Biological enzyme for catalyzing hydroxylation of c9 site in taxane compound, nucleic acid molecule, biological material and use of biological enzyme, nucleic acid molecule and biological material

Abstract

Provided are a biological enzyme for catalyzing hydroxylation of C9 site in a taxane compound, a nucleic acid molecule, a biological material and a use of the biological enzyme, the nucleic acid molecule and the biological material. The biological enzyme comprises at least one of amino acid sequences as shown in SEQ ID NOs: 1-14; and / or a fusion amino acid sequence thereof; and / or an amino acid sequence having the same function obtained by substitution and / or deletion and / or addition of one or more amino acid residues; and / or an amino acid sequence having at least 60% identity and having the same function.

Description

Biological enzymes, nucleic acid molecules, biomaterials and their applications for catalyzing C9 hydroxylation in taxane compounds Technical Field

[0001] The present application relates to the technical fields of plant metabolic biology and synthetic biology, and specifically to biological enzymes, nucleic acid molecules, biomaterials, and applications thereof that catalyze the hydroxylation of the C9 position in taxane compounds. Background Art

[0002] Taxanes are a series of derivatives synthesized from anti-tumor active ingredients isolated from plants through structural modification of the compounds of the obtained active ingredients. They are important first-line anti-tumor drugs. Taxanes mainly include paclitaxel (the compound shown in Formula III), docetaxel, cabazitaxel, and derivatives with a taxane skeleton structure. Currently, the main production method for obtaining paclitaxel APIs is semi-synthesis, which first extracts the natural precursors baccatin III and 10-deacetylbaccatin III (10-DAB) and then chemically synthesizes paclitaxel. However, the precursors used in this method still rely on extraction from the bark or leaf cells of the yew tree. Due to the scarcity of yew resources, it cannot completely solve the supply problem.

[0003] Baccatin III and 10-deacetylbaccatin III currently rely on extraction from scarce Taxus chinensis because several key enzymes in their synthesis pathway remain unresolved, one of which is the enzyme catalyzing the C9 oxidation reaction. Because Baccatin III contains seven single oxidation sites, the oxidation sequence is unknown, and the oxidized substrates are unknown, screening for the enzyme catalyzing the C9 oxidation reaction (i.e., C9 hydroxylase, T9αH) is extremely difficult, resulting in the enzyme's elusive identification over the past three decades.

[0004] Based on the above situation, there is an urgent need to comprehensively analyze the key enzymes in paclitaxel synthesis, especially the key enzymes in the synthesis of natural precursors baccatin III and 10-deacetylbaccatin III, such as C9 hydroxylase, and then use synthetic biology methods and metabolic engineering strategies to reconstruct the paclitaxel synthesis pathway in plants or microbial chassis, so as to mass produce paclitaxel and completely solve the problem of paclitaxel supply and demand.

[0005] Summary of the Invention

[0006] In light of this, the present application provides enzymes, nucleic acid molecules, biomaterials, and their applications for catalyzing the C9 hydroxylation of taxane compounds. The successful characterization of this enzyme (also known as C9 hydroxylase, T9αH) will facilitate the complete biosynthesis of paclitaxel, increase the yield of baccatin III biosynthesis, and address the challenge of paclitaxel's insufficient supply.

[0007] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0008] The present application provides a biological enzyme T9αH, whose amino acid sequence includes at least one of the following sequences:

[0009] a1) at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 14;

[0010] a2) a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 14;

[0011] a3) an amino acid sequence having the same function obtained by substitution and / or deletion and / or addition of one or more amino acid residues in at least one of the amino acid sequences shown in SEQ ID NOs: 1 to 14;

[0012] a4) an amino acid sequence that is at least 60% identical to at least one of the amino acid sequences of SEQ ID NOs: 1 to 14 and has the same function.

[0013] Optionally, a4) the amino acid sequence is derived from the genus Taxus, including but not limited to European yew Taxus baccata L., short-leaved yew Taxus brevifolia Nutt., limestone yew Taxus calcicola LMGao & Mich. Taxus canadensis Marshall, Taxus chinensis (Pilg.) Rehd., Taxus contorta Griff., Taxus cuspidata Siebold & Zucc., Taxus floridana Nutt.ex Chapm., Taxus florinii Spjut, Taxus globosa Schltdl., Taxus mairei (Lemée & Lév.) SYHu, Taxus phytonii Spjut, Taxus sumatrana (Miq.) de Laub., Taxus wallichiana Zucc., Taxus Huangshan type, Taxus Emei type, Taxus Qinling type., Taxus yunnanensis WCCheng&L.K.Fu, Taxus×media Rehder, Taxus×hunnewelliana Rehder, etc.

[0014] This application creatively identifies an enzyme from Taxus chinensis that catalyzes the hydroxylation reaction at the C9 position during the synthesis of taxane compounds, designated T9αH. Functional analysis revealed that T9αH can catalyze the hydroxylation of the C9 position in compounds with the carbon skeleton structure of paclitaxel but without hydroxylation at the C9 position, thereby opening up the biosynthetic pathway for taxane intermediates such as baccatin III or its precursors, and further enabling the complete biosynthesis of taxanes such as paclitaxel.

[0015] In a specific embodiment of the present application, when synthesizing taxin, the T9αH enzyme can catalyze the conversion of taxadiene-5α,10β,13α-triol into taxadiene-5α,9α,10β,13α-tetraol, thereby achieving a hydroxylation reaction at the C9 position.

[0016] In another specific embodiment of the present application, when synthesizing baccatin III (a paclitaxel precursor), the T9αH enzyme can catalyze the hydroxylation of the C9 position in a compound having a paclitaxel carbon skeleton structure and an unhydroxylated C9 position, thereby successfully synthesizing baccatin III.

[0017] Furthermore, the present application obtained homologous proteins and mutants of T9αH, demonstrating that the homologous proteins and mutants can also catalyze the hydroxylation of the C9 position.

[0018] In a specific embodiment of the present application, a2) in the fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1 to 14, the connecting tag is not specifically limited and can be added according to the purpose of use. For example, the following can be selected but are not limited to: HIS, GST, Flag, MBP, HA, c-Myc, eGFP, eYFP, and eCFP.

[0019] In an embodiment of the present application, the amino acid sequence in a3) is an amino acid sequence in which one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid residues are added, deleted, or replaced in the amino acid sequence shown in a1) and has the same biological enzyme activity function.

[0020] In an embodiment of the present application, the amino acid sequence in a3) is an amino acid sequence in which one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid residues are added, deleted, or substituted into the amino acid sequence shown in a1) and has similar or better biological enzyme activity function.

[0021] In an embodiment of the present application, the amino acid sequence in a4) is an amino acid sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in a1) and has the same biological enzyme activity function.

[0022] In an embodiment of the present application, the amino acid sequence in a4) is an amino acid sequence that is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in a1) and has similar or better biological enzyme activity.

