CN117721056B - Genetically engineered bacterium and application thereof in production of ergothioneine - Google Patents
Genetically engineered bacterium and application thereof in production of ergothioneine Download PDFInfo
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- CN117721056B CN117721056B CN202311814202.5A CN202311814202A CN117721056B CN 117721056 B CN117721056 B CN 117721056B CN 202311814202 A CN202311814202 A CN 202311814202A CN 117721056 B CN117721056 B CN 117721056B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 56
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 53
- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 52
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 20
- GPPYTCRVKHULJH-QMMMGPOBSA-N N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC=N1 GPPYTCRVKHULJH-QMMMGPOBSA-N 0.000 claims description 13
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- BNZPZVPHQDLRKJ-UHFFFAOYSA-N 3-[bis(2-carboxyethoxy)phosphoryloxy]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCOP(=O)(OCCC(O)=O)OCCC(O)=O BNZPZVPHQDLRKJ-UHFFFAOYSA-N 0.000 description 1
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- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
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- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 150000002410 histidine derivatives Chemical class 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000155 iron(II) phosphate Inorganic materials 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a genetically engineered bacterium and application thereof in preparation of ergothioneine, wherein the genetically engineered bacterium comprises a host cell and a target gene transferred into the host cell; the target gene consists of a target gene I and a target gene II; the target gene I is shown as SEQ ID NO. 1; the target gene II is shown as SEQ ID NO.2 or SEQ ID NO. 3. The genetically engineered bacteria obtained by taking the egt1 gene and the egt2 gene which are from specific sources as target genes not only have higher enzyme activity, but also can reach higher conversion rate in shorter reaction time, and the ee value is high.
Description
Technical Field
The invention relates to the technical field of construction of genetic engineering strains, in particular to a genetic engineering strain and application thereof in producing ergothioneine.
Background
Ergothioneine is a chiral histidine derivative with unique biological functions and pharmacological activities, and has wide biomedical application prospect. Due to the unique chemical property and biological activity, the ergothioneine has wide application prospect and market prospect in the fields of foods, cosmetics, medicines and the like.
The synthesis method of ergothioneine mainly comprises chemical synthesis method, extraction method, biological fermentation method and biological catalysis method. Among them, enzyme catalysis is the most widely studied method of ergothioneine production. Ergothioneine synthesis in eukaryotes mainly involves two key enzymes, egt1 and Egt2, egt1 catalyses the synthesis of sulfoxide intermediates from cysteine and histidine betaines (FIG. 1). Then, in a strongly reducing environment, egt2 cleaves the sulfoxide intermediate to form ergothioneine (fig. 2).
However, the current methods of enzymatic synthesis of ergothioneine still have some problems. For example: the engineering bacteria have long culture time and low protein expression quantity; the enzyme activity is not high, and the production efficiency is reduced; the enzyme substrate has poor tolerance and low conversion rate.
The invention patent with the application publication number of CN110551697B discloses application of Pleurotus ostreatus ergothioneine synthetase PEGT1 and PEGT2 in synthesis of ergothioneine, and the invention starts from Pleurotus ostreatus, pleurotus nebrodensis and Pleurotus eryngii, clones and functionally identifies the three genes related to synthesis and coding of the ergothioneine of Pleurotus ostreatus.
The invention patent application with the application publication number of CN116355820A discloses a high-yield ergothioneine engineering strain and a method for producing ergothioneine by using the engineering strain, wherein the engineering strain takes escherichia coli as a chassis strain, and one or more of an ergothioneine synthetic gene cluster egtBDE, an ergothioneine biosynthesis protein egt1 coding gene and an ergothioneine biosynthesis protein egt2 coding gene are overexpressed in the chassis strain.
Although the above patent application opens up a new synthesis path or reduces the addition of precursor substances, it is still necessary to further dig ergothioneine synthase with high catalytic activity, and it is of great significance to construct bioengineering bacteria with short culture time and high protein expression for catalytic synthesis of ergothioneine by using genetic engineering technology.
Disclosure of Invention
Aiming at the defects of the existing ergothioneine synthesis process, the invention provides a genetic engineering bacterium for high-yield ergothioneine and a method for preparing the ergothioneine by using the genetic engineering bacterium in one-pot method; the genetically engineered bacterium not only has higher enzyme activity, but also can reach higher conversion rate in shorter reaction time, and the ee value is high.
The specific technical scheme is as follows:
The invention provides a genetically engineered bacterium, which comprises a host cell and a target gene transferred into the host cell, wherein the target gene consists of a target gene I and a target gene II; the target gene I is shown as SEQ ID NO. 1; the target gene II is shown as SEQ ID NO.2 or SEQ ID NO. 3.
Wherein the target gene I, abbreviated as egt1 gene, is derived from myceliophthora thermophila (Myceliophthora thermophila); the target gene II, abbreviated as egt1 gene, is derived from trichoderma longibrachiatum (Trichoderma longibrachiatum) or trichoderma harzianum (Trichoderma harzianum).
The invention screens and groups egt1 genes (accession numbers of database UniProt are A0A0F9XQS5, G2Q5S1, G4UKZ4, A0A395SUF0, A0A439D8X 8) respectively from Trichoderma harzianum(Thegt1)、Myceliophthora thermophila(Mtegt1)、Neurospora tetrasperma(Ntegt1)、Fusarium longipes(Flegt1)、Xylaria grammica(Xgegt1) and other species, and egt2 genes (accession numbers of database UniProt are A0A2T4BWP1, A0A0G0A2B8 respectively) respectively from Trichoderma longibrachiatum (Tlegt 2), trichoderma harzianum (Thegt 2) and other species.
The discovery is as follows: only the egt1 gene from myceliophthora thermophila (Myceliophthora thermophila) and the egt2 gene from trichoderma longibrachiatum (Trichoderma longibrachiatum) and trichoderma harzianum (Trichoderma harzianum) are expressed, so that the genetically engineered bacteria with high enzyme activity, high conversion rate and high ee value and high yield of ergothioneine can be obtained.
