Methods and cells for producing paclitaxel precursors

Abstract

The present invention relates to the field of plant diterpene synthesis, in particular to host cells, methods and uses thereof. More specifically, the present invention relates to the production of taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, and enzymes and host cells for such production.

Description

Technical Field

[0001] This invention relates to the field of plant diterpene synthesis, and in particular to its host cells, methods, and uses. More specifically, this invention relates to the production of taxanes having side chains comprising a hydroxylated β-phenylalanine moiety, and enzymes and host cells for such production. Background of the Invention

[0003] Paclitaxel (also known as taxol) is one of the most effective anticancer drugs ever developed. It has been used to treat melanoma, ovarian cancer, breast cancer, bladder cancer, prostate cancer, and esophageal cancer (Cragg, GM, 1998).

[0004] However, its current production costs are particularly high, necessitating the development of sustainable and cost-effective production methods. Producing paclitaxel in microbial or plant cell factories would reduce its costs, increase its availability, and enable the efficient synthesis of derivatives with improved pharmacological properties. However, due to the limitations imposed on its natural host, the yew tree (Taxus chinensis), production methods remain challenging. Taxus spp. The lack of complete understanding of biosynthetic pathways in α-hydroxylamine has hindered progress in this direction.

[0005] One of the structurally crucial components for the anticancer activity of paclitaxel (taxol) is the hydroxyl group at the C2' position of the C13 side chain. However, an efficient enzyme catalyzing this reaction has not yet been reported, thus the complete biosynthesis of paclitaxel in a heterologous host has not yet been achieved. Summary of the Invention

[0006] To date, incomplete understanding of biosynthesis, lack of catalytic enzymes, or ineffective catalytic enzymes have hindered de novo (biosynthesis) synthesis. de novo Heterogeneous biosynthesis of paclitaxel has traditionally been a semi-synthetic process. The inventors of this disclosure have achieved efficient heterogeneous production of the direct paclitaxel precursor N-debenzoyl-paclitaxel and its precursor 10-deacetyl-N-debenzoyl-paclitaxel, both containing an important hydroxyl group (OH group) at the C2' position of the C13 side chain. This hydroxylation is crucial for the anticancer activity of paclitaxel. Therefore, this invention enables the efficient heterogeneous production of taxanes containing a hydroxyl group at the C2' position of the C13 side chain, i.e., taxanes with a side chain containing a hydroxylated β-phenylalanine moiety.

[0007] Therefore, this invention enables the heterogeneous production of taxanes having side chains comprising a hydroxylated β-phenylalanine moiety, such as direct precursors of paclitaxel, such as N-debenzoyl-paclitaxel and / or 10-deacetyl-N-debenzoyl-paclitaxel. This allows for the efficient production of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, paclitaxel, and / or other taxanes, reducing both production costs and the environmental footprint of producing these bioactive compounds. Furthermore, heterogeneous production using microorganisms allows for a controlled production process and potentially reduces the purification requirements and associated resources and costs required to obtain safely administerable drugs.

[0008] In particular, this disclosure provides novel host cells, enzymes, methods, and uses for the biosynthesis of taxanes (e.g., direct precursors of paclitaxel, such as N-debenzoyl-paclitaxel and / or 10-deacetyl-N-debenzoyl-paclitaxel) having side chains containing hydroxylated β-phenylalanine moieties.

[0009] A key aspect of this disclosure is providing a host cell comprising a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity). The host cell of the present invention is preferably capable of producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably, said taxane comprising or composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel.

[0010] Another key aspect is providing a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, the method comprising the following steps: i. Provide the host cell described herein; ii. Culture the host cells in a culture medium. This produces the taxane having a side chain containing a hydroxylated β-phenylalanine moiety. Preferably, the taxane having a side chain containing a hydroxylated β-phenylalanine moiety is produced in the presence of baccatin III, 10-deacetyl-baccatin III, β-phenylalanine, β-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and / or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol. Production in the presence of the above compounds can be achieved through various methods. Baccatin III, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel, and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel can be present in a culture medium; for example, the compounds can be added to the culture medium. Alternatively, the host cells can produce baccatin III, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel, and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0011] Another aspect of this disclosure is the provision of materials from Chinese yew (Taxus chinensis) TaxusChinensis The use of an oxidase from, for example, Taxus chinensis (Chinese yew) in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, in a process whereby said oxidase is derived. T. Chinensis 2-oxoglutarate-dependent oxygenase or derived from Taxus chinensis (Chinese yew) T. Chinensis 2-oxoglutarate-dependent dioxygenase, preferably derived from Chinese yew (Taxus chinensis). T. Chinensis 2-Oxyglutaric acid-dependent dioxygenase of . In a preferred embodiment, the Chinese yew ( T. ChinensisThe oxidase is OD3 as described in SEQ ID NO: 3 or has at least 70% sequence identity with it (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%). The functional homologs of the following: at least 85% sequence identity, at least 86% sequence identity, at least 87% sequence identity, at least 88% sequence identity, at least 89% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity.

[0012] The terms “2-oxoglutarate-dependent oxygenase”, “2-oxoglutarate-dependent dioxygenase”, “Fe(II) and 2-oxoglutarate-dependent dioxygenase”, “Fe(II) and α-ketoglutarate-dependent dioxygenase”, “α-ketoglutarate-dependent dioxygenase” and “α-ketoglutarate-dependent oxygenase” are interchangeable.

[0013] Another aspect of this disclosure is to provide a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity, such as SEQ ID NO: 8 or a homolog thereof having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0014] Also provided are isolated polypeptides as described in SEQ ID NO: 3 or their functional homologs or variants having at least 70% sequence identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or higher).

[0015] In addition, a vector comprising one or more of the above-described nucleic acid constructs is provided, as well as a host cell comprising the nucleic acid constructs and / or the vector.

[0016] This document also provides a kit of parts comprising the host cells described herein, and / or the nucleic acid constructs described herein, and / or the vectors described herein, as well as optional host cells to be modified, and optionally includes instructions for use.

[0017] It also provides the use of nucleic acid constructs, vectors and / or host cells in the production of taxanes having side chains containing hydroxylated β-phenylalanine moieties.

[0018] This document also provides taxanes, N-debenzoyl-paclitaxel, and / or 10-deacetyl-N-debenzoyl-paclitaxel having side chains comprising a hydroxylated β-phenylalanine moiety, obtained by means of the methods described herein, host cells, and / or uses.

[0019] Cell cultures obtained by the method described herein and / or containing the host cells described herein are also provided.

[0020] In addition, a fermentation broth comprising a taxane having a side chain containing a hydroxylated β-phenylalanine moiety is provided, wherein the fermentation broth is obtained by the method described herein, contained in a cell culture, and / or contained in and / or secreted by the host cells described herein.

[0021] Compositions are also provided comprising the fermentation broth described herein, and / or taxanes, N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel having side chains comprising hydroxylated β-phenylalanine moieties obtained by the methods described herein.

[0022] Methods for treating diseases such as cancer are also provided, comprising administering a medicament comprising a composition obtained by the methods, host cells, and / or uses described herein, said composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety. Preferably, said taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel. Attached Figure Description

[0023] Figure 1.a) By expressing the expression of Penicillium chrysogenum ( Penicillium chrysogenum The coenzyme A ligase mutant (CoAL(A312G), encoded by SEQ ID NO: 7 and SEQ ID NO: 2), from Taxus chinensis (… Taxus cuspidata The aminophenylpropionyltransferase (BAPT, encoded by SEQ ID NO: 1, SEQ ID NO: 6) and the aminophenylpropionyltransferase (BAPT) from Taxus chinensis (Sinocyclohexane) Taxus chinensisa) Biosynthesis of N-debenzoyl-paclitaxel in tobacco by the oxidase (OD3, encoded by SEQ ID NO: 3, SEQ ID NO: 8). b) Transient expression of CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) in tobacco. Nicotiana benthamiana UPLC-HRMS chromatogram (EIC, positive ion mode) of the methanol extract of the leaf showed the production of N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C 40 H 47 NO 13 Tobacco leaf extracts expressing only CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6) were used as negative controls and showed no production of compound 3. Conversely, the substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C ) accumulated OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8). 40 H 47 NO 12 Following agro-infiltration, tobacco leaves were fed 200 mg / L β-phenylalanine and 200 mg / L baccatin III (compound 1). Chemically synthesized compounds (compounds 2 and 3) or commercially available compound 1 were used as standards. P19 (SEQ ID NO: 5, encoded by SEQ ID NO: 10) was co-expressed in all samples to help suppress gene silencing.

[0024] Figure 2.a) By expressing the expression of Penicillium chrysogenum ( Penicillium chrysogenum The A312G mutant of the coenzyme A ligase (CoAL) (CoAL(A312G), encoded by SEQ ID NO: 7 and SEQ ID NO: 2), and the fusion of MBP with Taxus chinensis (Malica macrophylla) Taxus cuspidata The aminophenylpropionyltransferase (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) and the aminophenylpropionyltransferase from Chinese yew ( Taxus chinensis The oxidase (OD3, encoded by SEQ ID NO: 3, SEQ ID NO: 8) in Saccharomyces cerevisiae (Saccharomyces cerevisiae) S. cerevisiaeN-Debenzoyl-paclitaxel was produced from baker's yeast. BAPT was fused at the N-terminus with maltose-binding protein (MBP) from Escherichia coli to increase its solubility. b) UPLC-HRMS chromatograms (EIC, positive ion mode) of ethyl acetate extracts expressing CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) showed the production of N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C 40 H 47 NO 13 Yeast culture extracts expressing only CoAL(A312G) and MBPig3BAPT were used as negative controls and showed no production of compound 3. Instead, the substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C40H47NO12) accumulated OD3. Chemically synthesized compounds (2 and 3) or commercially available compound 1 were used as standards.

[0025] Figure 3.a) By expressing the expression of Penicillium chrysogenum ( Penicillium chrysogenum The A312G mutant of the coenzyme A ligase (CoAL) (CoAL(A312G), encoded by SEQ ID NO: 7 and SEQ ID NO: 2), from Taxus chinensis ( Taxus cuspidata The aminophenylpropionyltransferase (BAPT, encoded by SEQ ID NO: 6, SEQ ID NO: 1) and the aminophenylpropionyltransferase from Chinese yew (Taxus chinensis) Taxus chinensis (a) The oxidase (OD3, encoded by SEQ ID NO: 3, SEQ ID NO: 8) of the α-hydroxyl group produces 10-deacetylated-N-debenzoyl-paclitaxel in tobacco. (b) Transient expression of CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) in tobacco. Nicotiana benthamiana UPLC-HRMS chromatogram (EIC, positive ion mode) of the methanol extract of the leaf showed the production of 10-deacetylated-N-debenzoyl-paclitaxel (compound 6, [M+H]+ 708.3015±0.01, C 38 H 45 NO 12Tobacco leaf extracts expressing only CoAL (A312G) and BAPT were used as a negative control and showed no production of compound 6. Instead, the substrate 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5, [M+H]+ 692.3065±0.01, C) accumulated OD3. 38 H 45 NO 11 After Agrobacterium infiltration, tobacco leaves were supplemented with 200 mg / L β-phenylalanine and 200 mg / L 10-deacetylated-baccatin III (compound 4). Chemically synthesized compounds (5 and 6) or commercially available compound 4 were used as standards. P19 (SEQ ID NO: 5, encoded by SEQ ID NO: 10) was co-expressed in all samples to help suppress gene silencing.

[0026] Figure 4.a) By expressing the expression of Taxus chinensis ( Taxus cuspidata DBAT of ) (SEQ ID NO: 21 encoded by SEQ ID NO: 23) produces N-debenzoyl-2'-deoxy-paclitaxel and N-debenzoyl-paclitaxel in tobacco. b) Transient expression of yew (Taxus media) Taxus cuspidata The DBAT of the tobacco (encoded by SEQ ID NO: 21, SEQ ID NO: 23) Nicotiana benthamiana UPLC-HRMS chromatogram (EIC, positive ion mode) of the methanol extract of the leaf showed that when the substrate 10-deacetylated-N-debenzoyl-paclitaxel (compound 6, [M+H]+ 708.3015±0.01, C 38 H 45 NO 12 ) and 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5, [M+H]+ 692.3065±0.01, C 38 H 45 NO 11 When N-debenzoyl-paclitaxel (compound 3, [M+H]+750.3120±0.01, C) was produced, N-debenzoyl-paclitaxel was produced. 40 H 47 NO 13 ) and N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C 40 H 47 NO 12 Tobacco plants that did not express DBAT were used as a negative control, and the substrate for the supplemental feed was 10-deacetylated-N-debenzoyl-paclitaxel (compound 6, [M+H]+ 708.3015±0.01, C 38H 45 NO 12 ) and 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5, [M+H]+ 692.3065±0.01, C 38 H 45 NO 11 It was not acetylated.

[0027] Figure 5. Escherichia coli (E. coli) E. coli The production of N-debenzoyl-paclitaxel ([M+H)) + 750.3120±0.01, C 40 H 47 NO 13 ).

[0028] a). By expressing a coenzyme A ligase mutant (CoAL(A312G), encoded by SEQ ID NO: 7, SEQ ID NO: 2), and fusing it with maltose-binding protein via a four-amino acid linker IG3 (SEQ ID NO: 28), a strain derived from Taxus chinensis (Malica macrophylla) Taxus cuspidata The aminophenylpropionyltransferase (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4), and those from the Chinese yew (Taxus chinensis) Taxus chinensis The oxidase (OD3, encoded by SEQ ID NO: 3) of Escherichia coli (E. coli) E. coli The biochemical pathway for the production of N-debenzoyl-paclitaxel (compound 3) from baccatin III (BACIII, compound 1) was described.

[0029] b). *E. coli* expressing different enzymes of the N-debenzoyl-paclitaxel biosynthesis pathway and supplied with 100 mg / L baccatin III (compound 1) and 100 mg / L β-phenylalanine. E. coli UPLC-HRMS chromatogram (EIC, positive ion mode) of ethyl acetate extract of cells.

[0030] Chromatogram I: Escherichia coli expressing CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and BAPT (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) fused with the maltose-binding protein MBP using the IG3 amino acid linker (SEQ ID NO: 28). E. coli Cell (strain ETL001) extract showed no production of N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C40 H 47 NO 13 Conversely, N-debenzoyl-2'-deoxy-paclitaxel (compound 2) accumulated.

[0031] Chromatogram II: Escherichia coli co-expressing CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) fused with maltose-binding protein MBP via an IG4 amino acid linker (SEQ ID NO: 28), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) E. coli Analysis of cells (strain ETL002) showed that N-debenzoyl-paclitaxel (compound 3) was produced and the OD3 substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2) was consumed.

[0032] Chromatogram III: Chemically synthesized compounds 2 and 3, or commercially available compound 1 and paclitaxel, were used as standards.

[0033] Figure 6. Escherichia coli (E. coli) E. coli ) produces 10-deacetylated-N-debenzoyl-paclitaxel ([M+H) + 708.3015±0.01, C 38 H 45 NO 12 ).

[0034] a. By expressing a coenzyme A ligase mutant (CoAL(A312G), encoded by SEQ ID NO: 7, SEQ ID NO: 2), and fusing it with maltose-binding protein via a four-amino acid linker IG3 (SEQ ID NO: 28), a strain derived from Taxus chinensis (Malica macrophylla) Taxus cuspidata The aminophenylpropionyltransferase (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) and the aminophenylpropionyltransferase from Chinese yew ( T. chinensis The oxidase (OD3, encoded by SEQ ID NO: 3) of Escherichia coli (E. coli) E. coli The biochemical pathway for producing 10-deacetyl-N-debenzoyl-paclitaxel (compound 6) from 10-deacetyl-baccatin III (10-DAB, compound 4) was described.

[0035] b) *E. coli* expressing different enzymes of the N-debenzoyl-paclitaxel biosynthesis pathway and supplied with 100 mg / L 10-deacetylated-baccatin III (compound 4) and 100 mg / L β-phenylalanine (…) E. coli UPLC-HRMS chromatogram (EIC, positive ion mode) of ethyl acetate extract of cells.

[0036] Chromatogram I: Escherichia coli expressing CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and BAPT (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) fused with the maltose-binding protein MBP using the IG3 amino acid linker (SEQ ID NO: 28). E. coli Cell (strain ETL001) extracts showed no production of 10-deacetylated-N-debenzoyl-paclitaxel (compound 6, [M+H]+ 708.3015±0.01, C38H45NO12). Instead, 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5) accumulated.

[0037] Chromatogram II: Escherichia coli co-expressing CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (MBPig3BAPT, encoded by SEQ ID NO: 9, SEQ ID NO: 4) fused with maltose-binding protein MBP via an IG3 amino acid linker (SEQ ID NO: 28), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) E. coli Analysis of cells (strain ETL002) showed that 10-deacetylated-N-debenzoyl-paclitaxel (compound 6) was produced and the OD3 substrate 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5) was digested.

[0038] Chromatogram III: Chemically synthesized compounds 5 and 6, or commercially available compounds 4 and paclitaxel, were used as standards.

[0039] Figure 7. OD3 variants at amino acid sites 169 and 195 retain their ability to synthesize N-debenzoyl-paclitaxel.

[0040] a) By expressing a coenzyme A ligase mutant (CoAL(A312G), encoded by SEQ ID NO: 7, SEQ ID NO: 2), derived from Taxus chinensis (… Taxus cuspidataThe aminophenylpropionyltransferase BAPT (encoded by SEQ ID NO: 1, SEQ ID NO: 6) and the aminophenylpropionyltransferase BAPT from Taxus chinensis (Sinopsis thunbergii) Taxus chinensis The biochemical pathway of the oxidase (OD3, encoded by SEQ ID NO: 3, SEQ ID NO: 8) of baccatin III (BACIII, compound 1) to produce N-debenzoyl-paclitaxel (compound 3) in tobacco.

