Method and biomaterial for total biosynthesis of paclitaxel precursor substance baccatin iii, and use

Abstract

A method and a biomaterial for the total biosynthesis of a key paclitaxel precursor substance baccatin III, and the use. The biological enzyme composition used in the method comprises taxane 9α-hydroxylase (T9αH) and taxane C4-C20 oxetane-forming enzyme (TOT). The core ingredients of the biosynthetic pathway of baccatin III are identified, and an artificial synthesis route for baccatin III is constructed, which pave the way for the efficient development of a green and low-carbon production pathway for taxane products such as paclitaxel by means of synthetic biology.

Description

A method for biosynthesizing paclitaxel precursor baccatin III, biomaterials and applications Technical Field

[0001] The present application relates to the technical fields of plant metabolic biology and synthetic biology, and specifically to a method, biomaterial, and application of the biosynthesis of baccatin III, a key precursor of paclitaxel. Background Art

[0002] Paclitaxel is a diterpene alkaloid compound derived from the yew plant, with the structural formula shown in Formula I. It is widely used clinically to treat breast cancer, ovarian cancer, some head and neck cancers, and lung cancer. Paclitaxel is currently produced using a semi-chemical synthesis method, which involves first extracting the natural precursors baccatin III or 10-deacetylbaccatin III (10-DAB) from the leaves, followed by chemical synthesis of paclitaxel.

[0003] Since the structure of paclitaxel was first discovered in 1971, elucidation of its biosynthetic pathway has been a focal point in natural product research. As shown in Figure 1, the paclitaxel biosynthetic pathway begins with the linear coupling of isoprenyl precursors (IPP and DMAPP) with GGPP to form taxadiene. Taxadiene undergoes a series of oxidations, acylations, and epoxidations to form the key intermediate, baccatin III. Baccatin III is then conjugated to a C13 side chain to produce paclitaxel. Based on the intermediate structures, the synthesis of paclitaxel requires approximately 19 enzymatic steps, of which four oxidases and one mutase remain unidentified: C9 hydroxylase (Taxane9αhydroxylase, T9αH), C9 oxidase (Taxane9αdioxygenase, T9αO), C1 hydroxylase (Taxane1βhydroxylase, T1βH), C4-C20 epoxidase (C4β, C20-epoxidase, EPOX), and oxomutase (OXM). Because the key enzymes for C4-C20 oxygen heterocycle formation and C9 oxidation remain unknown, the biosynthesis of the paclitaxel intermediate baccatin III has been impossible, and a complete paclitaxel biosynthetic pathway has long been lacking.

[0004] Summary of the Invention

[0005] In light of this, the present application provides a method, biomaterial, and application for the biosynthesis of baccatin III, a key precursor of paclitaxel. This application successfully identified the genes for the taxane C4-C20 oxetane-forming enzyme (TOT) and the taxane 9α-hydroxylase (T9αH), identifying the core components of the baccatin III biosynthetic pathway. This paves the way for the efficient development of green, low-carbon production pathways for paclitaxel and other taxane products through synthetic biology.

[0006] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0007] The present application provides a biological enzyme composition, which includes taxane 9α hydroxylase (T9αH) and taxane C4-C20 oxetane forming enzyme (TOT);

[0008] The amino acid sequence of T9αH includes at least one of the following (a1) to (a4):

[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 of 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] The amino acid sequence of TOT includes at least one of the following (b1) to (b4):

[0014] (b1) at least one of the amino acid sequences shown in SEQ ID NOs: 29 to 52;

[0015] (b2) a fusion amino acid sequence obtained by linking a tag to the N-terminus and / or C-terminus of at least one of the amino acid sequences shown in SEQ ID NOs: 29 to 52;

[0016] (b3) 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 of SEQ ID NOs: 29 to 52;

[0017] (b4) an amino acid sequence that is at least 60% identical to at least one of the amino acid sequences of SEQ ID NOs: 29 to 52 and has the same function.

[0018] In (a4) or (b4) above, the amino acid sequences with at least 60% identity are exemplified by amino acid sequences with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0019] In a specific embodiment of the present application, the bioenzyme composition includes taxane 9α hydroxylase (T9αH), and taxane C4-C20 oxetane forming enzyme (TOT);

[0020] Including at least one of the following (a1) to (a4):

[0021] (a1) the amino acid sequence shown in SEQ ID NO: 1;

[0022] (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;

[0023] (a3) an amino acid sequence having the same function as that of the amino acid sequence of SEQ ID NO: 1 obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0024] (a4) an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 1 and has the same function;

[0025] The amino acid sequence of TOT includes at least one of the following (b1) to (b4):

[0026] (b1) the amino acid sequence set forth in SEQ ID NO: 29;

[0027] (b2) 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: 29;

[0028] (b3) an amino acid sequence having the same function as that of SEQ ID NO: 29 obtained by substitution and / or deletion and / or addition of one or more amino acid residues;

[0029] (b4) an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 29 and has the same function.

[0030] In a specific embodiment of the present application, in (a4), the amino acid sequence that has at least 60% identity with the amino acid sequence shown in SEQ ID NO: 1 and has the same function includes at least one of the amino acid sequences shown in SEQ ID NOs: 2 to 14; and / or, in (b4), the amino acid sequence that has at least 60% identity with the amino acid sequence shown in SEQ ID NO: 29 and has the same function includes at least one of the amino acid sequences shown in SEQ ID NOs: 30 to 52.

[0031] In a specific embodiment of the present application, the biological enzyme composition includes any one of the taxane T9αH enzyme with the amino acid sequence shown in SEQ ID NO: 1 and the TOT enzyme with the amino acid sequence shown in SEQ ID NO: 30-52.

[0032] In a specific embodiment of the present application, the bioenzyme composition includes any one of the taxane T9αH enzymes with amino acid sequences shown in SEQ ID NOs: 2 to 14 and the TOT enzyme with amino acid sequence shown in SEQ ID NO: 29.

[0033] In a specific embodiment of the present application, the bioenzyme composition includes any one of the taxane T9αH enzymes with amino acid sequences shown in SEQ ID NOs: 2 to 14 and any one of the TOT enzymes with amino acid sequences shown in SEQ ID NOs: 30 to 52.

[0034] Preferably, the biological enzyme composition comprises a combination of the taxane T9αH enzyme having the amino acid sequence shown in SEQ ID NO: 1 and the TOT enzyme having the amino acid sequence shown in SEQ ID NO: 29.

[0035] In the specific implementation of the present application, the above 14 sequences (SEQ ID The sequence similarity of the amino acid sequence of NO: 1-14 T9αH is 63.15%-100%. 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.

[0036] In a specific embodiment of the present application, the amino acid sequence in b3) is a mutant of the amino acid sequence shown in b1), and the mutant includes at least one of V237A, P167A, Q187A, Q6A, V129A, I172A, S314A, V319A, Q381A, E313A, V205A, R50A, S500A, P428A, H249A, S310A, K259A, R269A, M177A, D361A, V382A, I347A, V371A, G448A, P454AA, P490A, I285A, T410A, T231A, K360A, G455A, K94A and other mutants.

[0037] The present study found that in the baccatin III biosynthetic pathway, GGPP is converted to taxadiene under the catalysis of taxadiene synthase TS; subsequently, taxadiene is modified by the coordinated catalysis of six endoplasmic reticulum-associated p450 enzymes, including taxane 5α-hydroxylase T5αH, taxane 2α-hydroxylase T2αH, taxane 7β-hydroxylase T7βH, taxane 9α-hydroxylase T9αH, taxane 13α-hydroxylase T13αH and taxane C4-C20 oxetane-forming enzyme TOT, and is finally converted to baccatin III under the catalysis of two cytoplasmic acyltransferases (taxane 5α-acetyltransferase TAT and taxane 2α-oxy-benzoyltransferase TBT).

[0038] In one embodiment of the present application, when the starting material is geranylgeranyl pyrophosphate (GGPP) and baccatin III is synthesized, the bioenzyme composition of the present application further comprises at least one of taxadiene synthase (TS), taxane 5α hydroxylase (T5αH), taxane 13α hydroxylase (T13αH), taxane 2α hydroxylase (T2αH), taxane 7β hydroxylase (T7βH), taxane 5α acetyltransferase (TAT), and taxane 2α-oxy-benzoyltransferase (TBT).

[0039] Preferably, the biological enzyme composition includes all of the aforementioned biological enzymes: taxadiene synthase (TS), taxane 5α hydroxylase (T5αH), taxane 13α hydroxylase (T13αH), taxane 2α hydroxylase (T2αH), taxane 7β hydroxylase (T7βH), taxane 5α acetyltransferase (TAT), and taxane 2α-oxy-benzoyltransferase (TBT).

[0040] In some embodiments, the natural amino acid sequences of the aforementioned various biological enzymes may be variant sequences derived from different species and / or strains of Taxus. The artificial amino acid sequences of the aforementioned various biological enzymes may be variant sequences obtained by appropriate modification of the natural amino acid sequences, including but not limited to appropriate amino acid substitutions / additions / deletions that do not affect the biological activity of the target protein, truncation of the N-terminal amino acid, codon optimization suitable for host cell preference, addition of tags, fusion, etc.

[0041] The above-mentioned biological enzyme may be a biological enzyme that removes the signal peptide, or may be a biological enzyme that includes the signal peptide.