Further, the target gene I and the target gene II are jointly inserted into the same episomal plasmid I and transferred into the same host cell; or the target gene I and the target gene II are respectively and independently inserted into the episomal plasmid II and then transferred into the host cells of the same kind.
The invention provides an expression vector containing the ergothioneine synthase gene egt1. The expression vector is formed by inserting egt1 between BamHI and HindIII restriction enzyme sites of pET28-a (+) expression plasmids, including pET28a-Thegt1, pET28a-Mtegt1, pET28a-Ntegt1, pET28a-Flegt1 and pET28a-Xgegt1.
The invention provides an expression vector containing the ergothioneine synthase gene egt2. The expression vector is formed by inserting egt2 between BamHI and HindIII restriction enzyme sites of pET28-a (+) expression plasmids, including pET28a-Tlegt2 and pET28a-Thegt2.
The invention provides a co-expression vector comprising the ergothioneine synthase genes egt1 and egt 2. Inserting the ergothioneine synthesis gene egt1 between the multiple cloning sites BamHI and HindIII and the ergothioneine synthesis gene egt2 between the multiple cloning sites Nde I and Xho I using the pETDuet-1 co-expression plasmid, comprising pETDuet-Thegt1-Tlegt2、pETDuet-Mtegt1-Tlegt2、pETDuet-Ntegt1-Tlegt2、pETDuet-Flegt1-Tlegt2、pETDuet-Xgegt1-Tlegt2.
The invention provides ESCHERICHIA COLI BL (DE 3) engineering bacteria containing the egt1 gene: BL21-pET28a-Thegt1, BL21-28a-Mtegt1, BL21-28a-Ntegt1, BL21-28a-Flegt1, BL21-28a-Xgegt1.
The invention provides ESCHERICHIA COLI BL (DE 3) engineering bacteria containing the egt2 gene: BL21-28a-Tlegt2, BL21-28a-Thegt2.
The invention provides Vibrio natriegens VnDX engineering bacteria containing the egt1 gene: VNDX-pET28a-Thegt1, VNDX-28a-Mtegt1, VNDX-28a-Ntegt1, VNDX-28a-Flegt1, VNDX-28a-Xgegt1.
The invention provides Vibrio natriegens VnDX engineering bacteria containing the egt2 gene: VNDX-28a-Tlegt2, VNDX-28a-Thegt2.
The invention provides ESCHERICHIA COLI BL (DE 3) co-expression engineering bacteria containing egt1 and Tlegt genes BL21-Duet-Thegt1-Tlegt2、BL21-Duet-Mtegt1-Tlegt2、BL21-Duet-Ntegt1-Tlegt2、BL21-Duet-Flegt1-Tlegt2、BL21-Duet-Xgegt1-Tlegt2.
The invention provides ESCHERICHIA COLI BL (DE 3) co-expression engineering bacteria containing egt1 and Thegt genes ：BL21-Duet-Thegt1-Thegt2、BL21-Duet-Mtegt1-Thegt2、BL21-Duet-Ntegt1-Thegt2、BL21-Duet-Flegt1-Thegt2、BL21-Duet-Xgegt1-Thegt2.
The invention provides Vibrio natriegens VnDX co-expression engineering bacteria containing egt1 and Tlegt genes VNDX-Duet-Thegt1-Tlegt2、VNDX-Duet-Mtegt1-Tlegt2、VNDX-Duet-Ntegt1-Tlegt2、VNDX-Duet-Flegt1-Tlegt2、VNDX-Duet-Xgegt1-Tlegt2.
The invention provides Vibrio natriegens VnDX co-expression engineering bacteria containing egt1 and Thegt genes ：VNDX-Duet-Thegt1-Thegt2、VNDX-Duet-Mtegt1-Thegt2、VNDX-Duet-Ntegt1-Thegt2、VNDX-Duet-Flegt1-Thegt2、VNDX-Duet-Xgegt1-Thegt2.
Still further, the host cell is E.coli ESCHERICHIA COLI BL (DE 3) or Vibrio natrii Vibrio natriegens VnDX.
Further, the episomal plasmid I is a pETDuet-1 plasmid; episomal plasmid II is pET28-a (+) plasmid.
Further, the episomal plasmid I is pETDuet-1 plasmid, and the egt1 gene is inserted between BamHI and HindIII of the plasmid multicloning site; the egt2 gene is inserted between Nde I and Xho I of the plasmid multiple cloning site;
the episomal plasmid II is pET28-a (+) plasmid, and the egt1 gene and the egt2 gene are inserted between BamHI and HindIII of plasmid multiple cloning sites.
Further, the nucleotide sequence of the egt1 gene is shown as SEQ ID NO.1; the nucleotide sequence of the egt2 gene is shown as SEQ ID NO.2 or SEQ ID NO. 3.
Further, the Vibrio natrii is Vibrio natrii (Vibrio natriegens) ATCC14048.
Further, the Vibrio natrii is Vibrio natrii NATRIEGENS ATCC14048 with a T7 RNA polymerase expression cassette integrated in the dns site of the genome.
The invention also provides application of the genetically engineered bacterium in producing ergothioneine.
The invention also provides a production method of ergothioneine, which comprises the following steps: histidine betaine and L-cysteine are used as substrates, tris (2-carbonyl ethyl) phosphate hydrochloride or 1, 4-dithiothreitol is used as a reducing agent, pyridoxal phosphate and ferrous ions are used as coenzymes, and whole cells, wet thalli or crude enzyme liquid of the genetically engineered bacteria are used as catalysts for catalytic reaction to obtain the ergothioneine.
