and then the temperature was adjusted to 33 ° C for 60 h.
  • the fermentation supernatant was appropriately diluted and the Pfa expression level was determined according to the above enzyme activity detection method.
  • the recombinant bacteria obtained by transforming the plasmid pAD123 into the recombinant bacteria BSP without expressing PonA or DcPonA were used as the control strain.
  • the Pfa enzyme activity in the fermentation supernatant is shown in Figure 2. Except for PonA (Bl), the recombinant bacteria expressing other Bacillus-derived PonA proteins showed a significant increase in protein expression compared with the control strain.
  • the enzyme activities of the recombinant bacteria co-expressing PonA (Bs), PonA (Ba), PonA (Bm), and PonA (Gs) were measured after 60 hours of fermentation: 74.17U/mL, 82.07U/mL, 143.98U/mL, and 171.76U/mL, respectively. Compared with the control strain, they were increased by 1.71 times, 1.89 times, 3.32 times, and 3.96 times, respectively.
  • the Pfa enzyme activity of the recombinant bacteria co-expressing DcPonA was significantly improved compared with the control strain.
  • the enzyme activities of the recombinant bacteria co-expressing DcPonA(Bs), DcPonA(Bl), DcPonA(Ba), DcPonA(Bm), and DcPonA(Gs) were measured after 60 hours of fermentation: 90.48U/mL, 143.48U/mL, 94.04U/mL, 87.68U/mL, and 145.26U/mL, respectively.
  • the plasmid pAD123-P HpaII -PonA (Bs) constitutively expressing PonA was constructed, and the specific steps were as follows: the promoter P HpaII gene fragment with the nucleotide sequence shown in SEQ ID NO.32 was amplified from the B. subtilis 168 genome by PCR.
  • Example 7 The correctly constructed recombinant plasmid pAD123-P HpaII -PonA (Bs) in Example 7 was transformed into the recombinant bacterial BSP competent cells constructed in Example 1, ice bathed for 20 min, 37° C. water bathed for 20 min, revived at 37° C., 200 rpm for 3 h, spread on LB plates containing kanamycin resistance (40 ⁇ g/mL) and chloramphenicol resistance (10 ⁇ g/mL), and cultured overnight to obtain recombinant bacteria with constitutive PonA overexpression.
  • kanamycin resistance 40 ⁇ g/mL
  • chloramphenicol resistance 10 ⁇ g/mL
  • the strain freshly preserved in the glycerol tube was cultured at 37°C for 10h by plate streaking method, and a single colony was picked into 50mL LB medium containing kanamycin (40 ⁇ g/mL) and chloramphenicol (10 ⁇ g/mL), and cultured at 37°C, 200rpm for 12h, and then transferred to 900mL fermentation medium containing kanamycin (40 ⁇ g/mL) and chloramphenicol (10 ⁇ g/mL) at a transfer volume of 10%, and the temperature was adjusted to 33°C, and the pH was controlled at 7.0 with ammonia water and 20% phosphoric acid.
  • Feeding was started when dissolved oxygen rebound occurred at 6.5-8h of fermentation, and the feed flow rate was controlled so that the residual sugar content in the system was not higher than 0.1g/L.
  • Samples were taken regularly to determine the ultra-high temperature amylase activity in the bacterial concentration and fermentation broth.
  • the total enzyme activity of the control strain reached 1611.86U/mL at 90h of fermentation ( Figure 3a), and the PonA(Bs) co-expression strain reached 2041.73U/mL at 102h ( Figure 3b), which was 1.26 times higher than that of the control strain.
  • the bacterial concentrations of the two recombinant bacteria were basically the same during the high-density fermentation cycle, and SDS-PAGE showed that the protein bands were consistent with the enzyme activity trend (Figure 4).

