WO2024120509A1 - Nouvelles ent-caurène hydroxylases et leur utilisation - Google Patents

Nouvelles ent-caurène hydroxylases et leur utilisation Download PDF

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WO2024120509A1
WO2024120509A1 PCT/CN2023/137352 CN2023137352W WO2024120509A1 WO 2024120509 A1 WO2024120509 A1 WO 2024120509A1 CN 2023137352 W CN2023137352 W CN 2023137352W WO 2024120509 A1 WO2024120509 A1 WO 2024120509A1
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seq
ent
kaurene
sequence
protein
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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 novel ent-kaurene hydroxylase or a variant thereof, which specifically catalyzes the hydroxylation of ent-kaurene and its derivatives at the 15th, 7th or 14th positions.
  • Isodon rubescens also known as ice grass and ice flower, is a perennial herb of the Lamiaceae 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 diterpenoid compounds, and triterpenes, flavonoids and alkaloid compounds are also reported.
  • an ent-kaurene diterpenoid compound oridonin was isolated from the plant. The compound has very significant anti-tumor activity and is effective against a variety of transplanted tumors.
  • Oridonin is also distributed in other plants of the genus Amygdalus in the Lamiaceae family, such as Amygdalus amethystoides, Amygdalus neruosa, Amygdalus rabdosia, Amygdalus downsis and Amygdalus japonicus. So far, the research on Oridonin has involved many aspects such as phytochemistry, pharmacological effects, extraction technology and structural modification, but little is known about its complete biosynthetic pathway.
  • Oridonin and other kaurene-type diterpenoids have the same biosynthetic precursor ent-kaurene. It is currently speculated that Oridonin is formed by intramolecular cyclization of the precursor ent-kaurene after several steps of P450 enzyme oxidation reactions. So far, none of the various P450 oxidative modification enzymes involved in the biosynthesis of Oridonin has been identified and characterized, which is an important basic scientific issue that urgently needs to be broken through in this field.
  • Biosynthesis is expected to replace traditional plant extraction methods, but its prerequisite is the analysis of biosynthetic enzyme genes and the determination of synthesis pathways, which is urgently needed to be developed and researched in this field.
  • a method for hydroxylating ent-kaurene comprising: catalyzing the ent-kaurene with ent-kaurene hydroxylase to hydroxylate the 15th, 7th or 14th position of the ent-kaurene; wherein the hydroxyl
  • the hydroxylation product is 15 ⁇ -hydroxy en-kaurene (Kau-15 ⁇ -ol)
  • the en-kaurene hydroxylase is selected from CYP706V7, CYP706V4, CYP706V9, CYP706V15 or CYP706V16 or conservative variants thereof; or, the hydroxylation product is 7 ⁇ -hydroxy en-kaurene (Kau-7 ⁇ -ol), and the en-kaurene hydroxylase is selected from CYP706V2, CYP706V3 or CYP 706V8 or its conservative variant protein; or, the hydroxylation product is 14 ⁇ -hydroxy ent-
  • the ent-kaurene hydroxylase is (a) CYP706V7 shown in SEQ ID NO: 8, CYP706V4 shown in SEQ ID NO: 7, CYP706V9 shown in SEQ ID NO: 9, CYP706V15 shown in SEQ ID NO: 10, CYP706V16 shown in SEQ ID NO: 11, CYP706V2 shown in SEQ ID NO: 1, CYP706V3 shown in SEQ ID NO: 2, CYP706V8 shown in SEQ ID NO: 3, or CYP706V6 shown in SEQ ID NO: 17; or a conservative variant protein of the enzyme in (a);
  • the conservative variant protein of the enzyme of (a) includes a protein selected from:
  • a derivatized 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 sequence of the enzyme in (a) and having the function of hydroxylating ent-kaurene; or
  • the conservative variant protein of the enzyme (a) is a truncated variant; preferably, it is a truncated variant with the N-terminal transmembrane region truncated; more preferably, the truncated variant has: the sequence of SEQ ID NO: 8 after truncating positions 1-20, the sequence of SEQ ID NO: 7 after truncating positions 1-27, the sequence of SEQ ID NO: 9 after truncating positions 1-20, the sequence of SEQ ID NO: 10 after truncating positions 1-27, the sequence of SEQ ID NO: 11 after truncating positions 1-27, the sequence of SEQ ID NO: 1 after truncating positions 1-27, the sequence of SEQ ID NO: 2 after truncating positions 1-27, the sequence of SEQ ID NO: 3 after truncating positions 1-25, and/or the sequence of SEQ ID NO: 17 after truncating positions 1-24.
