WO2024120148A1 - Nouvelle diterpène synthase et son utilisation - Google Patents

Nouvelle diterpène synthase et son utilisation Download PDF

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WO2024120148A1
WO2024120148A1 PCT/CN2023/132096 CN2023132096W WO2024120148A1 WO 2024120148 A1 WO2024120148 A1 WO 2024120148A1 CN 2023132096 W CN2023132096 W CN 2023132096W WO 2024120148 A1 WO2024120148 A1 WO 2024120148A1
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nucleic acid
sequence
diterpene
ent
cyclase
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PCT/CN2023/132096
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Chinese (zh)
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王勇
孙雨伟
邵洁
刘海利
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中国科学院分子植物科学卓越创新中心
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  • the present invention belongs to the field of biotechnology, and more specifically, the present invention relates to a group of novel diterpene synthases derived from Rubescens rubescens, which can specifically synthesize ent-sandaracopimaradiene, palustradiene, abieta-7,13-diene or abietatriene skeletons.
  • Isodon rubescens also known as ice ling grass and ice ling flower, is a perennial herbaceous plant of the Labiatae family. The whole plant is used as medicine, which has good heat-clearing and detoxifying, blood-activating and analgesic, antibacterial and anti-tumor effects, and has great comprehensive development and utilization value.
  • the chemical components of Isodon rubescens are mainly diterpenoids, and triterpenoids, flavonoids and alkaloids are also reported.
  • the diterpenoids of Isodon rubescens include ent-kaurene type, abietane type, atane type and hemi-florane type.
  • Isodon rubescens In previous studies, several type I and type II diterpene cyclases have been reported in Isodon rubescens, involving the synthesis of skeletons such as ent-kaurene, tanshinone diene, nezukol, and isopimaradiene. In addition to the above diterpene skeletons, there are still a large number of diterpene cyclases in Isodon rubescens that need to be identified. By using these new diterpene cyclases, Isodon rubescens can have the potential to synthesize more diverse diterpene skeletons.
  • the present invention first provides a diterpene cyclase, which is a type II diterpene cyclase derived from the genus Isodon (preferably Isodon rubescens).
  • the diterpene cyclase is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-ase cyclase
  • the present invention also provides a fusion protein having the amino acid sequence of the diterpene cyclase of the present invention and other polypeptides.
  • the other polypeptides are located at the N-terminus and/or C-terminus of the diterpene cyclase.
  • the other polypeptides include a signal peptide, a tag for purification, or a tag for immunoblotting.
  • the first aspect of the present invention also provides a nucleic acid molecule comprising a sequence selected from the following:
  • nucleic acid sequence encoding a diterpene cyclase or a fusion protein as described herein;
  • the fragment is a primer.
  • nucleic acid sequence is codon-optimized for E. coli.
  • the nucleic acid sequence is DNA or RNA.
  • the nucleic acid sequence is as shown in SEQ ID NO: 2, 4, 6 or 8.
  • Another aspect of the present invention is to provide a nucleic acid construct comprising the nucleic acid molecule described herein.
  • the nucleic acid construct is a vector, such as a cloning vector, an integration vector, or an expression vector.
  • the nucleic acid molecule is operably linked to an expression control sequence.
  • the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.
  • the nucleic acid construct further contains any one or more of the coding sequences of GGPPS, ent-CPS, and CPS.
  • the present invention also provides a host cell, wherein:
  • nucleic acid molecule comprising a nucleic acid molecule and/or a nucleic acid construct as described in any embodiment of the present invention.
  • the host cell is an E. coli cell.
  • the E. coli is a B-lineage E. coli; more preferably, it is E. coli BL21 (DE3).
  • the host cell also expresses an enzyme that catalyzes the production of IPP or DMAPP from acetyl-CoA, or contains a nucleic acid construct encoding the enzyme.
  • the host cell further expresses an enzyme of the MVA pathway, or contains a nucleic acid construct encoding the enzyme.
  • the protein of the MVA pathway includes one or more selected from the following: MVD, PMK, MVK, HMGR, HMGS, AACT, preferably one or more selected from the following: AtoB, MvaS, MvaE, Mvk1, Mvk2, MvaD and Fni.
  • the host cell also expresses an enzyme of the MEP pathway, or contains a nucleic acid construct encoding the enzyme.