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Abstract

提供了一种共表达芽孢杆菌来源增强因子提高重组蛋白在枯草芽孢杆菌中表达量的方法，属于酶程技术领域。将编码重组蛋白基因分别与芽孢杆菌来源的PonA、PonA截短体、OppA或SppA共表达，以提高重组蛋白的表达量。结果显示，所述方法可使超高温淀粉酶、中温淀粉酶或蔗糖异构酶在摇瓶水平的表达量提高至3.96倍、1.49倍和2.26倍。在3L生物反应器高密度发酵中可将超高温淀粉酶的表达量提高26％。所述枯草芽孢杆菌作为外源蛋白表达宿主提供了新的可开发靶点，同时为增强因子的优化改造提供了技术支持。

Description

一种共表达芽孢杆菌来源增强因子提高重组蛋白在枯草芽孢杆菌中表达量的方法 技术领域
本发明涉及一种共表达芽孢杆菌来源增强因子提高重组蛋白在枯草芽孢杆菌中表达量的方法，属于酶工程技术领域。
背景技术
枯草芽孢杆菌(Bacillus subtilis)是芽孢杆菌属(Bacillus Cohn)的一种革兰氏阳性菌株。B.subtilis由于不产毒素和致热致敏蛋白，因此被列为美国FDA《GRAS(公认为安全物质)》清单认证菌株，其易于分离培养，可高密度发酵和蛋白分泌能力强的特性，使得枯草芽孢杆菌作为重要的工业菌株，用于生产多种外源重组蛋白和代谢产物，其优秀性状具备较高的鉴定价值。同时，枯草芽孢杆菌具有清晰的遗传背景和成熟的分子生物学基因编辑方法，这有利于对其进行基因改造以强化枯草芽孢杆菌作为重组蛋白生产宿主的表达能力。
进一步提高枯草芽孢杆菌的外源蛋白生产能力是当前研究的重中之重。目前已经采取了许多策略以改造这种模式微生物以获得更高的重组蛋白产量，例如过表达Sec途径相关因子，敲除胞内或胞外蛋白酶，增加伴侣蛋白含量等。然而，这些改造靶点的选取极大程度上依赖于现有的对于枯草芽孢杆菌功能基因注释，而蛋白的合成分泌过程并不是由单一途径参与调控。有研究指出，调控生理相关功能的基因在蛋白质表达或代谢物积累过程中发挥重要作用，如Jennifer Staudacher通过上调翻译起始因子表达水平将异源蛋白表达水平提高至3倍，Xiao-Ran Jiang通过调控细胞形态控制基因，将PHB的产量提高至1.8倍。因此，当前对于蛋白质合成分泌相关途径的注释并不完善，这意味着当前的改造策略是相对片面的，从而限制了枯草芽孢杆菌作为表达宿主的进一步开发。
目前针对蛋白质表达过程中其关键作用的因子的鉴定仍不完善，因此急需在现有基础上，开发其他新型能够增强重组蛋白在枯草芽孢杆菌中表达的因子。
发明内容
为解决现有技术中重组蛋白在枯草芽孢杆菌中表达量有限的问题，本发明提供了一种提高枯草芽孢杆菌中重组蛋白表达量的方法，所述方法是在表达重组蛋白的同时过表达芽孢杆菌A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
在一种实施方式中，所述的PonA是来源于枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、巨大芽孢杆菌 (Bacillus megaterium)或嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的A类青霉素结合蛋白。
在一种实施方式中，来源于枯草芽孢杆菌(Bacillus subtilis)的PonA蛋白为PonA(Bs)，具有SEQ ID NO.1所示的氨基酸序列，或具有与SEQ ID NO.1具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于地衣芽孢杆菌(Bacillus licheniformis)的PonA蛋白为PonA(Bl)，具有SEQ ID NO.2所示的氨基酸序列，或具有与SEQ ID NO.2具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的PonA蛋白为PonA(Ba)，具有SEQ ID NO.3所示的氨基酸序列，或具有与SEQ ID NO.3具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于巨大芽孢杆菌(Bacillus megaterium)的PonA蛋白为PonA(Bm)，具有SEQ ID NO.4所示的氨基酸序列，或具有与SEQ ID NO.4具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的PonA蛋白为PonA(Gs)，具有SEQ ID NO.5所示的氨基酸序列，或具有与SEQ ID NO.5具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，所述的PonA截短体是来源于枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、巨大芽孢杆菌(Bacillus megaterium)或嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的A类青霉素结合蛋白截短体。
