WO2024002131A1 - 一种高效表达d-阿洛酮糖3-差向异构酶的工程菌及应用 - Google Patents

一种高效表达d-阿洛酮糖3-差向异构酶的工程菌及应用 Download PDF

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WO2024002131A1
WO2024002131A1 PCT/CN2023/103067 CN2023103067W WO2024002131A1 WO 2024002131 A1 WO2024002131 A1 WO 2024002131A1 CN 2023103067 W CN2023103067 W CN 2023103067W WO 2024002131 A1 WO2024002131 A1 WO 2024002131A1
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nucleotide sequence
psicose
expression
expression vector
expression element
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French (fr)
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华君
裴亮
李政泓
潘月
刘超
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四川盈嘉合生科技有限公司
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/60—Vectors containing traps for, e.g. exons, promoters
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of bioengineering technology, and specifically relates to an engineering bacterium that efficiently expresses D-psicose 3-epimerase and its application.
  • Allulose also known as D-psicose, is the epimer corresponding to the third carbon of D-fructose. Allulose has a taste similar to sucrose, but its sweetness is equivalent to 70% of sucrose. %, the calories are equivalent to 0.3% of sucrose, and are almost not metabolized in the body. And because it can inhibit glucose by inhibiting glucose absorption, it has the effect of lowering blood sugar and is one of the most potential sucrose substitutes.
  • D-psicose naturally exists in foods such as raisins, figs, kiwis, and brown sugar. It is a rare monosaccharide with very little content in nature. The method of relying on natural product extraction is expensive and difficult to achieve mass production.
  • methods for producing D-psicose include chemical methods and biological methods. For example, Bilik and others have developed technology that uses the catalytic effect of molybdate ions to produce psicose from fructose. McDonald produced psicose through a three-step chemical process of 1,2:4,5-di-o-acetonylglycidyl- ⁇ -d-fructose.
  • the current bioconversion method mainly uses fructose as raw material and catalyzes D-psicose 3-epimerase to synthesize D-psicose. This method has a high catalytic synthesis efficiency. , is currently the main way to synthesize psicose.
  • D-psicose 3-epimerase disclosed in the application number 201710124243.
  • D-psicose 3-epimerase, and the D-psicose 3-epimerase is used to catalyze the production of psicose in industry, or is used to catalyze the production of psicose in industry. Catalyst for psicose.
  • This patent provides a mutant of D-psicose 3-epimerase, containing one or more mutation sites among the G86D, D164E, and W262S mutation sites, with catalytic activity
  • the catalytic activity was significantly improved, and the catalytic activity was more than 1.4 times that of the wild type, which reduced the amount of enzyme used in the synthesis of D-psicose.
  • the present invention provides an engineering bacterium that efficiently expresses D-psicose 3-epimerase and its application.
  • the present invention constructs an engineering bacterium that efficiently expresses D-psicose 3-epimerase.
  • the engineering bacterium contains a constructed recombinant expression vector and a co-expression vector, which improves the efficiency of expressing soluble proteins, thereby improving D-Allulone Expression levels of sugar 3-epimerase.
  • the present invention provides the application of any of the following items in the production of D-psicose:
  • the present invention also provides expression elements, which include a gene expressing D-psicose 3-epimerase and a constitutive promoter HCE gene.
  • the gene of D-psicose 3-epimerase is derived from Ruminococcus sp.; the HCE gene is derived from Bacillus Geobacillus sp.WCH70.
  • the expression element further includes an RBS sequence.
  • the RBS sequence is added at the 5' end.
  • the D-psicose 3-epimerase has:
  • the constitutive promoter HCE has:
  • the vector is a plasmid, and any one of pET21a, pET20b, pET22b, and pET28a is selected but not limited to ;
  • the vector used in the present invention is pET28a plasmid.
  • the present invention also provides co-expression elements, which include molecular chaperone protein genes and/or deleted lacZ genes;
  • the molecular chaperone proteins include dnaJ, dnaK, GroEL and/or grpE.
  • the genes of dnaJ, dnaK, GroEL and/or grpE are derived from Bacillus Geobacillus sp.WCH70.
  • the co-expression element includes any of the following and/or deletion of the lacZ gene:
  • the plasmid gRNA-1 used to delete the lacZ gene has:
  • (B) a nucleotide sequence encoding the same protein as the nucleotide sequence shown in (A), but different from the nucleotide sequence shown in (A) due to the degeneracy of the genetic code;
  • nucleotide sequence represented by (A) or (B) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (A) or (B), and being identical to the nucleotide sequence represented by (A) or (B)
  • the nucleotide sequences shown have the same or similar functions nucleotide sequence; or
  • (D) A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence described in any one of (A) to (C).
  • the co-expression element further includes an RBS sequence and/or lac promoter.
  • the co-expression elements include any of the following:
  • the nucleotide sequence of the dnaJ gene has:
  • nucleotide sequence encoding the same protein as the nucleotide sequence shown in (i), but different from the nucleotide sequence shown in (i) due to the degeneracy of the genetic code;
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (i) or (ii), and being the same as the nucleotide sequence indicated in (i) or (ii).
  • the nucleotide sequence of the dnaK gene has:
  • nucleotide sequence represented by (v) or (vi) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (v) or (vi), and being identical to the nucleotide sequence represented by (v) or (vi) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown; or
  • the nucleotide sequence of the GroEL gene has:
  • nucleotide sequence shown in (a) or (b) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (a) or (b), and being the same as the nucleotide sequence shown in (a) or (b).
  • the nucleotide sequence of the grpE gene has:
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence represented by (e) or (f), and being identical to the nucleotide sequence represented by (e) or (f) Nucleotides with the same or similar functions as the nucleotide sequences shown nucleotide sequence; or
  • the present invention also provides expression vectors, including any of the following:
  • the present invention also provides a method for constructing the expression vector, which includes: performing total synthesis of any of the following items, inserting into the multiple cloning site of the vector, and constructing the expression vector:
  • the present invention also provides a host, including any of the following:
  • the host includes engineered bacteria
  • the engineering bacteria include any of the following:
  • the chassis strain of the engineering bacteria is selected from E.coli K12, E.coli BL21, E.coli JM109, E.coli Rosetta, E.coli BL21 plysS, E.coli Top 10 , one of E.coli DH5 ⁇ .
  • the construction method of the engineering bacteria includes transferring any of the following items:
  • the present invention provides the application of any of the following items in the production of D-psicose:
  • the present invention provides the application of any of the following items in the production of D-psicose 3-epimerase:
  • the preparation of the crude enzyme solution of D-psicose 3-epimerase includes taking the engineering bacteria, inoculating it into LB liquid culture medium, and cultivating it to OD at 37°C. 600 reaches 0.7 ⁇ 0.8, join The final concentration is 0.5mmol/L IPTG, induce expression overnight at 22°C, 150rpm, centrifuge at 4°C, 6000r/min for 10-15min, collect the cells, and use buffer (20mmol/LTris-HCl, pH8.0) as follows Resuspend the bacterial cells at a ratio of 5:1 (add 5 mL of buffer to 1 gram of wet bacterial cells), ultrasonically disrupt, centrifuge at 10,000 r/min for 20 minutes, and collect the supernatant to obtain.
  • the present invention provides the use of any of the following items in the preparation of hypoglycemic drugs:
  • the invention provides medicines, including any of the following items and pharmaceutically acceptable excipients or auxiliaries;
  • the present invention provides pharmaceutical combinations, including the medicine and any other active ingredients.
  • the invention provides methods of treating disease, comprising administering any of the following:
  • the invention provides a preparation method of D-psicose, which can prepare D-psicose based on any of the following items:
  • the preparation method includes the following steps:
  • Step 1 Mix the host and inducer for fermentation and culture, separate and resuspend to obtain bacterial liquid;
  • Step 2 Take D-fructose solution and the bacterial liquid for biotransformation to obtain D-psicose.
  • the present invention provides a preparation method of D-psicose, which includes the following steps:
  • the fermentation medium in step 1) includes: yeast extract 20 ⁇ 25g/L, soy peptone 5 ⁇ 10g/L, glycerol 4 ⁇ 6g/L, potassium dihydrogen phosphate 2 ⁇ 3g/L, dipotassium hydrogen phosphate 12 ⁇ 16g/L;
  • the fermentation culture conditions include: initial pH 6-7, culture temperature 22-37°C, 200-600 rpm, culture 20-28 hours, and dissolved oxygen 20%-30%.
  • step 2) after fermentation and culture until OD 600 is greater than 20, the temperature is cooled to 22-25°C, and the inducer is added, and the amount of the inducer is 0.21-0.25 mmol/L final concentration;
  • the separation method includes centrifugation; the centrifugation speed is 6000r/min and the time is 10-15min;
  • Step 4) The D-fructose solution includes 500-800g/L, and the added amount of bacterial liquid includes 6-12g/L;
  • the conditions for the biotransformation are pH 6 to 7, 50 to 60°C, and transformation for 16 to 18 hours.
  • the present invention has the following advantages:
  • the present invention replaces the promoter for expressing the target gene in the vector expressing D-psicose 3-epimerase with the constitutive promoter HCE, thereby achieving more efficient expression by replacing the promoter.
  • the present invention integrates the molecular chaperone proteins dnaJ, dnaK, GroEL and grpE into the genome, allowing D-psicose 3-epimerase to co-express with the molecular chaperone, effectively reducing the formation of inclusion body proteins.
  • the molecular chaperone assists in folding to form a tetramer high-level structure, which improves the efficiency of expressing soluble proteins, thereby increasing the expression level of D-psicose 3-epimerase and reducing preparation costs.
  • the genes of the constitutive promoter HCE and the molecular chaperone protein of the present invention are derived from the same donor organism and have good homology, which contributes to the stability of the expression of the target gene and thereby improves the expression efficiency.