Further, the catalytic reaction takes water as a solvent, the reaction temperature is 25-40 ℃, and the pH value is 6-9;
The concentration of the histidine betaine is 50 mM-500 mM, the concentration of the L-cysteine is 75 mM-750 mM, the concentration of the tris (2-carbonyl ethyl) phosphate hydrochloride or the 1, 4-dithiothreitol is 25 mM-75 mM, the concentration of the pyridoxal phosphate is 50 mu M-200 mu M, and the concentration of the ferrous ion is 50 mu M-200 mu M.
Furthermore, the catalytic reaction takes water as a solvent, the reaction temperature is 30 ℃, and the pH value is 8;
The concentration of histidine betaine is 200mM, the concentration of L-cysteine is 300mM, the concentration of tris (2-carboxyethyl) phosphate hydrochloride or 1, 4-dithiothreitol is 50mM, the concentration of pyridoxal phosphate is 100 mu M, and the concentration of ferrous ions is 100 mu M.
Further, the genetically engineered bacterium is genetically engineered bacterium I or is formed by mixing genetically engineered bacterium II-1 and genetically engineered bacterium II-2;
The gene engineering bacteria I are obtained by jointly inserting an egt1 gene and an egt2 gene into the same episomal plasmid I and transferring the episomal plasmid I into the same host cell;
the gene engineering bacteria II-1 are obtained by independently inserting egt1 genes into episomal plasmid II and respectively transferring the episomal plasmid II into host cells of the same type; the gene engineering bacteria II-2 are obtained by separately inserting egt2 genes into episomal plasmid II and respectively transferring the episomal plasmid II into similar host cells.
Further, the volume ratio of the crude enzyme liquid of the genetically engineered bacterium II-1 to the crude enzyme liquid of the genetically engineered bacterium II-2 is 1-5: 0.3 to 1.
Further, the volume of the crude enzyme solution of the genetically engineered bacterium II-1 is 300-400 mL; the volume of the crude enzyme solution of the genetically engineered bacterium II-2 is 100-200 mL. The concentration of the crude enzyme solution is 8-12 g wet cells/L.
Compared with the prior art, the invention has the following beneficial effects:
(1) The genetically engineered bacteria obtained by taking the egt1 gene and the egt2 gene which are from specific sources as target genes not only have higher enzyme activity, but also can reach higher conversion rate in shorter reaction time, and the ee value is high.
(2) The genetically engineered bacterium has the advantages of high growth rate and high protein expression.
(3) The crude enzyme solution of the genetically engineered bacterium has high enzyme activity, short reaction time and mild reaction conditions.
(4) The method for preparing ergothioneine by the one-pot method has the advantages that the optical purity of the obtained product is high, and resolution is not required; the substrate concentration is high, and the conversion rate is high; the production process is environment-friendly, less in pollution and accords with the green chemical idea; the product is simple to separate and purify and low in cost.
Drawings
FIG. 1 is a reaction equation for Egt1 catalyzed histidine betaine and L-cysteine to intermediate sulfoxide.
FIG. 2 is a reaction equation for the formation of ergothioneine from Egt2 catalytic intermediate sulfoxide.
FIG. 3 shows the reaction equation for the one-pot process of ergothioneine.
FIG. 4 is a high performance liquid detection spectrum of the substrate and the product in the analysis reaction liquid after the ergothioneine is prepared by a one-pot method.
FIG. 5 is a mass spectrum of the substrate and the product in the analysis reaction liquid after the ergothioneine is prepared by the one-pot method.
Detailed Description
The invention will be further described with reference to the following examples, which are given by way of illustration only, but the scope of the invention is not limited thereto.
Reagents used in the catalytic process: histidine betaine, L-cysteine, ergothioneine, pyridoxal phosphate, TCEP, DTT, ferrous sulfate, phosphate, and the like are all commercially available analytical purity.
The structural formula of the histidine betaine is shown as a formula (1); the structural formula of the L-cysteine is shown as a formula (2); ergothioneine is shown in formula (3).
The reaction equation of the ergothioneine prepared by the one-pot method is shown in figure 3.
The invention monitors the progress of the reaction by analyzing the concentration of the substrate and the product in the reaction liquid through High Performance Liquid Chromatography (HPLC).
The HPLC analysis method is as follows: chromatographic column model: QS-C18, 5 μm, 4.6X1250 mm. Sodium acetate (50 mM): acetonitrile=99:1, ph=8.0; detection wavelength: 210nm, flow rate: 1.0mL/min, column temperature: 40 ℃. The peak is shown in FIG. 4, intermediate sulfoxide for 3.6min, ergothioneine for 4.1min, and histidine betaine for 5.2min.
The invention adopts LC-MS (Agilent 1260/6460LC/Triple Quadrupole MS, agilent Technologies) and Agilent ZORBAX NH chromatographic columns (4.6X1250 mm5 μm) to identify the substrate and the product in the reaction liquid. The mobile phase is acetonitrile: 4mmol/L ammonium acetate (70:30, v/v).
The parameters of the analysis method are as follows: the ion source is ESI; the scanning mode is positive mode scanning; ion scanning range 50-220; scanning time 500; the lysis voltage (frag) was 135V; the acceleration voltage (cell acc) is 5V; step size (step size): 0.1; capillary voltage; the drying Gas temperature (Gas Temp) was 325 ℃; dry air Flow (Gas Flow): 10L/min; spray gas pressure (Nebulizer): 20psi; sheath temperature (SHEATH GAS TEMP): 400 ℃; sheath airflow rate (SHEATH GAS Flow): 11L/min; capillary voltage (CAPILLARY): 4000V. The results are shown in FIG. 5.