在一种实施方式中，来源于枯草芽孢杆菌(Bacillus subtilis)的PonA蛋白截短体为DcPonA(Bs)，具有SEQ ID NO.6所示的氨基酸序列，或具有与SEQ ID NO.6具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于地衣芽孢杆菌(Bacillus licheniformis)的PonA蛋白截短体为DcPonA(Bl)，具有SEQ ID NO.7所示的氨基酸序列，或具有与SEQ ID NO.7具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于解淀粉芽孢杆菌(Bacillus amyloliquefaciens)的PonA蛋白截短体为DcPonA(Ba)，具有SEQ ID NO.8所示的氨基酸序列，或具有与SEQ ID NO.8具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸 序列。
在一种实施方式中，来源于巨大芽孢杆菌(Bacillus megaterium)的PonA蛋白截短体为DcPonA(Bm)，具有SEQ ID NO.9所示的氨基酸序列，或具有与SEQ ID NO.9具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，来源于嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的PonA蛋白截短体为DcPonA(Gs)，具有SEQ ID NO.10所示的氨基酸序列，或具有与SEQ ID NO.10具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，所述枯草芽孢杆菌来源的ABC途径转运蛋白OppA具有SEQ ID NO.21所示的氨基酸序列；所述枯草芽孢杆菌来源信号肽肽酶SppA，具有SEQ ID NO.22所示的氨基酸序列。
在一种实施方式中，所述的重组蛋白包括但不限于超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
在一种实施方式中，所述超高温淀粉酶具有SEQ ID NO.23所示的氨基酸序列，或具有与SEQ ID NO.23具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，所述中温淀粉酶具有SEQ ID NO.24所示的氨基酸序列，或具有与SEQ ID NO.24具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，所述蔗糖异构酶具有SEQ ID NO.25所示的氨基酸序列，或具有与SEQ ID NO.25具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
在一种实施方式中，以pUB110为表达载体表达超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
在一种实施方式中，以pAD123为表达载体表达所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
在一种实施方式中，所述方法是在同一宿主中表达重组蛋白和所述PonA、OppA以及SppA。
在一种实施方式中，所述宿主包括但不限于枯草芽孢杆菌SCK6。
在一种实施方式中，所述的重组蛋白通过组成型启动子PamyQ’进行表达，所述启动子PamyQ’ 的核苷酸序列如SEQ ID NO.31所示。
在一种实施方式中，所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA通过启动子Pglv或启动子PHpaII进行表达。
在一种实施方式中，所述启动子PHpaII的核苷酸序列如SEQ ID NO.32所示。
在一种实施方式中，所述启动子Pglv的核苷酸序列如SEQ ID NO.33所示。
本发明还提供了外源蛋白表达量提高的枯草芽孢杆菌重组菌，所述枯草芽孢杆菌重组菌在表达外源蛋白的同时分别共表达芽孢杆菌来源的A类青霉素结合蛋白PonA或DcPonA、枯草芽孢杆菌来源的ABC途径转运蛋白OppA或枯草芽孢杆菌来源的信号肽肽酶SppA。
在一种实施方式中，所述外源蛋白是指相对于表达和分泌该蛋白质的枯草芽孢杆菌而言的外源性(exogenous)的蛋白质。例如，外源蛋白可以是源自微生物的蛋白质、源自植物的蛋白质、源自动物的蛋白质、源自病毒的蛋白质、甚至是氨基酸序列由人工设计而成的蛋白质。特别是，异源蛋白质可以为源自人的蛋白质。异源蛋白质可以是单体蛋白质(monomeric protein)或多聚体蛋白质(multimeric protein)。“多聚体蛋白质”指可以作为包含两个或多个亚基的多聚体而存在的蛋白质。在多聚体中，各亚基可以通过二硫键等共价键实现连接；可以通过氢键和疏水性相互作用等非共价键实现连接，或者可以通过它们的组合实现连接。在多聚体中优选包含一个或多个分子间二硫键。多聚体可以是包含单一种类的亚基的同源多聚体，也可以是包含两种以上种类的亚基的异源多聚体。需要说明的是，在多聚体蛋白质是异源多聚体的情况下，构成多聚体的亚基中的至少一个亚基是异源蛋白质即可。即，所有亚基可以是异源的，或者仅一部分亚基是异源的。异源蛋白质可以是天然的具有分泌性的蛋白质，也可以是天然的不具有分泌性的蛋白质，优选其是天然的具有分泌性的蛋白质。此外，异源蛋白质可以是天然的Tat依赖性分泌性蛋白质，也可以是天然的Sec依赖性分泌性蛋白质。
