WO2026013281A1

WO2026013281A1 - Cell and methods for producing taxol and its analogs

Info

Publication number: WO2026013281A1
Application number: PCT/EP2025/069959
Authority: WO
Inventors: Sotirios KAMPRANIS; Feiyan LIANG; Chi Zhang
Original assignee: Københavns Universitet
Current assignee: Københavns Universitet
Priority date: 2024-07-11
Filing date: 2025-07-11
Publication date: 2026-01-15
Anticipated expiration: 2027-01-11

Abstract

The present invention relates to the field of plant diterpenoid synthesis, and in particular host cells, methods and uses therefor. More specifically, the invention relates to production of taxol and/or other taxanes having a side chain comprising an N- benzoylated or an N-acetylated β-phenylalanine moiety, as well as enzymes and host cells useful for such production.

Description

Cell and methods for producing taxol and its analogs

Technical field

The present invention relates to the field of plant diterpenoid synthesis, and in particular host cells, methods and uses therefor. More specifically, the invention relates to production of taxol and/or other taxanes having a side chain comprising an N- benzoylated or an N-acetylated -phenylalanine moiety, as well as enzymes and host cells useful for such production.

Background

Paclitaxel (taxol), is one of the most effective anticancer drugs ever developed. It is used for the treatment of melanoma, ovarian, breast, bladder, prostate and esophageal cancer (Cragg, G. M., 1998).

However, its current production is particularly costly, pressing for the development of sustainable and cost-effective production methods. Production of paclitaxel in microbial or plant cell factories will reduce its cost, increase its availability, and enable the efficient synthesis of derivatives with improved pharmacological properties. Nevertheless, progress in this direction has been hampered by incomplete knowledge of the biosynthetic pathway in the native host, the yew tree (Taxus spp.).

One of the structural elements important for the anti-cancer activity of taxol is the benzoyl group at the 3'-N position of the C13 side chain. An efficient enzyme that catalyses the addition of the benzoyl moiety to the 3'-N position of A/-debenzoyl-taxol in a non-native host has not yet been reported, which is one reason that the complete biosynthesis of taxol in a heterologous host has proven problematic. DBTNBT, an enzyme that has been reported to carry out this reaction and enable the synthesis of taxol in tobacco, is herein shown to be inefficient. Therefore, an efficient enzyme for the benzoylation of the 3 -N position of A/-debenzoyl-taxol is desirable to enable efficient biotechnological production of taxol in heterologous cells.

Summary

Hitherto taxol has been semi-synthesised due incomplete understanding of the biosynthesis, or lack of effective catalytic enzymes hampering de novo heterologous biosynthesis. The inventors of the present disclosure have realised efficient heterologous production of taxol and 10-deacetyl-taxol, both comprising the important benzoyl moiety on the 3'-N of the C13 side chain. This benzoylation is of great importance to the anti-cancer activity of taxol. Thus, the disclosure allows for efficient heterologous production of taxanes and taxoid compounds comprising a benzoyl group on the 3'-N position of the C13 side chain, in other words taxol and/or other taxanes or taxoid compounds having a side chain comprising an N-benzoylated. The disclosure also allows for efficient heterologous production of taxanes and taxoid compounds comprising a acetyl group on the 3'-N position of the C13 side chain, e.g. B7.

Thus, the disclosure allows for heterologous production of taxanes having a side chain comprising an N-benzoylated p-phenylalanine moiety.

In addition, the inventors have realized that by modification of the intracellular level of benzoyl-CoA and acetyl-CoA in the host cell, heterologous production of taxanes comprising an acetyl group on the 3'N position of the 013 side chain can also be realised. Thus, by adjusting the intracellular level of benzoyl-CoA and/or acetyl-CoA or the ratio between these two compounds, more or less amounts of either taxanes having an N-acetylated [3-phenylalanine moiety and taxanes having an N-acetylated [3- phenylalanine moiety can be produced.

In particular, the present disclosure provides novel host cells, enzymes, methods and uses for biosynthesis of taxanes comprising an N-benzoylated or an N-acetylated |3- phenylalanine moiety, for example taxol and/or 10-deacetyl-taxol that both comprises an N-benzoylated [3-phenylalanine moiety or the taxol analog B7 that comprises an N- acetylated [3-phenylalanine moiety.

It is a main aspect of the present disclosure to provide a host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 5.

It is also a main aspect of the present disclosure to provide a host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5, BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1 (C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61, BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 5.

A further main aspect is to provide methods for producing a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety, said method comprising the steps of: i. providing a host cell described herein; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N-benzoylated |3- phenylalanine moiety and/or said taxane having a side chain comprising an N- acetylated p-phenylalanine moiety. Preferably wherein said taxane having a side chain comprising a N-benzoylated [3-phenylalanine moiety comprises or consists of 10- deacetyl-taxol, and/or paclitaxel (taxol), and/or wherein said taxane having a side chain comprising an N-acetylated p-phenylalanine moiety comprises or consists of B7.

Production of aforementioned compounds may be obtained in multiple ways, for example by cultivating the host cell in the presence of one or more of benzoyl-CoA, acetyl-CoA, benzoic acid, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, [3-phenylalanine, [3-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10- deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol. Said compounds may be present in the cultivation medium, e.g. said compounds may be added to the cultivation medium. Alternatively, said host cells are capable of producing benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N- debenzoyl-taxol, a-phenylalanine, [3-phenylalanine, [3-phenylalanoyl-CoA, N- debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N- debenzoyl-2'-deoxy-taxol.

It is also an aspect of the disclosure to provide use of a benzoyl transferase from Taxus chinensis in a method for producing a taxane having a side chain comprising an N- benzoylated |3-phenylalanine moiety and/or a taxane having a side chain comprising N- acetylated p-phenylalanine moiety. In some embodiments, the T. chinensis benzoyl transferase is BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity thereto.

It is also an aspect of the present disclosure to provide use of benzoyl transferase in a method for producing a taxane having a side chain comprising an N-benzoylated p- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated P-phenylalanine moiety, wherein the benzoyl transferase is a benzoyl transferase from T. chinensis, from T. cuspidate, or BT6.1 as set forth in SEQ ID NO: 5, BT6.1 (T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61, BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

It is also an aspect of the present disclosure to provide a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto. For example, the nucleic acid construct may comprise or consist of SEQ ID NO: 11 or a homologue thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

Provided is also a compound having the structure (VII) (B7):

Structure (VII).

Provided is also cell cultures obtained by the methods, uses, and/or comprising the host cells described herein.

Furthermore, also provided is a fermentation liquid comprising a taxane having a side chain comprising an N-benzoylated -phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety, wherein said fermentation liquid is obtained by a method, comprised in a cell culture, and/or comprised within and/or secreted by a host cell described herein.

Provided is also compositions comprising a fermentation liquid and/or a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety, a taxane having a side chain comprising an N-acetylated -phenylalanine moiety, B7, and/or taxol obtained by a method and/or use described herein.

Further provided are methods for treating a disorder such as cancer, comprising administration of a medicament comprising a composition obtained by a method and/or a use described herein, said composition comprising a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety. Preferably said taxane having a side chain comprising an N-benzoylated -phenylalanine moiety comprises or consists of 10-deacetyl-taxol or taxol, and/or said taxane having a side chain comprising an N-acetylated p-phenylalanine moiety comprises or consists of B7.

Description of Drawings

Figure 1. Testing benzoyl transferase activity of DBTNBT from Taxus canadensis (TcaDBTNBT). a. Biochemical pathway for the production of N-debenzoyl-taxol, an immediate precursor of taxol, from baccatin III (BACIU, compound 1) in tobacco by expressing a phenylalanine aminomutase from T. canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G, SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from Taxus cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), and an oxidase from Taxus chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9). TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) has been reported to benzoylate N-debenzoyl- taxol to produce taxol by (Walker et al., 2002) and (Long et al., 2008). b. UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), and OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) revealed production of N-debenzoyl-taxol (compound 3, [M+H]+ 750.3120±0.01 , C40H47NO13) (chromatogram I). No detectable consumption of N- debenzoyl-taxol (compound 3) was observed, when TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) is co-infiltrated with PAM, CoAL(A312G), BAPT and OD3 (chromatogram II). c. Trace amounts of taxol are present in the extracts of the tobacco leaves, inferred from the peaks with the mass of taxol ([M+H]+ 854.3382±0.01, C47H51 NO14) in chromatogram I and chromatogram II. Trace amounts of taxol are detected in chromatogram I, where TcaDBTNBT was not present. Co-infiltrating TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) does not result in a detectable higher taxol peak (chromatogram II). In these experiments, the concentration of baccatin III (compound 1) fed into tobacco leaves after agro-infiltration was 200 mg/L. Chemically synthesized compounds (compound 2 (N-debenzoyl-2'-deoxy-taxol) and compound 3 (N-debenzoyl-taxol)) or commercially purchased compound 1 (baccatin III) and taxol were used as standards. P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) was coexpressed in all tobacco leaves to help suppress gene silencing.

Figure 2. Production of taxol ([M+H]+ 854.3382 0.01, C47H51 NO14) and an acetylated taxol analog, termed B7 herein, in tobacco leaves from baccatin III (BACIU; compound 1). a. Biochemical pathway for the production of taxol from baccatin III (BACIU, compound 1) in tobacco (N. benthamiana) leaves by expressing a phenylalanine aminomutase from Taxus canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from Taxus cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from Taxus chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 from T. chinensis (SEQ ID NO: 5 encoded by SEQ ID NO: 11) b. UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (A/. benthamiana) leaves transiently expressing taxol pathway enzymes. The extract of tobacco leaves only expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) showed production of N- debenzoyl-taxol but did not show taxol production above background levels (chromatogram I). The extract of tobacco leaves expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) revealed the production of considerable amounts of taxol ([M+H]+ 854.3382±0.01 , C47H51 NO14) or B7 (structure VII, chromatogram II). In chromatogram II, N-debenzoyl-taxol (compound 3, [M+H]+ 750.3120±0.01 , C40H47NO13) is dramatically consumed. In these experiments, the concentration of baccatin III (compound 1) fed into tobacco leaves after agro-infiltration was 200 mg/L. P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) was co-expressed in all samples to help suppress gene silencing. c. Quantification of taxol production. Taxol production in tobacco expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) (‘BT6.1’) is 280±30 times higher than that in tobacco expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), and TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24). To determine the relative taxol production, taxol peak area values were normalized to the taxol peak area value of the extract (sample) containing TcaDBTNBT. The latter was set to 1.

Figure 3. Structure derivation of the acetylated taxol analog referred to herein as B7. Mass spectra of taxol and B7 obtained from extracts of tobacco leaves expressing PAM, BAPT, CoAL(A312G), OD3 and BT6.1 , and a taxol standard. Structures of observed fragments F1 and F2 were predicted based on the structure of taxol and the exact mass of F1 and F2. In a similar way, F3 was predicted to contain an acetyl group instead of a benzoyl group at the 3'N position. Therefore, B7 was predicted to be an N- acetylated compound, from compound 3, with the structure shown here.

Figure 4. Production of 10-deacetyl-taxol ([M+H]+ 812.3277±0.01, C45H49NO13) in tobacco, a. Biochemical pathway for the production of 10-deacetyl-taxol in tobacco by expression of a phenylalanine aminomutase from Taxus canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G, SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from T. cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from T. chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9) and the benzoyl transferase BT6.1 from T. chinensis (SEQ ID NO: 5 encoded by SEQ ID NO: 11). b. A UPLC- HRMS chromatogram (EIC, positive mode) of methanol extracts of tobacco (N. benthamiana) leaves transiently expressing PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) revealed the production of 10- deacetyl-taxol (Chromatogram II). The extract of tobacco leaves expressing only PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), and OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), but not BT6.1 , revealed no production of 10- deacetyl-taxol (Chromatogram I). Instead, 10-deacetyl-N-debenzoyl-taxol (compound 6, [M+H]+708.3015±0.01, C38H45NO12), the precursor of 10-deacetyl-taxol, accumulated. In these experiments, the concentration of 10-deacetyl-baccatin III (compound 4) fed into tobacco leaves after agro-infiltration was 200 mg/L. Chemically synthesized compound 6 and 10-deacetyl-N-debenzoyl-2'-deoxy-taxol (compound 5) or commercially purchased compound 4 were used as standard. Taxus baccata bark extract shows the presence of 10-deacetyl-taxol. P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) was co-expressed in all samples to help suppress gene silencing.

Figure 5. Production of taxol ([M+H]+ 854.3382±0.01 , C47H51 NO14) and its analog B7 ([M+H]+ 792.3226±0.01, C42H49NO14) in tobacco from 10-deacetyl-baccatin III. a. Biochemical pathway for the production of taxol and its analog B7 in tobacco by expressing a 10-deacetyl-baccatin lll-10-O-acetyl-transferase from T. cuspidata (TCL/DBAT, SEQ ID NO: 25 encoded by SEQ ID NO: 26), a phenylalanine aminomutase from T. canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from T. cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from T. chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9) and the benzoyl transferase BT6.1 from Taxus chinensis (SEQ ID NO: 5 encoded by SEQ ID NO: 11). b. UPLC-HRMS chromatogram (EIC, positive mode) of methanol extracts of tobacco (A/, benthamiana) leaves transiently expressing a 10-deacetyl-baccatin 111-10-O-acetyl- transferase from T. cuspidata (TcuDBAT, SEQ ID NO: 25 encoded by SEQ ID NO: 26), a phenylalanine aminomutase from T. canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G, SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from T. cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from T. chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9), and the benzoyl transferase BT6.1 from T. chinensis (SEQ ID NO: 5 encoded by SEQ ID NO: 11) reveal the production of taxol ([M+H]+ 854.3382 0.01, C47H51 NO14) or B7 ([M+H]+ 792.322610.01, C42H49NO14). In this experiment, the concentration of 10-deacetyl-baccatin III (compound 4) fed into tobacco leaves after agro-infiltration was 200 mg/L. Chemically synthesized compounds (compound 2 and compound 3) or commercially purchased baccatin III (compound 1) and compound 4 and taxol were used as standards. P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) was co-expressed in all samples to help suppress gene silencing.

Figure 6. a. Scheme of benzoyl-CoA biosynthetic pathway containing four enzymes, PAL (phenylalanine ammonia-lyase 2, SEQ ID NO: 28 encoded by SEQ ID NO: 33), CNL (cinnamic acid:CoA ligase, SEQ ID NO: 29 encoded by SEQ ID NO: 34), CHD (cinnamoyl-CoA hydratase-dehydrogenase, SEQ ID NO: 30 encoded by SEQ ID NO: 35) and Kat1 (3-ketoacyl CoA thiolase, SEQ ID NO: 31 encoded by SEQ ID NO: 36). b. Co-infiltrating four benzoyl-CoA pathway enzymes (“Benzoyl-CoA pathway boost”), PAL, CNL, CHD, and Kat1 together with the taxol pathway enzymes PAM, BAPT, CoAL(A312G), OD3, and BT6.1 resulted in a shift in the taxoid product profile from B7 to taxol compared to the profile of extracts (samples) from tobacco leaves only expressing PAM, BAPT, CoAL(A312G), OD3, and BT6.1 (i.e. “No benzoyl-CoA pathway boost”). 3-oxo-3-phenylpropanoyl-CoA has been abbreviated as 3O3PP-CoA.

Figure 7. Production of taxol ([M+H]+ 854.3382±0.01 , C47H51 NO14) and its analog B7 in S. cerevisiae. a. Biochemical pathway for the production of taxol and its analog B7 from baccatin III (BACIU, compound 1) in S. cerevisiae by expressing a coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from T. cuspidata which is fused with a maltose binding protein through a four amino acid linker IGGG (MBPig3BAPT, SEQ ID NO: 27 encoded by SEQ ID NO: 32), an oxidase from T. chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11). b) UPLC-HRMS chromatograms (EIC, positive mode) of ethyl acetate extracts of S. cerevisiae cells expressing different enzymes of the taxol biosynthetic pathway and supplied with 100 mg/L baccatin III (compound 1) and 100 mg/L p-phenylalanine. Chromatogram I: Analysis of extract of S. cerevisiae strains only expressing CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), MBPig3BAPT (SEQ ID NO: 27 encoded by SEQ ID NO: 32), and OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) reveals no production of taxol or B7 (negative control). Instead, N-debenzoyl-taxol (compound 3, [M+H]+ 750.3120 0.01 , C40H47NO13), the substrate of BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11), accumulated. Chromatogram //. Analysis of extract of S. cerevisiae cells co-expressing CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), MBPig3BAPT (SEQ ID NO: 27 encoded by SEQ ID NO: 32), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) reveal the production of taxol ([M+H]+ 854.338210.01 , C47H51 NO14) and its analog B7 ([M+H]+ 792.322610.01, C42H49NO14). Chromatogram III: Analysis of extract of S. cerevisiae cells expressing CoAL(A312G), MBPig3BAPT, OD3, and BT6.1 together with four additional enzymes for overproduction of benzoyl-CoA, i.e. PAL (phenylalanine ammonia-lyase 2, SEQ ID NO: 28 encoded by SEQ ID NO: 33), CNL (cinnamic acid:CoA ligase, SEQ ID NO: 29 encoded by SEQ ID NO: 34), CHD (cinnamoyl-CoA hydratase-dehydrogenase, SEQ ID NO: 30 encoded by SEQ ID NO: 35) and Kat1 (3-ketoacyl CoA thiolase, SEQ ID NO: 31 encoded by SEQ ID NO: 36), resulted in an increase in the levels of taxol and a reduction in the levels of the N- acetylated analog B7 compared to corresponding S. cerevisiae strains not overexpressing PAL, CNL, CHD and Kat1. Chromatogram IV Chemically synthesized compounds 2 and 3 or commercially available compound 1 and taxol were used as standards.

Figure 8. Production of taxol ([M+H]+ 854.3382±0.01 , C47H51 NO14) and its analog B7 in E. coli. a. Biochemical pathway for the production of taxol and its analog B7 from baccatin III (BACIU, compound 1) in E. coli by expressing a coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from T. cuspidata which is fused with a maltose binding protein through a four amino acid linker IGGG (MBPig3BAPT, SEQ ID NO: 27 encoded by SEQ ID NO: 32), an oxidase from T. chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9), and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11). b) UPLC-HRMS chromatograms (EIC, positive mode) of ethyl acetate extracts of E. coli cells expressing different enzymes of the taxol biosynthetic pathway and supplied with 100 mg/L baccatin III (compound 1), 100 mg/L p-phenylalanine, and 100 mg/L benzoic acid. Chromatogram I: Analysis of extract of E. coli strains only expressing CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT fused with maltose binding protein MBP using an IGGG amino acid linker (MBPig3BAPT, SEQ ID NO: 27 encoded by SEQ ID NO: 32), and OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) revealed that these cells do not produce taxol or B7 (negative control).

Chromatogram II: Analysis of extract of E. coli cells co-expressing CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT fused with maltose binding protein MBP through an IGGG amino acid linker (MBPig3BAPT, SEQ ID NO: 27 encoded by SEQ ID NO: 32), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), and the benzoyl transferase BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) revealed the production of B7.

Chromatogram III: Benzoyl-CoA is a co-substrate of BT6.1. E.coli cells do not have a high content of benzoyl-CoA. Introducing one additional enzyme, AtBZOI (a benzoyl- CoA ligase, SEQ ID NO: 49 encoded by SEQ ID NO: 52) (Kliebenstein et al., 2007), enables E.coli to convert exogenous benzoic acid into benzoyl-CoA. Under this condition, E. coli cells expressing BT6.1 produce primarily taxol ([M+H]+ 854.3382 0.01 , C47H51 NO14) and considerably lower amounts of the N-acetylated analog B7 compared to corresponding cells not expressing AtBZOI .

