CN116790536B - Bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof - Google Patents

Bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof Download PDF

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CN116790536B
CN116790536B CN202210826807.5A CN202210826807A CN116790536B CN 116790536 B CN116790536 B CN 116790536B CN 202210826807 A CN202210826807 A CN 202210826807A CN 116790536 B CN116790536 B CN 116790536B
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黄胜雄
杨静
王永江
黄建萍
罗剑英
颜一军
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Kunming Institute of Botany of CAS
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Abstract

The invention discloses bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof. EnEMT1 the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 3; enEMT2 amino acid sequence is shown as SEQ ID NO.8, and nucleotide sequence is shown as SEQ ID NO. 7. The protein expressed by the escherichia coli can catalyze the ecgonine to generate methyl ecgonine. The discovery of the bud ketone methyltransferase provides more basic elements for the synthesis biology of natural products, provides guidance and basis for the rational design of the enzyme, and has good industrialized prospect.

Description

Bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof
Technical Field
The invention belongs to the technical field of plant molecular biology, and particularly relates to bud ketone methyltransferases EnEMT and EnEMT from lithocarpus coreanus, and genes and applications thereof.
Background
Tropane alkaloids (tropane alkaloids) are a class of alkaloids containing 8-azabicyclo [3.2.1] octane as a skeleton, and are mainly distributed in plants of the family of cocaceae (Erythroxylaceae), solanaceae (Solanaceae), convolvulaceae (Convolvulaceae), hongshui (Rhizophoraceae), brassicaceae (Brassicaceae) and the like, and more than 300 tropane alkaloids have been identified. The plant source tropane alkaloid has good physiological activity, such as: cocaine separated from the family of cocaceae can block conduction between nerve fibers and has postoperative vasoconstriction effect, and can be used as local anesthetic for nasal, throat and lower respiratory tract surgery; the hyoscyamine and scopolamine isolated from Solanaceae can block parasympathetic nerve or inhibit central nervous system, etc., thereby can be used for relieving pain and spasmolysis, etc.
The tropane alkaloid has a plurality of chiral centers, is synthesized by a chemical means directly, has complex process and high cost, and is mostly separated from plants in medical use at the present stage; however, the biosynthesis route of tropane alkaloids such as cocaine is not clear, the rate limiting step of in vivo synthesis of tropane alkaloids such as cocaine cannot be eliminated, and the improvement of the output of tropane alkaloids in plants is limited. In addition, the separation of tropane alkaloids by plants is affected by various factors such as plant growth and climate change, and the supply of tropane alkaloids such as cocaine is also limited. The development of modern molecular biology and synthetic biology has made it possible to produce cocaine and other natural medicinal products in microorganisms without the use of original plants. Analysis of the biosynthetic pathway of cocaine and excavation of biosynthetic genes are the most important basic elements in microbial production as the basis for metabolic engineering.
The biosynthesis pathway of cocaine is presumed to be as follows based on the existing isotopic precursor labeling experiments and the like: starting from ornithine, undergo N-methylpyrrole cations, 4- (1-methyl-2-pyrrole) -3-oxobutanoic acid, methyl bud ketone and other intermediates; finally, methyl ecgonine condenses with benzoyl CoA under the action of CS enzymes to form cocaine. However, in the prior art, there are no reports of the identification of enzymes EnEMT and EnEMT associated with methyl bud ketone synthesis and the genes encoding them, and the use of bud ketone methyltransferases (EnEMT 1 and EnEMT 2).
Disclosure of Invention
In view of the above-described deficiencies in the prior art, the present invention provides budesone methyltransferases EnEMT and EnEMT from lithocarpus, and genes and uses thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
Providing a buddleone methyltransferase which has a protein of one of the following amino acid residue sequences:
a) A protein having an amino acid residue sequence shown in SEQ ID NO.4 or SEQ ID NO. 8;
b) Amino acid sequence derived from a) with a) enzyme function by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid residues in SEQ ID NO. 4or SEQ ID NO. 8;
c) A protein derived from a) having 80% or more homology with the amino acid sequence defined in a) or b) and having a) an enzymatic function;