在一种实施方式中，以pUB110为表达载体表达超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
在一种实施方式中，以pAD123为表达载体表达所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
本发明还提供了构建所述枯草芽孢杆菌重组菌的构建方法，所述方法包括向枯草芽孢杆菌中导入重组蛋白的编码基因，和编码增强因子的基因。
在一种实施方式中，所述编码增强因子的基因包括但不限于A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA的编码基因。
在一种实施方式中，所述A类青霉素结合蛋白PonA的编码基因具有SEQ ID NO.11～SEQ ID NO.15任一所示的核苷酸序列。
在一种实施方式中，所述A类青霉素结合蛋白PonA截短体的编码基因具有SEQ ID NO.16～SEQ ID NO.20任一所示的核苷酸序列。
在一种实施方式中，所述重组蛋白的编码基因包括但不限于超高温淀粉酶、中温淀粉酶或蔗糖异构酶的编码基因。
在一种实施方式中，所述超高温淀粉酶的编码基因具有SEQ ID NO.28所示的核苷酸序列。
在一种实施方式中，所述中温淀粉酶的编码基因具有SEQ ID NO.29所示的核苷酸序列。
在一种实施方式中，所述蔗糖异构酶的编码基因具有SEQ ID NO.30所示的核苷酸序列。
本发明还提供所述枯草芽孢杆菌重组菌在生产超高温淀粉酶、中温淀粉酶或蔗糖异构酶中的应用。
在一种实施方式中，所述应用是将所述重组菌在发酵培养基中培养一段时间，收集超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
在一种实施方式中，所述应用是在30～40℃，或30～37℃，或35～40℃，或37～40℃，或35～37℃发酵。
在一种实施方式中，所述发酵是发酵至少24h，或至少36h，或至少42h，或至少48h，或至少60h。
在一种实施方式中，所述发酵培养基中包含酵母粉、大豆蛋白胨、柠檬酸铵和金属离子。
在一种实施方式中，所述发酵培养基含有酵母粉9.0g/L、大豆蛋白胨18g/L、柠檬酸铵1.0g/L、K2HPO4·3H2O 14.66g/L、(NH4)2SO4 2.68g/L、Na2SO3 2.0g/L、NaH2PO4 3.49g/L、MgSO4 0.49g/L、TES金属离子液0.3％v/v。
在一种实施方式中，所述TES金属离子液含有：CaCl2 0.5g/L、ZnSO4·7H2O 0.18g/L、MnSO4·H2O 0.1g/L、FeCl3 8.35g/L、CuSO4·5H2O 0.16g/L、CoCl2·6H2O 0.18g/L、Na2·EDTA 10.5g/L。
在一种实施方式中，所述发酵的过程中还进行了补料。
在一种实施方式中，所述补料是补加含葡萄糖的补料培养基。
在一种实施方式中，所述补料培养基含有葡萄糖、大豆蛋白胨、酵母粉和金属离子。
在一种实施方式中，所述补料培养基含有葡萄糖250.0g/L、大豆蛋白胨66.66g/L、酵母粉33.33g/L、MgSO4 0.39g/L、TES金属离子液4.0％v/v。
本发明还提供所述枯草芽孢杆菌重组菌或所述方法在食品、化工、制糖、饲料等领域生产产品中的应用。
有益效果：
(1)本发明通过过表达芽孢杆菌来源的增强因子PonA、OppA或SppA，显著提高了重组蛋白在枯草芽孢杆菌中的表达量，可使超高温淀粉酶酶活提高至野生型的2.03倍、1.42倍和1.92倍。
(2)本发明还筛选了不同来源的增强因子PonA蛋白，可使共表达超高温淀粉酶的表达量相比对照菌株提高至1.71～3.96倍。
(3)本发明对不同来源的PonA蛋白FN3结构域进行了截短，获得的截短体DcPonA与超高温淀粉酶共表达可使超高温淀粉酶表达量相比对照菌株提高2.02～3.35倍。
附图说明
图1为共表达枯草芽孢杆菌来源的PonA、OppA或SppA的重组菌对于超高温淀粉酶、中温淀粉酶和蔗糖异构酶酶活影响的示意图。
图2为共表达芽孢杆菌来源的PonA或其基因工程截短体DcPonA和超高温淀粉酶重组菌株的摇瓶菌体浓度和酶活示意图。
图3为共表达PonA(Bs)和超高温淀粉酶重组菌株在3L生物反应器中的菌体浓度和酶活曲线图；(a)SCK6/pfa-ck，(b)SCK6/pfa-ponA(Bs)。
图4为共表达PonA(Bs)和超高温淀粉酶重组菌株在3L生物反应器中发酵样品的SDS-PAGE分析电泳图。
图5为共表达枯草芽孢杆菌来源的PonA或其同类蛋白磷壁酸合酶LtaS和超高温淀粉酶重组菌株的摇瓶菌体浓度和酶活示意图。
具体实施方式
(一)本发明简写与全称，以及中英文对照：
PonA：A类青霉素结合蛋白；
OppA：ABC途径转运蛋白；
SppA：信号肽肽酶；
ponA(Bs)：Bacillus subtilis 168来源的A类青霉素结合蛋白；
ponA(Bl)：Bacillus licheniformis DSM13来源的A类青霉素结合蛋白；