[0042] In another embodiment of the present application, when the starting material is geranylgeranyl pyrophosphate (GGPP) and paclitaxel, a downstream product of baccatin III, is synthesized, in addition to the above-mentioned T9αH and TOT, and in addition to at least one of TS, T5αH, T13αH, T2αH, T7βH, TAT, and TBT, the bioenzyme composition further comprises at least one of phenylalanyl CoA ligase (PCL), phenylalanine aminomutase (PAM), baccatin III 3-amino-3-phenylpropanoyl transferase (BAPT), cytochrome P450 hydroxylase (T2′αH), and 3′N-debenzoyl 2′deoxytaxol N-benzoyl transferase (DBTNPT).

[0043] Preferably, the biological enzyme composition includes all the aforementioned biological enzyme types: TS, T5αH, T13αH, T2αH, T7βH, TAT, TBT, T9αH, TOT, phenylalanyl CoA ligase, phenylalanine aminomutase, baccatin III 3 amino 3 phenylpropanol transferase, cytochrome P450 hydroxylase, 3'N debenzoyl 2' deoxytaxel N benzoyl transferase.

[0044] In another embodiment of the present application, when the starting material is isopentenyl pyrophosphate (IPP) and / or dimethylallyl pyrophosphate (DMAPP) for synthesizing baccatin III, in this embodiment of the present application, the bioenzyme composition further comprises geranylgeranyl pyrophosphate synthase (GGPPS). The precursor molecules IPP and DMAPP for synthesizing GGPP can be provided by the host cell itself.

[0045] The present application also provides a nucleic acid molecule composition encoding the above-mentioned biological enzyme composition, which includes a nucleic acid molecule encoding T9αH and a nucleic acid molecule encoding TOT;

[0046] The nucleotide sequence of the nucleic acid molecule encoding T9αH includes at least one of the following sequences:

[0047] (c1) at least one of the nucleotide sequences shown in SEQ ID NOs: 15 to 28;

[0048] (c2) a complementary sequence, a degenerate sequence or a homologous sequence of at least one of the nucleotide sequences shown in SEQ ID NOs: 15 to 28, wherein a homologous sequence is a nucleotide sequence having at least 75% identity with the nucleotide sequence shown in SEQ ID NOs: 15 to 28; a nucleotide sequence having at least 75% identity is exemplified by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0049] (c3) a nucleotide sequence that hybridizes with the nucleotide sequence shown in (c1) or (c2) under stringent conditions and is capable of encoding a protein with the same function;

[0050] The nucleotide sequence of the nucleic acid molecule encoding TOT includes at least one of the following sequences:

[0051] (d1) at least one of the nucleotide sequences shown in SEQ ID NOs: 53 to 76;

[0052] (d2) a complementary sequence, a degenerate sequence or a homologous sequence of at least one of the nucleotide sequences shown in SEQ ID NOs: 53 to 76, wherein a homologous sequence is a nucleotide sequence having at least 74% identity with the nucleotide sequence shown in SEQ ID NOs: 53 to 76; a nucleotide sequence having at least 74% identity is exemplified by a nucleotide sequence having at least 74%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0053] (d3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (d1) or (d2) under stringent conditions and is capable of encoding a protein with the same function.

[0054] In a specific embodiment of the present application, the nucleic acid molecule composition of the above-mentioned biological enzyme composition includes a nucleic acid molecule encoding T9αH and a nucleic acid molecule encoding TOT;

[0055] The nucleotide sequence of the nucleic acid molecule encoding T9αH includes at least one of the following sequences:

[0056] (c1) the nucleotide sequence shown in SEQ ID NO: 15;

[0057] (c2) a complementary sequence, a degenerate sequence, or a homologous sequence of the nucleotide sequence of SEQ ID NO: 15, wherein the homologous sequence is a nucleotide sequence having at least 75% identity with the nucleotide sequence of SEQ ID NO: 15;

[0058] (c3) a nucleotide sequence that hybridizes with the nucleotide sequence shown in (c1) or (c2) under stringent conditions and is capable of encoding a protein with the same function;

[0059] The nucleotide sequence of the nucleic acid molecule encoding TOT includes at least one of the following sequences:

[0060] (d1) the nucleotide sequence shown in SEQ ID NO: 53;

[0061] (d2) a complementary sequence, a degenerate sequence, or a homologous sequence of the nucleotide sequence of SEQ ID NO: 53, wherein the homologous sequence is a nucleotide sequence having at least 74% identity with the nucleotide sequence of SEQ ID NO: 53;

[0062] (d3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (d1) or (d2) under stringent conditions and is capable of encoding a protein with the same function.

[0063] In a specific embodiment of the present application, in (c2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 28; and / or, in (d2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 54 to 76.

[0064] In a specific embodiment of the present application, the nucleic acid molecule composition encoding the above-mentioned biological enzyme composition includes: a nucleotide sequence encoding T9αH as shown in any one of SEQ ID NOs: 16 to 28, and a nucleotide sequence encoding TOT as shown in SEQ ID NO: 53.

[0065] In a specific embodiment of the present application, the nucleic acid molecule composition encoding the above-mentioned biological enzyme composition includes: a nucleotide sequence encoding T9αH as shown in SEQ ID NO: 15, and a nucleotide sequence encoding TOT as shown in any one of SEQ ID NOs: 54 to 76.

[0066] In a specific embodiment of the present application, the nucleic acid molecule composition encoding the above-mentioned biological enzyme composition includes: a nucleotide sequence encoding T9αH as shown in any one of SEQ ID NOs: 16 to 28, and a nucleotide sequence encoding TOT as shown in any one of SEQ ID NOs: 54 to 76.

[0067] Preferably, the nucleic acid molecule encoding T9αH comprises: a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 1 to 14. Preferably, the nucleic acid molecule encoding T9αH comprises: a nucleotide sequence shown in any one of SEQ ID NOs: 15 to 28.

[0068] More preferably, the nucleic acid molecule encoding T9αH is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1. More preferably, the nucleic acid molecule encoding T9αH includes the nucleotide sequence shown in SEQ ID NO: 15.

[0069] Preferably, the nucleic acid molecule encoding TOT comprises: a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOs: 29 to 52. Preferably, the nucleic acid molecule encoding TOT comprises: a nucleotide sequence shown in any one of SEQ ID NOs: 53 to 76.

[0070] More preferably, the nucleic acid molecule encoding TOT is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 29. More preferably, the nucleic acid molecule encoding TOT includes the nucleotide sequence shown in SEQ ID NO: 53.

[0071] Preferably, the nucleic acid molecule composition encoding the above-mentioned biological enzyme composition includes: a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1, and a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 29.

[0072] More preferably, the nucleic acid molecule composition encoding the above-mentioned biological enzyme composition includes: a nucleotide sequence encoding T9αH as shown in SEQ ID NO: 15, and a nucleotide sequence encoding TOT as shown in SEQ ID NO: 53.

[0073] In one embodiment of the present application, when baccatin III is synthesized from GGPP, the nucleic acid molecule composition further comprises a nucleic acid molecule encoding at least one of the following biological enzymes: taxadiene synthase (TS), taxane 5α hydroxylase (T5αH), taxane 13α hydroxylase (T13αH), taxane 2α hydroxylase (T2αH), taxane 7β hydroxylase (T7βH), taxane 5α acetyltransferase (TAT), and taxane 2α-oxy-benzoyltransferase (TBT).

[0074] Preferably, the nucleic acid molecule composition includes nucleic acid molecules encoding all the aforementioned enzyme types: taxadiene synthase (TS), taxane 5α hydroxylase (T5αH), taxane 13α hydroxylase (T13αH), taxane 2α hydroxylase (T2αH), taxane 7β hydroxylase (T7βH), taxane 5α acetyltransferase (TAT), and taxane 2α-oxy-benzoyltransferase (TBT).

[0075] In some embodiments, the natural nucleotide sequence of the nucleic acid molecule related to the present application may be a variant sequence derived from different species and / or strains of Taxus, and its artificial nucleotide sequence may be a variant sequence obtained by appropriate modification of the natural nucleotide sequence, and the modification includes but is not limited to appropriate nucleotide substitution / addition / deletion that does not affect the biological activity of the target protein, truncation of the N-terminal nucleotide, codon optimization suitable for host cell preferences, addition of tags, fusion, etc., corresponding nucleotide variant sequences.

[0076] In another embodiment of the present application, when paclitaxel, a downstream product of baccatin III, is synthesized from GGPP, the nucleic acid molecule composition further comprises at least one nucleic acid molecule encoding phenylalanyl CoA ligase (PCL), phenylalanine aminomutase (PAM), baccatin III 3-amino-3-phenylpropanol transferase (BAPT), cytochrome P450 hydroxylase (T2′αH), and 3′N-debenzoyl-2′-deoxypaclitaxel N-benzoyltransferase (DBTNPT).

[0077] Preferably, the nucleic acid molecule composition includes nucleic acid molecules encoding all the aforementioned biological enzyme types: phenylalanyl CoA ligase (PCL), phenylalanine aminomutase (PAM), baccatin III 3-amino-3-phenylpropanol transferase (BAPT), cytochrome P450 hydroxylase (T2′αH), and 3′N-debenzoyl 2′-deoxytaxol N-benzoyl transferase (DBTNPT).

[0078] In an embodiment of the present application, when baccatin III is synthesized from the precursors of GGPP, IPP and / or DMAPP, the nucleic acid molecule composition further includes a nucleic acid molecule encoding geranylgeranyl pyrophosphate synthase (GGPPS) and / or IPP isomerase.