Chromatogram IV Commercially available taxol is used as a standard. Figure 9. Functional homologues of BT6.1 carrying substitutions at amino acid positions 12, 15, 24, 29, 36, 44, 56, 62, 68, 165, 169, 171, 185, 187, 189, 199, 227, 239, 241 , 286, 294, 295, 314, 324, 328, 346, 362, 369, 372, 379, 398, and 432 still retain their ability to synthesize taxol and compound B7. a) Biochemical pathway for the production of taxol and its analog B7 in tobacco by expressing a 10-deacetyl-baccatin lll-10-O-acetyl-transferase from Taxus cuspidata (DBAT, SEQ ID NO: 25 encoded by SEQ ID NO: 26), a phenylalanine aminomutase from Taxus canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from Taxus cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from Taxus chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9) and a transferase from Taxus chinensis (BT6.1, SEQ ID NO: 5 encoded by SEQ ID NO: 11). b) and c) UPLC-HRMS chromatogram (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing a 10-deacetyl-baccatin lll-10-O-acetyl-transferase from Taxus cuspidata (DBAT, SEQ ID NO: 25 encoded by SEQ ID NO: 26), a phenylalanine aminomutase from Taxus canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), a coenzyme A ligase mutant (CoAL(A312G, SEQ ID NO: 2 encoded by SEQ ID NO: 8), an amino phenylpropanoyl transferase from Taxus cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), an oxidase from Taxus chinensis (OD3, SEQ ID NO: 3 encoded by SEQ ID NO: 9), and a transferase from Taxus chinensis (BT6.1, SEQ ID NO: 5 encoded by SEQ ID NO: 11), BT6.1(T294N) (SEQ ID NO: 57 encoded by SEQ ID NO: 56), BT6.1(C369W) (SEQ ID NO: 59 encoded by SEQ ID NO: 58), BT6.1(L199M) (SEQ ID NO: 61 encoded by SEQ ID NO: 60), BT6.1 (L199M, A372T) (SEQ ID NO: 63 encoded by SEQ ID NO: 62), BT6.1_21Xmut (SEQ ID NO: 65 encoded by SEQ ID NO: 64) or BT6.1_17Xmut (SEQ ID NO: 67 encoded by SEQ ID NO: 66) reveal the production of taxol ([M+H]+ 854.3382±0.01, C47H51 NO14) and B7 ([M+H]+ 792.3226±0.01, C42H49NO14). In this experiment, the concentration of 10- deacetyl-baccatin III (compound 4) fed into tobacco leaves after agro-infiltration was 200 mg/L. Chemically synthesized compounds (compound 2 and compound 3) or commercially purchased compound 1 and compound 4 and taxol were used as standards. P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) was co-expressed in all samples to help suppress gene silencing. Detailed description

Definitions

The term “as set forth in” is herein used as equivalent to “as set out in”, “as described in”, “as depicted in”, “being the same as set forth in SEQ ID NO: 5” herein means, that BT6.1 has the sequence of SEQ ID NO: 5. Another example is taxol as set forth in structure (VI)”, which herein implies that structure (VI) is the structure of the compound taxol. It follows that “as set forth in” denotes that something “is the same as” and is therefore to be construed as “being limited to”.

The term “in the presence of” with respect to a compound herein refers to that said compound is available to a host cell, either because it is provided to the host cell and/or because it is synthesised by said host cell. For example, “a host cell cultivated in the presence of baccatin III” or “in the presence of “baccatin III” herein implies that baccatin III is either provided to the host cell, such as supplied to or comprised in the cultivation medium, or that the host cell is producing or is capable of producing baccatin III.

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.

The terms “incubating” and “cultivating” are used interchangeably herein, when used in respect of host cells, and refers to maintaining host cells under culture conditions, which allow the cells to grow. Preferably, said culture conditions allow expression of the enzyme(s) encoded by any heterologous nucleic acids, such as the gene(s), contained in said host cells. Preferably, the host cells are incubated under culture conditions allowing said host cells to produce said taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety, for example 10-deacetyl-taxol or taxol, and/or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety, for example B7. In embodiments where the host cell is contained within a multicellular organism (e.g. a plant), “cultivating” or “incubation” refers to maintaining said multicellular organisms under conditions allowing said multicellular organism to grow, e.g. cultivating said plant. In embodiments where the host cell is a unicellular organism, “cultivating” or “incubating” refers to maintaining said unicellular organism under conditions allowing said unicellular organism to grow and/or multiply. The term "enzyme" as used herein refers to proteins or polypeptides, which are capable of catalysing biochemical reactions. Further, unless context dictates otherwise, as used herein "enzyme" includes protein fragments that retain the relevant catalytic activity, and may include artificial enzymes synthesized to retain the relevant catalytic activity.

The term "functional homologue" of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion. The term "homologue" of a nucleic acid encoding a polypeptide, refers to a nucleic acid comprising said nucleic acid sequence with the proviso that one or more nucleobases are substituted, deleted, added, and/or inserted, and which nucleic acid encodes a polypeptide, which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion as the polypeptide encoded by said nucleic acid. Nucleic acids or nucleic acid sequence may also be referred to as polynucleotides and the terms are used interchangeable herein. Preferably, a functional homologue shares at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence. Preferably, a homologue shares at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said nucleic acid sequence.

The term “heterologous nucleic acid” refers to a nucleic acid, which is not endogenous to a host cell. Preferably, a “heterologous nucleic acid” has been inserted into a host cell or into a progenitor of the host cell, e.g. by recombinant or transgenic methods. The respective protein or RNA encoded by a heterologous nucleic acid is also referred to as "heterologous”. The heterologous nucleic acid may be part of a non-integrated nucleic acid, e.g. a vector, including but not limited to a plasmid. Preferably, the heterologous nucleic acid(s) are integrated into the host cell genome.

The term "host cell" refers to a cell, which comprises one or more heterologous nucleic acids. The term "polypeptide" as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length. As is known to those skilled in the art, polypeptides may be processed and/or modified, and the term polypeptide may refer to both unmodified or modified polypeptides.

The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleic acid sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment. For purposes of the present disclosure, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.

For purposes of the present disclosure, the sequence identity between two nucleic acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment). Sequence identity is calculated over the entire length of the reference sequence.

The term “taxane” herein comprises a class of diterpenoid compounds comprising a common core skeleton set forth in (I): and wherein the core may further be substituted, preferably the core may be further substituted at positions 1 , 2, 4, 5, 7, 9, 10, 11, 13, and/or 20. With regards to the structure set forth in (I), the dashed lines indicates either a single or a double bond, with the proviso that only one of the bonds between position 11 and 12 or position 12 and 13 may be a double bond simultaneously and only one of the bonds between position 4 and 5 or position 4 and 20 may be a double bond simultaneously. The atom at position 20 may be carbon (C) or oxygen (O) and may further be substituted. In particular, the atoms 4, 5 and 20 may be connected to form a ring, preferably an oxetane ring. Taxoids are taxadiene-derived diterpenoids. A preferred type of taxanes are taxoid compounds and/or taxoids. Taxanes have various structures and may be substituted with different groups, such as different functional groups. Non-limiting examples of taxanes of structure (I) are taxol (paclitaxel, VI), N-debenzoyl-taxol (II), N- debenzoyl-2'-deoxy-taxol (III), 10-deacetyl-N-debenzoyl-2'-deoxy-taxol (IV), 10- deacetyl-N-debenzoyl-taxol (V), and/or baccatin III. Unless otherwise specified the numbering of atoms of taxanes, such as a taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety, a taxane having a side chain comprising an N- acetylated [3-phenylalanine moiety and/or a taxane having a side chain comprising a [3- phenylalanine moiety, used herein is as indicated in formula (I).

In agreement with the above definition, the term “taxane having a side chain comprising a |3-phenylalanine moiety” herein refers to a taxane of structure (I) substituted with a side chain comprising a (3-phenylalanine moiety, preferably at position 13, i.e. at C13. Herein [3-phenylalanine moiety refers to [3-phenylalanine covalently bound to another moiety. The structure of free p-phenylalanine is provided in Figure 1a. The terms “side chain comprising a (3-phenylalanine moiety”, “side chain comprising (3-phenylalanine”, and “(3-phenylalanine side chain”, are used interchangeably herein. Furthermore, the terms C13 side chain, C13 side chain comprising p-phenylalanine, and C13 side chain comprising a p-phenylalanine moiety all refer to a side chain comprising a P-phenylalanine moiety positioned at C13 of a taxane of structure (I). If nothing else is specified, the term “taxane side chain” is also used to refer to a C13 side chain comprising a (3-phenylalanine moiety.

The numbering of atoms in taxanes comprising a C13 side chain comprising a (3- phenylalanine moiety is depicted in structure (VI) below. The skilled person will appreciate that even though taxanes comprising a C13 side chain comprising a [3- phenylalanine moiety may be differentially substituted compared to structure (VI), the indicated numbering of atoms still applies.

In accordance with this, the term “taxane having a side chain comprising an N- benzoylated p-phenylalanine moiety” herein refers to a taxane of structure (I) substituted with a side chain comprising an N-benzoylated (3-phenylalanine moiety, preferably at position 13, i.e. at C13, of structure (I), wherein position 3'-N, also sometimes referred to as position C3'N, position 3' N, position N 3', position 3'N and/or the C3' amino group (amine), of said side chain is benzoylated. Thus, the terms side chain comprising an N-benzoylated p-phenylalanine moiety, side chain comprising an N-benzoylated (3-phenylalanine moiety, an N-benzoylated (3-phenylalanine side chain, and/or N-benzoylated taxane side chain all refer to a side chain comprising an N- benzoylated (3-phenylalanine moiety, preferably positioned at C13 of a taxane of structure (I), preferably position 3'-N, also sometimes referred to as position C3'N, position 3' N, position N 3', position 3'N and/or the C3' amino group (amine), of said side chain is benzoylated. Preferably said side chain comprising an N-benzoylated (3- phenylalanine moiety is a side chain comprising a 3'N-benzoylated-2'C-hydroxylated (3- phenylalanine moiety. Furthermore, the terms benzoylated C13 side chain, C13 side chain comprising benzoylated (3-phenylalanine, and/or C13 side chain comprising a benzoylated (3-phenylalanine moiety all refer to a side chain comprising an N- benzoylated |3-phenylalanine moiety positioned at C13 of a taxane of structure (I).

In accordance with this, the term “taxane having a side chain comprising an N- acetylated 0-phenylalanine moiety” herein refers to a taxane of structure (I) substituted with a side chain comprising an N-acetylated |3-phenylalanine moiety, preferably at position 13, i.e. at C13, of structure (I), wherein position 3 -N, also sometimes referred to as position C3'N, position 3' N, position N 3', position 3'N and/or the C3' amino group (amine), of said side chain is acetylated. Thus, the terms side chain comprising an N- acetylated 0-phenylalanine moiety, side chain comprising an N-acetylated 0- phenylalanine moiety, an N-acetylated 0-phenylalanine side chain, and/or N-acetylated taxane side chain all refer to a side chain comprising an N-acetylated 0-phenylalanine moiety, preferably positioned at C13 of a taxane of structure (I), preferably position 3'- N, also sometimes referred to as position C3'N, position 3' N, position N 3', position 3’N and/or the C3' amino group (amine), of said side chain is acetylated. Preferably said side chain comprising an N-acetylated 0-phenylalanine moiety is a side chain comprising a 3'N-acetylated-2'C hydroxylated 0-phenylalanine moiety. Furthermore, the terms acetylated C13 side chain, C13 side chain comprising acetylated 0- phenylalanine, and/or C13 side chain comprising an acetylated 0-phenylalanine moiety all refer to a side chain comprising an N-acetylated 0-phenylalanine moiety positioned at C13 of a taxane of structure (I).

The term “N-debenzoyl-taxol” refers to the chemical structure as set forth in (II): “Bz” of structure (II) denotes a benzoyl group and “Ac” denotes an acetyl group. N- debenzoyl-taxol may also be referred to as compound 3 herein, and the terms are used interchangeably.

The term “N-debenzoyl-2'-deoxy-taxol” refers to the chemical structure as set forth in (III):

“Bz” of structure (III) denotes a benzoyl group and “Ac” denotes an acetyl group. N- debenzoyl-2'-deoxy-taxol may also be referred to as compound 2 herein, and the terms are used interchangeably. The term “10-deacetyl-N-debenzoyl-2'-deoxy-taxol” refers to the chemical structure as set forth in (IV):

“Bz” denotes a benzoyl group and “Ac” denotes an acetyl group of structure (IV). 10- deacetyl-N-debenzoyl-2'-deoxy-taxol may also be referred to as compound 5 herein, and the terms are used interchangeably. The term “10-deacetyl-N-debenzoyl-taxol” refers to the chemical structure as set forth in (V):

“Bz” denotes a benzoyl group and “Ac” denotes an acetyl group of structure (V). 10- deacetyl-N-debenzoyl-taxol may also be referred to as compound 6 herein, and the terms are used interchangeably.

The term “taxol” herein refers to the compound of the chemical structure as set forth in (VI): Arrow pointing to the nitrogen referred to as 3 -N. “Bz” denotes a benzoyl group and “Ac” denotes an acetyl group of structure (VI). Taxol may also be referred to as paclitaxel and the terms are used interchangeably.

The term “B7” herein refers to the compound of the chemical structure as set forth in (VII):

“Bz” denotes a benzoyl group and “Ac” denotes an acetyl group of structure (VII). B7 may also be referred to as taxol B7 and the terms may be used interchangeably.

The term “10-deacetyl-taxol” herein refers to the compound of the chemical structure as set forth in (VIII):

“Bz” denotes a benzoyl group and “Ac” denotes an acetyl group of structure (VIII).

Benzoyl transferase

The present disclosure provides host cells, methods, uses and polypeptides for production of diterpenoids, notable taxanes having a side chain comprising an N- benzoylated |3-phenylalanine moiety, such as 10-deacetyl-taxol or taxol, or taxanes having a side chain comprising an N-acetylated 0-phenylalanine moiety, such as B7. The host cell to be used with the present disclosure comprises a heterologous nucleic acid encoding an enzyme capable of benzoylating or acetylating 3'-N of the C13 side chain, i.e. the so-called taxane side chain, or the side chain comprising a 0- phenylalanine moiety, of taxanes. Production of taxanes having a side chain comprising an N-benzoylated 0- phenylalanine moiety, for example 10-deacetyl-taxol or taxol, and/or taxanes having a side chain comprising an N-acetylated 0-phenylalanine moiety, for example B7, may be obtained by expression of a benzoyl transferase, either alone or together with one or more polypeptides of different activity as described herein, for example below in the sections “Host cell” or “Nucleic acids and expression systems”, in a host cell of the present disclosure. The benzoyl transferase preferably has the enzyme activity described in this section and may also sometimes be referred to as a benzoyltransferase, N-benzoyl transferase, N-benzoyltransferase, C13-side-chain N- benzoyltransferase, N-acyl-transferase, or alike terms.

The benzoyl transferase described herein is capable of catalysing formation of a benzoyl-group (Bz) at position 3'-N of the side chain comprising a 0-phenylalanine moiety, i.e. said benzoyl transferase is capable of benzoylating position 3 -N of the C13 side chain. In other words, said benzoyl transferase is capable of catalysing formation of taxanes having a side chain comprising an N-benzoylated 0-phenylalanine moiety from taxanes having a side chain comprising a 0-phenylalanine moiety.

Benzoyl-CoA is a preferred co-substrate of the benzoyl transferase when catalysing formation of a benzoyl-group (Bz) at position 3'-N of the side chain comprising a 0- phenylalanine moiety. Thus, it is to be understood throughout the present disclosure, even if not explicitly disclosed, that benzoyl-CoA preferably is present when said benzoyl transferase is benzoylating position 3'-N of the side chain comprising a 0- phenylalanine moiety of taxanes. Benzoyl-CoA may be added to the cultivation medium of said host cell and/or produced by the host cell. Preferably, the host cell is capable of overproducing benzoyl-CoA, and such host cells are described herein below.

The benzoyl transferase described herein may be capable of catalysing formation of a acetyl-group (Ac) at position 3'-N of the side chain comprising a 0-phenylalanine moiety, i.e. said benzoyl transferase is capable of acetylating position 3 -N of the C13 side chain. In other words, said benzoyl transferase is capable of catalysing formation of taxanes having a side chain comprising an N-acetylated 0-phenylalanine moiety from taxanes having a side chain comprising a 0-phenylalanine moiety. Acetyl-CoA is a preferred co-substrate of the benzoyl transferase when catalysing formation of an acetyl-group (Ac) at position 3'-N of the side chain comprising a 0- phenylalanine moiety. Thus, it is to be understood throughout the present disclosure, even if not explicitly disclosed, that acetyl-CoA preferably is present when said benzoyl transferase is acetylating position 3'-N of the side chain comprising a 0-phenylalanine moiety of taxanes. Acetyl-CoA may be added to the cultivation medium of said host cell and/or produced by the host cell. Typically, acetyl-CoA is produced by the host cell, and production may be increased in various ways known to the skilled person.

BT6.1

The host cell to be used with the present disclosure may preferably comprise a heterologous nucleic acid encoding an enzyme capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety. The disclosure also provides such enzyme as well as method using same.

It is preferred that said enzyme when capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety is capable of catalysing formation of taxol as set forth in structure (VI). In other words, it is preferred that said enzyme is capable of catalysing the following reaction (A):

In other words, the enzyme, preferably a benzoyl transferase, is capable of catalysing benzoylation of position 3'-N of the C13 side chain of taxanes may be capable of catalysing reaction A outlined above.

In another preferred embodiment, it is preferred that said enzyme when capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated 0- phenylalanine moiety is capable of catalysing formation of 10-deacetyl-taxol as set forth in structure (VIII). In other words, it is preferred that said enzyme is capable of catalysing the following reaction (B):

10-deacetyl- 10-deacetyl-taxol

N-debenzoyl- taxol

In other words, the enzyme, preferably a benzoyl transferase, is capable of catalysing benzoylation of position 3'-N of the C13 side chain of taxanes may be capable of catalysing reaction B outlined above.

In a another preferred embodiment, it is preferred that said enzyme when capable of catalysing formation of a taxane having a side chain comprising an N-acetylated 0- phenylalanine moiety is capable of catalysing formation of B7 as set forth in structure (VII). In other words, it is preferred that said enzyme is capable of catalysing the following reaction (C):

N-debenzoyl-taxol B7

(C)

In preferred embodiment, said enzyme capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety is capable of catalysing all of reactions A, B and C outlined above. In preferred embodiments, said enzyme is a benzoyl transferase. Thus, a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety may be taxol as set forth in structure (VI). In other embodiments, a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety may be 10- deacetyl-taxol as set forth in structure (VIII). In some embodiments, a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety may be B7 as set forth in structure (VII).

The enzyme capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety may be any useful enzyme with above-mentioned activities, in particular said enzyme may be a benzoyl transferase. In some embodiments, the enzyme is an N-benzoyltransferase, such as a C13-side-chain N-benzoyltransferase. The enzyme capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety may be derived from any suitable source, but in a preferred embodiment, said enzyme is an enzyme from Taxus chinensis or Taxus cuspidata, even more preferably it is an enzyme from Taxus chinensis. Thus, the enzyme capable of catalysing formation of a taxane having a side chain comprising an N-benzoylated |3- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety may be a benzoyl transferase from Taxus chinensis or Taxus cuspidata. The benzoyl transferase may be an N-benzoyltransferase, such as a 013- side-chain N-benzoyltransferase, from T. chinensis or T. cuspidata. In preferred embodiments, said benzoyl transferase is BT6.1 as disclosed herein or a functional homologue thereof and/or a functional variant thereof. In other preferred embodiments, said benzoyl transferase is BT6.1(L199M), BT6.1(L199M, A372T), BT6.1_21Xmut or BT6.1_17Xmut as disclosed herein or functional homologues thereof.