EXAMPLE 1 construction of wild-type enzyme engineering bacteria
Searching for ergothioneine synthase in the UniProt database and selecting for sources
Myceliophthora thermophila Myceliophthora thermophila (Mtegt, accession number G2Q5S1 of database UniProt), the base sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 4); trichoderma harzianum Trichoderma harzianum (Thegt 1, accession number A0A0F9XQS of database UniProt); neurospora tetraspora Neurospora tetrasperma (Ntegt, accession number G4UKZ, database UniProt); fusarium longum Fusarium longipes (Flegt, database UniProt accession number A0A395SUF 0); egt1 amino acid sequence of Xylaria stripe Xylaria grammica (Xgegt, accession number A0A439D8X8 of database UniProt).
And Egt2 amino acid sequences derived from Trichoderma longibrachiatum Trichoderma longibrachiatum (Tlegt, base sequence shown as SEQ ID NO.2, amino acid sequence shown as SEQ ID NO. 5) and Trichoderma harzianum Trichoderma harzianum (Thegt 2, base sequence shown as SEQ ID NO.3, amino acid sequence shown as SEQ ID NO. 6); the accession numbers of the UniProt in the database are A0A2T4BWP1 and A0A0G0A2B8 respectively.
Nine amino acid sequences were converted to nucleotide sequences by codon optimization. The nine nucleotide sequences are synthesized completely by chemical method (Optimus of Optimus), and integrated in the expression vector pET-28a (+) or pETDuet-1 multiple cloning site; and finally, introducing the constructed plasmid into a host cell of escherichia coli BL21 (DE 3) or Vibrio natriegens VnDX to construct the high-yield ergothioneine engineering bacteria.
Myceliophthora thermophila Myceliophthora thermophila
Base sequence (shown as SEQ ID NO. 1)
ATGCCCATAGCTCAAGAAACAACTGTAGTTCCGCAGGGCGCCACCCCGCGCTTGGGTGCCATCAAAGAAAAAAAGGGTGTTCCGCCGCTGAGAACTGCGGCGGCGACCGGACCGTATATTATCGATATTCGCCACGCGGTGGTGGAGATCAACTTGAAGGCGGAGGTGCTAGCGCAATTTAGAGCACGCGACGGTCCGCGTAAGCTGCCGACCCTGCTGTTATACGACGAGAACGGACTCCAACTGTTCGAGAAGATAACCTACCTTGAGGAGTACTACCTGACCAATGATGAGATCGCGGTACTGAAGTCCTATGCGGCTGACATCGTTAAATATATCCCGAGCGGTGCTATGGTAATTGAACTGGGCTCCGGTAACCTGCGCAAGGTTAATCTGCTGCTGCAGGCTTTGGAGGACGCGGCTAAGGACATTGATTATTACGCGTTGGACCTTAGCCAACAGGAGCTGGAGCGGACTCTGGCGCAGCTGCCACCCTACAAACACGTGCGCGCGCATGGTTTGCTGGGCACGTACGACGACGGTCGGCTCTGGCTGAAAGACCCTACGATTGCGACGCGCCAGAAATGCATTCTGTCCCTGGGCTCGAGCGTCGGTAATTTTGATCGCCGAGAAGCGGGTGCGTTCCTGAAGTCGTTCGCCGATGTTCTCGGTCCGGACGATACCATGTTAATTGGCCTGGACGCGTGTGATGATCCGGCAAAGGTCTACCATGCCTATAACGATAAAGAGGGCATCACCCACGAATTCATTTTGAATGGTCTGCGTCACGCAAATCGTATTCTGGGTGAAAACGCTTTTGTTGAGAAGGACTGGCGTGTTATTGGTCAATATGTGTACGATGCGGAAGGCGGTCGTCACCAGGCATTCTACGCACCGGTTCGCGAAACCATTGTTATGGGTGAGCGCATCCGTCCGCATGATCGTATCCAAGTTGAACAGTCTCTGAAGTACAGCGCTGCCGAAGCCGAAGAACTGTGGCGTCGTGCTGGCATGACCGAAATCGCCCAATGGCGTCACTTGAAGGAATATGGCCTTCATATGCTGGCGCGTCCTCGTATGGCATTCAGCTTGACTCCTAGTGTTTACGCACGCACCGCTCTGCCGTCCATGCGTGACTGGGAAGGTTTATGGGCCGCTTGGGACGTGGTTACCCGTGATATGCTGCCACCGGAGGAGATCTTGGAGAAACCGATTAAACTGCGCAACGCGTGTATCTTTTATCTGGGTCATATTCCGACCTTTCTGGACATCCAGCTGACCAAGACCACGAAACAGCCGCCGACGGACCCGGCGTATTACTACGGTATTTTTGAAAGAGGCATCGACCCCGACGTGGATAACCCGGAATTATGCCATGCGCATAGCGAAATTCCGGATGAGTGGCCGCCGGTCGATGAAATCCGTGCGTACCAGGGTCGTGTCCGCGCTAGACTGCAATCCCTGTACGCAGCAGGGATCGATGCAATTCCGCGTCACGTTGGTCGCGCGATCTGGGTTGGTTTTGAACACGAAGCCATGCACCTGGAAACCCTGCTGTACATGATGCTGCAATCCGACCGCACACGTCCGCCACCGCGTATTCCGGCTCCGGATTTTGAATCTTTGGCGGCGAAGGCTCGCTCGGAACGTGTACCGAATCAGTGGTTTGACATTCCGGAACAAGAGGTTGTGATCGGCCTGGACGACCCGGAAGATGGCACTGACCCGCATGCGCCATACGGTTGGGATAACGAGAAACCGCTGCGTCGTGTCAAGGTGCATGCGTTCCAAGCGCAAGGTCGCCCTATCAGCAATGAAGAGTATGCGCGTTATCTCTACAATACCCGCACGACCAAAATCCCGGCCAGCTGGGCACAGATCCCGGGTGGCACGGTTAAAACCTCCGATGAGACTGCTGCGGCGGGTGGTGGCACCGATAGCGCGGAGGCGAACAACAACAACAACGAGAACGGCCGTACCAACAGCCACTCTCTCGCAAACGGCGTGGCATTGCCGGAAAGCTTCCTGGACGATAAAGCCGTGCGTACGGTGTATGGCTTGGTTCCGCTGAAACACGCGTTGGACTGGCCAGTATTTGCAAGCTATGACGAGCTCGCTGGGTGCGCAGCGTGGATGGGAGGCCGTATACCGACCTTCGAAGAGGTGCGTTCAATTTATAAGCACGCCGAGGCTCTGAAGAAAGAACAGGCGGAGAATCAGCTGTCACAAACCGTCCCGGCGGTGAACGGCCACCTGACCAACAACGGCGTGGAGATCAGCCCGCCGGCAACCCCGCCAGGTTCGACCGACGCAGGCTCGGAGGCTGACAGCCGTGATCGCCGTTTGTCTCAAGAGGACCTGTTTATCGACCTGAATGGTGCGAACGTGTCCTTCAGCCATTGGCATCCGACTCCGGTAACAAGCCGTGGTAATCAGCTGGCCGGTCAGAGCGATGCTGGTGGCGTTTGGGAATGGACCAGCAGCGTTTTGAGACCTTGGGATGAGTTCCAGGCTATGAGCTTGTACCCGGGTTATACCGCAGACTTCTTTGATGAAAAGCACAACATGGTTTTGGGCGGTAGCTGGGCAACCCACCCGCGTTTGGCCGGCCGTAAAAGCTTCGTCAACTGGTATCAGCGTAATTACCCGTATGCCTGGATCGGCGCTCGCCTGGTTCGTGACGTGGAG.