[0079] The nucleic acid molecules of the present application can be obtained from a variety of sources. In some embodiments, some nucleic acid molecules are provided by the host cells themselves. For example, the nucleotide molecules required for the synthesis of GGPP can be provided by the host cells themselves, and the synthesis of taxadiene and / or taxadiene-5α-ol can be provided by transformed host cells as described previously, such as those described in Ajikumar PK, Xiao WH, Tyo KE, et al. Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli [J]. Science, 2010, 330(6000): 70-74.; Zhou K, Qiao K, Edgar S, et al. Distributing a metabolic pathway among a microbial consortium enhances production of natural products [J]. Nature Biotechnology, 2015, doi: 10.1038 / nbt.3095.

[0080] Optionally, the nucleic acid molecule is synthesized and / or isolated.

[0081] The present application also provides a biomaterial, which is any one of the following (e1) to (e3):

[0082] (e1) an expression cassette comprising the above-mentioned nucleic acid molecule composition or a combination thereof;

[0083] (e2) a vector or a combination thereof containing the above-mentioned nucleic acid molecule composition or the expression cassette (e1) or a combination thereof;

[0084] (e3) A host cell or a combination thereof containing at least one of the above-mentioned nucleic acid molecule composition, (e1) an expression cassette or a combination thereof, and (e2) a vector or a combination thereof.

[0085] In the embodiment of the present application, the (e1) is an expression cassette, in which all nucleic acid molecules are expressed; or is a composition composed of multiple expression cassettes, in which all nucleic acid molecules are co-expressed.

[0086] In an embodiment of the present application, the (e2) is a vector in which all nucleic acid molecules are expressed, or is a composition composed of multiple vectors in which all nucleic acid molecules are co-expressed.

[0087] In an embodiment of the present application, the (e3) is a host cell in which all nucleic acid molecules are expressed; or is a composition composed of multiple host cells, which co-express all nucleic acid molecules through co-culture and / or multi-stage culture.

[0088] In one embodiment of the present application, the biological material is an expression cassette or a composition thereof.

[0089] In an embodiment of the present application, when the nucleic acid molecule composition includes nucleic acid molecules encoding two biological enzymes, T9αH and TOT, it can be prepared into an expression cassette, expressing the T9αH gene and the TOT gene in one expression cassette, or it can be prepared into a composition consisting of multiple expression cassettes, expressing the T9αH gene and the TOT gene in two expression cassettes, respectively.

[0090] In an embodiment of the present application, when the nucleic acid molecule composition includes nucleic acid molecules encoding two biological enzymes, T9αH and TOT, and also includes nucleic acid molecules encoding at least one of TS, T5αH, T13αH, T2αH, T7βH, TAT, and TBT, it can be prepared into an expression cassette to express all nucleic acid molecules in one expression cassette; it can also be prepared into a composition consisting of multiple expression cassettes, expressing all nucleic acid molecules in multiple expression cassettes, and expressing one or more nucleic acid molecules in one expression cassette, as long as co-expression of all nucleic acid molecules can be achieved.

[0091] In an embodiment of the present application, the expression cassette further includes a regulatory element, and the regulatory element includes at least one of a promoter, an enhancer, a leader sequence, a transposon, a terminator, and a marker gene.

[0092] In an embodiment of the present application, the expression cassette includes a natural promoter and / or a heterologous promoter.

[0093] In embodiments of the present application, the selection of an operably linked heterologous promoter may depend on many factors, such as, for example, desired timing, localization, and expression pattern, as well as responsiveness to specific biotic or abiotic stimuli. Heterologous promoters include, but are not limited to, inducible promoters, constitutive promoters, tissue-specific promoters, wound-inducible promoters, and chemically regulated promoters. For example, Trc, T5, Tac, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, T3, GAL1, GAL10, MET17, CUP1, AOX1, polyhedrin, CMV, EF1A, EFS, CAG, PGK1, CBh, SFFV, MSCV, SV40, mPGK, hPGK, UBC, human beta actin, Actin, CaMV35S, TEF1, GPD, GDS, Ubi, ADH1, GAP, actin5C, Polyubiquitin, a1tubulin, TRE, TRE3G, UAS, Ac5, etc.

[0094] In an embodiment of the present application, the expression cassette also includes an expression cassette of a modified nucleic acid sequence of the enzyme in (a)-(d), wherein the modification may be to add a 5' leader sequence to enhance the translation expression of the sequence; the modification may also be to add a regulatory element or to modify the promoter, etc.

[0095] The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally should include 5' non-transcribed and 5' non-translated sequences for transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, and the like, as needed. In particular, such 5' non-transcribed regulatory sequences will include a promoter region, which includes a promoter sequence that controls transcriptional control of the operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as required. The expression cassettes of the present application may optionally include a 5' leader or signal sequence.

[0096] In an embodiment of the present application, the expression of a nucleic acid molecule can be regulated by manipulating the copy number of a gene or operon in a cell.

[0097] In some embodiments, the expression of nucleic acid molecules can be regulated by manipulating the order of nucleic acid molecules within a module.

[0098] In some embodiments, expression of a nucleic acid molecule is regulated by integrating one or more nucleic acid molecules or operons into a chromosome.

[0099] In another embodiment of the present application, the biological material is a carrier or a composition thereof.

[0100] In some embodiments, one or more nucleic acid molecules of interest are expressed in an expression vector. As used herein, a "vector" can be any of a variety of nucleic acids into which one or more desired sequences can be inserted by restriction enzyme digestion and ligation for transport or expression in a host cell in a variety of genetic environments. Vectors are typically composed of DNA but can also be composed of RNA.

[0101] In the present application embodiment, the vector includes a plasmid, a chloroplast, a viral vector, a phage, a phagemid, a clay, a fosmid, a bacteriophage or an artificial chromosome, and optionally, the viral vector includes an adenoviral vector, a retroviral vector or an adeno-associated viral vector, and optionally, the vector includes a bacterial artificial chromosome (BAC), a plasmid, a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome (YAC) or a mammalian artificial chromosome (MAC). For example, the vector includes pFastBac1, pYES2, pYES2.1, pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pGEX2T, pTAex3, pUSA, pYMB0, pHT43, pET28b, pIJ702, pUCP19, pYMB03, pHT43, pEAQ, etc.

[0102] Cloning vectors are capable of autonomous replication or integration into the host cell genome and are further characterized by one or more restriction endonuclease sites at which the vector can be cut in a deterministic manner and the desired DNA sequence can be ligated into the vector so that the new plasmid retains its ability to replicate in the host cell. Where the plasmid is present in a host cell, replication of the desired sequence can occur multiple times as the number of copies of the plasmid in the host cell (e.g., a bacterial host) increases, or only once per host before the host reproduces by mitosis. Where the plasmid is present in a bacteriophage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0103] Expression vectors can be inserted into the desired DNA sequence by restriction enzyme digestion and connection so that it is effectively connected to the regulatory sequence and can be expressed as an RNA transcript. The vector can also include one or more marker sequences suitable for identifying whether the cell has been transformed or transfected by the vector. The marker sequence includes genes such as proteins that increase or decrease its resistance or sensitivity to antibiotics or other compounds, genes (such as beta-galactosidase, luciferase or alkaline phosphatase) that encode enzymes that can detect activity by standard methods known in the art, and genes (such as green fluorescent protein) that have a visible effect on the phenotype of transformed or transfected cells, hosts, bacterium colonies or plaques. Preferred vectors are vectors that can autonomously replicate and express the structural gene products present in the DNA fragments that are effectively connected thereto.

[0104] In some embodiments, the nucleic acid molecule composition of the present application can be stably integrated into the genome (including the chloroplast genome) of the host cell.

[0105] In other embodiments, modified viruses and / or modified viral nucleic acids are applied to plants or parts thereof by microinjection, microparticle bombardment, viral vector infection, or by spraying, irrigation, dusting, etc., so that the nucleic acid molecules are not stably integrated into the genome of the host cell, causing the host or host cell to transiently express the target gene.

[0106] In an embodiment of the present application, when the nucleic acid molecule composition includes nucleic acid molecules encoding two biological enzymes, T9αH and TOT, it can be prepared into one vector to express the T9αH nucleic acid molecule and the TOT nucleic acid molecule in one vector, or it can be prepared into a composition consisting of multiple vectors to express the T9αH nucleic acid molecule and the TOT nucleic acid molecule in two vectors, respectively.

[0107] In an embodiment of the present application, when the nucleic acid molecule composition includes nucleic acid molecules encoding two biological enzymes, T9αH and TOT, and also includes at least one nucleic acid molecule encoding TS, T5αH, T13αH, T2αH, T7βH, TAT, and TBT, it can be prepared into a vector to express all nucleic acid molecules in one vector; it can also be prepared into a composition consisting of multiple vectors, expressing all nucleic acid molecules in multiple vectors, and expressing one or more nucleic acid molecules in one vector, as long as co-expression of all nucleic acid molecules can be achieved.

[0108] In an embodiment of the present application, the vector or its composition further includes at least one of the nucleic acid molecules encoding side chain esterification-related nucleic acids such as phenylalanyl CoA ligase (PCL), phenylalanine aminomutase (PAM), baccatin III 3-amino 3-phenylpropanol transferase (BAPT), cytochrome P450 hydroxylase (T2′αH), 3′N-debenzoyl 2′-deoxytaxol N-benzoyltransferase (DBTNPT).