[0023] In a specific embodiment of the present application, the sequence similarity of the above 14 T9αH amino acid sequences is 63.15%-100%, and the conserved domains / sites are the conserved PSRF motif (amino acids 423-426), the highly conserved heme-binding PFG element (amino acids 439-441), the ETMR salt bridge (amino acids 369-372), the EXXR motif and the key cysteine ​​(amino acid 447); the key amino acid sites of the mutants include L75, G128, P129, F132, V136, V142, L228, T231, L232, F305, H308, A309, D312, T313, S316, P376, A377, F378, G379, S380, F381, R382, M402, F485, P486, and P487. The above amino acids are represented by single-letter abbreviations. The amino acids, English names and English abbreviations are shown in Table 4.

[0024] In a specific embodiment of the present application, the above-mentioned biological enzyme is an isolated biological enzyme.

[0025] In a specific embodiment of the present application, the homologous sequences of the amino acid sequence shown in SEQ ID NO: 1 are the amino acid sequences shown in SEQ ID NO: 2 to 14. The homologous sequences shown in SEQ ID NO: 2 to 14 have a homology of 63.15% to 100% with the amino acid sequence shown in SEQ ID NO: 1.

[0026] The present application also provides a nucleic acid molecule encoding the above-mentioned biological enzyme, the nucleotide sequence of which includes at least one of the following sequences:

[0027] b1) at least one of the nucleotide sequences shown in SEQ ID NOs: 15 to 28;

[0028] b2) a complementary sequence, a degenerate sequence, or a homologous sequence of at least one of the nucleotide sequences of SEQ ID NOs: 15 to 28, wherein a homologous sequence is a nucleotide sequence that is at least 75% identical to the nucleotide sequence of SEQ ID NOs: 15 to 28;

[0029] b3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in b1) or b2) under stringent conditions and is capable of encoding a protein with the same function.

[0030] In the embodiment of the present application, the complementary sequence is a complementary sequence formed according to the principle of base complementary pairing, and the complementary sequence can be an incomplete complementary sequence or a complete complementary sequence having the same function as the nucleotide sequence shown in b1).

[0031] In the embodiments of the present application, a degenerate sequence means that after changing one or more nucleotides in the nucleotide sequence shown in b1), the type of amino acid encoded by the changed nucleotide sequence position remains unchanged, and the function and expression level of the nucleic acid molecule will not be affected.

[0032] In an embodiment of the present application, a homologous sequence is a nucleotide sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in b1) and has the same function, such as a mutant gene, allele or derivative.

[0033] Optionally, b2) the nucleotide sequence of the nucleic acid molecule is derived from Taxus, including but not limited to European yew Taxus baccata L., short-leaved yew Taxus brevifolia Nutt., limestone yew Taxus calcicola LMGao & Mich. Taxus canadensis Marshall, Taxus chinensis (Pilg.) Rehd., Taxus contorta Griff., Taxus cuspidata Siebold & Zucc., Taxus floridana Nutt.ex Chapm., Taxus florinii Spjut, Taxus globosa Schltdl., Taxus mairei (Lemée & Lév.) SYHu, Taxus phytonii Spjut, Taxus sumatrana (Miq.) de Laub., Taxus wallichiana Zucc., Taxus Huangshan type, Taxus Emei type, Taxus Qinling type., Taxus yunnanensis WC Heng & L.K. Fu, Taxus × media Rehder, Taxus × hunnewelliana Rehder, etc. Optionally, the nucleic acid molecule is a synthetic and / or isolated nucleic acid molecule.

[0034] Illustratively, "stringent conditions" refer to conditions under which a probe will hybridize to its target sequence to a detectable extent exceeding hybridization to other sequences (e.g., at least 2 times background). Stringent conditions are sequence-dependent and vary depending on the circumstances. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified. Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches, such that a lower degree of similarity is detected.

[0035] In a specific embodiment of the present application, the sequence similarity of the nucleotide sequence encoding T9αH is 75.48%-100%.

[0036] In a specific embodiment of the present application, the homologous sequences of the nucleotide sequence shown in SEQ ID NO: 15 are the nucleotide sequences shown in SEQ ID NO: 16 to 28. The homologous sequences shown in SEQ ID NO: 16 to 28 have a homology of 75.48% to 100% with the nucleotide sequence shown in SEQ ID NO: 15.

[0037] The present invention also provides a biological enzyme combination, which includes the above-mentioned biological enzyme T9αH and at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

[0038] Preferably, the biological enzyme composition includes all the aforementioned biological enzymes.

[0039] The present application also provides a biomaterial, which is any one of the following c1) to c3):

[0040] c1) an expression cassette containing the above nucleic acid molecule;

[0041] c2) a vector containing the above-mentioned nucleic acid molecule or the expression cassette described in c1);

[0042] c3) A host cell containing at least one of the above nucleic acid molecule, the expression cassette described in c1), and the vector described in c2).

[0043] Optionally, the host cell includes at least one of a microbial cell, a plant cell, an animal cell, and an algae cell.

[0044] In an embodiment of the present application, the expression cassette in c1) further comprises a promoter operably linked to the above nucleic acid molecule.

[0045] Optionally, the expression cassette in c1) further comprises other regulatory elements, such as enhancers, leader sequences, transposons, terminators, marker genes, and the like.

[0046] In the embodiments of the present application, the type of vector in c2) is not specifically limited, and a suitable vector can be selected as needed. For example, the vector includes but is not limited to pFastBac1, pYES2, pYES2.1, pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pGEX2T, pTAex3, pUSA, pYMB0, pHT43, pET28b, pIJ702, pUCP19, pEAQ-HT, pYMB03, or pHT43, preferably pEAQ-HT.

[0047] In an embodiment of the present application, the host cells in c3) include, but are not limited to, at least one of Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli.

[0048] In an embodiment of the present application, among the host cells in c3), the microbial cells include bacteria and / or fungi.

[0049] In the embodiments of the present application, bacteria include but are not limited to Escherichia cells, Lactobacillus cells, Lactococcus cells, Corynebacterium bacteria, Acetobacter bacteria, Acinetobacter bacteria, Pseudomonas cells, Streptomyces cells, Bacillus cells, Staphylococcus cells, Agrobacterium cells, and yew endophytes, such as Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, Agrobacterium rhizogenes, Lactococcus lactis, Bacillus cereus, Pseudomonas fluorescens, and the like.In some embodiments, bacterial cells such as Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp. spp.), Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp.), Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp., and Pantoea spp. The bacterial cell can be a Gram-negative cell, such as an E. coli cell, or a Gram-positive cell, such as a Bacillus species.

[0050] In an embodiment of the present application, the fungus includes but is not limited to yeast, filamentous fungi or mushrooms.

[0051] In a specific embodiment of the present application, yeast includes but is not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida utilis, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, A. adeninetolytica, Phaffia rhodozyma, and Candida albicans. Yeast cells are, for example, Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp., and industrial polyploid yeast strains. Preferably, the yeast strain is a S. cerevisiae strain or a Yarrowia strain.