  • Figure 1 shows the nucleic acid electrophoresis verification diagram of Example 1
  • Figure 2 shows the plasmid map of the recombinant expression vector pHCE-DPE constructed in Example 1;
  • Figure 3 shows the plasmid map of the co-expression vector pUCMOD-JKLE constructed in Example 2;
  • Figure 4 shows the plasmid map of the co-expression vector pUCMOD-JKEL constructed in Example 3;
  • Figure 5 shows the map of the pET28a-DPE expression vector in Comparative Example 1;
  • Figure 6 shows the CP-1 genome gene operation diagram in Example 5.
  • Figure 7 shows the CP-2 genome gene operation diagram in Example 6
  • Figure 8 shows the agarose gel electrophoresis patterns of CP-1 and CP-2
  • Figure 9 shows the protein electrophoresis pattern of the D-psicose-3-epimerase crude enzyme solution prepared by the five engineering bacteria in Example 7;
  • Figure 10 shows the sample HPLC chromatogram of Example 8.
  • Figure 11 shows the standard product control chart of psicose in Example 8.
  • Figure 12 shows the standard product comparison chart of fructose in Example 8.
  • the invention discloses an engineering bacterium that efficiently expresses D-psicose 3-epimerase and its application. Persons skilled in the art can learn from the contents of this article and appropriately improve the process parameters to achieve it. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
  • the present invention constructs an engineering bacterium that efficiently expresses D-psicose 3-epimerase.
  • the engineering bacterium contains a constructed recombinant expression vector and a co-expression vector, which improves the efficiency of expressing soluble proteins, thereby improving The expression level of D-psicose 3-epimerase.
  • the present invention utilizes the D-psicose3-epimerase D-psicose3-epimerase in Ruminococcus sp., which can catalyze the formation of psicose from fructose.
  • the accession Number of this gene is: ZP_04858451.
  • the present invention believes that the key issue affecting the expression efficiency of psicose isomerase is that after expression of the enzyme, a large number of molecular chaperone proteins are required to assist in its folding to form a tetramer high-level structure before it becomes active.
  • the present invention uses the modified Plac promoter to constitutively express these molecular chaperone proteins in E. coli.
  • the A2 strain was formed after transforming pHCEIB-psicose isomerase into the A1 strain.
  • One of the technical solutions provided by the present invention is to provide a recombinant expression vector, which includes a gene expressing D-psicose 3-epimerase and a constitutive promoter HCE gene.
  • D-psicose 3-epimerase is the following a) or b) protein:
  • amino acid sequence represented by SEQ ID NO: 1 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and has D-psicose 3-epimerase activity
  • SEQ ID NO: 1 has undergone substitution and/or deletion and/or addition of one or several amino acid residues and has D-psicose 3-epimerase activity
  • constitutive promoter HCE is the DNA fragment of the following c) or d):
  • the vector is any one of pET21a, pET20b, pET22b, and pET28a.
  • the second technical solution provided by the present invention is to provide a co-expression vector, and the co-expression vector contains the gene of a molecular chaperone protein, and the molecular chaperone protein includes dnaJ, dnaK, GroEL and grpE.
  • nucleotide sequence of the dnaJ gene is shown in the sequence list SEQ ID NO: 3.
  • nucleotide sequence of the dnaK gene is shown in the sequence list SEQ ID NO: 4.
  • nucleotide sequence of the GroEL gene is shown in the sequence list SEQ ID NO: 5.
  • nucleotide sequence of the grpE gene is shown in the sequence list SEQ ID NO: 6.
  • the construction method of the co-expression vector includes:
  • the gene sequences of dnaJ, dnaK, GroEL and grpE were fully synthesized in the order of dnaJ-dnaK-GroEL-grpE, and the fully synthesized sequences were inserted into the multiple cloning site of the vector to construct a co-expression vector.
  • the construction method of the co-expression vector includes:
  • the gene sequences of dnaJ, dnaK, GroEL and grpE were fully synthesized in the order of dnaJ-dnaK-grpE-GroEL, and the fully synthesized sequences were inserted into the multiple cloning site of the vector to construct a co-expression vector.
  • the third technical solution provided by the present invention is an engineering bacterium that efficiently expresses D-psicose 3-epimerase, and the chassis strain of the engineering bacterium contains the above-mentioned recombinant expression vector and co-expression vector.
  • the chassis strain is selected from one of E.coli K12, E.coli BL21, E.coli JM109, E.coli Rosetta, E.coli BL21 plysS, E.coli Top 10, and E.coli DH5 ⁇ .
  • the construction method of the engineering strain that efficiently expresses D-psicose 3-epimerase is: the above recombinant expression vector and co-expression vector are transferred into the chassis strain to obtain the engineering strain. bacteria.
  • the invention provides an application of an engineering bacterium that efficiently expresses D-psicose 3-epimerase: it is used for intracellular production of D-psicose 3-epimerase and catalyzes D-psicose 3-epimerase. Fructose produces D-psicose.
  • the fourth technical solution provided by the present invention is a method for preparing D-psicose, which includes the following steps:
  • the invention provides a recombinant expression vector, which includes a gene expressing D-psicose 3-epimerase and a constitutive promoter HCE gene.
  • D-psicose 3-epimerase is derived from Ruminococcus sp.
  • D-psicose 3-epimerase is the following a) or b) protein:
  • amino acid sequence represented by SEQ ID NO: 1 has undergone substitution of one or several amino acid residues and/or Proteins derived from (a) that are deleted and/or added and have D-psicose 3-epimerase activity.
  • the constitutive promoter HCE is derived from Bacillus (Geobacillus sp.WCH70), which is one of the thermophilic organisms isolated from the hot compost in Middleton, Wisconsin, and was preserved in December 2009 at NCBI (CP001638).
  • the above-mentioned constitutive promoter HCE is the DNA fragment of the following c) or d):
  • the vector is a plasmid, and is selected from but not limited to any one of pET21a, pET20b, pET22b, and pET28a.
  • the vector used in the present invention is pET28a plasmid.
  • the present invention also provides a co-expression vector comprising genes for molecular chaperone proteins including dnaJ, dnaK, GroEL and grpE.
  • the above molecular chaperone proteins dnaJ, dnaK, GroEL and grpE are derived from Bacillus Geobacillus sp.WCH70.
  • nucleotide sequence of the dnaJ gene is shown in the sequence list SEQ ID NO: 3;
  • the nucleotide sequence of the dnaK gene is shown in the sequence list SEQ ID NO: 4;
  • the nucleotide sequence of the GroEL gene is shown in the sequence list SEQ ID NO: 5;
  • the nucleotide sequence of the grpE gene is shown in the sequence list SEQ ID NO: 6.
  • the vector is a plasmid, and one of pUC18 and pUC19 is selected but not limited to.
  • the assembly order of dnaJ, dnaK, GroEL and grpE genes in the vector is different, which has a certain impact on expression.
  • the construction method of the co-expression vector includes:
  • the gene sequences of dnaJ, dnaK, GroEL and grpE were fully synthesized in the order of dnaJ-dnaK-GroEL-grpE, and the fully synthesized sequences were inserted into the multiple cloning site of the vector to construct a co-expression vector.
  • the construction method of the co-expression vector includes:
  • the gene sequences of dnaJ, dnaK, GroEL and grpE were fully synthesized in the order of dnaJ-dnaK-grpE-GroEL, and the fully synthesized sequences were inserted into the multiple cloning site of the vector to construct a co-expression vector.
  • the present invention also provides an engineering bacterium that efficiently expresses D-psicose 3-epimerase, and the chassis strain of the engineering bacterium contains the above-mentioned recombinant expression vector. vectors and co-expression vectors.
  • the chassis strain is selected from one of E.coli K12, E.coli BL21, E.coli JM109, E.coli Rosetta, E.coli BL21 plysS, E.coli Top 10, and E.coli DH5 ⁇ .
  • the construction method of the engineering strain that efficiently expresses D-psicose 3-epimerase is: the above recombinant expression vector and co-expression vector are transferred into the chassis strain to obtain the engineering strain. bacteria.
  • the invention provides an application of an engineering bacterium that efficiently expresses D-psicose 3-epimerase: it is used for intracellular production of D-psicose 3-epimerase and catalyzes D-psicose 3-epimerase. Fructose produces D-psicose.
  • the fourth technical solution provided by the present invention is a method for preparing D-psicose, which includes the following steps:
  • the fermentation medium is: yeast extract 20 ⁇ 25g/L, soy peptone 5 ⁇ 10g/L, glycerin 4 ⁇ 6g/L, potassium dihydrogen phosphate 2 ⁇ 3g/L, dipotassium hydrogenphosphate 12 ⁇ 16g/L.
  • the conditions for fermentation culture are: initial pH 6 to 7, culture temperature 22 to 37°C, 200 to 600 rpm, culture for 20 to 28 hours, and dissolved oxygen 20% to 30%.
  • step 2) after fermentation and culture until OD 600 is greater than 20, the temperature is cooled to 22-25°C, and the inducer is added.
  • the amount of the inducer is 0.21-0.25 mmol/L final concentration.
  • the inducer is IPTG.
  • step 4 the D-fructose solution is 500-800g/L, and the amount of bacterial liquid added is 6-12g/L.
  • the conditions for biotransformation are pH 6 to 7, 50 to 60°C, and transformation for 16 to 18 hours.
  • the engineering bacterium that efficiently expresses D-psicose 3-epimerase and the raw materials and reagents used in its application can be purchased from the market. Experimental methods without specifying specific conditions in the examples of the present invention are usually carried out according to conventional conditions, such as the conditions described in the "Molecular Cloning Experiment Guide: 4th Edition", or according to the instructions of the kit or the conditions recommended by the reagent manufacturer. .
  • the gene of D-psicose 3-epimerase is derived from Ruminococcus sp.