[0109] In an embodiment of the present application, the vector or its composition further comprises a nucleic acid molecule encoding geranylgeranyl pyrophosphate synthase (GGPPS).

[0110] In an embodiment of the present application, the vector further comprises a transcriptional regulatory factor, for example, a transcriptional regulatory factor including TcWRKY1, TcWRKY2, TcWRKY6, TcWRKY8, TcWRKY47, TcMYC2a, and the like.

[0111] In another embodiment of the present application, the biological material is a host cell or a composition thereof.

[0112] Optionally, the host cell includes at least one of a microbial cell, a plant cell, an animal cell, and an algae cell.

[0113] In an embodiment of the present application, the host cell is a microorganism.

[0114] In an embodiment of the present application, the microorganisms include bacteria and / or fungi.

[0115] In an embodiment of the present invention, the host cell can be a single host cell in which all nucleic acid molecules are expressed; or it can be a composition composed of multiple host cells in which all nucleic acid molecules are co-expressed through co-culture, multi-stage culture, etc.

[0116] In embodiments of the present application, the host cell compositions include, but are not limited to, combinations of bacteria, fungi, or bacteria and fungi. For example, combinations of Escherichia coli and yeast. Combinations composed of different host cells can be achieved by co-cultivation, multi-stage culture, or other methods to achieve the objectives of the present application.

[0117] 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.

[0118] In an embodiment of the present application, the fungus includes but is not limited to yeast, filamentous fungi or mushrooms.

[0119] 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.

[0120] 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.

[0121] In an embodiment of the present application, the host cell is a plant cell.

[0122] In a specific embodiment of the present application, plant cells include but are not limited to tobacco cells, white yew cells, Artemisia annua cells, Arabidopsis cells, Physcomitrella patens cells, liverwort cells, tomato cells, ginseng cells, cotton cells, sugarcane cells, potato cells, corn cells, wheat cells, rice cells, radish cells, lettuce cells, etc., and the cells include protoplasts, suspension cells, etc.

[0123] In an embodiment of the present application, the host cell is an animal cell.

[0124] In an embodiment of the present application, animal cells include insect cells, mammalian cells, nematode cells, and fish cells.

[0125] In a specific embodiment of the present application, insect cells include but are not limited to S2 Drosophila cells or Sf9 cells.

[0126] 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.

[0127] In a specific embodiment of the present application, fish cells include but are not limited to zebrafish cells.

[0128] 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.

[0129] Optionally, plant cells or animal cells are cultured to express biological enzymes and produce target products.

[0130] In an embodiment of the present application, the host cell is an algae cell.

[0131] In an embodiment of the present application, the algae cells include but are not limited to at least one of cyanobacteria, green algae, Synechococcus, Synechococcus elongatus, Synechocystis, Anabaena, Chlamydomonas, and Chlamydomonas reinhardtii.

[0132] 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.

[0133] 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.

[0134] The present application also provides a method for producing the above-mentioned biological enzyme composition, which comprises transforming the nucleic acid molecule composition of the present application, or at least one of the expression cassette or its composition, vector or its composition in the biological material into a host cell, so that the host cell produces the biological enzyme composition.

[0135] 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.

[0136] In the above methods, methods for transforming nucleic acid molecules, expression cassettes, and vectors into host cells include, but are not limited to, Agrobacterium-mediated transformation, gene gun transformation, electroporation, polyethylene glycol (PEG) transformation, lipofection, heat shock, calcium phosphate precipitation, virus-mediated, microinjection, and genetic engineering editing techniques. Host cells include microorganisms, plant cells, animal cells, and / or algae cells.

[0137] In a specific embodiment of the present application, the method for producing a biological enzyme composition is, for example: (1) constructing a vector comprising the nucleic acid molecule composition of the present application; (2) transforming the obtained vector into a host cell (including microbial cells, plant cells, animal cells, algae cells, etc.); (3) culturing the obtained host cell to co-express the genes to produce baccatin III.

[0138] In a specific embodiment of the present application, in the method for producing a biological enzyme composition, when a nucleic acid molecule is expressed in multiple vectors, the vector includes a vector expressing at least two nucleic acid molecules, and the construction of the vector containing multiple nucleic acid molecules can be constructed using multivariate modular metabolic engineering (MMME).

[0139] In some embodiments, one or more nucleic acid molecules related to the present application are recombinantly expressed in bacterial cells. The bacterial cells of the present application can be cultured in any type of culture medium (rich or basic) and any composition. It will be understood by those skilled in the art that routine optimization will allow the use of various types of culture medium. The selected culture medium can be supplemented with a variety of additional components. Some non-limiting examples of supplementary components include glucose, antibiotics, IPTG for gene induction, ATCC trace mineral supplements, and glycolic acid. Similarly, other aspects of the culture medium and the growth conditions of the cells of the present application can be optimized through routine experimentation. For example, pH and temperature are non-limiting examples of factors that can be optimized. In some embodiments, factors such as culture medium selection, culture medium supplementation, and temperature can affect the production level of taxane products such as baccatin III and paclitaxel. In some embodiments, the concentration and amount of supplementary components can be optimized. In some embodiments, the frequency of supplementing the culture medium with one or more supplementary components and the culture time of the culture medium before harvesting terpenoid compounds such as taxadiene are optimized.

[0140] Liquid cultures used to grow cells relevant to the present application can be stored in any culture vessel known and used in the art. In some embodiments, large-scale production in aerated reaction vessels (e.g., stirred tank reactors) can be used to produce large quantities of taxane product, which can be recovered from the cell culture. In some embodiments, the taxane product is recovered from the gas phase of the cell culture, for example, by adding an organic layer, such as dodecane, to the cell culture and recovering the taxane product from the organic layer.

[0141] In some embodiments, the taxane product is recovered from the cell culture, for example, by adding an organic layer, such as ethyl acetate, to the cell culture.

[0142] The present application also provides the use of any one of the bioenzyme composition, nucleic acid molecule composition, and biomaterial of the present application in the synthesis of Baccatin III and / or its intermediates.

[0143] In an embodiment of the present application, the baccatin III intermediate includes compounds shown in Formula II and / or Formula III:

[0144] Among them, R1, R3, and R4 are each independently selected from H, -OH, and -OAc (acetoxy, -OOCCH3); R2 and R6 are selected from H, -OH, -OAc, and -OBz ​​(benzoyl, C6H5-CO-CH2-); R5 and R7 are selected from H, -OH, =O, and -OAc.

[0145] In an embodiment of the present application, baccatin III also includes baccatin III derivatives.

[0146] The present application also provides a method for synthesizing baccatin III and / or its intermediates, using a precursor of baccatin III and / or its intermediates as a substrate, and under the action of the bioenzyme composition of the present application or the bioenzyme composition produced by the above method, catalyzing the hydroxylation of the C9 position of the taxane ring skeleton and catalyzing the formation of C4-C20 oxetane of the taxane ring skeleton, thereby synthesizing baccatin III and / or its intermediates.

[0147] In an embodiment of the present application, any one of the nucleic acid molecule composition and biological materials (e1) to (e2) of the present application is transformed into a host cell, so that the host cell produces a bioenzyme composition; the host cell comprises at least one of a microbial cell, a plant cell, an animal cell, and an algae cell; a precursor of baccatin III and / or its intermediates is used as a substrate, and under the action of the produced bioenzyme composition, the hydroxylation at the C9 position of the taxane ring skeleton and the formation of C4-C20 oxetanes of the taxane ring skeleton are catalyzed to synthesize baccatin III and / or its intermediates.

[0148] In an embodiment of the present application, the method for synthesizing Baccatin III and / or its intermediates further comprises any one of the following reactions (f1) to (f3):

[0149] (f1) hydroxylation of at least one carbon position among C1, C2, C5, C7, C10, and C13 of the taxane ring backbone;

[0150] (f2) acylation of at least one hydroxyl group among C2, C5, and C10;

[0151] (f3) Ketonization of the C9 hydroxyl group.

[0152] In certain embodiments of the present application, the host cell is a microbial cell, and the method for synthesizing Baccatin III and / or its intermediates further comprises a fermentation culture step.

[0153] In certain embodiments of the present application, the host cell is a plant cell or an animal cell, and the method for synthesizing Baccatin III and / or its intermediates further comprises a cell culture step.

[0154] Optionally, the culture method includes immobilized cell culture, two-stage culture, two-phase culture, addition of inducers (such as salicylic acid, silver nitrate, methyl jasmonate, arachidonic acid, ammonium citrate, etc.), addition of bypass inhibitors (such as chlormequat chloride), addition of substrates (GGPP, taxadiene and / or other baccatin III intermediates), etc.

[0155] In certain embodiments of the present application, the host cell is a plant cell. Optionally, the method for synthesizing Baccatin III and / or its intermediates further comprises the steps of planting the plant, harvesting the plant, and extracting the product.

[0156] In an embodiment of the present application, the method for synthesizing baccatin III comprises using taxadiene as a substrate and synthesizing baccatin III under the action of at least six p450 enzymes (i.e., T2αH, T5αH, T7βH, T9αH, T13αH, and TOT) and two cytoplasmic acyltransferases (TAT and TBT).

[0157] In certain specific embodiments of the present application, the substrate taxadiene is synthesized under the action of TS enzyme.

[0158] The present application also provides the use of any one of the bioenzyme composition, nucleic acid molecule composition, and biomaterial of the present application in the synthesis of taxane compounds and / or their intermediates.