[0052] In a specific embodiment of the present application, filamentous fungi include but are not limited to Monascus, Aspergillus oryzae, Aspergillus niger, Aspergillus flavus, and Penicillium. Fungi are, for example, Aspergillus spp., Penicillium spp., Fusarium spp., Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp., Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., and Trichoderma spp.

[0053] In an embodiment of the present application, the host cells in c3) include, but are not limited to, tobacco cells, Pseudomonas aeruginosa cells, Artemisia annua cells, Arabidopsis thaliana cells, Physcomitrella patens cells, Marchantia polymorpha cells, tomato cells, ginseng cells, cotton cells, sugarcane cells, potato cells, corn cells, wheat cells, rice cells, radish cells, lettuce cells, etc., and the plant cells include protoplasts, suspension cells, etc.

[0054] In an embodiment of the present application, among the host cells in c3), animal cells include but are not limited to at least one of insect cells, mammalian cells, fruit fly cells, nematode cells, and fish cells.

[0055] In a specific embodiment of the present application, insect cells include but are not limited to S2 Drosophila cells or Sf9 cells.

[0056] In a specific embodiment of the present application, mammalian cells include, but are not limited to, fibroblasts, lymphocytes, epithelial cells, and myeloblasts, such as HEK293 cells, CHO cells, COS cells, BHK cells, HeLa cells, Chinese hamster ovary cells, Vero cells, SP2 / 0 cells, NS / 0 myeloma cells, mini-hamster kidney cells, human B cells, human Jurkat T cells, neurons, CV-1 / EBNA cells, L cells, 3T3 cells, HEPG2 cells, and MDCK cells.

[0057] In a specific embodiment of the present application, fish cells include but are not limited to zebrafish cells.

[0058] In an embodiment of the present application, the host cell in c3) is an algae cell including but not limited to at least one of cyanobacteria, green algae, Synechococcus, Synechococcus elongatus, Synechocystis, Anabaena, Chlamydomonas, and Chlamydomonas reinhardtii.

[0059] In an embodiment of the present application, the host cell further contains at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

[0060] The present application also provides an engineered strain comprising at least one of the above-mentioned nucleic acid molecule, expression cassette, and vector. In an embodiment of the present application, the engineered strain may be an engineered yeast strain or an engineered Escherichia coli strain.

[0061] In an embodiment of the present application, the plant cells and / or animal cells are non-biologically transformed recombinant plant cells and / or recombinant animal cells.

[0062] In the embodiments of the present application, optionally, the plant cells and / or animal cells are non-biologically transformed (non-biologically transformed, i.e., produced by non-substantially biological methods) recombinant plant cells and / or recombinant animal cells.

[0063] Optionally, plant cells or animal cells can be cultured to express biological enzymes and produce target products.

[0064] Among the above-mentioned biomaterials, methods for transforming nucleic acid molecules, expression cassettes, and vectors into host cells in vivo and in vitro include but are not limited to electroporation, polyethylene glycol (PEG) transformation, lipofection, heat shock, calcium phosphate precipitation, virus-mediated, and microinjection.

[0065] The present application also provides a method for producing a host cell, which comprises transforming the host cell with at least one of the above-mentioned nucleic acid molecules, expression cassettes in biological materials or vectors.

[0066] In an embodiment of the present application, the method for producing a host cell further comprises transforming the host cell with at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

[0067] In an embodiment of the present application, the host cell includes a microbial cell, a plant cell, an animal cell and / or an algae cell.

[0068] The present application also provides a method for producing a plant or a plant cell, which comprises transforming the plant or the plant cell with at least one of the above-mentioned nucleic acid molecules, expression cassettes in biological materials or vectors.

[0069] In an embodiment of the present application, the method for producing a plant or plant cell further comprises transforming the plant or plant cell with at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

[0070] The present application also provides a method for producing the above-mentioned biological enzyme, comprising: transforming the above-mentioned nucleic acid molecule, or at least one of the expression cassettes and vectors in the biological material into a host cell, so that the host cell produces the biological enzyme.

[0071] In an embodiment of the present application, the host cell includes a microbial cell, a plant cell, an animal cell and / or an algae cell.

[0072] The present application also provides the use of any of the above-mentioned biological enzymes, nucleic acid molecules or biological materials in the following d1), d2), d3) and / or d4):

[0073] d1) catalyzing the hydroxylation of the C9 position in a precursor of a taxane compound and / or an intermediate thereof; the precursor of the taxane compound and / or an intermediate thereof is a precursor in which the C9 position has not been hydroxylated or oxidized;

[0074] d2) Use in the synthesis of paclitaxel and / or its intermediates;

[0075] d3) Use in the synthesis of Baccatin III and / or its intermediates;

[0076] d4) Use in the synthesis of taxane compounds and / or their intermediates.

[0077] In an embodiment of the present application, the taxane compound includes but is not limited to at least one of paclitaxel and its derivatives, taxine and its derivatives, docetaxel and its derivatives, cabazitaxel and its derivatives.

[0078] In an embodiment of the present application, the taxane compound intermediate includes but is not limited to at least one of taxadiene-hexol-hexaacetate and its derivatives, 1-dehydroxybaccatin IV and its derivatives, baccatin III and its derivatives, 10-deacetylbaccatin III and its derivatives, paclitaxel and its derivatives, baccatin IV and its derivatives, baccatin VI and its derivatives, baccatin VII and its derivatives, baccatin IX and its derivatives, baccatin V and its derivatives, taxane and its derivatives, baccatin I and its derivatives, taxadiene-2α,5α,7β,9β,10β,13α-hexol-5α-acetate and its derivatives, 5α-acetate-Taxuspine F and its derivatives, taxadiene-hexol-tetraacetate and its derivatives, and Taxuspine F and its derivatives.

[0079] In the implementation manner of this application, the application includes the following aspects:

[0080] (1) The amino acid sequence of the biological enzyme provided in this application or the polypeptide of at least a part of the sequence may still have biological activity or even new biological activity after removing or replacing certain amino acids, or increase the yield or optimize the protein kinetic characteristics or other properties that are intended to be obtained.

[0081] (2) The nucleotide sequence of the nucleic acid molecule provided in the present application or at least part of the nucleotide sequence is modified or mutated, and the modification or mutation methods include codon optimization, insertion, deletion, polymerase chain reaction (PCR), error-prone PCR, reconnection of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by chemical reagents, etc.

[0082] (3) The nucleotide sequence provided in this application or at least a portion of the cloned gene of the nucleotide sequence is expressed in an exogenous host through a suitable expression system to obtain the corresponding enzyme or other higher biological activity or yield.

[0083] (4) Overexpressing the polynucleotide or protein in a recipient host cell to obtain a target host cell, wherein the target host cell has an increased production of paclitaxel and / or its intermediates compared to the recipient host. Methods for overexpressing the polynucleotide or protein in the recipient host include one or more of promoter editing technology, codon optimization, use of a strong promoter, insertion of introns, use of viral vectors, fusion protein technology, and other methods that can achieve the purpose of overexpression.

