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WO2024251059A1 - Céphalotaxane diterpène synthase, son procédé de préparation et son utilisation - Google Patents

Céphalotaxane diterpène synthase, son procédé de préparation et son utilisation Download PDF

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Publication number
WO2024251059A1
WO2024251059A1 PCT/CN2024/096943 CN2024096943W WO2024251059A1 WO 2024251059 A1 WO2024251059 A1 WO 2024251059A1 CN 2024096943 W CN2024096943 W CN 2024096943W WO 2024251059 A1 WO2024251059 A1 WO 2024251059A1
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seq
cell
amino acid
acid sequence
cephalotaxane
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王勇
孙雨伟
毛亚平
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Center for Excellence in Molecular Plant Sciences of CAS
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Center for Excellence in Molecular Plant Sciences of CAS
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Definitions

  • the present invention relates to the field of synthetic biology and pharmaceutical technology, and in particular to a novel diterpene cyclase, a microorganism for synthesizing a novel cephalotaxane diterpene skeleton, a preparation method thereof and an application thereof.
  • Cephalotaxus truncatus is an endangered species.
  • the methods for obtaining plant compounds include plant extraction, chemical synthesis, cell tissue culture and plant endophytic fungi.
  • plant secondary metabolites are strictly regulated in plants, resulting in very low content in plants, and the long growth cycle of plants and the long extraction process have limited the development of plant extraction technology.
  • the chemical synthesis process is very mature and has low production costs, it has the advantage of saving resources; however, the chemical synthesis reaction conditions are difficult to control, the products are complex, and the pollution problems generated during the synthesis process have yet to be solved.
  • Plant cell tissue culture has the advantage of expanding the source of drugs, but the culture cycle is relatively long, the synthesis capacity is low and unstable.
  • the mechanism of action between endophytic fungi and host plants is still unclear and is in its infancy.
  • some medicinal or industrial ingredients are mainly produced by extracting their active ingredients from Cephalotaxus plants, which has put certain pressure on the wild resources and cultivation of Cephalotaxus plants.
  • the purpose of the present invention is to provide a novel cephalotaxane diterpene synthase and its application.
  • a method for preparing a diterpene core compound comprising: using cephalotane diterpene synthase (CS) to catalyze geranylgeranyl pyrophosphate (GGPP) to produce a diterpene core compound product; wherein the cephalotane diterpene synthase is an enzyme having an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a conservative variant protein thereof.
  • the conservative variant protein includes: (a) a derivative protein formed by replacing, deleting or adding one or more (such as 1-30 or 1-20; preferably 1-15; preferably 1-10; preferably 1-5, 1-3 or 1-2) amino acid residues of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, and having the catalytic function; (b) a derivative protein having an amino acid sequence that is more than 80% (preferably more than 85%, more than 90% or more than 95%; such as more than 98% or more than 99%) identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 and having the catalytic function; or (c) a protein formed by adding a tag or signal peptide sequence to the N or C terminus of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the conservative variant protein of the cephalotane diterpene synthase includes: a variant protein corresponding to the amino acid sequence shown in SEQ ID NO: 1, with a C to G mutation at position 833, a V to F mutation at position 718, and an M to F mutation at position 627, and the enzyme with the amino acid sequence shown in SEQ ID NO: 1 or the variant protein is used to prepare cephalot-12(13)-ene.
  • the conservative variant protein of the trifoliate diterpene synthase includes: a variant protein corresponding to the amino acid sequence shown in SEQ ID NO: 1, with a mutation from F to V at position 625, and the variant protein is used to prepare one or more diterpene core compounds including (but not limited to) the following: (1R)-verticillia-4(20), 7,11-triene, taxadiene, verticillia-3,7,12-triene, verticillia-3,7,12(18)-triene and (1S)-verticillia-4(20), 7,11-triene.
  • the taxane diterpene synthase or its conservative variant protein is a protein with a truncated N-terminus; preferably, it corresponds to the protein of the amino acid sequence shown in SEQ ID NO: 1, truncated from the 2nd to 54th amino acid residues at its N-terminus, or corresponds to SEQ ID NO: 2, truncated from the 2nd to 54th amino acid residues at its N-terminus.
  • the method is performed in vitro (eg, by catalyzing a substrate through a chemical reaction) or in a cell (eg, by biosynthesis through genetically engineered cells).