[0159] The present application also provides a method for synthesizing taxane compounds and / or their intermediates, comprising the following steps:

[0160] Baccatin III was synthesized using the aforementioned method;

[0161] Baccatin III is used as a substrate to further catalyze the synthesis of taxane compounds and / or their intermediates.

[0162] The above-mentioned method for synthesizing Baccatin III, taxane compounds and / or intermediates thereof further comprises an optimization step. The optimization step comprises at least one of the following optimization methods:

[0163] Gene expression optimization: for example, codon optimization, transcriptional regulatory factor optimization, promoter optimization, fusion protein optimization, etc. Codon optimization (including identification of optimal codons for a variety of organisms, and methods for achieving codon optimization) is well known to those of ordinary skill in the art and can be accomplished using standard methods. The transcriptional regulatory factor ORCA3 gene is a MeJA-induced transcriptional regulatory factor that regulates plant basal metabolism and secondary metabolism. Optimization of gene expression can also be achieved by selecting appropriate promoters and ribosome binding sites. In some embodiments, this can include selecting a plasmid with a high copy number, or a plasmid with a low or medium copy number. Gene expression can also be regulated by targeting the step of transcription termination, which is achieved by introducing or eliminating structures such as stem loops.

[0164] Metabolic engineering optimization: methods such as increasing secondary metabolite production through metabolic engineering by overcoming rate-limiting steps, reducing metabolic flux to competing pathways, reducing catabolism, and overexpressing regulatory genes;

[0165] Optimization of regulatory factors: Adding MeJA and its analogs, salicylic acid, arachidonic acid, coronatine, etc. to regulate biosynthesis;

[0166] Optimization of culture methods: for example, improving culture medium, adjusting environmental factors such as culture temperature and time, optimizing culture process, combined culture, etc.; for example, Zhou K et al. gave a culture example of co-culture of Escherichia coli and yeast (Zhou, K., Qiao, K., Edgar, S., and Stephanopoulos, G. (2015). Distributing a metabolic pathway among a microbial consortium enhances production of natural products. NAT BIOTECHNOL 33, 377-383.).

[0167] 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.

[0168] In an embodiment of the present application, the taxane compound intermediates include but are not limited to β-phenylalanoyl baccatin III and 3′-N-debenzoyltaxol.

[0169] The present application also provides a plant or a plant part thereof, wherein the plant is one of the following plants:

[0170] (g1) a plant formed by growing the above plant cells;

[0171] (g2) producing a plant obtained by the above-mentioned method for producing a biological enzyme composition in a plant;

[0172] (g3) progeny formed by self-pollination of any one of the plants (g1) to (g2), and plants grown from the progeny;

[0173] (g4) offspring formed by hybridization of any plant of (g1) to (g2) with other varieties, and plants grown from the offspring;

[0174] The above-mentioned plant parts are roots, stems, leaves, flowers, fruits, pollen or seeds.

[0175] The present application also provides a plant or a plant part thereof, wherein the plant includes a transgenic plant containing the above-mentioned nucleic acid molecule composition.

[0176] The present application also provides a method for preparing a plant or a plant part thereof, which comprises the steps of tissue culture and / or induction culture.

[0177] The present application also provides a method for producing a commercial product, comprising obtaining the plant or plant part thereof according to any one of the ninth and tenth aspects, 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, 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.

[0178] The present application also provides a product produced by any of the above methods, wherein the product contains at least one of the following ingredients: baccatin III and its derivatives, paclitaxel and its derivatives, taxane and its derivatives, docetaxel and its derivatives, and cabazitaxel and its derivatives.

[0179] Compared with the prior art, the present invention has the following beneficial effects:

[0180] This application successfully identified two essential genes for the synthesis of baccatin III: the taxane C4-C20 oxetane-forming enzyme gene (TOT) and the taxane 9α hydroxylase gene (T9αH). TOT, in particular, has the previously hypothesized functions of two enzymes, C4-C20 epoxidase (C4β, C20-epoxidase, EPOX) and oxomutase (OXM), catalyzing the oxidation of carbon-carbon double bonds in taxane molecules to oxetanes and propanes.

[0181] Furthermore, the present application co-expressed the two new genes TOT and T9αH with other known genes involved in the biosynthesis of baccatin III (TS, T5αH, T13αH, T2αH, T7βH, TAT and TBT), and surprisingly found that 9 genes were able to successfully reconstruct the biosynthetic pathway of baccatin III from GGPP to baccatin III in tobacco. The results showed that these 9 enzymes are the core components of the biosynthesis from GGPP to baccatin III, including taxadiene synthase (TS), 2 acyltransferases and 6 CYP450 enzymes. The C9 oxidase (T9αO) and C1 hydroxylase (T1βH) speculated by predecessors may be replaced by other enzymes in the gene combination of this application. The research results of this application revealed an unprecedented oxidative rearrangement reaction mechanism, identified the core components of the baccatin III biosynthetic pathway, and constructed a baccatin III artificial synthesis circuit.

[0182] Furthermore, T10βH and 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT), which have been considered essential genes for baccatin III biosynthesis in the prior art, can still be synthesized in the present baccatin III biosynthesis method even without these two enzymes. The present invention's research indicates that T10βH and DBAT are not essential, and their functions may be performed by other enzymes in the present gene combination.

[0183] Furthermore, by successfully constructing the complete biosynthetic pathway of baccatin III in tobacco, a new enzymatic mechanism in the biosynthesis of paclitaxel was revealed, filling the key gap in its complete biosynthetic pathway, and paving the way for the efficient development of green and low-carbon production pathways for paclitaxel and other taxane products through synthetic biology. BRIEF DESCRIPTION OF THE DRAWINGS

[0184] FIG1 is a schematic diagram of the biosynthetic pathway of paclitaxel.

[0185] FIG2 shows the synthesis of Baccatin III in tobacco using different gene combinations in Example 3;

[0186] Among them, baccatin III corresponds to the chromatographic elution curve of baccatin III reference substance; 7 represents product peak 7;

[0187] Group 1 corresponds to the chromatographic elution curve of the product synthesized by the combination of 9 genes (T9αH, TOT, TS, TAT, T5αH, T13αH, T2αH, T7βH, TBT);

[0188] Groups 2 to 10 correspond to the chromatographic elution curves of the products synthesized by deleting one of the nine genes.

[0189] FIG3 shows the tandem mass spectrometry (MS / MS) analysis results of the products synthesized by Group 1.

[0190] FIG4 is a graph showing the chromatographic elution curve of Baccatin III produced in yeast in Example 4, compared with the chromatographic elution curve of a Baccatin III reference substance and the chromatographic elution curve of a yeast control.

[0191] The English and Chinese interpretations of the attached drawings are as follows:

[0192] baccatin III: Baccatin III;

[0193] Group: group;

[0194] EIC: Extracted ion chromatography, the full name of which is Extract Ion Chromatogram;

[0195] Retention time: retention time;

[0196] Relative abundance: relative abundance;

[0197] GGPPS: geranylgeranyl pyrophosphate synthase;

[0198] TS: taxadiene synthase;

[0199] PAM: phenylalanine isomerase;

[0200] IPP: isopentenyl pyrophosphate;

[0201] DMAPP: dimethylallyl pyrophosphate;

[0202] GGPP: geranylgeranyl pyrophosphate;

[0203] Time, min: Time: minutes. DETAILED DESCRIPTION

[0204] This application discloses a method, biomaterial, and application for the complete biosynthesis of baccatin III, a key precursor of paclitaxel. Those skilled in the art can refer to the contents herein and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications will be apparent to those skilled in the art and are considered to be encompassed by this application. The methods and applications of this application have been described through preferred embodiments, and it is apparent that those skilled in the art can modify or appropriately 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.

[0205] Explanation of terms:

[0206] 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.

[0207] "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 of the same genus or different samples of the same species, 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 its ability to function identically or similarly, 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 exemplary 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.

[0208] 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.

[0209] In this application, the term "taxadiene synthase" refers to an enzyme that catalyzes the cyclization of GGPP to produce taxadiene (taxa-4(5), 11(12)-diene). Its exemplary amino acid sequence or nucleotide sequence is shown in Table 1. Those skilled in the art can also obtain other amino acid sequences or nucleotide sequences with the same function through databases or literature reports. This enzymatic reaction generates a unique skeleton structure of taxane compounds, so this enzyme is considered to be a key enzyme that determines whether the precursor complex can synthesize paclitaxel. In this application, the term "TS" can represent taxadiene synthase, or taxadiene synthase gene, or nucleotide molecule or sequence encoding taxadiene synthase. The specific meaning can be judged in combination with the context. The relevant content in patent CN202010647327.3 is incorporated herein by reference in its entirety.

[0210] As used herein, the term "taxane 5α-hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C5 position of the taxane backbone. An exemplary amino acid sequence or nucleotide sequence thereof is shown in Table 1. Those skilled in the art may also be aware of other amino acid sequences or nucleotide sequences with the same function through databases or literature reports. As used herein, the term "T5αH" may refer to taxane 5α-hydroxylase, a taxane 5α-hydroxylase gene, or a nucleotide molecule or sequence encoding taxane 5α-hydroxylase. The specific meaning may be determined based on the context.