[0084] (5) The nucleotide sequence or at least a portion of the gene or gene cluster provided in this application can be used to construct a recombinant plasmid through genetic recombination to obtain a new biosynthetic pathway, or can be used to obtain a new biosynthetic pathway through insertion, replacement, deletion or inactivation.

[0085] The present application also provides a method for synthesizing taxane compounds and / or intermediates thereof in vivo or in vitro, wherein at least one of the precursors of the taxane compounds and / or intermediates thereof is used as a substrate, and hydroxylation occurs at the C9 position under the catalytic action of the above-mentioned biological enzyme; the precursor of the taxane compounds and / or intermediates thereof is a precursor in which the C9 position has not been hydroxylated or oxidized.

[0086] In an embodiment of the present application, the precursors of the taxane compounds and / or their intermediates include compounds represented by Formula I and / or Formula II:

[0087] Wherein, R1, R3, and R4 are each independently selected from H, -OH, and -OAc (acetoxy, -OOCCH3); R2 and R6 are each independently selected from H, -OH, -OAc, and -OBz ​​(benzoyl, C6H5-CO-CH2-); R5 and R7 are each independently selected from H, -OH, -OAc, and =O.

[0088] In a specific embodiment of the present application, the precursors of the taxane compounds and / or their intermediates include but are not limited to at least one of taxa-4(5),11(12)-diene, taxa-4(20),11(12)-diene-5α-ol, taxadiene-5α,10β,13α-triol and / or their derivatives.

[0089] In an embodiment of the present application, the biological enzyme for synthesizing taxane compounds and / or their intermediates further comprises at least one of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, TBT, T10βH, T14βH, DBAT, BAPT, T2'OH, DBTNBT, PAM, CoA ligase, TAX9, TAX14, and TAX19.

[0090] Among them, the enzymes (T9αH), TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT mentioned in this application are required for synthesizing baccatin III from geranylgeranyl pyrophosphate (GGPP). DBAT, BAPT, T2'OH, DBTNBT, PAM, and CoA ligase are required for synthesizing taxanes such as paclitaxel from baccatin III. Enzymes for synthesizing other taxanes include T10βH, T14βH, TAX9, TAX14, and TAX19.

[0091] In the embodiments of the present application, in vivo includes within microbial cells, within algae cells, within plant cells and / or within animal cells.

[0092] In an embodiment of the present application, in vivo synthesis of taxane compounds and / or their intermediates comprises causing the aforementioned nucleic acid molecules to express the bioenzymes of the present application within microbial cells, algal cells, plant cells, and / or animal cells; and synthesizing the taxane compounds and / or their intermediates using precursors of the taxane compounds and / or their intermediates as substrates under the catalytic action of the aforementioned bioenzymes. Optionally, the substrates are produced within microbial cells, algal cells, plant cells, and / or animal cells. Optionally, the substrates may be artificially added.

[0093] In a specific embodiment of the present application, the method of expressing the biological enzyme of the present application is to transform the above-mentioned nucleic acid molecules, or at least one of the expression cassettes and vectors in the biological material into a host cell, so that the host cell produces the biological enzyme.

[0094] The present application also provides a method for synthesizing baccatin III, which uses geranylgeranyl pyrophosphate (GGPP) as a substrate and synthesizes baccatin III under the action of biological enzymes T9αH, TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

[0095] Preferably, the method for synthesizing Baccatin III comprises synthesizing Baccatin III using the above-mentioned host cell, wherein the host cell contains heterologous T9αH, TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT and TBT.

[0096] The present application also provides a plant or a plant part thereof, wherein the plant is one of the following plants:

[0097] e1) transgenic plants containing the above nucleic acid molecules;

[0098] e2) plants grown from the above plant cells;

[0099] e3) Plants produced by the above method;

[0100] e4) progeny formed by self-pollination of any plant described in e1) to e3), and plants grown from the progeny;

[0101] e5) Offspring formed by hybridization of any plant in e1) to e3) with other varieties, and plants grown from the offspring;

[0102] The above-mentioned plant parts are roots, stems, leaves, flowers, fruits, pollen or seeds.

[0103] The present application also provides a method for producing a commercial product, comprising obtaining the above-mentioned plant or plant part thereof, and producing the commercial product from the plant or plant part thereof, wherein the commercial product is selected from the group consisting of: a crude extract, a raw material drug, and / or a pharmaceutical preparation containing at least one of baccatin III and its derivatives, paclitaxel and its derivatives, taxane and its derivatives, docetaxel and its derivatives, and cabazitaxel and its derivatives.

[0104] Compared with the prior art, the present invention has the following beneficial effects:

[0105] This application creatively identifies T9αH from Taxus chinensis, which can catalyze the hydroxylation reaction at the C9 position during the synthesis of taxane compounds. For example, during the synthesis of paclitaxel, the T9αH enzyme can catalyze the conversion of taxadiene-5α,10β,13α-triol to taxadiene-5α,9α,10β,13α-tetraol, achieving the hydroxylation reaction at the C9 position.

[0106] Furthermore, the present application co-expresses the above-mentioned T9αH gene with other identified catalytic enzyme genes in model biological systems such as plant cells (such as tobacco, etc.) or microbial cells (such as Escherichia coli, Saccharomyces cerevisiae, etc.), and ultimately enables the de novo synthesis of the precursor substance baccatin III in plant cells or microbial cells.

[0107] Furthermore, this application fills a decades-long gap in the final enzymatic steps of the biosynthesis of paclitaxel and other taxane compounds, ultimately deciphering the complete biosynthetic pathway for paclitaxel and other taxane compounds. This not only provides a new industrial production route for paclitaxel and other taxane compounds, but also allows for efficient production of large quantities of paclitaxel and other taxanes from low-value precursors through modification of pathway enzymes. This not only meets environmental protection requirements but also significantly reduces the price of paclitaxel-related drugs, making them more affordable and reducing the burden on patients' families. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 is an extracted ion chromatogram of taxane synthesized by a combination of seven genes (TXS, T5αH, T13αH, T10βH, T9αH1, TAT, and TAX19), with GFP as a control;

[0109] FIG2 shows the results of characterizing the activity of T9αH in tobacco;

[0110] The structural formula represents taxol, and the bar graph shows the activity comparison of genes for known synthases, three T9αH homologous genes (T9αH1 / 2 / 3), and a control (GFP), that is, the comparison of taxol content produced by different hydroxylases, expressed as ion abundance.