  • the conservative variant protein of the enzyme (a) is a variant in which a tag is fused to the N-terminus of ent-kaurene hydroxylase or its truncated variant, and the tag can increase the solubility or stability of the protein; preferably, the tag is 17 ⁇ ; preferably, the amino acid sequence of the tag is as shown in SEQ ID NO: 19.
  • the method is carried out in vitro (such as catalyzing the substrate by a chemical reaction) or in a cell (such as biosynthesis by genetically engineered cells); preferably, the method is carried out in a cell; preferably, the cell is a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli or Bacillus subtilis, preferably Escherichia coli; preferably, the eukaryotic cell includes a fungal cell, an insect cell or a mammalian cell; preferably, the fungal cell is a yeast cell (such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces or Candida).
  • a yeast cell such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces or Candida).
  • the method is performed in a cell (host cell) comprising (natural Module I (precursor synthesis): including mevalonate pathway (MVA) and methylerythritol phosphate pathway (MEP), producing IPP and DMAPP; Module II (diterpene nucleus synthesis): using IPP and DMAPP as substrates, producing ent-kaurene.
  • a cell comprising (natural Module I (precursor synthesis): including mevalonate pathway (MVA) and methylerythritol phosphate pathway (MEP), producing IPP and DMAPP; Module II (diterpene nucleus synthesis): using IPP and DMAPP as substrates, producing ent-kaurene.
  • the cell further expresses one or more or all of the following proteins selected from the group consisting of GGPPS, ent-CPS, KS, CPR, or a nucleic acid construct encoding the protein.
  • the cell also expresses a protein of the mevalonate pathway or contains a nucleic acid construct encoding the protein.
  • the proteins of the mevalonate pathway include one or more selected from the following: MVD, PMK, MVK, HMGR, HMGS, AACT, preferably include one or more selected from the following: AtoB, MvaS, MvaE, Mvk1, Mvk2, MvaD and Fni.
  • the methylerythritol phosphate pathway naturally exists in the cell.
  • the Escherichia coli is a K-type Escherichia coli
  • the Escherichia coli is selected from JM109(DE3), HMS174(DE3) and NovaBlue(DE3).
  • the cells are cultured in TB medium.
  • the culture temperature is 20-25°C, preferably 22°C.
  • the culturing is for at least 24 hours, preferably at least 96 hours.
  • the method further comprises the step of isolating 15 ⁇ -hydroxy-ent-kaurene, 7 ⁇ -hydroxy-ent-kaurene, 14 ⁇ -hydroxy-ent-kaurene or 7 ⁇ ,15 ⁇ -dihydroxy-ent-kaurene from the cells.
  • the step of isolating 15 ⁇ -hydroxy-ent-kaurene, 7 ⁇ -hydroxy-ent-kaurene, 14 ⁇ -hydroxy-ent-kaurene or 7 ⁇ ,15 ⁇ -dihydroxy-ent-kaurene from cells comprises: crushing the cells, extracting with an organic solvent and vacuum drying, wherein the organic solvent is preferably ethyl acetate.
  • ent-kaurene hydroxylase for hydroxylating ent-kaurene; wherein the hydroxylation product is 15 ⁇ -hydroxy ent-kaurene (Kau-15 ⁇ -ol), and the ent-kaurene hydroxylase is selected from CYP706V7, CYP706V4, CYP706V9, CYP706V15 or CYP706V16 or conservative variant proteins thereof; or, the hydroxylation product is 7 ⁇ -hydroxy ent-kaurene (Kau-7 ⁇ -ol), and the ent-kaurene hydroxylase is selected from CYP706V7, CYP706V4, CYP706V9, CYP706V15 or CYP706V16 or conservative variant proteins thereof.