In preferred embodiments of the present disclosure, the host cell comprises a heterologous nucleic acid encoding the benzoyl transferase BT6.1. Said BT6.1 is preferably BT6.1 of SEQ ID NO: 5 or a functional homologue thereof. The person skilled in the art will appreciate that BT6.1 or a functional homologue thereof preferably has the ability to convert N-debenzoyl-taxol to taxol and/or 10-deacetyl-N-debenzoyl to 10-deacetyl-taxol as outlined in reactions A and B, respectively, depicted herein above. BT6.1 may also have the ability to convert the ability to convert N-debenzoyl-taxol to B7 as outlined in reaction C depicted herein above.

A functional homologue of BT6.1 of SEQ ID NO: 5 preferably has at least 70% sequence identity, preferably at least 75%, preferably at least 80%, preferably at least 81% sequence identity, preferably at least 82% sequence identity, preferably at least 83% sequence identity, preferably at least 84% sequence identity, preferably at least 85% sequence identity, preferably at least 86% sequence identity, preferably at least 87% sequence identity, preferably at least 88% sequence identity, preferably at least 89% sequence identity, preferably at least 90% sequence identity, preferably at least 91% sequence identity, preferably at least 92% sequence identity, preferably at least 93% sequence identity, preferably at least 94% sequence identity, preferably at least 95% sequence identity, preferably at least 96% sequence identity, preferably at least 97% sequence identity, more preferred at least 98% sequence identity, more preferred at least 99% sequence identity to SEQ ID NO: 5.

More preferably, a functional homologue of BT6.1 of SEQ ID NO: 5 has at least 80% to SEQ ID NO: 5.

Even more preferably, a functional homologue of BT6.1 as set forth in SEQ ID NO: 5 has at least 95% sequence identity to SEQ ID NO: 5.

A functional homologue of BT6.1 may be identified by expressing said functional homologue in a host cell, and either purifying the produced enzyme to perform an in vitro enzyme activity assay or by performing an in vivo enzyme activity assay, in order to measure the conversion of N-debenzoyl-taxol to taxol, 10-deacetyl-N-debenzoyl to 10-deacetyl-taxol, and/or N-debenzoyl-taxol to B7 using standard techniques. For example, the activity may be measured as described in Example 1 to 9 or 10 and 11.

The heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof, may have any sequence encoding said protein comprising or consisting of BT6.1. In some embodiments, the nucleic acid encoding said protein comprising or consisting of BT6.1 is a nucleic acid as set forth in SEQ ID NO: 11 , or a homologue thereof encoding a functional homologue of BT6.1 having at least 70% sequence identity to the BT6.1 encoded by SEQ ID NO: 11.

BT6.1 functional homologues

As illustrated in Example 10 herein, the amino acid sequence of BT6.1 may for example vary at position/residues 12, 15, 24, 29, 36, 44, 56, 62, 68, 165, 169, 171 , 185, 187, 189, 199, 227, 239, 241 , 286, 294, 295, 314, 324, 328, 346, 362, 369, 372, 379, 398, and 432 compared to the sequence as set forth in SEQ ID NO: 5, while still maintaining the ability to catalyse synthesis of taxol and/or B7.

Thus, the functional homologue of BT6.1 may be as set forth in SEQ ID NO: 5, with the exception of having an amino acid substitution at one or more of the positions 12, 15, 24, 29, 36, 44, 56, 62, 68, 165, 169, 171 , 185, 187, 189, 199, 227, 239, 241 , 286, 294, 295, 314, 324, 328, 346, 362, 369, 372, 379, 398, and 432 compared to the sequence as set forth in SEQ ID NO: 5. Said substitution may be conservative or nonconservative. Thus in some embodiments the substitution is a conservative substitution. In some embodiment the substitution is a radical substitution. Thus, the functional homologue may have one or more mutations, such as an amino acid substitutions, in the region:

- from position 10 to position 70, such as from position 12 to position 68;

- from position 160 to position 200, such as from position 165 to position 199;

- from position 225 to position 245, such as from position 227 to position 241;

- from position 285 to position 300, such as from position 286 to position 295;

- from position 310 to position 400, such as from position 314 to position 398; or

- from position 430 to position 439, such as from position 432 to position 439, compared to the sequence of SEQ ID NO: 5. For example, functional homologues of BT6.1 (SEQ ID NO: 5) may have one or more amino acid substitutions:

- from position 10 to position 70;

- from position 165 to position 200;

- from position 225 to position 400; or

- from position 430 to position 435, compared to the sequence of SEQ ID NO: 5.

For example, the functional homologue of BT6.1 may be as set forth in SEQ ID NO: 5 with the exception of having one or more of the following mutations V12A, A15T, I24V, G29S, Y36C, Y44C, A56V, G62A, M68V, V165A, L169V, C171G, E185D, 1187V, N189K, L199M, I227F, C241S, P286H, T294N, T295A, L314S, I324V, N328S, E239D, I346M, F362L, C369W, A372T, A372V, L379M, P398S and T432S.

Non-limiting examples of functional homologues of BT6.1 of SEQ ID NO: 5 are BT6.1(T294N) of SEQ ID NO: 57, BT6.1(C369W) of SEQ ID NO: 59, BT6.1(L199M) of SEQ ID NO: 61 , BT6.1(L199M, A372T) of SEQ ID NO: 63, BT6.1_21Xmut of SEQ ID NO: 65 or BT6.1_17Xmut of SEQ ID NO: 67.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1(T294N) is a nucleic acid as set forth in SEQ ID NO: 56, or a homologue thereof encoding a functional homologue of BT6.1(T294N) having at least 80%, preferably at least 90%, more preferably at least 95% sequence identity to the BT6.1(T294N) encoded by SEQ ID NO: 56.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1(C369W) is a nucleic acid as set forth in SEQ ID NO: 58, or a homologue thereof encoding a functional homologue of BT6.1(C369W) having at least 80%, preferably a at least 90%, more preferably at least 95% sequence identity to the BT6.1(C369W) encoded by SEQ ID NO: 58.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1(L199M) is a nucleic acid as set forth in SEQ ID NO: 60, or a homologue thereof encoding a functional homologue of BT6.1(L199M) having at least 80%, preferably a at least 90%, more preferably at least 95% sequence identity to the BT6.1(L199M) encoded by SEQ ID NO: 60.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1(L199M, A372T) is a nucleic acid as set forth in SEQ ID NO: 62, or a homologue thereof encoding a functional homologue of BT6.1(L199M, A372T) having at least 80%, preferably a at least 90%, more preferably at least 95% sequence identity to the BT6.1(L199M, A372T) encoded by SEQ ID NO: 62.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1_21Xmut is a nucleic acid as set forth in SEQ ID NO: 64, or a homologue thereof encoding a functional homologue of BT6.1_21Xmut having at least 80%, preferably a at least 90%, more preferably at least 95% sequence identity to the BT6.1_21Xmut encoded by SEQ ID NO: 64.

In some embodiments, the nucleic acid encoding the protein comprising or consisting of BT6.1_17Xmut is a nucleic acid as set forth in SEQ ID NO: 66, or a homologue thereof encoding a functional homologue of BT6.1_17Xmut having at least 80%, preferably a at least 90%, more preferably at least 95% sequence identity to the BT6.1_17Xmut encoded by SEQ ID NO: 66.

Host cells

The present disclosure relates to host cells comprising one or more nucleic acids, such as one or more heterologous nucleic acids, encoding enzymes of the biosynthetic pathway towards taxol, i.e. taxoid pathway.

A main aspect of the present disclosure is to provide host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 5.

Thus, in a preferred embodiment the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 5.

Most preferred, the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, said host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) comprises a nucleic acid as set forth in SEQ ID NO: 11 or a homologue thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

Instead of BT6.1 (SEQ ID NO: 5), the protein encoded by the heterologous nucleic acid may be a functional homologue thereof, and the host cell expresses said functional homologue. Thus, in some embodiments, disclosed herein is host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1(T294N) as set forth in SEQ ID NO: 57 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 57.

In other embodiments, disclosed herein is host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1(C369W) as set forth in SEQ ID NO: 59 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 59.

In some embodiments, the present disclosure relates to host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1(L199M) as set forth in SEQ ID NO: 61 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 61.

In other embodiments, disclosed herein is host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 63.

In some embodiments, the present disclosure relates to host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1_21Xmut as set forth in SEQ ID NO: 65 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 65.

In other embodiments, the present disclosure relates to host cells comprising a heterologous encoding a protein comprising or consisting of BT6.1_17Xmut of SEQ ID NO: 67 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 67.

In another preferred embodiment, the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, wherein said host cell is capable of producing a a taxane having a side chain comprising an N- benzoylated |3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety. Preferably said taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety comprises or consists of 10- deacetyl-taxol or taxol, and/or said taxane having a side chain comprising an N- acetylated [3-phenylalanine moiety comprises or consists of B7.

In other embodiments, the host cell is capable of producing a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety, preferably wherein said taxane comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol), and/or said host cell is capable of producing a taxane having a side chain comprising an N- acetylated [3-phenylalanine moiety, preferably wherein said taxane comprises or consists of B7. In some embodiments, the host cell is capable of producing a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety in the presence of, such as when cultivated in the presence of, one or more precursor compound(s), for example benzoyl-CoA, acetyl-CoA, benzoic acid, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, [3-phenylalanine, p-phenylalanoyl-CoA, N- debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N- debenzoyl-2'-deoxy-taxol. Said precursor compounds may be provided to the host cell, such as in the cultivation medium or with respect to plant cells be infiltrated in the leaves. In other embodiments, the host cell is capable of producing benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, [3-phenylalanine, P-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol. Host cells capable of producing said compounds are described in the sections herein below.

In addition to the heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, the host cell may comprise one or more nucleic acids, such as native nucleic acids and/or heterologous nucleic acids, encoding one or more of the following: an amino phenylpropanoyl transferase (EC: 2.3.1) capable of converting baccatin III and P-phenylalanoyl-CoA into N-debenzoyl-2'-deoxy-taxol, and/or 10-deacetyl-baccatin III and P-phenylalanoyl-CoA into 10-deacetyl-N- debenzoyl-2'-deoxy-taxol; a phenylalanine aminomutase (EC: 5.4.3.11) capable of converting a- phenylalanine to [3-phenylalanine; a coenzyme A ligase (CoAL, EC: 6.2.1) capable of converting [3-phenylalanine to [3-phenylalanoyl-CoA; a 10-deacetyl-baccatin lll-10-O-acetyltransferase (DBAT, EC: 2.3.1) capable of converting 10-deacetyl-N-debenzoyl-2'-deoxy-taxol to N-debenzoyl-2'-deoxy- taxol, and/or 10-deacetyl-N-debenzoyl-taxol to N-debenzoyl-taxol, and/or 10- deacetyl-baccatin III to baccatin III; an oxidase, such as OD3, capable of converting N-debenzoyl-2'-deoxy-taxol to N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol to 10- deacetyl-N-debenzoyl-taxol. In addition to the heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, as well as one or more of the above nucleic acids, the host cell may comprise one or more nucleic acids, such as native nucleic acids and/or heterologous nucleic acids, encoding one or more of the following: a benzoyl ligase (EC: 6.2.1 .25) capable of converting benzoic acid to benzoyl- CoA; and/or a phenylalanine ammonia-lyase (EC: 4.3.1.24) capable of converting a- phenylalanine to trans-cinnamic acid; a cinnamic acid:CoA ligase (EC: 6.2.1) capable of converting trans-cinnamic acid to cinnamoyl-CoA; a cinnamoyl-CoA hydratase-dehydrogenase (EC: 1.13.11) capable of converting cinnamoyl-CoA to 3-oxo-3-phenylpropanoyl-CoA (303PP-CoA); and a 3-ketoacyl CoA thiolase (EC: 2.3.1.16) capable of converting 3O3PP-CoA into benzoyl-CoA.

These enzyme activities and host cells comprising said activities are described further herein below.

In some embodiments, the nucleic acid encoding a protein comprising or consisting of a protein of having a specific enzyme activity and/or having a specific sequence or a functional homologue thereof may be an endogenous nucleic acid or a heterologous nucleic acid.

It is preferred that the host cell is capable of producing GGPP, a-phenylalanine, baccatin III, and/or 10-deacetyl-baccatin III.

In some embodiments the host cell may further comprise one or more of the nucleic acids and/or nucleic acid sequences described in patent application PCT/EP2024/065772, and/or EP23386140.0 or in a patent application claiming priority thereof, such as patent application PCT/EP2024/088095. Organisms

In some embodiments of the present disclosure, said host cell is selected from the group of plant cells, yeast cells, bacterial cells and fungal cells.

In some embodiments the host cell is comprised within a multicellular organism. In such embodiments, only some of the cells of said multicellular organism may comprise heterologous nucleic acid(s) and/or heterologous polypeptide(s). It is however preferred that all cells of said multicellular organism are host cells that comprise the same nucleic acids, such as the same heterologous nucleic acids(s), and/or polypeptides, such as heterologous polypeptide(s).

In some embodiments of the present disclosure, said host cell is plant cells, such as plant cells comprised within a plant, within a part of a plant and/or within the seeds of said plant. Preferably, all cells of said plant or part thereof are host cells comprising the same nucleic acids, such as the same heterologous nucleic acids(s), and/or polypeptides, such as heterologous polypeptide(s).

In some embodiments of the present disclosure, the host cells are plant cells, such as plant cells comprised within a plant or within a part of a plant. In some embodiments, said plant cells are from a species of Nicotiana, such as Nicotiana benthamiana or Nicotiana tabacum and optionally are comprised within a plant of the species of Nicotiana. The person skilled in the art will appreciate that a “plant cell” as used within the present invention refers to a structural and physiological unit of a plant, e.g. a tobacco plant. The plant cell may be in form of a protoplast without a cell wall, an isolated single cell or a cultured cell, or as a part of higher organized unit such as but not limited to, plant tissue, a plant organ, or a whole plant.

In some embodiments of the present disclosure, the host cell is a yeast cell, such as yeast cell belonging to the genus of Saccharomyces, Pichia, Candida, Cryptococcus, Pichia (Komagataella), Lipomyces, Pseudozyma, Rhodosporidium, Rhodotorula, Trichosporon, Trigonopsis, Yarrowia or Saccharomycopsis, such as a yeast cell of the species Saccharomyces cerevisiae Yarrowia lipolytica, Hansenula polymorpha (Ogataea polymorpha), Rhodotorula toruloides or Pichia pastoris (Komagataella phaffii). The person skilled in the art will appreciate that a fungi or fungal cell(s) as used herein refers to any cell present within or derived from an organism belonging to the Kingdom Fungi. The methods are applicable to all fungi and fungal cells that are susceptible of genetic modifications. Furthermore, the person skilled in the art will appreciate that a “yeast cell” is herein defined to include the group consisting of small, unicellular organisms capable of growth and reproduction through budding or direct division (fission), or by growth as simple irregular filaments (mycelium). The yeast cell may be transformed or transfected with a heterologous vector for expression of a nucleic acid and/or nucleic acid sequence inserted into the heterologous vector. Examples of a yeast cell include, but are not limited to Saccharomyces cerevisiae, Yarrowia lipolytica, Hansenula polymorpha (Ogataea polymorpha), Rhodotorula toruloides and/or Pichia pastoris (Komagataella phaffii), commonly used for transfection and expression of heterologous proteins.

In some embodiments of the present disclosure, the host cell is a bacterial cell, such as a bacterial cell belonging to the genus of Escherichia, Bacillus, Corynebacterium, Pseudomonas or Streptomyces, such as a bacterial cell of the species Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas putida or Streptomyces sp.

The person skilled in the art will appreciate that a bacterial cell includes prokaryotic cells that may be propagated in culture. The bacterial cell may act as a host cell for the recombinant expression of heterologous polypeptide(s). The bacterial cell may be transformed, transfected or infected with a vector for expression of a nucleic acid and/or nucleic acid sequence inserted into the vector. Examples of suitable bacterial cells include, but are not limited to E. coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas putida and/or Streptomyces sp.

The host cell or a progenitor thereof may be prepared by any useful method available to the skilled person. For example, the heterologous nucleic acid(s) may be inserted into a cell by direct uptake, transduction, f-mating, transfection, transformation, bacterial infiltration or any other methods known in the art useful for creating recombinant host cells. Amino phenylpropanoyl transferase

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may also comprise a nucleic acid encoding an amino phenylpropanoyl transferase as described in this section and/or elsewhere.

The amino phenylpropanoyl transferase may be an amino phenylpropanoyl transferase falling within EC number 2.3.1. The amino phenylpropanoyl transferase may be capable of converting baccatin III and p-phenylalanoyl-CoA to N-debenzoyl-2'-deoxy- taxol. Said amino phenylpropanoyl transferase may also be capable of converting 10- deacetyl-baccatin III and -phenylalanoyl-CoA to 10-deacetyl-N-debenzoyl-2'-deoxy- taxol. Hence, a host cell comprising a nucleic acid encoding said amino phenylpropanoyl transferase may be capable of producing N-debenzoyl-2'-deoxy-taxol and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol.

In some embodiments the host cell further comprises a nucleic acid encoding amino phenylpropanoyl transferase. Said amino phenylpropanoyl transferase may in some embodiments be native to a Taxus cell, such as a Taxus cuspidata cell.

Thus, in some embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of BAPT as set forth in SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 1. In other embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of MBPig3BAPT as set forth in SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 27.

Said amino phenylpropanoyl transferase may be BAPT (SEQ ID NO: 1) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 1 .

The amino phenylpropanoyl transferase may also be linked, such as fused to a solubility tag. Said solubility tag may for example be maltose-binding protein, such as MBP as set forth in SEQ ID NO: 54 or a or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 54. The linker peptide may for example be the IG3-linker (IG3, SEQ ID NO: 55), i.e. IGGG, or another suitable, flexible linker. Thus, said amino phenylpropanoyl transferase may be MBPig3BAPT (SEQ ID NO: 27) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 27. The polypeptide MBPig3BAPT (SEQ ID NO: 27) is a fusion protein of the MBP solubility taq and the amino phenylpropanoyl transferase BAPT.

The nucleic acid encoding BAPT (SEQ ID NO: 1) or encoding a protein comprising or consisting of BAPT (SEQ ID NO: 1) or a functional homologue may be the nucleic acid as set forth in SEQ ID NO: 7 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 7. The nucleic acid encoding MBPig3BAPT (SEQ ID NO: 27) or encoding a protein comprising or consisting of MBPig3BAPT (SEQ ID NO: 27) or a functional homologue thereof may be the nucleic acid as set forth in SEQ ID NO: 32 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 32.

Phenylalanine aminomutase

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may also comprise a nucleic acid encoding a phenylalanine aminomutase as described in this section and/or elsewhere. Preferably, said host cell further comprises a nucleic acid encoding an amino phenylpropanoyl transferase as described herein above.

The phenylalanine aminomutase may be a phenylalanine aminomutase falling within EC number 5.4.3.11. The phenylalanine aminomutase may be capable of converting a- phenylalanine to p-phenylalanine.

In some embodiments the host cell further comprises a nucleic acid encoding a phenylalanine aminomutase. Said phenylalanine aminomutase may in some embodiments be native to a Taxus cell, such as a Taxus canadensis cell.

Thus, in some embodiments the host cell further comprises a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4, or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 4. Said amino phenylalanine aminomutase may be PAM (SEQ ID NO: 4) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 4.

The nucleic acid encoding PAM (SEQ ID NO: 4) or encoding a protein comprising or consisting of PAM (SEQ ID NO: 4) or a functional homologue may be the nucleic acid as set forth in SEQ ID NO: 10 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 10.

CoAL

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may further comprise a nucleic acid encoding a coenzyme A ligase (CoAL) as described in this section and/or elsewhere. Preferably, said host cell also comprises a nucleic acid encoding an amino phenylpropanoyl transferase and/or a nucleic acid encoding a phenylalanine aminomutase as described herein above.