[0111] Figure 3 is the extracted ion chromatogram of the product baccatin III synthesized by a combination of 9 genes (TXS, T5αH, T13αH, T9αH1, T2αH, T7βH, TOT, TAT, TBT), with GFP as the control;

[0112] Figure 4 shows that tobacco can synthesize baccatin III de novo with the participation of T9αH;

[0113] The structural formula represents baccatin III, and the bar graph shows the activity comparison of genes for known synthases, three T9αH homologous genes (T9αH1 / 2 / 3), and a control (GFP), that is, the comparison of the content of baccatin III produced by different hydroxylases, expressed as ion abundance. DETAILED DESCRIPTION

[0114] This application discloses enzymes, nucleic acid molecules, biomaterials, and their applications for catalyzing the C9 hydroxylation of taxane compounds. Those skilled in the art can draw upon the disclosure herein and appropriately modify process parameters to achieve these goals. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by this application. The methods and applications of this application have been described through preferred embodiments. It is apparent that those skilled in the art can modify, alter, and combine the methods and applications described herein to implement and apply the technology of this application without departing from the content, spirit, and scope of this application.

[0115] Explanation of terms:

[0116] In this application, the term "biological enzyme" or "enzyme" is a protein (or "polypeptide" or "peptide composition") with biological activity, including both naturally occurring proteins and variants and modified forms thereof. As used herein, the terms "protein" and "polypeptide" are used interchangeably, so the term polypeptide can be used to refer to a full-length polypeptide or a fragment of a full-length polypeptide. The term "fragment" refers to a portion of a polypeptide sequence. "Fragments" or "biologically active portions" include polypeptides that contain a sufficient number of contiguous amino acid residues to retain biological activity, such as polypeptides in which the N-terminal amino acid is truncated.

[0117] "Variant" means a substantially similar sequence. As will be readily understood by those skilled in the art, naturally occurring proteins may have some differences between different species or samples, which may be deletions and / or additions of one or more amino acids at one or more internal sites and / or substitutions of one or more amino acids at one or more sites of a natural polypeptide. However, this does not affect the same or similar effects, and these proteins may be referred to as natural variants of exemplary proteins. Modified forms of proteins include, but are not limited to, appropriate amino acid substitutions / additions / deletions that do not affect the biological activity of the target protein, truncated N-terminal amino acids, codon optimization suitable for host cell preferences, additions of tags, fusions, etc. These proteins may be referred to as artificial variants of reference proteins. Guidance for appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), which is incorporated herein by reference. Conservative substitutions, such as replacing one amino acid with another amino acid having similar properties, may be optimal.

[0118] In this application, the term "amino acid" refers to any amino acid (both standard and non-standard amino acids), including but not limited to α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examples of suitable amino acids include but are not limited to alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.

[0119] In this application, the term "taxane 9α-hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C9 position of the taxane ring backbone. Based on the experimental results analyzed in this application, it may also catalyze the carbonylation of the C9 position of the taxane ring backbone. In this application, the term "T9αH" may refer to taxane 9α-hydroxylase, or taxane 9α-hydroxylase, or a nucleotide molecule or sequence encoding taxane 9α-hydroxylase. The specific meaning can be determined based on the context.

[0120] In some embodiments, the enzymes of interest herein can be isolated from materials containing the enzymes from any source. Any method for obtaining the enzymes of interest herein is compatible with the present invention.

[0121] As used herein, the term "isolated" means removed from its natural environment, or from other compounds present when the compound is first formed. The term "isolated" includes material isolated from a natural source as well as material recovered following preparation by recombinant expression in a host cell (e.g., nucleic acids and proteins), or chemically synthesized compounds, such as nucleic acid molecules, proteins, and peptides.

[0122] As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") may refer to a polymeric form of nucleotides, which may include sense and antisense strands of RNA, cDNA, genomic DNA, as well as synthetic forms and mixed polymers of the above. Nucleotides may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is typically at least 10 bases in length, unless otherwise indicated. The term may refer to RNA or DNA molecules of indefinite length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules may include one or both of naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0123] As will be readily appreciated by those skilled in the art, for nucleotide sequences, well-known molecular biology techniques, such as the polymerase chain reaction (PCR) and hybridization techniques outlined herein, can be used to identify naturally occurring variants, ie, substantially similar sequences.

[0124] As will be readily appreciated by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may comprise non-natural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications (e.g., uncharged bonds: such as methyl phosphonates, phosphotriesters, phosphoramidites, carbamates, etc.; charged bonds: such as phosphorothioates, phosphorodithioates, etc.; pendant moieties: such as peptides; intercalators: such as acridine, psoralen, etc.; chelating agents; alkylating agents; and modified bonds: such as α-anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and padlock conformations.

[0125] In some embodiments, the nucleic acid molecules associated with the present application can be cloned from DNA comprising a given nucleic acid molecule from any source, for example, by PCR amplification and / or restriction enzyme digestion. In some embodiments, the nucleic acid molecules associated with the present application are synthetic. Any method for obtaining the nucleic acid molecules associated with the invention is compatible with the present application.

[0126] In this application, the term "identity" refers to sequence similarity to a natural nucleic acid sequence or amino acid sequence. Identity can be evaluated by the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0127] Unless otherwise indicated, the sequence identity values ​​provided herein refer to the values ​​obtained using the full-length sequences of the present application by Jalview Version 2.11.2.7 (Waterhouse, AM, Procter, JB, Martin, DMA, Clamp, M. and Barton, GJ (2009) "Jalview Version 2-a multiple sequence alignment editor and analysis workbench" Bioinformatics 25(9)1189-1191 doi:10.1093 / bioinformatics / btp033) using the default parameters in the multiple alignment software package MUSCLE v3.8.31 ("MUSCLE: multiple sequence alignment with high accuracy and high throughput" Nucleic Acids Res. 32(5):1792 (2004)); or any equivalent program thereof).

[0128] In this application, the term "homology" is sometimes used to refer to the level of similarity between two or more nucleic acid or amino acid sequences (i.e., sequence similarity or identity) expressed as a percentage of positional identity. Homology also refers to the concept of evolutionary relatedness, typically demonstrated by similar functional properties between different nucleic acids or proteins that share similar sequences.

[0129] As those skilled in the art will readily appreciate, homologous sequences can be obtained by comparing known sequences with genomic or transcriptomic data from samples of evolutionarily closely related species. For example, between different species of the same genus or between different strains of the same species, by comparing a known sequence with the genomic or transcriptomic data of a sample, those skilled in the art can anticipate that the sequence has the same or similar function.

[0130] Other mathematical algorithms are known in the art and can be used to align two sequences. See, for example, the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. This algorithm was incorporated into the BLAST program of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed using the BLASTN program (nucleotide query for nucleotide sequence searches) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application, or using the BLASTX program (translated nucleotide query for protein sequence searches) to obtain protein sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed using the BLASTP program (protein query for protein sequence searches) to obtain amino acid sequences homologous to the protein molecules of the present application, or using the TBLASTN program (protein query for translated nucleotide sequence searches) to obtain nucleotide sequences homologous to the protein molecules of the present application. In order to obtain gap alignments for comparison purposes, Gapped BLAST (in BLAST2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the corresponding programs (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection.