[0041] b) UPLC-HRMS chromatogram (EIC, positive ion mode) of a methanol extract of tobacco leaves transiently expressing different enzymes of the N-debenzoyl-paclitaxel biosynthetic pathway and supplied with 200 mg / L baccatin III (compound 1) and 200 mg / L β-phenylalanine after Agrobacterium infiltration. P19 (SEQ ID NO: 5, encoded by SEQ ID NO: 10) was co-expressed in all samples to aid in suppressing gene silencing.

[0042] Chromatogram I: Tobacco leaf extracts expressing only CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6) were used as negative controls and showed no production of compound 3. Conversely, the substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C ) accumulated OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8). 40 H 47 NO 12 ).

[0043] Chromatogram II: The expression CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6), and those from the Chinese yew ( Taxus chinensis The OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) of tobacco leaf extract showed that compound 3 was produced and almost completely consumed the OD3 substrate compound 2.

[0044] Chromatograms III-IV : Expresses CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7), BAPT (SEQ ID NO: 1, encoded by SEQ ID NO: 6), and from Chinese yew ( Taxus chinensisTobacco leaf extracts of two OD3s (SEQ ID NO: 3) (namely, mutants (OD3(N195A), SEQ ID NO: 24 encoded by SEQ ID NO: 25, or OD3(Y169F), SEQ ID NO: 26 encoded by SEQ ID NO: 27) showed that compound 3 was produced and almost completely consumed the OD3 substrate compound 2.

[0045] Chromatogram V: Chemically synthesized compounds (compounds 2 and 3) or commercially available compound 1 were used as standards. Invention Details

[0047] definition

[0048] The term “as described in” is used herein as equivalent to “as listed in,” “as shown in,” “as depicted,” “identical to,” and / or “according to.” For example, “[..] OD3 as described in SEQ ID NO: 3” means OD3 having the sequence of SEQ ID NO: 3. Another example is “[..] paclitaxel as described in structure (VI),” which means in this document that structure (VI) is the structure of the compound paclitaxel. Thus, “as described in” indicates that something is “identical to” and should therefore be interpreted as “limited to.”

[0049] Regarding compounds, the term "in the presence of" herein means that the host cell can obtain the compound, either because the compound is provided to the host cell, and / or because the compound is synthesized by the host cell. For example, "host cells cultured in the presence of baccatin III" herein means that baccatin III is provided to the cells (e.g., supplied to or contained in the culture medium) or that the host cells are producing or are able to produce baccatin III.

[0050] As used in this article, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0051] The terms “incubation” and “culture” are used interchangeably herein and refer to maintaining a host cell under culture conditions that allow the host cell to grow. Preferably, the culture conditions allow the expression of an enzyme encoded by a heterologous gene contained in the host cell. Preferably, the host cell is incubated under culture conditions that allow the host cell to produce taxanes, N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel having a side chain containing a hydroxylated β-phenylalanine moiety. In embodiments where the host cell is contained in a multicellular organism (e.g., a plant), “culture” or “incubation” refers to maintaining the multicellular organism under conditions that allow the multicellular organism to grow. In embodiments where the host cell is a unicellular organism, “culture” or “incubation” refers to maintaining the unicellular organism under conditions that allow the unicellular organism to grow and / or reproduce.

[0052] As used herein, the term "enzyme" refers to a protein or polypeptide capable of catalyzing biochemical reactions. Furthermore, unless the context otherwise requires, "enzyme" as used herein includes protein fragments that retain the relevant catalytic activity, and may include synthetically produced artificial enzymes that retain the relevant catalytic activity.

[0053] The term "functional homolog" or "variant" for an amino acid sequence refers to a polypeptide containing the amino acid sequence, provided that one or more amino acids are substituted, deleted, added, and / or inserted, and that the polypeptide has the (qualitatively) identical enzymatic function for substrate transformation. The term "functional homolog" or "homolog" for a nucleic acid encoding a polypeptide refers to a nucleic acid containing the nucleic acid sequence, provided that one or more nucleobases are substituted, deleted, added, and / or inserted, and that the nucleic acid encodes a polypeptide having the (qualitatively) identical enzymatic function for substrate transformation to the polypeptide encoded by the nucleic acid. Nucleic acid or nucleic acid sequence may also be referred to as a polynucleotide. Preferably, the functional homolog or variant has at least 70% sequence identity with the amino acid sequence, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and more preferably at least 98%. Preferably, the functional homolog, homolog, or variant has at least 70% sequence identity with the amino acid sequence encoded by the nucleic acid sequence, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and more preferably at least 98%. Preferably, the functional homolog or homolog has at least 70% sequence identity with the nucleic acid sequence, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, and more preferably at least 98%.

[0054] The term "heterologous nucleic acid" refers to nucleic acid that has been inserted into a host cell or a host cell ancestor, for example, through recombination or transgenic methods. The corresponding protein or RNA encoded by the heterologous nucleic acid is also referred to as "heterologous." Heterologous nucleic acids can be part of non-integrating nucleic acids (e.g., vectors, including but not limited to plasmids). Preferably, the heterologous nucleic acid is integrated into the host cell genome.

[0055] The expressions “encoding…nucleic acid” and “encoding…nucleic acid sequence” are used interchangeably in this article.

[0056] The term "host cell" refers to a cell containing one or more heterologous nucleic acids.

[0057] As used herein, the term "peptide" refers to a continuous chain of amino acids linked together by peptide bonds. This term is used to refer to amino acid chains of any length. As those skilled in the art will appreciate, peptides can be processed and / or modified, and the term peptide can refer to an unmodified peptide or a modified peptide.

[0058] The term “sequence identity” as used in this article describes the correlation between two amino acid sequences or two nucleotide sequences, based on their paired candidate sequences (e.g., mutant sequences) and reference sequences (e.g., wild-type sequences). For the purposes of this invention, sequence identity between two amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology OpenSoftware Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The Needle output labeled “longest identity” (obtained using the -nobrief option) was used as the percentage identity, calculated as follows: (identical residues x 100) / (alignment length - total number of vacancies in the alignment) The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a non-reference sequence corresponds to a given position in a reference sequence.

[0059] For the purposes of this invention, sequence identity between two nucleotide sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.), implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or higher. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and a DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled “longest identity” (obtained using the -nobrief option) was used as the percentage identity, calculated as follows: (Identical deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment). Sequence identity is calculated over the full length of the reference sequence.

[0060] The term "taxane" in this document encompasses a class of diterpenoid compounds that contain the common core skeleton described in (I):

[0061] Furthermore, the core can be further substituted, preferably at positions 1, 2, 4, 5, 7, 9, 10, 11, 13, and / or 20. Regarding the structure described in (I), dashed lines indicate single or double bonds, provided that only one of the bonds between positions 11 and 12 or between positions 12 and 13 can be a double bond at any given time, and only one of the bonds between positions 4 and 5 or between positions 4 and 20 can be a double bond at any given time. The atom at position 20 can be carbon (C) or oxygen (O) and can be further substituted. In particular, atoms 4, 5, and 20 can connect to form a ring, preferably an oxetane ring. Taxoids are taxadiene-derived diterpenes. Preferred types of taxanes are taxoid compounds and / or taxanes. Taxanes have various structures and can be substituted with different groups (e.g., different functional groups). Non-limiting examples of taxanes of structure (I) are paclitaxel (or taxol, VI), N-debenzoyl-paclitaxel (II), N-debenzoyl-2'-deoxy-paclitaxel (III), 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel (IV), 10-deacetyl-N-debenzoyl-paclitaxel (V), and / or baccatin III. Unless otherwise stated, the atom numbering of taxanes used herein (e.g., taxanes having a side chain containing a hydroxylated β-phenylalanine moiety and / or taxanes having a side chain containing a β-phenylalanine moiety) is as shown in formula (I).

[0062] According to the above definition, the term "taxane having a side chain containing a β-phenylalanine moiety" herein refers to a taxane with structure (I) substituted with a side chain containing a β-phenylalanine moiety, preferably at position 13, i.e., C13. The β-phenylalanine moiety in this document refers to β-phenylalanine covalently bonded to another moiety. The structure of free β-phenylalanine is... Figure 1aProvided herein. The terms "side chain containing a β-phenylalanine moiety," "side chain containing β-phenylalanine," and "β-phenylalanine side chain" are used interchangeably herein. Furthermore, the terms "C13 side chain," "C13 side chain containing β-phenylalanine," and "C13 side chain containing a β-phenylalanine moiety" all refer to the side chain containing a β-phenylalanine moiety at C13 of the taxane in structure (I). Unless otherwise specified, the term "taxane side chain" is also used to refer to the C13 side chain containing a β-phenylalanine moiety. Accordingly, the term "taxane having a side chain containing a hydroxylated β-phenylalanine moiety" herein refers to the taxane of structure (I) substituted with a side chain containing a hydroxylated β-phenylalanine moiety, preferably at position 13, i.e., C13, and preferably at position 2' (i.e., C2' or 2'C) of the side chain in structure (I). Therefore, the terms "side chain containing a hydroxylated β-phenylalanine moiety," "side chain containing hydroxylated β-phenylalanine," "hydroxylated β-phenylalanine side chain," and / or "hydroxylated taxane side chain" all refer to a side chain containing a hydroxylated β-phenylalanine moiety, preferably located at C13 of the taxane in structure (I), and preferably, position 2' (i.e., C2' or 2'C) of the side chain is hydroxylated. Preferably, the side chain containing a hydroxylated β-phenylalanine moiety is a side chain containing a 2'C hydroxylated β-phenylalanine moiety or a side chain containing a 3'N-benzoyl-2'C hydroxylated β-phenylalanine moiety. Furthermore, the terms "hydroxylated C13 side chain," "C13 side chain containing hydroxylated β-phenylalanine," and / or "C13 side chain containing a hydroxylated β-phenylalanine moiety" all refer to a side chain containing a hydroxylated β-phenylalanine moiety located at C13 of the taxane in structure (I).

[0063] The term “N-debenzoyl-paclitaxel” refers to the chemical structure described in (II):

[0064] In structure (II), “Bz” represents benzoyl group and “Ac” represents acetyl group. N-debenzoyl-paclitaxel may also be referred to herein as compound 3 or (3), and these terms are used interchangeably.

[0065] The term “N-debenzoyl-2'-deoxy-paclitaxel” refers to the chemical structure described in (III):

[0066] In structure (III), “Bz” represents benzoyl group and “Ac” represents acetyl group. N-debenzoyl-2'-deoxy-paclitaxel may also be referred to herein as compound 2 or (2), and these terms are used interchangeably.

[0067] The term "10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel" refers to the chemical structure described in (IV):

[0068] In structure (IV), “Bz” represents benzoyl and “Ac” represents acetyl. 10-Deacetylated-N-debenzoyl-2'-deoxy-paclitaxel may also be referred to herein as compound 5 or (5), and these terms are used interchangeably.

[0069] The term "10-deacetylated-N-debenzoyl-paclitaxel" refers to the chemical structure described in (V):

[0070] In structure (V), “Bz” represents benzoyl group and “Ac” represents acetyl group. 10-Deacetylated-N-debenzoyl-paclitaxel may also be referred to herein as compound 6 or (6), and these terms are used interchangeably.

[0071] The term "paclitaxel" in this document refers to the chemical structure described in (VI):

[0072] In structure (VI), "Bz" represents benzoyl and "Ac" represents acetyl. Taxol is also known as paclitaxel, and these terms are used interchangeably.

[0073] Regarding host cells containing certain enzymes, the term "capable of producing" in this document means that, in the presence of a suitable / appropriate substrate / precursor, when the host cell expresses the desired enzyme as described herein, the host cell also converts the substrate / precursor into the intended product of the catalytic reaction.

[0074] Oxidase

[0075] This disclosure provides host cells, methods, uses, and polypeptides for the production of diterpenes (particularly taxanes having a side chain containing a hydroxylated β-phenylalanine moiety, such as N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel). The host cells used in this disclosure contain a heterologous nucleic acid encoding an enzyme capable of hydroxylating the C2' of the C13 side chain of taxanes (i.e., the so-called taxane side chain or the side chain containing the β-phenylalanine moiety).

[0076] The production of taxanes (e.g., N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel) having side chains comprising a hydroxylated β-phenylalanine moiety can be obtained by expressing an oxidase in a host cell of this disclosure. The oxidase can be expressed alone or together with one or more polypeptides with different activities described herein (e.g., in the “Host Cells” or “Polypeptides and Nucleic Acids” sections below). The oxidase preferably has the enzymatic activity described in this section.

[0077] The oxidase described herein catalyzes the formation of a hydroxyl group (OH) at position 2' of the side chain containing the β-phenylalanine moiety; that is, the oxidase is capable of hydroxylating the C13 side chain at position 2'. In other words, the oxidase is capable of catalyzing the formation of a taxane having a side chain containing a hydroxylated β-phenylalanine moiety from a taxane having a side chain containing the β-phenylalanine moiety.

[0078] OD3

[0079] The host cells used in this disclosure preferably contain a heterologous nucleic acid encoding an enzyme capable of catalyzing the formation of a taxane having a side chain containing a hydroxylated β-phenylalanine moiety. The present invention also provides such enzymes and methods of using the enzyme.

[0080] Preferably, the enzyme capable of catalyzing the formation of taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety is capable of catalyzing the formation of N-debenzoyl-paclitaxel as described in structure (II). In other words, preferably, the enzyme is capable of catalyzing the following reaction (A):

[0081] In other words, an oxidase capable of catalyzing the 2' hydroxylation of the C13 side chain of taxane can catalyze reaction A above. This reaction can also be referred to as 2' α-hydroxylation.

[0082] N-Debenzoyl-2'-deoxy-paclitaxel can also be called 3'N-dehydroxydebenzoyltaxel, β-phenylalanoyl baccatin III, and / or 13-O-β-phenylalanoyl baccatin III, and these names are used interchangeably.

[0083] In another preferred embodiment, an enzyme capable of catalyzing the formation of taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety is capable of catalyzing the formation of 10-deacetylated-N-debenzoyl-paclitaxel as described in structure (V). In other words, preferably, the enzyme is capable of catalyzing the following reaction (B):

[0084] In other words, an oxidase capable of catalyzing the 2' hydroxylation of the C13 side chain of taxane can catalyze the above reaction B.

[0085] In a preferred embodiment, the enzyme capable of catalyzing the formation of taxanes having side chains containing hydroxylated β-phenylalanine moieties is capable of catalyzing reactions A and B.

[0086] Therefore, a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety can be N-debenzoyl-paclitaxel as described in structure (II) and / or referred to herein as compound 3. In other embodiments, a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety can be 10-deacetyl-N-debenzoyl-paclitaxel as described in structure (V) and / or referred to herein as compound 6. In a further embodiment, a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety can be paclitaxel as described in structure (VI).

[0087] The enzyme capable of catalyzing the formation of taxanes having a side chain containing a hydroxylated β-phenylalanine moiety can be any useful enzyme with the above-described activity, and in particular, the enzyme can be an oxidase. In some embodiments, the enzyme is a 2-oxoglutarate-dependent dioxygenase. The enzyme capable of catalyzing the formation of taxanes having a side chain containing a hydroxylated β-phenylalanine moiety can be derived from any suitable source, but in a preferred embodiment, the enzyme is derived from Taxus chinensis (Chinese yew). Taxus chinensis Therefore, an enzyme capable of catalyzing the formation of taxanes with side chains containing hydroxylated β-phenylalanine moieties could be derived from the Chinese yew (Taxus chinensis). Taxus chinensis Oxidases derived from the Chinese yew (Taxus chinensis). T. chinensis 2-Oxyglutaric acid-dependent dioxygenase.

[0088] In a preferred embodiment of this disclosure, the host cell contains a heterologous nucleic acid encoding the oxidase OD3. The OD3 is preferably the OD3 of SEQ ID NO: 3 or a functional homolog thereof. Those skilled in the art will understand that the OD3 or its functional homolog preferably has the ability to convert N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-paclitaxel and 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel to 10-deacetyl-N-debenzoyl-paclitaxel, as shown in reactions A and B above herein, respectively.

[0089] The functional homolog of OD3 of SEQ ID NO: 3 preferably has at least 70% sequence identity, for example, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, preferably at least 80%, at least 81%, at least 82%, at least 83%, or at least 84%. Sequence identity, preferably at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, preferably at least 95%, at least 96%, at least 97%, more preferably at least 98%, and most preferably at least 99%.

[0090] Functional homologs of OD3 can be identified by expressing the homologs in host cells and purifying the resulting enzymes for in vitro enzyme activity assays, or by performing in vivo enzyme activity assays, to measure the conversion of N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-paclitaxel and / or the conversion of 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel to 10-deacetyl-N-debenzoyl-paclitaxel using standard techniques. For example, activity can be measured as described in Examples 1 to 4. Non-limiting examples of functional homologs of OD3 of SEQ ID NO: 3 are OD3 (Y169F) as described in SEQ ID NO: 26 and OD3 (N195A) as described in SEQ ID NO: 24.

[0091] The heteronucleotide encoding OD3 or a functional homolog thereof of SEQ ID NO: 3 may have any sequence encoding said OD3. In some embodiments, the nucleic acid encoding OD3 is the nucleic acid as described in SEQ ID NO: 8, or a functional homolog thereof encoding an OD3 functional homolog having at least 70% sequence identity with the OD3 encoded by SEQ ID NO: 8.

[0092] host cells

[0093] This disclosure relates to a host cell containing a heterologous nucleic acid that encodes one or more enzymes encoding a paclitaxel biosynthetic pathway (i.e., a paclitaxel-like pathway).

[0094] A key aspect of this disclosure is providing a host cell comprising a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or having at least 70% sequence identity with SEQ ID NO: 3 (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0095] Therefore, in a preferred embodiment, the host cell contains a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog of SEQ ID NO: 3 having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0096] A heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity is preferably “a heterologous nucleic acid encoding a protein comprising SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity, or a protein composed of SEQ ID NO: 3 or the functional homolog thereof.” Similarly, a nucleic acid sequence encoding an enzyme or a functional homolog thereof disclosed herein is preferably a nucleic acid encoding a protein comprising the sequence or a functional homolog thereof, or a nucleic acid encoding a protein composed of the sequence or a functional homolog thereof.