  • the method is carried out in a cell; preferably, the cell is a eukaryotic cell or a prokaryotic cell (such as Escherichia coli or Bacillus subtilis); preferably, the eukaryotic cell includes: a plant cell, a fungal cell, an insect cell or a mammalian cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, the plant cell is a tobacco cell, or the fungal cell is a yeast cell (such as Pichia pastoris).
  • the cell is a eukaryotic cell or a prokaryotic cell (such as Escherichia coli or Bacillus subtilis); preferably, the eukaryotic cell includes: a plant cell, a fungal cell, an insect cell or a mammalian cell; preferably, the prokaryotic cell includes Escherichia coli; preferably, the plant cell is a tobacco cell, or the fungal cell is a yeast cell (such as Pichia pastoris
  • the cell is a cell containing (including naturally containing or exogenously/heterologously introduced) IPP (isopentenyl pyrophosphate) and DMAPP (dimethylallyl pyrophosphate) synthesis modules.
  • IPP isopentenyl pyrophosphate
  • DMAPP dimethylallyl pyrophosphate
  • the cell containing IPP and DMAPP synthesis modules includes (naturally occurring or exogenous/heterologously introduced): module I (precursor synthesis): including mevalonate (MVA) pathway and/or methylerythritol phosphate pathway (MEP), producing IPP and DMAPP; module II (diterpene nucleus synthesis): using IPP and DMAPP as substrates to produce GGPP (geranylgeranyl pyrophosphate).
  • MVA mevalonate
  • MEP methylerythritol phosphate pathway
  • module II diterpene nucleus synthesis
  • the cell also expresses (preferably exogenously/heterologously introduced): geranylgeranyl diphosphate synthase (GGPPS), cephalotaxane diterpene synthase (CS), or contains a nucleic acid construct encoding the protein.
  • GGPPS geranylgeranyl diphosphate synthase
  • CS cephalotaxane diterpene synthase
  • the cell (such as a eukaryotic cell, in particular a tobacco cell) further expresses HMG-CoA-reductase (HMGR).
  • HMGR HMG-CoA-reductase
  • the cell (such as a prokaryotic cell, specifically in Escherichia coli) also expresses proteins of the MVA pathway and/or the MEP pathway, including proteins selected from: AtoB acetyl-CoA acetyltransferase), MvaS (mevalonyl-CoA synthetase), MvaE (mevalonyl-CoA reductase), Mvk1 (mevalonate kinase), Mvk2 (phosphomevalonate kinase), MvaD (mevalonate pyrophosphate decarboxylase) and Fni (isopentenyl pyrophosphate ⁇ -isomerase).
  • proteins of the MVA pathway and/or the MEP pathway including proteins selected from: AtoB acetyl-CoA acetyltransferase), MvaS (mevalonyl-CoA synthetase), MvaE (mevalonyl-CoA reductas
  • the MVA pathway and/or the MEP pathway naturally occurs in the cell.
  • the cell also expresses other proteins of the MVA pathway and/or the MEP pathway or contains a nucleic acid construct encoding the proteins.
  • the cell also expresses other proteins of the MVA pathway and/or the MEP pathway or contains a nucleic acid construct encoding the proteins.
  • further recombinant expression in the presence of the naturally occurring proteins/enzymes can increase the amount of the proteins/enzymes involved in the pathway, thereby promoting the corresponding catalytic steps in the pathway.
  • the cell is a tobacco cell, and the expression is performed in a tobacco plant.
  • the method further comprises the step of isolating a diterpene core compound (such as Cephalot-12(13)-ene) or a cephalotane diterpene compound (such as Harringtonolide) formed therefrom from cells or plants containing the cells.
  • a diterpene core compound such as Cephalot-12(13)-ene
  • a cephalotane diterpene compound such as Harringtonolide
  • the step of isolating the compound from cells comprises: disrupting the cells, extracting with an organic solvent, concentrating and/or drying, and the organic solvent preferably comprises n-hexane.
  • the obtained diterpene core compound includes Cephalot-12(13)-ene, which can further form Harringtonolide.
  • the application of the cephalotaxane diterpene synthase is provided for catalyzing geranylgeranyl pyrophosphate (GGPP) to form a diterpene core compound; wherein the cephalotaxane diterpene synthase is an enzyme having an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a conservative variant protein thereof.
  • GGPP geranylgeranyl pyrophosphate
  • a cephalotaxane diterpene synthase is provided, which is an enzyme having an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or a conservative variant protein thereof.