[0211] As used herein, the term "taxane 13α-hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C13 position of the taxane backbone. An exemplary amino acid sequence or nucleotide sequence thereof is shown in Table 1. Those skilled in the art may also be aware of other amino acid sequences or nucleotide sequences with the same function through databases or literature reports. As used herein, the term "T13αH" may refer to taxane 13α-hydroxylase, a taxane 13α-hydroxylase gene, or a nucleotide molecule or sequence encoding taxane 13α-hydroxylase. The specific meaning may be determined based on the context.

[0212] In this application, the term "taxane 2α-hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C2 position of the taxane backbone. An exemplary amino acid sequence or nucleotide sequence thereof is shown in Table 1. Those skilled in the art may also be aware of other amino acid sequences or nucleotide sequences with the same function through databases or literature reports. In this application, the term "T2αH" may refer to taxane 2α-hydroxylase, a taxane 2α-hydroxylase gene, or a nucleotide molecule or sequence encoding taxane 2α-hydroxylase. The specific meaning can be determined based on the context.

[0213] In this application, the term "taxane 7β hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C7 position of the taxane backbone. An exemplary amino acid sequence or nucleotide sequence thereof is shown in Table 1. Those skilled in the art may also be aware of other amino acid sequences or nucleotide sequences with the same function through databases or literature reports. In this application, the term "T7βH" may refer to taxane 7β hydroxylase, or the taxane 7β hydroxylase gene, or a nucleotide molecule or sequence encoding taxane 7β hydroxylase. The specific meaning can be determined based on the context.

[0214] As used herein, the term "taxane 5α acetyltransferase" refers to an enzyme that catalyzes the acetylation of taxane C5 oxidation products. Example amino acid or nucleotide sequences are shown in Table 1. It may also catalyze the acetylation of taxane C10 oxidation products. Those skilled in the art can also identify other amino acid or nucleotide sequences with the same function through databases or literature reports. As used herein, the term "TAT" may refer to taxane 5α acetyltransferase, the taxane 5α acetyltransferase gene, or a nucleotide molecule or sequence encoding taxane 5α acetyltransferase. The specific meaning can be determined based on the context.

[0215] As used herein, the term "taxane 2α-O-benzoyltransferase" refers to an enzyme that catalyzes the benzoylation of C2-oxidized products of taxanes. An exemplary amino acid sequence or nucleotide sequence is shown in Table 1. Those skilled in the art can also obtain other amino acid sequences or nucleotide sequences with the same function from databases or literature reports. As used herein, the term "TBT" may refer to taxane 2α-O-benzoyltransferase, a taxane 2α-O-benzoyltransferase gene, or a nucleotide molecule or sequence encoding taxane 2α-O-benzoyltransferase. The specific meaning can be determined based on the context.

[0216] In this application, the term "taxane 9α hydroxylase" refers to an enzyme that catalyzes the hydroxylation of the C9 position of the taxane backbone. Based on the experimental results of this application, it can also catalyze the carbonylation of the C9 position of the taxane ring backbone. Its exemplary amino acid sequence or nucleotide sequence is shown in Table 1. In this application, the term "T9αH" can 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 in the context. The relevant content of patent CN202310961179.6 is incorporated herein by reference in its entirety.

[0217] In this application, the term "taxane C4-C20 oxetane-forming enzyme" refers to an enzyme that catalyzes the oxidation of carbon-carbon double bonds in taxane molecules to oxetanes and oxiranes, such as catalyzing the conversion of taxadiene-hexol-hexaacetate to 1-dehydroxybaccatin IV and baccatin I. Example amino acid or nucleotide sequences are shown in Table 1. In this application, the term "TOT" may refer to a taxane C4-C20 oxetane-forming enzyme, a taxane C4-C20 oxetane-forming enzyme gene, or a nucleotide molecule or sequence encoding a taxane C4-C20 oxetane-forming enzyme. The specific meaning can be determined in the context. The relevant content of patent CN202310496624.6 is incorporated herein by reference in its entirety.

[0218] In some embodiments, the enzymes of interest can be isolated from materials containing the enzymes from any source. Any method of obtaining the enzymes of interest is compatible with the present invention.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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 including, 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.), and 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.

[0223] 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.

[0224] 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).

[0225] 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.

[0226] 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.

[0227] In some embodiments, homologous sequences related to the present application can be obtained by comparing the exemplary sequences with the genome or transcriptome data of 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 the exemplary sequences with the genome or transcriptome data of the samples, a person skilled in the art can expect that the homologous sequences of the sequences will have the same or similar functions.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] The expression cassette can also include a selective marker gene for selecting transformed cells. The selective marker gene is used to select the cells or tissues of transformation. The marker gene includes a gene encoding antibiotic resistance, such as the gene encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and a gene that confers resistance to herbicidal compounds (such as glufosinate, bromoxynil, imidazolinone and 2,4-dichlorophenoxyacetic acid ester (2,4-d)). Other selective markers include phenotypic markers such as beta-galactosidase and fluorescent proteins, such as green fluorescent protein (GFP), cyan fluorescent protein (CYP) and yellow fluorescent protein.

[0234] 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 contain 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 selectable marker gene. Examples include, but are not limited to, plasmids, cosmids, bacteriophages, bacterial artificial chromosomes (BACs), or viruses that carry exogenous DNA into cells. Vectors can also include one or more genes, antisense molecules, and / or selectable marker genes and other genetic elements known in the art. Vectors can transduce, transform, or infect cells, thereby causing the cells to express nucleic acid molecules and / or proteins encoded by the vector. The term "plasmid" refers to a circular chain of nucleic acids that can replicate autosomes in prokaryotic or eukaryotic host cells. The term includes nucleic acids, which can be DNA or RNA, and can be single-stranded or double-stranded. The plasmid defined in this definition can also include sequences corresponding to bacterial replication origins.

[0235] In some embodiments, a "cloning vector" can replicate or integrate autonomously in the host cell genome and is further characterized by one or more restriction endonuclease sites at which the vector can be cut in a defined manner and a desired DNA sequence can be ligated into the vector so that the new recombinant plasmid retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the number of copies of the plasmid in the host cell (e.g., a bacterial host) increases, or only once per host before the host reproduces by mitosis. In the case of a bacteriophage, replication can occur actively during the lytic phase or passively during the lysogenic phase.

[0236] In some embodiments, an "expression vector" can be inserted into a desired DNA sequence by restriction enzyme digestion and ligation so that it is effectively linked to a regulatory sequence and can be expressed as an RNA transcript. The vector may also contain one or more marker sequences suitable for identifying whether a cell has been transformed or transfected by the vector. Markers include genes that encode proteins that increase or decrease their resistance or sensitivity to antibiotics or other compounds, genes that encode enzymes whose activity can be detected by standard methods known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that have a visible effect on the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). Preferred vectors are those that are capable of autonomous replication and expression of the products of the structural genes present in the DNA fragments to which they are effectively linked.

[0237] 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.

[0238] Gene expression can be influenced by external signals, such as exposure of a cell, tissue, or organism to a substance that increases or decreases gene expression. Gene expression can also be regulated anywhere along the pathway from DNA to RNA to protein. Regulation of gene expression can be achieved through control of transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production, or a combination thereof. The exact nature of the regulatory sequences required for gene expression may vary between species or cell types, but generally should include, as needed, 5′ non-transcribed and 5′ non-translated sequences involved in the initiation of transcription and translation, respectively, such as TATA boxes, capping sequences, CAAT sequences, and the like. In particular, such 5′ non-transcribed regulatory sequences will include a promoter region, which includes a promoter sequence that controls transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. The vectors herein may optionally include a 5′ leader or signal sequence. The selection and design of appropriate vectors are within the ability and judgment of those skilled in the art.

[0239] In some embodiments, when the nucleic acid molecule encoding any enzyme of the present application is expressed in a cell, a variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be a natural promoter, i.e., the promoter of a gene in its endogenous environment, which provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter continuously transcribes its associated gene without regulation. A variety of conditional promoters can also be used, such as promoters controlled by the presence or absence of a molecule. Chemically regulated promoters can be used to regulate the expression of genes in a host by applying exogenous chemical regulators. Depending on the purpose of use, the promoter can be a chemically induced promoter, wherein the application of the chemical induces gene expression, or a chemically induced repressible promoter, wherein the application of the chemical represses gene expression. Chemically induced promoters are known in the art, including but not limited to the corn In2-2 promoter (which is activated by benzenesulfonamide herbicide safeners), the corn GST promoter (which is activated by hydrophobic electrophilic compounds used as pre-germination herbicides), and the tobacco PR-1a promoter (which is activated by salicylic acid). Other chemically regulated promoters of interest include glucocorticoid-inducible promoters among the steroid-responsive promoters, as well as tetracycline-inducible and tetracycline-repressible promoters.

[0240] 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.

[0241] 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 mediated transformation; and microprojectile bombardment. These techniques can be used for both stable and transient transformation of host 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.

[0242] 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.

[0243] In the present application, term " plant " comprises seed, vegetable cell, plant protoplast, can be from the plant cell tissue culture, plant callus, plant piece of its regenerated plant and in plant or plant part such as embryo, pollen, ovule, seed, tuber, propagule, leaf, flower, branch, fruit, root, root tip, anther etc., be complete plant cell.The offspring, variant and mutant of regenerated plant are also included in the scope of the present application, and condition is that these parts comprise the polynucleotide of introducing.As used herein, unless otherwise clearly stated or apparent from the background of use, otherwise " offspring " and " offspring plant " comprise any subsequent generation of plant, no matter be produced by sexual reproduction and / or asexual reproduction.