[0097] In another preferred embodiment, the host cell comprises a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity, wherein the host cell is capable of producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably wherein the taxane comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel.

[0098] In other embodiments, the host cell comprises a heterologous nucleic acid sequence encoding OD3 (N195A) as described in SEQ ID NO: 24 or having at least 70% sequence identity with SEQ ID NO: 24 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 99%). In some embodiments, the host cell comprises a heterologous nucleic acid sequence encoding OD3 (Y169F) as described in SEQ ID NO: 26 or having at least 70% sequence identity with SEQ ID NO: 26 (e.g., at least 75%, at least 80%, at least 90%, at least 95%, at least 99%).

[0099] In other embodiments, the host cell comprises a heterologous nucleic acid sequence encoding OD3(N195A) (SEQ ID NO: 24) or OD3(Y169F) (SEQ ID NO: 26), or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 24 or SEQ ID NO: 26, respectively, wherein the host cell is capable of producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably wherein the taxane comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel.

[0100] In some embodiments, host cells can produce taxanes having side chains comprising a hydroxylated β-phenylalanine moiety in the presence of baccatin III, 10-deacetylated-baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel, and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel. The precursor compounds can be provided to host cells, for example, in a culture medium, or, in the case of plant cells, by infiltration into leaves. In other embodiments, the host cell is capable of producing baccatin III, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel, and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel. Host cells capable of producing these compounds are described below.

[0101] Therefore, the reaction products catalyzed by OD3 are determined to some extent by the available substrates / precursors. For example, in the presence of 2'-deoxy-N-debenzoyl-paclitaxel, host cells containing a heterologous nucleic acid sequence encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof with at least 70% sequence identity can produce N-debenzoyl-paclitaxel. In the presence of 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel, host cells containing a heterologous nucleic acid sequence encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof with at least 70% sequence identity can produce 10-deacetyl-N-debenzoyl-paclitaxel. The resulting N-debenzoyl-paclitaxel or 10-deacetyl-N-debenzoyl-paclitaxel can be converted into paclitaxel and 10-deacetylpaclitaxel, respectively, either enzymatically or chemically after purifying the precursors of these compounds (e.g., N-debenzoyl-paclitaxel or 10-deacetyl-N-debenzoyl-paclitaxel). Similarly, the substrates 2'-deoxy-N-debenzoyl-paclitaxel and 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel can be produced by host cells via enzymatic catalysis or provided to host cells, for example, in a culture medium.

[0102] Therefore, in addition to heterologous nucleic acids encoding OD3 (SEQ ID NO: 3) or its functional homologs, host cells may also contain nucleic acids encoding one or more of the following enzymes: - Aminophenylpropionyltransferase (EC: 2.3.1) can convert baccatin III and β-phenylalanyl-CoA to N-debenzoyl-2'-deoxy-paclitaxel, and 10-deacetyl-baccatin III and β-phenylalanyl-CoA to 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel; - Coenzyme A ligase (CoAL, EC: 6.2.1) can convert β-phenylalanine to β-phenylalanyl-CoA; -10-Deacetylated-baccatin III-10-O-acetyltransferase (DBAT, EC: 2.3.1) is capable of converting 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-2'-deoxy-paclitaxel, and / or converting 10-deacetylated-N-debenzoyl-paclitaxel to N-debenzoyl-paclitaxel, and / or converting 10-deacetylated-baccatin III to baccatin III; and / or - 3'-N-debenzoyl-2'-deoxypaclitaxel-N-benzoyltransferase (DBTNBT, EC: 2.3.1), as described in the "DBTNBT" section of this article.

[0103] These enzyme activities and the host cells containing said activities will be described further below.

[0104] The preferred host cells are capable of producing GGPP, baccatin III, 10-deacetylated baccatin III, and / or β-phenylalanine.

[0105] In some embodiments, the host cell may further comprise one or more heterologous nucleic acids as described in patent application EP23386045.1 or a patent application claiming priority thereto (e.g., PCT / EP2024 / 065772, published as WO2024 / 251968).

[0106] organism

[0107] In some embodiments of this disclosure, the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells, and fungal cells.

[0108] In some embodiments, the host cell is contained within a multicellular organism. In such embodiments, only a portion of the cells of the multicellular organism may contain heterologous nucleic acids and / or heterologous polypeptides. However, it is preferable that all cells of the multicellular organism are host cells containing the same heterologous nucleic acids and / or heterologous polypeptides.

[0109] In some embodiments of this disclosure, the host cell is a plant cell, such as a plant cell contained in a plant, in a part of a plant, and / or in the seed of the plant. Preferably, all cells of the plant or a part thereof are host cells containing the same heterologous nucleic acids and / or polypeptides.

[0110] In some embodiments of this disclosure, the host cell is a plant cell, such as one derived from the genus *Nicotiana*. Nicotiana Plant cells of species, such as tobacco Benzoinus ( ) Nicotiana benthamiana ) or safflower tobacco ( Nicotiana tabacum Those skilled in the art will understand that, as used in this invention, "plant cell" refers to the structural and physiological unit of a plant, such as a tobacco plant. A plant cell may be in the form of a cell wall-less protoplast, an isolated single cell, or a cultured cell, or as part of a higher tissue unit, such as, but not limited to, plant tissue, plant organ, or the whole plant.

[0111] In some embodiments of this disclosure, the host cell is a yeast cell, for example, belonging to the genus *Saccharomyces* (Yeast). Saccharomyces ), Pichia pastoris ( Pichia ), Candida genus ( Candida Cryptococcus ( Cryptococcus Pichia pastoris (Pichia pastoris) genus Pichia (Komagataella) )), genus *Olecosomyces* ( Lipomyces ), *Pseudomycium* ( Pseudozyma ), genus *Rhodotorula* ( Rhodosporidium ), Rhodotorula genus ( Rhodotorula ), genus *Mycosaccharomyces* ( Trichosporon ), Trichosaccharomyces ( Trigonopsis ), Yersinia ( Yarrowia ) or *Cryptotympany* genus ( Saccharomycopsis Yeast cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha (Hansenula polymorpha) (Ogaria polymorpha) Hansenula polymorpha (Ogataea polymorpha ), Rhodotorula buergerianum ( Rhodotorula toruloides ) or Pichia pastoris (Pichia pastoris) ( Pichia pastor i s( Komagataella phaffii Yeast cells of type ))

[0112] Those skilled in the art will understand that the fungi or fungal cells used herein refer to any cell present in or derived from an organism belonging to the kingdom Fungi. These methods are applicable to all genetically modified fungi and fungal cells. Furthermore, those skilled in the art will understand that “yeast cell” herein is defined as comprising the group of small, single-celled organisms capable of growth and reproduction by budding or direct division (fission) or by growth into simple, irregular filaments (mycelium). Yeast cells can be transformed or transfected with a heterologous vector to express nucleic acid sequences inserted into the heterologous vector. Examples of yeast cells include, but are not limited to, *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha (Hansenula polymorpha) (Ogaria polymorpha) Hansenula polymorpha (Ogataea polymorpha ), Rhodotorula buergerianum ( Rhodotorula toruloides ) and / or Pichia pastoris (Pichia pastoris) ( Pichia pastoris(Komagataella phaffii These are typically used for the transfection and expression of heterologous proteins.

[0113] In some embodiments of this disclosure, the host cell is a bacterial cell, such as one belonging to the genus Escherichia (Escherichia). Escherichia ), Bacillus spp. Bacillus Corynebacterium spp. Corynebacterium ), Pseudomonas spp. Pseudomonas ) or Streptomyces ( Streptomyces Bacterial cells, such as Escherichia coli (E. coli) Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Pseudomonas putida ( Pseudomonas putida ) or Streptomyces ( Streptomyces sp .) bacterial cells.

[0114] Those skilled in the art will understand that bacterial cells include prokaryotic cells that can proliferate in cultures. Bacterial cells can serve as host cells for the recombinant expression of heterologous polypeptides. Bacterial cells can be transformed, transfected, or infected with vectors to express nucleic acid sequences inserted into the vectors. Examples of suitable bacterial cells include, but are not limited to, *Escherichia coli*. E. coli Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Pseudomonas putida ( Pseudomonas putida ) and / or Streptomyces ( Streptomyces sp. ).

[0115] The host cell or its ancestor can be prepared by any useful method available to those skilled in the art. For example, heterologous nucleic acids can be inserted into the cell by direct uptake, transduction, f-mating, transfection, transformation, bacterial infiltration, or any other method known in the art for creating recombinant host cells.

[0116] In a preferred embodiment, the host cell is a yeast cell (e.g., Saccharomyces cerevisiae). S. cerevisiae (yeast cells) or bacterial cells (e.g., Escherichia coli) E. coli (bacterial cells of a certain species).

[0117] aminophenylpropionyltransferase

[0118] Host cells containing heterologous nucleic acids encoding OD3 (SEQ ID NO: 3) or its functional homologs may further contain nucleic acids encoding aminophenylpropionyltransferases as described in this section and elsewhere.

[0119] The aminophenylpropionyltransferase may be an aminophenylpropionyltransferase belonging to EC no. 2.3.1. The aminophenylpropionyltransferase is capable of converting baccatin III and β-phenylalanyl-CoA to N-debenzoyl-2'-deoxy-paclitaxel. The aminophenylpropionyltransferase is also capable of converting 10-deacetyl-baccatin III and β-phenylalanyl-CoA to 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel. Therefore, a host cell containing nucleic acid encoding the aminophenylpropionyltransferase can produce N-debenzoyl-2'-deoxy-paclitaxel and / or 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0120] In some embodiments, the aminophenylpropionyltransferase is yew (Taxus chinensis) Taxus ) cells (e.g., Taxus chinensis) Taxus cuspidata (Cellular) Natural. In other embodiments, the aminophenylpropionyltransferase is BAPT as described in SEQ ID NO: 1 or MBPig3BAPT as described in SEQ ID NO: 4, or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, respectively.

[0121] The nucleic acid encoding BAPT (SEQ ID NO: 1) or its functional homolog can be the nucleic acid described in SEQ ID NO: 6 or a homolog having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity with it. The nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or its functional homolog can be the nucleic acid described in SEQ ID NO: 9 or a homolog having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity with it.

[0122] In some embodiments, the host cell comprises a heteronucleotide encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively. In the presence of β-phenylalanyl-CoA and baccatin III, the host cell is capable of producing N-debenzoyl-paclitaxel from β-phenylalanyl-CoA and baccatin III. If 10-deacetylated baccatin III is present instead of baccatin III, the host cell is capable of producing 10-deacetylated-N-debenzoyl-paclitaxel from β-phenylalanyl-CoA and 10-deacetylated baccatin III. Preferably, the host cell is capable of producing β-phenylalanyl-CoA, baccatin III, and / or 10-deacetylated baccatin III, but the compounds may also be supplied to the host cell, for example, in the culture medium in which the host cell is cultured.

[0123] CoAL

[0124] Host cells containing heterologous nucleic acids encoding OD3 (SEQ ID NO: 3) or its functional homologs may further contain nucleic acids encoding coenzyme A ligases (CoAL) as described herein and elsewhere. Preferably, the host cells further contain nucleic acids encoding aminophenylpropionyltransferases as described above herein.

[0125] The CoAL can be CoAL belonging to EC no. 6.2.1. CoAL can convert β-phenylalanine to β-phenylalanyl-CoA. Therefore, a host cell containing nucleic acid encoding the CoAL can produce β-phenylalanyl-CoA.

[0126] In some implementations, the CoAL is a Penicillium species ( Penicillium) cells (e.g., Penicillium chrysogenum) P. chrysogenum ) cells) natural CoAL or Taxus genus ( Taxus ) cells (e.g., Chinese yew) T. chinensis (Cellular) native CoAL. In other embodiments, the CoAL is CoAL (A312G) as described in SEQ ID NO: 2 or TchiAAE5 as described in SEQ ID NO: 22, or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 22, respectively. In other embodiments, the CoAL is AAE-867.5 as described in SEQ ID NO: 29 or a functional homolog thereof having at least 70% sequence identity with it.

[0127] This document also discloses a host cell comprising a heterologous nucleic acid sequence encoding TchiAAE5 as described in SEQ ID NO: 22 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity). In some embodiments, the host cell comprises a heterologous nucleic acid sequence encoding TchiAAE5 as described in SEQ ID NO: 22 or a functional homolog thereof having at least 70% sequence identity, wherein the host cell is capable of producing β-phenylalanine-CoA. Preferably, the host cell is capable of converting β-phenylalanine to β-phenylalanine-CoA.

[0128] In some implementations, the CoAL is the CoAL described in GenBank accession number XP_002569052.1 or its functional homologs having at least 70% sequence identity.

[0129] CoAL(A312G) may sometimes also be referred to as CoAL_A312G and / or CoALA312G, and these terms are used interchangeably.

[0130] The nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof may be the nucleic acid described in SEQ ID NO: 7 or a homolog thereof having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity. The nucleic acid encoding TchiAAE5 (SEQ ID NO: 22) or a functional homolog thereof may be the nucleic acid encoding the polypeptide described in SEQ ID NO: 22 or a functional homolog thereof having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity.

[0131] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), and further contains a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) and a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1), or a functional homolog of either of the above with at least 70% sequence identity. In other embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), and further contains a nucleic acid encoding TchiAAE5 (SEQ ID NO: 22) and a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1), or a functional homolog of either of the above with at least 70% sequence identity. In other embodiments, the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), and further comprises a nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) and a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1), or a functional homolog of either of the above with at least 70% sequence identity. In the presence of β-phenylalanine and baccatin III, the host cell is capable of producing N-debenzoyl-paclitaxel from β-phenylalanine and baccatin III. If 10-deacetylated baccatin III is present instead of baccatin III, the host cell is capable of producing 10-deacetylated-N-debenzoyl-paclitaxel from β-phenylalanine and 10-deacetylated baccatin III. Preferably, the host cell is capable of producing β-phenylalanine, baccatin III, and / or 10-deacetylated baccatin III, but the compounds may also be supplied to the host cell, for example, in the culture medium in which the host cell is cultured.

[0132] DBAT

[0133] Host cells containing heterologous nucleic acids encoding OD3 (SEQ ID NO: 3) or its functional homologs may further contain nucleic acids encoding 10-deacetylated baccatin III-10-O-acetyltransferase (DBAT) as described in this section and elsewhere herein. Preferably, the host cells further contain nucleic acids encoding CoAL and / or aminophenylpropionyltransferases as described above herein.

[0134] The DBAT may be a DBAT belonging to EC no. 2.3.1. The DBAT may acetylate 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-2'-deoxy-paclitaxel. The DBAT may also acetylate 10-deacetyl-N-debenzoyl-paclitaxel to N-debenzoyl-paclitaxel. The DBAT may also acetylate 10-deacetyl-baccatin III to baccatin III. The DBAT may also acetylate 10-deacetyl-paclitaxel to paclitaxel. Therefore, host cells containing nucleic acids encoding the DBAT can produce baccatin III, paclitaxel, N-debenzoyl-2'-deoxy-paclitaxel, and / or N-debenzoyl-paclitaxel in the presence of 10-deacetylated baccatin III, paclitaxel, 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel, and / or 10-deacetylated-N-debenzoyl-paclitaxel, respectively. The DBAT may be... Tcu DBAT (SEQ ID NO: 21) or its functional homologs having at least 70% sequence identity with SEQ ID NO: 21.

[0135] In some implementations, the DBAT is yew ( Taxus ) cells (e.g., Taxus chinensis) Taxus cuspidata (Cellular) Natural. In other embodiments, DBAT is as described in SEQ ID NO: 21. Tcu DBAT or its functional homologs that have at least 70% sequence identity.

[0136] Tcu The activities of DBAT in the production of baccatin III from 10-deacetylated baccatin III and in the production of paclitaxel from 10-deacetylated paclitaxel have been described elsewhere (Li et al., 2016 and Walker et al., 2000).

[0137] The nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or its functional homologs may be the nucleic acid described in SEQ ID NO: 23 or a homolog having at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity with it.

[0138] For example, host cells containing nucleic acids encoding DBAT (e.g., TcuDBAT (SEQ ID NO: 21) or its functional homologs having at least 70% sequence identity) can produce N-debenzoyl-paclitaxel in the presence of 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel.

[0139] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), and further contains a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) and a nucleic acid encoding TcuDBAT (SEQ ID NO: 21), or a functional homolog of either of the above with at least 70% sequence identity. In the presence of β-phenylalanyl-CoA and 10-deacetylated-baccatin III, the host cell is capable of producing N-debenzoyl-paclitaxel from β-phenylalanyl-CoA and 10-deacetylated-baccatin III. Preferably, the host cell is capable of producing β-phenylalanyl-CoA and / or 10-deacetylated-baccatin III, but the compounds may also be supplied to the host cell, for example, in the culture medium in which the host cell is cultured.

[0140] In other embodiments, the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. Encoding Tcu DBAT (SEQ ID NO: 21) or a nucleic acid of its functional homolog that has at least 70% sequence identity with it.

[0141] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding TchiAAE5 (SEQ ID NO: 22) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. Nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0142] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. Nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0143] In the presence of β-phenylalanine and 10-deacetylated baccatin III, the host cells are capable of producing N-debenzoyl-paclitaxel from β-phenylalanine and 10-deacetylated baccatin III. Preferably, the host cells are capable of producing β-phenylalanine and / or 10-deacetylated baccatin III, but the compounds may also be supplied to the host cells, for example, in the culture medium in which the host cells are cultured.

[0144] DBTNBT

[0145] Host cells containing heterologous nucleic acids encoding OD3 (SEQ ID NO: 3) or its functional homologs may further contain nucleic acids encoding 3'-N-debenzoyl-2'-deoxypaclitaxel-N-benzoyltransferase (DBTNBT) as described in this section and elsewhere. Preferably, the host cells further contain nucleic acids encoding DBAT, CoAL, and / or aminophenylpropionyltransferase, or nucleic acids encoding CoAL and / or aminophenylpropionyltransferase, as described above.

