Enzymes for heterologous taxane production

Abstract

As provided herein, many embodiments are directed to polynucleotides, expression cassettes, vectors, system, cells, tissues, and organisms to be used in the heterologous production of taxanes. In various instances, such polynucleotides, expression cassettes, vectors, cells, tissues, and organisms may be configured to produce one or more enzymes in a biosynthetic pathway for such taxanes. Such enzymes may be isolated for use in exogenous reactions (e.g., outside of or independent of a biological cell) to produce taxanes. Alternatively or additionally, such enzymes may be used to produce taxanes within a cell, tissue, or organism in in vitro and / or in vivo reactions. Additional embodiments include methods of making and / or using such polynucleotides, expression cassettes, vectors, systems, cells, tissues, and organisms for the production of taxanes.

Description

BFF Ref: STAN-2224WO Clt Ref: S24-220 ENZYMES FOR HETEROLOGOUS TAXANE PRODUCTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The current application claims priority to U.S. Provisional Patent Application No. 63 / 703,747, filed October 4, 2024, entitled “Enzymes for Heterologous Taxane Production” to Sattely, et al. and U.S. Provisional Patent Application No. 63 / 720,610, filed November 14, 2024, entitled “Enzymes for Heterologous Taxane Production” to Sattely, et al; the disclosures of which are hereby incorporated by reference in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under contracts # AT010593 and AT012787 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention relates to the production of taxanes, more specifically, polynucleotides, expression cassettes, and organisms for the expression of one or more proteins for use in the production of taxanes.SEQUENCE LISTING

[0004] This application hereby incorporates by reference the material of the electronic Sequence Listing filed concurrently herewith. The material in the electronic Sequence Listing is submitted as an xml (.xml) file entitled “STAN-2224WO_SeqList.xml” created on September 30, 2025, which has a file size of approximately 20,480 bytes, and is herein incorporated by reference in its entirety.BACKGROUND

[0005] Plants synthesize potent small molecules, but disentangling or engineering their biosynthetic capacity remains a significant challenge. Taxol (paclitaxel) is a prominent chemotherapeutic agent used in the treatment of various cancers, including ovarian, breast, and lung cancer. Sales for this therapeutic reached $2 billion in 2001 (when it was the bestselling therapeutic on the market). By 2019, paclitaxel and its derivatives constituted approximately $15 billion in annual sales. Despite its clinical importance, the production of paclitaxel is challenged by its low natural abundance and the complex, costly processes currently required for its synthesis and extraction fromBFF Ref: STAN-2224WO Clt Ref: S24-220 natural sources. These methods are not only inefficient but also environmentally demanding, as they rely on the harvesting of bark from the yew tree, which is slow- growing and environmentally sensitive. Thus, there is a need for efficient methods for synthesizing paclitaxel and its derivatives, rather than relying on isolation and / or purification.SUMMARY OF THE INVENTION

[0006] As provided herein, many embodiments are directed to polynucleotides, expression cassettes, vectors, system, cells, tissues, and organisms to be used in the heterologous production of taxanes. In various instances, such polynucleotides, expression cassettes, vectors, cells, tissues, and organisms may be configured to produce one or more enzymes in a biosynthetic pathway for such taxanes. Such enzymes may be isolated for use in exogenous reactions (e.g., outside of or independent of a biological cell) to produce taxanes. Alternatively or additionally, such enzymes may be used to produce taxanes within a cell, tissue, or organism in in vitro and / or in vivo reactions. Additional embodiments include methods of making and / or using such polynucleotides, expression cassettes, vectors, systems, cells, tissues, and organisms for the production of taxanes.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The description and claims will be more fully understood with reference to the following figures and data graphs, which are presented as exemplary embodiments of the invention and should not be construed as a complete recitation of the scope of the invention.

[0008] Figures 1 A-1 F illustrate exemplary data showing a platform combining snRNA- seq and multiplexed perturbation to identify plant biosynthesis pathways in accordance with various embodiments of the invention.

[0009] Figures 2A-2I illustrate exemplary data showing identification of taxane biosynthesis gene modules in accordance with various embodiments of the invention.

[0010] Figures 3A-3Q illustrate exemplary data showing the characterization of FoTO1 (facilitator of taxane oxidization) in accordance with various embodiments of the invention.

[0011] Figures 4A-4L illustrate exemplary data showing the discovery and characterization of T9aH, T7AT, and T1 H in accordance with various embodiments of the invention.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0012] Figures 5A-5O illustrate exemplary data showing the discovery and characterization of two hydrolases in Nicotiana benthamiana in accordance with various embodiments of the invention.

[0013] Figures 6A-6D illustrate exemplary data showing newly identified genes for baccatin biosynthesis in accordance with various embodiments of the invention.

[0014] Figure 7 illustrates exemplary data showing a biosynthetic pathway in Nicotiana benthamiana and proposed final steps to synthesize Taxol in accordance with various embodiments of the invention.

[0015] Figure 8 illustrates exemplary data showing FoTO1 corrects the product profile of T5oH in yeast in accordance with various embodiments of the invention.

[0016] Figure 9 illustrates exemplary data showing that BCL boosts benzoyltransferase activity of DBTNBT in accordance with various embodiments of the invention.

[0017] Figure 10 illustrates exemplary data showing de novo biosynthesis of Taxol in N. benthamiana in accordance with various embodiments of the invention.

[0018] Figure 11 illustrates exemplary data showing identification of two putative transporters that boost yield of Taxol de novo biosynthesis in accordance with various embodiments of the invention.DETAILED DESCRIPTION

[0019] Paclitaxel is a taxane with a common use as a chemotherapeutic agent. Paclitaxel is commonly known as “Taxol” and the terms “paclitaxel” and “Taxol” are used interchangeably within this document. The biosynthetic pathway for paclitaxel, commonly known as, and interchangeably referred to as as “Taxol.” has eluded discovery despite intensive research in the past half-century. To address the complexity, redundancy, and scale of plant biosynthetic machinery, many embodiments utilized a multiplexed perturbation strategy to transcriptionally profile cell states spanning tissues, cell types, developmental stages, and elicitation conditions. This strategy identified biosynthetic genes segregated into consecutive expression modules.

[0020] Turning now to the drawings, many embodiments are directed to polynucleotides, expression cassettes, vectors, system, cells, tissues, and organisms to be used in the heterologous production of taxanes. In various instances, such polynucleotides, expression cassettes, vectors, cells, tissues, and organisms may be configured to produce one or more enzymes in a biosynthetic pathway for such taxanes. Such enzymes may be isolated for use in exogenous reactions (e.g., outside of orBFF Ref: STAN-2224WO Clt Ref: S24-220 independent of a biological cell) to produce taxanes. Alternatively or additionally, such enzymes may be used to produce taxanes within a cell, tissue, or organism in in vitro and / or in vivo reactions. Additional embodiments include methods of making and / or using such polynucleotides, expression cassettes, vectors, systems, cells, tissues, and organisms for the production of taxanes.

[0021] Before the present polynucleotides, vectors, systems, cells, and methods are described, it is to be understood that this invention is not limited to particular methods or components described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the presentBFF Ref: STAN-2224WO Clt Ref: S24-220 invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0025] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the vector" includes reference to one or more vectors and equivalents thereof, such as viral vectors, plasmids, constructs, and the like, known to those skilled in the art, and so forth.

[0026] It must be noted that as used herein and in the appended claims, references to numerical order — e.g., “first,” “second,” “third,” etc. — may be used for convenience to differentiate between similar components or features. Unless mandated by the text or context, such references to order should not be considered to force order on any such component.

[0027] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0028] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains.

[0029] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of up to plus or minus five percent.

[0030] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the doublestranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0031] As used herein, the term “polynucleotide construct” refers to a DNA segment of any size that includes one or more sequences encoding an RNA or protein and at least one promoter for driving expression from the one or more sequences. A polynucleotide construct may be a circular DNA or a linear DNA. A polynucleotide construct may be single stranded or double stranded.

[0032] As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which may transfer gene sequences into and between cells. Thus, the term includes cloning and expression vehicles (e.g., expression cassettes), as well as bacterial or viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A vector may be linear or circular, single stranded or double stranded, DNA or RNA. In certain aspects, the vector may be circular, double stranded DNA.

[0033] As used herein, the term “vector system” refers to two or more vectors that are used together, e.g., by simultaneous or sequential introduction into a cell, to provide at least two different components into the cell. The two different components may then work together in the cell.

[0034] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. The term percent “sequence identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “sequence identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0035] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A havingBFF Ref: STAN-2224WO Clt Ref: S24-220 a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows:[(Number of Identical Positions) / (Total Number of Positions in the Test Sequence)] x 100%

[0036] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match / mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value andBFF Ref: STAN-2224WO Clt Ref: S24-220 a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows:Percent Sequence Identity = (“Percent Identity” output value) x (“Query Coverage” output value)

[0037] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions) / (total number of nucleotides in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 1 : AAAAAGGGGG (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5) / (10)] x100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10) / (20)] x100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10) / (10)] x100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6.