Said CoAL may be a CoAL falling within EC number 6.2.1. The CoAL may be capable of converting p-phenylalanine to p-phenylalanoyl-CoA. Thus, a host cell comprising a nucleic acid encoding said CoAL may be capable of producing p-phenylalanoyl-CoA.

In some embodiments the host cell further comprises a nucleic acid encoding a protein comprising or consisting of CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, HcAAEI as set forth in SEQ ID NO: 51, or functional homologues of any of the aforementioned having at least 70% sequence identity to any of SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 51.

In some embodiments, said CoAL is a CoAL native to a Penicillium cell, such as a P. chrysogenum cell, a CoAL native to a Taxus cell, such as a T. chinensis cell, or a CoAL native to a Hypericum cell, such as a Hypericum calycinum cell. In other embodiments, said CoAL is CoAL(A312G) (SEQ ID NO: 2), TchiAAE5 (SEQ ID NO: 48), sbCoAL (SEQ ID NO: 50), HcAAEI (SEQ ID NO: 51), or functional homologues of any of the aforementioned having at least 70% sequence identity to any of SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 51 . The activity of HcAAEI (SEQ ID NO: 51) of converting -phenylalanine to - phenylalanoyl-CoA has been described elsewhere (Singh et al., 2020).

Disclosed herein is also a host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of sbCoAL as set forth in SEQ ID NO: 50 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 50. In some embodiments, the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of sbCoAL (SEQ ID NO: 50) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 50, wherein said host cell is capable of producing p-phenylalanoyl-CoA. Preferably, said host cell is capable of converting p-phenylalanine to p-phenylalanoyl-CoA. In some embodiments, said CoAL is the CoAL as set forth in GenBank accession no.: KAH9288255.1 or a functional homologue thereof having at least 70% sequence identity thereto.

CoAL(A312G) may also sometimes be referred to as CoAL_A312G and/or CoALA312G and the terms may be used interchangeably.

The nucleic acid encoding CoAL(A312G) (SEQ ID NO: 2) or a functional homologue thereof may be the nucleic acid as set forth in SEQ ID NO: 8 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 8.

DBAT

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may further comprise a nucleic acid encoding a 10-deacetyl-baccatin lll-10-O-acetyltransferase (DBAT) as described in this section and elsewhere herein. Preferably, said host cell also comprises one or more of a nucleic acid encoding a CoAL, a nucleic acid encoding an amino phenylpropanoyl transferase, and/or a nucleic acid encoding a phenylalanine aminomutase as described herein above. Said DBAT may be a DBAT falling within EC number 2.3.1. The DBAT may be capable of acetylating 10-deacetyl-baccatin III to baccatin III. The DBAT may also be capable of acetylating 10-deacetyl-taxol to taxol. The DBAT may be capable of acetylating 10- deacetyl-N-debenzoyl-2'-deoxy-taxol to N-debenzoyl-2 '-deoxy- taxol. The DBAT may be capable of acetylating 10-deacetyl-N-debenzoyl-taxol to N-debenzoyl-taxol. Hence, a host cell comprising a nucleic acid encoding said DBAT may be capable of producing baccatin III, taxol, N-debenzoyl-2 '-deoxy-taxol and/or N-debenzoyl-taxol in the presence of 10-deacetyl-baccatin III, 10-deacetyl-taxol, 10-deacetyl-N-debenzoyl-2'- deoxy-taxol and/or 10-deacetyl-N-debenzoyl-taxol, respectively. Said DBAT may be Tct/DBAT (SEQ ID NO: 25) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 25.

The activities of producing baccatin III from 10-deacetyl-baccatin III, and taxol from 10- deacetyl-taxol, by TcuDBAT has been described elsewhere (Li et al., 2016 and Walker et al., 2000).

In some embodiments, the host cell further comprises a nucleic acid encoding a DBAT. Said DBAT may in some embodiments be native to a Taxus cell, such as a Taxus cuspidata cell.

In some embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 25.

Said DBAT may be TcuDBAT (SEQ ID NO: 25) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 25.

The nucleic acid encoding TcuDBAT (SEQ ID NO: 25) or a functional homologue thereof may be the nucleic acid as set forth in SEQ ID NO: 26 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 26.

Oxidase

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may further comprise a nucleic acid encoding an oxidase as described in this section and elsewhere herein. Preferably, said host cell also comprises one or more of a nucleic acid encoding a CoAL, a nucleic acid encoding an amino phenyl propanoyl transferase, a nucleic acid encoding a phenylalanine aminomutase and/or a nucleic acid encoding a DBAT as described herein above.

Said oxidase may be an oxidase falling within EC number EC 1.14.11. The oxidase may be capable of converting N-debenzoyl-2'-deoxy-taxol to N-debenzoyl-taxol. The oxidase may be capable of converting 10-deacetyl-N-debenzoyl-2'-deoxy-taxol to 10- deacetyl-N-debenzoyl-taxol. Hence, a host cell comprising a nucleic acid encoding said oxidase may be capable of producing to N-debenzoyl-taxol and/or 10-deacetyl-N- debenzoyl-taxol in the presence of N-debenzoyl-2'-deoxy-taxol and/or 10-deacetyl-N- debenzoyl-2'-deoxy-taxol, respectively.

In some embodiments, the host cell further comprises a nucleic acid encoding said oxidase. Said oxidase may in some embodiments be native to a Taxus cell, such as a Taxus chinensis cell.

In some embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 3.

The activities of OD3 producing N-debenzoyl-taxol and/or 10-deacetyl-N-debenzoyl- taxol from N-debenzoyl-2'-deoxy-taxol and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol, respectively, has been described in the patent application EP23386140.0 or in a patent application claiming priority thereof, such as patent application PCT/EP2024/088095.

The nucleic acid encoding OD3 (SEQ ID NO: 3) or a functional homologue thereof may be the nucleic acid as set forth in SEQ ID NO: 9 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 9. Benzoyl-CoA overproduction

The host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, may further comprise one or more nucleic acids encoding one or more proteins enabling the host cell to produce, such as overproduce, benzoyl-CoA. As disclosed herein above, benzoyl-CoA is a co-substrate of the benzoyl transferase described herein, preferably BT6.1 , when catalysing formation of a benzoyl-group (Bz) at position 3'-N of the side chain comprising a p-phenylalanine moiety of taxanes.

In some embodiments, benzoyl-CoA is comprised within the cultivation medium. In other embodiments, the host cell is capable of producing benzoyl-CoA. Host cell capable of producing, such as overproducing, benzoyl-CoA are described herein below.

Benzoyl-CoA may be produced by the host cell from benzoic acid or benzoate. Benzoic acid or benzoate may be produced natively by the host cell, such as plant cells or fungal cells, and/or supplied to the host cell, such as comprised in the cultivation medium of said host cell, such as yeast cells, bacterial cells, or fungal cells.

Benzoic acid may be converted to benzoyl-CoA by a benzoyl-CoA ligase. Said benzoyl-CoA ligase may be a benzoyl-CoA ligase falling within EC number 6.2.1.25. In some embodiments, the host cell further comprises a nucleic acid encoding a benzoyl- CoA ligase, such as a benzoyl-ligase capable of converting benzoic acid into benzoyl- CoA. Said benzoyl-CoA ligase may in some embodiments be native to an Arabidopsis cell, such as an Arabidopsis thaliana cell.

Some host cells comprise an endogenous benzoyl-CoA ligase capable of converting benzoic acid to benzoyl-CoA. For example, plant cells usually comprises a benzoyl- CoA ligase and benzoyl-CoA may be overproduced by feeding the plant cells benzoic acid or boosting the native benzoic acid synthesis of said plant cells, in instances where the plant cells are capable of producing benzoic acid. Other host cells, such as most bacterial cells and most yeast cells do not comprise an endogenous benzoyl-CoA ligase. In such instances, for increasing production of benzoyl-CoA from benzoic acid requires introducing a heterologous nucleic acid encoding a benzoyl-CoA ligase into the host cell and supplying benzoic acid. Irrespective of whether the host cell comprises a native benzoyl-CoA ligase or not, it may comprise a nucleic acid, such as a heterologous nucleic acid, encoding a benzoyl- CoA ligase. In some embodiments, the host cell further comprises nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 49. The nucleic acid encoding AtBZOI (SEQ ID NO: 49) or a functional homologue thereof may be the nucleic acid as set forth in SEQ ID NO: 52 or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 52.

Benzoyl-CoA may also be produced from a-phenylalanine via a four step enzymatic pathway comprising the enzymes: cinnamic acid:CoA ligase (EC: 6.2.1) capable of converting trans-cinnamic acid to cinnamoyl-CoA; cinnamoyl-CoA hydratase-dehydrogenase (EC: 1.13.11) capable of converting cinnamoyl-CoA to 3O3PP-CoA; and

3-ketoacyl CoA thiolase (EC: 2.3.1.16) capable of converting 3O3PP-CoA into benzoyl-CoA.

In some embodiments, the host cell further comprises a nucleic acid encoding a phenylalanine ammonia-lyase (EC: 4.3.1.24), such as a phenylalanine ammonia-lyase 2. Said phenylalanine ammonia-lyase 2 may in some embodiments be native to an Arabidopsis cell, such as an Arabidopsis thaliana cell. In other embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28, or a functional homologue thereof having at least 70% sequence identity thereto.

In some embodiments, the host cell further comprises a nucleic acid encoding a cinnamic acid:CoA ligase (EC: 6.2.1). Said cinnamic acid:CoA ligase may in some embodiments be native to a Petunia cell, such as an Petunia x hybrid cell. In other embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29, or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments, the host cell further comprises a nucleic acid encoding a cinnamoyl-CoA hydratase-dehydrogenase (EC: 1.13.11). Said cinnamoyl-CoA hydratase-dehydrogenase may in some embodiments be native to a Petunia cell, such as an Petunia x hybrid cell. In other embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30, or a functional homologue thereof having at least 70% sequence identity thereto.

In some embodiments, the host cell further comprises a nucleic acid encoding a 3- ketoacyl CoA thiolase (EC: 2.3.1.16). Said 3-ketoacyl CoA thiolase may in some embodiments be native to a Petunia cell, such as an Petunia x hybrid cell. In other embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31, or a functional homologue thereof having at least 70% sequence identity thereto.

In further embodiments, the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of a phenylalanine ammonia-lyase, such as a phenylalanine ammonia-lyase 2, for example PAL (SEQ ID NO: 28); ii. a nucleic acid encoding a protein comprising or consisting of a cinnamic acid:CoA ligase, such as CNL (SEQ ID NO: 29);

Hi. a nucleic acid encoding a protein comprising or consisting of a cinnamoyl-CoA hydratase-dehydrogenase, such as CHD (SEQ ID NO: 30); and iv. a nucleic acid encoding a protein comprising or consisting of a 3-ketoacyl CoA thiolase, such as Kat1 (SEQ ID NO: 31); or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In other embodiments, the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

The host cell described in this section may also comprise one or more of a nucleic acid encoding a CoAL, a nucleic acid encoding an amino phenylpropanoyl transferase, a nucleic acid encoding a phenylalanine aminomutase, a nucleic acid encoding a DBAT, and/or a nucleic acid encoding an oxidase, as described herein above, in addition to the heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof, and the one or more nucleic acids encoding one or more proteins making the host cell capable of producing, such as overproducing, benzoyl-CoA, as disclosed in the present section.

Host cells for taxol production

The host cells disclosed herein are in particular useful for producing paclitaxel (taxol) or derivatives thereof. This sections discloses examples of host cells, such as host cell designs, for producing taxol from various substrates/precursors. Host cells useful for producing taxol are also described in the section “Methods”, in particular in the section “Methods for taxol production”, herein below.

In some embodiments, the host cell is capable of producing taxol in the presence of: i. N-debenzoyl-taxol, optionally wherein the host cell is capable of producing of N-debenzoyl-taxol; or ii. N-debenzoyl-taxol and benzoyl-CoA, optionally wherein the host cell is capable of producing of N-debenzoyl-taxol and/or benzoyl-CoA; and said host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the host cell is capable of producing taxol in the presence of baccatin III, a-phenylalanine, and benzoyl-CoA, and optionally said host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto, or optionally said host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. In some embodiments, the host cell is capable of producing taxol in the presence of: i. baccatin III, optionally wherein the host cell is capable of producing baccatin HI; ii. baccatin III and 0-phenylalanoyl-CoA, optionally wherein the host cell is capable of producing baccatin III and/or |3-phenylalanoyl-CoA; iii. baccatin III and benzoyl-CoA, optionally wherein the host cell is capable of producing baccatin III and/or benzoyl-CoA; or iv. baccatin III, benzoyl-CoA, and 0-phenylalanoyl-CoA, optionally wherein the host cell is capable of producing baccatin III, benzoyl-CoA, and/or 0- phenylalanoyl-CoA; and said host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; ii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iii. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In some embodiments, the host cell is capable of producing taxol in the presence of: i. 10-deacetyl-baccatin III, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III; ii. 10-deacetyl-baccatin III and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and/or benzoyl-CoA; iii. 10-deacetyl-baccatin III, 0-phenylalanoyl-CoA, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, 0- phenylalanoyl-CoA, and/or benzoyl-CoA; or iv. 10-deacetyl-baccatin III and 0-phenylalanoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and 0- phenylalanoyl-CoA; and said host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iv. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In some embodiments, the host cell is capable of producing taxol in the presence of 10- deacetyl-baccatin III, a-phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl- CoA. Thus, in some other embodiments, the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, a-phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In some other embodiments, the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, a-phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

Any of the host cells described in this section may further be capable of producing said benzoyl-CoA in the presence of a-phenylalanine.

Thus, in some embodiments, the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

Any of the host cells described in this section may further be capable of producing said benzoyl-CoA in the presence of benzoic acid.

Thus, in some embodiments, the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

As described elsewhere herein, functional homologues of BT6.1 (SEQ ID NO: 5) include, but are not limited to BT6.1(T294N) (SEQ ID NO: 57), BT6.1(C369W) (SEQ ID NO: 59), BT6.1(L199M) (SEQ ID NO: 61), BT6.1(L199M, A372T) (SEQ ID NO: 63), BT6.1_21Xmut (SEQ ID NO: 65) and BT6.1_17Xmut (SEQ ID NO: 67). Thus, with respect to the host cells for taxol production described in this section, the nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5), may be exchanged for a nucleic acid encoding a protein comprising or consisting of any one of BT6.1(T294N) (SEQ ID NO: 57), BT6.1(C369W) (SEQ ID NO: 59), BT6.1(L199M) (SEQ ID NO: 61), BT6.1(L199M, A372T) (SEQ ID NO: 63), BT6.1_21Xmut (SEQ ID NO: 65) and BT6.1_17Xmut (SEQ ID NO: 67), or functional homologues thereof having at least 80% sequence identity thereto.

Enzyme activities for cte novo baccatin III or 10-deacetyl-baccatin III production In addition to any one of the above described nucleic acids, the host cell may also comprise nucleic acids encoding enzymes for production of other taxoid compounds, such as baccatin III from the common diterpene precursor geranylgeranyl diphosphate (GGPP), in order to obtain complete biosynthesis of taxol in host cell, such as in a heterologous host cell. In other words, the host cell may comprise enzyme activities enabling de novo production of said taxane having a side chain comprising an N-benzoylated 0- phenylalanine moiety from GGPP and/or said taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety from GGPP. Enzyme activities required for complete biosynthesis of baccatin III from GGPP may include one terpene synthase, such as taxadiene synthase, two acetyltransferases, a benzoyl transferase, an epoxidase and seven cytochrome P450. Enzyme activities required for complete biosynthesis of 10-deacetyl-baccatin III from GGPP may include one terpene synthase, such as taxadiene synthase, an acetyltransferases, a benzoyl transferase, an epoxidase and seven cytochrome P450.

In particular, it may be the enzymes described in Zhang et al., 2023, and/or it may be the enzymes described in or encoded by any of the nucleic acids and/or nucleic acid sequences described in patent application PCT/EP2024/065772, and/or EP23386140.0 or in a patent application claiming priority thereof, such as patent application PCT/EP2024/088095.

Methods

In another main aspect, the present disclosure concerns methods for producing a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety. The methods of the invention generally comprises the steps of: i. providing a host cell as described herein; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N-benzoylated 0- phenylalanine moiety and/or said taxane having a side chain comprising an N- acetylated 0-phenylalanine moiety.

The host cell may be any of the host cells described herein, for example herein above in the section “Host cells”.

Thus, in some embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, said method comprising the steps of: i. providing a host cell comprising a heterologous encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 5; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N-benzoylated |3- phenylalanine moiety and/or said taxane having a side chain comprising an N- acetylated [3-phenylalanine moiety.

In preferred embodiments of the method, the functional homologue of BT6.1 has at least 80% sequence identity to SEQ ID NO: 5. In even more preferred embodiments of the method, the functional homologue of BT6.1 has at least 95% sequence identity to SEQ ID NO: 5.

Functional homologues of BT6.1 (SEQ ID NO: 5) include, but are not limited to BT6.1(T294N) (SEQ ID NO: 57), BT6.1(C369W) (SEQ ID NO: 59), BT6.1(L199M) (SEQ ID NO: 61), BT6.1(L199M, A372T) (SEQ ID NO: 63), BT6.1_21Xmut (SEQ ID NO: 65) and BT6.1_17Xmut (SEQ ID NO: 67).

Thus, in other embodiments, the method comprises the steps of: i. providing a host cell comprising a heterologous encoding a protein comprising or consisting BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity, such as at least 99% sequence identity thereto; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N-benzoylated |3- phenylalanine moiety and/or said taxane having a side chain comprising an N- acetylated p-phenylalanine moiety.

In preferred embodiments, said taxane having a side chain comprising a N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol). In preferred embodiments, said taxane having a side chain comprising an N- acetylated p-phenylalanine moiety comprises or consists of B7.

Cultivation

The step of cultivating and/or incubating the host cell in a cultivation medium may be under conditions enabling growth may be performed by any method known to the skilled person. The term “growth” of a host cell or a multicellular organism comprising a host cell should be understood as a reference to proliferation, multiplication, differentiation and/or maintenance of viability of the subject host cell, or multicellular organism.

If the host cell is comprised in a multicellular organism, said conditions are usually conditions enabling maintenance of viability and/or growth of the multicellular organism. Thus, if the host cells are plant cells comprised in a plant, the cultivation conditions are conditions suitable for maintenance and/or growth of said plant. That could e.g. be sowing seeds or other regenerative parts of the said plant in a field or in a green house. Cultivation may further comprise watering and/or fertilising.

If the host cell is a unicellular organism, such as a bacterium or yeast cell, cultivation may be incubation in a medium comprising at least a carbon source and a nitrogen source at a temperature suitable for growth of said unicellular organism. The carbon source may e.g. be a carbohydrate, such as sugars or polysaccharides. The nitrogen source may for example be amino acids or polypeptides.

The skilled person is well able of selecting a suitable cultivation medium based on the particular host cell. Culture conditions

In some embodiments, said step of cultivating said host cell is performed in the presence of benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, -phenylalanine, [3-phenylalanoyl-CoA, N- debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N- debenzoyl-2'-deoxy-taxol.

Cultivating and/or incubating said host cell in the presence of benzoic acid, benzoyl- CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, -phenylalanine, |3-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10- deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol may be obtained in several manners. For example, said compounds may be added or supplied to the host cell, for example they may be present in the cultivation medium. If the host cell is a microorganism, such as bacterial cells, yeast cells, and/or fungal cells, then said compounds may be added to the cultivation medium of said microorganism, such as comprised in the cultivation medium of said microorganism. If the host cell is a plant, then said compounds may be added to the soil of the plant or it may be introduced into the plant by infiltration. Thus, if the nucleic acid(s), such as the heterologous nucleic acid(s), are introduced into the plant by infiltration, then said compounds may be coinfiltrated together with the nucleic acid(s), such as the heterologous nucleic acid(s).