[0146] The DBTNBT may be DBTNBT belonging to EC no. 2.3.1. DBTNBT is capable of converting N-debenzoyl-paclitaxel to paclitaxel and / or N-debenzoyl-2'-deoxy-paclitaxel to 2'-deoxy-paclitaxel. In other words, DBTNBT is capable of benzoyl substitution at the 3' position of the side chain containing the β-phenylalanine moiety or at the amino group attached to the 3' position of the side chain containing the hydroxylated β-phenylalanine moiety. Therefore, host cells containing nucleic acids encoding the DBTNBT can be able to produce paclitaxel. When benzoylating its substrates (e.g., N-debenzoyl-paclitaxel and N-debenzoyl-2'-deoxy-paclitaxel), DBTNBT preferably uses benzoyl-CoA as a co-substrate. The benzoyl-CoA may be contained in the culture medium or produced by the host cells.

[0147] In some implementations, the DBTNBT is yew ( Taxus ) cells (e.g., Canada yew) Taxus canadensis (Cellular) Natural. In other embodiments, the DBTNBT is TcaDBTNBT as described in SEQ ID NO: 20 or a functional homolog thereof having at least 70% sequence identity.

[0148] The activity of TcaDBTNBT has been described elsewhere (Walker, K. et al, 2002, Long, RM. et al, 2009).

[0149] N-Debenzoyl-paclitaxel can also be known as 3'-N-debenzoylpaclitaxel, N-debenzoylpaclitaxel, N-debenzoylpaclitaxel A, 3'-N-debenzoylpaclitaxel, N-debenzoylpaclitaxel, 3'-N-debenzoylpaclitaxel, 3'-debenzoylpaclitaxel, and Baccatin III 13-[β(S)-amino-α(R)-hydroxyphenylpropionate] (Baccatin III) 13-[β(S)-amino-α(R)-hydroxyphenylpropionate] and 13-O-3-phenylisoserinoyl baccatin III, and these names are used interchangeably.

[0150] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1); and ii. Encoding DBTNBT (EC: 2.3.1), optionally a nucleic acid of TcaDBTNBT as described in SEQ ID NO: 20; Or functional homologs of any of the above, whose sequence identity is at least 70%.

[0151] In other embodiments, the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2); ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1); and iii. Nucleic acids encoding DBTNBT, such as TcaDBTNBT (SEQ ID NO: 20); Or functional homologs of any of the above, whose sequence identity is at least 70%.

[0152] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding TchiAAE5 (SEQ ID NO: 22); ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1); and iii. Nucleic acids encoding DBTNBT, such as TcaDBTNBT (SEQ ID NO: 20); Or functional homologs of any of the above, whose sequence identity is at least 70%.

[0153] In some embodiments, the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding AAE-867.5 (SEQ ID NO: 29); ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1); and iii. Nucleic acids encoding DBTNBT, such as TcaDBTNBT (SEQ ID NO: 20); Or functional homologs of any of the above, whose sequence identity is at least 70%.

[0154] In the presence of β-phenylalanine and baccatin III, the host cells are capable of producing paclitaxel from β-phenylalanine and baccatin III. Preferably, the host cells are capable of producing β-phenylalanine and baccatin III, but the compounds can also be supplied to the host cells, for example, in the culture medium in which the host cells are cultured.

[0155] If the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) and further contains nucleic acids encoding TchiAAE5 (SEQ ID NO: 22), AAE-867.5 (SEQ ID NO: 29), or CoAL(A312G) (SEQ ID NO: 2), nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1), and nucleic acids encoding DBTNBT (e.g., TcaDBTNBT (SEQ ID NO: 20)), it also contains nucleic acids encoding... TcuThe host cell is capable of producing paclitaxel in the presence of 10-deacetylated-baccatin III and β-phenylalanine, and is a functional homolog of the nucleic acid of DBAT (SEQ ID NO: 21) or any of the above-mentioned sequences having at least 70% sequence identity.

[0156] From the beginning ( de novo Enzyme activity for producing baccatin III or 10-deacetylated baccatin III

[0157] The host cell may also contain nucleic acids encoding enzyme activities for the production of other paclitaxel compounds, such as baccatin III from the common diterpenoid precursor catechol diphosphate (GGPP), in order to obtain the complete biosynthesis of paclitaxel.

[0158] In other words, the host cell can contain enzymatic activity capable of de novo production of the taxane having a side chain containing a hydroxylated β-phenylalanine moiety from GGPP. The enzymatic activity required for the complete biosynthesis of baccatin III from GGPP may include a terpene synthase (e.g., taxadiene synthase), two acetyltransferases, a benzoyltransferase, a cyclooxygenase, and seven cytochrome P450s. The enzymatic activity required for the complete biosynthesis of 10-deacetylated-baccatin III from GGPP may include a terpene synthase (e.g., taxadiene synthase), acetyltransferases, benzoyltransferases, cyclooxygenases, and seven cytochrome P450s.

[0159] Specifically, it can be the enzyme described in Zhang, YJ. et al, 2023 and / or McClure, CJ. et al, 2024.

[0160] method

[0161] In another key aspect, this disclosure relates to a method for preparing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety. The method of the invention generally comprises the following steps: i. Provide the host cell described herein; ii. Culture the host cells in a culture medium. This produces the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0162] A method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety and / or a fermentation broth comprising said taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is also provided, the method comprising the following steps: i. Provide the host cell described herein; ii. The host cells are cultured in a culture medium to obtain a fermentation broth containing the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety; and iii. Optionally, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is recovered. This produces a fermentation broth containing the taxane having a side chain containing a hydroxylated β-phenylalanine moiety and / or the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0163] A method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety and / or a fermentation broth comprising said taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is also provided, the method comprising the following steps: i. Provide the host cell described herein; ii. Incubating and optionally proliferating the host cells in a culture medium to obtain a fermentation broth containing the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety; and iii. Optionally, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is recovered. This produces a fermentation broth containing the taxane having a side chain containing a hydroxylated β-phenylalanine moiety and / or the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0164] The host cell can be any host cell described herein, such as the cells described in the “Host Cell” section above.

[0165] Therefore, in some embodiments, the method is a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, the method comprising the following steps: i. Provide a host cell containing a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog of SEQ ID NO: 3 having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity); ii. Culture the host cells in a culture medium. This produces the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0166] As described above, non-limiting examples of functional homologs of OD3 of SEQ ID NO: 3 are OD3 (Y169F) of SEQ ID NO: 26 and OD3 (N195A) of SEQ ID NO: 24, and OD3 of SEQ ID NO: 3 can be replaced with these functional homologs in the methods described herein, particularly in the production of N-debenzoyl-paclitaxel or 10-deacetyl-N-debenzoyl-paclitaxel.

[0167] In a preferred embodiment, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel.

[0168] Therefore, the method can be a method for producing N-debenzoyl-paclitaxel, the method comprising the following steps: i. Provide a host cell containing a heterologous nucleic acid sequence encoding OD3 (SEQ ID NO: 3) or a functional homology thereof having at least 70% sequence identity with SEQ ID NO: 3; ii. The host cells are cultured and optionally proliferated in a culture medium; This leads to the production of N-desbenzoyl-paclitaxel. OD3 or its functional homologs can convert N-desbenzoyl-2'-deoxy-paclitaxel to N-desbenzoyl-paclitaxel. The culture medium may contain N-desbenzoyl-2'-deoxy-paclitaxel, but preferably the host cells are capable of producing N-desbenzoyl-2'-deoxy-paclitaxel.

[0169] The method may be a method for producing 10-deacetyl-N-debenzoyl-paclitaxel, the method comprising the following steps: i. Provide a host cell containing a heterologous nucleic acid sequence encoding OD3 (SEQ ID NO: 3) or a functional homology thereof having at least 70% sequence identity with SEQ ID NO: 3; ii. The host cells are cultured and optionally proliferated in a culture medium; This produces 10-deacetyl-N-debenzoyl-paclitaxel. OD3 or its functional homologs can convert 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel to 10-deacetyl-N-debenzoyl-paclitaxel. The culture medium may contain 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel, but preferably the host cells are capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0170] nourish

[0171] The steps of culturing and / or incubating host cells in a culture medium may be performed by any method known to those skilled in the art, under conditions that allow for growth. The term “growth” of host cells or multicellular organisms containing host cells should be understood to mean the proliferation, multiplication, differentiation, and / or maintenance of vitality of the tested host cells or multicellular organisms.

[0172] If the host cell is contained within a multicellular organism, the conditions are generally those that enable the multicellular organism to maintain its viability and / or growth. Therefore, if the host cell is a plant cell contained within a plant, the culture conditions are those suitable for the maintenance and / or growth of said plant. For example, this could be sowing seeds or other regenerated parts of said plant in a field or greenhouse. Culture may further include watering and / or fertilization.

[0173] If the host cell is a single-celled organism (e.g., a bacterial or yeast cell), cultivation can be carried out in a medium (e.g., a culture medium) containing at least a carbon source and a nitrogen source at a temperature suitable for the growth of the single-celled organism. The carbon source can be, for example, a carbohydrate, such as a sugar or polysaccharide. The nitrogen source can be, for example, an amino acid or a polypeptide. Those skilled in the art are fully capable of selecting a suitable culture medium based on the specific host cell.

[0174] Cultivation conditions

[0175] In some embodiments, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is produced in the presence of baccatin III, benzoyl-CoA, 10-deacetylated-baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel.

[0176] The host cells cultured and / or incubated in the presence of baccatin III, benzoyl-CoA, 10-deacetylated-baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel, and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel can be obtained in a variety of ways. For example, the compounds can be added to the host cells. If the host cells are microorganisms, the compounds can be added to the culture medium of the microorganisms. If the host organism (e.g., the host cells) is a plant, the compounds can be added to the soil of the plant, or introduced into the plant by infiltration. Thus, if a heterologous nucleic acid is introduced into a plant by infiltration, the compounds can be co-infiltrated with the heterologous nucleic acid.

[0177] In other words, in some embodiments, the culture medium is suitable for producing the taxane having a side chain containing a hydroxylated β-phenylalanine moiety, preferably the culture medium comprising baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0178] Therefore, in some embodiments, the method is a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, wherein the step of culturing the host cells is performed in the presence of baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel.

[0179] Therefore, the method may further include the step of supplying host cells with the baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel, for example, by incubating the host cells in a culture medium containing the compounds.

[0180] This disclosure also includes the following: host cells are capable of and / or able to produce baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel, and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel. Host cells capable of producing said compounds are described elsewhere herein, for example, in the “Host Cells” section above. In such embodiments, incubating said host cells in the presence of taxadiene only requires culturing said host cells.

[0181] Therefore, in other embodiments, the method is a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, wherein the host cell is capable of producing baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0182] Production of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel method

[0183] The methods disclosed herein are particularly applicable to the production of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, paclitaxel, and / or its derivatives. Host cells suitable for producing these compounds are also described in various sections of this document, particularly the “Host Cells” section above. This section discloses examples of the production of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel from various substrates / precursors, and examples of host cells suitable for this purpose. Depending on which enzymes the host cell contains, it may preferably be cultured in the presence of a suitable enzyme substrate that produces the product of the enzyme-catalyzed reaction, or the host cell itself may be capable of producing the enzyme substrate. Suitable substrates for the enzymes disclosed herein are described above and in this section.

[0184] For example, in some embodiments, the method is a method for producing N-debenzoyl-paclitaxel, and the culture medium contains baccatin III. In other embodiments, the method is a method for producing N-debenzoyl-paclitaxel, and the host cells are producing baccatin III and / or are capable of producing baccatin III.

[0185] In some embodiments, the method is a method for producing N-debenzoyl-paclitaxel, and: i. The culture medium contains N-desbenzoyl-2'-deoxy-paclitaxel and / or the host cells are producing N-desbenzoyl-2'-deoxy-paclitaxel and / or are capable of producing N-desbenzoyl-2'-deoxy-paclitaxel; ii. The culture medium contains baccatin III and / or the host cells are producing baccatin III or are capable of producing baccatin III; iii. The culture medium contains baccatin III and the host cells are producing β-phenylalanine-CoA or are capable of producing β-phenylalanine-CoA; iv. The culture medium contains baccatin III and the host cells are producing β-phenylalanine or are capable of producing β-phenylalanine; v. The culture medium contains baccatin III and / or β-phenylalanyl-CoA, and / or the host cells are producing baccatin III and / or β-phenylalanyl-CoA or are capable of producing baccatin III and / or β-phenylalanyl-CoA; and / or vi. The culture medium contains baccatin III and / or β-phenylalanine, and / or the host cells are producing baccatin III and / or β-phenylalanine or are capable of producing baccatin III and / or β-phenylalanine.

[0186] In other embodiments, the method is a method for producing 10-deacetyl-N-debenzoyl-paclitaxel, and: i. The culture medium contains 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel and / or the host cells are producing or capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel; ii. The culture medium contains 10-deacetylated baccatin III and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III; iii. The culture medium contains 10-deacetylated baccatin III and / or β-phenylalanyl-CoA, and / or the host cells are producing 10-deacetylated baccatin III and / or β-phenylalanyl-CoA or are capable of producing 10-deacetylated baccatin III and / or β-phenylalanyl-CoA; iv. The culture medium contains 10-deacetylated baccatin III, and the host cells are producing β-phenylalanyl-CoA or are capable of producing β-phenylalanyl-CoA; v. The culture medium contains 10-deacetylated baccatin III, and the host cells are producing or are capable of producing β-phenylalanine; and / or vi. The culture medium contains 10-deacetylated baccatin III and / or β-phenylalanine, and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III and / or β-phenylalanine.

[0187] In some embodiments, the method is a method for producing N-desbenzoyl-paclitaxel or 10-desacetyl-N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) and a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), or a functional homolog of either of the above with at least 70% sequence identity. In other embodiments, the method is a method for producing N-desbenzoyl-paclitaxel or 10-desacetyl-N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) and a nucleic acid encoding BAPT (SEQ ID NO: 1), or a functional homolog of either of the above with at least 70% sequence identity. In other embodiments, the method is a method for producing N-desbenzoyl-paclitaxel or 10-desacetyl-N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2), and a nucleic acid encoding BAPT (SEQ ID NO: 1), or a functional homolog thereof with at least 70% sequence identity. In other embodiments, the method is a method for producing N-desbenzoyl-paclitaxel or 10-desacetyl-N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) or TchiAAE5 (SEQ ID NO: 22), and a nucleic acid encoding BAPT (SEQ ID NO: 1), or a functional homolog thereof with at least 70% sequence identity.

[0188] In some embodiments, the method is a method for producing N-debenzoyl-paclitaxel, and: i. The culture medium contains 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel and / or the host cells are producing or capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel; ii. The culture medium contains 10-deacetylated baccatin III and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III; iii. The culture medium contains 10-deacetylated baccatin III, and the host cells are producing β-phenylalanyl-CoA or are capable of producing β-phenylalanyl-CoA; iv. The culture medium contains 10-deacetylated baccatin III, and the host cells are producing or are capable of producing β-phenylalanine; v. The culture medium contains 10-deacetylated baccatin III and β-phenylalanyl-CoA, and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III and β-phenylalanyl-CoA; and / or vi. The culture medium contains 10-deacetylated baccatin III and β-phenylalanine, and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III and β-phenylalanine.

[0189] In some embodiments, the method is a method for producing N-debenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), and a nucleic acid encoding... TcuThe method comprises a nucleic acid encoding DBAT (SEQ ID NO: 21), or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding TcuDBAT (SEQ ID NO: 21), or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2), a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), and a nucleic acid encoding TcuDBAT (SEQ ID NO: 21), or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding TcuDBAT (SEQ ID NO: 21), or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing N-desbenzoyl-paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) or TchiAAE5 (SEQ ID NO: 22), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding TcuDBAT (SEQ ID NO: 21), or a functional homolog of any of the above with at least 70% sequence identity.

[0190] In some embodiments, the method is a method for producing paclitaxel, and: i. The culture medium contains N-desbenzoyl-2'-deoxy-paclitaxel and / or the host cells are producing N-desbenzoyl-2'-deoxy-paclitaxel or are capable of producing N-desbenzoyl-2'-deoxy-paclitaxel; and / or ii. The culture medium contains N-debenzoyl-2'-deoxy-paclitaxel and / or benzoyl-CoA, and / or the host cells are producing N-debenzoyl-2'-deoxy-paclitaxel and / or benzoyl-CoA or are capable of producing N-debenzoyl-2'-deoxy-paclitaxel and / or benzoyl-CoA; and / or iii. The culture medium contains baccatin III and / or the host cells are producing baccatin III or are capable of producing baccatin III; iv. The culture medium contains 10-deacetylated baccatin III and / or the host cells are producing or are capable of producing 10-deacetylated baccatin III; v. The culture medium contains baccatin III and the host cells are producing β-phenylalanine or are capable of producing β-phenylalanine; vi. The culture medium contains baccatin III and the host cells are producing β-phenylalanyl-CoA or are capable of producing β-phenylalanyl-CoA; vii. The culture medium contains baccatin III and β-phenylalanyl-CoA and / or the host cells are producing baccatin III and / or β-phenylalanyl-CoA or are capable of producing baccatin III and / or β-phenylalanyl-CoA; and / or viii. The culture medium contains baccatin III and β-phenylalanine and / or the host cells are producing baccatin III and / or β-phenylalanine or are capable of producing baccatin III and / or β-phenylalanine.

[0191] When producing paclitaxel using the method disclosed herein, benzoyl-CoA is preferably present. Benzoyl-CoA can be produced by host cells or contained in the culture medium.

[0192] In some embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) and a nucleic acid encoding DBTNBT (EC: 2.3.1), optionally as TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog of either of the above with at least 70% sequence identity.