  • the gene of constitutive promoter HCE is derived from Bacillus Geobacillus sp.WCH70.
  • the nucleotide sequence of the D-psicose 3-epimerase gene was optimized by Genescript.
  • the constitutive promoter HCE and ribosome binding site (RBS) sequences were added to the 5' end, and XbaI was introduced at both ends. and XhoI restriction site, and perform total synthesis to obtain a fully synthetic gene fragment.
  • the nucleotide sequence of the above-mentioned fully synthetic gene fragment is shown in the sequence list SEQ ID NO: 7.
  • the fully synthetic gene fragment and pET28a vector were digested with XbaI and XhoI respectively, and the target fragment was recovered and purified using a gel recovery kit. The two products were ligated with T4 ligase to obtain the recombinant plasmid.
  • This recombinant plasmid was transformed into E.coli DH5 ⁇ competent cells, spread on kanamycin-resistant plates, and cultured at 37°C overnight. Pick the grown positive clones, extract the plasmid and conduct sequencing verification to obtain the recombinant expression vector pHCE-DPE, whose map is shown in Figure 2.
  • the plasmid is extracted, the target gene fragment is obtained by PCR, and nucleic acid electrophoresis is performed for verification.
  • the PCR primers are as follows:
  • Nucleic acid electrophoresis verification is shown in Figure 1.
  • the size of the target gene is 873bp.
  • the electrophoresis verification result is consistent with the size of the target gene, indicating that the recombinant expression vector was successfully constructed.
  • genes of the four molecular chaperone proteins dnaJ, dnaK, GroEL, and grpE are derived from Bacillus Geobacillus sp.WCH70.
  • the fully synthetic gene fragment and pUC 19 vector were digested with XbaI and KpnI respectively, and the target fragment was recovered and purified using a gel recovery kit. The two products were ligated with T4 ligase to obtain the recombinant plasmid.
  • This recombinant plasmid was transformed into E.coli DH5 ⁇ competent cells, spread on ampicillin-resistant plates, and cultured at 37°C overnight. Pick the grown positive clones, extract the plasmid and conduct sequencing verification to obtain the co-expression vector pUCMOD-JKLE, whose map is shown in Figure 3.
  • genes of the four molecular chaperone proteins dnaJ, dnaK, GroEL, and grpE are derived from Bacillus Geobacillus sp.WCH70.
  • the molecular chaperone proteins dnaJ, dnaK, grpE, and GroEL were assembled in the order of dnaJ-dnaK-grpE-GroEL.
  • the RBS sequence was inserted after dnaJ and grpE, and the lac promoter and RBS sequence were inserted after dnaK.
  • XbaI and KpnI restriction sites were introduced at both ends of the sequence, and total synthesis was performed by Genescript Company to obtain a fully synthetic gene fragment.
  • the nucleotide sequence of the gene fragment is shown in the sequence list SEQ ID NO: 9.
  • the fully synthetic gene fragment and pUC 19 vector were digested with XbaI and KpnI respectively, and the target fragment was recovered and purified through a gel recovery kit. The two products were connected with T4 ligase to obtain the recombinant plasmid.
  • This recombinant plasmid was transformed into E.coli DH5 ⁇ competent cells, spread on ampicillin-resistant plates, and cultured at 37°C overnight. The grown positive clones were picked, and the plasmid was extracted for sequencing verification to obtain the co-expression vector pUCMOD-JKEL, whose map is shown in Figure 4.
  • the recombinant expression plasmid pHCE-DPE prepared in Example 1 was transformed into E.coli BL21 (DE3) competent cells, cultured on a plate containing kanamycin, positive clones were picked, plasmids were extracted, and after sequencing and verification, Strain DPE-A was obtained.
  • DPE-A strain to prepare competent cells, transform the molecular chaperone plasmid pUCMOD-JKLE prepared in Example 2 into the DPE-1 competent cells, culture them on a plate containing ampicillin, pick positive clones, extract the plasmid, and sequence After verification, the engineering bacterium DPE-B1 was obtained.
  • DPE-A strain to prepare competent cells, transform the molecular chaperone plasmid pUCMOD-JKEL prepared in Example 3 into the DPE-1 competent cells, culture them on a plate containing ampicillin, pick positive clones, extract the plasmid, and sequence After verification, the engineering bacterium DPE-B2 was obtained.
  • Example 5 Construction of a high-yield psicose strain to obtain a chassis strain DPE-D for psicose production
  • the genes of the molecular chaperone proteins dnaJ, dnaK, GroEL, and grpE were sequentially inserted into the lacZ position of the JM109 strain genome through CRISPR-Cas9 system editing. Among them, the required CP-1 fragment (linear DNA fragment) and plasmid pgRNA-1 need to be constructed.
  • Plasmid pgRNA-1 was fully synthesized by Genescript Company, and its sequence is shown in SEQ ID NO: 15.
  • CP-1 linear fragment The construction method of CP-1 linear fragment is as follows. Using two pairs of PCR primers pCP1-1 and pCP1-2 and pCP1-5 and pCP1-6, and using the E. coli JM109 genome as a template, the CP-1U and CP-1D fragments were amplified. Using pCP1-3 and pCP1-4 primers, and using the vector pUCMOD-JKLE constructed in Example 2 as a template, amplification was performed to obtain the CP-1M fragment. 1.0% agarose gel electrophoresis was used to detect and cut the gel to recover the three fragments, and fusion PCR was performed. The obtained bands were then recovered using 1.0% agarose gel electrophoresis to obtain the fusion fragment CP-1.
  • the genome gene operation diagram is shown in Figure 6 below.
  • the CP-1 fragment (400ng) and plasmid pgRNA-1 (100ng) were electrotransformed into the strain JM109 containing the pCas9 vector. After overnight culture, a single colony was picked as a template, and PCR was performed with primer pCP1-1 and primer pCP1-6, and the dnaJ, dnaK, GroEL, and grpE genes were confirmed by observing the presence of a 6500-bp DNA band in a 1.0% agarose gel. have been integrated into the chromosome.
  • the PCR product was sequenced and verified, and the sequencing results confirmed through BLAST sequence comparison that the position of the lacZ gene had been successfully replaced by the dnaJ, dnaK, GroEL, and grpE genes.
  • the constructed strain was designated as DPE-D.
  • Example 6 Construction of a high-yield psicose strain to obtain a chassis strain DPE-E for psicose production
  • the genes of the molecular chaperone proteins dnaJ, dnaK, grpE, and GroEL were sequentially inserted into the lacZ position of the JM109 strain genome through CRISPR-Cas9 system editing. Among them, the required CP-2 fragment (linear DNA fragment) and plasmid pgRNA-1 need to be constructed.
  • Plasmid pgRNA-1 has been described in Example 5 above.
  • CP-2 linear fragments are constructed as follows. Using two pairs of PCR primers pCP2-1 and pCP2-2 and pCP2-5 and pCP2-6, and using the E. coli JM109 genome as a template, the CP-2U and CP-2D fragments were amplified. Using pCP2-3 and pCP2-4 primers, and using the vector pUCMOD-JKEL constructed in Example 3 as a template, amplification was performed to obtain the CP-2M fragment. 1.0% agarose gel electrophoresis was used to detect and cut the gel to recover the three fragments, and fusion PCR was performed. The obtained bands were then recovered using 1.0% agarose gel electrophoresis to obtain the fusion fragment CP-2.
  • the genome gene operation diagram is shown in Figure 7 below.
  • the CP-2 fragment (400ng) and plasmid pgRNA-1 (100ng) were electrotransformed into the strain JM109 containing the pCas9 vector. After overnight culture, a single colony was picked as a template, and PCR was performed with primer pCP2-1 and primer pCP2-6, and the dnaJ, dnaK, grpE, and GroEL genes were confirmed by observing the presence of a 6500-bp DNA band in a 1.0% agarose gel. have been integrated into the chromosome.
  • the PCR product was sequenced and verified, and the sequencing results confirmed through BLAST sequence comparison that the position of the lacZ gene had been successfully replaced by the dnaJ, dnaK, grpE, and GroEL genes.
  • the constructed strain was designated as DPE-E.
  • the gene of D-psicose 3-epimerase is derived from Ruminococcus sp.
  • the gene of constitutive promoter HCE is derived from Bacillus Geobacillus sp.WCH70.
  • the nucleotide sequence of the D-psicose 3-epimerase gene was optimized by Genescript, and XbaI and XhoI enzyme cutting sites were introduced at both ends for total synthesis to obtain a fully synthetic gene fragment (SEQ ID NO: 14).
  • the fully synthetic gene fragment and pET28a vector were digested with XbaI and XhoI respectively, and the target fragment was recovered and purified using a gel recovery kit. The two products were ligated with T4 ligase to obtain the recombinant plasmid.
  • This recombinant plasmid was transformed into E.coli DH5 ⁇ competent cells, spread on kanamycin-resistant plates, and cultured at 37°C overnight. The grown positive clones were picked, the plasmid was extracted for sequencing verification, and the recombinant expression vector pET28a-DPE was obtained.
  • the map of pET28a-DPE expression vector is shown in Figure 5.
  • the recombinant expression vector pET28a-DPE was transformed into E.coli BL21 (DE3) competent cells, and cells containing card were used. Culture the plate of namycin, pick the positive clones, extract the plasmid, and sequence it to verify that it is correct, and then obtain the engineering strain DPE-C.
  • Ultrasonic fragmentation running time 1s; interval time 2s; total time 5min; temperature 10-15°C; power 600w
  • centrifuge at 10000r/min for 20min collect the supernatant to obtain D-psicose-3-epimer Crude enzyme solution of enzyme.
  • Lane 1 is the crude enzyme prepared from the empty chassis strain.