[0131] In this application, the term "synthetic" refers to polynucleotide (i.e., DNA or RNA) molecules produced by chemical synthesis as an in vitro process. For example, TM Synthetic DNA is produced during the reaction process in the tube so that the synthetic DNA is enzymatically produced from natural DNA or RNA chains. Other laboratory methods can be used to synthesize polynucleotide sequences. Oligonucleotides can be chemically synthesized on an oligonucleotide synthesizer using solid phase synthesis using phosphoramidites. The synthesized oligonucleotides can be annealed to each other as complexes to produce "synthetic" polynucleotides. Other methods for chemically synthesizing polynucleotides are known in the art and can be easily implemented for use in the present disclosure.

[0132] As used herein, the term "gene" refers to a nucleic acid fragment that expresses a specific protein. A "gene" includes the DNA region that encodes the gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding and / or transcribed sequence. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, introns, and locus control regions.

[0133] As used herein, the term "gene product" refers to any product produced by a gene. For example, a gene product can be a direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA, or any other type of RNA), or a protein produced by translation of an mRNA.

[0134] In this application, the term "expression cassette" refers to a DNA fragment that can be inserted into a nucleic acid or polynucleotide at a specific restriction site or by homologous recombination. As used herein, the DNA fragment comprises a polynucleotide encoding a polypeptide of interest, and the expression cassette and restriction sites are designed to ensure that the expression cassette is inserted into an appropriate reading frame for transcription and translation. In one embodiment, the expression cassette can include a polynucleotide encoding a polypeptide of interest and, in addition to the polynucleotide, also has elements that promote specific host cell transformation. In one embodiment, the expression cassette can also include elements that allow the expression of the polynucleotide encoding the polypeptide of interest to be enhanced in the host cell. These elements can include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, etc.

[0135] In this application, the terms "vector" and "construct", "cloning vector" and "expression vector" are used interchangeably, and mean a vector that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. "Non-viral vector" refers to any vector that does not comprise a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence that comprises at least one DNA replication origin and at least one selective marker gene. Examples include, but are not limited to, plasmids, cosmids, phages, bacterial artificial chromosomes (BACs) or viruses that carry exogenous DNA into cells. Vectors can also include one or more genes, antisense molecules and / or selective marker genes and other genetic elements known in the art. Vectors can transduce, transform or infect cells, thereby causing cells to express nucleic acid molecules and / or proteins encoded by the vector.

[0136] In this application, the term "expression" refers to the biosynthesis of a gene product, including the transcription and / or translation of the gene product. "Expressing" or "producing" a protein or polypeptide from a DNA molecule refers to transcribing and translating the coding sequence to produce the protein or polypeptide, while "expressing" or "producing" a protein or polypeptide from an RNA molecule refers to translating the RNA coding sequence to produce the protein or polypeptide.

[0137] Expression vectors containing all necessary expression elements are commercially available and are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by introducing exogenous DNA (RNA) into the cells. The exogenous DNA (RNA) is placed under the effective control of transcriptional elements to allow expression of the exogenous DNA in the host cell.

[0138] As used herein, the term "transformation" includes all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors; transformation with plasmid vectors; electroporation; adipose infection; microinjection; Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS TM Transgenic plants are characterized by transgenic genes, including genomic DNA-mediated transformation and microprojectile bombardment. These techniques can be used for both stable and transient transformation of plant cells. "Stable transformation" refers to the introduction of a nucleic acid fragment into the genome of a host organism, resulting in genetic stability. Once stably transformed, the nucleic acid fragment is stably integrated into the genome of the host organism and any subsequent generations. Host organisms containing the transformed nucleic acid fragment are referred to as "transgenic" organisms. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelles of a host organism, resulting in gene expression without genetically stable inheritance.

[0139] In some embodiments, to transform hosts and host cells, the nucleotide sequences of the present application can be inserted into any vector known in the art that is suitable for expressing nucleotide sequences in hosts or host cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target host species to be transformed. The transformation method depends on the host cell to be transformed, the stability of the vector used, the expression level of the gene product, and other parameters.

[0140] In this application, the term "stringent conditions" may refer to any of low, moderate, and high stringency conditions. "Low stringency conditions" include, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Moderate stringency conditions" include, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" include, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Among the above conditions, increasing the temperature increases the likelihood of obtaining DNA with high homology. Hybridization stringency can be affected by a variety of factors, including temperature, probe concentration, probe length, ionic strength, time, and salt concentration. Those skilled in the art can achieve similar stringency conditions by appropriately selecting these factors.

[0141] Table 1 Chinese-English comparison table

[0142] Note: The protein sequence numbers of the identified genes in Table 1 are from Uniprot. The protein and gene sequences of T9αH and TOT provided in Table 1 are from Taxus spp.

[0143] Table 2 Amino acid sequence similarity of enzymes

[0144] Table 3 Nucleotide sequence similarity of biological enzymes

[0145] Table 4 Amino acids and their English names and abbreviations

[0146] The reagents, instruments, and biological materials used in this application can all be obtained through commercial channels.

[0147] The present application is further described below with reference to the embodiments:

[0148] Example 1. Expression of Taxane C9 Hydroxylase T9αH Gene in Tobacco

[0149] 1. cDNA Acquisition

[0150] For laboratory preservation, young leaves of Taxus chinensis were collected and immediately frozen in liquid nitrogen. A portion of the sample was ground in a mortar and pestle, and 100 mg was added to a 1.5 mL centrifuge tube. Lysis buffer was then added, and RNA was extracted (Plant Total RNA Isolation Kit Plus, FOREGENE, China). The mRNA was reverse transcribed into cDNA using a reverse transcription kit (HiScript III 1st Strand cDNA Synthesis Kit, Vazyme, China).

[0151] 2. Cloning of Taxane C9 Hydroxylase T9αH

[0152] The genome of Taxus chinensis var. mairei was analyzed, and the amino acid sequence and nucleotide sequence of the T9αH reading frame were shown in SEQ ID NO: 1 (its homologous sequences are shown in SEQ ID NO: 2-14 in the sequence listing) and SEQ ID NO: 15 (its homologous sequences are shown in SEQ ID NO: 16-28 in the sequence listing), respectively.

[0153] T9αH1 open reading frame amino acid sequence (SEQ ID NO: 1):

[0154] T9αH1 open reading frame nucleotide sequence (SEQ ID NO: 15):

[0155] Based on the nucleotide sequence of the T9αH1 gene, primers P1 and P2 were designed to amplify the T9αH1 gene. These primers contain a portion of the sequence of the tobacco expression vector pEAQ-HT. Using cDNA from Taxus chinensis leaves as a template, the T9αH1 gene was amplified by PCR. The PCR product was recovered by gel excision and ligated into the linearized pEAQ-HT vector digested with RruI and XhoI. The recombinant plasmid was cloned and sequenced using the ClonExpress One-Step Cloning Kit (Novozymes). The positive recombinant plasmid was designated pEAQ-HT-T9αH1.

[0156] Using the same method, pEAQ-HT-T9αH2 and pEAQ-HT-T9αH3 were obtained.