Amino acid sequence (shown as SEQ ID NO. 4)
MPIAQETTVVPQGATPRLGAIKEKKGVPPLRTAAATGPYIIDIRHAVVEINLKAEVLAQFRARDGPRKLPTLLLYDENGLQLFEKITYLEEYYLTNDEIAVLKSYAADIVKYIPSGAMVIELGSGNLRKVNLLLQALEDAAKDIDYYALDLSQQELERTLAQLPPYKHVRAHGLLGTYDDGRLWLKDPTIATRQKCILSLGSSVGNFDRREAGAFLKSFADVLGPDDTMLIGLDACDDPAKVYHAYNDKEGITHEFILNGLRHANRILGENAFVEKDWRVIGQYVYDAEGGRHQAFYAPVRETIVMGERIRPHDRIQVEQSLKYSAAEAEELWRRAGMTEIAQWRHLKEYGLHMLARPRMAFSLTPSVYARTALPSMRDWEGLWAAWDVVTRDMLPPEEILEKPIKLRNACIFYLGHIPTFLDIQLTKTTKQPPTDPAYYYGIFERGIDPDVDNPELCHAHSEIPDEWPPVDEIRAYQGRVRARLQSLYAAGIDAIPRHVGRAIWVGFEHEAMHLETLLYMMLQSDRTRPPPRIPAPDFESLAAKARSERVPNQWFDIPEQEVVIGLDDPEDGTDPHAPYGWDNEKPLRRVKVHAFQAQGRPISNEEYARYLYNTRTTKIPASWAQIPGGTVKTSDETAAAGGGTDSAEANNNNNENGRTNSHSLANGVALPESFLDDKAVRTVYGLVPLKHALDWPVFASYDELAGCAAWMGGRIPTFEEVRSIYKHAEALKKEQAENQLSQTVPAVNGHLTNNGVEISPPATPPGSTDAGSEADSRDRRLSQEDLFIDLNGANVSFSHWHPTPVTSRGNQLAGQSDAGGVWEWTSSVLRPWDEFQAMSLYPGYTADFFDEKHNMVLGGSWATHPRLAGRKSFVNWYQRNYPYAWIGARLVRDVE.
Trichoderma longibrachiatum Trichoderma longibrachiatum
Base sequence (shown as SEQ ID NO. 2)
ATGGCTAGTCTACCCGTAAGGCAAAGAGAAGAGGGCGAGGCGAAAGTTGGCGAGGACGGTTTTAAAGTGTTCGGTGGCGAGATGACCAAAGATTTTCTGTTTGCGCCAAATTGGACCAATCTGAACCACGGTAGCTATGGCAGCATTCCGCGTGCGATTCAGGCAAAGCTGCGCTCTTACCAGGATGACATCGAAGCCCGTCCGGACCCGTTCGTGCGCTTCGAACACGCCCGTCTGACCGATGAAAGCCGTGCGGCGGTTGCTGGTGTGCTCAATGCGCCAGTTGAGACTGTCGTGTTCGTGAATAACGCGACCGAAGGTGTTAATACGGTTTTCCGCAACATCAAATGGGATGCTGATGGCAAGGACGTGGCGCTGTACTTCACCACCGTTTACGAGGCATGTGGTAAGGCGATCGATTTCCTGTACGATTACCACGGTGAGGGCCGTCTGTCGTCCCGCGAGATCGAGATTGCCTATCCGATTGAGGATGACGAAATTCTGCGTCGCTTCCGTGCGGCGGTGGAGCAGGTACGTTCCGAAGGTAAGCGGGCTAAAATCTGCATTTTTGATGTGGTGAGCAGCCGCCCGGGTGTTGTGTTCCCGTGGGAACGTATGGTCGCGGCTTGCCGTGAATTGGGCGTTCTGAGCTTGGTTGATGGCGCTCAAGGCATTGGTATGGTCCGTCTCGACCTGGGTGCGGCGGACCCGGATTTCTTTGTGAGCAACTGCCACAAATGGTTGTTCGCGCCGCGTGGCTGCGCGGTTTTTTATGTTCCGGTGCGTAACCAGGGTCTGTTACCGAGCACGTTGGCGACCAGCCACGGTTATGCGTCCCTGACCGGCAAGCGCCGTGTGGTCCTGCCTCCGCATGTTGGGGACGGCGGTGCCGGTAAATCCGCATTTGTCTCGAACTTTGAGTTCACCGGTACGCGTGACTACGCCCCGAACTATTGCGTGAAAGATGCAGTTGCGTACCGCCGCGACGTACTTGGCGGTGAGGAACGCATCCTGGGCTATCTGTGGGAGCTGAATAAGAAAGGTTCACGGCTCGTCGCTGAACGTTTGGGTACTGAGGTTCTGGAAAACAAGAAGGGCACCCTGACGAACTGCGCTATGTCTAACATCGCAATGCCGCTTTGGAAAGGTGAAAAAGAGGGTAAAGAAGGCGACGATGTCGTAGTGCCGGAAGAGGACGGAGACCGTGTGGTTGCATGGATGATGAGCACGATGGCGCGTGACTACGACACCATTGTTCCGATGTTTTGGTTGGGCAGACGCTTCTGGGTTCGAATCGCGGCCCAAGTGTACCTGGACTTAGGTGACTATGAGTACGGCGCTGAAGTGTTGAAAAAGCTGGTTGAACGTGTTGGTAAGGGCGAGTATAAGAGCGGTCAGCAAAACTAA.