d) Derived proteins comprising the amino acid sequences of a), b) or c) are included in the sequences.
Providing a buddleone methyltransferase gene having a polynucleotide of one of the following nucleotide sequences:
a) A polynucleotide with a nucleotide sequence shown as SEQ ID NO.3 or SEQ ID NO. 7;
b) A polynucleotide encoding an amino acid sequence shown as SEQ ID NO.4 or SEQ ID NO. 8;
c) A polynucleotide having more than 80% homology to the nucleotide sequence defined in a) or b) and encoding a function of the bud ketomethyltransferase of claim 1;
d) A polynucleotide complementary to the sequence of a), b) or c).
Recombinant expression vectors comprising the buddleone methyltransferase gene are provided.
Further, the recombinant expression vector is obtained by inserting the buddleone methyltransferase gene into a prokaryotic or eukaryotic expression vector.
Further, the bud ketone methyltransferase gene is constructed into a pET28a vector.
Transgenic recombinant bacteria or transgenic cell lines comprising a buddlenone methyltransferase gene are provided.
Further, the transgenic recombinant bacteria are bacteria and fungi, including but not limited to, escherichia coli, bacillus subtilis, pichia pastoris, saccharomyces cerevisiae.
Provides the application of the methyl bud ketone methyltransferase in the in-vivo or in-vitro catalysis generation of methyl bud ketone and derivatives thereof.
There is provided the use of a budesonide methyltransferase in the catalytic production of tropane alkaloids, including but not limited to cocaine, in vivo or in vitro.
Provides the application of a recombinant expression vector containing the methyl bud ketone transferase gene in constructing the synthesis path of methyl bud ketone and derivatives thereof in prokaryotes or eukaryotes without the synthesis path of methyl bud ketone and derivatives thereof or in improving the yield of methyl bud ketone and derivatives thereof in prokaryotes or eukaryotes with the synthesis path of methyl bud ketone and derivatives thereof.
There is provided the use of a recombinant expression vector comprising a budesonide-methyl transferase gene in the construction of a tropane alkaloid biosynthesis pathway in a prokaryote or eukaryote not having a tropane alkaloid biosynthesis pathway, or in the enhancement of the yield of tropane alkaloid in a prokaryote or eukaryote having a tropane alkaloid biosynthesis pathway.
The use of transgenic recombinant bacteria or transgenic cell lines comprising a buddleone methyltransferase gene for catalyzing methylation of buddleones and derivatives thereof is provided.
The beneficial effects of the invention are as follows:
The invention discloses a bud ketone methyltransferase, wherein the amino acid sequence of the bud ketone methyltransferase is EnEMT shown in SEQ ID NO.4, enEMT is shown in SEQ ID NO.8, the coded nucleotide is EnEMT shown in SEQ ID NO.3, enEMT is shown in SEQ ID NO.7, and the bud ketone methyltransferase can catalyze bud ketone to generate methyl bud ketone after prokaryotic expression. The discovery of the bud ketone methyltransferase provides more basic elements for the synthesis biology of natural products, provides guidance and basis for the rational design of the enzyme, and has good industrialized prospect.
Drawings
FIG. 1 is a schematic diagram of the equations for the enzymatic chemical reactions catalyzed by EnEMT and EnEMT2 according to an example of the present invention;
FIG. 2 is a plasmid map of EnEMT-pET 28a provided in an example of the present invention;
FIG. 3 is a plasmid map of EnEMT-pET 28a provided in an example of the present invention;
FIG. 4 is a diagram showing the result of SDS-polyacrylamide gel electrophoresis of EnEMT and EnEMT2 proteins provided in the examples of the present invention;
FIG. 5 is a diagram of an LC-MS analysis of the catalytic formation of methyl bud ketone from bud ketone using EnEMT and EnEMT2 provided in the examples of the present invention.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
Example 1
Cloning of the buddleone methyltransferase (ecgonone methyltransferase, EMT) gene.
(1) Extraction of total RNA of the lithocarpus sprout and synthesis of the first strand of cDNA.
Taking a proper amount of lithocarpus leaf bud tissue, grinding in liquid nitrogen, and extracting total RNA by using biotek polysaccharide polyphenol plant total RNA rapid extraction kit according to instruction. RNA concentration and quality were measured using Thermo Scientific NanoDrop spectrophotometer and RNA quality was measured using agarose gel electrophoresis.
CDNA was synthesized using the Rev company HISCRIPT III, no. 1, st Strand cDNA Synthesis Kit reverse transcription kit, using total RNA as template, as indicated by the product instructions.
(2) Cloning of EnEMT and EnEMT genes.
Specific primers were designed, the specific primer sequences were as follows:
EnEMT1-F:5’-atggcaattgaacaagtgcttcacat-3’(SEQ ID NO.1)