  • the enzyme having the amino acid sequence shown in SEQ ID NO: 1 is derived from Cephalotaxus harringtonia;
  • the enzyme with the amino acid sequence shown in SEQ ID NO: 1 is derived from Cephalotaxus hainanensis.
  • a nucleic acid molecule which encodes the above-mentioned cephalotaxane diterpene synthase.
  • nucleic acid construct comprising the nucleic acid molecule.
  • the nucleic acid construct is a vector, such as a cloning vector, an integration vector or an expression vector.
  • a host cell which: (1) expresses the above-mentioned cephalotaxane diterpene synthase, or (2) contains the above-mentioned nucleic acid molecule and/or the above-mentioned nucleic acid construct.
  • the host cell is provided as a cell containing (including naturally containing or exogenously/heterologously introduced) IPP and DMAPP synthesis modules; preferably, the cell containing the IPP and DMAPP synthesis modules includes: Module I (precursor synthesis): including the mevalonate (MVA) pathway and/or the methylerythritol phosphate pathway (MEP), producing IPP and DMAPP; Module II (diterpene nucleus synthesis): using IPP and DMAPP as substrates to produce GGPP (geranylgeranyl pyrophosphate).
  • MVA mevalonate
  • MEP methylerythritol phosphate pathway
  • the cell also expresses (preferably exogenously/heterologously introduced): geranylgeranyl diphosphate synthase (GGPPS) or contains a nucleic acid construct encoding the protein; preferably, the cell also expresses HMG-CoA-reductase (HMGR).
  • GGPPS geranylgeranyl diphosphate synthase
  • HMGR HMG-CoA-reductase
  • a kit for preparing a diterpene core compound comprising: the above-mentioned cephalotaxane diterpene synthase, or a nucleic acid molecule encoding the enzyme, or a nucleic acid construct containing the nucleic acid molecule; preferably, it also comprises: geranylgeranyl diphosphate synthase (GGPPS), or a nucleic acid molecule encoding the enzyme, or a nucleic acid construct containing the nucleic acid molecule; preferably, it also comprises a nucleic acid molecule expressing HMG-CoA-reductase (HMGR), or a nucleic acid molecule encoding the enzyme, or a nucleic acid construct containing the nucleic acid molecule;
  • GGPPS geranylgeranyl diphosphate synthase
  • HMGR HMG-CoA-reductase
  • kit for preparing a diterpene core compound comprising: any of the host cells described above.
  • the kit further comprises a cell culture medium.
  • the kit further includes instructions for use describing the method for preparing the diterpene core compound.
  • FIG1 Schematic diagram of the predicted reaction pathway for the biosynthesis of cephalotaxane diterpenes.
  • Fig. 2 GC-MS analysis of heterologous expression products of strains s6070 and s3840 in Nicotiana benthamiana.
  • Figure 4 Inhibitory effect of Harringtonolide on various tumor cell lines (human oral epidermal carcinoma cell line KB, HCT-116 cells, HepG2 cells, A-549 cells, Hela cells, SGC-7901 cells).
  • FIG. 7 Determination of product yields of some mutants transiently expressed in tobacco.
  • harringtonia diterpene synthases After extensive research and screening, and mining of plant genome and transcriptome information, the inventors have obtained a group of novel harringtonia diterpene synthases from Cephalotaxus harringtonia and Cephalotaxus hainanensis.
  • the harringtonia diterpene synthases can catalyze geranylgeranyl pyrophosphate (GGPP) to produce diterpene nucleus compounds (such as cephalot-12(13)-ene) compound products, which can further generate harringtonolide compounds (such as harringtonolide).
  • GGPP geranylgeranyl pyrophosphate
  • the novel cephalotaxane diterpene synthase isolated from Cephalotaxus harringtonia and Cephalotaxus hainanensis of the present invention has an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively.
  • the present invention also includes their conservative variant polypeptides (proteins).
  • the conservative variant polypeptides are also referred to as "variants”.
  • the "conservative variant polypeptide” refers to a polypeptide that substantially maintains the same biological function or activity as the polypeptide.
  • the “conservative variant polypeptide” may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with an antigen IgG fragment).
  • the "conservative variant polypeptide” may include (but is not limited to): a sequence having at least 80% (e.g., at least 90%, 96%, at least 98%, at least 99%) sequence identity with any one of SEQ ID NOs: 1-2 while retaining its biological activity.