[0157] Among the primers used, P1 and P2 were used to amplify T9αH1, P3 and P4 were used to amplify T9αH2, and P5 and P6 were used to amplify T9αH3. The primer sequences are shown in Table 5.

[0158] Table 5 Primer sequences

[0159] Note: The lowercase nucleotide sequence is the vector sequence, and the uppercase nucleotide sequence is the T9αH specific primer sequence.

[0160] 3. Functional Verification of the Taxane C9 Hydroxylase T9αH Gene in Tobacco

[0161] The constructed expression vector pEAQ-HT-T9αH was transformed into Agrobacterium GV3101 to obtain the GV3101 / pEAQ-HT-T9αH transgenic strain; a positive single clone was picked and inoculated into LB medium (containing 50 μg / mL kanamycin and 25 μg / mL rifampicin) and cultured at 28°C for 24 h; the cultured bacterial liquid was taken and diluted in 10 mL LB medium (containing 50 μg / mL kanamycin and 25 μg / mL rifampicin) at a ratio of 1:100 and cultured at 28°C overnight until the OD 600 Add resuspension MMA (10mM MES, 10mM MgCl2, 150μM acetosyringone) to the final OD 600 The viscosity is 0.8-1.0, and it is allowed to stand at room temperature for 1-2 hours.

[0162] The resuspended Agrobacterium combination (known gene + T9αH homolog or GFP) was injected onto the underside of 4-6 week-old Nicotiana benthamiana leaves using a 1mL syringe without the needle. After drying under light for 1-2 hours, the leaves were transferred to darkness and incubated for 24 hours. The leaves were then returned to normal light. Five days after Agrobacterium injection, samples were collected and leaves injected with GFP served as controls. The known genes mentioned above include TXS (U48796), T5αH (AY289209), T13αH (AY056019), TAX19 (AY628434), TAT (AF190130), and T10βH (AF318211).

[0163] The tobacco sample was ground with a grinder, and the sample powder was transferred to a 5 mL centrifuge tube. After freeze-drying, 1 mL of methanol was added, ultrasonic extraction was performed for 30 min, and centrifugation was performed at 13,000 rpm for 15 min. The supernatant was transferred to a new centrifuge tube and centrifuged at 13,000 rpm for 15 min. 200 μL was taken out and transferred to an injection bottle for detection in LC-MS. By comparing with the paclitaxel standard, it was found that substances with the same retention time and molecular weight as paclitaxel could be detected from tobacco.

[0164] As shown in Figures 1 and 2 , in the combination containing T9αH, taxane can be synthesized de novo in tobacco leaves, and the activities of its homologous genes have certain differences.

[0165] Example 2. Taxane C9 Hydroxylase T9αH Gene Involved in the Synthesis of Baccatin III

[0166] 1. Taxane C9 hydroxylase T9αH gene is involved in the de novo synthesis of baccatin III in tobacco

[0167] The constructed expression vector pEAQ-HT-T9αH was transformed into Agrobacterium GV3101 to obtain the GV3101 / pEAQ-HT-T9αH transgenic strain; a positive single clone was picked and inoculated into LB medium (containing 50 μg / mL kanamycin and 25 μg / mL rifampicin) and cultured at 28°C for 24 h; the cultured bacterial liquid was taken and diluted in 10 mL LB medium (containing 50 μg / mL kanamycin and 25 μg / mL rifampicin) at a ratio of 1:100 and cultured at 28°C overnight until the OD 600 Add resuspension MMA (10mM MES, 10mM MgCl2, 150μM acetosyringone) to the final OD 600 The viscosity is 0.8-1.0, and it is allowed to stand at room temperature for 1-2 hours.

[0168] In the same manner, CV3101 Agrobacterium containing genes such as TXS (U48796), TAT (AF190130), T5αH (AY289209), T13αH (AY056019), T2αH (AY518383), T7βH (AY307951), TOT (SEQ ID NO: 29), and TBT (AF297618) were cultured and resuspended in MMA to an OD of 600 The value was 0.8-1.0, and then mixed with Agrobacterium containing T9αH at a ratio of 1:1, and Agrobacterium containing fluorescent protein GFP was used as a control.

[0169] Use a 1 mL syringe with the needle removed to inject the resuspended Agrobacterium combination into the underside of 4-6 week-old Nicotiana benthamiana leaves. After drying under light for 1-2 hours, transfer to a dark place and culture for 24 hours. Then transfer to normal light for culture. 5 days after Agrobacterium injection, collect samples and use leaves injected with GFP as a control.

[0170] The tobacco sample was ground with a grinder, and the sample powder was transferred to a 5 mL centrifuge tube. After freeze-drying, 1 mL of methanol was added, ultrasonic extraction was performed for 30 minutes, and centrifugation was performed at 13,000 rpm for 15 minutes. The supernatant was transferred to a new centrifuge tube and centrifuged at 13,000 rpm for 15 minutes. 200 μL was taken out and transferred to an injection bottle for detection in LC-MS. By comparison with the Baccatin III standard, it was found that substances with the same retention time and molecular weight as Baccatin III could be detected from tobacco.

[0171] As shown in Figures 3 and 4 , in the combination containing T9αH, baccatin III can be synthesized de novo in tobacco leaves, and the activities of its homologous genes have certain differences.

[0172] The above is only a preferred embodiment of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A biological enzyme T9αH, characterized in that Its amino acid sequence includes at least one of the following sequences: a1) the amino acid sequence shown in SEQ ID NO: 1; a2) a fusion amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 1; a3) an amino acid sequence having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; a4) an amino acid sequence that is at least 60% identical to at least one of the amino acid sequences shown in SEQ ID NO: 1 and has the same function.

2. The biological enzyme T9αH according to claim 1, characterized in that In a4), the amino acid sequence having at least 60% identity with the amino acid sequence shown in SEQ ID NO: 1 and having the same function includes at least one of the amino acid sequences shown in SEQ ID NO: 2 to 14.

3. A nucleic acid molecule encoding the biological enzyme according to claim 1, characterized in that: The nucleotide sequence thereof includes at least one of the following sequences: b1) the nucleotide sequence shown in SEQ ID NO: 15; b2) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO: 15, wherein the homologous sequence is a nucleotide sequence having at least 75% identity with the nucleotide sequence shown in SEQ ID NO: 15; b3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in b1) or b2) under stringent conditions and is capable of encoding a protein with the same function.

4. The nucleic acid molecule according to claim 3, characterized in that b2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 28.

5. A biological enzyme combination, characterized in that: The invention comprises the biological enzyme T9αH as described in claim 1 or 2, and further comprises at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT and TBT.

6. A biomaterial, characterized in that The biological material is any one of the following c1) to c3): c1) an expression cassette containing the nucleic acid molecule according to claim 3 or 4; c2) a vector containing the nucleic acid molecule according to claim 3 or 4 or the expression cassette according to c1); c3) a host cell containing at least one of the nucleic acid molecule according to claim 3 or 4, the expression cassette according to c1), and the vector according to c2); Optionally, the host cell includes at least one of a microbial cell, a plant cell, an animal cell, and an algae cell.