[0193] In other embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), and a nucleic acid encoding DBTNBT (EC: 2.3.1), optionally as TcaDBTNBT described in SEQ ID NO: 20, or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding DBTNBT (EC: 2.3.1), optionally as TcaDBTNBT described in SEQ ID NO: 20, or a functional homolog of any of the above with at least 70% sequence identity. In other embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2), a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), and a nucleic acid encoding DBTNBT (EC: 2.3.1), optionally as TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog of any of the above with at least 70% sequence identity. In some embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding DBTNBT (EC: 2.3.1), optionally as TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog of any of the above with at least 70% sequence identity. In other embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) or TchiAAE5 (SEQ ID NO: 22), a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4), and a nucleic acid encoding DBTNBT (EC: 2.3.1) optionally as TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog of any of the above with a sequence identity of at least 70%.In some embodiments, the method is a method for producing paclitaxel, and the host cell contains a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3), a nucleic acid encoding AAE-867.5 (SEQ ID NO: 29) or TchiAAE5 (SEQ ID NO: 22), a nucleic acid encoding BAPT (SEQ ID NO: 1), and a nucleic acid encoding DBTNBT (EC: 2.3.1) optionally as TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog of any of the above with a sequence identity of at least 70%.

[0194] In some implementations, the host cell may further contain nucleic acids encoding DBAT, for example. Tcu DBAT (SEQ ID NO: 21), or its functional homologs having at least 70% sequence identity.

[0195] Separation

[0196] The method may further include the step of separating and / or recovering the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0197] These compounds can be separated and / or recovered by any useful method known to those skilled in the art. For example, the recovery of said taxanes having side chains containing hydroxylated β-phenylalanine moieties can be achieved by separating and / or recovering them by methods including one or more of the following: - Extraction, such as solvent extraction; - Precipitation; and / or - Chromatography, such as liquid chromatography (LC).

[0198] In some embodiments of this disclosure, the steps of separating and / or recovering the taxane having a side chain containing a hydroxylated β-phenylalanine moiety include an extraction step, such as extraction with a solvent, for example methanol (MeOH) and / or ethyl acetate (EtOAc).

[0199] In other embodiments, the steps of separating and / or recovering the taxane having a side chain containing a hydroxylated β-phenylalanine moiety include a chromatographic step, such as liquid chromatography (LC), column chromatography, or preparative / semi-preparative high-performance liquid chromatography (HPLC).

[0200] polypeptides and nucleic acids

[0201] In addition to the method and host cell, the present invention also provides an enzyme that can be used to produce taxanes having a side chain containing a hydroxylated β-phenylalanine moiety.

[0202] This document provides a nucleic acid construct for expression in host cells, comprising a nucleic acid encoding OD3 as described in SEQ ID NO:3 or a functional homolog thereof having at least 70% sequence identity, such as SEQ ID NO:8 or a homolog thereof having at least 70% sequence identity (e.g., at least 65%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%). Furthermore, a nucleic acid encoding OD3(N195A) as described in SEQ ID NO:24 or a functional homolog thereof having at least 70% sequence identity, such as SEQ ID NO:25 or a homolog thereof having at least 70% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%). Also provided is a nucleic acid encoding OD3(Y169F) as described in SEQ ID NO: 26 or a functional homolog thereof having at least 70% sequence identity, such as SEQ ID NO: 27 or a homolog thereof having at least 70% (e.g., at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity) of SEQ ID NO: 27.

[0203] In some embodiments, the nucleic acid construct comprising a nucleic acid encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof (e.g., SEQ ID NO: 8 or a homolog having at least 70% sequence identity with it) further comprises one or more of the following: i. Nucleic acids encoding BAPT as described in SEQ ID NO: 1 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 6; ii. Nucleic acids encoding MBPig3BAPT as described in SEQ ID NO: 4 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 9; iii. Nucleic acids encoding CoAL(A312G) as described in SEQ ID NO: 2 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 7; iv. Nucleic acid encoding TchiAAE5 as described in SEQ ID NO: 22 or a functional homolog thereof having at least 70% sequence identity; v. Nucleic acid encoding AAE-867.5 as described in SEQ ID NO: 29 or a functional homolog thereof having at least 70% sequence identity; vi. Nucleic acids encoding TcuDBAT as described in SEQ ID NO: 21 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 23; and / or vii. Nucleic acids encoding TcaDBTNBT as described in SEQ ID NO: 20 or its functional homologs having at least 70% sequence identity, Or homologs of any of the above with a sequence identity of at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%).

[0204] The functional homolog of OD3 (SEQ ID NO: 3) can be OD3 (N195A) of SEQ ID NO: 24 or OD3 (Y169F) of SEQ ID NO: 26. The nucleic acid encoding OD3 (N195A) (SEQ ID NO: 24) can be as described in SEQ ID NO: 25, or a homolog thereof having at least 70% sequence identity with SEQ ID NO: 25 (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity). The nucleic acid encoding OD3 (Y169F) (SEQ ID NO: 26) can be as described in SEQ ID NO: 27, or a homolog thereof having at least 70% sequence identity with SEQ ID NO: 27 (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity).

[0205] In some implementations, the nucleic acid construct further includes promoters, such as constitutive promoters and / or inducible promoters, which are operatively linked to any one or more of the nucleic acid sequence.

[0206] This document also provides an isolated polypeptide as described in SEQ ID NO: 3 or its functional homologs having at least 70% sequence identity with SEQ ID NO: 3 (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, e.g., at least 90%, at least 95%, e.g., at least 99% sequence identity).

[0207] In addition, this article provides an isolated polypeptide as described in SEQ ID NO: 24 or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 24 (e.g., at least 71% sequence identity, at least 72% sequence identity, at least 73% sequence identity, at least 74% sequence identity, at least 75% sequence identity, at least 90%, at least 95%, at least 99% sequence identity).

[0208] Also provided is an isolated polypeptide as described in SEQ ID NO: 26 or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 26 (e.g., at least 71% sequence identity, at least 72% sequence identity, at least 73% sequence identity, at least 74% sequence identity, at least 75% sequence identity, e.g., at least 90%, at least 95%, e.g., at least 99% sequence identity).

[0209] This document also provides a vector comprising at least one of the nucleic acid constructs described herein, such as a vector comprising a nucleic acid encoding OD3 (SEQ ID NO: 3), OD3(N195A) (SEQ ID NO: 24), or OD3(Y169F) (SEQ ID NO: 26), or a homolog thereof having at least 70% sequence identity with SEQ ID NO: 3, SEQ ID NO: 24, or SEQ ID NO: 26, respectively. Therefore, in some embodiments, the vector comprises at least one nucleic acid construct comprising or consisting of the following nucleic acid sequences: SEQ ID NO: 8, SEQ ID NO: 25, or SEQ ID NO: 27, or a homolog thereof having at least 70% (e.g., at least 65% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity) of SEQ ID NO: 8, SEQ ID NO: 25, or SEQ ID NO: 27.

[0210] A host cell, as described elsewhere herein, is also provided, comprising the nucleic acid constructs described in this section or the vectors according to this section. Preferably, the host cell is the host cell described in the "Host Cell" section above.

[0211] A kit of parts is also provided, comprising: i. The host cells described in this section, and optional instructions for use, and / or ii. One or more nucleic acid constructs or vectors described herein, and optionally instructions for use, further optionally including a host cell to be modified. Preferably, the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells and fungal cells.

[0212] use

[0213] This article also discloses information from the Chinese yew (Taxus chinensis) Taxus chinensis The use of an oxidase from, for example, Taxus chinensis (Chinese yew) in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, wherein the oxidase is... T. chinensis 2-Oxyglutaric acid-dependent oxygenase from yew trees (Taxus chinensis) or from Chinese yew trees (Taxus chinensis) T. chinensis 2-Oxyglutaric acid-dependent dioxygenases, preferably derived from Chinese yew (Taxus chinensis). T. chinensis 2-Oxyglutaric acid-dependent dioxygenase. Preferably, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel.

[0214] In a preferred embodiment, the Chinese yew ( T. chinensis The oxidase is OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 80%, at least 90%, at least 95%). Therefore, the use of OD3 of SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity is disclosed in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety. Similarly, the use of OD3 (N195A) of SEQ ID NO: 24 or OD3 (Y169F) of SEQ ID NO: 26 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 80%, at least 90%, at least 95%) with SEQ ID NO: 24 or SEQ ID NO: 26, respectively, in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety is also disclosed herein. In some implementations, the method is the method described herein, for example, in the "Methods" section.

[0215] In other embodiments, the polypeptide or oxidase comprises the sequence as described in SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 26, but with a maximum of 30 residues mutated, such as a maximum of 29 residues, a maximum of 28 residues, a maximum of 27 residues, a maximum of 26 residues, a maximum of 25 residues, a maximum of 20 residues, a maximum of 15 residues, a maximum of 10 residues, a maximum of 5 residues or fewer residues mutated.

[0216] In some embodiments, the use includes expressing a polypeptide as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity) in a host cell, preferably wherein the host cell is as described herein, for example in the “Host Cell” section.

[0217] Compounds, Compositions and Pharmaceutical Uses

[0218] The host cells, methods, and / or uses described herein can be used to produce various plant diterpenoid compounds efficiently and / or at high titers. In particular, this disclosure provides host cells, methods, and / or uses for producing one or more taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably wherein said taxane comprises N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel. The methods are described elsewhere herein, for example in the “Oxidases” and “Methods” sections above. The host cells are described elsewhere herein, for example in the “Oxidases” and “Host Cells” sections above. The uses are described elsewhere herein, for example in the “Oxidases” section above and the “Uses” section below.

[0219] In addition, this section provides a fermentation broth, cell culture, and / or composition comprising a taxane having a side chain containing a hydroxylated β-phenylalanine moiety, wherein the taxane having the side chain containing the hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel.

[0220] compound

[0221] Therefore, this document provides a composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety, obtained through the methods, host cells, and / or uses described herein. N-Debenzoyl-paclitaxel, obtained through the methods, host cells, and / or uses described herein, is also provided. 10-Deacetyl-N-Debenzoyl-paclitaxel, obtained through the methods, host cells, and / or uses described herein, is also provided. N-Debenzoyl-2'-Deoxy-paclitaxel, obtained through the methods, host cells, and / or uses described herein, is also provided. N-Debenzoyl-paclitaxel, obtained through the methods, host cells, and / or uses described herein, is also provided.

[0222] Cell cultures, fermentation broth and titers

[0223] This article also provides a cell culture obtained by the method described herein. A cell culture comprising the host cells described herein and optionally a culture medium is also provided.

[0224] This document further provides a fermentation broth comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety. In some embodiments, the fermentation broth is obtained by the methods and / or uses described herein. In other embodiments, the method may further include the step of obtaining the fermentation broth, optionally wherein the fermentation broth comprises the host cells described herein and / or a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety. In some embodiments, the fermentation broth is contained in the cell culture described herein. In further embodiments, the fermentation broth is contained within and / or secreted by the host cells described herein, for example, secreted into the fermentation broth, culture medium, or broth. In some embodiments, the fermentation broth contains the host cells described herein, and at least 50% of the host cells are lysed, for example, at least 75%, at least 95%, or at least 99% of the host cells are lysed. In other embodiments, at least 50% of the solid cell material has been separated from the liquid, for example, at least 75%, at least 95%, or at least 99% of the solid cell material has been separated from the liquid.

[0225] As described above, taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety can be secreted by host cells and thus exist in the extracellular portion (supernatant), or they can be retained in the host cells and thus exist in the intracellular portion. The total titer of the compound is the sum of the intracellular and extracellular titers of the compound. In some embodiments, the host cell can be able to produce taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety at a titer of at least 50 μg / L, for example at least 0.75 μg / L, for example at least 100 μg / L, for example at least 250 μg / L, for example at 500 μg / L, for example at least 750 μg / L, for example at least 900 μg / L, for example at least 1000 μg / L, for example at least 2.5 mg / L, for example at least 5 mg / L, for example at least 7.5 mg / L, for example at least 10 mg / L or more.

[0226] In some embodiments, the yield of taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is at least 0.2 μg / mg wet weight.

[0227] Methods for determining the titer of plant diterpenoid compounds are known in the art. For example, the titer can be determined by UPLC-HRMS, as shown in embodiments of this disclosure.

[0228] Composition

[0229] Diterpenoid compounds (more particularly taxanes having a side chain containing a hydroxylated β-phenylalanine moiety) obtainable by this method and / or use can be used to obtain compositions containing any of the compounds produced by the host cells of this disclosure.

[0230] Therefore, this document provides a composition comprising one or more taxanes (e.g., N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel, and / or N-debenzoyl-2'-deoxy-paclitaxel and / or N-debenzoyl-paclitaxel) having a side chain comprising a hydroxylated β-phenylalanine moiety, obtained through the methods and / or uses described herein, and optionally one or more reagents, additives, and / or excipients. In particular, this document provides a composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety, obtained through the host cells, methods, and / or uses described herein. A composition comprising paclitaxel obtained through the host cells, methods, and / or uses described herein, and optionally one or more reagents, additives, and / or excipients is also disclosed.

[0231] A composition comprising the fermentation broth described herein is also provided.

[0232] In some embodiments, the composition has been processed into a semi-dry or dry solid form, optionally in the form of powder, tablets, capsules, chewable tablets, gels, and / or chewing gum. In other embodiments, the composition is in a liquid form, optionally in a stabilized liquid form.

[0233] Drug Use

[0234] Plant diterpenoids (more particularly taxanes having a side chain containing a hydroxylated β-phenylalanine moiety) that are available through the method and / or using the host cells can be used to manufacture pharmaceutical compounds and / or compositions, particularly taxol compounds such as N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel.

[0235] Therefore, the method may further include the step of producing a drug and / or composition from any compound produced by the host cells of this disclosure.

[0236] A method for treating diseases such as cancer is also provided, the method comprising administering a medicament comprising a composition obtained by the methods described herein, a host cell, and / or the use thereof, the composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety. Preferably, the method of treating the disease comprises administering a therapeutically sufficient amount of the medicament. Preferably, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel.

[0237] Non-limiting examples of this type of cancer include melanoma, ovarian cancer, breast cancer, bladder cancer, prostate cancer, and / or esophageal cancer.

[0238] Example

[0239] Example 1: Materials and methods of Examples 2, 3 and 4 to 9

[0240] This embodiment includes the materials and methods of embodiments 2-4 and 5-9 below.

[0241] CDS Acquisition and Gene Cloning

[0242] CoAL (A312G), BAPT, and MBP (maltose-binding protein) (from E. coli) are synthesized from ThermoFisher and target Saccharomyces cerevisiae (Saccharomyces cerevisiae). S. cerevisiae Codon optimization was performed. OD3 was synthesized from TWIST as the natural cDNA sequence.

[0243] Using specific primers (USER-GeneName-FP and USER-GeneName-RP, Table 1), a gene for the production of tobacco Benzodiazepines was produced via USER cloning. N. benthamiana Vectors for transient and yeast expression were used. All primers were ordered from TAG in Copenhagen, Denmark. The pLIFE33 vector (Forman et al., 2022) was used for transient expression in tobacco.

[0244] Table 1. Primers used to construct vectors for gene expression in tobacco and yeast.

[0245]

[0246] Ben's tobacco ( N. benthamiana Transient co-expression of target genes in leaves

[0247] Electroporation transformation of the construct for transient tobacco expression into Agrobacterium was performed. ( Agrobacterium ( A. tumefaciens In strain AGL-1-GV3850), overnight cultures of engineered Agrobacterium strains were used for Agrobacterium infiltration. In short, OD... 600 A culture mixture of 1:1 was used in equal proportions for each Agrobacterium strain. Four-week-old tobacco leaves were used for inoculation. After inoculation, the plants were placed in a greenhouse before supplementing with substrate. ( Exposure to light for 16 hours at 20°C, followed by darkness for 8 hours at 19°C for 2 days. The substrate was dissolved in 5% methanol.

[0248] Heterologous expression of candidate genes in yeast

[0249] brewing yeast ( S. cerevisiae Yeast strain EGY48 was used as the parent strain for gene expression. Yeast transformation was performed using a lithium acetate protocol. To prepare for the detection of the produced compounds, the yeast strain was cultured overnight in selective glucose medium at 30°C and 150 rpm and used as seed cultures. To induce the production of paclitaxel derivatives, each seed culture was washed three times with sterile MQ water and transferred to 100 mL glass bottles containing 10 mL of galactose / raffinose medium. The yeast cultures were incubated prior to ethyl acetate extraction. 3 days at 20℃ and 150 rpm.

[0250] yeast culture mediumYeast glucose medium: 2% (w / v) glucose, 0.13% (w / v) Yeast Synthetic Dropout Medium Supplements (all essential amino acids), 0.67% (w / v) Yeast Nitrogen Base w / o AA. Yeast galactose / raffinose medium: 2% (w / v) galactose, 1% (w / v) raffinose, 0.13% (w / v) Yeast Synthetic Dropout Medium Supplements (all essential amino acids), 0.67% (w / v) Yeast Nitrogen Base w / o AA (Y2025, US Biologicals). Yeast pH buffer medium: Yeast galactose / raffinose medium containing 10% phosphate buffer (1M) and pH adjusted to 7.

[0251] Table 2. Yeast strains. Abbreviations: MBP stands for maltose-binding protein, and IG3 stands for IGGG-linker (SEQ ID NO: 28). SEQ ID NOs of nucleic acids and / or polypeptides contained in yeast are also shown.

[0252]

[0253] Candidate genes in E. coli ( Heterologous expression in E. coli

[0254] Escherichia coli ( E. coli The strain BL21(DE3) (Studier et al, 1986) was used for gene expression. *Escherichia coli* (…) E. coli The conversion was performed using a heat shock protocol. To prepare for the detection of the produced compounds, *E. coli* strains were cultured overnight in LB broth (Miller) at 37°C and 150 rpm and used as seed cultures. To induce production, each seed culture was diluted 1:100 into fresh LB broth (Miller) and incubated at 37°C for 1–2 hours until the OD600 reached 0.5. At this point, production was induced by adding 200 µM isopropyl-β-D-1-thiogalactoside (IPTG), 100 mg / L baccatin III, and 100 mg / L β-phenylalanine. The *E. coli* cultures were incubated (18°C and 150 rpm) for 1 day prior to ethyl acetate extraction.