  • the present invention also provides a method for catalyzing copalyl pyrophosphate (CPP) or enantio-copalyl pyrophosphate (ent-CPP) to generate a product, comprising:
  • diterpene cyclase or a fusion protein containing the diterpene cyclase to catalyze the enantiomeric CPP, wherein the diterpene cyclase has a sequence as shown in SEQ ID NO: 1 or a sequence having at least 70% identity thereto and retaining diterpene cyclase activity,
  • the product is a diterpene core skeleton compound.
  • the product of (1) is ent-sandamaradiene.
  • the product of (2) is parrustadiene, abietadiene-7,13-diene or abietadiene.
  • the method further comprises:
  • a step of catalyzing the formation of ent-copalyl pyrophosphate or copalyl pyrophosphate from (E,E,E)-geranylgeranyl pyrophosphate preferably using ent-CPS or CPS catalysis, and/or
  • step of catalyzing IPP or DMAPP to generate (E,E,E)-geranylgeranyl pyrophosphate which step is preferably catalyzed by GGPPS, and/or
  • the step of catalyzing acetyl-CoA to produce IPP or DMAPP is preferably catalyzed by one or more enzymes selected from the group consisting of MVD, PMK, MVK, HMGR, HMGS, and AACT.
  • the method comprises the step of culturing the host cell described in any of the embodiments herein under conditions suitable for catalyzing copalyl pyrophosphate or ent-copalyl pyrophosphate to produce a product.
  • the conditions include TB medium.
  • the TB medium contains an initial carbon source, preferably glucose; preferably, the concentration of glucose is 2%.
  • the culturing temperature is 20-30°C, preferably 28°C.
  • the culturing is for at least 24 hours, preferably at least 96 hours.
  • the conditions include an inducing agent, preferably IPTG; preferably, the concentration of IPTG is at least 0.05 mM.
  • the method further comprises the step of isolating ent-sandamaradiene, palustadiene, abietriene-7,13-diene and abietriene from the host cells; specifically comprising: crushing the cells, extracting with an organic solvent and vacuum drying, and the organic solvent is preferably ethyl acetate.
  • FIG. 1 Biosynthetic pathway of ent-sandamaradiene.
  • Module I precursor synthesis
  • MVA mevalonic acid pathway
  • MEP intrinsic methylerythritol phosphate pathway
  • module II diterpene nucleus synthesis
  • GGPPS GGPPS
  • ent-CPS ent-CPS
  • IrubKSL4 synthesize ent-sandamaradiene using IPP and DMAPP as substrates.
  • Figure 2 Gas chromatography analysis confirms that strain sIrubDiT1 is capable of producing ent-sandaracopimaradiene.
  • Figure 3 Nuclear magnetic resonance spectroscopy (A, hydrogen spectrum; B, carbon spectrum) results and gas chromatography-mass spectrometry (C) detection, confirming that the produced compound is ent-sandamaradiene.
  • FIG. 4 Biosynthetic pathways of parrustadiene, abiet-7,13-diene and abiettriene.
  • Module I precursor synthesis
  • MVA mevalonate pathway
  • MEP intrinsic methylerythritol phosphate
  • IPP and DMAPP is used as substrate
  • GGPPS and CPS synthesize the intermediate CPP
  • IrubKSL7, IrubKSL8 and IrubKSL9 catalyze CPP to produce palustradiene, abieta-7,13-diene and abietatriene, respectively.
  • Figure 5 Gas chromatography analysis confirmed that strains sIrubDiT2, sIrubDiT3, and sIrubDiT4 were able to produce parrustadiene, abietriene-7,13-diene, and abietriene, respectively.
  • Figure 7 Nuclear magnetic resonance spectroscopy (A, hydrogen spectrum; B, carbon spectrum) results and gas chromatography-mass spectrometry (C) detection confirmed that the compound produced by sIrubDiT3 is arosin-7,13-diene.
  • Figure 8 Nuclear magnetic resonance spectroscopy (A, hydrogen spectrum; B, carbon spectrum) results and gas chromatography-mass spectrometry (C) detection confirmed that the compound produced by sIrubDiT4 is abietriene.
  • the present invention relates to a group of novel diterpene cyclases encoded by nucleotide sequences derived from Isodon rubescens.