7. The biomaterial according to claim 6, characterized in that The vector includes pFastBac1, pYES2, pYES2.1, pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pGEX2T, pTAex3, pUSA, pYMB0, pHT43, pET28b, pIJ702, pUCP19, pEAQ-HT, pYMB03 or pHT43; And / or, the microbial cells include at least one of Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli; And / or, the plant cell comprises at least one of tobacco cell, Artemisia annua cell, Arabidopsis thaliana cell, Physcomitrella patens cell, Marchantia polyphylla cell, tomato cell, ginseng cell, cotton cell, sugarcane cell, potato cell, corn cell, wheat cell, rice cell, radish cell and lettuce cell; And / or, the animal cell comprises at least one of an insect cell, a mammalian cell, a fruit fly cell, a nematode cell, and a fish cell; And / or, the algae cells include at least one of cyanobacterial cells and green algae cells.

8. The biomaterial according to claim 5 or 6, characterized in that: The host cell also contains at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

9. A method for producing a host cell, the method comprising transforming the host cell with at least one of the nucleic acid molecule of claim 3 or 4, the expression cassette or vector in the biological material of any one of claims 6 to 8; optionally, the method further comprises transforming the host cell with at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, TBT.

10. A method for producing a plant or a plant cell, the method comprising transforming the plant or the plant cell with at least one of the nucleic acid molecule of claim 3 or 4, the expression cassette or the vector in the biological material of any one of claims 6 to 8; optionally, the method further comprises transforming the plant or the plant cell with at least one or all of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, TBT.

11. A method for producing the biological enzyme according to claim 1 or 2, characterized in that: The nucleic acid molecule according to claim 3 or 4, or at least one of the expression cassettes and vectors in the biological material according to any one of claims 6 to 8, is used to transform a host cell, so that the host cell produces the biological enzyme.

12. Use of any of the biological enzyme according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, or the biological material according to any one of claims 6 to 8 in the following d1), d2), d3) and / or d4): d1) catalyzing the hydroxylation of the C9 position in a precursor of a taxane compound and / or an intermediate thereof; the precursor of the taxane compound and / or an intermediate thereof is a precursor in which the C9 position has not been hydroxylated or oxidized; d2) Use in the synthesis of paclitaxel and / or its intermediates; d3) Use in the synthesis of baccatin III and / or its intermediates; d4) Use in the synthesis of taxane compounds and / or their intermediates.

13. The use according to claim 12, characterized in that: The taxane compound includes at least one of paclitaxel and its derivatives, taxine and its derivatives, docetaxel and its derivatives, cabazitaxel and its derivatives; And / or, the taxane compound intermediate includes at least one of taxadiene-hexaol-hexaacetate and its derivatives, 1-dehydroxybaccatin IV and its derivatives, baccatin III and its derivatives, 10-deacetylbaccatin III and its derivatives, paclitaxel and its derivatives, baccatin IV and its derivatives, baccatin VI and its derivatives, baccatin VII and its derivatives, baccatin IX and its derivatives, baccatin V and its derivatives, taxane and its derivatives, baccatin I and its derivatives, taxadiene-2α,5α,7β,9β,10β,13α-hexaol-5α-acetate and its derivatives, 5α-acetate-Taxuspine F and its derivatives, taxadiene-hexaol-tetraacetate and its derivatives, and Taxuspine F and its derivatives.

14. A method for synthesizing taxane compounds and / or their intermediates in vivo or in vitro, characterized in that: At least one of the precursors of taxane compounds and / or their intermediates is used as a substrate, and under the catalytic action of the biological enzyme described in claim 1 or 2 and / or the biological enzyme produced by the method described in claim 11, hydroxylation occurs at the C9 position; the precursor of the taxane compounds and / or their intermediates is a precursor whose C9 position is not hydroxylated and oxidized.

15. The method according to claim 14, characterized in that The precursors of the taxane compounds and / or their intermediates include compounds shown in Formula I and / or Formula II: Wherein, R1, R3, and R4 are each independently selected from H, -OH, and -OAc; R2 and R6 are each independently selected from H, -OH, -OAc, and -OBz; R5 and R7 are each independently selected from H, -OH, -OAc, and =O.

16. The method according to claim 14 or 15, characterized in that The precursor of the taxane compound and / or its intermediate includes at least one of taxa-4(5),11(12)-diene, taxa-4(20),11(12)-diene-5α-ol, taxadiene-5α,10β,13α-triol and / or their derivatives.

17. The method according to any one of claims 14 to 16, characterized in that: The biological enzyme for synthesizing taxane compounds and / or their intermediates also includes at least one of TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, TBT, T10βH, T14βH, DBAT, BAPT, T2'OH, DBTNBT, PAM, CoA ligase, TAX9, TAX14, and TAX19.

18. The method according to any one of claims 14 to 17, characterized in that: The in vivo includes within microbial cells, within algae cells, within plant cells and / or within animal cells.

19. The method according to any one of claims 14 to 18, characterized in that: The in vivo synthesis of taxane compounds and / or intermediates thereof comprises causing the nucleic acid molecule of claim 3 or 4 to express a biological enzyme in a microbial cell, an algae cell, a plant cell and / or an animal cell; and using a precursor substance of the taxane compounds and / or intermediates thereof as a substrate, under the catalytic action of the biological enzyme, synthesizing the taxane compounds and / or intermediates thereof.

20. A method for synthesizing Baccatin III, characterized in that: Using geranylgeranyl pyrophosphate (GGPP) as a substrate, baccatin III is synthesized under the action of biological enzymes T9αH, TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT; preferably, the method comprises synthesizing baccatin III using the host cell according to claim 6, wherein the host cell contains heterologous T9αH, TXS, TAT, T5αH, T13αH, T2αH, T7βH, TOT, and TBT.

21. A plant or a plant part thereof, characterized in that The plant is one of the following plants: e1) A transgenic plant containing the nucleic acid molecule according to claim 3 or 4; e2) A plant formed by growing the plant cell according to any one of claims 6 to 8; e3) A plant obtained by the method of claim 9; e4) progeny formed by self-pollination of any plant described in e1) to e3), and plants grown from the progeny; e5) offspring formed by hybridization of any plant in e1)-e3) with other varieties, and plants grown from the offspring; Preferably, the plant part is a root, stem, leaf, flower, fruit, pollen or seed.

22. A method of producing a commercial product, comprising obtaining the plant or plant part thereof as claimed in claim 21, and producing the commercial product from the plant or plant part thereof; Preferably, the commercial product is selected from the group consisting of: a crude extract, a raw material and / or a pharmaceutical preparation containing at least one of baccatin III and its derivatives, paclitaxel and its derivatives, taxane and its derivatives, docetaxel and its derivatives, cabazitaxel and its derivatives.

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