[0255] Table 3. Escherichia coli ( E. coli ) strain. Abbreviations: MBP is maltose-binding protein, IG3 is IGGG-linker (SEQ ID NO: 28). Also shown are the SEQ ID NOs of nucleic acids and / or polypeptides contained in the bacteria.

[0256]

[0257] Sample extraction and UPLC-HRMS analysis

[0258] For tobacco metabolite extraction, two leaf discs (Ø3 cm, one disc per leaf) were used for each sample, and the samples were ground in liquid nitrogen and then extracted with 1 mL of methanol (MeOH). For yeast samples, 5 mL of ethyl acetate (EtOAc) was added to each induced yeast culture (10 mL of culture in a 100 mL flask). The mixture was centrifuged at 4000 RPM for 10 min. Subsequently, 1 mL of the EtOAc supernatant was dried under nitrogen, and the dried metabolites were redissolved in 100 μL of MeOH. All samples were filtered through a 0.22 μM filter before ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) analysis. The final samples were diluted 200-fold before HPLC-HRMS analysis.

[0259] UPLC-HRMS analysis was performed on a Dionex UltiMate® 3000 quaternary rapid separation UHPLC system (Thermo Fisher Scientific, 732 Germering, Germany) equipped with a Phenomenex Kinetex XB-C18 column (100 mm × 2.1 mm i.d., 1.7 µM particle size, 100 Å pore size) (Phenomenex, Inc., Torrance, CA, USA). The column temperature was maintained at 40 °C and the flow rate at 0.3 mL / min. -1 The mobile phase consisted of water (A) and 100% acetonitrile (B), both acidified with 0.05% formic acid. Separation was achieved using the following gradient curves: 0 min, 20% B; 11 min, 80% B; 21 min, 90% B; 22 min, 100% B; 27 min, 100% B; 28 min, 20% B. The column outlet was connected to a Bruker Daltonics Compact QqTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with an electrospray ionization (ESI) interface. Mass spectra were acquired in positive ion mode using a capillary voltage of 4000 V, an end plate offset of –500 V, a drying temperature of 220 °C, a nebulizer pressure of 2.0 bar, and 8 L / min. -1A dry gas stream was used. Sodium formate solution (internal standard) was injected at the start of each chromatography run, and the raw UPLC–HRMS data were calibrated against these sodium clusters using the Data Analysis 4.3 (BrukerDaltonics) software program.

[0260] Example 2: Production of N-debenzoyl-paclitaxel in tobacco

[0261] This embodiment demonstrates that N-debenzoyl-paclitaxel can be synthesized in vivo by expressing BAPT, CoAL(A312G) and OD3, and by feeding host cells with β-phenylalanine and baccatin III.

[0262] The materials and methods are described in Example 1 above.

[0263] Agrobacterium-mediated tobacco ( Nicotiana benthamiana Transient gene expression is an efficient and reliable method for producing terpenoids. To reconstruct the bioreaction pathway for the production of N-debenzoyl-paclitaxel, we impregnated tobacco leaves with Agrobacterium carrying nucleic acids encoding BAPT (SEQ ID NO: 1), CoAL (A312G) (SEQ ID NO: 2), OD3 (SEQ ID NO: 3), and P19 (SEQ ID NO: 5). P19 was used to suppress gene silencing. Prior to co-expression, samples from Taxus chinensis (Malica spp.) were... Taxus cuspidata The nucleic acid sequence encoding BAPT (SEQ ID NO: 6) and the sequence from Penicillium chrysogenum ( ) Penicillium chrysogenum The CoAL A312G mutant (CoAL(A312G), SEQ ID NO: 7) targets *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae The codons used were optimized and synthesized by Thermo Fisher. The encoding is derived from the Chinese yew (Taxus chinensis). Taxus chinensis The OD3 nucleic acid (SEQ ID NO: 8) of Agrobacterium was synthesized by TWIST without codon optimization. Two days after Agrobacterium infiltration, 200 mg / L β-phenylalanine and 200 mg / L baccatin III dissolved in 5% methanol were injected into leaves that had previously been infiltrated with the corresponding Agrobacterium.

[0264] Tobacco that transiently expresses CoAL (A312G) (SEQ ID NO: 2, encoded by 7), BAPT (SEQ ID NO: 1, encoded by 6), and OD3 (SEQ ID NO: 3, encoded by 8) N. benthamiana UPLC-HRMS analysis of the methanol extract of the leaves showed the production of N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C 40 H 47 NO13 Extracts from tobacco leaves expressing only CoAL (A312G) (SEQ ID NO: 2, encoded by 7) and BAPT (SEQ ID NO: 1, encoded by 6) were used as negative controls and showed no production of N-debenzoyl-paclitaxel (compound 3). Conversely, the substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+734.3171±0.01, C...) accumulated OD3 (SEQ ID NO: 3, encoded by 8). 40 H 47 NO 12 See Figure 1 above for further details.

[0265] Conclusion: Experiments show that OD3 (SEQ ID NO: 3, encoded by 8) expression together with BAPT (SEQ ID NO: 1, encoded by 6) and CoAL (A312G) (SEQ ID NO: 2, encoded by 7) is sufficient to synthesize the paclitaxel intermediate N-debenzoyl-paclitaxel (compound 3) in tobacco (Figure 1).

[0266] Example 3: Production of N-debenzoyl-paclitaxel in yeast

[0267] This embodiment demonstrates that by expressing BAPT, CoAL(A312G), and OD3, and in yeast (Saccharomyces cerevisiae), Saccharomyces cerevisiae The supplementation of β-phenylalanine and baccatin III led to the in vivo synthesis of N-debenzoyl-paclitaxel.

[0268] Materials and methods See Example 1 above.

[0269] To investigate whether OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) could catalyze the hydroxylation of the C2' site on the C13 side chain of the paclitaxel intermediate in another heterologous bioproduction system, we introduced OD3 into *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae In strain TL001, CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9) were expressed. This strain was engineered to produce a C13 side-chain non-hydroxylated paclitaxel intermediate, thus obtaining strain TL002 (Table 2).

[0270] UPLC-HRMS analysis showed that Saccharomyces cerevisiae (Saccharomyces cerevisia S. cerevisiaeTL002 cells produced N-desbenzoyl-paclitaxel (compound 3) in different yeast cultures, but TL001 cells, lacking OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8), did not produce compound 3 (Figure 2). Instead, TL001 cells accumulated the OD3 substrate N-desbenzoyl-2'-deoxy-paclitaxel (compound 2, Figure 1). See the description of Figure 2 above for more details.

[0271] Conclusion: These results indicate that OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) expression, together with CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9), is sufficient to be expressed in yeast (here, Saccharomyces cerevisiae). S. cerevisiae BAPT is used in the synthesis of the paclitaxel intermediate N-debenzoyl-paclitaxel from baker's yeast (Figure 2). BAPT binds at its N-terminus with paclitaxel derived from *Escherichia coli*. E. coli The maltose-binding protein (MBP) was fused to increase solubility. Extracts from yeast cultures expressing only CoAL (A312G) and MBPig3BAPT were used as a negative control, showing no production of compound 3, but instead accumulation of N-debenzoyl-2'-deoxy-paclitaxel (compound 2).

[0272] Example 4. Production of 10-deacetylated-N-debenzoyl-paclitaxel in tobacco

[0273] This embodiment demonstrates that 10-deacetylated-N-debenzoyl-paclitaxel was synthesized in vivo in tobacco by expressing BAPT, CoAL(A312G) and OD3, and feeding β-phenylalanine and 10-deacetylated-baccatin III.

[0274] Materials and methods See Example 1 above.

[0275] To synthesize 10-deacetylated-N-debenzoyl-paclitaxel in tobacco, we impregnated tobacco leaves with Agrobacterium carrying nucleic acids encoding BAPT, CoAL (A312G), OD3, and P19 (for suppressing gene silencing). Prior to co-expression, samples derived from Taxus chinensis (… Taxus cuspidata BAPT (SEQ ID NO: 1, encoded by 6), derived from Penicillium chrysogenum ( Penicillium chrysogenum The coding nucleic acid sequence of CoAL(A312G) (SEQ ID NO: 2, encoded by 7) targets Saccharomyces cerevisiae (Saccharomyces cerevisiae). S. cerevisiae The codons were optimized and synthesized from Thermo Fisher. (From Chinese yew) Taxus chinensisThe OD3 (SEQ ID NO: 3, encoded by 8) of the agrobacterium was synthesized by TWIST without codon optimization. Two days after Agrobacterium infiltration, 200 mg / L β-phenylalanine and 200 mg / L 10-deacetylated baccatin III dissolved in 5% methanol were injected into leaves that had previously been infiltrated with the corresponding Agrobacterium.

[0276] Tobacco that transiently expresses CoAL (A312G) (SEQ ID NO: 2, encoded by 7), BAPT (SEQ ID NO: 1, encoded by 6), and OD3 (SEQ ID NO: 3, encoded by 8) N. benthamiana UPLC-HRMS analysis of the methanol extract of the leaves of [compound 6, [M+H]+ 708.3015±0.01, C] showed the production of 10-deacetylated-N-debenzoyl-paclitaxel (C) 38 H 45 NO 12 Tobacco leaf extracts expressing only CoAL (A312G) and BAPT were used as negative controls, showing no production of 10-deacetylated-N-debenzoyl-paclitaxel (compound 6). Conversely, the OD3 substrate 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5, [M+H]+692.3065±0.01, C) accumulated. 38 H 45 NO 11 See Figure 3 above for further details.

[0277] in conclusion: These results demonstrate that by expressing the expression of Penicillium chrysogenum (… P. chrysogenum The A312G mutant of CoAL (CoAL(A312G), SEQ ID NO: 2, encoded by 7), BAPT from Taxus cuspidata (SEQ ID NO: 1, encoded by 6), and Taxus chinensis (… T. chinensis The OD3 of ) (SEQ ID NO: 3, encoded by 8) is capable of producing N-debenzoyl-2'-deoxy-paclitaxel (compound 2) in tobacco.

[0278] Example 5: Production of N-desbenzoyl-2'-deoxy-paclitaxel and N-desbenzoyl-paclitaxel in tobacco

[0279] This embodiment demonstrates that by expressing... TcuDBAT (SEQ ID NO: 21, encoded by SEQ ID NO: 23) was fed with 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel or 10-deacetyl-N-debenzoyl-paclitaxel to synthesize N-debenzoyl-2'-deoxy-paclitaxel in vivo in tobacco.

[0280] Materials and methods See Example 1 above.

[0281] Tcu DBAT catalyzes C10 acetylation, with a diverse range of substrates including 10-deacetylated baccatin III, 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel, and 10-deacetylated-N-debenzoyl-paclitaxel. We impregnated tobacco leaves with Agrobacterium carrying DBAT and P19 (used to suppress gene silencing) encoding nucleic acids. Prior to co-expression, Tcu DBAT (SEQ ID NO: 21, encoded by SEQ ID NO: 23) was synthesized by Thermo Fisher and targets *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). S. cerevisiae Codons were optimized. Two days after Agrobacterium infiltration, 100 mg / L of 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel or 100 mg / L of 10-deacetylated-N-debenzoyl-paclitaxel dissolved in 5% methanol was injected into the leaves infiltrated with the corresponding Agrobacterium (see above).

[0282] Instant expression Tcu DBAT (SEQ ID NO: 21, encoded by SEQ ID NO: 23) tobacco ( N. benthamiana UPLC-HRMS analysis of the methanol extract of the leaves of [compound 5, [M+H]+] showed that when supplied with 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (C15), the [M+H]+ 692.3065±0.01, C 38 H 45 NO 11 When N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C) was produced. 40 H 47 NO 12 Only 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel is supplied without expression. Tcu Extracts from tobacco leaves obtained from DBAT were used as a negative control, showing no production of N-debenzoyl-2'-deoxy-paclitaxel (compound 2, [M+H]+ 734.3171±0.01, C 40H 47 NO 12 Conversely, 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5, [M+H]+692.3065±0.01, C 38 H 45 NO 11 It remained unconsumed in the control group. See Figure 4 for further details.

[0283] Instant expression Tcu DBAT (SEQ ID NO: 21, encoded by SEQ ID NO: 23) tobacco ( N. benthamiana UPLC-HRMS analysis of the methanol extract of the leaves of [compound 6, [M+H]+] showed that when 10-deacetylated-N-debenzoyl-paclitaxel (C6, [M+H]+) was supplied, the yield was 708.3015±0.01, C 38 H 45 NO 12 When N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C) was produced. 40 H 47 NO 13 Only 10-deacetylated-N-debenzoyl-paclitaxel is supplied without expression. Tcu Extracts from tobacco leaves of DBAT were used as a negative control, showing no production of N-debenzoyl-paclitaxel (compound 3, [M+H]+ 750.3120±0.01, C 40 H 47 NO 13 Conversely, 10-deacetylated-N-debenzoyl-paclitaxel (compound 6, [M+H]+ 708.3015±0.01, C 38 H 45 NO 12 It remained unconsumed in the control group. See Figure 4 for further details.

[0284] Conclusion: These results demonstrate that N-debenzoyl-paclitaxel (compound 3) can also be produced in tobacco using either 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel or 10-deacetyl-N-debenzoyl-paclitaxel as substrates. Therefore, the biosynthetic routes or reactions are diverse. See Figure 4 for further details, particularly... Figure 4a .

[0285] Example 6: Production of N-debenzoyl-paclitaxel in Escherichia coli (E. coli)

[0286] This example demonstrates that OD3 can be used to synthesize N-debenzoyl-paclitaxel in Escherichia coli (E. coli) cells.

[0287] Materials and methods See Example 1 above.

[0288] To investigate whether OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) can catalyze Escherichia coli ( E. coli The C13 side chain of the paclitaxel pathway intermediate in paclitaxel was oxidized. We introduced OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) along with CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9) into *E. coli*. E. coli In cells, strain ETL002 was obtained (Table 3). E. coli expressing only CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9) were also constructed. E. coli Cells were used as a negative control (named strain ETL001). Strain ETL002 (Table 3) was used to test OD3 in Escherichia coli ( E. coli ) Functions within cells.

[0289] Results: UPLC-HRMS analysis showed that E. coli fed with 100 mg / L baccatin III (compound 1) and 100 mg / L β-phenylalanine (…) E. coli ETL002 cells produced N-debenzoyl-paclitaxel (compound 3). When fed 100 mg / L baccatin III (compound 1) and 100 mg / L β-phenylalanine, OD3-deficient ETL001 cells did not produce 3 (Figure 5). Conversely, in ETL001 cells, the substrate N-debenzoyl-2'-deoxy-paclitaxel (compound 2), which contains OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8), accumulated.

[0290] Conclusion: These results indicate that OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) is sufficient to [contain OD3 in Escherichia coli]. E. coli N-Debenzoyl-2'-deoxy-paclitaxel (compound 3) was synthesized from N-debenzoyl-2'-deoxy-paclitaxel (compound 2).

[0291] Example 7: Production of 10-deacetylated-N-debenzoyl-paclitaxel in Escherichia coli (E. coli)

[0292] This embodiment demonstrates that OD3 can be used in Escherichia coli (E. coli) E. coli 10-Deacetyl-N-Debenzoyl-paclitaxel is synthesized in cells.

[0293] Materials and methods See Example 1 above.

[0294] To investigate whether OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) can catalyze Escherichia coli ( E. coli The C13 side chain of the paclitaxel intermediate in paclitaxel was oxidized. OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) was introduced into *E. coli* along with CoAL (A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9). E. coli In cells, strain ETL002 was obtained (Table 3). E. coli expressing only CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 7) and MBPig3BAPT (SEQ ID NO: 4, encoded by SEQ ID NO: 9) were also constructed. E. coli Cells were used as a negative control (named strain ETL001). Strain ETL002 (Table 3) was used to test OD3 in Escherichia coli ( E. coli ) Functions within cells.

[0295] Results: UPLC-HRMS analysis showed that *E. coli* fed with 100 mg / L 10-deacetylated baccatin III (compound 4) and 100 mg / L β-phenylalanine (…) E. coli ETL002 cells produced 10-deacetylated-N-debenzoyl-paclitaxel (compound 6). When fed 100 mg / L 10-deacetylated-baccatin III (compound 4) and 100 mg / L β-phenylalanine, OD3-deficient ETL001 cells did not produce 6 (Figure 6). Conversely, in ETL001 cells, the substrate 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel (compound 5), which contains OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8), accumulated.

[0296] Conclusion: These results indicate that OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 8) is sufficient to [contain OD3 in Escherichia coli]. E. coli10-Deacetyl-N-Debenzoyl-2'-Deoxy-paclitaxel (compound 6) was synthesized from 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel (compound 5) (Figure 6).

[0297] Example 8: Other Chinese yew (T. chinensis) OD enzymes cannot produce N-debenzoyl-paclitaxel.

[0298] This embodiment demonstrates that the Chinese yew (Taxus chinensis) T. chinensis Other 2-oxoglutarate-dependent dioxygenases (ODs) that share up to 71% identity with OD3 cannot produce N-debenzoyl-paclitaxel in tobacco.

[0299] Materials and methods See Example 1 above.

[0300] In order to study the yew ( Taxus To determine if plants possess another OD enzyme capable of producing N-debenzoyl-paclitaxel using 2'-deoxy-N-debenzoyl-paclitaxel as a substrate, we selected a plant from the Chinese yew (Taxus chinensis). Taxus chinensis Eight candidate ODs were identified (Table 4). These eight candidate ODs shared 18% to 71% sequence identity with OD3 (SEQ ID NO: 3) at the amino acid level. However, none of the candidate ODs could produce N-debenzoyl-paclitaxel in tobacco. Furthermore, OD candidate 8 shared 92% sequence identity with KP178205 (TB328) of Ramírez-Estrada K. et al., 2016.