[0038] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions) / (total number of amino acids in the test sequence)] x 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5) / (10)] x100% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10) / (20)] x100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity isBFF Ref: STAN-2224WO Clt Ref: S24-220 calculated to compare test sequence 1 1 : FFFFFYYYYY (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10) / (10)] x100% = 100%. Test sequence 1 1 has 100% sequence identity to reference sequence 12.

[0039] For purposes herein, reference to a polynucleotide sequence (e.g., a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG also encompasses a sequence of CCCCCTTTTT.

[0040] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0041] The term "host cell" denotes, for example, microorganisms, yeast cells, insect cells, plant cells, and mammalian cells, that may be, or have been, used as recipients of a vector or vector system as described herein, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.

[0042] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.

[0043] The term "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0044] The terms “constitutive” or “constitutive expression” are used interchangeably herein. They refer to genes that are transcribed in an ongoing manner. Such genes are driven by a constitutive promoter. In some embodiments, the terms refer to the expression of a protein or enzyme, or a nucleic acid sequence that is not conditioned on addition of an expression triggering agent to the cell culture medium. A constitutive promoter is capable of directing continuous gene expression in a cell. Constitutive promoters regulate expression of basal genes, like housekeeping genes. In contrast, an inducible promoter directs gene expression in the presence of particular transcription activator(s) or absence of a transcription repressor(s). Thus, an inducible promoter can be controlled by controlling the level of the transcription activator(s) or transcription repressor(s).

[0045] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. A recombinant bacterium is a bacterium comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original construct.

[0046] A "control element” or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. A promoter is usually upstream of a gene whose expression is controlled by the promoter.

[0047] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0048] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter. Thus, for example, a coding sequence for a taxadiene synthase coding sequence or protein operatively linked to a heterologous promoter refers to a taxadiene synthase coding sequence operatively linked to a nonnative promoter.

[0049] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.

[0050] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a payload to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a payload to a mammalian subject (which may be referred to as "transgenes" to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

[0051] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substanceBFF Ref: STAN-2224WO Clt Ref: S24-220 may be prepared by using a purification technique to enrich it from a source mixture. Enrichment may be measured on an absolute basis, such as weight per volume of solution, or it may be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some cases purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.

[0052] The terms "hybridize" and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.

[0053] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule may have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.).

[0054] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands may base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or betweenBFF Ref: STAN-2224WO Clt Ref: S24-220 an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non- complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.

[0055] As used herein, the term “recombination site” denotes a region of a nucleic acid molecule comprising a binding site or sequence-specific motif recognized by a sitespecific recombinase that binds at the target site and catalyzes recombination of specific sequences of DNA at the target site. Site-specific recombinases catalyze recombination between two such target sites. The relative orientation of the target sites determines the outcome of recombination. For example, translocation occurs if the recombination sites are on separate DNA molecules. DNA between two recombination sites oriented in the same direction on the same DNA molecule will be excised as a circular loop of DNA. DNA between two recombination sites that are orientated in the opposite direction on the same DNA molecule will be inverted.

[0056] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-.

[0057] The nucleotide sequences of the nucleic acids may be codon-optimized. “Codon-optimized” refers to changes in the codons of the polynucleotide encoding aBFF Ref: STAN-2224WO Clt Ref: S24-220 protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, a nucleic acid of the present disclosure encoding the taxadiene synthase may be codon-optimized for optimal production from the host organism selected for expression, e.g., plant cells, such as cells form Nicotiana benthamiana.

[0058] The term “transfection” or “transduction” is used to refer to the introduction of foreign DNA into a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rdedition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw- Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material.

[0059] The term “affinity tag,” as used herein, refers to a chemical moiety that functions as, or contains, an affinity ligand that is capable of binding (e.g., non-covalently or covalently) to a second, “capture” chemical moiety, such that the nucleic acid complex or derivative thereof can be selected (or “captured”) from a mixture using the capture moiety. In some embodiments, the capture moiety is bound to a solid support, e.g., a bead (e.g., a magnetic bead), planar surface, or the like. Non-limiting examples of affinity tags that may be included in the oligonucleotide probe, the probe complement oligonucleotide, or both, include biotin, avidin, streptavidin, an aptamer (see, e.g., Wilson & Szostak (1999) Anna Rev Biochem. 68:611-647), an MS2 coat protein-interacting sequence, a U1 A protein-interacting sequence, etc. Nucleic acid affinity tags that find use in the oligonucleotides and methods of the present disclosure are described, e.g., in Walker et al. (2008) Methods Mol Biol. 488:23-40. Interactions between the affinity tag and the capture moiety may be specific and reversible (e.g., non-covalent binding or hydrolyzable covalent linkage), but if desired, may be (or subsequently may be made)BFF Ref: STAN-2224WO Clt Ref: S24-220 irreversible, e.g., a non-hydrolyzable covalent linkage between the affinity tag and the capture moiety.

[0060] Non-limiting examples of tags which may be employed in the single-chain proteins include a Flag tag (e.g., DYKDDDDK), HA tag (e.g., YPYDVPDYA), HA 12Ca5 tag (e.g., CYPYDVPDYA), Myc tag (e.g., EQKLISEEDL), His tag (e.g., HHHHHH), V5 tag (e.g., GKPIPNPLLGLDST), GST tag, biotin tag, and the like.Enzymes for Taxane Synthesis

[0061] Various embodiments are directed to one or more enzymes for taxane synthesis. In some instances, the enzyme is selected from one or more of the enzymes listed in Tables 1 and 2. Table 1 above provides a list of enzymes used in Taxol production, while Table 2 above provides a list of enzymes that are used in taxanes other than Taxol . Thus, the enzymes in Table 2 can be considered optional for production of Taxol .

[0062] In some preferred embodiments, the sequence encoding an enzyme for taxane production is selected from one or more of: FoTO1 , T9aH1 , T7AT, T1 [3H-686, DeAc898, T9ox, DeAc1023, POL, BCL, ABC transporter, lipid transfer protein, T1 pH-184, and / or T1 pH-246. In some instances, the sequence encoding an enzyme for taxane production is selected from one or more of: SEQ ID NOs: 1 -13. In some instances, the sequence encoding an enzyme for taxane production produces an enzyme that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more of: SEQ ID NOs: 1 -13. In some instances, the sequence encoding an enzyme for taxane production produces an enzyme that has approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% sequence identity to one or more of: SEQ ID NOs: 1 -13. In some instances, the sequence encoding an enzyme for taxane production comprises the sequence of one or more of SEQ ID NOs: 1 -13.

[0063] In some cases, the sequence encoding an enzyme for taxane production is selected includes FoTO1 (SEQ ID NO: 1 ). In some instances, the sequence encoding an enzyme for taxane production is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some instances, the sequence encoding an enzyme for taxane production is approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% sequence identity to SEQ ID NO: 1. In some instances, theBFF Ref: STAN-2224WO Clt Ref: S24-220 sequence encoding an enzyme for taxane production comprises the sequence of SEQ ID NO: 9.

[0064] In some instances, the one or more enzyme may include a tag, such as an affinity tag. In such instances, the affinity tag may be used to isolate or purify the enzyme after production from a supernatant, culture, or lysate, or other liquid in which the enzyme is (or enzymes are) present. Such affinity tags are known in the art and may be placed in any appropriate location (e.g., N-terminus, C-terminus, etc.) of the enzyme to allow isolation or purification while maintaining enzymatic activity. The isolation or purification can utilize tools, such as affinity resin, pulldown, and / or other known methodologies.

[0065] Additional modifications to an enzymes amino acid sequence can include a signaling sequence and / or export sequence. Such sequences may allow for surface display and / or exportation of the enzyme.Polynucleotide and Polynucleotide Systems

[0066] Various embodiments are directed to one or more polynucleotides, where each polynucleotide comprises a sequence encoding an enzyme for taxane synthesis. A collection of polynucleotides may be considered a “polynucleotide system,” “a polynucleotide set,” and / or “a set of polynucleotides.”

[0067] In various instances, a polynucleotide is provided that includes a first promoter operably linked to a first sequence encoding an enzyme for taxane synthesis. In some embodiments, the first promoter is heterologous to the first sequence encoding an enzyme for taxane synthesis. In some embodiments, the first promoter has higher promoter activity as compared to the native promoter of the first sequence encoding an enzyme for taxane synthesis. In some instances, the first promoter is inducible. In some instances, the first promoter operably is constitutive.