  • the inventors cloned and heterologously expressed kaurene synthase-like (KSL) diterpene cyclase encoding genes IrubKSL4, IrubKSL7, IrubKSL8 and IrubKSL9 in Escherichia coli BL21 (DE3) by mining the genome and transcriptome information of Isodon rubescens.
  • KSL kaurene synthase-like
  • the generated recombinant proteins can specifically cyclize the diterpene intermediates ent-copalyl pyrophosphate or copalyl pyrophosphate, thereby producing ent-sandaracopimaradiene, palustradiene, abieta-7,13-diene or abietatriene skeletons.
  • the present invention first provides a group of novel diterpene cyclases, named IrubKSL4, IrubKSL7, IrubKSL8 and IrubKSL9 derived from Isodon rubescens, and their amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, respectively.
  • the present invention also includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 has at least 80% (e.g., at least 90%, 96%, at least 98%, at least 99%) sequence identity while retaining its biological activity.
  • the "biological activity" of a diterpene cyclase generally refers to its ability to specifically catalyze the synthesis of enantio-copalyl pyrophosphate or copalyl pyrophosphate into a diterpene nucleus skeleton compound.
  • Exemplary such variants include biologically active fragments of the enzyme and variants of the enzyme or its biologically active fragment.
  • the meaning of a biologically active fragment of an enzyme refers to a polypeptide that still retains all or part of the functions of a full-length enzyme or protein. Typically, the biologically active fragment retains at least 98% or 99% activity.
  • “diterpene core skeleton compounds” mainly include ent-pimaradiene diterpene compounds, abietane diterpene compounds, and ent-kaurane diterpene compounds.
  • ent-pimaradiene includes ent-sandamaradiene
  • abietane includes palustadiene, abietane-7,13-diene, and abietriene.
  • a mutant of the enzyme or its biologically active fragment refers to an amino acid sequence formed by substitution, deletion or addition of one or more amino acid residues.
  • Appropriate replacement of amino acids is a technique well known in the art, which can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques have made those skilled in the art realize that, in general, changing a single amino acid in a non-essential region of a polypeptide will not substantially change the biological 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 is from the same or similar source (such as the same plant), such as the diterpene cyclase is from Rubescens rubescens, so its variant is preferably also from Rubescens rubescens or its co-genus, the genus Ipomoea.
  • the present invention also provides a fusion polypeptide comprising the diterpene cyclase described herein and other polypeptides.
  • polypeptides include polypeptides that localize the diterpene cyclase to different organelles or sub-organelles, tags for purification, or tags for immunoblotting.
  • the present invention also provides a polynucleotide encoding a diterpene cyclase or a variant thereof as described herein.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or artificially synthesized DNA.
  • the DNA may be single-stranded or double-stranded.
  • the DNA may be a coding strand or a non-coding strand.
  • nucleic acids As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be produced. They all encode the antibodies or antigen-binding fragments thereof of the present invention. Therefore, in the case where a specific amino acid sequence has been identified, a person skilled in the art can make 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, a nucleic acid sequence is optimized using a species (e.g., E. coli) preferred codon to make the sequence more easily expressed in the species, such as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. The E.
  • E. coli preferred codon to make the sequence more easily expressed in the species
  • the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) addition of denaturants during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • 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 host cells after proliferation by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules 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 (or vectors) and cells known in the art. In addition, 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 may be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear or circular sequences obtained from any source.
  • 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.
  • the vector generally contains sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences.
  • the sequence generally includes one or more of the following nucleotide sequences: promoter, one or more enhancer sequences, replication origin, transcription termination sequence, complete intron sequence containing donor and acceptor splice sites, sequence encoding leader sequence for polypeptide secretion, ribosome binding site, polyadenylation sequence, multiple linker regions and selectable marker elements for inserting nucleic acids encoding antibodies to be expressed.
  • exemplary nucleic acid constructs include pET21a.
  • the nucleic acid construct may also contain any one or more of the coding sequences of GGPPS, ent-CPS, and CPS.
  • 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 (e.g., LB or TB supplemented with glucose). Culture is carried out under conditions suitable for the growth of the host cells (e.g., 37° C.). When the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction), and the cells are cultured for a period of time (e.g., 28° C., more than 96 hours).