  • the second lane is the crude enzyme solution prepared by the engineering strain DPE-B1; the third lane is the crude enzyme solution prepared by the engineering strain DPE-B2; the fourth lane is the crude enzyme solution prepared by the engineering strain DPE-D;
  • the 5th lane is the crude enzyme solution prepared by the engineering strain DPE-E;
  • the 6th lane is the engineering strain DPE-C.
  • the protein electrophoresis verification results were consistent with the size of the target gene, indicating that the above five engineering bacteria successfully expressed the target gene, and the efficiency of the expressed protein was verified by the depth of the bands.
  • HPLC chromatography conditions are as follows:
  • Injection volume 10 ⁇ L.
  • the engineering strain JM109dnaJ dnaK grpE GroEL(lac) ⁇ lacZ with the best conversion rate in Example 7 was selected as the strain for biosynthetic preparation of D-psicose.
  • the above genetically engineered strain was inoculated into the fermentation medium for fermentation culture.
  • the fermentation medium It is yeast extract 25g/L, soy peptone 8g/L, glycerin 6g/L, potassium dihydrogen phosphate 2g/L, and potassium dihydrogen phosphate 15g/L.
  • the initial pH of the fermentation culture was 7, the culture temperature was 26°C, the dissolved oxygen was controlled at 20% to 30%, and the culture was carried out for 24 hours.
  • the temperature is lowered to 22°C and the inducer is added.
  • the inducer is IPTG, and the added amount of IPTG is 0.25 mmol/L final concentration.
  • the fermentation liquid is obtained and centrifuged at 6000r/min for 10-15min to obtain bacterial cells.
  • Use buffer (20mmol/LTris-HCl, pH 8.0) according to the ratio of 5:1 (resuspend the bacterial cells to obtain bacterial liquid, add 12g/L bacterial liquid to 700g/L D-fructose solution, pH is 6, Bioconversion was carried out at 60°C, and the conversion time was 18 hours. After the conversion, samples were taken for HPLC to determine the D-psicose content, and the conversion rate was calculated. The results are shown in Figure 10. The results show that the peak area of D-psicose It is 431106.
  • the standard product control chart of psicose ( Figure 11) and the standard product control chart of fructose ( Figure 12) are used to calculate the conversion rate through the external standard method based on the standard product data.
  • cx is the sample concentration; cr is the control concentration; Ax is the sample peak area; Ar is the control peak area.

Abstract

本发明属于生物工程技术领域，具体涉及一种高效表达D-阿洛酮糖3-差向异构酶的工程菌，该工程菌中包含构建的重组表达载体和共表达载体，所述重组表达载体包含表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE基因，所述共表达载体包含分子伴侣蛋白的基因，所述分子伴侣蛋白包括dnaJ、dnaK、GroEL和grpE。本发明通过替换启动子更高效的进行表达，并使D-阿洛酮糖3-差向异构酶与分子伴侣共表达，有效减少包涵体蛋白的形成，分子伴侣进行辅助折叠形成四聚体高级结构，提高表达出可溶蛋白的效率，从而提高D-阿洛酮糖3-差向异构酶的表达水平，降低制备成本。

Description

一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用
本申请要求于2022年07月01日提交中国专利局、申请号为202210768159.2、发明名称为“一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物工程技术领域，具体涉及一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用。
背景技术
阿洛酮糖又称D-阿洛酮糖，为D-果糖三号位碳所对应的差向异构体，阿洛酮糖具有与蔗糖相似的口感，但其甜度相当于蔗糖的70％，热量相当于蔗糖的0.3％，且几乎不在体内代谢。并且由于可以通过抑制葡萄糖的吸收来抑制葡萄糖，因此具有降血糖的功效，是最具潜力的蔗糖替代品之一。
阿洛酮糖天然存在于葡萄干、无花果、猕猴桃和红糖等食物中，是自然界中含量极少的稀有单糖，依靠天然产物提取的方法成本高，且很难达到量产。现有技术中，制造D-阿洛酮糖的方法中有化学方法和生物方法。如比利克(bilik)等人曾研发利用钼酸离子的催化作用来由果糖生产阿洛酮糖的技术。麦克唐纳(mcdonald)通过对1,2:4,5-二-o-丙酮缩甘油-β-d-果糖进行三步骤化学处理过程来生产了阿洛酮糖。
但化学方法会产生大量的副产物和使用大量化学试剂，且生产效率并不高。近年来，生物方法制备阿洛酮糖以其安全、绿色、环保的优势而逐渐成为未来的主流趋势。早期，肯·伊兹莫里(kenizumori)等人证实了利用微生物细胞反应以半乳糖醇、d-塔格糖或d-塔罗糖醇为原料生产阿洛酮糖，但原料不易取得，成本高。经过前人的不断研究，现阶段的生物转化法主要是以果糖为原料，经D-阿洛酮糖3-差向异构酶催化合成D-阿洛酮糖，该方法催化合成效率较高，是目前合成阿洛酮糖的主要方式。
如申请号为201710124243.X公开的一种D-阿洛酮糖3-差向异构酶的应用，其中记载的D-阿洛酮糖3-差向异构酶为类芽孢杆菌(Paenibacillus senegalensis)的D-阿洛酮糖3-差向异构酶，并且该D-阿洛酮糖3-差向异构酶用于在工业上催化生产阿洛酮糖，或用作工业上催化生产阿洛酮糖的催化剂。
但由于来源于野生菌株未经改造的D-阿洛酮糖3-差向异构酶在热稳定性、催化活性等方面存在一定的局限性，导致酶的应用成本偏高，限制了其工业应用范围。现有技术常通过改造菌体来提高D-阿洛酮糖3-差向异构酶的催化活性，如申请号为201610818847.X公开的一种D-阿洛酮糖3-差向异构酶的突变体及其应用，该专利提供了D-阿洛酮糖3-差向异构酶的突变体，含有G86D、D164E、W262S突变位点中的一个或多个突变位点，催化活性得到显著提高，催化活性为野生型的1.4倍以上，降低了合成D-阿洛酮糖过程中的酶的用量。
虽然现有技术通过突变等方式提高了酶的催化活性，但仍然存在用大肠杆菌或芽孢杆菌表达蛋白时有大量包涵体蛋白，表达出可溶蛋白效率较低的问题。且由于阿洛酮糖与果糖的差价较低，因此制备阿洛酮糖的关键因素在于酶的制备成本。基于此，如何构建出高效可溶性表达D-阿洛酮糖3-差向异构酶的工程菌株成为低成本制备阿洛酮糖的关键点。
发明内容
有鉴于此，本发明提供一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用。本发明构建了高效表达D-阿洛酮糖3-差向异构酶的工程菌，该工程菌中包含构建的重组表达载体和共表达载体，提高了表达出可溶蛋白的效率，从而提高了D-阿洛酮 糖3-差向异构酶的表达水平。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了如下任意项在生产D-阿洛酮糖中的应用：
(Ⅰ)、共表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE；和/或
(Ⅱ)、共表达D-阿洛酮糖3-差向异构酶的基因、组成型启动子HCE和RBS序列；和/或
(Ⅲ)、共表达dnaJ、dnaK、GroEL和/或grpE；和/或
(Ⅳ)、共表达dnaJ、dnaK、GroEL、grpE、RBS序列和/或lac启动子；
(Ⅴ)、缺失lacZ基因和共表达dnaJ、dnaK、GroEL和/或grpE。
本发明还提供了表达元件，所述表达元件包括表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE基因。
在本发明的一些具体实施方案中，所述D-阿洛酮糖3-差向异构酶的基因来源于瘤胃球菌Ruminococcus sp.；所述HCE基因来源于芽孢杆菌Geobacillus sp.WCH70。
在本发明的一些具体实施方案中，所述表达元件还包括RBS序列。
在本发明的一些具体实施方案中，所述RBS序列于5’端添加。
在本发明的一些具体实施方案中，所述D-阿洛酮糖3-差向异构酶具有：
(Ⅰ)、如SEQ ID NO：1所示的氨基酸序列；
(Ⅱ)、在如(Ⅰ)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
(Ⅲ)、与如(Ⅰ)所示的氨基酸序列具有至少90％序列同源性的氨基酸序列；
所述组成型启动子HCE具有：
(Ⅳ)、如SEQ ID NO:2所示核苷酸序列的DNA片段；或
(Ⅴ)、与(Ⅳ)所述核苷酸序列具有至少90％序列同源性，且具有启动子功能的DNA片段。
在本发明的一些具体实施方案中，常用的载体均适用于该重组表达载体，没有特殊限制，优选的，所述载体为质粒，选择但不限于pET21a、pET20b、pET22b、pET28a中的任意一种；
进一步优选的，本发明采用的载体为pET28a质粒。
在上述研究的基础上，本发明还提供了共表达元件，所述共表达元件包括分子伴侣蛋白的基因和/或缺失lacZ基因；