[0068] In certain embodiments, a polynucleotide includes a second sequence encoding an enzyme for taxane synthesis. A second sequence encoding an enzyme for taxane synthesis may be operably linked to the first promoter (e.g., polycistronic open reading frames). In some instances, the second sequence encoding an enzyme for taxane synthesis is operably linked to a second promoter. In various instances, the second promoter is heterologous to the second sequence encoding an enzyme for taxane synthesis. In some embodiments, the second promoter has higher promoter activity as compared to the native promoter of the second sequence encoding an enzyme for taxane synthesis. In some instances, the second promoter is inducible. In some instances, the second promoter operably is constitutive.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0069] Additional embodiments may include three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or greater number of sequences encoding an enzyme for taxane synthesis. As with a first and / or second sequences, each additional (e.g., third or greater) sequence encoding an enzyme for taxane synthesis may be operably linked to a previous promoter (e.g., polycistronic) or operably linked to its own promoter.

[0070] In various instances, the enzyme for taxane synthesis is selected from one or more of the enzymes listed in Tables 1 and 2. In some embodiments, such enzymes may include modifications, such as described previously.

[0071] While the promoters have been described as first promoter, second promoter, etc., the promoters may have the same sequence — e.g., the first promoter and the second promoter may each be independent constitutive promoters (e.g., cauliflower mosaic virus 35S) or an inducible promoter, such as an antibiotic-inducible promoter. Exemplary (non-limiting) promoters are described in Villao-Uzho L, et al. Plant Promoters: Their Identification, Characterization, and Role in Gene Regulation. Genes (Basel). 2023 Jun 6;14(6):1226; the disclosure of which is hereby incorporated by reference in its entirety.

[0072] In some embodiments, the polynucleotide may include a sequence encoding a selectable marker. In such instances, the sequence encoding a selectable marker may be operably linked to a promoter. In some preferred embodiments, the promoter is a constitutive promoter. In many instances, the selectable marker is an antibiotic resistance protein. Such antibiotic resistance proteins can provide resistance to one or more of hygromycin, penicillin, tetracycline, ampicillin, amoxicillin, streptomycin, gentamicin, kanamycin, doxycycline, vancomycin, rifampin, and / or any other antibiotic.

[0073] The sequence encoding for the selectable marker may be located on the same strand of a polynucleotide or the complimentary strand of a polynucleotide. Additionally, the sequence encoding for the selectable marker may be located upstream (i.e., 5’) or downstream (i.e., 3’) of a sequence encoding an enzyme for taxane synthesis.

[0074] As noted previously, some embodiments are directed to systems of polynucleotides. In such instances, a first polynucleotide may include one or more sequences encoding an enzyme for taxane synthesis, while a second polynucleotide may include one or more additional sequences encoding an enzyme for taxane synthesis. In various instances, the number of sequences encoding an enzyme for taxane synthesis and / or the identity of the enzyme may be different on each polynucleotide within theBFF Ref: STAN-2224WO Clt Ref: S24-220 system of polynucleotides. Additionally, each polynucleotide in the system of polynucleotides may include a sequence encoding for a different selectable marker — e.g., a first polynucleotide comprises a sequence encoding an ampicillin resistance gene, while the second polynucleotide comprises a sequence encoding a rifampin resistance gene. The foregoing example listing ampicillin and rifampin resistance genes is merely exemplary, and any combination of selectable markers may be used.Vectors and Vector Systems

[0075] A vector system for taxane synthesis may include a vector for producing an enzyme for taxane synthesis as provided herein and described in the preceding section. In various instances, the vector may include a sequence to assist with integration into the genome of a cell — e.g., for stable integration. Other embodiments may be configured for transient expression.

[0076] As noted, some embodiments are directed to vector systems — i.e., a system that comprises multiple vectors. In such instances, one or more vectors may include sequences to allow for genomic integration while one or more vectors may be configured for transient expression. In certain instances, all vectors in a vector system may include sequences for stable integration. In other instances, all vectors in a vector system may be configured for transient expression.Cells, Tissues, and Organisms

[0077] Various embodiments are directed to a cell comprising one or more polynucleotide, vector, or other construct as described herein. The cell may be a bacterial cell (e.g., Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus), a yeast cell (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans), a plant cell (e.g., Nicotiana benthamiana, Arabidopsis thaliana, Glycine max, Zea mays), an algal cell (e.g., Chlamydomonas spp., Spirulina spp., Chlorella spp.), a mammal cell, and / or any other applicable cell. In some embodiments, the cell is a cell line, such that the cell line is stably propagated in culture. Such cell lines may include a stably integrated construct — e.g., one or more polynucleotides or vectors described herein is integrated into the nuclear genome of the cell.

[0078] In various instances, the cell is part of a tissue. In some instances, a tissue comprises a cell comprising one or more polynucleotide, vector, or other construct as described herein. In various instances, a tissue is transfected with one or more polynucleotide, vector, or other construct as described herein, such that at least one cell of the tissue comprises one or more polynucleotide, vector, or other construct asBFF Ref: STAN-2224WO Clt Ref: S24-220 described herein. Such tissue can be isolated from an organism and / or produced in culture. Such tissue can arise from plant, mammal, avian, reptilian, amphibian, and / or other tissue source. Exemplary mammalian tissues include (but are not limited to) muscle, adipose, neural, dermal, hepatic, nephrotic, and / or any other tissue. Exemplary plant tissue include (but are not limited to) leaf, floral, germ, stem, meristem, root, root hair, and / or any other plant tissue.

[0079] In various instances, an organism comprises at least one cell comprising one or more polynucleotide, vector, or other construct as described herein. In some embodiments, every cell in an organism comprises one or more polynucleotide, vector, or other construct as described herein. In some instances, an organism, where every cell comprises one or more polynucleotide, vector, or other construct as described herein, the one or more polynucleotide, vector, or other construct can be under control of inducible promoters, such that the encoded enzymes are expressed when a triggering agent is applied. Such organisms can include a plant (including but not limited to, Nicotiana benthamiana, Arabidopsis thaliana, Glycine max, Zea mays), a mammal (e.g., murine, caprine, ovine, bovine, human, non-human primate, canine, feline, and / or any other mammal), bird, fish, reptile, amphibian, and / or any other organism.

[0080] In various embodiments, a cell, tissue, or organism is present within a culture. In such instances, the culture comprises a vessel, such as a petri dish, a flask, a welled plate (e.g., multi-well plate), a bioreactor, a vat, a pot, and / or any other appropriate culturing vessel. In some instances, the culture further comprises a media, such as a growth media. Such media are known in the art and include solid or liquid media depending on desired conditions. Exemplary media includes (but is not limited to) Eagle’s minimum essential medium, Dulbecco's modified eagle medium, Luria-Bertani broth, potting media, soil, and / or any other appropriate media for growth. In some instances, the media may contain a selective reagent, such as an antibiotic, to prevent growth of cells, tissue, or organisms lacking a polynucleotide, vector, or other construct as described herein.Methods of Making a Cell, Tissue, or Organism for Taxane Synthesis

[0081] Various embodiments provide for methods to produce a cell, tissue, and / or organism for taxane synthesis. In some instances, the cell, tissue, and / or organism comprises one or more gene to synthesize a taxane — e.g., the cell, tissue, and / or organism manufactures one or more enzymes that may be isolated or used to produce a taxane. In various instances, the cell, tissue, and / or organism comprises all of the genesBFF Ref: STAN-2224WO Clt Ref: S24-220 to synthesize a taxane — e.g., all enzymes within a biosynthetic pathway for a particular taxane.

[0082] One or more polynucleotide, vector, or other construct as described herein may be introduced into such a cell, tissue, and / or organism may be introduced by any relevant method, such as viral mediated transfection, bacterial mediated transfection (e.g., using Agrobacterium spp.), electroporation, microinjection, particle bombardment (e.g., biolistics), heat shock, sonoporation, lipofection, and / or any other relevant methodology.

[0083] Once the one or more polynucleotide, vector, or other construct are introduced, the cell, tissue, or organism can be selected for the presence of the polynucleotide, vector, or other construct. Such selection can include growth on a media comprising an antibiotic. For example, if the one or more polynucleotide, vector, or other construct comprise a gene for rifampin resistance, the media may contain rifampin to prevent growth of a cell, tissue, or organism lacking the one or more polynucleotide, vector, or other construct. In instances, where multiple polynucleotides, vectors, or other constructs are introduced, the media may contain multiple antibiotics. For example, if a first polynucleotide includes a rifampin resistance gene and a second polynucleotide includes an ampicillin gene, the media may contain both rifampin and ampicillin, thus selecting for cells, tissue, or organisms including both polynucleotides. Alternatively, the introduction of one or more polynucleotides, vectors, or other constructs may occur sequentially, each with an independent selection step.