[0301] Table 4. Amino acid sequence identity of the 8 candidate ODs with OD3 (SEQ ID NO: 3).

[0302]

[0303] Results: Neither the candidate with the highest identity to OD3 (OD candidate 8) nor the other OD candidates 1 to 7 could produce detectable levels of N-debenzoyl-paclitaxel in tobacco.

[0304] Conclusion: These results indicate that although OD3 can synthesize N-debenzoyl-paclitaxel, other enzymes in the same family that share up to 71% amino acid sequence identity with OD3 cannot catalyze the production of N-debenzoyl-paclitaxel to any significant extent.

[0305] Example 9: The OD3 variants at amino acid sites 169 and 195 retained their synthetic N-debenzoyl-paclitaxel. The ability.

[0306] This example demonstrates that the OD3 variants at amino acid sites 169 and 195 retain their ability to synthesize N-debenzoyl-paclitaxel (3).

[0307] Materials and methods See Example 1 above.

[0308] Obtaining OD3 variants: Variants OD3(Y169F) (SEQ ID NO: 26, encoded by SEQ ID NO: 27) and OD3(N195A) (SEQ ID NO: 24, encoded by SEQ ID NO: 25) were generated by mutating amino acid residues Y169 and N195 to phenylalanine (F) and alanine (A), respectively.

[0309] Results: We tested the ability of these two variants to produce N-desbenzoyl-paclitaxel (3) in tobacco. To this end, we transiently expressed CoAL (A312G) and BAPT, as well as wild-type OD3 (SEQ ID NO: 3) or OD3 (Y169F) (SEQ ID NO: 26) or OD3 (N195A) (SEQ ID NO: 24) (Figure 7) in tobacco by Agrobacterium infiltration, followed by feeding with 200 mg / L β-phenylalanine and 200 mg / L baccatin III (BACIII) two days after Agrobacterium infiltration. After 5 days of biotransformation, we extracted metabolites from tobacco leaves and performed LC-qTOF analysis to determine the activity of the expressed enzymes. The results showed that OD3 (Y169F) and OD3 (N195A) had similar catalytic activity to wild-type OD3 because the amount of N-desbenzoyl-paclitaxel (3) they produced was similar to that of wild-type OD3 (Figure 7).

[0310] Conclusion: These results indicate that the OD3 variant at amino acid sites 169 or 195 retains the ability to synthesize N-debenzoyl-paclitaxel (3) from β-phenylalanine and baccatin III (BACIII).

[0311] Sequence Overview

[0312] References

[0313] Forman, V., et al. A gene cluster in Ginkgo biloba encodes uniquemultifunctional cytochrome P450s that initiate ginkgolide biosynthesis. Nat.Commun. 13, 5143(2022).

[0314] Cragg, G. M. Paclitaxel(Taxol): a success story with valuable lessonsfor natural product drug discovery and development. Medicinal research reviews 18, 315-331(1998).

[0315] Walker, K., et al. Molecular cloning and heterologous expression ofthe C-13 phenylpropanoid side chain-CoAacyltransferase that functions inTaxol biosynthesis. Proceedings of the National Academy of Sciences , 99:12715-12720(2002)Koetsier, M., et al. Aminoacyl-coenzyme A synthesiscatalyzed by a CoA ligase from Penicillium chrysogenum . FEBS Letters, 585:893-898(2011)

[0316] Sanchez-Munoz, R., et al. A novel Hydroxylation step in the TaxaneBiosynthetic Pathway: A New Approach to Paclitaxel Production by SyntheticBiology. Frontiers in Bioengineering and Biotechnology . 8: 410(2020)

[0317] Zhang, YJ., et al. Synthetic biology identifies the minimal gene setrequired for Paclitaxel biosynthesis in a plant chassis. Molecular Plant .Pre-print(2023)

[0318] Walker, K., et al. The final acylation step in taxol biosynthesis:cloning of the taxoid C13-side-chainN-benzoyltransferase from Taxus . ProcNatl Acad Sci U S A. 99: 9166-9171(2002)

[0319] Long, RM., et al. Specificiyt of theN-benzoyl transferase responsiblefor the last step of Taxol biosynthesis. Arch Biochem Biophys. 477: 384–389(2009)

[0320] Li, BH., et al. Improving10-deacetylbaccatin III-10-b-O-acetyltransferase catalytic fitness for Taxol production. Nat. Commun. 8:15544(2016)

[0321] Walker, K., et al. Molecular cloning of a10-deacetylbaccatin III-10-O-acetyl transferasecDNA from Taxus and functional expression in Escherichiacoli. Proc Natl Acad Sci U S A. 97: 583-587(2000)

[0322] Studier, FW, et al. Use of bacteriophage T7 RNA polymerase to directselective high-level expression of cloned genes. JMB. 189, 113-130(1986)

[0323] Ramírez-Estrada K., et al. “Transcript profiling of jasmonate-elicited Taxus cells reveal a β-phenylalanine-CoAligase." Plant BiotechnolJ. 2016 Jan;14(1):85-96.

[0324] McClune, CJ., et al. Multiplexed perturbation of yew reveals crypticproteins that enable a total biosynthesis of baccatin III and Taxolprecursors. BioRxiv (2024)

[0325] project

[0326] 1. A host cell comprising a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 71% sequence identity, at least 72% sequence identity, at least 73% sequence identity, at least 74% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0327] 2. A host cell comprising a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3, OD3(N195A) as described in SEQ ID NO: 24, or OD3(Y169F) as described in SEQ ID NO: 26, or a homolog thereof having at least 70% sequence identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%).

[0328] 3. The host cell according to any of the preceding items, wherein the host cell is capable of producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably wherein the taxane comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel (taxol).

[0329] 4. The host cell according to any of the preceding items, wherein in the presence of baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel, the host cell is capable of producing taxanes having side chains comprising a hydroxylated β-phenylalanine moiety.

[0330] 5. The host cell according to any of the preceding items, wherein the host cell is capable of producing baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0331] 6. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of N-debenzoyl-2'-deoxy-paclitaxel, optionally wherein the host cell is capable of producing N-debenzoyl-2'-deoxy-paclitaxel.

[0332] 7. The host cell according to any of the preceding items, wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-paclitaxel in the presence of 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel, optionally wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0333] 8. A host cell according to any of the foregoing items, wherein the host cell further comprises nucleic acid encoding aminophenylpropionyltransferase (EC: 2.3.1), optionally wherein the aminophenylpropionyltransferase is a yew (Taxus chinensis) Taxus ) cells, such as Taxus chinensis ( Taxus cuspidata (Cellular) Natural.

[0334] 9. The host cell according to Item 8, wherein the aminophenylpropionyltransferase is BAPT as described in SEQ ID NO: 1 or MBPig3BAPT as described in SEQ ID NO: 4, or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 4, respectively.

[0335] 10. A host cell according to any of the foregoing items, wherein the host cell further comprises nucleic acid encoding coenzyme A ligase (CoAL, EC: 6.2.1), optionally wherein the CoAL is a Penicillium genus (… Penicillium ) cells, such as Penicillium chrysogenum ( P. chrysogenum ) cells naturally occurring in the CoAL or Taxus genus ( Taxus Cells such as those of the Chinese yew (T. chinensis) naturally contain CoAL.

[0336] 11. The host cell according to item 10, wherein the CoAL is CoAL(A312G) as described in SEQ ID NO: 2, AAE-867.5 as described in SEQ ID NO: 29, or TchiAAE5 as described in SEQ ID NO: 22, or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 2, SEQ ID NO: 29, or SEQ ID NO: 22, respectively.

[0337] 12. The host cell according to any of the preceding items, wherein the host cell further comprises encoding 10-deacetylated-baccatin III-10- O - Acetyltransferase (DBAT) nucleic acid, optionally, wherein said DBAT is yew (Taxus chinensis) Taxus ) cells, such as Taxus chinensis ( Taxus cuspidata ) Cellular natural.

[0338] 13. The host cell according to item 12, wherein the DBAT is TcuDBAT as described in SEQ ID NO: 21 or its functional homolog having at least 70% sequence identity with it.

[0339] 14. A host cell according to any of the preceding items, wherein the host cell further comprises nucleic acid encoding 3'-N-debenzoyl-2'-deoxypaclitaxel-N-benzoyltransferase (DBTNBT, EC: 2.3.1), optionally wherein the DBTNBT is a yew (Taxus chinensis) Taxus ) cells, such as Canada yew ( Taxus (canadensis) cells are natural.

[0340] 15. The host cell according to item 14, wherein the DBTNBT is TcaDBTNBT as described in SEQ ID NO: 20 or its functional homolog having at least 70% sequence identity with it.

[0341] 16. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of baccatin III, optionally wherein the host cell is capable of producing baccatin III.

[0342] 17. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of baccatin III and β-phenylalanyl-CoA, optionally wherein the host cell is capable of producing baccatin III and / or β-phenylalanyl-CoA.

[0343] 18. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of baccatin III and β-phenylalanine, optionally wherein the host cell is capable of producing baccatin III and / or β-phenylalanine.

[0344] 19. The host cell according to any of the preceding items, wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-paclitaxel in the presence of 10-deacetyl-baccatin III, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III.

[0345] 20. The host cell according to any of the preceding items, wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-paclitaxel in the presence of 10-deacetyl-baccatin III and β-phenylalanyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and / or β-phenylalanyl-CoA.

[0346] 21. The host cell according to any of the preceding items, wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-paclitaxel in the presence of 10-deacetyl-baccatin III and β-phenylalanine, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and / or β-phenylalanine.

[0347] 22. The host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively.

[0348] 23. A host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding CoAL(A312G) (SEQ ID NO: 2) or its functional homologs having at least 70% sequence identity; and ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively.

[0349] 24. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel, optionally wherein the host cell is capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0350] 25. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of 10-deacetylated baccatin III, optionally wherein the host cell is capable of producing 10-deacetylated baccatin III.

[0351] 26. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of 10-deacetylated-baccatin III and β-phenylalanyl-CoA, optionally wherein the host cell is capable of producing 10-deacetylated-baccatin III and / or β-phenylalanyl-CoA.

[0352] 27. The host cell according to any of the preceding items, wherein the host cell is capable of producing N-debenzoyl-paclitaxel in the presence of 10-deacetylated baccatin III and β-phenylalanine, optionally wherein the host cell is capable of producing 10-deacetylated baccatin III and / or β-phenylalanine.

[0353] 28. A host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and ii. Encoding Tcu DBAT (SEQ ID NO: 21) or a nucleic acid of its functional homolog that has at least 70% sequence identity with it.

[0354] 29. A host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. Nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0355] 30. A host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; ii. A nucleic acid encoding DBTNBT (EC: 2.3.1), optionally TcaDBTNBT as described in SEQ ID NO: 20, or a functional homolog thereof having at least 70% sequence identity; and iii. Optionally, a nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0356] 31. A host cell according to any of the preceding items, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; iii. A nucleic acid encoding DBTNBT (EC: 2.3.1), optionally TcaDBTNBT (SEQ ID NO: 20) as described in SEQ ID NO: 20, or a functional homolog thereof having at least 70% sequence identity; and iv. Optionally, a nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0357] 32. The host cell according to any of the preceding items, wherein the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells and fungal cells.

[0358] 33. The host cell according to any of the preceding items, wherein the host cell is a plant cell, such as a plant cell contained in or in a part of a plant.

[0359] 34. The host cell according to item 33, wherein the plant cell is derived from the genus *Nicotiana* (…). Nicotiana ) species, such as Nicotiana benthamiana ( Nicotiana benthamiana ) or safflower tobacco ( Nicotiana tabacum ).

[0360] 35. The host cell according to any of the foregoing items, wherein the host cell belongs to the genus *Yeast* ( Saccharomyces ), Pichia pastoris ( Pichia ), Candida genus ( Candida Cryptococcus ( Cryptococcus Pichia pastoris (Pichia pastoris) genus Pichia ( Komagataella )), genus *Olecosomyces* ( Lipomyces ), *Pseudomycium* ( Pseudozyma ), genus *Rhodotorula* ( Rhodosporidium ), Rhodotorula genus ( Rhododendron ), genus *Mycosaccharomyces* ( Trichosporon ), Trichosaccharomyces ( Trigonopsis ), Yersinia ( Yarrow ) or *Cryptotympany* genus ( Saccharomycopsis Yeast cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha (Hansenula polymorpha) (Ogaria polymorpha) Hansenula polymorpha ( Ogataea polymorpha ), Rhodotorula buergerianum ( Rhodotorula toruloides ) or Pichia pastoris (Pichia pastoris) ( Shepherd's pie ( Komagataella phaffii The host cell is a type of yeast cell, preferably a brewer's yeast (Saccharomyces cerevisiae). S. yeast ) types of yeast cells.

[0361] 36. The host cell described in any of the preceding items, wherein the host cell belongs to the genus *Escherichia* (…). Escherichia ), Bacillus spp. Bacillus Corynebacterium spp. Corynebacterium ), Pseudomonas spp. Pseudomonas ) or Streptomyces (St r Bacterial cells of eptomyces, such as Escherichia coli (E. coli) Escherichia to be cultivated Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamic ), Pseudomonas putida ( Pseudomonas putida ) or species of the genus Streptomyces ( Streptomyces sp. The host cell is a bacterial cell of the species *Escherichia coli*. E. coli. ) species of bacterial cells.

[0362] 37. A method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, the method comprising the following steps: i. Provide a host cell according to any one of items 1 to 36 or item 85; ii. Culture the host cells in a culture medium. This produces the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0363] 38. A method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, the method comprising the following steps: i. Provide a host cell according to any one of items 1 to 36 or item 85; ii. The host cells are cultured in a culture medium to obtain a fermentation broth containing the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety; and iii. Optionally, the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is recovered. This produces a fermentation broth containing the taxane having a side chain containing a hydroxylated β-phenylalanine moiety and / or the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

[0364] 39. The method according to any one of items 37 to 38, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is produced in the presence of baccatin III, benzoyl-CoA, 10-deacetyl-baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0365] 40. The method according to any one of items 37 to 39, wherein the step of culturing the host cells is performed in the presence of baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0366] 41. The method according to any one of items 37 to 40, wherein the host cell is capable of producing baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

[0367] 42. The method according to any one of items 37 to 41, wherein the culture medium is suitable for producing the taxane, preferably wherein the culture medium comprises baccatin III, benzoyl-CoA, 10-deacetyl-baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0368] 43. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises N-debenzoyl-2'-deoxy-paclitaxel and / or wherein the host cells are capable of producing N-debenzoyl-2'-deoxy-paclitaxel.

[0369] 44. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises baccatin III and / or wherein the host cells are capable of producing baccatin III.

[0370] 45. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises baccatin III and / or β-phenylalanyl-CoA, and / or wherein the host cells are capable of producing baccatin III and / or β-phenylalanyl-CoA.

[0371] 46. ​​The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises baccatin III and / or β-phenylalanine, and / or wherein the host cells are capable of producing baccatin III and / or β-phenylalanine.

[0372] 47. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises baccatin, and wherein the host cells are capable of producing β-phenylalanyl-CoA.

[0373] 48. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetyl-N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel, and / or wherein the host cells are capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0374] 49. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetylated-N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated-baccatin III, and / or wherein the host cells are capable of producing 10-deacetylated-baccatin III.

[0375] 50. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetylated-N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated-baccatin III and / or β-phenylalanyl-CoA, and / or wherein the host cells are capable of producing 10-deacetylated-baccatin III and / or β-phenylalanyl-CoA.

[0376] 51. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetylated-N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated-baccatin III and / or β-phenylalanine, and / or wherein the host cells are capable of producing 10-deacetylated-baccatin III and / or β-phenylalanine.

[0377] 52. The method according to any one of items 37 to 42, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetylated-N-debenzoyl-paclitaxel, and wherein the culture medium comprises baccatin, and wherein the host cells are capable of producing β-phenylalanyl-CoA.

[0378] 53. The method according to any one of items 37 to 52, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises a nucleic acid encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively.

[0379] 54. The method according to any one of items 37 to 53, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding CoAL(A312G) (SEQ ID NO: 2) or its functional homologs having at least 70% sequence identity; and ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively.

[0380] 55. The method according to any one of items 37 to 54, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel or wherein the host cells are capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

[0381] 56. The method according to any one of items 37 to 55, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated baccatin III or wherein the host cells are capable of producing 10-deacetylated baccatin III.

[0382] 57. The method according to any one of items 37 to 56, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetyl-baccatin III and / or β-phenylalanyl-CoA, and / or wherein the host cells are capable of producing 10-deacetyl-baccatin III and / or β-phenylalanyl-CoA.

[0383] 58. The method according to any one of items 37 to 57, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated baccatin, and wherein the host cells are capable of producing β-phenylalanyl-CoA.

[0384] 59. The method according to any one of items 37 to 58, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetylated baccatin III and / or β-phenylalanine, and / or wherein the host cells are capable of producing 10-deacetylated baccatin III and / or β-phenylalanine.

[0385] 60. The method according to any one of items 37 to 59, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and ii. Nucleic acid encoding TcuDBAT (SEQ ID NO: 21) or a functional homolog thereof having at least 70% sequence identity.

[0386] 61. The method according to any one of items 37 to 59, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. Encoding Tcu DBAT (SEQ ID NO: 21) or a nucleic acid of its functional homolog that has at least 70% sequence identity with it.

[0387] 62. The method according to any one of items 37 to 61, wherein the culture medium comprises N-desbenzoyl-2'-deoxy-paclitaxel or wherein the host cells are capable of producing N-desbenzoyl-2'-deoxy-paclitaxel.

[0388] 63. The method according to any one of items 37 to 62, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is paclitaxel, and wherein the host cell comprises a heteronucleotide encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity with it, and further comprises a nucleic acid encoding DBTNBT (EC: 2.3.1) optionally as TcaDBTNBT as described in SEQ ID NO: 20 or a functional homolog thereof having at least 70% sequence identity with it.