  • the hydroxylation product is 14 ⁇ -hydroxy ent-kaurene (Kau-14 ⁇ -ol), and the ent-kaurene hydroxylase is CYP706V6 or conservative variants thereof; or, the hydroxylation product is 7 ⁇ , 15 ⁇ -dihydroxy ent-kaurene (Kau-7 ⁇ , 15 ⁇ -diol), and the ent-kaurene hydroxylase is a combination of CYP706V7 and CYP706V2 or conservative variants thereof.
  • an ent-kaurene hydroxylase for hydroxylating ent-kaurene wherein the enzyme is selected from: CYP706V7, CYP706V4, CYP706V9, CYP706V15, CYP706V16, CYP706V2, CYP706V3, CYP706V8, CYP706V6 or conservative variants thereof.
  • the enzyme is an enzyme derived from Isodon rubescens
  • the enzyme is selected from: (a) CYP706V7 shown in SEQ ID NO: 8, SEQ CYP706V4 shown in SEQ ID NO: 7, CYP706V9 shown in SEQ ID NO: 9, CYP706V15 shown in SEQ ID NO: 10, CYP706V16 shown in SEQ ID NO: 11, CYP706V2 shown in SEQ ID NO: 1, CYP706V3 shown in SEQ ID NO: 2, CYP706V8 shown in SEQ ID NO: 3, CYP706V6 shown in SEQ ID NO: 17; or a conservative variant protein of the enzyme (a).
  • the conservative variant protein of the enzyme of (a) includes a protein selected from:
  • a derivatized 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 sequence of the enzyme in (a) and having the function of hydroxylating ent-kaurene; or
  • the conservative variant protein of the enzyme (a) is a truncated variant; preferably, it is a truncated variant with the N-terminal transmembrane region truncated; more preferably, the truncated variant has: the sequence of SEQ ID NO: 7 after truncating positions 1-27, the sequence of SEQ ID NO: 8 after truncating positions 1-20, the sequence of SEQ ID NO: 9 after truncating positions 1-20, the sequence of SEQ ID NO: 10 after truncating positions 1-27, the sequence of SEQ ID NO: 11 after truncating positions 1-27, the sequence of SEQ ID NO: 1 after truncating positions 1-27, the sequence of SEQ ID NO: 2 after truncating positions 1-27, the sequence of SEQ ID NO: 3 after truncating positions 1-25, and/or the sequence of SEQ ID NO: 17 after truncating positions 1-24.
  • the conservative variant protein of the enzyme (a) is a variant in which a tag is fused to the N-terminus of ent-kaurene hydroxylase or its truncated variant, and the tag can increase the solubility or stability of the protein; preferably, the tag is 17 ⁇ ; preferably, the amino acid sequence of the tag is as shown in SEQ ID NO: 19.
  • a nucleic acid molecule which encodes any one of the above-mentioned ent-kaurene hydroxylase for hydroxylating ent-kaurene; preferably, the nucleic acid molecule is codon-optimized.
  • the nucleic acid has a sequence selected from one or more of the following:
  • nucleic acid construct comprising the nucleic acid molecule; preferably, the nucleic acid construct is a vector, such as a cloning vector, an integration vector or an expression vector.
  • a host cell in another aspect of the present invention, is provided, wherein the host cell: (1) expresses the ent-kaurene hydroxylase for hydroxylating ent-kaurene, or (2) contains the nucleic acid molecule and/or the nucleic acid construct.
  • the host cell also expresses one or more or all of the following proteins: GGPPS, ent-CPS, KS, CPR, or nucleic acid constructs encoding the proteins; and/or, the host cell also expresses a protein of the mevalonate pathway or contains a nucleic acid construct encoding the protein.