[0084] In some embodiments, the cell is expanded to produce a population of cells. In some embodiments, the population of cells produces a stable cell line wherein the polynucleotide as described herein is in integrated into the genome of the cells. In some embodiments, the cell is passaged at least three times. In some embodiments, the cell can be passaged up to 60 times. In some embodiments, the cell can be passage more than 60 times. In some embodiments, expanding the population of cells comprises passaging the population of cells at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 times. In some embodiments, the cell maintains the ability to be conditionally induced after each passage.Methods of Producing an Enzyme for Taxane Synthesis

[0085] Additional embodiments are directed to the production of an enzyme for taxane synthesis. In such embodiments, a cell, tissue, or organism comprising a polynucleotide, vector, or other construct produces one or more enzymes. In some instances, a triggeringBFF Ref: STAN-2224WO Clt Ref: S24-220 agent is provided to the cell, tissue, or organism to induce expression of a gene encoding the enzyme — e.g., such as when the gene is operably linked to an inducible promoter.

[0086] After the cell, tissue, or organism have been allowed to express the enzyme, the enzyme may be isolated or purified from cell, tissue, or organism. In some instances, the enzyme is tagged with an exportation signal sequence, such that the enzyme is released into media. In some instances, the cell, tissue, or organism are pulverized and / or lysed to allow release of the enzyme into solution, such as a buffer. Once the enzyme is in solution (e.g., media and / or buffer), the enzyme may be purified or isolated, such as by column purification (e.g., size exclusion, ion affinity), centrifugation (e.g., sucrose gradient, bead / microparticle pulldown, and / or to remove cellular debris), affinity purification (e.g., when the enzyme includes an affinity tag). Various other steps or methods may be used to isolate the enzyme produced by the cell, tissue, or organism.Methods of Synthesizing Taxanes

[0087] In some instances, one or more isolated enzymes may be combined in vitro to produce a desired taxane. In some instances, all of the isolated enzymes are combined in a single reaction vessel along with the proper precursors. In other instances, a reaction vessel comprises one enzyme or a subset of enzymes. In such instances, individual reactions or subsets or reactions are allowed to proceed and limiting possibly erroneous byproducts or undesirable compounds. In such instances, the intermediate compounds may be isolates and / or the preceding enzymes may be deactivated prior to introduction of the next enzymes or proceeding with the next reaction in the pathway.

[0088] In some embodiments, all or part of the synthesis occurs within a cell, tissue, or organism comprising a polynucleotide, vector, or other construct as described herein. In such instances, precursor molecules and / or intermediate molecules may be provided to the cell, tissue, or organism to allow the desired reaction or reactions to occur. A resulting compound may be isolated or purified from the cell, tissue, or organism for purification and / or subsequent reactions.Taxanes Produced By Methods of the Present Disclosure

[0089] In some aspects, a taxane (e.g., Taxol and its derivatives) is produced by the methods described herein.Pharmaceutical Compositions

[0090] Additional embodiments are directed to pharmaceutical compositions comprising a therapeutically effective amount of a taxane as produced by the methods described herein. In many instances, a pharmaceutical composition can include one orBFF Ref: STAN-2224WO Clt Ref: S24-220 more of: a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some cases, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human. Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that may be administered without undue toxicity.

[0091] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Certain facilitators of nucleic acid uptake and / or expression may also be included in the compositions or coadministered.Methods of Treatment

[0092] In another aspect, methods of treatment are provided. In various embodiments, the method comprises administering a taxane produced by the methods described herein to a patient in need thereof. In some embodiments, the administering is by intravenous administration, intramuscular administration, intrathecal administration, intracisternal administration, orally, rectally, nasally, and / or any other appropriate methodology to administer a pharmaceutical composition of a taxane.

[0093] In some embodiments, a method of treating a condition or disorder comprises administering a therapeutically effective amount of the pharmaceutical composition of as disclosed herein to a patient in need thereof. In some embodiments, the disorder is a cancer. In some embodiments, the treatment results in at least one undesirable side effect and wherein the undesirable side effect is reduced relative to administering a daily dose that deviates more than 50%, 40%, 30%, 30%, 15%, 10%, 5%, or 2% from an expected dose. In some embodiments, the administering is by injection. In some embodiments, the injection is an infusion. In some embodiments, the daily dose isBFF Ref: STAN-2224WO Clt Ref: S24-220 administered to the patient once. In some embodiments, the daily dose is administered to the patient two or more times.Kits

[0094] Also provided are kits comprising the polynucleotide, vector system, cells, tissue, organisms, or enzymes for taxane synthesis, as described herein. In some embodiments, a polynucleotide, vector, polynucleotide system, and / or vector system is provided with cells (e.g., already transfected with one or more polynucleotide constructs of the vector system or separately). Other agents may also be included in the kit such as transfection agents, suitable media for culturing cells, buffers, antibiotics, agents for inducing production of an enzyme, and the like.

[0095] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. In some embodiments, instructions for using the vector systems or cell lines to inducibly produce one or more enzymes for taxane synthesis. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, SD drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.ASPECTS OF THE INVENTION

[0096] The below items disclose various aspects of the invention. Each of the aspects described below can be combined with other aspects and embodiments disclosed elsewhere herein, including the claims, where the combinations are clearly compatible. Certain aspects include:

[0097] Aspect 1 . A vector comprising a sequence encoding FoTO1 operably linked to a promoter.

[0098] Aspect 2. The vector of Aspect 1 , wherein the promoter is heterologous to FoTO1 .

[0099] Aspect 3. The vector of Aspect 1 or 2, wherein the sequence encoding FoTO1 is from a Taxus species.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0100] Aspect 4. The vector of any one of Aspects 1 -3, wherein the sequence encoding FoTOI is from T. chinensis or T. media.

[0101] Aspect 5. The vector of any one of Aspects 1 -4, wherein the sequence encoding FoTOI is codon optimized.

[0102] Aspect 6. The vector of any one of Aspects 1 -5, wherein the promoter is selected from an inducible promoter, a constitutive promoter.

[0103] Aspect 7. The vector of any one of Aspects 1 -6, further comprising a sequence encoding one or more of: T9aH, T7AT, T 1 pH, DeAc898, DeAc1023,T9ox, and PCL.

[0104] Aspect 8. The vector of any one of Aspects 1 -6, further comprising a sequence encoding one or more of: TS, T5aH, TAT, T10 H, DBAT, T13aH, T2aH, TBT, T7 H, and TOT.

[0105] Aspect 9. A cell comprising the vector of any one of Aspects 1 -8.

[0106] Aspect 10. The cell of Aspect 9, wherein the cell is selected from a bacterium, a yeast cell, or a plant cell.

[0107] Aspect 11. The cell of Aspect 9 or 10, wherein the vector is stably integrated into the genome of the cell.

[0108] Aspect 12. A culture comprising a vessel and the cell of any one of Aspects 9-11.

[0109] Aspect 13. The culture of Aspect 12, wherein the vessel is selected from a petri dish, a flask, a bioreactor, and a vat.

[0110] Aspect 14. The culture of Aspect 12 or 13, wherein the culture is a solid culture or a liquid culture.

[0111] Aspect 15. A plant tissue comprising the cell of any one of Aspects 9-1 1.

[0112] Aspect 16. The plant tissue of Aspect 15, wherein the tissue is in the culture of any one of Aspects 12 14.

[0113] Aspect 17. A plant comprising the cell of any one of Aspects 9-11 or the tissue of Aspect 15.

[0114] Aspect 18. A method of producing a taxane, comprising: transfecting a cell with the vector of any one of Aspects 1 -8; harvesting a taxane from the cell.

[0115] Aspect 19. The method of Aspect 18, further comprising providing a triggering agent to induce expression of one or more sequences operably linked to a promoter.

[0116] Aspect 101 . A method of producing a taxane, comprising:BFF Ref: STAN-2224WO Clt Ref: S24-220 obtaining a cell comprising an expression cassette, wherein the expression cassette comprises a sequence encoding FoTOI operably linked to a promoter; and harvesting a taxane from the cell.

[0117] Aspect 102. The method of Aspect 101 , wherein FoTO1 has at least 75% sequence identity to SEQ ID NO: 1 .

[0118] Aspect 103. The method of Aspect 101 or 102, wherein FoTOI has at least 95% sequence identity to SEQ ID NO: 1 .

[0119] Aspect 104. The method of any one of Aspects 101 -103, wherein the sequence encoding FoTOI is from a Taxus species.

[0120] Aspect 105. The method of any one of Aspects 101 -104, wherein the sequence encoding FoTOI is from T. chinensis or T. media.

[0121] Aspect 106. The method of any one of Aspects 101 -105, wherein the sequence encoding FoTOI is codon optimized for the cell.

[0122] Aspect 107. The method of any one of Aspects 101 -106, wherein the promoter is heterologous to FoTOI .

[0123] Aspect 108. The method of any one of Aspects 101 -107, wherein the promoter is selected from an inducible promoter, a constitutive promoter.

[0124] Aspect 109. The method of any one of Aspects 101 -108, wherein the promoter is an inducible promoter, and the method further comprises providing a triggering agent to induce expression of the sequence encoding FoTOI .