Amino acid sequence (shown as SEQ ID NO. 5)
MASLPVRQREEGEAKVGEDGFKVFGGEMTKDFLFAPNWTNLNHGSYGSIPRAIQAKLRSYQDDIEARPDPFVRFEHARLTDESRAAVAGVLNAPVETVVFVNNATEGVNTVFRNIKWDADGKDVALYFTTVYEACGKAIDFLYDYHGEGRLSSREIEIAYPIEDDEILRRFRAAVEQVRSEGKRAKICIFDVVSSRPGVVFPWERMVAACRELGVLSLVDGAQGIGMVRLDLGAADPDFFVSNCHKWLFAPRGCAVFYVPVRNQGLLPSTLATSHGYASLTGKRRVVLPPHVGDGGAGKSAFVSNFEFTGTRDYAPNYCVKDAVAYRRDVLGGEERILGYLWELNKKGSRLVAERLGTEVLENKKGTLTNCAMSNIAMPLWKGEKEGKEGDDVVVPEEDGDRVVAWMMSTMARDYDTIVPMFWLGRRFWVRIAAQVYLDLGDYEYGAEVLKKLVERVGKGEYKSGQQN.
Trichoderma harzianum Trichoderma harzianum
Base sequence (shown as SEQ ID NO. 3)
GTATCACTAACAATAAGGGAAAGAGATGGAGAGGAAGCTAAGGTTGGTGAGGACGGTTTTAAAGTGTTCGGCCGTGAGATGCGTAAGGACTTCCTGTTTGCACCGGACTGGACCAACCTGAACCACGGCTCTTACGGCTCAATTCCGCGTGCAATTCAAGATAAGCTGCGTGGTTACCAGGACGACATCGAGGCGAGACCAGACCCGTTTATCCGTTTCGCCCTGGCCCGTCTTACGGATGAAAGCCGGGAGGCCGTTGCGGGTGTTGTGAACGCTCCGGTGGAAACCGTTGTTTTCGTCAACAACGCTACCGAAGGCGTTAACACCGTTTTTCGTAACTTGAAGTGGAATCCGGATGGGAAGGACGTTGCCTTGTGCTTCAGCACGGTCTATGATGCGTGCGGTAAAGTTATCGATTTCCTGTACGACTACCACGGTGAGGGCCGTTTCACCTCGCGCGAGATCCCGATCACCTATCCGATCGAGGACGACGAAATTCTGCAACGTTTCCGCGATACCGTCAAGTCCGTGCAGGATGAGGGTAAGCGCGCGAAAGTCTGTATTTTTGATGTCGTTAGCAGCCGTCCGGGTGTGGTGTTCCCGTGGGAACGCATGGTTAAAGCGTGTCGTGAGCTGGGCGTGCTTAGTCTGGTCGATGGTGCGCAAGGTATTGGCATGGTGCGCCTGAACTTGTCCGAAGCGGACCCGGATTTCTTTGTGTCTAATTGCCATAAATGGTTATTTACCCCGCGTGGCTGCGCGGTGTTCTACGTTCCGGTTCGCAACCAGCATCTGCTGCCGACCACCTTAGCGACTTCTCACGGTTACAGCAGCCAGAGCGGTGAGCACAGCATCCGTCCGGAACCGCCATATAAACCGAAAGATAAATCCTTTTTCGTAAATAATTTTGAGTTCACGGGTACGCGTGACTACGCTCCCAACTTGTGTGTTAAGGACGCGGTTAAGTATCGCAAAGAAGTGCTCGGTGGTGAGGAACGTATCCTGAGCTATCTGTGGGATCTGAACAAAAAGGGTTCGAAATTGGTAGCTGAGAAATTGGGTACTGAGGTTCTGGAAAATAACAAAGGCACATTGACCAATTGCAGCATGGCAAATATTTCCATCCCGCTGTGGCGTGGCGACAAAGGTGAGGCGAAGAAGGGCGACGTTGTGGTGCCGGTCGAAGACGGTGACCGCATCGTGGTGTGGATGATGAGCACGATGGCGAGCGATTACAAGACCATTGTTCCTATGTTTTGGCTGGGAAACCGTTTCTTCGTTCGCATGAGCGCACAGATTTATCTGGATCTGGACGACTACGAATTTGGTGCGGAAACCCTGAAAAAGCTCGTGGATCGTGTGGGCAAGGGCGAGTATAAGGCATAA.