ponA(Ba)：Bacillus amyloliquefaciens DSM7来源的A类青霉素结合蛋白；
ponA(Bm)：Bacillus megaterium DSM319来源的A类青霉素结合蛋白；
ponA(Gs)：Geobacillus stearothermophilus ATCC 7953来源的A类青霉素结合蛋白；
DcPonA(Bs)：Bacillus subtilis 168来源的A类青霉素结合蛋白截短体；
DcPonA(Bl)：Bacillus licheniformis DSM13来源的A类青霉素结合蛋白截短体；
DcPonA(Ba)：Bacillus amyloliquefaciens DSM7来源的A类青霉素结合蛋白截短体；
DcPonA(Bm)：Bacillus megaterium DSM319来源的A类青霉素结合蛋白截短体；
DcPonA(Gs)：Geobacillus stearothermophilus ATCC 7953来源的A类青霉素结合蛋白截短体；
LB：Luria-Bertani培养基，用于枯草芽孢杆菌的培养。
TB：Terrific Broth，超级肉汤培养基。
(二)本发明具体实施方式中涉及的检测方法：
(1)生物量测定：
采用菌浓度(OD600)表示。取样后将样品用去离子水稀释适当倍数后，置于分光光度计中测量600nm处吸光值。
(2)超高温淀粉酶酶活测定：
具体操作见文献(檀瑞婷,张康,吴敬.嗜热古菌超高温α-淀粉酶在枯草芽孢杆菌中的分泌表达[J].基因组学与应用生物学)。酶活测定公式为：U(U·mL-1)＝n×(6.0539×△540+0.3494)，其中U为酶活力单位，n为酶液稀释倍数，△540为样品在OD540下测出的吸光值减去空白组的吸光值。
(3)中温淀粉酶酶活测定：
具体操作见文献(Yao D,Zhang K,Zhu X,et al.Enhanced extracellularα-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment[J].Journal of Industrial Microbiology and Biotechnology,2021(1):1.)。酶活测定公式为：U(U·mL-1)＝n×(6.0539×△540+0.3494)，其中U为酶活力单位，n为酶液稀释倍数，△540为样品在OD540下测出的吸光值减去空白组的吸光值。
(4)蔗糖异构酶酶活测定：
具体操作见文献(刘军彤,吴敬,陈晟.分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化[J].生物工程学报,2016,032(008):1070-1080)。酶活测定公式为：U(U·mL-1)＝n×(6.0539×△540+0.3494)，其中U为酶活力单位，n为酶液稀释倍数，△540为样品在OD540下测出的吸光值减去空白组的吸光值。
(三)本发明具体实施方式中涉及的培养基：
培养基均使用ddH2O配制，配制完成后121℃灭菌20min。
LB液体培养基：酵母粉5.0g/L、胰蛋白胨10.0g/L、NaCl 10.0g/L。
TB液体培养基：酵母粉24.0g/L、胰蛋白胨12.0g/L、甘油5.0g/L、K2HPO4·3H2O 16.43g/L、KH2PO4 2.31g/L。
3L发酵罐基础培养基：酵母粉9.0g/L、大豆蛋白胨18g/L、柠檬酸铵1.0g/L、K2HPO4·3H2O 14.66g/L、(NH4)2SO4 2.68g/L、Na2SO3 2.0g/L、NaH2PO4 3.49g/L、MgSO4 0.49g/L、TES金属离子液0.3％v/v。
3L发酵罐补料培养基：葡萄糖250.0g/L、大豆蛋白胨66.66g/L、酵母粉33.33g/L、MgSO40.39g/L、TES金属离子液4.0％v/v。
TES金属离子液：CaCl2 0.5g/L、ZnSO4·7H2O 0.18g/L、MnSO4·H2O 0.1g/L、FeCl3 8.35g/L、CuSO4·5H2O 0.16g/L、CoCl2·6H2O 0.18g/L、Na2·EDTA 10.5g/L。
实施例1表达超高温淀粉酶、中温淀粉酶或蔗糖异构酶的重组菌的构建
利用PCR方法分别从超高温淀粉酶、中温淀粉酶和蔗糖异构酶表达菌株中扩增得到核苷酸序列分别如SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30所示的基因片段pfa、amyS和si。经过胶回收后通过poe-pcr的方法将其连接至pUB110载体，重组蛋白的表达由SEQ ID NO.31所示的组成型启动子PamyQ’调控。随后将poe-pcr产物转化枯草芽孢杆菌SCK6，冰浴20min，37℃水浴20min，37℃，200rpm复苏3h，涂布在含有卡那霉素抗性(40μg/mL)的LB平板上，过夜培养经过筛选验证获得重组菌，重组菌分别命名为BSP、BSA、BSS。其中，用于获取pfa、amyS及si基因的重组菌为发明人团队前期构建得到(公开于：檀瑞婷,张康,吴敬.嗜热古菌超高温α-淀粉酶在B.subtilis中的分泌表达[J].基因组学与应用生物学；Yao D,Zhang K,Zhu X,et al.Enhanced extracellularα-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment[J].Journal of Industrial Microbiology and Biotechnology,2021(1):1.；刘军彤,吴敬,陈晟.分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化[J].生物工程学报,2016,032(008):1070-1080)。