[0125] Aspect 110. The method of any one of Aspects 101 -109, wherein the expression cassette further comprises a sequence encoding one or more of: T9oH, T7AT, T1 PH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

[0126] Aspect 111 . The method of Aspect 1 10, further wherein the expression cassette further comprises a sequence encoding one or more of: TS, T5aH, TAT, T 10 H, DBAT, T13aH, T2aH, TBT, T7 H, and TOT.

[0127] Aspect 112. The method of any one of Aspects 101 -111 , wherein obtaining the cell comprises transfecting the cell with vector comprising the expression cassette.

[0128] Aspect 113. The method of Aspect 1 12, wherein the vector integrates the expression cassette into the genome of the cell.

[0129] Aspect 114. The method of Aspect 1 12, wherein the vector is nonintegrating.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0130] Aspect 115. The method of any one of Aspects 101 -109, wherein the cell comprises a second expression cassette, wherein the second expression cassette comprises a sequence encoding one or more of: T9oH, T7AT, T1 H, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

[0131] Aspect 116. The method of Aspect 115, wherein the second expression cassette comprises a sequence encoding one or more of: TS, T5aH, TAT, T 10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

[0132] Aspect 117. The method of Aspect 1 15 or 1 16, wherein obtaining the cell comprises transfecting the cell with a vector comprising the second expression cassette.

[0133] Aspect 118. The method of any one of Aspect 101 -117, wherein the cell is a plant cell, an animal cell, a bacterial cell, or a yest cell.

[0134] Aspect 119. The method of any one of Aspects 101 -1 18, wherein the cell is from Nicotiana benthamiana or Saccharomyces cerevisiae.

[0135] Aspect 120. The method of any one of Aspects 101 -1 19, wherein the cell is comprised in a cell culture.

[0136] Aspect 121 . The method of any one of Aspects 101 -120, wherein the cell is comprised in a tissue.

[0137] Aspect 122. The method of Aspect 121 , wherein the tissue is an N. benthamiana leaf.

[0138] Aspect 123. The method of any one of Aspects 101 -122, wherein the cell is comprised within a vessel.

[0139] Aspect 124. The method of Aspect 123, wherein the vessel is selected from a petri dish, a flask, a bioreactor, and a vat.

[0140] Aspect 125. A cell comprising an expression cassette, wherein the expression cassette comprises a sequence encoding FoTO1 operably linked to a promoter.

[0141] Aspect 126. The cell of Aspect 125, wherein FoTO1 has at least 75% sequence identity to SEQ ID NO: 1 .

[0142] Aspect 127. The cell of Aspect 125 or 126, wherein FoTO1 has at least 95% sequence identity to SEQ ID NO: 1 .

[0143] Aspect 128. The cell of any one of Aspects 125-128, wherein the sequence encoding FoTO1 is from a Taxus species.

[0144] Aspect 129. The cell of any one of Aspects 125-128, wherein the sequence encoding FoTO1 is from T. chinensis or T. media.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0145] Aspect 130. The cell of any one of Aspects 125-129, wherein the sequence encoding FoT01 is codon optimized for the cell.

[0146] Aspect 131 . The cell of any one of Aspects 125-130, wherein the promoter is heterologous to FoTO1 .

[0147] Aspect 132. The cell of any one of Aspects 125-131 , wherein the promoter is selected from an inducible promoter, a constitutive promoter.

[0148] Aspect 133. The cell of any one of Aspects 125-132, wherein the promoter is an inducible promoter, and the method further comprises providing a triggering agent to induce expression of the sequence encoding FoTO1 .

[0149] Aspect 134. The cell of any one of Aspects 125-133, wherein the expression cassette further comprises a sequence encoding one or more of: T9aH, T7AT, Ti pH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

[0150] Aspect 135. The cell of Aspect 134, further wherein the expression cassette further comprises a sequence encoding one or more of: TS, T5aH, TAT, T 10 H, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

[0151] Aspect 136. The cell of any one of Aspects 125-135, wherein the expression cassette is integrated into the genome of the cell.

[0152] Aspect 137. The cell of any one of Aspects 125-135, wherein the expression cassette is not integrated into the genome of the cell.

[0153] Aspect 138. The cell of any one of Aspects 125-133, wherein the cell comprises a second expression cassette, wherein the second expression cassette comprises a sequence encoding one or more of: T9aH, T7AT, Ti pH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

[0154] Aspect 139. The cell of Aspect 138, wherein the second expression cassette comprises a sequence encoding one or more of: TS, T5aH, TAT, T 10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

[0155] Aspect 140. The cell of any one of Aspect 125-139, wherein the cell is a plant cell, an animal cell, a bacterial cell, or a yest cell.

[0156] Aspect 141 . The cell of any one of Aspects 125-140, wherein the cell is from Nicotiana benthamiana or Saccharomyces cerevisiae.

[0157] Aspect 142. The cell of any one of Aspects 125-141 , wherein the cell is comprised in a cell culture.

[0158] Aspect 143. The cell of any one of Aspects 125-142, wherein the cell is comprised in a tissue.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0159] Aspect 144. The cell of Aspect 143, wherein the tissue is an N. benthamiana leaf.

[0160] Aspect 145. The cell of any one of Aspects 125-144, wherein the cell is comprised within a vessel.

[0161] Aspect 146. The cell of Aspect 145, wherein the vessel is selected from a petri dish, a flask, a bioreactor, and a vat.EXEMPLARY EMBODIMENTS

[0162] Although the following embodiments provide details on certain embodiments of the inventions, it should be understood that these are only exemplary in nature and are not intended to limit the scope of the invention.Example 1 : A platform combining snRNA-seq and multiplexed perturbation to identify plant biosynthesis pathways

[0163] Figures 1 A-1 F illustrate various aspects to identify biosynthetic pathways for taxanes. Figure 1 A illustrates a proposed biosynthesis pathway for Taxol with gaps notated (e.g., by “+ ? Unknown”). In addition to an incomplete knowledge of the biosynthetic gene set, significant inefficiencies (e.g., arrows pointing to side products) of the first oxidase, taxadiene 5a-hydroxylase (T5aH), prevent Taxol pathway reconstitution and discovery. In Figure 1 A, “TDS” refers to taxadiene synthase. Figure 1 B provides prominent classes of taxane metabolites beyond Taxol that have been isolated from Taxus species. The tailoring acyl groups on taxanes include acetyl, benzoyl, small chain fatty acid residues, or phenylisoserine derivatives Figure 1 C provides the number of enzymes in the Taxus chinensis genome belonging to secondary metabolism-related families. In Figure 1 C, 2-ODD refers to 2-oxoglutarate dependent dioxygenase, P450 refers to cytochrome P450, ap-hydro refers to ap-hydrolase, AcyIT refers to acyltransferase, and TPS refers to terpene synthase. Figure 1 D provides an overview of the differences between conventional co-expression approaches and an mpXsn (multiplexed elicitation with single nuclei sequencing) methodology. Dot networks are visualizations of the co-expression network (nodes linked when mutual rank <20) using either bulk RNA-seq or mpXsn data. For visual clarity, only cytochrome P450s are displayed. Figure 1 E illustrates an experimental overview of mpXsn with UMAP of single nuclei transcriptomes. Figure 1 F provides a rank of each known Taxol gene by Pearson correlation coefficient (PCC) to taxadiene synthase, TDS (the first enzyme in the Taxol pathway), using either bulk (n=79) or mpXsn (n=17,143 cells across three experiments) data.BFF Ref: STAN-2224WO Clt Ref: S24-220Example 2: Identification of taxane biosynthesis gene modules

[0164] Figures 2A-2I provide details regarding additional genes and enzymes involved in taxane biosynthesis that have been discovered. Figure 2A illustrates a schematic of Taxol biosynthesis and previously hypothesized gene order. Figure 2B illustrates a correlation between known Taxol genes using mpXsn data. To identify substructures, genes were hierarchically clustered (scipy fcluster, Euclidean distance) on both axes. Figure 2C provides a schematic for matrix factorization. For this data, mpXsn data was factorized with consensus non-negative matrix factorization (cNMF). Figure 2D illustrates a heatmap showing known Taxol biosynthesis genes’ rank in each of the modules produced by matrix factorization. Figure 2E illustrates similar data as Figure 2D, but it displays only three modules enriched in Taxol genes (further referred to as Modules 1 , 2, and 3, respectively). Figure 2F provides a heat map of Taxol Modules 1 , 2, and 3 from Figure 2E, for the two isoprenoid pathways in the primary metabolism potentially upstream of the Taxol biosynthesis.. Only the MEP pathway is co-expressed with the first Taxol module, supporting its role in synthesizing Taxol precursors. In Figure 2G, all gene modules are ranked by the total number of 2-ODD, P450, and acetyltransferase genes in the top 100 genes of each module. Figure 2H illustrates module usage (analogous to expression) of each cell, which is analogous to gene expression, plotted onto the single nuclei transcriptomic UMAP. Taxol Modules 1 -3 are expressed in non-overlapping cell states and were primarily identified in different experiments. Figure 2I illustrates unfiltered lists of top genes in each module, plotted as module rank and score. As noted, darker dots represent previously identified genes associated with Taxol biosynthesis, while medium dark dots represent new biosynthetic genes identified in this study.Example 3: Characterization of FoTO1 (Facilitator of Taxane Oxidization 1)