  • the proteins of the mevalonate pathway include one or more selected from the following: MVD, PMK, MVK, HMGR, HMGS, AACT, preferably include one or more selected from the following: AtoB, MvaS, MvaE, Mvk1, Mvk2, MvaD and Fni.
  • kits for hydroxylating ent-kaurene comprising: the ent-kaurene hydroxylase for hydroxylating ent-kaurene; or, the nucleic acid molecule; or, the nucleic acid construct; or, the host cell.
  • the kit further comprises a cell culture medium.
  • the kit further includes instructions for use describing a method for hydroxylating ent-kaurene.
  • FIG3 The results of nuclear magnetic resonance (A, hydrogen spectrum; B, carbon spectrum) and gas chromatography-mass spectrometry (C) confirmed that the generated compound was 7 ⁇ -hydroxy-ent-kaurene.
  • FIG4 Gas chromatography analysis results confirm that strains sIrubOx4, sIrubOx5, sIrubOx6, sIrubOx7, and sIrubOx8 are capable of producing 15 ⁇ -hydroxy-ent-kaurene (Kau-15 ⁇ -ol).
  • FIG5 The results of nuclear magnetic resonance (A, hydrogen spectrum; B, carbon spectrum) and gas chromatography-mass spectrometry (C) confirmed that the generated compound was 15 ⁇ -hydroxy-ent-kaurene.
  • FIG6 Gas chromatography analysis results confirm that strain sIrubOx9 is capable of producing 14 ⁇ -hydroxy-ent-kaurene (Kau-14 ⁇ -ol).
  • FIG7 The results of nuclear magnetic resonance (A, hydrogen spectrum; B, carbon spectrum) and gas chromatography-mass spectrometry (C) confirmed that the generated compound was 14 ⁇ -hydroxy-ent-kaurene.
  • FIG8 Gas chromatography analysis results confirm that strain sIrubOx10 is capable of producing 7 ⁇ , 15 ⁇ -dihydroxy-ent-kaurene (Kau-7 ⁇ , 15 ⁇ -diol).
  • FIG9 The results of nuclear magnetic resonance (A, hydrogen spectrum; B, carbon spectrum) and gas chromatography-mass spectrometry (C) confirmed that the generated compound was 7 ⁇ ,15 ⁇ -dihydroxy-ent-kaurene.
  • the inventors have obtained a group of novel ent-kaurene hydroxylases from Isodon rubescens through extensive research, screening, and mining of plant genome and transcriptome information, including CYP706V2, CYP706V3, CYP706V4, CYP706V6, CYP706V7, CYP706V8, CYP706V9, CYP706V15, and CYP706V16.
  • the ent-kaurene hydroxylases catalyze ent-kaurene to cause hydroxylation at specific sites.
  • the CYP706V2, CYP706V3, CYP706V4, CYP706V6, CYP706V7, CYP706V8, CYP706V9, CYP706V15 and CYP706V16 have the amino acid sequences shown in SEQ ID NO: 1-3, SEQ ID NO: 7-11 and SEQ ID NO: 17, respectively.
  • the present invention also includes the conservative variant polypeptides (proteins) thereof.
  • 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 NO: 1-3, SEQ ID NO: 7-11 and SEQ ID NO: 17, while retaining its biological activity.
  • the "biological activity" of ent-kaurene hydroxylase generally refers to its ability to specifically catalyze the hydroxylation of ent-kaurene at position 15, position 7 or position 14.
  • Exemplary such variants include biologically active fragments of the enzyme and variants of the enzyme or its biologically active fragment.
  • 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 the activity.
  • the variant is a truncated variant, such as a truncated variant with the N-terminal transmembrane region truncated. More preferably, the variant is a variant in which a solubility-promoting tag is fused to the N-terminus of ent-kaurene hydroxylase or a truncated variant thereof, and the tag can increase protein solubility, stability, etc.
  • the tag is 17 ⁇ .
  • 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 IrCYP706V family P450 oxidase is from wintergrass, so its variant is preferably also from wintergrass or its same genus, the ...
  • the present invention also provides polynucleotides encoding ent-kaurene hydroxylase or variants thereof as described herein.