  • the "biological activity" of the cephalotane diterpene synthase generally refers to its ability to specifically catalyze GGPP and produce diterpene core compounds (such as cephalotane-12(13)-ene).
  • Exemplary such variants include biologically active fragments of the enzyme and variants of the enzyme or its biologically active fragments.
  • the meaning of a biologically active fragment of an enzyme refers to a polypeptide that can still maintain all or part of the functions of a full-length enzyme or protein. Typically, the biologically active fragment retains at least 98% or 99% of its activity.
  • the present invention includes a protein or enzyme whose amino acid sequence has at least 98%, at least 99% sequence identity with the enzyme while retaining the biological activity of the enzyme.
  • the variant may also be from a species of the same or similar origin (e.g. same plant).
  • the cephalotaxane diterpene synthase or its conservative variant protein is a protein with N-terminal truncated; preferably, the protein corresponding to the amino acid sequence shown in SEQ ID NO: 1, with the 2nd to 54th amino acid residues of the N-terminal truncated, or corresponding to SEQ ID NO: 2, with the 2nd to 54th amino acid residues of the N-terminal truncated.
  • Removing the polypeptide fragment can increase the soluble expression of the truncated protein and significantly improve the intracellular expression efficiency, and when applied to the biosynthetic pathway, it can exert better activity.
  • the conservative variant protein of the cephalotane diterpene synthase of the present invention includes: a variant protein corresponding to the amino acid sequence shown in SEQ ID NO: 1, with a C to G mutation at position 833, a V to F mutation at position 718, and an M to F mutation at position 627, and the enzyme or the variant protein of the amino acid sequence shown in SEQ ID NO: 1 is used to prepare cephalotane cephalot-12(13)-ene.
  • the mutant has higher catalytic activity than the wild type.
  • the cephalot-12(13)-ene can be (but not limited to) used as an intermediate for synthesizing downstream products (such as Hainan crude quercetin compounds).
  • the conservative variant protein of the trihalomethane diterpene synthase includes: a variant protein corresponding to the amino acid sequence shown in SEQ ID NO: 1, with a mutation from F to V at position 625, and the variant protein is used to prepare one or more diterpene core compounds including (but not limited to) the following: (1R)-verticillia-4(20), 7,11-triene, taxadiene, verticillia-3,7,12-triene, verticillia-3,7,12(18)-triene and (1S)-verticillia-4(20), 7,11-triene.
  • the present invention also provides a polynucleotide encoding the cephalotaxane diterpene synthase or a variant thereof described herein.
  • the polynucleotide of the present invention can be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.
  • the DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • nucleic acids due to the degeneracy of genetic code, a large amount of nucleic acids can be obtained, which all encode enzymes of the present invention. Therefore, when identifying a specific amino acid sequence, those skilled in the art can obtain any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. For example, species (e.g. tobacco) preference codons are used to optimize nucleic acid sequences so that the sequence is easier to express in the species.
  • the full-length nucleotide sequence of the protein or enzyme of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short.
  • a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • the biological molecules (nucleic acids, proteins, etc.) involved in the present invention include biological molecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules known in the art (or such as vectors).
  • mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.
  • the present invention also relates to a nucleic acid construct comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence
  • the nucleic acid construct usually carries an extrachromosomal element of a gene that is not part of the central metabolism of the cell, and is often in the form of a circular double-stranded DNA molecule.
  • Such elements can be autonomous replication sequences, genome integration sequences, phage or nucleotide sequences, linear or circular single-stranded or double-stranded DNA or RNA obtained from any source, many of which have been joined or recombined into a specific construct that is capable of introducing the promoter fragment and DNA sequence of the selected gene product together with the appropriate 3' non-translated sequence into the cell.
  • nucleic acid constructs include expression vectors and recombinant vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences.
  • the sequences typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding an antibody to be expressed, and a selectable marker element.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2 . If necessary, transformation can also be carried out by electroporation.
  • the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.
  • the protein or enzyme of the sequence of the present invention can be expressed in a heterologous host.
  • the host is, for example, a bacterial cell, a fungal cell, such as a yeast cell, a mammalian cell, an insect cell and a plant cell.
  • the heterologous host cell used to express the nucleic acid molecule of the present invention is preferably a cell in which the IPP and DMAPP synthesis modules are endogenously present, so that it can produce the precursors IPP and DMAPP for the reaction in which the enzyme of the present invention participates.