[0165] Figures 3A-3Q illustrate data for the characterization of FoT01. Figure 3A illustrates an early Taxol biosynthetic pathway and the T5aH product divergence. The shaded area highlights the biosynthetic pathway toward Taxol. The diamond indicates the structure is supported by NMR. Figure 3B provides a rank and score of genes in Taxol Module 1. Figure 3C provides a bar graph showing the FoTO1 -induced fold change in end products' peak area of subpathways when transiently expressed in N. benthamiana leaves. Fold change is calculated by quantifying 2~4 GCMS total ion chromatogram (TIC) peak area and normalized to a -FoTO1 condition. Data are shown as the mean ± standard deviation (n = 3 biological, independent leaf samples). Statistical analyses were performed using a two-sided, unpaired Welch’s t-test. Figure 3D shows GCMS TIC of N. benthamiana leaves transiently expressing the indicated genes. Figure 3E provides a barBFF Ref: STAN-2224WO Clt Ref: S24-220 graph of total oxidized taxanes for +T5aH and +T5aH+FoTO1 condition. Data are shown as the mean ± standard deviation (n = 6 biological, independent leaf samples). Figure 3F provides phylogenetic tree of FoTO1 homologs identified by HMMER. The tree was produced with FastTree, rooted with red algae homologs. Figure 3G provides a structural model of FoTO1 generated by AlphaFold3 and aligned to the Arabidopsis thaliana ortholog with FoldSeek. Figure 3H provides bar graphs showing integrated peak area of taxadien-5a-ol (2) and side product OCT (2’a) when N- or C-terminal truncated FoTO1 is transiently expressed in N. benthamiana leaves together with TDS and T5aH. Data are shown as the mean ± standard deviation (n = 3 biological replicates). Figure 3I illustrates quantification of binding between purified T5aH and FoTO1 or AC-FoTO1 , using microscale thermophoresis. Data are shown as the mean ± standard deviation (n = 3 biological replicates). Figure 3J illustrates quantification of binding between purified TDS2 and FoT01 or AC-FoTO1 , using microscale thermophoresis. Data are shown as the mean ± standard deviation (n = 3 biological replicates). In Figures 3I and 3J, N- terminal transmembrane domains of T5aH and TDS2 are removed for purification purposes. Data are shown as the mean ± standard deviation (n = 3). Figure 3K illustrates an immunoblot of the co-immunoprecipitation (Co-IP) of T5aH-HA (prey) by FoTO1 -V5 (bait) in N. benthamiana leaves expressing both proteins.

[0166] Figures 3L & 3M illustrate further characterization of FoTO1 homologs. Figure 3L illustrates how FoT01 homologs from Taxus media and Arabidopsis thaliana have no effect on taxane yield increases. At the top of Figure 3L, TDS2 and the first three pathway oxidases (T5aH, T 1 OPH, T 13aH) were coexpressed in N. benthamiana with one of GFP, FoTO1 , At5g04830 (A. thaliana homolog), or ctg8326.3 (T. media homolog). Masses corresponding to the expected products, 5a, 10 ,13a-triol and 5a, 1 op-diol 13-one, are displayed as EICs and bar graphs quantified across three independent replicate leaves. Unlike FoTO1 , neither FoTO1 homolog (At5g04830 and ctg8326.3) alters the product profile of this early subpathway. Data are shown as the mean ± standard deviation, n=3. Figure 3M shows FoT01 structural homologs identified in other land plants. Using FoldSeek v4, all pre-folded proteins databases were searched for full-length structural homologs of FoTO1 . The top five hits were proteins of model plants Oryza sativa Japonica (rice), Zea mays (corn), A. thaliana, and Glycine max (soybean). After the NTF2 domain, each of these homologs contain the alpha-helical C-terminus (highlighted) that were found to be crucial for FoT01 ’s phenotype in vivo and for binding to TDS and T5aH. The identification of these structural homologs indicates that FoTO1 is not just restricted to gymnosperms, but has structural analogs across angiosperms, too.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0167] Figures 3N-3Q show that FoTO1 does not affect the production of taxadiene by TDS. Figure 3N illustrates GCMS total ion content (TIC) traces of N. benthamiana expressing boost (tHMGR, GGPPS) and taxadiene synthase (TDS1 or TDS2) with and without FoTO1. When FoTO1 is coexpressed, the formations of taxadiene [1 , taxa- 4(5), 1 1 (12)-diene] or iso-taxadiene [taxa-4(20),11 (12)-diene] remain the same and there are no observed new peaks. Representative traces of three biological replicates for each condition are shown. Figures 30 & 3P show relative yields of taxadien-5a-ol (2, desired product) and iso-OCT (2’b, an undesired product) measured by GCMS when TDS2 and T5aH were coexpressed with various FoTO1 mutants in N. benthamiana. Candidate residues for catalytic or substrate interaction in the cavity of the NTF2-like fold and regions of truncation were selected based on the AlphaFold3 structure. Data are shown as the mean ± standard deviation, n=3. Figure 3Q illustrates FoTO1 structure predicated by AlphaFold3.

[0168] FoTO1 was an unprecedented and unexpected component of the Taxol biosynthetic pathway that is important for efficient production of taxanes. Without FoT01 , early oxidases in the pathway (T5oH, T 13aH) primarily produce unwanted side products. By co-expressing FoTO1 with other biosynthetic enzymes, yield increases 10-17 fold (see Figure 3C). FoTO1 is the first described member of its family (the NTF2 protein superfamily) that has been found to have a role in plant metabolism, so it may be one of many examples of this type of activity.Example 4: Discovery and characterization of T9aH, T7AT, and two TipHs

[0169] Figures 4A-4J illustrate data showing the discovery and characterization of additional enzymes used in Taxol biosynthesis. Figure 4A provides a heat map showing the ranks of new T9aH and T1 Hs and other Taxol biosynthetic genes in the three modules. T9aH-725A is the T9aH independently reported by other research groups. Figure 4B illustrates a proposed biosynthetic pathway from compound 4, the latest intermediate was reported recently to taxusin (6) and the corresponding extracted ion chromatograms (EICs) of product 4-6 when the indicated sets of genes were expressed in N. benthamiana leaves. Shading highlights the biosynthetic pathway toward Taxol, which only involves 4 and 5 . The illustration was created using BioRender.com. Figure 2C provides a proposed biosynthetic pathway from compound 5 to baccatin VI (11-Bz) and the corresponding EICs of intermediates when the indicated sets of genes were expressed in N. benthamiana leaves. Shading highlights the biosynthetic pathway toward Taxol . Structures of 5, 7-Ac, 7-Bz, 8-Ac, 8-Bz, 9-Ac, 9-Bz, 10-Ac, 10-Bz, 11-Ac are proposed based on functions of enzymes previously characterized (TAT, TAX19, T2aH,BFF Ref: STAN-2224WO Clt Ref: S24-220 TBT, T7pH, and TOT) and described in this study (T9aH-750C, T7AT, and T1 pH). TAX19 are used to generate known 13-O-acetylated products, including taxusin (6) and baccatin VI (11 -Bz), for structural analysis. The diamond indicates the structure is supported by NMR. Figure 4D illustrates EICs of expected products when the pathway to 6O4A is expressed with TOT, T7AT, or both in / . benthamiana. In the absence of T7AT, no significant 6O4A depletion or product formation by TOT is observed. Figure 4E illustrates MSMS fragmentation patterns of heterologously produced baccatin VI (11 -Bz) in N. benthamiana compared to that of an 11-Bz standard. MSMS fragmentations were generated using [M+Na]+(m / z = 737.2788) as the precursor ion and fragmented with a collision energy of 30 eV.