  • the polynucleotides of the present invention may be in the form of DNA or RNA.
  • the DNA forms include 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 can be produced, all of which encode the antibodies or antigen-binding fragments thereof of the present invention.
  • a nucleic acid sequence can be optimized using a species-preferred codon such as E. coli to make the sequence more amenable to expression in that species, e.g., the E.
  • 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 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.
  • 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 splicing 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 GGPPS, ent-CPS, KS, and CPR.
  • 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 E. 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 glycerol). 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., 22° 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 growing within a wide range of temperature, pH value, and solvent tolerance.
  • any bacteria, yeast, and filamentous fungi can be a suitable host for expressing the nucleic acid molecule of the present invention.
  • the host cell is Escherichia coli, such as K-series Escherichia coli, including JM109 (DE3), HMS174 (DE3), and NovaBlue (DE3); more preferably Escherichia coli JM109 (DE3).
  • these novel ent-kaurene hydroxylases are applied to an artificially constructed recombinant Escherichia coli system to produce 7 ⁇ -hydroxy ent-kaurene, 15 ⁇ -hydroxy ent-kaurene, or 14 ⁇ -hydroxy ent-kaurene by fermentation engineering.
  • heterologous or “exogenous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a target gene sequence is not normally found in nature, then the promoter A particular sequence is “heterologous/foreign” to the cell or organism into which it is inserted.
  • a host cell wherein:
  • the present invention also provides a host cell, the host cell also expresses other enzymes in the hydroxylated ent-kaurene metabolic pathway, or comprises the coding sequence of the enzyme or its expression vector.
  • enzymes include enzymes that catalyze IPP or DMAPP to generate ent-kaurene, including: synthases (such as KS, preferably SrKS) that catalyze ent-copalyl pyrophosphate (ent-CPP) to generate ent-kaurene, pyrophosphate synthases (such as ent-CPS; preferably SrCPS) that catalyze (E, E, E)-geranylgeranyl pyrophosphate (GGPP) to generate ent-copalyl pyrophosphate (ent-CPP), and pyrophosphate synthases (such as GGPPS, preferably TcGGPPS) that catalyze IPP or DMAPP to generate (E, E, E)-GGPP.
  • synthases such as KS, preferably SrKS
  • ent-CPP ent-copalyl pyrophosphate
  • pyrophosphate synthases such as ent-CPS; preferably SrCPS
  • the other enzymes include enzymes that catalyze acetyl-CoA to generate IPP or DMAPP, including: MVD, PMK, MVK, HMGR, HMGS, AACT, such as AtoB (WP_077475940.1, Escherichia coli, acetyl-CoA acetyltransferase), MvaS (WP_002361740.1, Enterococcus faecalis, meglutaryl-CoA synthetase), MvaE (WP_002361740.1, Enterococcus faecalis, , mevalonyl-CoA reductase), Mvk1 (WP_000197034.1, derived from Staphylococcus aureus, mevalonate kinase), Mvk2 (WP_000616885.1, derived from Staphylococcus aureus, phosphomevalonate kinase
  • the host cell can express these enzymes by incorporating their coding sequences into a nucleic acid construct (e.g., an expression vector and introducing it into the cell).
  • a nucleic acid construct e.g., an expression vector and introducing it into the cell.
  • the nucleic acid construct is described elsewhere herein.
  • the host cell is an Escherichia coli cell.
  • the Escherichia coli is a K-series Escherichia coli, including JM109 (DE3), HMS174 (DE3) and NovaBlue (DE3); more preferably, it is Escherichia coli JM109 (DE3).
  • the cloned and heterologously expressed fusion protein with a tag (e.g., N-terminus) produced can specifically and efficiently catalyze the hydroxylation of the 7, 15 or 14 position of the tetracyclic diterpenoid compound ent-kaurene, thereby producing a class of 7 ⁇ -hydroxy ent-kaurene (Kau-7 ⁇ -ol), 15 ⁇ -hydroxy ent-kaurene (Kau-15 ⁇ -ol), 14 ⁇ -hydroxy ent-kaurene (Kau-14 ⁇ -ol) or 7 ⁇ , 15 ⁇ -dihydroxy ent-kaurene (Kau-7 ⁇ , 15 ⁇ -diol).