EnEMT1-R:5’-tcaatccaaatcaactgcagttctt-3’(SEQ ID NO.2)
EnEMT2-F:5’-atggcaattgatcaagtacttcacatg-3’(SEQ ID NO.5)
EnEMT2-R:5’-tcaatccatatcaactacagttctc-3’(SEQ ID NO.6)
Amplifying EnEMT genes and EnEMT genes by PCR with cDNA of tender shoot tissues as templates, and sequencing to obtain nucleotide sequences of EnEMT genes and EnEMT genes, wherein EnEMT1 is shown as SEQ ID NO.3, an initiation codon is ATG, and a termination codon is TGA; the translated protein coding sequence is shown as SEQ ID NO. 4; enEMT2 is shown as SEQ ID NO.7, the start codon is ATG, and the stop codon is TGA; the translated protein has a coding sequence shown in SEQ ID NO. 8.
Example 2
Prokaryotic expression verifies EnEMT and EnEMT gene function.
EnEMT1 and EnEMT genes were introduced at the cleavage site. Designing primers for EnEMT and EnEMT genes, wherein a forward primer of EnEMT1 has a homology arm sequence shown as SEQ ID NO.9, and a reverse primer has a homology arm sequence shown as SEQ ID NO. 10; enEMT2 the forward primer has homology arm sequence shown in SEQ ID NO.11, and the reverse primer has homology arm sequence shown in SEQ ID NO. 12. After EnEMT and EnEMT genes with homologous arm sequences at two ends are obtained through PCR amplification, the complete sequences of the coding regions of EnEMT and EnEMT are respectively connected with a plasmid pET28a by using recombinase (ClonExpress Ultra One Step Cloning Kit) to obtain recombinant expression vectors EnEMT1-pET28a and EnEMT-pET 28a. As shown in fig. 2-3.
The primer sequences were as follows:
EnEMT1-AF:5’-tggtgccgcgcggcagccatatggcaattgaacaagtgcttc-3’(SEQ ID NO.9)
EnEMT1-AR:5’-cggagctcgaattcggatcctcaatccaaatcaactgcagtt-3’(SEQ ID NO.10)
EnEMT2-AF:5’-tggtgccgcgcggcagccatatggcaattgatcaagtacttc-3’(SEQ ID NO.11)
EnEMT2-AR:5’-cggagctcgaattcggatcctcaatccatatcaactacagtt-3’(SEQ ID NO.12)
The prokaryotic expression strains BL21 (DE 3) are transformed by the constructed EnEMT-pET 28a and EnEMT-pET 28a plasmids, LB solid plates containing kanamycin are coated, single colony culture is selected, plasmids are extracted for enzyme digestion verification, positive clones are screened, and prokaryotic expression engineering bacteria EnEMT1-pET28a-BL21 (DE 3) and EnEMT-pET 28a-BL21 (DE 3) are obtained. 20. Mu.L of the bacterial liquid was inoculated into 20mL of LB liquid medium containing 50. Mu.g/L kanamycin, and cultured overnight at 37℃and 200 rpm. Then according to the following steps of 1:100 proportion of inoculum size was inoculated into 2L of LB liquid medium at 37℃and 200rpm, and cultured until OD 600 =0.8 or so, and then ice-cooled for ten minutes, and IPTG was added to a final concentration of 0.2mM. The culture was continued at 16℃and 200rpm for 18 hours. The harvested bacterial liquid was centrifuged at 4000rpm and the supernatant removed. The cells were resuspended in protein purification buffer and, after sonication, protein purification was performed with nickel columns to obtain EnEMT and EnEMT proteins. The resulting protein was detected by SDS-polyacrylamide gel electrophoresis, as shown in FIG. 4.
The equations of the enzymatic chemistry reactions catalyzed by EnEMT and EnEMT are shown in FIG. 1, and the system of the in vitro enzymatic reactions catalyzed by EnEMT and EnEMT are as follows: 50mM potassium phosphate buffer (pH 7.5), 1mM bud ketone, 1mM SAM,300ng u L -1 EnEMT or EnEMT protein, total volume 100 u L,30 degrees C, after 1h incubation, adding equal acetonitrile to terminate the reaction.
The enzyme reaction products were analyzed by LC-MS with an LC-MS chromatography-mass spectrometer (agilent 1290/6530 system), a chromatographic column (YMC-Triart C 18 (I.D. 4.6X1250 mm), a column temperature of 30℃and a flow rate of 1mL/min, and a mobile phase elution procedure: 90% phase A (water with 0.1% formic acid), 10% phase B (acetonitrile), isocratic elution for 6min, mass spectrometer for electrospray ion source (ESI), positive ion mode scan, scan range (m/z): 50-400.
The results of the assay showed that EnEMT and EnEMT2 catalyzed the production of methyl budesone from budesone (m/z: 198.1125[ M+H ] +) with a retention time of 3.7min under the assay conditions as shown in FIG. 5.
The invention discloses a bud ketone methyltransferase, wherein the amino acid sequence of the bud ketone methyltransferase is EnEMT shown in SEQ ID NO.4, enEMT is shown in SEQ ID NO.8, the coded nucleotide is EnEMT shown in SEQ ID NO.3, enEMT is shown in SEQ ID NO.7, and the bud ketone methyltransferase can catalyze bud ketone to generate methyl bud ketone after prokaryotic expression. The discovery of the bud ketone methyltransferase provides more basic elements for the synthesis biology of natural products, provides guidance and basis for the rational design of the enzyme, and has good industrialized prospect.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (9)