[0170] Figures 4F-4J illustrate product profiles of T1 pH-184 and T1 pH-686 with different upstream pathways and the proposed mechanism for 1 p-hydroxylation and abeotaxane rearrangement. In these figures, two 2-ODDs, T1 pH-184 and T1 pH-686, were expressed in N. benthamiana with four different background conditions: Feeding taxusin (Figure 4F); feeding taxusin and co-expressing T2aH, TBT, T7PH, and T7Ac (Figure 4G); co-expressing full biosynthetic pathway to 7O5A, (tHMGR, GGPPS, TDS, FOTO, T5aH, TAT, T10pH, DBAT, T13aH, T2aH, TBT, T7pH, T7AT, TOT) (Figure 4H); and co-expressing full biosynthetic pathway to 7O6A, 7O5A pathway and TAX19 (Figure 4I). Figures 4F-4I provide EICs of mono-oxidized products on both C-2a-O-acetyl and - benzoyl precursors are shown as well as their potential structures. Among all products, i p-hydroxytaxusin (6-01 ) and 15-hydroxy-1 1 (15— >1 )abeo-taxusin (6-02) are confirmed by NMR, while baccatin VI (11-Bz) is confirmed by comparing to a chemical standard (see Figures 4C & 4E). The observed product diversity (formation of multiple peaks) likely arises from the dual-function of T1 PH and TOT: T1 pH performs both 1 p-hydroxylation and rearrangement to 1 1 (15— >1 )abeotaxane, and TOT generates both epoxide and oxetane products. Interestingly, C-2a-O-benzoylated products mostly show as a single dominant peak. This suggests that the T1 pH 1 p-hydroxylation activity is selective toward C-2a-0-benzoylated / oxetane intermediates. Figure 4J illustrates a proposed mechanism of T 1 pH. Formation of the two characterized products 1 p-hydroxytaxusin (6-01 ) and 15- hydroxy-11 (15^1 )abeo-taxusin (6-02) from taxusin (6) by T1 pH-184 can be explained by the different fates of the C-1 radical after the first proton abstraction: direct hydroxyl radical rebound from the enzyme would yield 1 p-hydroxytaxusin (6-01) while radical rearrangement forming C-1 / 1 1 bond followed by ring opening and hydroxyl radical rebound would give 15-hydroxy-11 (15^1 )abeo-taxusin (6-02). The rearrangement routeBFF Ref: STAN-2224WO Clt Ref: S24-220 likely leads to many non-classical abeotaxanes, like brevifoliol, while the 1 p-hydroxylation leads to classical taxanes like baccatin VI (16).Example 5: T1 pH-184 and T1 pH-246 for the production of abeotaxane scaffold

[0171] In furtherance of Example 4. 1 p-hydroxytaxusin was produced using T1 pH- 184 and T1 pH-246. This process can potentially lead to the production of a taxane analog, TPI-287, that entered clinical trials as of July 2025.

[0172] As the second largest class of taxane scaffold (among the eleven scaffold classes), more than 120 taxanes with the 1 1 (15^1 )abeotaxane scaffold have been reported. In T. chinensis, 1 1 (15— >1 )abeotaxane like taxchinin A and brevifoliol (see Figure 4K) have been found to be one of the most abundant toxoids in the leaves and stems tissue. In T. brevifolia, brevifoliol accounts for an astonishing 0.14% fresh weight of the needles. Despite the scaffold difference to Taxol, brevifoliol and its derivatives have demonstrated potent anticancer activity comparable to Taxol. Unlike the classical 6 / 8 / 6- scaffold of Taxol, the 5 / 7 / 6-scaffold of 1 1 (15->1 )abeotaxane is a poor substrate for efflux pumps like P-glycoprotein (Pgp). This property makes 1 1 (15— >1 )abeotaxane derivatives promising candidates for the treatment of central nervous system (CNS) tumors, where penetration of the Pgp-expressing blood-brain barrier (BBB) is essential, and multi-drug resistant (MDR) cancers, where P-gp overexpression and limits the efficacy of conventional chemotherapeutics. A notable semi-synthetic 1 1 (15— >1 )abeotaxane analog, TPI-287 (see Figure 4K), has successfully completed preclinical and phase I clinical trials, demonstrating good CNS penetration and anticancer activity across various models, including glioblastoma, neuroblastoma, medulloblastoma, and metastatic breast cancer. In 2024, TPI-287 received orphan drug designation for gliomas, pediatric neuroblastoma, and progressive supranuclear palsy. As of July 2025, it is in phase II clinical trials for glioblastoma multiforme.

[0173] The formation of 1 1 (15— >1 )abeotaxane scaffold has previously been proposed to occur via a Wagner-Weerwein rearrangement under acidic conditions. Here, 2-ODDs were discovered to enzymatically catalyze this transformation, representing a serendipitous and mechanistically novel discovery. Further characterization of the diverse products generated by T1 pH-184, T1 pH-246, and T1 pH-686, along with phylogenetics analysis (specifically Ti pH variants in T. brevifolia), and site-directed mutagenesis of the active site may elucidate the determinants for 1 -hydroxylation vs 11 (15— >1 )abeotaxane specificity for these 2-ODDs. These insights could ultimately inform the engineering of biocatalysis for TPI-287.BFF Ref: STAN-2224WO Clt Ref: S24-220

[0174] T1 pH-246 is a homolog of T1 pH-184 and T1 pH-686, and show product preference toward 11 (15— >1 )abeotaxane scaffold. As shown in Figure 4L, when T1 pH- 246 is expressed with the taxusin biosynthetic pathway, only 15-hydroxy- 11 (15 >1 )abeotaxusin is observed. This might be useful for the production of taxane analogs with 1 1 (15->1 )abeotaxane scaffold.Example 6: Total biosynthesis of baccatin III (16) and 3’-N-debenzoyl-2'- deoxypaclitaxel (17) in N. benthamiana

[0175] Figures 5A-5O provide data showing the discovery and characterization of taxane 9-oxidase (T9ox), and p-phenylalanine-CoA ligase (PCL) that complete the total biosynthesis of 3’-N-debenzoyl-2'-deoxypaclitaxel (17) in N. benthamiana. Figure 5A illustrates a simplified representation of biosynthetic transformations (acetylation and oxidation) from taxadiene (1) to baccatin III (16) Figure 5B illustrates a proposed biosynthetic pathway from compound 10-Bz to Taxol (18). Figure 5C shows EICs of intermediates when the indicated sets of genes were expressed in N. benthamiana leaves. Figure 5D illustrates MSMS of heterologously produced baccatin III (16) in N. benthamiana compared to that of a 16 standard. Figure 5E illustrates a heatmap showing ranks of PCL candidates in Taxol gene modules. Figure 5F illustrates EICs of compounds 16 and 17 from feeding 16 to N. benthamiana leaves expressing PAM, PCL, and BAPT or from expressing the complete gene set. Figure 5G illustrates ranks of new Taxol biosynthetic genes discovered in this study by PCC to TDS using either bulk (n=79 samples) or mpXsn (n=17,143 cells across three experiments) data. The lighter shaded dots (e.g., PCL, T9aH, T 1 pH, and T9ox) indicate anticipated discovery while darker dots (e.g., FoT01 , T9dA, T7dA, and T7AT) indicate unexpected discovery. Figure 5H provides a heatmap showing ranks of updated Taxol biosynthetic genes in the modules. Figure 5I illustrates baccatin III (16) yields in ug / g dried weight (DW), quantified with standards, of the 17-gene pathway with each single-gene dropout tested in N. benthamiana. Yields from replicating published gene sets and from T. media needles are shown for comparison. Data are show as the mean ± standard deviation, n=3 biological replicates. Figure 5J provides an EIC showing the proposed taxadien-5o, 13a-diol produced by T13aH and facilitated by FoTO1 . Over-oxidized derivative and TAX19-acetylated product are confirmed by NMR (noted by diamond). Figure 5k provides a phylogenetic tree of Taxus P450s. Figure 5L shows single cell expression data of T5oH and T13oH. Figure 5M shows Taxol biosynthesis (shading) and the reconstituted 13a-acetoxy taxane biosynthesis. Structures were assigned based on HRMS (high-resolution mass-BFF Ref: STAN-2224WO Clt Ref: S24-220 spectrometry predicted chemical formula), NMR (NMR analysis followed by purification), and / or HRMS / MS (MS / MS spectra comparison to authentic standards.

[0176] Figures 5N & 50 demonstrate the characterization of deacetylases T7dA and T9dA. Specifically, Figure 5N illustrates conversion of baccatin VI (11 -Bz) to 9-dihydro- 13a-acetylbaccatin III (9DHAB, 13) by T7dA and T9dA in N. benthamiana leaves. T7dA and T9dA were heterologously expressed in N. benthamiana via Agrobacterium- mediated transient expression and baccatin VI (11 -Bz) 20 pM was fed to the leaves three days after infiltration. Figure 50 demonstrates reconstitution of 9DHAB (13) biosynthetic pathway in N. benthamiana. EICs of leaves expressing the reconstituted pathway compared to the 9DHAB standard are shown. MSMS fragmentations were generated using [M+Na]+(m / z = 653.2576) as the precursor ion and fragmented with a collision energy of 30 eV. Regions between m / z 100 to 800 are shown.