  • the precursors IPP and DMAPP are catalyzed by GGPPS to form GGPP, which can be catalyzed by the enzyme of the present invention to produce a diterpene core compound (such as cephalot-12(13)-ene).
  • the IPP and DMAPP can be the products of MVA or MEP pathways.
  • the MVA pathway is present in higher eukaryotic organisms, such as but not limited to tobacco, yeast, etc. Therefore, in the present invention, it is preferred that the cell/plant body with the MVA pathway exists.
  • acetyl-CoA is the starting substrate, and under the catalysis of acetoacetyl-CoA synthetase, HMG-CoA synthetase and HMG-CoA-reductase (HMGR), other two molecules of acetyl-CoA are used for condensation and reduction to generate the intermediate product mevalonate. Subsequently, through kinase (mevalonate enzyme, phosphomevalonate kinase, mevalonate diphosphate carboxylase) activation and decarboxylation, the IPP and DMAPP are generated.
  • kinase mevalonate enzyme, phosphomevalonate kinase, meval
  • pathway As used herein, the "pathway” and “pathway” are used interchangeably.
  • heterologous or “foreign” refers to the relationship between two or more nucleic acid or protein sequences that are derived from different sources.
  • a promoter is heterologous/foreign to a gene of interest if the combination of the promoter and the gene of interest sequence does not normally occur in nature.
  • a particular sequence is “heterologous/foreign” to the cell or organism into which it is inserted.
  • the coding sequence of the novel cephalotane diterpene synthase of the present invention is introduced into tobacco cells/tobacco plants that endogenously contain IPP and DMAPP synthesis modules, resulting in the isolation of diterpene core compounds (such as cephalot-12(13)-ene) from tobacco leaves.
  • the diterpene core compound (such as cephalot-12(13)-ene) of the present invention can further generate cephalotane diterpene compounds (such as harringtonolide).
  • Hainan Torrone is a diterpene compound isolated from Cephalotaxus plants, with a unique and complex carbon skeleton and significant anti-tumor activity. Hainan Torrone has significant anti-tumor activity against oral epidermoid cancer cells, HCT-116 colon cancer cells, HepG2 liver cancer cells, A-549 lung adenocarcinoma cells, cervical cancer cells and SGC-7901 gastric cancer cells. The IC 50 value of Hainan Torrone against oral epidermoid cancer cells is 40nM, indicating that Hainan Torrone has great drug potential.
  • Hainan quinoline lactone compounds include plant extraction, chemical synthesis, cell tissue culture and plant endophytic fungi.
  • Hainan quinoline lactone is strictly regulated in plants as a secondary metabolite of plants, resulting in a very low content in plants, and the growth cycle of plants is long, and the extraction process is time-consuming, which limits the development of plant extraction technology.
  • the present invention uses biosynthesis technology for the first time to achieve the production of the precursor nucleus of such compounds.
  • Torreya grandis and Torreya grandis were collected from Shanghai Chenshan Botanical Garden.
  • Oligonucleotide primers were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.
  • the synthetic optimized sequence was purchased from GenScript Biotech Corp.
  • AxyPrep total RNA miniprep kit, polymerase chain reaction (PCR) gel recovery kit, and plasmid extraction kit are all American Axygen products; PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) polymerase kit and polymerase chain reaction (PCR) high-fidelity enzyme PrimeSTAR Max DNA Polymerase are products of Takara Bio (TAKARA) of Japan. Seamless cloning use II One Step Cloning Kit (Nanjing Novogene Biotech Co., Ltd.).
  • Escherichia coli DH10B, Agrobacterium tumefaciens GV3101 strain, plant Nicotiana benthamiana and pET-28a, pJF25 (a plasmid obtained by modifying pET-21a to make it resistant to apramycin), and pEAQ-HT vectors were used for gene cloning and protein expression.
  • the molecular chaperone plasmid pGro7 was a product of Takara Bio Co., Ltd. of Japan.
  • GGPP geranylgeranyl pyrophosphate
  • reagents were domestic analytical or chromatographic grade reagents purchased from Sinopharm Chemical Reagent Co., Ltd.
  • Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR.
  • the gas chromatography-mass spectrometry was performed using the Thermo Trace GC ultra-ISQ spectrometer system (Thermo Fisher Scientific).