所述分子伴侣蛋白包括dnaJ、dnaK、GroEL和/或grpE。
在本发明的一些具体实施方案中，所述dnaJ、dnaK、GroEL和/或grpE的基因来源于芽孢杆菌Geobacillus sp.WCH70。
在本发明的一些具体实施方案中，所述共表达元件包括如下任意项和/或缺失lacZ基因：
(Ⅰ)、依次包括dnaJ、dnaK、GroEL和grpE；和/或
(Ⅱ)、依次包括dnaJ、dnaK、grpE和GroEL。
在本发明的一些具体实施方案中，所述缺失lacZ基因采用的质粒gRNA-1具有：
(A)、如SEQ ID NO:15所示的核苷酸序列；或
(B)、与(A)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(A)所示的核苷酸序列不同的核苷酸序列；或
(C)、与(A)或(B)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(A)或(B)所示的核苷酸序列功能相同或相似的 核苷酸序列；或
(D)、与(A)～(C)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列。
在本发明的一些具体实施方案中，所述共表达元件还包括RBS序列和/或lac启动子。
在本发明的一些具体实施方案中，所述共表达元件包括如下任意项：
(Ⅰ)、依次包括dnaJ、RBS序列、dnaK、lac启动子、RBS序列、GroEL、RBS序列和grpE；和/或
(Ⅱ)、依次包括dnaJ、RBS序列、dnaK、lac启动子、RBS序列、grpE、RBS序列和GroEL。
在本发明的一些具体实施方案中，所述dnaJ基因的核苷酸序列具有：
(i)、如SEQ ID NO:3所示的核苷酸序列；或
(ii)、与(i)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(i)所示的核苷酸序列不同的核苷酸序列；或
(iii)、与(i)或(ii)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(i)或(ii)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(iv)、与(i)～(iv)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
所述dnaK基因的核苷酸序列具有：
(v)、如SEQ ID NO:4所示的核苷酸序列；或
(vi)、与(v)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)所示的核苷酸序列不同的核苷酸序列；或
(vii)、与(v)或(vi)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(v)或(vi)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(viii)、与(v)～(vii)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
所述GroEL基因的核苷酸序列具有：
(a)、如SEQ ID NO:5所示的核苷酸序列；或
(b)、与(a)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(a)所示的核苷酸序列不同的核苷酸序列；或
(c)、与(a)或(b)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(a)或(b)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(d)、与(a)～(c)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
所述grpE基因的核苷酸序具有：
(e)、如SEQ ID NO:6所示的核苷酸序列；或
(f)、与(e)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(e)所示的核苷酸序列不同的核苷酸序列；或
(g)、与(e)或(f)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(e)或(f)所示的核苷酸序列功能相同或相似的核 苷酸序列；或
(h)、与(e)～(g)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列。
本发明还提供了表达载体，包括如下任意项：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件。
本发明还提供了所述表达载体的构建方法，包括：将如下任意项进行全合成，插入载体的多克隆位点，构建所述表达载体：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件。
本发明还提供了宿主，包括如下任意项：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体。
在本发明的一些具体实施方案中，所述宿主包括工程菌；
所述工程菌包括如下任意项：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体。
在本发明的一些具体实施方案中，所述工程菌的底盘菌株选自E.coli K12、E.coli BL21、E.coli JM109、E.coli Rosetta、E.coli BL21 plysS、E.coli Top 10、E.coli DH5α中的一种。
在本发明的一些具体实施方案中，所述工程菌的构建方法包括转入如下任意项：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体。
本发明提供了如下任意项在生产D-阿洛酮糖中的应用：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体；和/或
(Ⅴ)、所述宿主。
本发明提供了如下任意项在生产D-阿洛酮糖3-差向异构酶中的应用：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体；和/或
(Ⅴ)、所述宿主。
在本发明的一些具体实施方案中，所述D-阿洛酮糖3-差向异构酶的粗酶液制备包括取所述工程菌，接种于LB液体培养基中，37℃培养至OD600达到0.7～0.8，加入 终浓度为0.5mmol/L IPTG，在22℃、150rpm条件下诱导表达过夜，4℃，6000r/min离心10-15min，收集菌体，用缓冲液(20mmol/LTris-HCl，pH8.0)按照5：1的比例(1克湿菌体添加5mL缓冲液)重悬菌体，超声破碎，10000r/min离心20min，收集上清液，得到。
本发明提供了如下任意项在制备降血糖药物中的应用：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体；和/或
(Ⅴ)、所述宿主。
本发明提供了药物，包括如下任意项与药学上可接受的辅料或助剂；
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述质粒；和/或
(Ⅳ)、所述宿主。
本发明提供了药物组合，包括所述药物以及其他任意有效成分。
本发明提供了治疗疾病的方法，包括施用如下任意项：
(Ⅰ)、所述药物；和/或
(Ⅱ)、所述药物组合。
本发明提供了D-阿洛酮糖的制备方法，基于如下任意项制得D-阿洛酮糖：
(Ⅰ)、所述表达元件；和/或
(Ⅱ)、所述共表达元件；和/或
(Ⅲ)、所述表达载体；和/或
(Ⅳ)、所述构建方法获得的表达载体；和/或
(Ⅴ)、所述宿主。
在本发明的一些具体实施方案中，所述制备方法包括如下步骤：
步骤1：将所述宿主与诱导剂混合发酵培养，分离重悬得到菌液；
步骤2：取D-果糖溶液与所述菌液进行生物转化，得到D-阿洛酮糖。
具体地，本发明提供了D-阿洛酮糖的制备方法，包括如下步骤：
1)将所述工程菌接种至发酵培养基中进行发酵培养；
2)在发酵培养基中添加诱导剂进行诱导表达，得到发酵液；
3)分离发酵液，获得菌体，加入缓冲液重悬，得到菌液；
4)向D-果糖溶液中加入菌液进行生物转化，得到D-阿洛酮糖。
在本发明的一些具体实施方案中，步骤1)所述发酵培养基包括：酵母浸膏20～25g/L，大豆蛋白胨5～10g/L，甘油4～6g/L，磷酸二氢钾2～3g/L，磷酸氢二钾12～16g/L；
所述发酵培养的条件包括：初始pH6～7，培养温度22～37℃，200～600rpm，培养20～28h，溶氧20％～30％。
在本发明的一些具体实施方案中，步骤2)中发酵培养至OD600大于20后，降温至22～25℃，开始加入诱导剂，诱导剂的加入量为0.21～0.25mmol/L终浓度；所述分离采用方式包括离心；所述离心的转速为6000r/min，时间为10-15min；
步骤3)所述缓冲液选用Tris-HCl缓冲液，pH＝8.0；所述缓冲液与菌体的添加比例为5：1；
步骤4)所述D-果糖溶液包括500～800g/L，菌液加入量包括6-12g/L；
所述生物转化的条件为pH6～7，50～60℃，转化16～18h。
现有技术相比，本发明具有以下优点：
本发明一方面将表达D-阿洛酮糖3-差向异构酶的载体中表达目的基因的启动子替换为组成型启动子HCE，通过替换启动子更高效的进行表达。另一方面，本发明将分子伴侣蛋白dnaJ、dnaK、GroEL和grpE整合在了基因组上，使D-阿洛酮糖3-差向异构酶与分子伴侣共表达，有效减少包涵体蛋白的形成，分子伴侣进行辅助折叠形成四聚体高级结构，提高表达出可溶蛋白的效率，从而提高D-阿洛酮糖3-差向异构酶的表达水平，降低制备成本。且本发明的组成型启动子HCE和分子伴侣蛋白的基因来源于同一供体生物，同源性好，有助于目的基因表达的稳定性，进而提升表达效率。
附图说明
图1示实施例1的核酸电泳验证图；
图2示实施例1构建的重组表达载体pHCE-DPE的质粒图谱；
图3示实施例2构建的共表达载体pUCMOD-JKLE的质粒图谱；
图4示实施例3构建的共表达载体pUCMOD-JKEL的质粒图谱；
图5示对比例1中pET28a-DPE表达载体图谱；
图6示实施例5中CP-1染色体组基因操作图；
图7示实施例6中CP-2染色体组基因操作图；
图8示CP-1、CP-2的琼脂糖凝胶电泳图；
图9示实施例7中五种工程菌制备的D-阿洛酮糖-3-差向异构酶粗酶液的蛋白电泳图；
图10示实施例8的样品HPLC色谱图；
图11示实施例8的阿洛酮糖的标品对照图；
图12示实施例8的果糖的标品对照图。
具体实施方式
本发明公开了一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
为了实现上述发明目的，本发明提供以下技术方案：
本发明构建了高效表达D-阿洛酮糖3-差向异构酶的工程菌，该工程菌中包含构建的重组表达载体和共表达载体，提高了表达出可溶蛋白的效率，从而提高了D-阿洛酮糖3-差向异构酶的表达水平。
本发明利用的是Ruminococcus sp.中的D-阿洛酮糖3-差向异构酶D-psicose3-epimerase，该酶可以催化果糖形成阿洛酮糖。该基因的accession Number是：ZP_04858451。参考文献请见：
Zhu Y,Men Y,Bai W,Li X,Zhang L,Sun Y et al.,Overexpression of D-psicose3-epimerase from Ruminococcus sp.in Escherichia coli and its potential application in D-psicose production.Biotechnol Lett 34:1901–1906(2012).
Li C,Lin J,Guo Q,et al.D-Psicose 3-epimerase secretory overexpression,immobilization,and d-psicose biotransformation,separation and crystallization[J].Journal  of Chemical Technology & Biotechnology,2018,93(2).