Thus, the method may further comprise a step of supplying said benzoic acid, benzoyl- CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, p-phenylalanine, p-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10- deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol to the host cell, such as cultivating and/or incubating said host cell in a cultivation medium comprising said compounds.

It is also comprised within this disclosure, that the host cell is producing and/or is capable of producing benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N- debenzoyl-taxol, a-phenylalanine, -phenylalanine, -phenylalanoyl-CoA, N-debenzoyl- 2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'- deoxy-taxol. Host cells capable of producing said compounds are described elsewhere herein, for example in section “Host cells” herein above. In other embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, wherein said cultivation medium is suitable for producing said taxane. In preferred embodiments, said cultivation medium comprises benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10- deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, p-phenylalanine, 0- phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacety I- N-debenzoy I-2 '-deoxy- taxol .

The skilled person is well able of selecting suitable culture conditions based on the particular host cell.

Methods for taxol production

The methods disclosed herein are in particular useful for producing paclitaxel (taxol) or derivatives thereof. Host cells useful for producing taxol are also described in the sections “Host cells” herein above. This section discloses examples of methods for producing taxol from various substrates/precursors and examples of suitable host cells therefor.

For example, in some embodiments, the method is a method for producing taxol and said cultivation medium comprises baccatin III. In other embodiments, the method is a method for producing taxol and said host cell is producing baccatin III and/or is capable of producing baccatin III.

In some embodiments, the method is a method for producing taxol, and said cultivation medium comprises baccatin III, and/or said host cell is producing baccatin III and/or is capable of producing baccatin III, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In other embodiments, the method is a method for producing taxol, and said cultivation medium comprises baccatin III, and/or said host cell is producing baccatin III and/or is capable of producing baccatin III, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In further some embodiments, the method is a method for producing taxol and said cultivation medium comprises 10-deacetyl-baccatin III. In other embodiments, the method is a method for producing taxol and said host cell is producing 10-deacetyl- baccatin III and/or is capable of producing 10-deacetyl-baccatin III. In other embodiments, the method is a method for producing taxol, and said cultivation medium comprises 10-deacetyl-baccatin III, and/or said host cell is producing 10- deacetyl-baccatin III and/or is capable of producing 10-deacetyl-baccatin III, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In further other embodiments, the method is a method for producing taxol, and said cultivation medium comprises 10-deacetyl-baccatin III, and/or said host cell is producing 10-deacetyl-baccatin III and/or is capable of producing 10-deacetyl-baccatin III, and the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; vi. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; vii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; viii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; ix. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and x. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In further some embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety, wherein the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine and wherein said cultivation medium comprises a-phenylalanine and/or the host cell is capable of producing said a-phenylalanine.

In preferred embodiments, the method is a method for producing taxol and/or 10- deacetyl-taxol, wherein the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine and wherein said cultivation medium comprises a- phenylalanine and/or the host cell is capable of producing said a-phenylalanine.

In some embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

In some embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated -phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety, and the host cell is capable of producing said benzoyl-CoA in the presence of benzoic acid and wherein said cultivation medium comprises benzoic acid.

In further some embodiments, the method is a method for producing a taxane having a side chain comprising an N-benzoylated -phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety, and the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI (SEQ ID NO: 49), or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 49.

In some embodiments, the method is a method for producing taxol, comprising: i. providing a host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 5, optionally wherein the host cell is capable of producing of N-debenzoyl-taxol and/or benzoyl-CoA; and ii. cultivating said host cell in a cultivation medium comprising: a. N-debenzoyl-taxol; or b. N-debenzoyl-taxol and benzoyl-CoA; thereby producing taxol.

In some embodiments, the method is a method for producing taxol in the presence of baccatin III, a-phenylalanine, and benzoyl-CoA, comprising: i. providing a host cell, wherein said host cell comprises: a. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; b. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; c. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; d. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and e. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto, or wherein said host cell comprises: a. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; b. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; c. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; d. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and e. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto; and ii. cultivating said host cell in a cultivation medium, optionally wherein said cultivation comprises baccatin III, a-phenylalanine, and/or benzoyl-CoA; thereby producing taxol.

In some embodiments, the method is a method for producing taxol, comprising: i. providing a host cell comprising: ii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iv. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto, optionally wherein the host cell is capable of producing baccatin III, benzoyl- CoA, and/or p-phenylalanoyl-CoA; and i. cultivating said host cell in a cultivation medium comprising: a. baccatin III; b. baccatin III and p-phenylalanoyl-CoA; c. baccatin III and benzoyl-CoA; or d. baccatin III, benzoyl-CoA, and p-phenylalanoyl-CoA; thereby producing taxol.

In some embodiments, the method is a method for producing taxol, comprising: i. providing a host cell comprising: a. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; b. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; c. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and d. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, benzoyl-CoA, and/or p-phenylalanoyl-CoA; and ii. cultivating said host cell in a cultivation medium comprising: a. 10-deacetyl-baccatin III; b. 10-deacetyl-baccatin III and benzoyl-CoA; c. 10-deacetyl-baccatin III, [3-phenylalanoyl-CoA, and benzoyl-CoA; or d. 10-deacetyl-baccatin III and p-phenylalanoyl-CoA; thereby producing taxol.

In some embodiments, the method is a method for producing taxol, comprising i. providing a host cell capable of producing taxol in the presence of 10- deacetyl-baccatin III, a-phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA; and ii. cultivating said host cell in a cultivation medium; thereby producing taxol.

In some embodiments, the method is a method for producing taxol, comprising i. providing a host cell comprising: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA; and vii. cultivating said host cell in a cultivation medium, optionally wherein cultivation medium comprises 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA; thereby producing taxol.

In some other embodiments, the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, a-phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA.

In some embodiments, the method is a method for producing taxol, comprising i. providing a host cell comprising: a. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; b. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; c. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; d. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; e. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and f. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA; and ii. cultivating said host cell in a cultivation medium comprising 10-deacetyl- baccatin III, a-phenylalanine, and/or benzoyl-CoA; thereby producing taxol.

In some embodiments, the method is a method for producing taxol, and the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine. In some embodiments, the method is a method for producing taxol, and the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity thereto.

In some embodiments, the method is a method for producing taxol, and the host cell is capable of producing said benzoyl-CoA in the presence of benzoic acid or benzoate, optionally wherein the cultivation medium comprises benzoic acid, and/or the host cell is capable of producing benzoic acid or benzoate.

In further some embodiments, the method is a method for producing taxol, and the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI (SEQ ID NO: 49), or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 49. For example, in some embodiments, the method is for producing taxol, and the host cell comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI (SEQ ID NO: 49), or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 49, and the host cell is cultivated in a cultivation medium comprising benzoic acid.

As described elsewhere herein, functional homologues of BT6.1 (SEQ ID NO: 5) include, but are not limited to BT6.1 (T294N) (SEQ ID NO: 57), BT6.1(C369W) (SEQ ID NO: 59), BT6.1(L199M) (SEQ ID NO: 61), BT6.1(L199M, A372T) (SEQ ID NO: 63), BT6.1_21Xmut (SEQ ID NO: 65) and BT6.1_17Xmut (SEQ ID NO: 67). Thus, with respect to the methods described herein, the nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5), may be exchanged for a nucleic acid encoding a protein comprising or consisting of any one of BT6.1(T294N) (SEQ ID NO: 57), BT6.1(C369W) (SEQ ID NO: 59), BT6.1(L199M) (SEQ ID NO: 61), BT6.1(L199M, A372T) (SEQ ID NO: 63), BT6.1_21Xmut (SEQ ID NO: 65) and BT6.1_17Xmut (SEQ ID NO: 67), or functional homologues thereof having at least 80% sequence identity thereto.

Isolation

The method may further comprise a step of isolating and/ a step of recovering said taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety.

The compounds may be isolated and/or recovered through any useful method known to the skilled person. For example, said taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety may be isolated and/or recovered by a method comprising one or more of the following:

• extraction, e g. solvent extraction;

• precipitation; and/or

• chromatography, e.g. liquid chromatography (LC).

In some embodiments of the present disclosure, the step of isolating and/or recovering said taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety comprises a step of extraction, such as extraction with a solvent, for example methanol (MeOH) and/or ethyl acetate (EtOAc).

In other embodiments, the step of isolating and/or recovering said taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety comprises a step of chromatography, such as liquid chromatography (LC), for example column chromatography or preparative/semi-preparative high performance LC (HPLC).

Nucleic acids and expression systems

In addition to the methods and host cells, the invention also provides nucleic acids and nucleic acid constructs useful in the production of said taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated -phenylalanine moiety.

Provided herein is a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 5, for example, the nucleic acid construct comprises or consists of SEQ ID NO: 11 or a homologue thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 11 .

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (T294N) as set forth in SEQ ID NO: 57 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 56 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 56.

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (C369W) as set forth in SEQ ID NO: 59 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 58 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 58.

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (L199M) as set forth in SEQ ID NO: 61 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 60 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 60.

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 62 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 62.

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1_21Xmut as set forth in SEQ ID NO: 65 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 64 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 64.

Provided is also a nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1_17Xmut as set forth in SEQ ID NO: 67 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 66 or a homologue thereof having at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 66.

In some embodiments, the nucleic acid construct comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 5, such as SEQ ID NO: 11 or a homologue thereof having at least 70% sequence identity to SEQ ID NO: 11 , further comprises one or more of: i. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 9; ii. a nucleic acid encoding a protein comprising or consisting of BAPT as set forth in SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 7; iii. a nucleic acid encoding a protein comprising or consisting of MBPig3BAPT as set forth in SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 32; iv. a nucleic acid encoding a protein comprising or consisting of CoAL(A312G) as set forth in SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 8; v. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 10; vi. a nucleic acid encoding a protein comprising or consisting of TchiAAE5 as set forth in SEQ ID NO: 48 or a functional homologue thereof having at least 70% sequence identity thereto; vii. a nucleic acid encoding a protein comprising or consisting of sbCoAL as set forth in SEQ ID NO: 50 or a functional homologue thereof having at least 70% sequence identity thereto; viii. a nucleic acid encoding a protein comprising or consisting of HcAAEI as set forth in SEQ ID NO: 51 or a functional homologue thereof having at least 70% sequence identity thereto, and/or ix. a nucleic acid encoding a protein comprising or consisting of TCL/DBAT as set forth in SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 26; or homologues of any of the aforementioned nucleic acids having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity thereto.

In some embodiments, said nucleic acid construct further comprises one or more of: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 33; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 34; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 35; and/or iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 36; or homologues of any of the aforementioned nucleic acids having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity thereto. Thus, the nucleic acid construct comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 5, for example the nucleic acid as set forth in SEQ ID NO: 11 or a homologue thereof having at least 70% sequence identity to SEQ ID NO: 11 , may further comprise one or more of: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 33; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 34; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 35; and/or iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 36; or homologues of any of the aforementioned nucleic acids having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity thereto.

In other embodiments, the nucleic acid construct further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 49, such as SEQ ID NO: 52, or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 52.

Thus, the nucleic acid construct comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (SEQ ID NO: 5) or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 5, such as SEQ ID NO: 11 or a homologue thereof having at least 70% sequence identity to SEQ ID NO: 11 , may further comprise a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49 or a functional homologue thereof having at least 70% sequence identity to SEQ ID NO: 49, such as SEQ ID NO: 52, or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 52.

In some embodiments, the nucleic acid construct further comprises a promotor, a promotor, such as a constitutive promoter and/or an inducible promoter, operably linked to any one or more of the nucleic acids.

Provided herein is also an isolated polypeptide as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 5.

Furthermore, provided is also an isolated polypeptide as set forth in SEQ ID NO: 57 (BT6.1(T294N)), SEQ ID NO: 59 (BT6.1(C369W)), SEQ ID NO: 61 (BT6.1(L199M)), SEQ ID NO: 63 (BT6.1(L199M, A372T)), SEQ ID NO: 65 (BT6.1_21Xmut) or SEQ ID NO: 67 (BT6.1_17Xmut), or functional homologues thereof having at least 80%, for example at least 90%, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95%, for example at least 99% sequence identity thereto.

Also provided herein is vector comprising at least one of the nucleic acids and/or nucleic acid constructs described herein.

Provided is also a host cell as described else wherein herein, comprising one or more nucleic acids or nucleic acid constructs as described herein, and/or a vector as described herein. Preferably, said host cell is a host cell as described in the section “Host cells” herein above.

Provided is also a kit of parts comprising: i. the host cell as described herein, and optionally instructions for use, and/or ii. one or more nucleic acids and/or nucleic acid constructs as described herein or a vector as described herein, and optionally instructions for use, and further optionally a host cell to be modified, preferably wherein the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells, and fungal cells.

Uses

Disclosed herein is also the use a benzoyl transferase from Taxus chinensis in a method for producing a taxane having a side chain comprising an N-benzoylated p- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated P-phenylalanine moiety. Also disclosed is the use of a benzoyl transferase from Taxus cuspidata in a method for producing a taxane having a side chain comprising an N- benzoylated |3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety. Preferably, said taxane having a side chain comprising a N-benzoylated p-phenylalanine moiety comprises or consists of 10- deacetyl-taxol, and/or paclitaxel (taxol). Preferably, said taxane having a side chain comprising an N-acetylated P-phenylalanine moiety comprises or consists of B7 of structure VII.

In preferred embodiments, said T. chinensis benzoyl transferase is BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity to SEQ ID NO: 5. In some embodiments, the functional homologue of BT6.1 (SEQ ID NO: 5) is BT6.1(T294N) (SEQ ID NO: 57) or BT6.1(C369W) (SEQ ID NO: 59), or functional homologues thereof having at least 80% sequence identity thereto. In preferred embodiments, said T. cuspidata benzoyl transferase is BT6.1(L199M) as set forth in SEQ ID NO: 61, BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. In some embodiments, the method is a method described herein, for example in the section ’’Method”.

In other embodiments, the benzoyl transferase, such as the benzoyl transferase from T. chinensis or T. cuspidata, comprises or consists of the sequence as set forth in SEQ ID NO: 5, with the exception that at the most 44 residues are mutated, such as at the most 40 residues, such as at the most 35 residues, such as at the most 30 residues, such as at the most 25 residues, such as at the most 20 residues, such as at the most 15 residues, such as at the most 10 residues, such as at the most 5 residues, or less residues are mutated.

In some embodiments, said use comprises expressing the benzoyl transferase comprising or consisting of the sequence as set forth in SEQ ID NO: 5, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65 or SEQ ID NO: 67 in a host cell, preferably wherein the host cell is as described herein, for example in the section “Host Cells”.

Compounds, compositions, and pharmaceutical use

The host cells, methods, and/or uses described herein can be used to produce different plant diterpenoid compounds efficiently. In particular, the present disclosure provides host cells, methods, and/or uses for production of one or more taxanes having a side chain comprising an N-benzoylated -phenylalanine moiety, preferably wherein said taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or taxol, and/or one or more taxanes having a side chain comprising an N-acetylated p-phenylalanine moiety, preferably wherein said taxane having a side chain comprising an N-acetylated p-phenylalanine moiety comprises or consists of B7.

Said methods are described elsewhere herein, for example in the sections “Benzoyl transferase” and “Methods” herein above. Said host cells are described elsewhere herein, for example in the sections “Benzoyl transferase” and “Host Cells” herein above. Said uses are described elsewhere herein, for example in the sections “Benzoyl transferase” herein above and/or “Uses” herein below.

Furthermore, provided in this section is a fermentation liquid, cell culture and/or composition, comprising:

- a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety, wherein said taxane having said side chain an N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or taxol, and/or

- a taxanes having a side chain comprising an N-acetylated p-phenylalanine moiety, preferably wherein said taxane having a side chain comprising an N-acetylated p- phenylalanine moiety comprises or consists of B7. Provided herein is a compound having the structure (VII) (B7):

Structure (VII)

Cell culture and fermentation liquid

Herein provided is also a cell culture obtained by a method and/or a use described herein. Provided is also a cell culture, comprising a host cell described herein, and optionally a cultivation medium.

Further provided herein is a fermentation liquid comprising a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety. In some embodiments, said fermentation liquid is obtained by a method and/or a use described herein. In other embodiments, the method may further comprise a step of obtaining a fermentation liquid, optionally wherein said fermentation liquid comprise the host cell as described herein and/or a taxane having a side chain comprising an N-benzoylated p- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated P-phenylalanine moiety. In some embodiments, said fermentation liquid is comprised in a cell culture described herein. In further embodiments, said fermentation liquid is comprised within and/or secreted by a host cell described herein, for example to the fermentation liquid, cultivation medium or broth. In some embodiments, the fermentation liquid comprises host cells described herein, and at least 50% of the host cells are lysed, such as at least 75%, such as at least 95%, such as at least 99% of the host cells are lysed. In other embodiments, at least 50% of solid cellular material has been separated from the liquid, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the liquid. Compositions

The diterpenoid compounds, and more particular the taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or the taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety obtainable by the present methods and/or uses may be useful for obtaining compositions comprising any of said compounds produced by a host cell of the present disclosure.

Provided herein is composition comprising a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety obtained by a method, a host cell and/or a use described herein.

In preferred embodiments, said taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or taxol. In preferred embodiments, said taxane having a side chain comprising an N- acetylated [3-phenylalanine moiety comprises or consists of B7.

Provided herein is a composition comprising one or more of a taxane having a side chain comprising an N-benzoylated |3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated |3-phenylalanine moiety obtained by a method and/or a use described herein, and optionally one or more agents, additives and/or excipients. Provided is also, a composition comprising B7 obtained by a method and/or a use described herein, and optionally one or more agents, additives and/or excipients. Also provided is a composition comprising taxol obtained by a method and/or a use described herein, and optionally one or more agents, additives and/or excipients. Also provided is a composition comprising 10-deacetyl-taxol obtained by the m a method and/or a use described herein, and optionally one or more agents, additives and/or excipients.

Also provided is a composition comprising:

B7 and taxol, optionally obtained by a method and/or a use described herein; B7 and 10-deacetyl-taxol, optionally obtained by a method and/or a use described herein;

10-deacetyl-taxol and taxol, optionally obtained a method and/or a use described herein. Provided is also a composition comprising the fermentation liquid described herein above. Indeed, any of the compositions described herein may in addition to comprising the specified compounds also comprises the fermentation liquid described herein, such as trace amounts of the fermentation liquid described herein.

In some embodiments, said composition have been processed into in a semi-dry or dry solid form, optionally in form of a powder, tablet, capsule, chewable, gel and/or gum.

In other embodiments, said composition is in a liquid form, optionally in a stabilized liquid form.

Pharmaceutical use

The plant diterpenoid compounds, and more particular the taxane having a side chain comprising an N-benzoylated -phenylalanine moiety or the taxane having a side chain comprising an N-acetylated -phenylalanine moiety, obtainable by the present methods and/or with the present host cells may be useful for manufacturing pharmaceuticals compounds and/or compositions, in particular taxoid compounds, such as 10-deacetyl- taxol, paclitaxel (taxol), and/or B7.

Thus, the methods may further comprise a step of producing a medicament and/or composition from any of the compounds produced by the host cell of the present disclosure.

Provided is also a method for treating a disorder such as cancer, comprising administration of a medicament comprising a composition obtained by a method and/or a use described herein, said composition comprising a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety. Preferably said taxane having a side chain comprising an N-benzoylated -phenylalanine moiety comprises or consists of 10-deacetyl-taxol or taxol, and/or said taxane having a side chain comprising an N-acetylated p-phenylalanine moiety comprises or consists of B7.

Non-limited examples of such cancer are melanoma cancer, ovarian cancer, breast cancer, bladder cancer, prostate cancer and/or esophageal cancer. Examples

Example 1: Materials and Methods for Examples 2 to 9

This example comprises the materials and methods for examples 2-4 herein below.