  • a suitable method e.g., temperature conversion or chemical induction
  • 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 protein precipitants (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 cell, such as a bacterial cell, a fungal cell, such as a yeast cell, a mammalian cell, an insect cell and a plant cell.
  • a heterologous host cell for expressing the nucleic acid molecule of the present invention can be a microbial host present in a fungus or bacterial family and grown in a wide temperature, pH value and solvent tolerance range.
  • any bacterium, yeast and filamentous fungi can be a suitable host for expressing the nucleic acid molecule of the present invention.
  • the host cell is an Escherichia coli, such as a B-series Escherichia coli, preferably an Escherichia coli BL21 (DE3).
  • Escherichia coli such as a B-series Escherichia coli, preferably an Escherichia coli BL21 (DE3).
  • these novel diterpene cyclases are applied to an artificially constructed recombinant Escherichia coli system to produce enantio-sandarama-piperadiene, palustadiene, abietic acid-7,13-diene and abietic acid triene by fermentation engineering.
  • a host cell which: (1) expresses a diterpene cyclase or a variant thereof described herein, or (2) contains a nucleic acid molecule and/or a nucleic acid construct described herein.
  • the host cell may also express other enzymes in the diterpene nucleus synthesis pathway, or contain the coding sequence of the enzyme or its expression vector.
  • other enzymes include enzymes that catalyze IPP or DMAPP to generate enantio-copalyl pyrophosphate (ent-CPP) or copalyl pyrophosphate (CPP), including: pyrophosphate synthase (e.g., ent-CPS; preferably SrCPS) that catalyzes (E, E, E)-geranylgeranyl pyrophosphate (GGPP) to generate enantio-copalyl pyrophosphate (ent-CPP), pyrophosphate synthase (e.g., CPS; preferably SmCPS1) that catalyzes (E, E, E)-geranylgeranyl pyrophosphate (GGPP) to generate copalyl pyrophosphate (
  • ent-CPP e.g., ent-
  • the host cell may also express an enzyme that catalyzes acetyl-CoA to generate IPP or DMAPP, including: MVD, PMK, MVK, HMGR, HMGS, AACT, such as AtoB (WP_077475940.1, from Escherichia coli, acetyl-CoA acetyltransferase), MvaS (WP_002361740.1, from Enterococcus faecalis, meglutaryl-CoA synthetase), MvaE (WP_002361740.1, from Enterococcus faecalis, mevalonyl-CoA reductase), Mvk1 (WP_000197034.1, from Staphylococcus aureus, mevalonate kinase), Mvk2 (WP_000616885.1, from Staphylococcus aureus, phosphomevalonate kinase),
  • Homologous sequences or variants of these enzymes in various species are easily obtained by those skilled in the art. See, for example: CN202210947431, CN202210948412, CN202210947464.
  • Host cells can express these enzymes by incorporating their coding sequences into nucleic acid constructs (e.g., expression vectors) and introducing them into cells. Nucleic acid constructs are described elsewhere herein.
  • the present invention also provides a method for catalyzing the production of a diterpene core skeleton compound, comprising the step of using the diterpene cyclase described herein in a catalytic system to catalyze enantio-copalyl pyrophosphate or copalyl pyrophosphate.
  • the method comprises the step of using the diterpene cyclase shown in SEQ ID NO: 1 or a sequence having at least 70% identity thereto to catalyze the enantio-CPP to produce enantio-sandamaradiene.
  • the method comprises the step of using the diterpene cyclase shown in SEQ ID NO: 3, 5, 7 or a sequence having at least 70% identity thereto to catalyze the CPP to produce parrustadiene, abietadiene-7,13-diene or abietadiene.
  • the method comprises incubating a host cell expressing a diterpene cyclase or a variant thereof as described herein under conditions suitable for producing a diterpene core skeleton compound. For example, culturing in TB medium containing 2% glucose and 0.1 mM IPTG at 28°C for 96 hours. Thereafter, the diterpene core skeleton compound can be enriched from the cell or cell culture by conventional methods of the present invention, such as by crushing the cells, extracting with ethyl acetate and vacuum drying.
  • a diterpene cyclase which is a type II diterpene cyclase derived from the genus Isodonis, wherein the diterpene cyclase:
  • Item 2 A fusion protein comprising the diterpene cyclase described in Item 1 and other polypeptides,
  • the other polypeptide includes a signal peptide, a tag for purification or a tag for immunoblotting.