  • the present invention obtains two new types of cephalotaxane diterpene synthases CharCS and ChaiCS by mining the transcriptome information of Cephalotaxus harringtonia and Cephalotaxus hainanensis.
  • amino acid sequences of the cephalotaxane diterpene synthase are as follows:
  • the reaction pathway mainly includes:
  • IPP and DMAPP produced by the MEP and MVA pathways
  • DMAPP and IPP are condensed to form the diterpene precursor GGPP via geranylgeranyl diphosphate synthase (GGPPS);
  • Diterpene synthase CS converts the diterpene precursor GGPP into cephalotaxane.
  • the rate-limiting step of the MVA pathway is the catalytic stage of hydroxymethylglutaryl (HMG)-CoA-reductase (HMGR), and the expression of this enzyme can be enhanced.
  • HMG hydroxymethylglutaryl
  • HMGR hydroxymethylglutaryl
  • the artificially synthesized gene sequence was constructed into the pET28a vector to obtain pET28a-ScHMGR, pET28a-TcGGPPS, pET28a-CharCS, and pET28a-ChaiCS, respectively.
  • ScHMGR1 GenBank accession number CAD6640909.1;
  • TcGGPPS GenBank accession number QUF98547.1;
  • CharCS is derived from the Torreya grandis transcriptome NCBI accession number PRJNA948641;
  • ChaiCS is derived from the transcriptome of Torreya grandis L. (NCBI accession number SRR1509462).
  • the PCR primers used to construct the tobacco transient protein expression plasmid of the gene related to cephalotaxane synthesis are shown in Table 1.
  • the ScHMGR fragment was amplified and ligated with the pEAQ-HT vector double-digested with XhoI/SmaI to obtain ptHMGR.
  • TcGGPPS fragment was amplified and ligated with pEAQ-HT double-digested with XhoI/SmaI to obtain ptGGPPS.
  • CharCS fragment was amplified and ligated with pEAQ-HT double-digested with XhoI/SmaI to obtain plasmid p6070.
  • the ChaiCS fragment was amplified and ligated with pEAQ-HT double-digested with XhoI/SmaI to obtain plasmid p3840.
  • the obtained plasmid was used for transient protein expression in Nicotiana benthamiana.
  • the successfully constructed ptHMGR, ptGGPPS, p6070 and p3840 were transformed into competent cells of Agrobacterium GV3101 to obtain expression strains, which were named sHMGR, sGGPPS, s6070 and s3840, respectively.
  • the strains were cultured at 30°C for 48 h using LB solid medium (kanamycin 50 ⁇ g/mL, gentamicin 25 ⁇ g/mL, rifampicin 25 ⁇ g/mL).
  • a single Agrobacterium clone was picked and transferred to 2 mL LB liquid medium (kanamycin 50 ⁇ g/mL, gentamicin 25 ⁇ g/mL, rifampicin 25 ⁇ g/mL), shaken and cultured for 24 h, 50 ⁇ L of the overnight cultured bacterial solution was transferred to new 20 mL LB liquid medium (kanamycin 50 ⁇ g/mL, gentamicin 25 ⁇ g/mL, rifampicin 25 ⁇ g/mL) and shaken and cultured at 30°C for 10 h until the OD600 value reached 1, centrifuged at 5000g for 15 min to collect the bacteria, washed once with resuspension solution (10 mM MgCl2 , 10 mM MES, 0.2 mM acetosyringone) to remove antibiotics and culture medium, centrifuged at 5000g for 15 min to collect the bacteria, and resuspended the bacteria.
  • sHMGR, sGGPPS, s6070 or s3840 were mixed in equal proportions to a final OD 600 of 1.
  • the infection solution was placed in a 1 ml syringe and the liquid was injected into the tobacco leaves from the lower epidermis of the leaves. 5 days after the injection, 0.5 g of the tobacco leaves were sampled and ground with liquid nitrogen and 0.5 mL of n-hexane was added to them for extraction three times. The residue obtained by concentrating the organic phase was dissolved in 100 ⁇ L of n-hexane and 1 ⁇ L was taken for GC-MS analysis.
  • Cephalotaxane and unconverted GGPP were detected in Nicotiana benthamiana leaves, as shown in Figure 2.
  • the product was isolated and purified. After chemistry, mass spectrometry analysis was performed.