[0244] The terms "transgenic plant" and "transformed plant" refer to equivalent terms for "plant" as described above, wherein the plant comprises a heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct introduced into the plant by, for example, any stable and transient transformation method disclosed elsewhere herein or otherwise known in the art. Such transgenic plants and transformed plants also refer, for example, to the plant into which the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct was first introduced, as well as any progeny plants thereof that comprise the heterologous nucleic acid molecule, heterologous polynucleotide, or heterologous polynucleotide construct.

[0245] In this application, the use of the term "DNA" or "RNA" is not intended to limit the application to polynucleotide molecules comprising DNA or RNA. It will be appreciated by those of ordinary skill in the art that the methods and compositions of the present application include polynucleotide molecules consisting of deoxyribonucleotides (i.e., DNA), ribonucleotides (i.e., RNA), or a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs, including but not limited to nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, having binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include but are not limited to phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA). The polynucleotide molecules of the present application also include all forms of polynucleotide molecules, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc. In addition, it will be understood by those of ordinary skill in the art that the nucleotide sequences disclosed herein also include the complementary sequences of the exemplary nucleotide sequences.

[0246] 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.

[0247] Table 1 Chinese-English comparison table

[0248] Note: The protein sequence numbers of the identified genes in Table 1 are all from Uniprot.

[0249] The protein and gene sequences of T9αH and TOT provided in Table 1 are all derived from the genus Taxus.

[0250] Table 2 Amino acid sequence similarity of T9αH enzyme

[0251] Table 3 Nucleotide sequence similarity of T9αH enzyme

[0252] Table 4 Amino acid sequence similarity of TOT enzyme

[0253] Table 5 Nucleotide sequence similarity of TOT enzyme

[0254] Table 6 Amino acids and their English names and abbreviations

[0255] The reagents, instruments, and biological materials used in this application can all be obtained through commercial channels.

[0256] The present application is further described below with reference to the embodiments:

[0257] Example 1. Cloning of the Taxane 9α Hydroxylase T9αH Gene

[0258] 1. cDNA Acquisition

[0259] Taxus chinensis was preserved in the laboratory. Young leaves 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 added, and RNA was extracted (Plant Total RNA Isolation Kit Plus, FOREGENE, China). mRNA was reverse transcribed into cDNA using a reverse transcription kit (HiScript III 1st Strand cDNA Synthesis Kit, Vazyme, China).

[0260] 2. Cloning of Taxane 9α Hydroxylase T9αH

[0261] 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.

[0262] T9αH open reading frame amino acid sequence (SEQ ID NO: 1):

[0263] T9αH open reading frame nucleotide sequence (SEQ ID NO: 15):

[0264] Based on the nucleotide sequence of the T9αH gene, primers P1 and P2 were designed to amplify the T9αH 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 T9αH gene was amplified by PCR. The PCR product was recovered by gel excision and recombined with the linearized pEAQ-HT vector digested with RruI and XhoI. The recombinant plasmid was cloned and sequenced using the ClonExpress One-Step Cloning Kit (Novagen). The positive recombinant plasmid was designated pEAQ-HT-T9αH.

[0265] Primer sequences are shown in the table below.

[0266] Table 7 Primer sequences

[0267] Note: The lowercase nucleotide sequence is the vector sequence, and the uppercase nucleotide sequence is the T9αH specific primer sequence.

[0268] Example 2. Cloning of the Taxane C4-C20 Oxetane-Forming Enzyme TOT Gene

[0269] 1. cDNA Acquisition

[0270] Taxus chinensis was preserved in the laboratory. Young leaves 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 added, and RNA was extracted (Plant Total RNA Isolation Kit Plus, FOREGENE, China). mRNA was reverse transcribed into cDNA using a reverse transcription kit (HiScript III 1st Strand cDNA Synthesis Kit, Vazyme, China).

[0271] 2. Cloning of Taxane C4-C20 Oxetane-Forming Enzyme TOT

[0272] The genome of Taxus chinensis var. mairei was analyzed, and the amino acid sequence and nucleotide sequence of the TOT reading frame were shown as SEQ ID NO: 31 (its homologous sequences are shown in SEQ ID NO: 29, 30, 32-52 in the sequence listing) and SEQ ID NO: 55 (its homologous sequences are shown in SEQ ID NO: 53, 54, 56-76 in the sequence listing), respectively.

[0273] The sequence of SEQ ID NO: 31 is as follows:

[0274] The sequence of SEQ ID NO: 55 is as follows:

[0275] Based on the nucleotide sequence of the TOT gene, primers P3 and P4 were designed to amplify the TOT 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 TOT gene was amplified by PCR. The PCR product was recovered by gel excision and recombined with the linearized pEAQ-HT vector digested with RruI and XhoI. The recombinant plasmid was cloned and sequenced using the ClonExpress One-Step Cloning Kit (Novagen). The positive recombinant plasmid was designated pEAQ-HT-TOT3.

[0276] The primer sequences are shown in the table below.

[0277] Table 8 Primer sequences

[0278] Note: The lowercase nucleotide sequence is the vector sequence, and the uppercase nucleotide sequence is the TOT-specific primer sequence.

[0279] Example 3. Functional Verification of the Involvement of T9αH and TOT Genes in the Synthesis of Baccatin III in Tobacco

[0280] 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 OD600 The viscosity is 0.8-1.0, and it is allowed to stand at room temperature for 1-2 hours.

[0281] The constructed expression vector pEAQ-HT-TOT was transformed into Agrobacterium GV3101 to obtain the GV3101 / pEAQ-HT-TOT3 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.

[0282] In the same manner, CV3101 Agrobacterium containing genes such as TS (U48796), TAT (AF190130 or AY628434, preferably AY628434), T5αH (AY289209), T13αH (AY056019), T2αH (AY518383), T7βH (AY307951), and TBT (AF297618) were cultured and resuspended in MMA to an OD of 600 The value was 0.8-1.0, and then they were mixed with Agrobacterium containing T9αH and Agrobacterium containing TOT in a ratio of 1:1, and Agrobacterium containing fluorescent protein GFP was used as a control.

[0283] 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.

[0284] 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, and ultrasonic extraction was performed for 30 minutes. After centrifugation 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.

[0285] The results showed that when T9αH and TOT were co-expressed with seven known genes in the baccatin III biosynthesis pathway, a new product peak 7 could be detected, which had the same mass as baccatin III (7) (m / z 609 [M+Na] + ) and retention time (Group 1 in Figure 2). Tandem mass spectrometry (MS / MS) analysis further confirmed that the new product 7 was baccatin III (Figure 3).

[0286] We further investigated whether these nine genes constitute the minimal gene set required for the biosynthesis of baccatin III in tobacco. As shown in Figure 2, when any of the nine genes in the co-expression system were missing, baccatin III production was undetectable (Groups 2 to 10 in Figure 2). These results suggest that these nine genes constitute the core components of baccatin III biosynthesis.

[0287] Furthermore, T10βH has long been considered an essential gene for the biosynthesis of baccatin III. The results of this application show that baccatin III can still be synthesized in the absence of T10βH (Figures 2 and 3).

[0288] In addition, 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) is considered to be the key enzyme for C10 acylation in baccatin III. The results of this application show that baccatin III can still be synthesized in the absence of DBAT (Figures 2 and 3), indicating that TAT can replace its function in tobacco.

[0289] Example 4. Construction of a synthetic pathway for Baccatin III in yeast and realization of heterologous production

[0290] The T9αH (SEQ ID NO: 1) and TOT (SEQ ID NO: 31) as well as TS (U48796), TAT (AY628434), T5αH (AY289209), T13αH (AY056019), T2αH (AY518383), T7βH (AY307951), and TBT (AF297618) genes were constructed into the expression vector pESC-LEU. The expression cassettes were amplified using primers containing yeast genomic integration sites, and these genes were integrated into the yeast genome using PEG / lithium acetate transformation to obtain the cpt10.1 strain.

[0291] The cpt10.1 strain was inoculated into 2-3 mL YPD (containing 2% glucose) and cultured overnight at 30°C and 200 rpm. 600The initial ratio was inoculated into 20 mL of YPD (containing 2% glucose), cultured for 2 days, then centrifuged at 4000 rpm for 10 min, the supernatant removed, and the cells were resuspended in 20 mL of sterile water and centrifuged at 4000 rpm for 10 min. After removing the supernatant, the cells were resuspended in 20 mL of YPL (containing 2% galactose) and cultured at 30°C, 200 rpm for 6-7 days. Wild-type yeast was used as a control.

[0292] After the incubation, an equal volume of ethyl acetate was added and the mixture was extracted by shaking at low temperature overnight. The mixture was then transferred to a 50 mL centrifuge tube and centrifuged at 4000 rpm for 10 min. The upper organic phase was transferred to a new 50 mL centrifuge tube, dried under vacuum, and re-dissolved in 200 μL of methanol. 100 μL was transferred to an injection vial for LC-MS detection.

[0293] The results showed that in the yeast strain cpt10.1 containing nine genes involved in the biosynthesis pathway of baccatin III, a new product peak 7 could be detected, and the peak 7 product had the same retention time as baccatin III ( Figure 4 ).