1. The bud ketone methyltransferase is characterized in that the amino acid sequence is shown as SEQ ID NO.4 or SEQ ID NO. 8.
2. The bud ketone methyl transferase gene is characterized in that the nucleotide sequence is shown as SEQ ID NO.3 or SEQ ID NO. 7.
3. A recombinant expression vector comprising the bud ketomethyltransferase gene of claim 2.
4. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is obtained by inserting a budesone methyltransferase gene into a prokaryotic or eukaryotic expression vector to obtain the recombinant expression vector for expressing budesone methyltransferase.
5. The recombinant expression vector according to claim 3, wherein the buddleone methyltransferase gene is constructed into pET28a vector.
6. A transgenic recombinant bacterium comprising the bud ketomethyltransferase gene of claim 2.
7. The transgenic recombinant strain of the buddlenone methyltransferase gene according to claim 6, wherein the transgenic recombinant strain is any one of escherichia coli, bacillus subtilis, pichia pastoris and saccharomyces cerevisiae.
8. Use of the ecgonione methyltransferase of claim 1 for in vitro catalytic production of methyl ecgonione;
the chemical formula of the bud ketone is as follows: 。
9. The use of the transgenic recombinant bacterium of claim 6 for catalyzing bud ketone methylation in vitro;
the chemical formula of the bud ketone is as follows: 。
CN202210826807.5A 2022-07-13 2022-07-13 Bud ketone methyltransferases EnEMT and EnEMT from lithocarpus erythropolis and genes and applications thereof Active CN116790536B (en)

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PCT/CN2022/122698 WO2024011761A1 (en) 2022-07-13 2022-09-29 Ecgonone transmethylases enemt1 and enemt2 from erythroxylaceae, and genes thereof and use thereof

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S-adenosyl-methionine 4-1-methyl-2-pyrrolidinyl-3-oxobutanic acid methyltransferase {ECO:0000313|;UNIPROT;UNIPROT;20230503;A0A9E9KUV1 *
salicylate carboxymethyltransferase [Jatropha curcas];NCBI;GenBank DataBase;20201116;Accession No.XP_020532383.1 *
可卡因毒性研究;夏瑞等;现代预防医学;20070605(第11期);摘要 *
植物次生代谢途径与调控研究进展;朱三明等;植物生理学报;20231201;第59卷(第12期);摘要 *

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