Amino acid sequence (shown as SEQ ID NO. 6)
MVSLTIRERDGEEAKVGEDGFKVFGREMRKDFLFAPDWTNLNHGSYGSIPRAIQDKLRGYQDDIEARPDPFIRFALARLTDESREAVAGVVNAPVETVVFVNNATEGVNTVFRNLKWNPDGKDVALCFSTVYDACGKVIDFLYDYHGEGRFTSREIPITYPIEDDEILQRFRDTVKSVQDEGKRAKVCIFDVVSSRPGVVFPWERMVKACRELGVLSLVDGAQGIGMVRLNLSEADPDFFVSNCHKWLFTPRGCAVFYVPVRNQHLLPTTLATSHGYSSQSGEHSIRPEPPYKPKDKSFFVNNFEFTGTRDYAPNLCVKDAVKYRKEVLGGEERILSYLWDLNKKGSKLVAEKLGTEVLENNKGTLTNCSMANISIPLWRGDKGEAKKGDVVVPVEDGDRIVVWMMSTMASDYKTIVPMFWLGNRFFVRMSAQIYLDLDDYEFGAETLKKLVDRVGKGEYKA.
EXAMPLE 2 cultivation of cell and preparation of crude enzyme solution
1. Culture of bacterial cells
Liquid medium composition: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl, and is dissolved in deionized water, then the volume is fixed, and the solution is sterilized at 115 ℃ for 30min for later use.
After streaking and activating engineering bacteria containing egt1 and egt2 genes on a plate, single colonies are selected and inoculated into 5mL LB liquid medium containing 50 mug/mL kanamycin, and shake culture is performed at 37 ℃ for 8 hours. Transferring into 50mL fresh LB liquid medium containing 50 mug/mL Kan according to 2% inoculum size, shake culturing at 37deg.C until OD600 reaches about 0.6, adding IPTG to final concentration of 0.5mM, and inducing culturing at 28deg.C for 18h. After the completion of the culture, the culture broth was centrifuged at 10000rpm for 5 minutes, the supernatant was discarded, and the cells were collected and washed twice with 50mM phosphate buffer pH 8.0. Storing in a refrigerator at the ultralow temperature of 80 ℃ below zero for later use.
2. Preparation of crude enzyme solution
The cells collected after the completion of the culture were suspended 10 times in 50mM phosphate buffer solution at pH 8.0, and sonicated 30 times at 400W power for 3s each time and intermittently for 7s. The cell disruption solution was centrifuged at 12000rpm for 10min at 4℃to remove the precipitate, and the obtained supernatant was a crude enzyme solution (10 g wet cell/L) containing recombinant ergothioneine synthase.
Example 3 one pot method of ergothioneine using single gene expression engineering bacteria using pET28-a (+) as a vector, ESCHERICHIA COLI BL (DE 3) as a host cell
Engineering bacteria (BL 21-pET28a-Thegt1, BL21-28a-Mtegt1, BL21-28a-Ntegt1, BL21-28a-Flegt1, BL21-28a-Xgegt 1) expressing Egt1 and engineering bacteria (BL 21-28a-Tlegt2, BL21-28a-Thegt 2) expressing Egt2 were cultured respectively as in example 2 to obtain crude enzyme solutions, and the two crude enzyme solutions were combined with each other to perform 14 reactions in total.
In 1L of the reaction solution, histidine betaine concentration was 200mM, L-cysteine concentration was 300mM, TCEP or DTT concentration was 50mM, pyridoxal phosphate concentration was 100. Mu.M, and ferrous ion concentration was 100. Mu.M. 350mL of Egt1 crude enzyme solution and 150mL of Egt2 crude enzyme solution. The reaction temperature was controlled to 30℃by means of a water bath, pH 8.0, stirring speed 150-300rpm, conversion and ee value were checked every 10 minutes until the reaction was completed. The reaction end data are shown in Table 1.
Example 4 one pot method of ergothioneine using single gene expression engineering bacteria using pET28-a (+) as a vector and Vibrio natriegens VnDX as a host cell
Engineering bacteria (VNDX-pET 28a-Thegt1, VNDX-28a-Mtegt1, VNDX-28a-Ntegt1, VNDX-28a-Flegt1, VNDX-28a-Xgegt 1) expressing Egt1 and engineering bacteria (VNDX-28 a-Tlegt2, VNDX-28a-Thegt 2) expressing Egt2 were cultured respectively as in example 2 to obtain crude enzyme solutions, and the two crude enzyme solutions were combined with each other to perform 14 reactions in total.
In 1L of the reaction solution, histidine betaine concentration was 200mM, L-cysteine concentration was 300mM, TCEP or DTT concentration was 50mM, pyridoxal phosphate concentration was 100. Mu.M, and ferrous ion concentration was 100. Mu.M. 350mL of Egt1 crude enzyme solution and 150mL of Egt2 crude enzyme solution. The reaction temperature was controlled to 30℃by means of a water bath, pH 8.0, stirring speed 150-300rpm, conversion and ee value were checked every 10 minutes until the reaction was completed. The reaction end data are shown in Table 1.
Example 5 one pot method of ergothioneine by using a polygene co-expression engineering bacterium using pETDuet-1 as a vector, ESCHERICHIA COLI BL (DE 3) as a host cell
ESCHERICHIA COLI BL21 (DE 3) engineering bacteria (BL21-Duet-Thegt1-Tlegt2、BL21-Duet-Mtegt1-Tlegt2、BL21-Duet-Ntegt1-Tlegt2、BL21-Duet-Flegt1-Tlegt2、BL21-Duet-Xgegt1-Tlegt2) co-expressing Egt1 and TlEgt2 and engineering bacteria (BL21-Duet-Thegt1-Thegt2、BL21-Duet-Mtegt1-Thegt2、BL21-Duet-Ntegt1-Thegt2、BL21-Duet-Flegt1-Thegt2、BL21-Duet-Xgegt1-Thegt2) co-expressing Egt1 and ThEgt were cultured separately as in example 2 to obtain crude enzyme solutions, and 14 reactions were performed in total.