[0294] 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 composition, characterized in that: The biological enzyme composition comprises taxane 9α hydroxylase T9αH and taxane C4-C20 oxetane forming enzyme TOT; Including at least one of the following (a1) to (a4): (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 as that of SEQ ID NO: 1 obtained by substitution and / or deletion and / or addition of one or more amino acid residues; (a4) an amino acid sequence that has at least 60% identity to the amino acid sequence of SEQ ID NO: 1 and has the same function; The amino acid sequence of the TOT includes at least one of the following (b1) to (b4): (b1) the amino acid sequence shown in SEQ ID NO: 29; (b2) 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: 29; (b3) an amino acid sequence having the same function as that of SEQ ID NO: 29 obtained by substitution and / or deletion and / or addition of one or more amino acid residues; (b4) an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 29 and has the same function.

2. The bioenzyme composition according to claim 1, characterized in that: In (a4), the amino acid sequence having at least 60% identity with the amino acid sequence of SEQ ID NO: 1 and having the same function includes at least one of the amino acid sequences of SEQ ID NO: 2 to 14; And / or, in (b4), the amino acid sequence having at least 60% identity with the amino acid sequence of SEQ ID NO: 29 and having the same function includes at least one of the amino acid sequences of SEQ ID NOs: 30 to 52.

3. The bioenzyme composition according to claim 1 or 2, characterized in that: The bio-enzyme composition further comprises at least one of taxadiene synthase TS, taxane 5α hydroxylase T5αH, taxane 13α hydroxylase T13αH, taxane 2α hydroxylase T2αH, taxane 7β hydroxylase T7βH, taxane 5α acetyltransferase TAT, and taxane 2α-oxy-benzoyltransferase TBT.

4. The bio-enzyme composition according to any one of claims 1 to 3, characterized in that: The biological enzyme composition also includes at least one of phenylalanyl CoA ligase PCL, phenylalanine aminomutase PAM, baccatin III 3-amino 3-phenylpropanol transferase BAPT, cytochrome P450 hydroxylase T2′αH, 3′N-debenzoyl 2′deoxytaxel N-benzoyl transferase DBTNPT, and geranylgeranyl pyrophosphate synthase GGPPS.

5. A nucleic acid molecule composition encoding the biological enzyme composition according to any one of claims 1 to 4, characterized in that: It includes a nucleic acid molecule encoding T9αH and a nucleic acid molecule encoding TOT; The nucleotide sequence of the nucleic acid molecule encoding T9αH includes at least one of the following sequences: (c1) the nucleotide sequence shown in SEQ ID NO: 15; (c2) 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; (c3) a nucleotide sequence that hybridizes with the nucleotide sequence shown in (c1) or (c2) under stringent conditions and is capable of encoding a protein with the same function; The nucleotide sequence of the nucleic acid molecule encoding POT includes at least one of the following sequences: (d1) the nucleotide sequence shown in SEQ ID NO: 53; (d2) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO: 53, wherein the homologous sequence is a nucleotide sequence having at least 74% identity with the nucleotide sequence shown in SEQ ID NO: 53; (d3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in (d1) or (d2) under stringent conditions and is capable of encoding a protein with the same function.

6. The nucleic acid molecule composition according to claim 5, characterized in that (c2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 16 to 28; And / or, in (d2), the homologous sequence includes at least one of the nucleotide sequences shown in SEQ ID NOs: 54 to 76.

7. The nucleic acid molecule composition according to claim 5 or 6, characterized in that: The nucleic acid molecule composition also includes a nucleic acid molecule encoding at least one of the following biological enzymes: taxane disynthase TS, taxane 5α hydroxylase T5αH, taxane 13α hydroxylase T13αH, taxane 2α hydroxylase T2αH, taxane 7β hydroxylase T7βH, taxane 5α acetyltransferase TAT, and taxane 2α-oxy-benzoyltransferase TBT.

8. The nucleic acid molecule composition according to any one of claims 5 to 7, characterized in that: The nucleic acid molecule composition also includes at least one nucleic acid molecule encoding phenylalanyl CoA ligase PCL, phenylalanine aminomutase PAM, baccatin III 3-amino 3-phenylpropanol transferase BAPT, cytochrome P450 hydroxylase T2′αH, 3′N-debenzoyl 2′deoxytaxel N-benzoyl transferase DBTNPT, and geranylgeranyl pyrophosphate synthetase GGPPS.

9. A biomaterial, characterized in that: The biological material is any one of the following (e1) to (e3): (e1) an expression cassette comprising the nucleic acid molecule composition according to any one of claims 5 to 8 or a combination thereof; (e2) a vector or a combination thereof containing the nucleic acid molecule composition according to any one of claims 5 to 8 or the expression cassette according to (e1); (e3) A host cell or a combination thereof containing at least one of the nucleic acid molecule composition according to any one of claims 5 to 8, the expression cassette or a combination thereof according to (e1), and the vector or a combination thereof according to (e2).

10. The biomaterial according to claim 9, characterized in that Said (e1) is one expression cassette, in which all nucleic acid molecules are expressed; or said (e1) is a composition consisting of multiple expression cassettes, in which all nucleic acid molecules are co-expressed; Said (e2) is a vector, in which all nucleic acid molecules are expressed, or said (e2) is a composition consisting of multiple vectors, in which all nucleic acid molecules are co-expressed; The (e3) is a host cell, in which all nucleic acid molecules are expressed; or the (e3) is a composition composed of multiple host cells, in which all nucleic acid molecules are co-expressed by co-culture and / or multi-stage culture.

11. A method for producing a host cell, characterized in that Transform a host cell with at least one of the nucleic acid molecule composition according to any one of claims 5 to 8, or the expression cassette or the composition thereof, the vector or the composition thereof in the biological material according to claim 9 or 10.

12. A method for producing a plant or a plant cell, characterized in that Transform a plant or plant cell with at least one of the nucleic acid molecule composition according to any one of claims 5 to 8, or the expression cassette or the composition thereof, the vector or the composition thereof in the biological material according to claim 9 or 10.

13. A method for producing the bioenzyme composition according to any one of claims 1 to 4, characterized in that: The nucleic acid molecule composition described in any one of claims 5 to 8, or at least one of the expression cassette or its composition, the vector or its composition in the biological material described in claim 9 or 10, is transformed into a host cell or its composition, so that the host cell or its composition produces the biological enzyme composition.

14. Use of the bioenzyme composition according to any one of claims 1 to 4, the nucleic acid molecule composition according to any one of claims 5 to 8, and the biomaterial according to claim 9 or 10 in the synthesis of baccatin III and / or its intermediates.

15. The use according to claim 14, characterized in that: The baccatin III intermediates include compounds shown in formula II and / or formula III: Wherein, R1, R3, and R4 are each independently selected from H, -OH, and -OAc; R2 and R6 are selected from H, -OH, -OAc, and -OBz; and R5 and R7 are selected from H, -OH, =O, and -OAc.

16. A method for synthesizing Baccatin III and / or its intermediates, characterized in that: Using the precursor of baccatin III and / or its intermediate as a substrate, under the action of the bioenzyme composition according to any one of claims 1 to 4 and / or the bioenzyme composition produced by the method according to claim 13, catalyzing the hydroxylation of the C9 position of the taxane ring skeleton and catalyzing the formation of C4-C20 oxetane of the taxane ring skeleton, to synthesize baccatin III and / or its intermediate; and / or, The nucleic acid molecule composition according to any one of claims 5 to 8 and any one of the biological materials (e1) to (e2) according to claim 9 or 10 are transformed into a host cell, so that the host cell produces a biological enzyme composition; the host cell comprises at least one of a microbial cell, a plant cell, an animal cell, and an algae cell; using a precursor of baccatin III and / or its intermediates as a substrate, under the action of the produced biological enzyme composition, catalyzing the hydroxylation of the C9 position of the taxane ring skeleton and catalyzing the formation of C4-C20 oxetane of the taxane ring skeleton, thereby synthesizing baccatin III and / or its intermediates.

17. The method according to claim 16, characterized in that It also includes any of the following reactions (f1) to (f3): (f1) hydroxylation of at least one carbon position among C1, C2, C5, C7, C10, and C13 of the taxane ring backbone; (f2) acylation of at least one hydroxyl group among C2, C5, and C10; (f3) Ketonization of the C9 hydroxyl group.

18. The method according to claim 16 or 17, characterized in that With taxadiene as a substrate, baccatin III is synthesized under the action of a biological enzyme composition comprising at least T2αH, T5αH, T7βH, T9αH, T13αH, TOT, TAT and TBT.

19. The method according to claim 18, characterized in that The substrate taxadiene is synthesized under the action of TS enzyme.

20. Use of the bioenzyme composition according to any one of claims 1 to 4, the nucleic acid molecule composition according to any one of claims 5 to 8, or the biomaterial according to claim 9 or 10 in the synthesis of taxane compounds and / or their intermediates.

21. A method for synthesizing taxane compounds and / or their intermediates, characterized in that: The steps include: Synthesizing Baccatin III using the method described in any one of claims 16-19; Using baccatin III as a substrate, the taxane compounds and / or their intermediates are further catalyzed to synthesize.

22. The method according to claim 21, 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; The taxane compound intermediate includes at least one of β-phenylalanyl baccatin III and 3'-nitrogen-debenzoyl paclitaxel.

23. A method of manufacturing a commercial product, characterized in that Obtaining a transgenic plant or plant part thereof containing the nucleic acid molecule composition of claims 5-8, 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 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.

24. The product obtained by the method of any one of claims 16-19, 21-23, characterized in that: The product contains the following ingredients: 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.

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