  • the structure of the product was determined to be cephalot-12(13)-ene (the left structural formula below) by GC-MS and NMR ( Figure 3). As shown in Figure 1, the cephalot-12(13)-ene further formed the harringtonolide (the right structural formula below) (CAS No. 64761-48-4):
  • Harringtonolide was used to treat various tumor cell lines (human oral epidermal cancer cell KB, HCT-116 cells, HepG2 cells, A-549 cells, Hela cells, SGC-7901 cells) and its inhibitory effect was measured. The results are shown in Figure 4, and Harringtonolide has a very significant inhibitory effect on all these tumor cells, among which the inhibition rate on KB cells is the best.
  • the synthetic gene sequence of the mevalonate pathway was integrated into the position of the lacY gene on the chromosome of the Escherichia coli BL21 (DE3) strain to ensure sufficient supply of DMAPP/IPP precursors, and strain sZG37 was obtained.
  • the genes used to strengthen the MVA pathway in Escherichia coli include AtoB (WP_077475940.1, from Escherichia coli, acetyl-CoA acetyltransferase), MvaS (WP_002361740.1, from Enterococcus faecalis, mevalonyl-CoA synthetase), MvaE (WP_002382276.1, from Enterococcus faecalis, mevalonyl-CoA reductase), Mvk1 (WP_0001 97034.1, from Staphylococcus aureus, mevalonate kinase), Mvk2 (WP_000616885.1, from Staphylococcus aureus, phosphomevalonate kinase), MvaD (WP_000597335.1, from Staphylococcus aureus, mevalonate pyrophosphate decarboxylase) and F
  • the TcGGPPS fragment was amplified and ligated with pJF25 double-digested with XbaI/SpeI to obtain pMYP100.
  • the obtained plasmid was used for heterologous synthesis in E. coli.
  • the successfully constructed pMYP100, pET28a-CharCS and pGro7 were co-transformed into Escherichia coli BL21 (DE3) strain sZG37 to obtain an expression strain named sBM2.
  • the strain was picked and cloned into 2ml LB (with kanamycin, apramycin and chloramphenicol), cultured at 37°C, 250rpm for 12h, transferred to 100ml TB (with kanamycin, apramycin and chloramphenicol) at 1% (v/v), and cultured at 37°C, 250rpm until OD 600 was about 0.6.
  • IPTG final concentration of 0.1mM
  • L-arabinose final concentration of 10mM
  • 2 ml of bacterial solution was centrifuged, the cells were collected, 1 ml of ddH 2 O was added for re-suspending, high-pressure ultrasonication was performed for 1 min, 1 ml of ethyl acetate was added for extraction, vortexed at 12000 rpm, centrifuged for 20 min, the organic phase was transferred to a clean EP tube, vacuum concentrated and dried, and re-dissolved in 100 ⁇ L of n-hexane.
  • GC-MS test results showed that strain sBM2 could ferment cephalot-12-ene with a yield of about 25 mg/L ( Figure 5).
  • the inventors further attempted to predict the key sites for interaction with the substrate GGPP through molecular modeling and molecular docking, and to modify these key amino acid residues through site-directed mutagenesis to enhance the catalytic activity of CS.
  • primer pair 6070-F625x-F/6070-F625x-R ( Table 3 ) was used to perform saturation mutagenesis on F625, and the obtained mutants were verified by sequencing to construct plasmid pMYPM17-33.
  • Plasmids pMYPM17-33 were transformed into E. coli BL21 (DE3) strains together with pMYP100 and pGro7 to obtain strains sMYPM17-33.
  • the aforementioned strain sBM2 containing wild-type CharCS (codon-optimized) was used as a positive control.
  • the mutants at positions 627, 718, and 833 as shown in Table 3 were co-infected with sHMGR and sGGPPS in Nicotiana benthamiana, and transient expression was performed in tobacco according to the method described in Example 2, and GC-MS detection was performed. After the strains sMYPM17-33 were cultured and the fermentation products were extracted according to the method described in Example 2, GC-MS detection was performed.

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Abstract

L'invention concerne une céphalotaxane diterpène synthase, son procédé de préparation et son utilisation. La céphalotaxane diterpène synthase est séparée pour la première fois, et peut catalyser le géranylgéranyl pyrophosphate (GGPP) pour produire un composé de céphalotaxane céphalot-12(13)-ène.
PCT/CN2024/096943 2023-06-05 2024-06-03 Céphalotaxane diterpène synthase, son procédé de préparation et son utilisation Pending WO2024251059A1 (fr)

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