本发明用于解决上述技术问题的技术方案如下：
1、提供可以高效组成型表达的组成型表达启动子
利用Geobacillus sp.WCH70的HCE promoter为启动子对阿洛酮糖的酶进行表达：
promoter序列
2、构建高表达Geobacillus分子伴侣蛋白的重组表达工程菌株
经过分析本发明认为影响阿洛酮糖异构酶表达效率的关键问题在于该酶表达后需要大量的分子伴侣蛋白对其进行辅助折叠形成四聚体高级结构后方有活性。
为了配合地芽孢杆菌Geobacillus的HCE启动子工作，本发明在大肠杆菌中利用修改后的Plac启动子对这些分子伴侣蛋白进行组成型表达。
A、从Geobacillus sp.WCH70基因组中将分子伴侣蛋白
dnaJ(1143bp，381aa，42.15kDa)SEQ ID NO：3：
dnaK(1830bp，610aa，66.21kDa)SEQ ID NO：4：

GroEL(1620aa，540aa，57.3kDa)SEQ ID NO：5：
grpE(627bp，209aa，24.12kDa)SEQ ID NO：6：
(atggaaaaagagcagaaagcagcacaagaacaggctacatacgaacaggaaccgttaaatacggaaccgcaagaggaaaaagtagagcaacatgaagtaaatgaacatcaagaggaaatcgaaatagaagggcaagaaaaagcacaagaagagcaaaacgatgaattggcggcggcaaacgcaaaaattgcggaactagaagcgaaaataaaagaaatggagaaccgctatcttcgtttatacgccgattttgaaaatttccgccgtcgtacaaaaatggaaatggaagcagctgaaaaatatcgcgcccaaagcttggttagcgatcttttgcctgctttggacaactttgagcgtgcgttaaagatagaggctgataacgaacaagcaaaatcgattctgcaaggaatggaaatggtgtatcgctccgtgttggatgcgctgaaaaaagaaggagtagaagcgatcgaagcggttggcaaaccgtttgatccgaacttgcatcaagccgtgatgcaagtagaagacagcaattatgagccgaatacagttgtggaagaatttcagaaaggttataaactgaaagatcgtgtcattcgtccagcaatggtgaaagtaagccaataa)的组成型表达质粒。
B、构建大肠杆菌基因编辑系统将组成型表达质粒整合至大肠杆菌的基因组中：
利用基因编辑技术在大肠杆菌JM109的recA处切开；利用同源重组将四个分子伴侣蛋白重组入大肠杆菌的基因组中构建出A1菌株。
3、构建出可以高效可溶性表达阿洛酮糖酶的工程菌株：
将pHCEIIB-阿洛酮糖异构酶转化至A1菌株后形成A2菌株。
本发明提供的技术方案之一，是提供重组表达载体，所述重组表达载体包含表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE基因。
进一步的，所述D-阿洛酮糖3-差向异构酶是如下a)或b)蛋白质：
a)具有SEQ ID NO：1所示氨基酸序列的蛋白质；
b)由SEQ ID NO：1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有D-阿洛酮糖3-差向异构酶活性的由(a)衍生的蛋白质。
进一步的，所述组成型启动子HCE是如下c)或d)的DNA片段：
c)具有SEQ ID NO：2所示核苷酸序列的DNA片段；
d)与SEQ ID NO：2所示核苷酸序列具有90％以上同源性，且具有启动子功能的DNA片段。
优选的，所述载体为pET21a、pET20b、pET22b、pET28a中的任意一种。
本发明提供的技术方案之二，是提供共表达载体，所述共表达载体包含分子伴侣蛋白的基因，所述分子伴侣蛋白包括dnaJ、dnaK、GroEL和grpE。
进一步的，所述dnaJ基因的核苷酸序列如序列表SEQ ID NO：3所示。
进一步的，所述dnaK基因的核苷酸序列如序列表SEQ ID NO：4所示。
进一步的，所述GroEL基因的核苷酸序列如序列表SEQ ID NO：5所示。
进一步的，所述grpE基因的核苷酸序列如序列表SEQ ID NO：6所示。
在一种具体实施方式中，所述的共表达载体的构建方法，包括：
将dnaJ、dnaK、GroEL和grpE的基因序列按照dnaJ-dnaK-GroEL-grpE的顺序进行全合成，将全合成的序列插入到载体的多克隆位点，构建成共表达载体。
在另一种具体实施方式中，所述的共表达载体的构建方法，包括：
将dnaJ、dnaK、GroEL和grpE的基因序列按照dnaJ-dnaK-grpE-GroEL的顺序进行全合成，将全合成的序列插入到载体的多克隆位点，构建成共表达载体。
本发明提供的技术方案之三，是一种高效表达D-阿洛酮糖3-差向异构酶的工程菌，所述工程菌的底盘菌株中包含上述重组表达载体和共表达载体。
优选的，述底盘菌株选自E.coli K12、E.coli BL21、E.coli JM109、E.coli Rosetta、E.coli BL21 plysS、E.coli Top 10、E.coli DH5α中的一种。
在一种具体实施方式中，所述高效表达D-阿洛酮糖3-差向异构酶的工程菌的构建方法为：将上述重组表达载体和共表达载体转入底盘菌株中，得到工程菌。
本发明提供的一种高效表达D-阿洛酮糖3-差向异构酶的工程菌的应用：是用于胞内生产D-阿洛酮糖3-差向异构酶，催化D-果糖生产D-阿洛酮糖。
本发明提供的技术方案之四，是一种制备D-阿洛酮糖的方法，包括如下步骤：
1)将所述基因工程菌株接种至发酵培养基中进行发酵培养；
2)在发酵培养基中添加诱导剂进行诱导表达，得到发酵液；
3)分离发酵液，获得菌体，加入缓冲液重悬，得到菌液；
4)向D-果糖溶液中加入菌液进行生物转化，得到D-阿洛酮糖。
本发明提供的重组表达载体，所述重组表达载体包含表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE基因。
其中，所述D-阿洛酮糖3-差向异构酶来源于瘤胃球菌(Ruminococcus sp.)。
上述D-阿洛酮糖3-差向异构酶是如下a)或b)蛋白质：
a)具有SEQ ID NO：1所示氨基酸序列的蛋白质；
b)由SEQ ID NO：1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或 缺失和/或添加且具有D-阿洛酮糖3-差向异构酶活性的由(a)衍生的蛋白质。
所述组成型启动子HCE来源于芽孢杆菌(Geobacillus sp.WCH70)，芽孢杆菌(Geobacillus sp.WCH70)是从威斯康星州Middleton的热堆肥中分离出来的嗜热生物之一，于2009年12月保藏在NCBI(CP001638)。
上述组成型启动子HCE是如下c)或d)的DNA片段：
c)具有SEQ ID NO：2所示核苷酸序列的DNA片段；
d)与SEQ ID NO：2所示核苷酸序列具有90％以上同源性，且具有启动子功能的DNA片段。
常用的载体均适用于该重组表达载体，没有特殊限制。优选的，载体为质粒，选择但不限于pET21a、pET20b、pET22b、pET28a中的任意一种。
进一步优选的，本发明采用的载体为pET28a质粒。
本发明还提供共表达载体，所述共表达载体包含分子伴侣蛋白的基因，所述分子伴侣蛋白包括dnaJ、dnaK、GroEL和grpE。
上述分子伴侣蛋白dnaJ、dnaK、GroEL和grpE来源于芽孢杆菌Geobacillus sp.WCH70。
其中，dnaJ基因的核苷酸序列如序列表SEQ ID NO：3所示；
dnaK基因的核苷酸序列如序列表SEQ ID NO：4所示；
GroEL基因的核苷酸序列如序列表SEQ ID NO：5所示；
grpE基因的核苷酸序列如序列表SEQ ID NO：6所示。
用的载体均适用于该共表达载体，没有特殊限制。优选的，载体为质粒，选择但不限于pUC18、pUC19中的一种。
dnaJ、dnaK、GroEL和grpE基因在载体中的组装顺序不同，对表达有一定影响。
优选的，在一种具体实施方式中，所述的共表达载体的构建方法，包括：
将dnaJ、dnaK、GroEL和grpE的基因序列按照dnaJ-dnaK-GroEL-grpE的顺序进行全合成，将全合成的序列插入到载体的多克隆位点，构建成共表达载体。
在另一种具体实施方式中，所述的共表达载体的构建方法，包括：
将dnaJ、dnaK、GroEL和grpE的基因序列按照dnaJ-dnaK-grpE-GroEL的顺序进行全合成，将全合成的序列插入到载体的多克隆位点，构建成共表达载体。
在上述重组表达载体和共表达载体的基础上，本发明还提供一种高效表达D-阿洛酮糖3-差向异构酶的工程菌，所述工程菌的底盘菌株中包含上述重组表达载体和共表达载体。
优选的，所述底盘菌株选自E.coli K12、E.coli BL21、E.coli JM109、E.coli Rosetta、E.coli BL21 plysS、E.coli Top 10、E.coli DH5α中的一种。
在一种具体实施方式中，所述高效表达D-阿洛酮糖3-差向异构酶的工程菌的构建方法为：将上述重组表达载体和共表达载体转入底盘菌株中，得到工程菌。
本发明提供的一种高效表达D-阿洛酮糖3-差向异构酶的工程菌的应用：是用于胞内生产D-阿洛酮糖3-差向异构酶，催化D-果糖生产D-阿洛酮糖。
本发明提供的技术方案之四，是一种制备D-阿洛酮糖的方法，包括如下步骤：
1)将所述基因工程菌株接种至发酵培养基中进行发酵培养；
2)在发酵培养基中添加诱导剂进行诱导表达，得到发酵液；
3)分离发酵液，获得菌体，加入缓冲液重悬，得到菌液；
4)向D-果糖溶液中加入菌液进行生物转化，得到D-阿洛酮糖。
进一步的，步骤1)中，所述发酵培养基为：酵母浸膏20～25g/L，大豆蛋白胨5～ 10g/L，甘油4～6g/L，磷酸二氢钾2～3g/L，磷酸氢二钾12～16g/L。
发酵培养的条件为：初始pH6～7，培养温度22～37℃，200～600rpm，培养20～28h，溶氧20％～30％。
进一步的，步骤2)中，发酵培养至OD600大于20后，降温至22～25℃，开始加入诱导剂，诱导剂的加入量为0.21～0.25mmol/L终浓度。
优选的，所述诱导剂为IPTG。
进一步的，步骤3中的缓冲液选用Tris-HCl缓冲液，pH＝8.0，缓冲液与菌体的添加比例为5：1。
进一步的，步骤4)中，D-果糖溶液为500～800g/L，菌液加入量为6-12g/L。