实施例2增强因子共表达质粒的构建
(1)重组质粒pAD123-PonA的构建
利用PCR方法从枯草芽孢杆菌基因组中扩增如SEQ ID NO.11所示的ponA基因片段。经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)将其连接至pAD123载体，由SEQ ID NO.33所示的麦芽糖诱导型启动子Pglv调控表达，随后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒。
(2)重组质粒pAD123-OppA的构建
利用PCR方法从枯草芽孢杆菌基因组中扩增核苷酸序列如SEQ ID NO.26所示oppA基因片段，其序列，经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)将其连接至pAD123载体，由SEQ ID NO.33所示的麦芽糖诱导型启动子Pglv调控表达，随后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒。
(3)重组质粒pAD123-SppA的构建
利用PCR方法从B.subtilis 168基因组中扩增sppA基因片段，其序列如SEQ ID NO.22所示，经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)将其连接至pAD123载体，由SEQ ID NO.33所示的麦芽糖诱导型启动子Pglv调控表达，随后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒。
实施例3产超高温淀粉酶、中温淀粉酶或蔗糖异构酶的共表达重组菌的构建
将实施例2构建正确的重组质粒pAD123-PonA、pAD123-OppA和pAD123-SppA分别转化至含有实施例1构建的重组菌BSP、BSA、BSS感受态细胞中。冰浴20min，37℃水浴20min，37℃，200rpm复苏3h，涂布在含有卡那霉素抗性(40μg/mL)及氯霉素抗性(10μg/mL)的LB平板上，过夜培养，获得同时表达重组蛋白和增强因子的重组菌。将验证正确的重组菌以2‰的接种量接种于10mL含有卡那霉素抗性(40μg/mL)及氯霉素抗性(10μg/mL)的LB培养基中，于37℃，200rpm下培养10h作为发酵种子液。随后以5％的接种量转接至50mL含有相同浓度抗生素的TB培养基中，同时加入5g/L麦芽糖进行诱导表达，先于37℃培养2h后调节温度至33℃直至发酵终点。超高温淀粉酶表达菌株培养至60h，中温淀粉酶和蔗糖异构酶表达菌株培养至48h后取发酵上清适当稀释后测定酶活。
发酵上清中的三种重组蛋白酶活如图1所示，当模型酶为超高温淀粉酶时，PonA、OppA和SppA的共表达分别将酶活提高至野生型的2.03倍、1.42倍和1.92倍，菌体OD没有显著差异。当模型酶为中温淀粉酶时，PonA和OppA的共表达分别将酶活提高至野生型的1.49倍和1.38倍，且PonA和OppA过表达菌株发酵过程中的菌体OD没有显著差异。当模型酶为蔗糖异构酶时，PonA和SppA的共表达分别将酶活提高至野生型的1.52倍和2.26倍，PonA的过表达使菌体OD显著降低，而SppA的过表达显著提高了菌浓。
实施例4表达不同来源PonA的重组质粒的构建
利用PCR方法分别从B.subtilis 168、Bacillus licheniformis DSM13、Bacillus amyloliquefaciens DSM7、Bacillus megaterium DSM319和Geobacillus stearothermophilus ATCC7953的基因组中扩增ponA(Bs)、ponA(Bl)、ponA(Ba)、ponA(Bm)和ponA(Gs)基因片段，其核苷酸序列分别如SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15所示，经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)将其连接至pAD123载体，由麦芽糖诱导型启动子Pglv调控表达，随后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒。
实施例5表达FN3结构域截短体DcPonA的重组质粒的构建