Synthetic genes and gene cloning

Nucleic acids encoding PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), and MBP (SEQ ID NO: 54 encoded by SEQ ID NO: 53) were synthesized from Thermo Fisher and codon optimized for yeast. Nucleic acids encoding OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) were synthesized from TWIST as native cDNA seguence.

Vectors for both N. benthamiana (tobacco) transient expression and yeast expression were produced using USER cloning with specific primers (USER-GeneName-FP and USER-GeneName-RP, Table 1). All of the primers were ordered from TAG Copenhagen, Denmark. pLIFE33 vector (Forman et al., 2022) was used in tobacco transient expression.

Table 1. Primers used for construction of vectors for gene expression in tobacco and yeast.

Heterologous expression of candidate genes in yeast

The S. cerevisiae strain EGY48 was used as the parent strain for gene expression. Yeast transformation was carried out using a lithium-acetate protocol. To prepare for the detection of produced compounds, the yeast strains were cultivated overnight in selective glucose media at 30°C and 150 rpm and used as seed cultures. To induce the production of taxol and taxol derivatives, each seed culture was washed three times with sterile deionized water and transferred into a 100 mL glass flask containing 10 mL galactose/raffinose media. The yeast cultures were incubated (20°C and 150 rpm) for 3 days before ethyl acetate extraction.

Yeast media: Yeast glucose media: 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 buffered media:yeast galactose/raffinose media containing 10% phosphate buffer solution (1M) and the pH adjusted to 7.

Table 2. Yeast strains. Abbreviations: MBP; maltose-binding protein, IG3: IGGG-linker (SEQ ID NO: 55). SEQ ID NOs of nucleic acids and/or polypeptides comprised in the yeasts are indicated as well.

Table 3. E.coli strains. Abbreviations: MBP; maltose-binding protein, IG3: IGGG-linker (SEQ ID NO: 55). SEQ ID NOs of nucleic acids and/or polypeptides introduced into the bacteria are also indicated.

Transient co-expression of genes of interest in N.benthamiana leaves

Constructs for tobacco transient expression were electro-transformed into agrobacteria (Agrobacterium tumefaciens strain AGL-1-GV3850). The overnight cultures of engineered agrobacteria strains were used for tobacco infiltration. Briefly, ODeoo of 1 of the culture was used to create mixtures of agrobacteria containing an equal ratio of each of the designated agrobacterial strains. For infiltration, four-week-old tobacco leaves were used. After infiltration, the plants were kept in the greenhouse (16 h light at 20°C, 8 h dark at 19 °C) for 2 days before substrates feeding. Substrates were dissolved in 5% methanol. After substrates feeding, the plants were kept in greenhouse for 5 days before metabolite extraction.

Sample extraction and UPLC-HRMS analysis

For tobacco metabolites extraction, two leaf discs (03 cm, 1 disc per leaf) were used for each sample and ground in liquid nitrogen before extracting with 1 ml_ methanol (MeOH). For yeast samples, 5 mL ethyl acetate (EtOAc) was added to each induced yeast culture (10 mL cultures in 100 mL flasks). The mixtures were centrifuged at 4000 RPM for 10 min. Afterwards, 1 mL EtOAc supernatant was dried by blowing nitrogen and the dried metabolites were re-dissolved in 100 LIL MeOH. All the samples were filtered through a 0.22 |iM filter before Ultra-Performance-Liquid-Chromatography-High-Resolution-Mass- Spectrometry (UPLC-HRMS) analysis. The final samples were diluted 200 times before UPLC-HRMS analysis.

UPLC-HRMS analysis was performed on a 731 Dionex UltiMate® 3000 Quaternary Rapid Separation UPLC-focused system (Thermo Fisher Scientific, 732 Germering, Germany) equipped with a Phenomenex Kinetex XB-C18 column (100 mm 2.1 mm i.d., 1.7 pm 733 particle size, 100 A pore size) (Phenomenex, Inc., Torrance, CA, USA). The column was operated at 40 °C, and the flow rate was maintained at 0.3 mL min^-1. The mobile phases were water (A) and 100% acetonitrile (B), both acidified with 0.05% formic acid. Separations were performed using the following gradient profile: 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 equipped with an electrospray ionization (ESI) interface (Bruker Daltonics, Bremen, Germany). Mass spectra were acquired in positive ion mode, using a capillary voltage of 4000V, an end plate offset of -500V, a drying temperature of 220 °C, a nebulizer pressure of 2.0 bar, and a drying gas flow of 8 L min^-1. Sodium formate solution (internal standard) was injected at the beginning of each chromatographic run, and the UPLC-HRMS raw data was calibrated against these sodium clusters using the Data Analysis 4.3 (Bruker Daltonics) software program.

Example 2 TcaDBTNBT is not an efficient N-debenzoyl-taxol benzoyltranferase The present example demonstrates that the enzyme TcaDBTNBT, which has been proposed to catalyze the synthesis of taxol through the benzoylation of N-debenzoyl- taxol is not an efficient enzyme in a heterologous host.

See Example 1 herein above for Material and Methods.

Agrobacterium-mediated transient gene expression in tobacco (Nicotiana benthamiana) is an efficient and reliable method for producing terpenoid compounds. To reconstruct the biosynthetic pathway to produce taxol, we infiltrated tobacco leaves with agrobacteria carrying nucleic acids encoding phenylalanine aminomutase from T. canadensis (PAM, SEQ ID NO: 4 encoded by SEQ ID NO: 10), coenzyme A ligase mutant (CoAL(A312G), SEQ ID NO: 2 encoded by SEQ ID NO: 8) derived from CoAL of Penicillium chrysogenum, amino phenylpropanoyl transferase from T. cuspidata (BAPT, SEQ ID NO: 1 encoded by SEQ ID NO: 7), oxidase OD3 from T. chinensis (SEQ ID NO: 3 encoded by SEQ ID NO: 9), TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24), the latter which has previously been reported to benzoylate N- debenzoyl-taxol to produce taxol, and P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12) (Figure 1a). P19 was introduced to suppress gene silencing. 2 days after agroinfiltration, 200 mg/L baccatin III dissolved in 5% methanol were injected into leaves which were earlier infiltrated with corresponding agrobacteria. Results

UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing the biosynthetic enzymes PAM, CoAL(A312G), BAPT, and OD3 revealed production of N-debenzoyl-taxol (compound 3, [M+H]+ 750.3120±0.01 , C40H47NO13) (Figure 1b, chromatogram I). No detectable consumption of compound 3 was observed when TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) was co-infiltrated with PAM, CoAL(A312G), BAPT and OD3 (Figure 1b, chromatogram II). However, trace amounts of taxol were present in chromatogram I and chromatogram II when [M+H]+ 854.3382±0.01 was extracted. Coinfiltrating TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24) did not result in a higher taxol peak (chromatogram II).

Conclusion

Using tobacco as heterologous production system, we initially attempted the reconstruction of the late steps of taxol biosynthesis from baccatin III to taxol using the previously characterized enzyme TcaDBTNBT for the catalysing the last step of this pathway (Walker et al., 2002; Long et al., 2008). We found that TcaDBTNBT was not able to synthesize taxol in sufficient amounts in this heterologous production system (Figure 1, Chromatogram II). Since an earlier publication claimed that taxol was produced in tobacco using TcaDBTNBT (Zhang, et al., 2023, Molecular Plant), we investigated further and found that when the rest of the pathway was assembled without TcaDBTNBT, a similarly low amount of taxol was also present (Figure 1 , Chromatogram I). This finding revealed that TcaDBTNBT is inefficient for catalyzing the final step of taxol biosynthesis in tobacco. Therefore, we set out to discover a benzoyl transferase that efficiently catalyzes the last step.

Example 3: BT6.1 enables the efficient production of taxol in tobacco

Using the available genomic and transcriptomic information of different Taxus tissues (Xiong, et al., 2021), we carried out a co-expression analysis and were successful in identifying genes that exhibit a similar expression pattern to taxadiene synthase (the first step of the pathway), PAM, or OD3. Based on the identified co-expression profiles, a total of 25 benzoyl transferase candidates were selected and tested for benzoyl transferase activity towards N-debenzoyl-taxol (Example 3 and Example 4) and/or 10- deacetyl-N-debenzoyl-taxol (Example 5). Among these, only BT6.1 (SEQ ID NO: 5) was found to possess benzoyl transferase activity towards these two taxoid compounds. The present example demonstrates that taxol is efficiently synthesized in vivo by expressing PAM, BAPT, CoAL(A312G), OD3, and BT6.1 from T. chinensis, and feeding baccatin III (BACIU) (Figure 2).

See Example 1 herein above for Material and Methods.

Results

We infiltrated tobacco leaves with agrobacteria carrying nucleic acids encoding PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) or TcaDBTNBT (SEQ ID NO: 23 encoded by SEQ ID NO: 24), and P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12). The latter was introduced to suppress gene silencing. Tobacco leaves were fed with 200 mg/L of baccatin III (compound 1) two days after agroinfiltration.

UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing PAM, CoAL(A312G), BAPT, OD3 and TcaDBTNBT showed production of N-debenzoyl-taxol, but did not show taxol production above background levels (Figure 2b, chromatogram I). The extract of tobacco leaves expressing PAM, CoAL(A312G), BAPT, OD3, and the benzoyl transferase BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) revealed the production of considerable amounts of taxol (Figure 2b, chromatogram II). In chromatogram II, N- debenzoyl-taxol (compound 3) is dramatically consumed.

We then proceeded to quantify the amount of taxol produced. Taxol production in tobacco expressing PAM, CoAL(A312G), BAPT, OD3 and BT6.1 was 280±30 times higher than that in tobacco expressing PAM, CoAL(A312G), BAPT, OD3, and TcaDBTNBT (Figure 2c).

Conclusion

These results demonstrate that BT6.1 is an efficient enzyme for the synthesis of taxol from N-debenzoyl-taxol and supersedes the activity of TcaDBTNBT by several hundred fold. Example 4: Production of the taxol analog B7 in tobacco

The present example demonstrates, that B7 is synthesized in vivo by expressing PAM, BAPT, CoAL(A312G), OD3, and BT6.1 , and feeding baccatin III (BACIU) in tobacco (Figure 2 and Figure 3).

See Example 1 herein above for Material and Methods.

Results

We infiltrated tobacco leaves with agrobacteria carrying nucleic acids encoding PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9), BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11), and P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12). The latter was used to suppress gene silencing. Tobacco leaves were fed with 200 mg/L of baccatin III (compound 1) two days after agro-infiltration.

UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing PAM, CoAL(A312G), BAPT, OD3, and the benzoyl transferase BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) revealed the production of considerable amounts of an analog of taxol, herein referred to as B7 (Figure 2b, chromatogram II). Based on the HRMS fragmentation data obtained for B7 and shown in Figure 3, we concluded that B7 is an analog of taxol with an acetyl group instead of a benzoyl group at the 3'-N position. The structure of compound B7 is shown in Figure 3, and herein elsewhere as structure VII.

Conclusion

These results show that BT6.1 can be used for the production of the taxol analog B7.

Example 5: Production of 10-deacetyl-taxol in tobacco

The present example demonstrates that 10-deacetyl-taxol is synthesized in vivo by expressing PAM, BAPT, CoAL(A312G), OD3 and BT6.1, and feeding 10-deacetyl- baccatin III (also sometimes referred to as 10-DAB) (Figure 4).

See Example 1 herein above for Material and Methods. To synthesize 10-deacetyl-taxol in tobacco, we infiltrated tobacco leaves with agrobacteria carrying nucleic acids encoding PAM (SEQ ID NO: 4 encoded by SEQ ID NO: 10), CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), BAPT (SEQ ID NO: 1 encoded by SEQ ID NO: 7), OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) and BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) and P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12), which was used to suppress gene silencing. Two days after agro-infiltration, 200 mg/l 10-deacetyl-baccatin III (10-DAB) dissolved in 5% methanol were injected to leaves which were infiltrated with corresponding agrobacteria earlier.

Results

UPLC-HRMS chromatograms (EIC, positive mode) of methanol extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing only PAM, BAPT, CoAL(A312G), OD3, but not BT6.1 , revealed no production of 10-deacetyl-taxol (Figure 4b, chromatogram I). Instead, 10-deacetyl-N-debenzoyl-taxol (compound 6, [M+H]+708.3015±0.01 , C38H45NO12), the precursor of 10-deacetyl-taxol, accumulated. However, extracts of tobacco (Nicotiana benthamiana) leaves transiently expressing PAM, BAPT, CoAL(A312G), OD3, and BT6.1 revealed the production of 10-deacetyl- taxol (Figure 4b, chromatogram II).

Conclusion

The experiments of example 5 show that BT6.1 can be used to produce 10-deacetyl- taxol.

Example 6: Production of taxol and its analog B7 in tobacco from 10-deacetyl-baccatin III.

The present example demonstrates that BT6.1 is efficient in synthesizing taxol and its analog B7 in tobacco cells using 10-deacetyl-baccatin III (10-DAB, compound 4) as starting compound.

See Example 1 herein above for Material and Methods.

To reconstruct the pathway to produce taxol from 10-deacetyl-baccatin III (10-DAB, compound 4), we infiltrated tobacco leaves with agrobacteria carrying nucleic acids encoding TcuDBAT (SEQ ID NO: 25 encoded by SEQ ID NO: 26), PAM, BAPT, CoAL(A312G), OD3, BT6.1 and P19 (SEQ ID NO: 6 encoded by SEQ ID NO: 12). The latter was used to suppress gene silencing. Two days after agro-infiltration, 200 mg/l 10- deacetyl-baccatin III dissolved in 5% methanol were injected to leaves which had been infiltrated with corresponding agrobacteria earlier.

Results

UPLC-HRMS analysis showed that tobacco expressing TcuDBAT, PAM, BAPT, CoAL(A312G), OD3, and BT6.1 consumes 10-DAB and produces taxol and its analog B7. See the drawing description in Figure 5 herein above for further details.

Conclusion

This results demonstrate that enzyme set including TcuDBAT, PAM, BAPT, CoAL(A312G), OD3, and BT6.1 is sufficient to synthesized taxol and its N-acetylated analog B7 from the readily available substrate 10-deacetyl-baccatin III (10-DAB, compound 4) in tobacco. This will enable the biotechnological production of taxol and its analogs in heterologous organisms.

Example 7 Product profile shifts from B7 to taxol by boosting benzoyl-CoA content in tobacco.

The present example demonstrates that the BT6.1 product profile shifts from B7 to taxol when benzoyl-CoA levels in tobacco are elevated.

Results

Benzoyl-CoA is a co-substrate of the benzoylation reaction. Four enzymes, PAL (phenylalanine ammonia-lyase 2, SEQ ID NO: 28 encoded by SEQ ID NO: 33), CNL (cinnamic acid:CoA ligase, SEQ ID NO: 29 encoded by SEQ ID NO: 34), CHD (cinnamoyl-CoA hydratase-dehydrogenase, SEQ ID NO: 30 encoded by SEQ ID NO: 35) and Kat1 (3-ketoacyl CoA thiolase, SEQ ID NO: 31 encoded by SEQ ID NO: 36), are responsible for converting endogenous a-phenylalanine into benzoyl-CoA (Figure 6a).

Without increasing benzoyl-CoA levels in tobacco by infiltrating PAL, CNL, CHD and Kat1 , the four enzymes that synthesizing benzoyl-CoA from a-phenylalanine, PAM, BAPT, CoAL(A312G), OD3, and BT6.1 produce taxol and B7 in tobacco in a ratio 51%±2% taxol and 48%±2% B7 (Figure 6b). However, after increasing benzoyl-CoA levels by co-infiltrating PAL, CNL, CHD and Kat1 together with the taxol pathway enzymes PAM, BAPT, CoAL(A312G), OD3, and BT6.1, the taxol percentage among the taxoid products increased to 70%±1% and the B7 percentage decreased to 30%±2%. This suggests that BT6.1 primarily functions as a benzoyl transferase when sufficient amounts of the substrate benzoyl-CoA are present, for example when benzoyl-CoA is overproduced as in this example.

Conclusion

This example demonstrates that the activity of BT6.1 can be controlled by the availability of the co-substrates, i.e. benzoyl-CoA or acetyl-CoA. When benzoyl-CoA is readily available, such upon overexpression of four biosynthetic enzymes for synthesizing it from a-phenylalanine, BT6.1 primarily synthesizes taxol. However, BT6.1 can also accept acetyl-CoA as co-substrate instead of benzoyl-CoA, and convert N-debenzoyl-taxol to B7, for example when benzoyl-CoA is not overproduced .

Example 8: Production of taxol and its analog B7 in yeast

The present example demonstrates that BT6.1 can be used to synthesize taxol and its analog B7 in yeast cells, illustrated herein with S. cerevisiae.

See Example 1 herein above for Material and Methods.

To investigate whether BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) can catalyze the benzoylation of the C13 side chain of taxol intermediates in yeast, we introduced BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) in the S. cerevisiae strain TL002, which expresses CoAL(A312G) (SEQ ID NO: 2, encoded by SEQ ID NO: 8), MBPig3BAPT (SEQ ID NO: 27, encoded by SEQ ID NO: 32), and OD3 (SEQ ID NO: 3, encoded by SEQ ID NO: 9) to obtain strain TL003 (Table 2). Additionally, since benzoyl- CoA is a co-substrate of the benzoylation reaction, we introduced four additional enzymes into yeast strain TL003, i.e. PAL (phenylalanine ammonia-lyase 2, SEQ ID NO: 28 encoded by SEQ ID NO: 33), CNL (cinnamic acid:CoA ligase, SEQ ID NO: 29 encoded by SEQ ID NO: 34), CHD (cinnamoyl-CoA hydratase-dehydrogenase, SEQ ID NO: 30 encoded by SEQ ID NO: 35) and Kat1 (3-ketoacyl CoA thiolase, SEQ ID NO: 31 encoded by SEQ ID NO: 36), to convert endogenous a-phenylalanine into benzoyl-CoA, resulting strain TL004. Strain TL004 (Table 2) was used to test the function BT6.1 in yeast cells where the availability of the co-substrate benzoyl-CoA was increased. Results

UPLC-HRMS analysis showed that S. cerevisiae TL003 cells are able to produce taxol and B7, but TL002 cells, which lack BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11), do not produce taxol or B7 (Figure 7). Instead, in TL002 cells, N-debenzoyl-taxol (compound 3, Figure 7), which is a substrate of BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11), accumulates and is not consumed.

Moreover, in TL004 yeast cells, where the level of benzoyl-CoA is high due to overexpression of PAL, CNL, CHD and Kat1,an increase in the levels of taxol and a reduction in the levels of the N-acetylated analog B7 are observed. This result confirms that BT6.1 is primarily a benzoyl transferase, but when benzoyl-CoA levels are low, for example when benzoyl-CoA is not overproduced, it can also accept acetyl-CoA as a cosubstrate to synthesize N-acetylated taxoid compounds, such as the taxol analog B7. See the drawing description in Figure 7 herein above for further details.

Conclusion

These results demonstrate that BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) is sufficient to synthesize taxol and its N-acetylated analog B7 in yeast, here exemplified by S. cerevisiae (baker’s yeast) (Figure 7). Furthermore, when there is a high level of benzoyl-CoA in the yeast cells, e.g. when benzoyl-CoA is overproduced, increased levels of taxol and decreased levels of B7 are obtained.

Example 9: Production of taxol and its analog B7 in E. coli

The present example demonstrates that BT6.1 can be used to synthesize taxol and its analog B7 in bacterial host cells, illustrated herein with E. coli cells.

See Example 1 herein above for Material and Methods.