生物转化的条件为pH6～7，50～60℃，转化16～18h。
本发明提供的一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用中所用原料及试剂均可由市场购得。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如《分子克隆实验指南：第4版》中所述的条件进行，或按照试剂盒说明书、试剂生产厂商建议的条件进行。
下面结合实施例，进一步阐述本发明：
实施例1重组表达载体的构建
本实施例中，D-阿洛酮糖3-差向异构酶的基因来源于瘤胃球菌Ruminococcus sp.，组成型启动子HCE的基因来源于芽孢杆菌Geobacillus sp.WCH70。
D-阿洛酮糖3-差向异构酶基因的核苷酸序列由Genescript公司优化，在5’端添加组成型启动子HCE及核糖体结合位点(RBS)序列，两端分别引入XbaI和XhoI酶切位点，并进行全合成，得到全合成基因片段。上述全合成基因片段的核苷酸序列如序列表SEQ ID NO：7所示。
分别将全合成基因片段和pET28a载体进行XbaⅠ和XhoⅠ双酶切，通过胶回收试剂盒回收并纯化目的片段，用T4连接酶将两个产物进行连接，得到重组质粒。
将此重组质粒转化入E.coli DH5α感受态细胞，涂布卡那霉素抗性平板，37℃培养过夜。挑取长出的阳性克隆，提取质粒进行测序验证，获得重组表达载体pHCE-DPE，其图谱如图2所述。
进一步，提取质粒，用PCR获取目的基因片段，进行核酸电泳验证。
PCR引物如下：
p1：ATGAAGTACGGTATCTACTATGCTTATTGGG(SEQ ID NO：10)
p2：CCTCGAAAACGTGTTTAACAAAATGCAAC(SEQ ID NO：11)
核酸电泳验证如图1所示，目的基因的大小为873bp，电泳验证结果与目的基因的大小一致，表明成功构建了该重组表达载体。
实施例2 dnaJ-dnaK-GroEL-grpE共表达载体的构建
本实施例中，4个分子伴侣蛋白dnaJ、dnaK、GroEL、grpE的基因来源于芽孢杆菌Geobacillus sp.WCH70。
将分子伴侣蛋白dnaJ、dnaK、grpE、GroEL按照dnaJ-dnaK-GroEL-grpE的顺序组装，在dnaJ和GroEL之后插入RBS序列(SEQ ID NO：12)，在dnaK之后插入lac启动子(SEQ ID NO：13)和RBS序列。整个序列两端分别引入XbaI和KpnI酶切位点，由Genescript公司进行全合成，得到全合成基因片段，该基因片段的核苷酸序列如序列表SEQ ID NO：8所示。
分别将全合成基因片段和pUC 19载体进行XbaI和KpnI双酶切，通过胶回收试剂盒回收并纯化目的片段，用T4连接酶将两个产物进行连接，得到重组质粒。
将此重组质粒转化入E.coli DH5α感受态细胞，涂布氨苄抗性平板，37℃培养过夜。挑取长出的阳性克隆，提取质粒进行测序验证，获得共表达载体pUCMOD-JKLE，其图谱如图3所述。
实施例3 dnaJ-dnaK-grpE-GroEL共表达载体的构建
本实施例中，4个分子伴侣蛋白dnaJ、dnaK、GroEL、grpE的基因来源于芽孢杆菌Geobacillus sp.WCH70。
将分子伴侣蛋白dnaJ、dnaK、grpE、GroEL按照dnaJ-dnaK-grpE-GroEL的顺序组装，在dnaJ和grpE之后分别插入RBS序列，在dnaK之后插入lac启动子和RBS序列。序列两端分别引入XbaI和KpnI酶切位点，由Genescript公司进行全合成，得到全合成基因片段，该基因片段的核苷酸序列如序列表SEQ ID NO：9所示。
分别将全合成基因片段和pUC 19载体进行XbaI和KpnI双酶切，通过胶回收试剂盒回收并纯化目的片段，用T4连接酶将两个产物进行连接，得到重组质粒。
将此重组质粒转化入E.coli DH5α感受态细胞，涂布氨苄抗性平板，37℃培养过夜。挑取长出的阳性克隆，提取质粒进行测序验证，获得共表达载体pUCMOD-JKEL，其图谱如图4所述。
实施例4工程菌的构建
将实施例1制备得到的重组表达质粒pHCE-DPE转化入E.coli BL21(DE3)感受态细胞，用含有卡那霉素的平板进行培养，挑取阳性克隆，提取质粒，测序验证正确后，获得菌株DPE-A。
用DPE-A菌株制备感受态细胞，将实施例2制备得到的分子伴侣质粒pUCMOD-JKLE转化入DPE-1感受态细胞，用含有氨苄青霉素的平板进行培养，挑取阳性克隆，提取质粒，测序验证正确后，获得工程菌DPE-B1。
用DPE-A菌株制备感受态细胞，将实施例3制备得到的分子伴侣质粒pUCMOD-JKEL转化入DPE-1感受态细胞，用含有氨苄青霉素的平板进行培养，挑取阳性克隆，提取质粒，测序验证正确后，获得工程菌DPE-B2。
实施例5阿洛酮糖高产菌株的构建以获得阿洛酮糖生产的底盘菌株DPE-D
通过CRISPR-Cas9系统编辑将分子伴侣蛋白dnaJ、dnaK、GroEL、grpE的基因按照顺序插入到JM109菌株染色体组的lacZ位置。其中需要构建所需要的CP-1片段(线性DNA片段)以及质粒pgRNA-1。
质粒pgRNA-1由Genescript公司进行全合成，其序列如SEQ ID NO：15所示。
CP-1线性片段的的构建方法如下。通过两对PCR引物pCP1-1和pCP1-2以及pCP1-5和pCP1-6，以大肠杆菌JM109染色体组为模板，扩增得到CP-1U和CP-1D片段。使用pCP1-3和pCP1-4引物，并以实施例2中构建的载体pUCMOD-JKLE为模板进行扩增，得到CP-1M片段。利用1.0％琼脂糖凝胶电泳检测并切胶回收三个片段，并进行融合PCR，所得到的条带再利用1.0％琼脂糖凝胶电泳进行回收得到融合片段CP-1。染色体组基因操作图如下图6所示。
将CP-1片段(400ng)与质粒pgRNA-1(100ng)一同电转化至有pCas9载体的菌株JM109菌株。过夜培养后挑取单菌落作为模板，以引物pCP1-1和引物pCP1-6进行PCR，并且通过观察到在1.0％琼脂糖凝胶中存在6500bp的DNA条带确认dnaJ、dnaK、GroEL、grpE基因的已整合到染色体组上。并将PCR产物进行测序验证，测序结果通过BLAST序列比对确认lacZ基因的位置已经被成功替换成dnaJ、dnaK、GroEL、grpE基因。构建的菌株记为DPE-D。
实施例6阿洛酮糖高产菌株的构建以获得阿洛酮糖生产的底盘菌株DPE-E
通过CRISPR-Cas9系统编辑将分子伴侣蛋白dnaJ、dnaK、grpE、GroEL的基因按照顺序插入到JM109菌株染色体组的lacZ位置。其中需要构建所需要的CP-2片段(线性DNA片段)以及质粒pgRNA-1。
质粒pgRNA-1在上述实施例5中已经描述。
CP-2线性片段的构建方法如下。通过两对PCR引物pCP2-1和pCP2-2以及pCP2-5和pCP2-6，以大肠杆菌JM109染色体组为模板，扩增得到CP-2U和CP-2D片段。使用pCP2-3和pCP2-4引物，并以实施例3中构建的载体pUCMOD-JKEL为模板进行扩增，得到CP-2M片段。利用1.0％琼脂糖凝胶电泳检测并切胶回收三个片段，并进行融合PCR，所得到的条带再利用1.0％琼脂糖凝胶电泳进行回收得到融合片段CP-2。染色体组基因操作图如下图7所示。
将CP-2片段(400ng)与质粒pgRNA-1(100ng)一同电转化至有pCas9载体的菌株JM109菌株。过夜培养后挑取单菌落作为模板，以引物pCP2-1和引物pCP2-6进行PCR，并且通过观察到在1.0％琼脂糖凝胶中存在6500bp的DNA条带确认dnaJ、dnaK、grpE、GroEL基因的已整合到染色体组上。并将PCR产物进行测序验证，测序结果通过BLAST序列比对确认lacZ基因的位置已经被成功替换成dnaJ、dnaK、grpE、GroEL基因。构建的菌株记为DPE-E。
实施例5、6中CP-1和CP-2的1.0％琼脂糖凝胶电泳图见图8，分别做了两次平行(第一泳道和第二泳道为DPE-D，第三泳道和第四泳道为DPE-E)。
对比例1
本实施例中，D-阿洛酮糖3-差向异构酶的基因来源于瘤胃球菌Ruminococcus sp.，组成型启动子HCE的基因来源于芽孢杆菌Geobacillus sp.WCH70。
D-阿洛酮糖3-差向异构酶基因的核苷酸序列由Genescript公司优化，两端分别引入XbaI和XhoI酶切位点，进行全合成，得到全合成基因片段(SEQ ID NO：14)。分别将全合成基因片段和pET28a载体进行XbaⅠ和XhoⅠ双酶切，通过胶回收试剂盒回收并纯化目的片段，用T4连接酶将两个产物进行连接，得到重组质粒。
将此重组质粒转化入E.coli DH5α感受态细胞，涂布卡那霉素抗性平板，37℃培养过夜。挑取长出的阳性克隆，提取质粒进行测序验证，获得重组表达载体pET28a-DPE。pET28a-DPE表达载体图谱如图5所示。
将重组表达载体pET28a-DPE转化入E.coli BL21(DE3)感受态细胞，用含有卡 那霉素的平板进行培养，挑取阳性克隆，提取质粒，测序验证正确后，获得工程菌DPE-C。
实施例7菌株培养及表达
实验菌株：
实施例4构建的工程菌DPE-B1；
实施例4构建的工程菌DPE-B2；
实施例5构建的工程菌DPE-D；
实施例6构建的工程菌DPE-E；
对比例1构建的工程菌DPE-C。
挑取上述工程菌DPE-B1、工程菌DPE-B2、工程菌DPE-C、工程菌DPE-D和工程菌DPE-E，分别接种在含有相应抗性的LB液体培养基中，37℃培养至OD600达到0.7-0.8，加入终浓度为0.5mmol/L IPTG，在22℃、150rpm条件下诱导表达过夜。4℃，6000r/min离心10-15min，收集菌体，用缓冲液(20mmol/LTris-HCl，pH 8.0)按照5：1的比例(1克湿菌体添加5mL缓冲液)重悬菌体，超声破碎(运行时间1s；间隔时间2s；总时间5min；温度10-15℃；功率600w)，10000r/min离心20min，收集上清液，得到D-阿洛酮糖-3-差向异构酶的粗酶液。
将收集到的三种D-阿洛酮糖-3-差向异构酶的粗酶液进行蛋白电泳验证，结果如图9所示，其中，第1泳道为空底盘菌株制备出的粗酶液；第2泳道为工程菌DPE-B1制备出的粗酶液；第3泳道为工程菌DPE-B2制备出的粗酶液；第4泳道为工程菌DPE-D制备出的粗酶液；第5泳道为工程菌DPE-E制备出的粗酶液；第6泳道为工程菌DPE-C。蛋白电泳验证结果与目的基因的大小一致，表明上述5种工程菌均成功表达了该目的基因，且通过条带深浅情况验证了表达蛋白的效率。
D-阿洛酮糖-3-差向异构酶转化率测定：