利用PCR方法通过同源重组截短PonA的C端FN3结构域，分别获得核苷酸序列如SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20所示的DcPonA(Bs)、DcPonA(Bl)、DcPonA(Ba)、DcPonA(Bm)、DcPonA(Gs)片段；按照实施例4相同策略将PCR产物经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)连接至pAD123载体，再经过dpnI消化模板后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒。
实施例6摇瓶发酵PonA或DcPonA共表达重组菌合成超高温淀粉酶
将实施例4、实施例5构建正确的重组质粒分别转化至实施例1构建的重组菌BSP中，冰浴20min，37℃水浴20min，37℃，200rpm复苏3h，涂布在含有卡那霉素抗性(40μg/mL)及氯霉素抗性(10μg/mL)的LB平板上，过夜培养，获得同时表达超高温淀粉酶Pfa和PonA蛋白的重组菌，或同时表达超高温淀粉酶Pfa和DcPonA蛋白的重组菌。将验证正确的重组菌以2‰的接种量接种于10mL含有卡那霉素抗性(40μg/mL)及氯霉素抗性(10μg/mL)的LB培养基中，于37℃，200rpm下培养10h作为发酵种子液。随后以5％的接种量转接至50mL含有相同浓度抗生素的TB培养基中，同时加入5g/L麦芽糖进行诱导表达，先于37℃培养2h后调节温度至33℃培养至60h。随后取发酵上清适当稀释后按照上述酶活检测方法测定Pfa表达量。以不表达PonA或DcPonA，仅向重组菌BSP中转化质粒pAD123获得的重组菌作为对照菌株。
发酵上清中的Pfa酶活如图2所示，除PonA(Bl)外，表达其他芽孢杆菌来源PonA蛋白的重组菌相较对照菌株均表现出了蛋白表达量的显著提高。共表达PonA(Bs)、PonA(Ba)、PonA(Bm)、PonA(Gs)的重组菌在发酵60h后测得酶活分别为：74.17U/mL、82.07U/mL、143.98U/mL、171.76U/mL。相比对照菌株分别提高至1.71倍、1.89倍、3.32倍和3.96倍。 共表达DcPonA的重组菌相较对照菌株的Pfa酶活均表现出显著提升。共表达DcPonA(Bs)、DcPonA(Bl)、DcPonA(Ba)、DcPonA(Bm)、DcPonA(Gs)的重组菌在发酵60h后测得酶活分别为：90.48U/mL、143.48U/mL、94.04U/mL、87.68U/mL、145.26U/mL。相比对照菌株分别提高至2.09倍、3.31倍、2.17倍、2.02倍、3.35倍。
实施例7启动子优化
构建组成型表达PonA的质粒pAD123-PHpaII-PonA(Bs)，具体步骤为：利用PCR方法从B.subtilis 168基因组中扩增得到核苷酸序列如SEQ ID NO.32所示的启动子PHpaII基因片段。经过胶回收后通过一步克隆试剂盒(ClonExpress II One Step Cloning Kit)将其连接至实施例4构建的携带PonA(Bs)的载体pAD123-PonA(Bs)上，随后转化至大肠杆菌JM109，冰浴30min，42℃水浴90s，冰浴3min，37℃，200rpm复苏1h后涂布在含有氨苄青霉素抗性(100μg/mL)的LB平板上，过夜培养。经过筛选验证获得重组质粒pAD123-PHpaII-PonA(Bs)。
实施例8 3L发酵罐水平发酵合成超高温淀粉酶
将实施例7构建正确的重组质粒pAD123-PHpaII-PonA(Bs)转化至实施例1构建的重组菌BSP感受态细胞中，冰浴20min，37℃水浴20min，37℃，200rpm复苏3h，涂布在含有卡那霉素抗性(40μg/mL)及氯霉素抗性(10μg/mL)的LB平板上，过夜培养，获得组成型PonA过表达的重组菌。将甘油管新鲜保藏的菌株用平板划线法于37℃培养10h，挑取单菌落至含有卡那霉素(40μg/mL)和氯霉素(10μg/mL)的50mL装LB培养基中，37℃，200rpm条件下培养12h，然后以10％的转接量转接至900mL含有卡那霉素(40μg/mL)和氯霉素(10μg/mL)的发酵培养基中，调节温度为33℃，用氨水和20％磷酸将pH控制在7.0，在发酵6.5～8h出现溶氧反弹时开始补料，控制补料流速使体系中的残糖含量不高于0.1g/L。定时取样测定菌浓和发酵液中的超高温淀粉酶酶活。对照菌株的总酶活在发酵90h时达到1611.86U/mL(图3a)，PonA(Bs)共表达菌株则于102h达到2041.73U/mL(图3b)，相较对照菌株酶活提高至1.26倍。两种重组菌的菌浓在高密度发酵周期中基本一致，SDS-PAGE显示蛋白条带与酶活趋势相符(图4)。
对比例1：
具体实施方式同实施例5～6，区别在于，替换调节因子，具体为：将PonA替换为枯草芽孢杆菌中主要的磷壁酸合酶LtaS(氨基酸序列如SEQ ID NO.34所示，基因序列如SEQ ID NO.35所示)，结果显示，LtaS过表达仅将Pfa的酶活提高至1.52倍，显著低于PonA对Pfa表达的提高效果(2.03倍)。在同样的发酵条件下，OD600没有显著变化(图5)。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (20)

  1. 重组枯草芽孢杆菌，其特征在于，表达外源蛋白和(1)～(4)任一蛋白：
    (1)芽孢杆菌来源的A类青霉素结合蛋白PonA；