First, we introduced OD3 (SEQ ID NO: 3 encoded by SEQ ID NO: 9) in E. coli together with CoAL(A312G) (SEQ ID NO: 2 encoded by SEQ ID NO: 8), MBPig3BAPT (SEQ ID NO: 27 encoded by SEQ ID NO: 32) to obtain strain ETL002 (Table 3). Subsequently, to investigate whether BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) can catalyze the benzoylation of the C13 side chain of taxol intermediates in E. coli, we introduced BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) into strain ETL002 to obtain strain ETL003 (Table 3). Additionally, since benzoyl-CoA is a co-substrate of the benzoylation reaction catalyzed by BT6.1 and E. coli cells do not have a high content of benzoyl-CoA, we constructed strain ETL004 (Table 3) by introducing an additional enzyme, AtBZOI (benzoyl-CoA ligase, SEQ ID NO: 49 encoded by SEQ ID NO: 52), into E. coli strain ETL003, to enable conversion of exogenous benzoic acid into benzoyl-CoA.

Results

UPLC-HRMS analysis showed that E. coli ETL003 cells are able to produce the taxol analog B7, but ETL002 cells, which lack BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11), do not produce B7 (Figure 8). Moreover, in ETL004 cells, where benzoyl-CoA is available, taxol is produced as the main product. This result confirms that BT6.1 is primarily a benzoyl transferase, but when benzoyl-CoA levels are low, for example when benzoyl-CoA is not overproduced, it can also accept acetyl-CoA as a cosubstrate to synthesize N-acetylated taxoid compounds, such as the taxol analog B7. See the drawing description in Figure 8 herein above for further details.

Conclusion

The results of Example 9 demonstrate that BT6.1 (SEQ ID NO: 5 encoded by SEQ ID NO: 11) is sufficient to synthesize taxol and its N-acetylated analog B7 in a bacterial host, such as E. coli (Figure 8). Furthermore, when benzoyl-CoA is overproduced in the bacterial host cell increased levels of taxol are produced compared to the production with corresponding cells not overproducing benzoyl-CoA (in this example not expressing AtBZOI).

Together with examples 7 and 8, this example further confirms that BT6.1 is an efficient benzoyl transferase of the taxol biosynthesis irrespective of the heterologous host cell, but also that when benzoyl-CoA levels are low, BT6.1 can accept acetyl-CoA as a cosubstrate and synthesize N-acetylated taxoid compounds, such as the taxol analog B7.

Example 10: Testing catalytic activity of functional homologues of BT6.1

The present example demonstrates that functional homologues of BT6.1 varying at amino acid positions 12, 15, 24, 29, 36, 44, 56, 62, 68, 165, 169, 171 , 185, 187, 189, 199, 227, 239, 241 , 286, 294, 295, 314, 324, 328, 346, 362, 369, 372, 379, 398, and 432 still retain their ability to synthesize taxol and compound B7. See Example 1 herein above for Material and Methods.

BT6.1 belongs to the BAHD family. Crystal structure of another BAHD family enzyme vinorine synthase is available in the Protein Data Bank (PDB, entry code 2BGH) (Ma et al. 2005). We aligned the BT6.1 sequence to the vinorine synthase sequence and found out that BT6.1 did not contain all the conserved amino acids identified using vinorine synthase crystal structure. Among these amino acids, we mutated two, T294 and C369, which correspond to N293 and W365 in vinorine synthase. As a result, we obtained two functional homologues of BT6.1, i.e. BT6.1 (T294N) (SEQ ID NO: 57 encoded by SEQ ID NO: 56) and BT6.1(C369W) (SEQ ID NO: 59 encoded by SEQ ID NO: 58). Besides, we amplified functional homologues of BT6.1 from Taxus cuspidata cDNA. As a result, we got four functional homologues of BT6.1 , i.e. BT6.1(L199M) (SEQ ID NO: 61 encoded by SEQ ID NO: 60), BT6.1(L199M, A372T) (SEQ ID NO: 63 encoded by SEQ ID NO: 62), BT6.1_21Xmut (SEQ ID NO: 65 encoded by SEQ ID NO: 64) and BT6.1_17Xmut (SEQ ID NO: 67 encoded by SEQ ID NO: 66).

Results

We tested the taxol and B7 producing activity of these six functional homologues of BT6.1 in tobacco. We transiently expressed PAM, DBAT, CoAL(A312G), BAPT and OD3 together with wild type BT6.1, BT6.1(T294N), BT6.1(C369W), BT6.1(L199M), BT6.1 (L199M, A372T), BT6.1_21Xmut or BT6.1_17Xmut individually (Figure 9a) in tobacco through agro-infiltration and fed substrate, 200 mg/L 10-deacetyl baccatin (10-DAB), after two days of agro-infiltration. After 5 days of bioconversion, we extracted metabolites from tobacco leaves and did LC-qTOF analysis.

We found that these six functional homologues can produce taxol or B7 (Figure 9b and 9c) in a similar manner compared with wild type BT6.1. This indicates that even though BT6.1 does not have all the conserved amino acids of BAHD family, it does not hamper its activity, and functional homologues of BT6.1 containing the conserved amino acids do not behave better than BT6.1. Moreover, the tested functional homologues of BT6.1 from Taxus cuspidata behave similarly with BT6.1 from T. chinensis.

Conclusion

These results show that functional homologues of BT6.1 varying at amino acid positions 12, 15, 24, 29, 36, 44, 56, 62, 68, 165, 169, 171, 185, 187, 189, 199, 227, 239, 241 , 286, 294, 295, 314, 324, 328, 346, 362, 369, 372, 379, 398, and 432 still retain their ability to synthesize taxol and compound B7.

Example 11: Testing of benzoyl transferase candidates in tobacco

This example demonstrates that the benzoyl transferase candidates TAX9 and TAX13cannot synthesize taxol or B7 in tobacco.

See Example 1 herein above for Material and Methods.

Results

To investigate if Taxus plants possess another BAHD enzyme that is able to produce taxol and B7 using N-debenzoyl-taxol as substrate, we tested TAX9 and TAX13 from Taxus media previously studied in WO 2001/23586 (Table 4):

- TAX13 having nucleic acid sequence SEQ ID NO: 57 and amino acid sequence SEQ ID NO: 58 in WO 2001/23586 and

- TAX9 having amino acid sequence SEQ ID NO: 60 in WO 2001/23586.

Table 4: Nucleic acid and amino acid sequence identity of TAX9 and TAX13 compared to BT6.1 of SEQ ID NO: 5 and encoded by SEQ ID NO: 11.

We found that none of TAX9 and TAX13 produces taxol or B7 in tobacco in this assay.

Sequence overview

References

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Walke, et al. Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis. Proceedings of the National Academy of Sciences, 99: 12715-12720 (2002)

Koetsier, et al. Aminoacyl-coenzyme A synthesis catalyzed by a CoA ligase from Penicillium chrysogenum. FEBS Letters, 585: 893-898 (2011)

Sanchez-Munoz, et al. A novel Hydroxylation step in the Taxane Biosynthetic Pathway: A New Approach to Paclitaxel Production by Synthetic Biology. Frontiers in Bioengineering and Biotechnology. 8: 410 (2020)

Zhang, et al. Synthetic biology identifies the minimal gene set required for Paclitaxel biosynthesis in a plant chassis. Molecular Plant. 16: 1951-1961 (2023)

Walker, et al. The final acylation step in taxol biosynthesis: cloning of the taxoid C13- side-chain N-benzoyltransferase from Taxus. Proc Natl Acad Sci U S A. 99: 9166-9171 (2002)

Long, et al. Specificiyt of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis. Arch Biochem Biophys. 477: 384-389 (2009) Li, et al. Improving 10-deacetylbaccatin lll-10-b-O- acetyltransferase catalytic fitness for

Taxol production. Nat. Commun. 8:15544 (2016)

Walker, et al. Molecular cloning of a 10-deacetylbaccatin lll-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli. Proc Natl Acad Sci U S A. 97: 583-587 (2000)

Huang, et al. A peroxisomal heterodimeric enzyme is involved in benzaldehyde synthesis in plants. Nat. Commun. 13, 1352 (2022).

Xiong, et al. The Taxus genome provides insights into paclitaxel biosynthesis. Nature Plants. 7, 1026-1036 (2021)

Kliebenstein, et al., Characterization of seed-specific benzoyloxyglucosinolate mutations in Arabidopsis thaliana, Plant J, 51: 1062-1076 (2007)

Singh, et al., A promiscuous coenzyme A ligase provides benzoyl-coenzyme A for xanthone biosynthesis in Hypericum, Plant J, 104: 1472-1490 (2020)

Ma et al., Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily. Journal of Biological Chemistry 280(14): 13576-83 (2005)

WO 2001/23586

Items

1 . A host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 75% sequence identity, such as at least 80% sequence identity, such as at least 81% sequence identity, such as at least 82% sequence identity, such as at least 83% sequence identity, such as at least 84% sequence identity, such as at least 85% sequence identity, such as at least 86% sequence identity, such as at least 87% sequence identity, such as at least 89% sequence identity, such as at least 90% sequence identity, such as at least 91% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 94% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity, such as at least 100% sequence identity to SEQ ID NO: 5. A host cell comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5, BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 81% sequence identity, such as at least 82% sequence identity, such as at least 83% sequence identity, such as at least 84% sequence identity, such as at least 85% sequence identity, such as at least 86% sequence identity, such as at least 87% sequence identity, such as at least 89% sequence identity, such as at least 90% sequence identity, such as at least 91 % sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 94% sequence identity, such as at least 95% sequence identity, such as at least 96% sequence identity, such as at least 97% sequence identity, such as at least 99% sequence identity, such as at least 100% sequence identity thereto. The host cell according to any one of the preceding items, wherein the nucleic acid is a heterologous nucleic acid. The host cell according to any one of the preceding items, wherein the heterologous nucleic acid encodes a protein comprising or consisting of BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1 (L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 92% sequence identity thereto, such as at least 93% sequence identity thereto. The host cell according to any one of the preceding items, wherein the heterologous nucleic acid encodes a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein said host cell is capable of producing a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety, preferably wherein said taxane comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol), and/or said host cell is capable of producing a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, preferably wherein said taxane comprises or consists of B7. The host cell according to any one of the preceding items, wherein the host cell is capable of producing a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety in the presence of benzoyl- CoA, acetyl-CoA, benzoic acid, baccatin III, 10-deacetyl-baccatin III, N- debenzoyl-taxol, a-phenylalanine, 0-phenylalanine, 0-phenylalanoyl-CoA, N- debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl- N-debenzoyl-2'-deoxy-taxol. The host cell according to any one of the preceding items, wherein the host cell is capable of producing benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10- deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, 0-phenylalanine, 0- phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl- taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and/or benzoyl-CoA, wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and/or benzoyl-CoA, wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 (T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61, BT6.1 (L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises a nucleic acid as set forth in SEQ ID NO: 11 or a homologue thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises a nucleic acid as set forth in SEQ ID NO: 56, 58, 60, 62, 64 or 66, or homologues thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding an oxidase (EC 1.14.11), optionally wherein said oxidase is native to a Taxus cell, such as a Taxus chinensis cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding an amino phenylpropanoyl transferase (EC: 2.3.1), optionally wherein said amino phenylpropanoyl transferase is native to a Taxus cell, such as a Taxus cuspidata cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27, or functional homologues of any of the aforementioned having at least 70% sequence identity to any of SEQ ID NO: 1 or SEQ ID NO: 27, respectively. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a coenzyme A ligase (CoAL, EC: 6.2.1), optionally wherein said CoAL is a CoAL native to a Penicillium cell, such as a P. chrysogenum cell, a CoAL native to a Taxus cell, such as a T. chinensis cell, or a CoAL native to a Hypericum cell, such as a Hypericum calycinum cell. The host cell according any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, HcAAEI as set forth in SEQ ID NO: 51, or functional homologues of any of the aforementioned having at least 70% sequence identity to any of SEQ ID NO: 2, SEQ ID NO: 48, SEQ ID NO: 50, or SEQ ID NO: 51. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a 10-deacetyl-baccatin lll-10-Q-acetyltransferase (DBAT), optionally wherein said DBAT is native to a Taxus cell, such as a Taxus cuspidata cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25, or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a phenylalanine aminomutase (EC: 5.4.3.11), optionally wherein said phenylalanine aminomutase is native to a Taxus cell, such as a Taxus canadensis cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4, or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a phenylalanine ammonia-lyase (EC: 4.3.1.24), such as a phenylalanine ammonia-lyase 2, optionally wherein said phenylalanine ammonia-lyase 2 is native to an Arabidopsis cell, such as an Arabidopsis thali ana cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28, or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a cinnamic acid:CoA ligase (EC: 6.2.1), optionally wherein said cinnamic acid:CoA ligase is native to a Petunia cell, such as an Petunia x hybrid cell. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29, or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a cinnamoyl-CoA hydratase-dehydrogenase (EC: 1.13.11), optionally wherein said cinnamoyl-CoA hydratase-dehydrogenase is native to a Petunia cell, such as an Petunia x hybrid cell. 8. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30, or a functional homologue thereof having at least 70% sequence identity thereto. 9. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a 3-ketoacyl CoA thiolase (EC: 2.3.1.16), optionally wherein said 3-ketoacyl CoA thiolase is native to a Petunia cell, such as an Petunia x hybrid cell. 0. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31 , or a functional homologue thereof having at least 70% sequence identity thereto. 1. The host cell according to any one of the preceding items, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of a phenylalanine ammonia-lyase, such as a phenylalanine ammonia-lyase 2, for example PAL (SEQ ID NO: 28); ii. a nucleic acid encoding a protein comprising or consisting of a cinnamic acid:CoA ligase, such as CNL (SEQ ID NO: 29);

Hi. a nucleic acid encoding a protein comprising or consisting of a cinnamoyl- CoA hydratase-dehydrogenase, such as CHD (SEQ ID NO: 30); and iv. a nucleic acid encoding a protein comprising or consisting of a 3-ketoacyl CoA thiolase, such as Kat1 (SEQ ID NO: 31); or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. . The host cell according to any one of the preceding items, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

33. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a benzoyl-ligase, such as a benzoyl-ligase capable of converting benzoic acid into benzoyl-CoA, optionally wherein said benzoyl- ligase is native to an Arabidopsis cell, such as an Arabidopsis thaliana cell.

34. The host cell according to any one of the preceding items, further comprising a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity thereto.

35. The host cell according to any one of the preceding items, wherein said host cell is selected from the group of plant cells, yeast cells, bacterial cells and fungal cells.

36. The host cell according to any one of the preceding items, wherein said host cell is plant cells, such as plant cells comprised within a plant or within a part of a plant.

37. The host cell according to item 36, wherein said plant cells are from a species of Nicotiana, such as Nicotiana benthamiana or Nicotiana tabacum and optionally are comprised within a plant of the species of Nicotiana.

38. The host cell according to any one of the preceding items, wherein said host cell is a yeast cell belonging to the genus of Saccharomyces, Pichia, Candida, Cryptococcus, Pichia (Komagataella), Lipomyces, Pseudozyma, Rhodosporidium, Rhodotorula, Trichosporon, Trigonopsis, Yarrowia or Saccharomycopsis, such as a yeast cell of the species Saccharomyces cerevisiae Yarrowia lipolytica, Hansenula polymorpha Ogataea polymorpha), Rhodotorula toruloides or Pichia pastoris (Komagataella phaffii).

39. The host cell according to any one of the preceding items, wherein said host cell is a bacterial cell belonging to the genus of Escherichia, Bacillus, Corynebacterium, Pseudomonas or Streptomyces, such as a bacterial cell of the species Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Pseudomonas putida or Streptomyces sp.

40. The host cell according to any one of the preceding items, wherein said nucleic acid is a heterologous nucleic acid.

41. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of baccatin III, a-phenylalanine, and benzoyl-CoA.

42. The host cell according to any one of the preceding items, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

43. The host cell according to any one of the preceding items, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

44. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of baccatin III, optionally wherein the host cell is capable of producing baccatin III.

45. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of baccatin III and [3- phenylalanoyl-CoA, optionally wherein the host cell is capable of producing baccatin III and/or p-phenylalanoyl-CoA.

46. The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of baccatin III and benzoyl-CoA, optionally wherein the host cell is capable of producing baccatin III and/or benzoyl-CoA. . The host cell according to any one of the preceding items, wherein the host cell is capable of producing taxol in the presence of baccatin III, benzoyl-CoA, and P-phenylalanoyl-CoA, optionally wherein the host cell is capable of producing baccatin III, benzoyl- CoA, and/or P-phenylalanoyl-CoA. . The host cell according to any one of items 44 to 47, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; ii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iii. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. . The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol, optionally wherein the host cell is capable of producing of N-debenzoyl-taxol. . The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and benzoyl- CoA, optionally wherein the host cell is capable of producing of N-debenzoyl- taxol and/or benzoyl-CoA. 1. The host cell according to any one of items 49 to 50, wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. 52. The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III.

53. The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and/or benzoyl-CoA.

54. The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, p- phenylalanoyl-CoA, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, P-phenylalanoyl-CoA, and/or benzoyl-CoA.

55. The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III and - phenylalanoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III and p-phenylalanoyl-CoA.

56. The host cell according to any one of items 52 to 55, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iv. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

57. The host cell according to any one of items 1 to 43, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, a- phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA.

58. The host cell according to item 57, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

59. The host cell according to item 57, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. 0. The host cell according to any one of items 1 to 59, wherein the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine. 1. The host cell according to any one of items 41 to 60, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. 2. The host cell according to any one of items 41 to 61 , wherein the host cell is capable of producing said benzoyl-CoA in the presence of benzoic acid. 3. The host cell according to any one of items 42 to 62, wherein the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

64. A method for producing a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, said method comprising the steps of: i. providing a host cell according to any one of items 1 to 63; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, preferably wherein said taxane having a side chain comprising a N-benzoylated 0-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol), and/or wherein said taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety comprises or consists of B7.

65. The method according to item 64, wherein said step of cultivating said host cell is performed in the presence of benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, 0- phenylalanine, 0-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl- N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol.

66. The method according to any one of items 64 to 65, wherein said host cell is capable of producing benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl- baccatin III, N-debenzoyl-taxol, a-phenylalanine, 0-phenylalanine, 0- phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl- taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol.

67. The method according to any one of items 64 to 66, wherein said cultivation medium is suitable for producing said taxane, preferably wherein said cultivation medium comprises benzoic acid, benzoyl- CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, p-phenylalanine, -phenylalanoyl-CoA, N-debenzoyl-2'-deoxy- taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'- deoxy-taxol.

68. The method according to any one of items 64 to 67, wherein said taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety is taxol and wherein said cultivation medium comprises baccatin III.

69. The method according to any one of items 64 to 68, wherein said taxane having a side chain comprising an N-benzoylated P-phenylalanine moiety is taxol and wherein said host cell is capable of producing baccatin III.

70. The method according to any one of items 68 to 69, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

71. The method according to any one of items 68 to 69, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. 2. The method according to any one of items 64 to 67, wherein said taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety is taxol and wherein said cultivation medium comprises 10-deacetyl-baccatin III. 3. The method according to item 72, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

74. The method according to item 72, wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

75. The method according to any one of items 64 to 74, wherein the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine and wherein said cultivation medium comprises a-phenylalanine and/or the host cell is capable of producing said a-phenylalanine.

76. The method according to any one of items 64 to 75, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto 7. The method according to any one of items 64 to 76, wherein the host cell is capable of producing said benzoyl-CoA in the presence of benzoic acid and wherein said cultivation medium comprises benzoic acid. 8. The method according to any one of items 64 to 77, wherein the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto. 9. The method according to any one of items 64 to 78, further comprising a step of recovering said taxane having a side chain comprising an N-benzoylated |3- phenylalanine moiety and/or said taxane having a side chain comprising an N- acetylated p-phenylalanine moiety. 0. The method according to any one of items 64 to 79, wherein said step of recovering comprises: i. a step of extraction, such as extraction with a solvent, for example methanol (MeOH) and/or ethyl acetate (EtOAc); and/or ii. a step of chromatography, such as liquid chromatography (LC), for example column chromatography or preparative/semi-preparative high performance LC (HPLC). 1. A method for producing a taxane having a side chain comprising an N- benzoylated -phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety, said method comprising the steps of: i. contacting a benzoyl transferase with a substrate, such as N-debenzoyl- taxol or 10-deacetyl-N-debenzoyl-taxol, wherein said benzoyl transferase is a benzoyl transferase from T. chinensis, such as BT6.1 as set forth in SEQ ID NO: 5, from T. cuspidata, such as BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

82. The method according to item 81 , wherein said taxane having a side chain comprising a N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol), and/or wherein said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety comprises or consists of B7.