[0389] 64. The method according to any one of items 37 to 63, wherein the culture medium comprises baccatin III, β-phenylalanyl-CoA and / or β-phenylalanine, and / or wherein the host cells are capable of producing baccatin III, β-phenylalanyl-CoA and / or β-phenylalanine.

[0390] 65. The method according to any one of items 37 to 64, wherein the culture medium comprises baccatin III and wherein the host cells are capable of producing β-phenylalanyl-CoA.

[0391] 66. The method according to any one of items 37 to 65, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is paclitaxel, and wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity with it, and further comprises: i. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; ii. Encoding DBTNBT (EC: 2.3.1) optionally as described in SEQ ID NO: 20, TcaDBTNBT or a functional homolog thereof having at least 70% sequence identity.

[0392] 67. The method according to any one of items 37 to 66, wherein the culture medium comprises baccatin III and wherein the host cells are capable of producing β-phenylalanine.

[0393] 68. The method according to any one of items 37 to 67, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is paclitaxel, and wherein the host cell comprises a heteronucleotide encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity with it, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; and iii. A nucleic acid encoding DBTNBT (EC: 2.3.1) optionally as described in SEQ ID NO: 20, TcaDBTNBT (SEQ ID NO: 20), or a functional homolog thereof having at least 70% sequence identity with it.

[0394] 69. The method according to any one of items 37 to 68 further includes the step of recovering the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety.

[0395] 70. The method according to any one of items 37 to 69, wherein the recycling step comprises: i. Extraction steps, such as extraction with solvents like methanol (MeOH) and / or ethyl acetate (EtOAc); and / or ii. Chromatographic steps, such as liquid chromatography (LC), column chromatography, or preparative / semi-preparative high-performance liquid chromatography (HPLC).

[0396] 71. From Chinese yew ( Yew Oxidase of *Taxus chinensis* (preferably derived from *Taxus chinensis*). T. Chinese Use of 2-oxoglutarate-dependent oxygenase or 2-oxoglutarate-dependent dioxygenase in the production of taxanes having a side chain containing a hydroxylated β-phenylalanine moiety. Optionally, the Chinese yew ( T. chinensis Oxidase is OD3 as described in SEQ ID NO: 3 or its functional homologs having at least 70% sequence identity (e.g., at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity).

[0397] 72. Use of OD3 (N195A) as described in SEQ ID NO: 24 or OD3 (Y169F) as described in SEQ ID NO: 26, or their functional homologs having at least 70% sequence identity with SEQ ID NO: 24 or SEQ ID NO: 26, respectively, in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety.

[0398] 73. The use according to any one of items 71 or 72, wherein the polypeptide comprises the sequence as described in SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 26, or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 26, but with a maximum of 30 residues mutated.

[0399] 74. The use according to any one of items 71 to 73, wherein the use comprises expressing, in a host cell, a polypeptide as described in SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 26 or a functional homolog thereof having at least 70% sequence identity with SEQ ID NO: 3, SEQ ID NO: 24 or SEQ ID NO: 26, preferably, wherein the host cell is defined as in any one of items 1 to 36.

[0400] 75. The use according to any one of items 71 to 74, wherein the method is as defined in any one of items 37 to 70.

[0401] 76. A nucleic acid construct for expression in host cells, comprising: i. Nucleic acids encoding OD3 as described in SEQ ID NO: 3 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 8 or its homologs having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0402] 77. A nucleic acid construct for expression in host cells, comprising: i. Nucleic acids encoding OD3(N195A) as described in SEQ ID NO: 24 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 25 or its homologs having at least 70% sequence identity (e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity).

[0403] 78. A nucleic acid construct for expression in host cells, comprising: i. Nucleic acids encoding OD3(Y169F) as described in SEQ ID NO: 26 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 27 or its homologs having at least 70% (e.g., at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 99% sequence identity) of SEQ ID NO: 27.

[0404] 79. The nucleic acid construct according to any one of items 76 to 78 further comprises one or more of the following: i. Nucleic acids encoding BAPT as described in SEQ ID NO: 1 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 6; ii. Nucleic acids encoding MBPig3BAPT as described in SEQ ID NO: 4 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 9; iii. Nucleic acids encoding CoAL(A312G) as described in SEQ ID NO: 2 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 7; iv. Nucleic acid encoding TchiAAE5 as described in SEQ ID NO: 22 or a functional homolog thereof having at least 70% sequence identity; v. Nucleic acid encoding AAE-867.5 as described in SEQ ID NO: 29 or a functional homolog thereof having at least 70% sequence identity; vi. Nucleic acids encoding TcuDBAT as described in SEQ ID NO: 21 or its functional homologs having at least 70% sequence identity, such as SEQ ID NO: 23; and / or vii. Nucleic acids encoding TcaDBTNBT as described in SEQ ID NO: 20 or its functional homologs having at least 70% sequence identity, Or a homolog of any of the above nucleic acids that has at least 70% (e.g., at least 80%, at least 90%, at least 95%, at least 99%) sequence identity with the above nucleic acids.

[0405] 80. The nucleic acid construct according to any one of items 76 to 79 further comprises a promoter, such as a constitutive promoter and / or an inducible promoter, operatively linked to any one or more of the nucleic acid sequence.

[0406] 81. An isolated polypeptide as described in SEQ ID NO: 3 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 90%, at least 95%, at least 99%).

[0407] 82. An isolated polypeptide as described in SEQ ID NO: 24 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 90%, at least 95%, at least 99%) with SEQ ID NO: 24.

[0408] 83. An isolated polypeptide as described in SEQ ID NO: 26 or a functional homolog thereof having at least 70% sequence identity (e.g., at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 90%, at least 95%, at least 99%) with SEQ ID NO: 26.

[0409] 84. A vector comprising at least one of the nucleic acid constructs according to any one of items 76 to 80.

[0410] 85. A host cell according to any one of items 1 to 36, comprising a nucleic acid construct according to any one of items 76 to 80 or a vector according to item 84.

[0411] 86. A kit of parts comprising: i. The host cell as described in item 85, and optionally the instruction manual, and / or ii. The nucleic acid construct according to any one of items 76 to 80 or the vector according to item 84, and optionally an instruction manual, also optionally comprising a host cell to be modified. Preferably, the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells and fungal cells.

[0412] 87. Taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, obtained by means of any one of items 37 to 70 and / or by means of any one of items 71 to 75.

[0413] 88. N-Debenzoyl-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75.

[0414] 89. 10-Deacetyl-N-debenzoyl-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75.

[0415] 90. N-Debenzoyl-2'-deoxy-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75.

[0416] 91. N-Debenzoyl-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75.

[0417] 92. Cell cultures obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75.

[0418] 93. A cell culture comprising a host cell according to any one of items 1 to 36 or 85 and optionally a culture medium.

[0419] 94. A fermentation broth comprising a taxane having a side chain containing a hydroxylated β-phenylalanine moiety, wherein the fermentation broth is: i. Obtained by the method described in any one of items 37 to 70 and / or the use described in any one of items 71 to 75; ii. The cell culture contained in any one of items 92 to 93; and / or iii. Contained in and / or secreted by the host cell according to any one of items 1 to 36 or 85.

[0420] 95. The fermentation broth according to item 94, wherein at least 50% (e.g., at least 75%, at least 95%, at least 99%) of the host cells are lysed.

[0421] 96. The fermentation broth according to any one of items 94 to 95, wherein at least 50% (e.g., at least 75%, at least 95%, at least 99%) of solid cell material has been separated from the broth.

[0422] 97. A composition comprising one or more of the following: i. Fermentation broth according to any one of items 94 to 96; ii. Taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, obtained by the method of any one of items 37 to 70 and / or by the use of any one of items 71 to 75; iii. N-Debenzoyl-paclitaxel obtained by the method of any one of items 37 to 70 and / or the use of any one of items 71 to 75; iv. N-Debenzoyl-2'-deoxy-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75; v. N-debenzoyl-paclitaxel obtained by the method according to any one of items 37 to 70 and / or the use according to any one of items 71 to 75; vi. 10-Deacetyl-N-descarboxyl-paclitaxel obtained by the method of any one of items 37 to 70 and / or the use of any one of items 71 to 75; and / or vii. Paclitaxel obtained by the method of any one of items 37 to 70 and / or the use of any one of items 71 to 75; And optionally one or more reagents, additives and / or excipients.

[0423] 98. The composition according to item 97, wherein the composition has been processed into a semi-dry or dry solid form, optionally in the form of powder, tablet, capsule, chewing agent, gel and / or gum.

[0424] 99. The composition according to item 97, wherein the composition is in liquid form, optionally in a stabilized liquid form.

[0425] 100. A method of treating a disease such as cancer, comprising administering a medicament comprising a composition obtained by any one of the methods of items 37 to 70 and / or by any one of the uses of items 71 to 75, said composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety.

[0426] 101. The host cell, method, use, fermentation broth, cell culture and / or composition according to any of the foregoing items, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel and / or paclitaxel.

[0427] 102. The host cell, method, use, and / or fermentation broth according to any of the foregoing items, wherein the OD3 or its functional homolog having at least 70% sequence identity with it is capable of catalyzing the conversion of N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-paclitaxel, and / or capable of catalyzing the conversion of 10-deacetyl-N-debenzoyl-paclitaxel to 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

Claims

1. A host cell comprising a heterologous nucleic acid sequence encoding OD3 as described in SEQ ID NO: 3 or having at least 75% sequence identity with it, such as at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity.

2. The host cell according to any of the preceding claims, wherein, in the presence of baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated-N-debenzoyl-paclitaxel and / or 10-deacetylated-N-debenzoyl-2'-deoxy-paclitaxel, the host cell is capable of producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety. Optionally, the host cell is capable of producing baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

3. The host cell according to any of the preceding claims, wherein the host cell further comprises nucleic acid encoding aminophenylpropionyltransferase (EC: 2.3.1), Optionally, said aminophenylpropionyltransferase is yew (Taxus chinensis) Taxus ) cells, such as Taxus chinensis ( Taxus cuspidata Cellular natural, Preferably, the aminophenylpropionyltransferase is BAPT as described in SEQ ID NO: 1 or MBPig3BAPT as described in SEQ ID NO: 4, or a functional homolog of SEQ ID NO: 1 or SEQ ID NO: 4, respectively, having at least 70% sequence identity.

4. The host cell according to any of the preceding claims, wherein the host cell further comprises nucleic acid encoding coenzyme A ligase (CoAL, EC: 6.2.1). Optionally, the CoAL mentioned herein is a Penicillium genus ( Penicillium ) cells, such as Penicillium chrysogenum ( P. chrysogenum ) cells naturally contain CoAL, or the genus Taxus ( Taxus ) cells, such as the Chinese yew ( T. chinensis Cellular natural CoAL, Preferably, the CoAL is CoAL(A312G) as described in SEQ ID NO: 2, AAE-867.5 as described in SEQ ID NO: 29, or TchiAAE5 as described in SEQ ID NO: 22, or a functional homolog of the same as SEQ ID NO: 2, SEQ ID NO: 29, or SEQ ID NO: 22, respectively.

5. The host cell according to any of the preceding claims, wherein the host cell further comprises nucleic acid encoding 10-deacetylated baccatin III-10-O-acetyltransferase (DBAT). Optionally, the DBAT mentioned therein is yew ( Taxus ) cells, such as Taxus chinensis ( Taxus cuspidata Cellular natural, Preferably, the DBAT is TcuDBAT as described in SEQ ID NO: 21 or its functional homolog having at least 70% sequence identity.

6. The host cell according to any of the preceding claims, wherein the host cell further comprises nucleic acid encoding 3'-N-debenzoyl-2'-deoxypaclitaxel-N-benzoyltransferase (DBTNBT, EC: 2.3.1), Optionally, the DBTNBT mentioned therein is yew ( Taxus ) cells, such as Canada yew ( Taxus The DBTNBT is native to canadensis cells, preferably, and is TcaDBTNBT as described in SEQ ID NO: 20 or a functional homolog thereof having at least 70% sequence identity with it.

7. The host cell according to any of the preceding claims, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acids encoding CoAL(A312G) (SEQ ID NO: 2) or its functional homologs having at least 70% sequence identity; and ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively.

8. The host cell according to any of the preceding claims, wherein the host cell comprises a heterologous nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homolog thereof having at least 70% sequence identity, and further comprises: i. Nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homolog thereof having at least 70% sequence identity; ii. Nucleic acids encoding MBPig3BAPT (SEQ ID NO: 4) or BAPT (SEQ ID NO: 1) or their functional homologs having at least 70% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 1, respectively; iii. The encoding DBTNBT (EC: 2.3.1) is optionally TcaDBTNBT (SEQ ID NO: 20) or a nucleic acid of its functional homolog having at least 70% sequence identity with it.

9. The host cell according to any of the preceding claims, wherein the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells and fungal cells.

10. The host cell according to any of the preceding claims, wherein the host cell belongs to the genus *Yeast* (…). Saccharomyces ), Pichia pastoris ( Pichia ), Candida genus ( Candida Cryptococcus ( Cryptococcus Pichia pastoris (Pichia pastoris) genus Pichia ( Komagataella )), genus *Olecosomyces* ( Lipomyces ), *Pseudomycium* ( Pseudozyma ), genus *Rhodotorula* ( Rhodosporidium ), Rhodotorula genus ( Rhodotorula ), genus *Mycosaccharomyces* ( Trichosporon ), Trigonopsis, Yersinia ( Yarrowia ) or *Cryptotympany* genus ( Saccharomycopsis Yeast cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Hansenula polymorpha (Hansenula polymorpha) (Ogaria polymorpha) Hansenula polymorpha ( Ogataea polymorpha ), Rhodotorula buergerianum ( Rhodotorula toruloides ) or Pichia pastoris (Pichia pastoris) ( Pichia pastoris ( Komagataella phaffii The host cell is a type of yeast cell, preferably a brewer's yeast (Saccharomyces cerevisiae). S. cerevisiae ) types of yeast cells.

11. The host cell according to any of the preceding claims, wherein the host cell belongs to the genus *Escherichia* (…). Escherichia ), Bacillus spp. Bacillus Corynebacterium spp. Corynebacterium ), Pseudomonas spp. Pseudomonas ) or Streptomyces ( Streptomyces Bacterial cells, such as Escherichia coli (E. coli) Escherichia coli ), Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Pseudomonas putida ( Pseudomonas putida ) or Streptomyces ( Streptomyces sp The host cell is a bacterial cell of the species *Escherichia coli*. E. coli. ) species of bacterial cells.

12. A method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, the method comprising the following steps: i. Providing a host cell according to any one of claims 1 to 11; ii. Culture the host cells in a culture medium. This produces the taxane having a side chain containing a hydroxylated β-phenylalanine moiety. Optionally, it may further include the step of recovering the taxane having a side chain containing a hydroxylated β-phenylalanine moiety.

13. The method of claim 12, wherein the step of culturing the host cells is performed in the presence of baccatin III, benzoyl-CoA, 10-deacetylated baccatin III, β-phenylalanine, β-phenylalanyl-CoA, N-debenzoyl-2'-deoxy-paclitaxel, 10-deacetylated N-debenzoyl-paclitaxel and / or 10-deacetylated N-debenzoyl-2'-deoxy-paclitaxel.

14. The method according to any one of claims 12 to 13, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is N-debenzoyl-paclitaxel, and wherein the culture medium comprises N-debenzoyl-2'-deoxy-paclitaxel or baccatin III, and / or wherein the host cells are capable of producing N-debenzoyl-2'-deoxy-paclitaxel or baccatin III.

15. The method according to any one of claims 12 to 14, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety is 10-deacetyl-N-debenzoyl-paclitaxel, and wherein the culture medium comprises 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel or 10-deacetyl-baccatin III and / or wherein the host cells are capable of producing 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel or 10-deacetyl-baccatin III.

16. The method according to any one of claims 12 to 15, wherein the step of culturing the host cells is performed in the presence of baccatin III, and wherein the host cells, as defined in claim 7, thereby produce N-debenzoyl-paclitaxel, wherein the culture medium contains baccatin III and / or wherein the host cells are capable of producing baccatin III.

17. The method according to any one of claims 12 to 16, wherein the step of culturing the host cells is performed in the presence of baccatin III, and wherein the host cells, as defined in claim 8, thereby produce paclitaxel, wherein the culture medium contains baccatin III and / or wherein the host cells are capable of producing baccatin III.

18. Use of OD3 as described in SEQ ID NO: 3, or its functional homologs having at least 75% sequence identity, such as at least 80%, at least 90%, or at least 95% sequence identity, in a method for producing taxanes having a side chain comprising a hydroxylated β-phenylalanine moiety, preferably, wherein said method is defined as in any one of claims 12 to 13.

19. An isolated polypeptide as described in SEQ ID NO: 3, or a functional homolog thereof having at least 75% sequence identity, such as at least 80% sequence identity, at least 85% sequence identity, such as at least 90%, at least 95%, or at least 99% sequence identity.

20. A fermentation broth comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety, wherein the fermentation broth is obtained by the method of any one of claims 12 to 17 and / or by use according to claim 18.

21. A method of treating a disease such as cancer, comprising administering a medicament comprising a composition obtained by the method of any one of claims 12 to 17 and / or by use according to claim 18, said composition comprising a taxane having a side chain comprising a hydroxylated β-phenylalanine moiety.

22. The host cell, method, use, and / or fermentation broth according to any of the preceding claims, wherein the taxane having a side chain comprising a hydroxylated β-phenylalanine moiety comprises N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel, or is composed of N-debenzoyl-paclitaxel, 10-deacetyl-N-debenzoyl-paclitaxel, and / or paclitaxel.

23. The host cell, method, use, and / or fermentation broth according to any of the preceding claims, wherein the OD3 or its functional homolog having at least 75% sequence identity with it is capable of catalyzing the conversion of N-debenzoyl-2'-deoxy-paclitaxel to N-debenzoyl-paclitaxel, and / or capable of catalyzing the conversion of 10-deacetyl-N-debenzoyl-paclitaxel to 10-deacetyl-N-debenzoyl-2'-deoxy-paclitaxel.

Images