反应体系如下：10mL反应体积，100μL粗酶液、50mmol/L Tris-HCl缓冲液(pH＝8.0)、700mg/mL D-果糖，60℃条件下反应2、4、8、12、16小时，分别取样，HPLC检测果糖和阿洛酮糖的含量，计算转化率。
HPLC色谱条件如下：
分析柱：ChromCore Sugar-10Ca；
流动相：去离子水
柱温：80℃；
示差检测器温度：50℃；
流速：0.5mL/min；
进样量：10μL。
反应结果如下表1所示，从表1所列出的数据可以看出，实施例6构建的工程菌的D-阿洛酮糖3-差向异构酶与高浓度的果糖反应时，仅仅经过约18个小时转化率就达到33％的最大转化率：
表1

实施例8制备D-阿洛酮糖
选择实施例7中转化率最好的工程菌JM109dnaJ dnaK grpE GroEL(lac)ΔlacZ为生物合成制备D-阿洛酮糖用菌株，上述基因工程菌株接种至发酵培养基中进行发酵培养，发酵培养基为酵母浸膏25g/L，大豆蛋白胨8g/L，甘油6g/L，磷酸二氢钾2g/L，磷酸氢二钾15g/L。发酵培养的初始pH为7，培养温度26℃，溶氧控制在20％～30％，培养24h。当发酵培养至OD600大于20后，降温至22℃，开始加入诱导剂，诱导剂选用IPTG，IPTG的加入量为0.25mmol/L终浓度。发酵结束，得到发酵液，6000r/min离心10-15min，获得菌体。
用缓冲液(20mmol/LTris-HCl，pH 8.0)按照5：1的比例(重悬菌体，得到菌液，在700g/L的D-果糖溶液中加入12g/L菌液，pH为6，60℃进行生物转化，转化时间为18h，转化结束后，取样进行HPLC测定D-阿洛酮糖含量，计算转化率，结果如图10所示。结果显示，D-阿洛酮糖的峰面积为431106。阿洛酮糖的标品对照图(图11)和果糖的标品对照图(图12)，根据标品数据通过外标法计算转化率。
阿洛酮糖样品采用外标法计算含量：
公式为：含量(cx)＝cr*Ax/Ar
其中：
cx为样品浓度；cr为对照浓度；Ax为样品峰面积；Ar为对照峰面积。
以上对本发明所提供的一种高效表达D-阿洛酮糖3-差向异构酶的工程菌及应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。










Claims (19)

  1. 如下任意项在生产D-阿洛酮糖中的应用：
    (Ⅰ)、共表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE；和/或
    (Ⅱ)、共表达D-阿洛酮糖3-差向异构酶的基因、组成型启动子HCE和RBS序列；和/或
    (Ⅲ)、共表达dnaJ、dnaK、GroEL和/或grpE；和/或
    (Ⅳ)、共表达dnaJ、dnaK、GroEL、grpE、RBS序列和/或lac启动子；
    (Ⅴ)、缺失lacZ基因和共表达dnaJ、dnaK、GroEL和/或grpE。
  2. 表达元件，其特征在于，所述表达元件包括表达D-阿洛酮糖3-差向异构酶的基因和组成型启动子HCE基因。
  3. 如权利要求2所述的表达元件，其特征在于，还包括RBS序列。
  4. 如权利要求2或3所述的表达元件，其特征在于，所述D-阿洛酮糖3-差向异构酶具有：
    (Ⅰ)、如SEQ ID NO：1所示的氨基酸序列；
    (Ⅱ)、在如(Ⅰ)所示的氨基酸序列的基础上经取代、缺失、添加和/或替换1个或多个氨基酸的序列；或
    (Ⅲ)、与如(Ⅰ)所示的氨基酸序列具有至少90％序列同源性的氨基酸序列；
    所述组成型启动子HCE具有：
    (Ⅳ)、如SEQ ID NO:2所示核苷酸序列的DNA片段；或
    (Ⅴ)、与(Ⅳ)所述核苷酸序列具有至少90％序列同源性，且具有启动子功能的DNA片段。
  5. 共表达元件，其特征在于，所述共表达元件包括分子伴侣蛋白的基因和/或缺失lacZ基因；
    所述分子伴侣蛋白包括dnaJ、dnaK、GroEL和/或grpE。
  6. 如权利要求5所述的共表达元件，其特征在于，包括如下任意项和/或缺失lacZ基因：
    (Ⅰ)、依次包括dnaJ、dnaK、GroEL和grpE；和/或
    (Ⅱ)、依次包括dnaJ、dnaK、grpE和GroEL。
  7. 如权利要求5或6所述的共表达元件，其特征在于，还包括RBS序列和/或lac启动子。
  8. 如权利要求5至7任一项所述的共表达元件，其特征在于，包括如下任意项：
    (Ⅰ)、依次包括dnaJ、RBS序列、dnaK、lac启动子、RBS序列、GroEL、RBS序列和grpE；和/或
    (Ⅱ)、依次包括dnaJ、RBS序列、dnaK、lac启动子、RBS序列、grpE、RBS序列和GroEL。
  9. 如权利要求5至8任一项所述的共表达元件，其特征在于，所述dnaJ基因的核苷酸序列具有：
    (i)、如SEQ ID NO:3所示的核苷酸序列；或
    (ii)、与(i)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(i)所示的核苷酸序列不同的核苷酸序列；或
    (iii)、与(i)或(ii)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(i)或(ii)所示的核苷酸序列功能相同或相似的核 苷酸序列；或
    (iv)、与(i)～(iv)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
    所述dnaK基因的核苷酸序列具有：
    (v)、如SEQ ID NO:4所示的核苷酸序列；或
    (vi)、与(v)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)所示的核苷酸序列不同的核苷酸序列；或
    (vii)、与(v)或(vi)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(v)或(vi)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (viii)、与(v)～(vii)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
    所述GroEL基因的核苷酸序列具有：
    (a)、如SEQ ID NO:5所示的核苷酸序列；或
    (b)、与(a)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(a)所示的核苷酸序列不同的核苷酸序列；或
    (c)、与(a)或(b)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(a)或(b)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (d)、与(a)～(c)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列；
    所述grpE基因的核苷酸序具有：
    (e)、如SEQ ID NO:6所示的核苷酸序列；或
    (f)、与(e)所示的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(e)所示的核苷酸序列不同的核苷酸序列；或
    (g)、与(e)或(f)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(e)或(f)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (h)、与(e)～(g)任一项所述核苷酸序列具有至少90％序列同源性的核苷酸序列。
  10. 表达载体，其特征在于，包括如下任意项：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件。
  11. 如权利要求10所述的表达载体的构建方法，其特征在于，包括：将如下任意项进行全合成，插入载体的多克隆位点，构建所述表达载体：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件。
  12. 宿主，其特征在于，包括如下任意项：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体。
  13. 如权利要求12所述的宿主，其特征在于，包括工程菌；
    所述工程菌包括如下任意项：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体。
  14. 如权利要求13所述的宿主，其特征在于，所述工程菌的构建方法包括转入如下任意项：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体。
  15. 如下任意项在生产D-阿洛酮糖中的应用：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体；和/或
    (Ⅴ)、如权利要求12至14任一项所述的宿主。
  16. 如下任意项在生产D-阿洛酮糖3-差向异构酶中的应用：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体；和/或
    (Ⅴ)、如权利要求12至14任一项所述的宿主。
  17. 如下任意项在制备降血糖药物中的应用：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体；和/或
    (Ⅴ)、如权利要求12至14任一项所述的宿主。
  18. D-阿洛酮糖的制备方法，其特征在于，基于如下任意项制得D-阿洛酮糖：
    (Ⅰ)、如权利要求2至4任一项所述的表达元件；和/或
    (Ⅱ)、如权利要求5至9任一项所述的共表达元件；和/或
    (Ⅲ)、如权利要求10所述的表达载体；和/或
    (Ⅳ)、如权利要求11所述构建方法获得的表达载体；和/或
    (Ⅴ)、如权利要求12至14任一项所述的宿主。
  19. 如权利要求18所述的制备方法，其特征在于，包括如下步骤：
    步骤1：将如权利要求12至14任一项所述的宿主与诱导剂混合发酵培养，分离重悬得到菌液；
    步骤2：取D-果糖溶液与所述菌液进行生物转化，得到D-阿洛酮糖。
PCT/CN2023/103067 2022-07-01 2023-06-28 一种高效表达d-阿洛酮糖3-差向异构酶的工程菌及应用 WO2024002131A1 (zh)

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