    (2)芽孢杆菌来源的A类青霉素结合蛋白截短体DcPonA；
    (3)枯草芽孢杆菌来源的ABC途径转运蛋白OppA；
    (4)枯草芽孢杆菌来源的信号肽肽酶SppA；
    所述的PonA是来源于枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、巨大芽孢杆菌(Bacillus megaterium)或嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的A类青霉素结合蛋白；
    所述的PonA截短体是来源于枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、巨大芽孢杆菌(Bacillus megaterium)或嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus)的A类青霉素结合蛋白截短体。
  2. 根据权利要求1所述的重组枯草芽孢杆菌，其特征在于，所述PonA蛋白具有SEQ ID NO.1～5任一所示的氨基酸序列，或具有与SEQ ID NO.1～5所示序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
  3. 根据权利要求1所述的重组枯草芽孢杆菌，其特征在于，所述PonA蛋白截短体为具有SEQ ID NO.6～10任一所示的氨基酸序列，或具有与SEQ ID NO.6～10所示序列具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或99.9％同一性的氨基酸序列。
  4. 根据权利要求1～3任一所述重组枯草芽孢杆菌，其特征在于，所述的外源蛋白包括超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
  5. 根据权利要求4所述的重组枯草芽孢杆菌，其特征在于，以pUB110为表达载体表达超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
  6. 根据权利要求1～5任一所述的重组枯草芽孢杆菌，其特征在于，以pAD123为表达载体表达所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
  7. 根据权利要求根据权利要求1～6任一所述的重组枯草芽孢杆菌，其特征在于，所述的外源蛋白通过组成型启动子PamyQ’进行表达。
  8. 根据权利要求根据权利要求1～7任一所述的重组枯草芽孢杆菌，其特征在于，所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA通过启动子Pglv或启动子PHpaII进行表达。
  9. 根据权利要求1～8任一所述的重组枯草芽孢杆菌，其特征在于，宿主包括枯草芽孢杆菌SCK6。
  10. 一种提高枯草芽孢杆菌中重组蛋白表达量的方法，其特征在于，所述方法是在表达重组蛋白的同时过表达芽孢杆菌A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
  11. 根据权利要求10所述的方法，其特征在于，所述的重组蛋白包括超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
  12. 根据权利要求11所述的方法，其特征在于，以pUB110为表达载体表达超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
  13. 根据权利要求10～12任一所述的方法，其特征在于，以pAD123为表达载体表达所述A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA。
  14. 构建权利要求1所述枯草芽孢杆菌重组菌的方法，其特征在于，所述方法包括向枯草芽孢杆菌中导入编码重组蛋白的基因和编码增强因子的基因；所述编码增强因子的基因包括A类青霉素结合蛋白PonA、A类青霉素结合蛋白PonA截短体、ABC途径转运蛋白OppA或信号肽肽酶SppA的编码基因。
  15. 根据权利要求14所述的方法，其特征在于，所述A类青霉素结合蛋白PonA的编码基因具有SEQ ID NO.11～SEQ ID NO.15任一所示的核苷酸序列。
  16. 根据权利要求14所述的方法，其特征在于，所述A类青霉素结合蛋白PonA截短体的编码基因具有SEQ ID NO.16～SEQ ID NO.20任一所示的核苷酸序列。
  17. 权利要求1～9任一所述重组枯草芽孢杆菌在生产超高温淀粉酶、中温淀粉酶或蔗糖异构酶中的应用。
  18. 根据权利要求17所述的方法，其特征在于，所述应用是将所述重组菌在发酵培养基中培养一段时间，收集超高温淀粉酶、中温淀粉酶或蔗糖异构酶。
  19. 根据权利要求17或18所述的方法，其特征在于，在发酵的过程中还进行了补料。
  20. 权利要求1～9任一所述重组枯草芽孢杆菌或权利要求10～19任一所述方法在食品、化工、制糖、饲料等领域生产产品中的应用。
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