  • a tag e.g., N-terminus
  • the present invention also provides a method for producing 7 ⁇ -hydroxy-ent-kaurene, 15 ⁇ -hydroxy-ent-kaurene and/or 14 ⁇ -hydroxy-ent-kaurene based on the method described herein, comprising: using an ent-kaurene hydroxylase or a variant thereof described herein to catalyze the production of ent-kaurene in a catalytic system.
  • the method comprises starting with the production of ent-kaurene.
  • the host cell expressing ent-kaurene hydroxylase or a variant thereof as described herein is incubated under conditions that ent-kaurene is synthesized.
  • Rabdosia rubescens was collected from Jiyuan County, Henan province. Oligonucleotide primers were purchased from Sangon Biotech (Shanghai) Co., Ltd. (Sangon Biotech) and GenScript Biotech. First-generation Sanger sequencing was commissioned to Sangon Biotech. Gene synthesis was commissioned to GenScript Biotech to perform full synthesis of related genes and clone them into target vectors. AxyPrep total RNA miniprep kit, polymerase chain reaction (PCR) gel recovery kit, and plasmid extraction kit were all products of Axygen, USA.
  • PCR polymerase chain reaction
  • PrimeSTAR Max DNA Polymerase were products of Takara Biotechnology Co., Ltd. of Japan; restriction endonucleases were all products of NEB.
  • Terrific Broth was purchased from Sangon Biotech.
  • Seamless cloning kit was purchased from Novozyme Biotechnology Co., Ltd.
  • E. coli DH10B was used for cloning and construction, JM109 (DE3) was used for de novo synthesis and fermentation testing.
  • 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 RV10digital 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).
  • IrCYP706V2 and IrCYP706V7 are P450s highly expressed in the apical tissue of the stem, and are speculated to be the key genes for the biosynthesis of Rubescens rubescens A.
  • Module I precursor synthesis
  • MVA mevalonic acid pathway
  • MEP intrinsic methylerythritol phosphate pathway of Escherichia coli
  • module II diterpene nucleus synthesis
  • GGPPS GGPPS
  • ent-CPS ent-CPS
  • KS methylerythritol phosphate
  • module III oxidative modification
  • the ent-kaurene nucleus is oxidatively modified by ent-kaurene-7 ⁇ -hydroxylase (IrCYP706V2, IrCYP706V3, and IrCYP706V8) to produce 7 ⁇ -hydroxy-ent-kaurene (Kau-7 ⁇ -ol).
  • the previously constructed plasmid pSY414 (Wang, et al., Cell Res (2016) 26, 258-261. https://doi.org/10.1038/cr.2015.111) was used as a template and the pSY414 portion was linearized by PCR amplification using primer pair 80V-F/80V-R (primers are shown in Table 1).
  • the genes TcGGPPS, SrCPS, SrKS, SrCPR, etc. contained in the linearized pSY414 fragment were all codon-optimized for E. coli.
  • the N-terminal signal peptide of TcGGPPS was truncated.
  • IrCYP706V2, IrCYP706V3, and IrCYP706V8 (all codon-optimized in E. coli) from Rubescens were amplified (primers are shown in Table 1).
  • amino acids 1-27 at the N-terminus of IrCYP706V2, 1-27 at the N-terminus of IrCYP706V3, and 1-25 at the N-terminus of IrCYP706V8 were replaced with 17 ⁇ tags, and connected to the linearized pSY414 fragment using seamless cloning to form plasmids pSYW497 (introducing IrCYP706V2), pSYW534 (introducing IrCYP706V3), and pSYW535 (introducing IrCYP706V8), respectively.
  • the pSYW497, pSYW534 and pSYW535 plasmids were co-transformed into Escherichia coli JM109 (DE3) with the previously constructed plasmid pCZ153 (Sun, et al., Metab Eng (2022) 73, 201-213. https://doi.org/10.1016/j.ymben.2022.08.001) to form strains sIrubOx1 (containing pSYW497), sIrubOx2 (containing pSYW534), and sIrubOx3 (containing pSYW535).