  • Item 3 A nucleic acid molecule comprising a sequence selected from the following:
  • the fragment is a primer.
  • Item 4 The nucleic acid molecule according to Item 3, wherein: (1) the nucleic acid sequence is codon-optimized for Escherichia coli;
  • nucleic acid sequence is as shown in SEQ ID NO: 2, 4, 6 or 8.
  • nucleic acid construct comprising the nucleic acid molecule described in Item 3 or 4,
  • the nucleic acid construct is a vector, such as a cloning vector, an integration vector or an expression vector.
  • the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector and a viral vector.
  • the nucleic acid molecule is operably linked to an expression control sequence, and/or
  • the nucleic acid construct also contains any one or more of the coding sequences of GGPPS, ent-CPS and CPS.
  • Item 7 A host cell, wherein:
  • the host cell is an Escherichia coli cell; more preferably, the Escherichia coli is a B strain Escherichia coli,
  • the host cell also expresses an enzyme that catalyzes acetyl-CoA to produce IPP or DMAPP, or contains a nucleic acid construct encoding the enzyme.
  • the host cell also expresses an enzyme of the MVA pathway, or contains a nucleic acid construct encoding the enzyme.
  • the host cell also expresses an enzyme of the MEP pathway, or contains a nucleic acid construct encoding the enzyme.
  • Item 8 A method for catalyzing copalyl pyrophosphate or enantio-copalyl pyrophosphate to produce a product, comprising:
  • diterpene cyclase or a fusion protein comprising the same to catalyze ent-copalyl pyrophosphate, wherein the diterpene cyclase has a sequence as shown in SEQ ID NO: 1 or has at least 70% identity thereto and Sequences retaining diterpene cyclase activity,
  • the product is a diterpene core skeleton compound; more preferably, the product of (1) is ent-sandaracopimaradiene, and the product of (2) is palustradiene, abieta-7,13-diene or abietatriene.
  • Item 9 The method according to Item 8, characterized in that the method further comprises:
  • a step of catalyzing the formation of ent-copalyl pyrophosphate or copalyl pyrophosphate from (E,E,E)-geranylgeranyl pyrophosphate preferably using ent-CPS or CPS catalysis, and/or
  • step of catalyzing IPP or DMAPP to generate (E,E,E)-geranylgeranyl pyrophosphate which step is preferably catalyzed by GGPPS, and/or
  • the step of catalyzing acetyl-CoA to produce IPP or DMAPP is preferably catalyzed by one or more enzymes selected from the group consisting of MVD, PMK, MVK, HMGR, HMGS, and AACT.
  • Item 10 The method according to Item 8 or 9, characterized in that the method further comprises:
  • the method comprises the step of culturing the host cell of item 7 under conditions suitable for catalyzing copalyl pyrophosphate or antero-copalyl pyrophosphate to produce a product,
  • the method further comprises the step of isolating ent-sandamaradiene, palustadiene, abietriene-7,13-diene and abietriene from the host cell.
  • the step comprises: crushing the cells, extracting with an organic solvent and vacuum drying, and the organic solvent is preferably ethyl acetate.
  • Rubescens rubescens was collected from Jiyuan County, Henan province. Oligonucleotide primers were purchased from Shenggong Biotechnology (Shanghai) Co., Ltd. Sangon Biotech and GenScript Biotech. First-generation Sanger sequencing was commissioned to Sangon Biotech. GenScript Biotech was commissioned to perform full synthesis of related genes and clone them into the target vector.
  • AxyPrep total RNA miniprep kit, polymerase chain reaction (PCR) gel recovery kit, and plasmid extraction kit are all products of Axygen in the United States.
  • PrimeSTAR Max DNA Polymerase are products of TAKARA in Japan; restriction endonucleases are all products of NEB.
  • Terrific Broth was purchased from Sangon Biotech. Seamless cloning kit was purchased from Novozyme Biotech.
  • Escherichia coli DH10B was used for cloning construction, and BL21 (DE3) was used for de novo synthesis fermentation test.
  • pET21a and pACYCDuet-1 vectors were used for gene cloning and tandem construction of genes required for the pathway.