83. The method according to any one of items 81 to 82, wherein the substrate is N- debenzoyl-taxol.

84. Use of a benzoyl transferase from Taxus chinensis in a method for producing a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3- phenylalanine moiety, optionally wherein the T. chinensis benzoyl transferase is BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

85. Use of benzoyl transferase in a method for producing a taxane having a side chain comprising an N-benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety, wherein the benzoyl transferase is a benzoyl transferase from T. chinensis, from T. cuspidata, or BT6.1 as set forth in SEQ ID NO: 5, BT6.1 (T294N) as set forth in SEQ ID NO: 57, BT6.1 (C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto. The use according to any one of items 84 to 85, wherein the benzoyl transferase is BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 95% sequence identity thereto. The use according to any one of items 84 to 86, wherein the benzoyl transferase is BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 92% sequence identity, such as at least 93% sequence identity thereto. The use according to any one of items 84 to 87, wherein said taxane having a side chain comprising a N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or paclitaxel (taxol) or said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety comprises or consists of B7. The use according to any one of items 84 to 88, wherein the benzoyl transferase comprises or consists of the sequence as set forth in SEQ ID NO: 5, with the exception that at the most 44 residues are mutated, such as at the most 40 residues, such as at the most 35 residues, such as at the most 30 residues, such as at the most 25 residues, such as at the most 20 residues, such as at the most 15 residues, such as at the most 10 residues, such as at the most 5 residues, or less residues are mutated. The use according to any one of items 84 to 89, wherein said use comprises expressing the benzoyl transferase comprising or consisting of the sequences as set forth in SEQ ID NO: 5 in a host cell, preferably wherein the host cell is as defined in any one of items 1 to 63. The use according to any one of items 84 to 90, wherein said method is as defined in any one of items 64 to 80. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity, such as at least 95% sequence identity thereto, such as SEQ ID NO: 11 or a homologue thereof having at least 70%, such as at least 80% sequence identity, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1(T294N) as set forth in SEQ ID NO: 57 or a functional homologue thereof having at least 80% sequence identity thereto, such as at least 92% sequence identity, such as at least 93% sequence identity thereto, such as SEQ ID NO: 56 or a homologue thereof having at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 (C369W) as set forth in SEQ ID NO: 59 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 58 or a homologue thereof having at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto. 95. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1(L199M) as set forth in SEQ ID NO: 61 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 60 or a homologue thereof having at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

96. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 62 or a homologue thereof having at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

97. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1_21Xmut as set forth in SEQ ID NO: 65 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 64 or a homologue thereof having at least 80%, for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

98. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1_17Xmut as set forth in SEQ ID NO: 67 or a functional homologue thereof having at least 80% sequence identity thereto, such as SEQ ID NO: 66 or a homologue thereof having at least 80%, such for example at least 90% sequence identity, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

99. The nucleic acid construct according to any one of items 92 to 98, further comprising one or more of: i. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 9; ii. a nucleic acid encoding a protein comprising or consisting of BAPT as set forth in SEQ ID NO: 1 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 7; iii. a nucleic acid encoding a protein comprising or consisting of MBPig3BAPT as set forth in SEQ ID NO: 27 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 32; iv. a nucleic acid encoding a protein comprising or consisting of CoAL(A312G) as set forth in SEQ ID NO: 2 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 8; v. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 10; vi. a nucleic acid encoding a protein comprising or consisting of TchiAAE5 as set forth in SEQ ID NO: 48 or a functional homologue thereof having at least 70% sequence identity thereto; vii. a nucleic acid encoding a protein comprising or consisting of sbCoAL as set forth in SEQ ID NO: 50 or a functional homologue thereof having at least 70% sequence identity thereto; viii. a nucleic acid encoding a protein comprising or consisting of HcAAEI as set forth in SEQ ID NO: 51 or a functional homologue thereof having at least 70% sequence identity thereto, and/or ix. a nucleic acid encoding a protein comprising or consisting of TcuDBAT as set forth in SEQ ID NO: 25 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 26; or homologues of any of the aforementioned nucleic acids having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity thereto. 00. The nucleic acid construct according to any one of items 92 to 99, further comprising one or more of: i. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 33; ii. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 34; iii. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 35; and/or iv. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 36; or homologues of any of the aforementioned nucleic acids having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity thereto.

101. The nucleic acid construct according to any one of items 92 to 100, further comprising a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49 or a functional homologue thereof having at least 70% sequence identity thereto, such as SEQ ID NO: 52, or a homologue thereof having at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 99% sequence identity to SEQ ID NO: 52.

102. The nucleic acid construct according to any one of items 92 to 101 , further comprising a promotor, such as a constitutive promoter and/or an inducible promoter, operably linked to any one or more of the nucleic acids.

103. An isolated polypeptide as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 70% sequence identity thereto, such as at least 80% sequence identity thereto, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto. 104. An isolated polypeptide as set forth in SEQ ID NO: 57 (BT6.1(T294N)), SEQ ID NO: 59 (BT6.1(C369W)), SEQ ID NO: 61 (BT6.1(L199M)), SEQ ID NO: 63 (BT6.1(L199M, A372T)), SEQ ID NO: 65 (BT6.1_21Xmut) or SEQ ID NO: 67 (BT6.1_17Xmut), or functional homologues thereof having at least 80% sequence identity thereto, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

105. A vector comprising at least one of the nucleic acid constructs according to any one of items 92 to 102.

106. A host cell according to any one of items 1 to 63, comprising the nucleic acid construct according to any one of items 92 to 102, or a vector according to item 105.

107. A kit of parts comprising: i. a host cell or the host cell according to item 106, and optionally instructions for use, and/or ii. the nucleic acid construct according to any one of items 92 to 102 or the vector according to item 105, and optionally instructions for use, and further optionally a host cell to be modified, preferably wherein the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells, and fungal cells.

108. A compound having the structure of structure (VII) (B7):

Structure (VII). . A cell culture obtained by a method according to any one of items 64 to 80 and/or the use according to any one of items 84 to 91 . . A cell culture, comprising a host cell according to any one of items 1 to 63 or 106, and optionally a cultivation medium. . A fermentation liquid comprising a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated |3-phenylalanine moiety, wherein said fermentation liquid is: i. obtained by the method of any one of items 64 to 80 and/or the use according to any one of items 84 to 91 ; ii. comprised in the cell culture according to any one of items 109 to 110, and/or iii. comprised within and/or secreted by the host cell according to any one of items 1 to 63 or 106. . The fermentation liquid of item 111 , wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the host cells are lysed. . The fermentation liquid according to any one of items 111 to 112, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has been separated from the liquid. . A composition comprising one or more of: i. the fermentation liquid according to any one of items 111 to 113; ii. a taxane having a side chain comprising an N-benzoylated [3- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated -phenylalanine moiety obtained by the method of any one of items 64 to 80 and/or the use according to any one of items 84 to 91 ; iii. B7 obtained by the method of any one items 64 to 80 and/or the use according to any one of items 84 to 91 ; and/or iv. taxol obtained by the method of any one of items 64 to 80 and/or the use according to any one of items 84 to 91, and optionally one or more agents, additives and/or excipients. . The composition of item 114, wherein the composition have been processed into in a semi-dry or dry solid form, optionally in form of a powder, tablet, capsule, chewable, gel and/or gum. . The composition of item 114, wherein the composition is in a liquid form, optionally in a stabilized liquid form. . A method for treating a disorder such as cancer, comprising administration of a medicament comprising a composition obtained by the method of any one of items 64 to 80 and/or the use according to any one of items 84 to 91 , said composition comprising a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety. . The host cell, the method, the use, the fermentation liquid, the cell culture and/or the composition according to any one of the preceding items, wherein said taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety comprises or consists of 10-deacetyl-taxol, and/or taxol. . The host cell, the method, the use, the fermentation liquid, the cell culture and/or the composition according to any one of the preceding items, wherein said taxane having a side chain comprising an N-acetylated [3-phenylalanine moiety comprises or consists of B7.

Claims

1 . A host cell comprising a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5, BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1 (C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity, such as at least 98% sequence identity, such as at least 99% sequence identity to SEQ ID NO: 5.

2. The host cell according to claim 1, wherein said host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 95% sequence identity to SEQ ID NO: 5.

3. The host cell according to any one of the preceding claims, wherein said host cell is selected from the group of plant cells, yeast cells, bacterial cells and fungal cells, optionally wherein said host cell is plant cells, such as plant cells comprised within a plant or within a part of a plant.

4. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing a taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety in the presence of benzoyl- CoA, acetyl-CoA, benzoic acid, baccatin III, 10-deacetyl-baccatin III, N- debenzoyl-taxol, a-phenylalanine, [3-phenylalanine, [3-phenylalanoyl-CoA, N- debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl- N-debenzoyl-2'-deoxy-taxol, optionally wherein said host cell is capable of producing benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl- baccatin III, N-debenzoyl-taxol, a-phenylalanine, [3-phenylalanine, [3- phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl- taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol.

5. The host cell according to any one of the preceding claims, further comprising one or more of: i. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; ii. a nucleic acid encoding a protein comprising or consisting of BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iii. a nucleic acid encoding a protein comprising or consisting of CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50 or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; v. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

6. The host cell according to any one of the preceding claims, wherein the host cell further comprises: i. a nucleic acid encoding a protein comprising or consisting of a phenylalanine ammonia-lyase, such as a phenylalanine ammonia-lyase 2, for example PAL (SEQ ID NO: 28); ii. a nucleic acid encoding a protein comprising or consisting of a cinnamic acid:CoA ligase, such as CNL (SEQ ID NO: 29); iii. a nucleic acid encoding a protein comprising or consisting of a cinnamoyl- CoA hydratase-dehydrogenase, such as CHD (SEQ ID NO: 30); and iv. a nucleic acid encoding a protein comprising or consisting of a 3-ketoacyl CoA thiolase, such as Kat1 (SEQ ID NO: 31); and/or a. a nucleic acid encoding a benzoyl-ligase, such as a benzoyl-ligase capable of converting benzoic acid into benzoyl-CoA, optionally wherein said benzoyl-ligase is native to an Arabidopsis cell, such as an Arabidopsis thaliana cell, further optionally wherein said nucleic acid encoding a protein comprises or consists of AtBZOI as set forth in SEQ ID NO: 49; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

7. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and/or benzoyl-CoA, optionally wherein the host cell is capable of producing of N- debenzoyl-taxol and/or benzoyl-CoA, preferably wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

8. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and/or benzoyl-CoA, wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

9. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of N-debenzoyl-taxol and/or benzoyl-CoA, wherein the host cell comprises a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61, BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

10. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of baccatin III, a- phenylalanine, and benzoyl-CoA, preferably wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; ii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iv. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and v. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

11. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of baccatin III, benzoyl-CoA, and/or p-phenylalanoyl-CoA, optionally wherein the host cell is capable of producing baccatin III, benzoyl- CoA, and/or p-phenylalanoyl-CoA, preferably wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; ii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iii. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

12. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, p- phenylalanoyl-CoA, and/or benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, -phenylalanoyl-CoA, and/or benzoyl-CoA, preferably wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; iii. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and iv. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

13. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing taxol in the presence of 10-deacetyl-baccatin III, a- phenylalanine, and benzoyl-CoA, optionally wherein the host cell is capable of producing 10-deacetyl-baccatin III, a-phenylalanine, and/or benzoyl-CoA, preferably wherein the host cell comprises: i. a nucleic acid encoding a protein comprising or consisting of Tct/DBAT as set forth in SEQ ID NO: 25; ii. a nucleic acid encoding a protein comprising or consisting of PAM as set forth in SEQ ID NO: 4; iii. a nucleic acid encoding a protein comprising or consisting of a CoAL, such as CoAL(A312G) as set forth in SEQ ID NO: 2, TchiAAE5 as set forth in SEQ ID NO: 48, sbCoAL as set forth in SEQ ID NO: 50, or HcAAEI as set forth in SEQ ID NO: 51 ; iv. a nucleic acid encoding a protein comprising or consisting of an amino phenylpropanoyl transferase, such as BAPT as set forth in SEQ ID NO: 1 or MBPig3BAPT as set forth in SEQ ID NO: 27; v. a nucleic acid encoding a protein comprising or consisting of OD3 as set forth in SEQ ID NO: 3; and vi. a heterologous nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5; or functional homologues of any of the aforementioned having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

14. The host cell according to any one of the preceding claims, i. wherein the host cell is capable of producing said benzoyl-CoA in the presence of a-phenylalanine, optionally wherein the host cell further comprises: a. a nucleic acid encoding a protein comprising or consisting of PAL as set forth in SEQ ID NO: 28; b. a nucleic acid encoding a protein comprising or consisting of CNL as set forth in SEQ ID NO: 29; c. a nucleic acid encoding a protein comprising or consisting of CHD as set forth in SEQ ID NO: 30; and d. a nucleic acid encoding a protein comprising or consisting of Kat1 as set forth in SEQ ID NO: 31 ; and/or ii. wherein the host cell is capable of producing said benzoyl-CoA in the presence of benzoic acid, optionally wherein the host cell further comprises a nucleic acid encoding a protein comprising or consisting of AtBZOI as set forth in SEQ ID NO: 49, or a functional homologue thereof having at least 70% sequence identity, such as at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

15. A method for producing a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, said method comprising the steps of: i. providing a host cell according to any one of claims 1 to 14; ii. cultivating said host cell in a cultivation medium, thereby producing said taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or said taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety.

16. The method according to claim 15, wherein said step of cultivating said host cell is performed in the presence of benzoic acid, benzoyl-CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, 0- phenylalanine, 0-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl- N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol, optionally wherein: i. said host cell is capable of producing benzoyl-CoA, acetyl-CoA, baccatin III, 10- deacetyl-baccatin III, N-debenzoyl-taxol, a-phenylalanine, 0-phenylalanine, 0- phenylalanoyl-CoA, N-debenzoyl-2'-deoxy-taxol, 10-deacetyl-N-debenzoyl- taxol, and/or 10-deacetyl-N-debenzoyl-2'-deoxy-taxol; and/or ii. said cultivation medium is suitable for producing said taxane, preferably wherein said cultivation medium comprises benzoic acid, benzoyl- CoA, acetyl-CoA, baccatin III, 10-deacetyl-baccatin III, N-debenzoyl-taxol, a- phenylalanine, 0-phenylalanine, 0-phenylalanoyl-CoA, N-debenzoyl-2'-deoxy- taxol, 10-deacetyl-N-debenzoyl-taxol, and/or 10-deacetyl-N-debenzoyl-2'- deoxy-taxol.

17. A method for producing a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated 0-phenylalanine moiety, said method comprising the steps of: i. contacting a benzoyl transferase with a substrate, such as N-debenzoyl- taxol or 10-deacetyl-N-debenzoyl-taxol, wherein said benzoyl transferase is a benzoyl transferase from T. chinensis, such as BT6.1 as set forth in SEQ ID NO: 5, from T. cuspidata, such as BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

18. Use of benzoyl transferase in a method for producing a taxane having a side chain comprising an N-benzoylated p-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety, wherein the benzoyl transferase is a benzoyl transferase from T. chinensis, from T. cuspidata, or BT6.1 as set forth in SEQ ID NO: 5, BT6.1 (T294N) as set forth in SEQ ID NO: 57, BT6.1 (C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1(L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

19. The use according to claim 18, wherein the T. chinensis benzoyl transferase is BT6.1 as set forth in SEQ ID NO: 5 or a functional homologue thereof having at least 80% sequence identity, such as at least 90% sequence identity, such as at least 95% sequence identity thereto.

20. A nucleic acid construct for expression in a host cell, comprising a nucleic acid encoding a protein comprising or consisting of BT6.1 as set forth in SEQ ID NO: 5, BT6.1(T294N) as set forth in SEQ ID NO: 57, BT6.1(C369W) as set forth in SEQ ID NO: 59, BT6.1(L199M) as set forth in SEQ ID NO: 61 , BT6.1 (L199M, A372T) as set forth in SEQ ID NO: 63, BT6.1_21Xmut as set forth in SEQ ID NO: 65 or BT6.1_17Xmut as set forth in SEQ ID NO: 67, or functional homologues thereof having at least 80% sequence identity thereto, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity thereto, such as at least 95% sequence identity, for example at least 99% sequence identity thereto, such as SEQ ID NO: 11, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64 or SEQ ID NO: 66, respectively, or homologues thereof having at least 80%, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity thereto, such as at least 95% sequence identity, for example at least 99% sequence identity thereto.

21. An isolated polypeptide as set forth in SEQ ID NO: 5, SEQ ID NO: 57 (BT6.1(T294N)), SEQ ID NO: 59 (BT6.1(C369W)), SEQ ID NO: 61 (BT6.1(L199M)), SEQ ID NO: 63 (BT6.1 (L199M, A372T)), SEQ ID NO: 65 (BT6.1_21Xmut) or SEQ ID NO: 67 (BT6.1_17Xmut), or functional homologues thereof having at least 80% sequence identity thereto, such as at least 85% sequence identity, such as at least 90% sequence identity, such as at least 92% sequence identity, such as at least 93% sequence identity, such as at least 95% sequence identity thereto.

22. A vector comprising at least one of the nucleic acid constructs according to claim 20.

23. A host cell according to any one of claims 1 to 14, comprising the nucleic acid construct according to claim 20, or a vector according to claim 22.

24. A kit of parts comprising: i. a host cell or the host cell according to claim 23, and optionally instructions for use, and/or ii. the nucleic acid construct according to claim 20, or a vector according to claim 22, and optionally instructions for use, and further optionally a host cell to be modified, preferably wherein the host cell is selected from the group consisting of plant cells, yeast cells, bacterial cells, and fungal cells.

25. A compound having the structure of structure (VII) (B7):

Structure (VII).

26. A cell culture obtained by a method according to any one of claims 15 to 17 and/or the use according to any one of claims 18 to 19.

27. A cell culture, comprising a host cell according to any one of claims 1 to 14, and optionally a cultivation medium.

28. A fermentation liquid comprising a taxane having a side chain comprising an N- benzoylated 0-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated |3-phenylalanine moiety, wherein said fermentation liquid is: i. obtained by the method of any one of claims 15 to 17 and/or the use according to any one of claims 18 to 19; ii. comprised in the cell culture according to any one of claims 26 to 27, and/or iii. comprised within and/or secreted by the host cell according to any one of claims 1 to 14.

29. A composition comprising one or more of: i. the fermentation liquid according to claim 28; ii. a taxane having a side chain comprising an N-benzoylated [3- phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety obtained by the method of any one of claims 15 to 17 and/or the use according to any one of claims 18 to iii. B7 obtained by the method of any one of claims 15 to 17 and/or the use according to any one of claims 18 to 19; and/or iv. taxol obtained by the method of any one of claims 15 to 17 and/or the use according to any one of claims 18 to 19, and optionally one or more agents, additives and/or excipients.

30. A method for treating a disorder such as cancer, comprising administration of a medicament comprising a composition obtained by the method of any one of claims 15 to 17 and/or the use according to any one of claims 18 to 19 said composition comprising a taxane having a side chain comprising an N- benzoylated [3-phenylalanine moiety and/or a taxane having a side chain comprising an N-acetylated p-phenylalanine moiety.

31. The host cell, the method, the use, the nucleic acid, the composition, the kit, the cell culture, the fermentation liquid according to any one of the preceding claims, wherein said taxane having a side chain comprising an N-benzoylated P-phenylalanine moiety comprises or consists of taxol.