In 1L of the reaction solution, the concentration of histidine betaine was 200mM, the concentration of L-cysteine was 300mM, the concentration of TCEP was 50mM, the concentration of pyridoxal phosphate was 100. Mu.M, the concentration of ferrous ion was 100. Mu.M, and the concentration of crude enzyme was 500mL. The reaction temperature was controlled to 30℃by means of a water bath, pH 8.0, stirring speed 150rpm, and conversion and ee value were measured every 10 minutes until the reaction was completed. The reaction end data are shown in Table 1.
Example 6 one pot method of ergothioneine by using a polygene co-expression engineering bacterium using pETDuet-1 as a vector and Vibrio natriegens VnDX as a host cell
The Vibrio natriegens VnDX engineering bacteria (VNDX-Duet-Thegt1-Tlegt2、VNDX-Duet-Mtegt1-Tlegt2、VNDX-Duet-Ntegt1-Tlegt2、VNDX-Duet-Flegt1-Tlegt2、VNDX-Duet-Xgegt1-Tlegt2) co-expressing Egt1 and TlEgt2 and the engineering bacteria (VNDX-Duet-Thegt1-Thegt2、VNDX-Duet-Mtegt1-Thegt2、VNDX-Duet-Ntegt1-Thegt2、VNDX-Duet-Flegt1-Thegt2、VNDX-Duet-Xgegt1-Thegt2) co-expressing Egt1 and ThEgt2 were cultured respectively as in example 2 to obtain crude enzyme solutions, and 14 reactions were performed in total.
In 1L of the reaction solution, histidine betaine concentration was 200mM, L-cysteine concentration was 300mM, DTT concentration was 50mM, pyridoxal phosphate concentration was 100. Mu.M, ferrous ion concentration was 100. Mu.M, and crude enzyme solution was 500mL. The reaction temperature was controlled to 30℃by means of a water bath, pH 8.0, stirring speed 150rpm, and conversion and ee value were measured every 10 minutes until the reaction was completed. The reaction end data are shown in Table 1.
TABLE 1 conversion and ee% values of ergothioneine prepared by one-pot method
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Claims (5)
1. A genetically engineered bacterium comprising a host cell and a target gene transferred into the host cell, wherein the target gene consists of a target gene I and a target gene II; the target gene I is shown as SEQ ID NO. 1; the target gene II is shown as SEQ ID NO.2 or SEQ ID NO. 3; the host cell is Escherichia coli (ESCHERICHIA COLI) BL21 (DE 3) or Vibrio natrium (Vibrio natriegens VnDX) ATCC14048.
2. The genetically engineered bacterium of claim 1, wherein the gene of interest I and the gene of interest II are co-inserted on the same episomal plasmid and transformed into the same host cell.
3. The genetically engineered bacterium of claim 2, wherein the episomal plasmid is a petdeut-1 plasmid or a pET28-a (+) plasmid.
4. The use of a genetically engineered bacterium according to any one of claims 1 to 3 for the production of ergothioneine.
5. A method for producing ergothioneine, comprising: preparing a catalyst by using histidine betaine and L-cysteine as substrates, using tris (2-carbonyl ethyl) phosphate hydrochloride or 1, 4-dithiothreitol as a reducing agent, using pyridoxal phosphate and ferrous ions as coenzymes, and using the whole cell, wet thalli or crude enzyme solution of the genetically engineered bacterium of any one of claims 1-3 to obtain ergothioneine through catalytic reaction;
The catalytic reaction takes water as a solvent, the reaction temperature is 25-40 ℃, and the pH value is 6-9; the concentration of the histidine betaine is 50 mM-500 mM, the concentration of the L-cysteine is 75 mM-750 mM, the concentration of the tris (2-carbonyl ethyl) phosphate hydrochloride or the 1, 4-dithiothreitol is 25 mM-75 mM, the concentration of the pyridoxal phosphate is 50 mu M-200 mu M, and the concentration of the ferrous ion is 50 mu M-200 mu M.
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| WO2022174177A1 (en) * | 2021-02-15 | 2022-08-18 | Conagen Inc. | Microbial ergothioneine biosynthesis |
| CN115976129A (en) * | 2023-01-05 | 2023-04-18 | 上海锐康生物技术研发有限公司 | Method for preparing ergothioneine |
| CN116286421A (en) * | 2023-03-23 | 2023-06-23 | 广州悦荟化妆品有限公司 | Pichia pastoris strain for producing ergothioneine and construction method and application thereof |
| WO2023208146A1 (en) * | 2022-04-27 | 2023-11-02 | 武汉合生科技有限公司 | Method and carrier for biosynthesis of ergothioneine |
| WO2023213276A1 (en) * | 2022-05-03 | 2023-11-09 | 中国科学院上海有机化学研究所 | Chemical-enzyme coupling method for synthesizing ergothioneine |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022174177A1 (en) * | 2021-02-15 | 2022-08-18 | Conagen Inc. | Microbial ergothioneine biosynthesis |
| WO2023208146A1 (en) * | 2022-04-27 | 2023-11-02 | 武汉合生科技有限公司 | Method and carrier for biosynthesis of ergothioneine |
| WO2023213276A1 (en) * | 2022-05-03 | 2023-11-09 | 中国科学院上海有机化学研究所 | Chemical-enzyme coupling method for synthesizing ergothioneine |
| CN115976129A (en) * | 2023-01-05 | 2023-04-18 | 上海锐康生物技术研发有限公司 | Method for preparing ergothioneine |
| CN116286421A (en) * | 2023-03-23 | 2023-06-23 | 广州悦荟化妆品有限公司 | Pichia pastoris strain for producing ergothioneine and construction method and application thereof |
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