  • Arktik Thermal Cycler (Thermo Fisher Scientific) was used for PCR; ZXGP-A2050 constant temperature incubator (Zhicheng) and ZWY-211G constant temperature culture oscillator (Zhicheng) were used for constant temperature culture; 5418R high-speed refrigerated centrifuge and 5418 small centrifuge (Eppendorf) were used for centrifugation. Concentrator plus concentrator (Eppendorf) was used for vacuum concentration; OD600 was detected by UV-1200 ultraviolet visible spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.).
  • the rotary evaporation system consisted of IKA RV 10digital rotary evaporator (IKA), MZ 2C NT chemical diaphragm pump, and CVC3000 vacuum controller (vacuubrand). JY92-IIN ultrasonic cell crusher (Ningbo Xinzhi Biotechnology) was used for cell disruption.
  • Thermo Trace GC ultra-ISQ gas chromatography-mass spectrometry analysis was performed using a Thermo Fisher Scientific gas chromatography-mass spectrometry instrument (Thermo Fisher Scientific). Silica gel column chromatography used 200-300 mesh silica gel (Qingdao Ocean Chemical).
  • Module I specifically synthesizes ent-sandaracopimaradiene, and its biosynthetic pathway is shown in Figure 1.
  • Module I precursor synthesis
  • MVA mevalonic acid pathway
  • MEP intrinsic methylerythritol phosphate
  • module II diterpene nucleus synthesis
  • IPP and DMAPP are used as substrates
  • GGPPS and ent-CPS synthesize the intermediate ent-copalyl pyrophosphate (ent-CPP)
  • IrubKSL4 catalyzes ent-CPP to synthesize the diterpene nucleus ent-sandaracopimaradiene.
  • the plasmid pSY400 constructed in the early stage was used as a template, and the pSY400 part was linearized by PCR amplification using primer pair 358V-F/358V-R (primers are shown in Table 1).
  • the genes such as TcGGPPS and SrCPS contained in the linearized pSY400-1 fragment were all optimized by E. coli codons.
  • TcGGPPS truncated the N-terminal signal peptide.
  • IrubKSL4 codon optimized by E.
  • coli from winter rubescens was amplified (primers are shown in Table 1), and connected to the linearized pSY400 fragment using a seamless cloning method to form a plasmid pSYW541.
  • the above plasmid and the plasmid pCZ153 constructed in the early stage were co-transformed into E. coli BL21 (DE3) to form the strain sIrubDiT1.
  • the pCZ153 contains all the biosynthetic enzymes required for the MVA synthesis pathway: AtoB (WP_077475940.1, from Escherichia coli), MvaS (WP_002361740.1, from Enterococcus faecalis), MvaE (WP_002361740.1, from Enterococcus faecalis), Mvk1 (WP_000197034.1, from Staphylococcus aureus), Mvk2 (WP_000616885.1, from Staphylococcus aureus), MvaD (WP_000597335.1, from Staphylococcus aureus) and Fni (WP_004399098.1, from Bacillus subtilis), which are used to enhance the production of precursor DMAPP/IPP.
  • AtoB WP_077475940.1, from Escherichia coli
  • MvaS WP_002361740.1, from Enterococcus fa
  • a single clone of the engineered strain sIrubDiT1 was picked and cultured overnight in LB medium containing appropriate resistance.
  • the seeds of the overnight culture were transferred to 50 mL of TB medium (containing 2% glucose) at 1% v/v and cultured at 37°C and 200 rpm until OD 600 ⁇ 0.5-0.8.
  • IPTG with a final concentration of 0.1 mM was used for induction, and the fermentation broth was collected after 96 hours of culture at 28°C. 500 ⁇ L of the fermentation broth was ultrasonically broken and extracted three times with an equal volume of ethyl acetate. The organic layers were combined and concentrated to dryness in vacuo.
  • the extract was reconstituted with 100 ⁇ L of ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS).
  • GC-MS gas chromatography-mass spectrometry
  • HP-5MS glass capillary column (0.25 mm id ⁇ 30 m, 0.25 ⁇ m film thickness) (Agilent Technologies, USA) was used for gas chromatography analysis.
  • the chromatographic conditions were set as follows: initial 100°C for 3 minutes, rising to 268°C at 14°C/min and then maintained for 4 minutes, and the carrier gas flow rate was 36.9 cm s -1 .