  • 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
  • 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: initially at 100°C for 3 minutes, then at 14°C/min to 268°C and then held for 4 minutes, with a carrier gas flow rate of 36.9 cm s -1 .
  • the injection temperature was 280°C, and the injection mode was splitless mode.
  • the electron impact ionization was set to 70 eV, the ion source temperature was set to 280°C, and the mass spectrum was collected in the range of m/z 30 to 550.
  • Example 3 Functional identification of ent-kaurene-15 ⁇ -hydroxylases IrCYP706V4, IrCYP706V7, IrCYP706V9, IrCYP706V15 and IrCYP706V16
  • 15 ⁇ -Hydroxy-ent-kaurene (Kau-15 ⁇ -ol) is also a key intermediate in the biosynthesis of Rubescensine A, and its biosynthetic pathway is shown in Figure 1.
  • Module I precursor synthesis
  • module II diterpene nucleus synthesis
  • the ent-kaurene nucleus is oxidatively modified by ent-kaurene-15 ⁇ -hydroxylase (IrCYP706V4, IrCYP706V7, IrCYP706V9, IrCYP706V15 and IrCYP706V16) to produce 15 ⁇ -hydroxy-ent-kaurene (Kau-15 ⁇ -ol).
  • the previously constructed plasmid pSY414 was used as a template, and the primer pair 80V-F/80V-R (primers are shown in Table 1) was used to linearize the pSY414 part by PCR amplification.
  • the linearized pSY414 was as described in Example 2.
  • the cloning vector was used as a template to amplify IrCYP706V4, IrCYP706V7, IrCYP706V9, IrCYP706V15, and IrCYP706V16 (all codon-optimized in E. coli) from Rubescens rubescens (primers are shown in Table 2).
  • amino acids 1-27 of the N-terminus of IrCYP706V4, 1-20 of the N-terminus of IrCYP706V7, 1-20 of the N-terminus of IrCYP706V9, 1-27 of the N-terminus of IrCYP706V15, and 1-27 of the N-terminus of IrCYP706V16 were replaced with 17 ⁇ tags, and ligated to the linearized pSY414 fragment using seamless cloning to form plasmids pSYW498 (containing IrCYP706V7), pSYW536 (containing IrCYP706V4), pSYW537 (containing IrCYP706V9), pSYW538 (containing IrCYP706V15), and pSYW539 (containing IrCYP706V16), respectively.
  • the above plasmids and the previously constructed plasmid pCZ153 were co-transformed into E. coli JM109 (DE3) to form strains sIrubOx4 (containing pSYW536), sIrubOx5 (containing pSYW498), sIrubOx6 (containing pSYW537), sIrubOx7 (containing pSYW538), and sIrubOx8 (containing pSYW539).
  • pCZ153 was used to enhance the production of precursors DMAPP and IPP.
  • strains sIrubOx4, sIrubOx5, sIrubOx6, sIrubOx7, and sIrubOx8 were able to produce 15 ⁇ -hydroxy ent-kaurene (Kau-15 ⁇ -ol), and some substrate ent-kaurene (Kau) that had not been completely converted was present.
  • the engineered strain sIrubOx5 was expanded to 1 L scale culture. After the culture was completed, separation and purification were performed in the manner described in Example 2. The fraction containing 15 ⁇ -hydroxy ent-kaurene (Kau-15 ⁇ -ol) was detected by GC-MS, and the chromatographic conditions were as described above.
  • 14 ⁇ -Hydroxy-ent-kaurene may be a key intermediate in the biosynthesis of Rubescensine A, and its biosynthetic pathway is shown in Figure 1.
  • Module I precursor synthesis
  • module II diterpene nucleus synthesis
  • the ent-kaurene nucleus is oxidatively modified by ent-kaurene-14 ⁇ -hydroxylase IrCYP706V6 to produce 14 ⁇ -hydroxy-ent-kaurene (Kau-14 ⁇ -ol).
  • the previously constructed plasmid pSY414 was used as a template, and the pSY414 was partially linearized by PCR amplification using the primer pair 80V-F/80V-R (primers are shown in Table 1).
  • the linearized pSY414 was described in Example 2.
  • the IrCYP706V6 from Rubescens rubescens was amplified using the cloning vector as a template (after Escherichia coli codon optimization) (primers are shown in Table 3).
  • the N-terminal 1-24 amino acids of IrCYP706V6 were replaced with a 17 ⁇ tag and connected to the linearized pSY414 fragment using a seamless cloning method to form the plasmid pSYW540.
  • a single clone of the engineered strain sIrubOx9 was selected and cultured overnight in LB medium containing appropriate resistance.
  • the fermentation culture was carried out as described. 500 ⁇ L of the fermentation broth was ultrasonically broken and extracted three times with an equal volume of ethyl acetate, and the organic layers were combined and concentrated to dryness in vacuo. The extract was re-dissolved with 100 ⁇ L of ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS).
  • the engineered strain sIrubOx9 was expanded to 1 L scale culture. After the culture was completed, separation and purification were performed in the manner described in Example 2. The fraction containing 14 ⁇ -hydroxy ent-kaurene (Kau-14 ⁇ -ol) was detected by GC-MS, and the chromatographic conditions were as described above.
  • Example 5 Production of 7 ⁇ ,15 ⁇ -dihydroxy-ent-kaurene using IrCYP706V7 and IrCYP706V2 in combination
  • IrCYP706V7 and IrCYP706V2 can oxidatively modify the ent-kaurene nucleus to produce 7 ⁇ ,15 ⁇ -dihydroxyent-kaurene (Kau-7 ⁇ ,15 ⁇ -diol).
  • the plasmid pSYW498 (containing IrCYP706V7) described in Example 3 was linearized by double restriction digestion with SpeI and SalI, and the amino acids 1-27 at the N-terminus were replaced with IrCYP706V2 (codon-optimized for E. coli) with a 17 ⁇ tag, which was ligated to the linearized vector to construct plasmid pSYW510.
  • a single clone of the engineered strain sIrubOx10 was picked and cultured overnight in LB medium containing appropriate resistance. Fermentation was performed as described in Example 2. 500 ⁇ L of the fermentation broth was ultrasonically broken and extracted three times with an equal volume of ethyl acetate, and the organic layers were combined and concentrated to dryness in vacuo. The extract was re-dissolved with 100 ⁇ L of ethyl acetate and analyzed by gas chromatography-mass spectrometry (GC-MS). The gas chromatography-mass spectrometry analysis conditions were as described in Example 2.
  • GC-MS gas chromatography-mass spectrometry
  • strain sIrubOx10 was able to produce 7 ⁇ ,15 ⁇ -dihydroxy-ent-kaurene (Kau-7 ⁇ ,15 ⁇ -diol), and that some substrates ent-kaurene (Kau) and 15 ⁇ -hydroxy-ent-kaurene (Kau-15 ⁇ -ol) that had not been completely converted were also present.
  • the engineered strain sIrubOx10 was expanded to 1 L scale culture. After the culture was completed, it was separated and purified in the manner described in Example 2. The fraction containing 7 ⁇ , 15 ⁇ -dihydroxy-ent-kaurene (Kau-7 ⁇ , 15 ⁇ -diol) was detected by GC-MS. The chromatographic strips Items as shown above.

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  • Enzymes And Modification Thereof (AREA)
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Abstract

L'invention concerne un ensemble d'ent-caurène hydroxylases dérivées d'Isodon rubescens, ou des variants associés. Les hydroxylases peuvent catalyser spécifiquement l'hydroxylation de position 15, 7 ou 14 d'ent-caurène ou d'un dérivé associé.
PCT/CN2023/137352 2022-12-09 2023-12-08 Nouvelles ent-caurène hydroxylases et leur utilisation WO2024120509A1 (fr)

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