  • the injection temperature was 280°C
  • the injection mode was splitless mode
  • the electron impact ionization was set to 70 eV
  • the ion source temperature was set to 280°C
  • the mass spectrum was collected in the range of m/z 30-550.
  • the engineered strain sIrubDiT1 was amplified to 1L scale culture. After the culture was completed, the fermentation liquid was ultrasonically crushed, extracted three times with equal volume of ethyl acetate, and the organic layers were combined and concentrated to dryness in vacuo. The crude extract was separated and purified by silica gel column chromatography (200-300 mesh), eluted with n-hexane. The fraction containing enantio-sandamaradiene was detected by GC-MS, and the chromatographic conditions were as shown above.
  • IrubKSL7, IrubKSL8 and IrubKSL9 specifically synthesize parrustadiene, abieta-7,13-diene and abietatriene, respectively.
  • the biosynthetic pathway is shown in Figure 4.
  • Module I (precursor synthesis) includes the heterologously introduced mevalonic acid pathway (MVA) and the intrinsic methylerythritol phosphate (MEP) pathway of Escherichia coli; in module II (diterpene nucleus synthesis), IPP and DMAPP are used as substrates, GGPPS and CPS synthesize the intermediate copalyl pyrophosphate (CPP), IrubKSL7 catalyzes the synthesis of diterpene nucleus palustadiene from CPP, IrubKSL8 catalyzes the synthesis of diterpene nucleus abietariene from CPP, and IrubKSL9 catalyzes the synthesis of diterpene nucleus abietariene from CPP.
  • MVA mevalonic acid pathway
  • MEP intrinsic methylerythritol phosphate pathway of Escherichia coli
  • IPP and DMAPP are used as substrates
  • GGPPS and CPS synthesize the
  • the pSY400 part was linearized by PCR amplification using primers pGGPPS-EcoRI-revF2/pGGPPS-SpeI-revRn2 (primers are shown in Table 2).
  • the TcGGPPS contained in the linearized pSY400-2 fragment was optimized by Escherichia coli codons and the N-terminal signal peptide was removed.
  • the SmCPS1 (codon optimized by Escherichia coli) from the salvia miltiorrhiza source was amplified using primers pGGPPS-SynSmCPS1-F/pGGPPS-SynSmCPS1-R, and connected to the linearized pSY400-2 fragment using seamless cloning to form plasmid pSYW542.
  • the pSYW542 part was linearized by PCR amplification using primers AX2-SalI-F/SynSmCPS1-R (primers are shown in Table 2).
  • IrubKSL7, IrubKSL8 and IrubKSL9 (codon optimized in E. coli) from Rubescens were amplified (primers are shown in Table 2), and connected to the linearized pSYW542 fragment using seamless cloning to form plasmids pSYW543, pSYW544 and pSYW545.
  • the above plasmids and the previously constructed plasmid pCZ153 were co-transformed into E. coli BL21 (DE3) to form strains sIrubDiT2-4.
  • the pCZ153 was used to enhance the production of precursor DMAPP/IPP as described in Example 2.
  • strain sIrubDiT2 can produce parustierene and abietriene
  • strain sIrubDiT3 can more specifically produce abietriene-7,13-diene (with a trace amount of abietriene)
  • strain sIrubDiT4 can produce abietriene and two unknown diterpene core products.
  • the engineered strains sIrubDiT2-4 were scaled up to 1L scale culture. After the culture was completed, the fermentation broth was ultrasonically broken, extracted three times with an equal volume of ethyl acetate, and the organic layers were combined and concentrated to dryness in vacuo. The crude extract was separated and purified by silica gel column chromatography (200-300 mesh) and eluted with n-hexane. The fractions containing the target diterpene nucleus were detected by GC-MS, and the chromatographic conditions were as shown above.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne une diterpène cyclase de classe II dérivée d'Isodon rubescens, qui possède une séquence choisie parmi l'une quelconque de SEQ ID NO : 1, 3, 5 et 7 ou une séquence identique à au moins 70 % à celle-ci, et conservant l'activité d'une diterpène cyclase.
PCT/CN2023/132096 2022-12-09 2023-11-16 Nouvelle diterpène synthase et son utilisation WO2024120148A1 (fr)

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