[0177] The new genes discovered include an unanticipated path that involves the addition and then removal of intermediate acetylations at carbons 9 and 7 (see Figures 5A & 5B). In this study, these acetylations are important for the pathway to proceed one of the intermediate step (oxidation TOT). However, since these two acetylations are not in the Taxol structure, two deacetylases (DeAc 1023, 898) were also discovered, which remove these acetylations. They are part of the 9 newly discovered genes.Example 7: Heterologous production of baccatin III (16) in N. benthamiana

[0178] Figures 6A-6D illustrate data showing genes for heterologous baccatin production. Figure 6A shows an untargeted analysis shows baccatin III (16) as a major product in the final step of pathway. Linear volcano plot comparing N. benthamiana leaves expressing the full baccatin III (16) pathway and full pathway without the penultimate enzyme T7dA (AT7dA). Metabolomic features with putative taxane masses are shown as dots whose sizes indicate EIC integrated area. P-values calculated by two- sided t-test with a Bonferroni correction for multiple hypothesis testing. The full baccatin III (16) pathway include: tHMGR, GGPPS, TDS, FoTO1 , T5aH, TAT, T10pH, DBAT, T13aH, T9aH-750C, T2aH, TBT, T7pH, T7AT, TOT, T1 pH-686, T9dA, T7dA, and T9ox. Figure 6B shows1H-NMR spectra of partially purified baccatin III (16) from N. benthamiana and baccatin III (16) standard (CDCI3, 500 MHz, 298 K). The spectra of the partially purified baccatin III (16) align with the standard, exhibiting all characteristic peaks (labeled with carbon number) as well as the H-20 coupling constant (J = 8.3 Hz) of the oxetane. The full baccatin III (16) pathway excluding T5aH was used to infiltrate 53 N. benthamiana plants [30.70 g dry weight (DW)] to yield baccatin III (16), whose yield (-270 pg) is derived from the total yield (1 .33 mg) with an estimated 20% purity. Figure 6CBFF Ref: STAN-2224WO Clt Ref: S24-220 illustrates that T9aH-725A cannot complement T9aH-750C for baccatin III (16) production. Representative EIC of N. benthamiana leaves expressing the full baccatin III pathway gene set, compared to full gene set without TDS (ATDS) or an exchange of our T9aH-750C for the recently reported T9aH-725A. A bar graph of integrated EICs for baccatin III (16) is shown. T9aH-725A pathway yields negligible baccatin III, statistically indistinguishable to a ATDS negative control. This would be expected from the finding that T9aH-725A appears to require a 13a-O-acetylation (see Figure 3B) that is absent from the baccatin III (16) pathway and from Taxol. Data are shown as the mean, n = 3 biological replicates. Significance indicates results of an ordinary one-way ANOVA comparison to the full pathway (““ indicates p = 5.63e-6). Figure 6D illustrates variations in baccatin III (16) production. The bar graph shows baccatin III (16) yields from N. benthamiana leaves expressing the full 17-gene pathway from different plants, calculated based on a baccatin III standard curve. Each sample (dot) was harvested from the 7th or 8th leaf counted from the bottom from separate plants under the same experimental conditions. The yields range from 1 .7-31.5 pg / g DW, comparable to those reported for the twig and leaf samples from T. chinensis, T. cupsidata, and T. media. Furthermore, the isolated yield (-8.7 pg / g DW) aligns well with the calculated yield. Data are shown as the mean ± standard deviation, n = 6.Example 8: Confirmation of baccatin III production in our heterologous system

[0179] The production of baccatin III — a direct industrial semi-synthesis precursors for Taxol — was further confirmed in the heterologous production system (see Example 7) after extraction, purification, and NMR.

[0180] In the reconstituted pathway in N. benthamiana, baccatin III is one of the most abundant and significant products. The yields range from 1.7 - 31 .5 pg / g dry weight (DW), comparable to those reported for the twig and leaf samples from T. chinensis, T. cupsidata, and T. media. To demonstrate that the reconstituted pathway in N. benthamiana can be a viable source for baccatin III, heterologously produced baccatin III (estimated isolation yield = 8.7 pg / g DW; see Figure 6B) were purified and its identity we purified by NMR.Example 9: Biosynthetic pathway reconstituted in N. benthamiana and proposed final steps to Taxol

[0181] Figure 7 illustrates the complete transformation from taxadiene (1) to 3’-N- debenzoyl-2’-deoxypaclitaxel, which is proposed to be two steps away to Taxol, in N. benthamiana. While T2’aH has been previously reported, its activity was unable to beBFF Ref: STAN-2224WO Clt Ref: S24-220 reconstituted. In Figure 7, the first oxidation of taxadiene (1) can be conducted by either T5aH or T 13aH, under the modulation by FoT01 . Branching of the Taxol pathway occurs at multiple enzymatic steps, including previously characterized T14PH and more recently reported TBT T7AT, TOT, and T1 pH-184, leading to other taxanes (shown in the last panel). The structural differences of other taxanes to Taxol are noted with lighter colored markings. Furthermore, TAX1940 allows the reconstitution of branching pathway to 13a- acetoxy taxanes including taxusin (6), baccatin IV, baccatin VI (11-Bz), and 9-dihydro- 13-acetylbaccatin III (9DHAB, 13) that can be confirmed with chemical standards (see Figures 4E & 50). However, the potential roles of these 13a-acetoxy taxanes as alternative intermediates toward baccatin III remain to be explored. The prevalence of these 13a-acetoxy taxanes in Taxus plants suggests it is difficult to rule out that the natural biosynthetic pathway might proceed through 13a-acetoxy intermediates. However, a corresponding C-13a-0-deacetylase (question mark from 13 to 16) was not able to be identified, which prevented access to baccatin III (16) via this putative, alternative route. Similarly, 10-deacetylbaccatin III, an abundant taxane in most Taxus species, might be an alternative biosynthetic intermediate with DBAT catalyzing a latestage acetylation (question mark from 13 to 10-deacetylbaccatin III). Full enzyme names are provided in Tables 1 & 2.Example 10: FoTO1 corrects the product profile of T5aH in yeast

[0182] Figure 8 illustrates expression of full-length FoTO1 in a Saccharomyces cerevisiae strain expressing taxadiene synthase (TDS) and T5aH enables production of the correct product (2). Expression of truncated FoTO1 does not yield this phenotype; and full length FoTO1 is necessary to produce correct product, taxadien-5a-ol (2), instead of rearranged side products (2a-c).Example 11 : BCL boosts benzoyltransferase activity of DBTNBT

[0183] Figure 9 illustrates expression of side chain enzymes (PAM, PCL, BAPT, DBTNBT) in N. benthamiana leaves infiltrated with baccatin III enables production of deoxy-paclitaxel. Inclusion of a co-expressed T. media benzoyl-CoA ligase (BCL) increases deoxy-paclitaxel yield by 33-fold.Example 12: De novo biosynthesis of Taxol in N. benthamiana

[0184] Figure 10 illustrates reconstitution of the final steps of Taxol biosynthesis in N. benthamiana. SC indicates side chain enzymes (PAM, PCL, BAPT). T2’aH and 3NBT indicate enzymes recently published by Liang, F., et. al. (Nat. Synth 2025; the disclosure of which is hereby incorporated by reference in its entirety). Intermediate taxane baccatinBFF Ref: STAN-2224WO Clt Ref: S24-220 III is either fed directly to N. benthamiana leaves (top 3 rows) or biosynthesized by the inclusion of 19 enzymes (tHMGR, GGPPS, TDS, FoT01 , T5aH, TAT, T10PH, DBAT, T13aH, T9aH-750C, T2aH, TBT, T7pH, T7AT, TOT, T1 pH-686, T9dA, T7dA, and T9ox) (bottom 5 rows). While the pathway up to T2’aH is capable of synthesizing debenzoyl- Taxol, the final enzyme, 3NBT, primarily synthesizes an acetylated product (column 3) instead of the benzoylated product Taxol. However, inclusion of the discovered T. media benzoyl-CoA ligase (BCL) corrects this, enabling de novo biosynthesis of Taxol (bottom row).Example 13: Identification of two putative transporters that boost yield of Taxol de novo biosynthesis

[0185] Figure 1 1 illustrates two transporters (“transporter-2” and “transporter-3”) increase yield of Taxol biosynthesis when combined with upstream pathway enzymes (tHMGR, GGPPS, TDS, FoTO1 , T5aH, TAT, T1 opH, DBAT, T13aH, T9aH-750C, T2aH, TBT, T7pH, T7AT, TOT, T1 pH-686, T9dA, T7dA, T9ox, T2’aH, N3’BT, BCL) in N. benthamiana transient expression assays.DOCTRINE OF EQUIVALENTS

[0186] Having described several embodiments, it will be recognized by those skilled in the art that various modifications, alternative constructions, and equivalents may be used without departing from the spirit of the invention. Additionally, a number of well- known processes and elements have not been described in order to avoid unnecessarily obscuring the present invention. Accordingly, the above description should not be taken as limiting the scope of the invention.

[0187] Those skilled in the art will appreciate that the foregoing examples and descriptions of various preferred embodiments of the present invention are merely illustrative of the invention as a whole, and that variations in the components or steps of the present invention may be made within the spirit and scope of the invention. Accordingly, the present invention is not limited to the specific embodiments described herein, but, rather, is defined by the scope of the appended claims.BFF Ref: STAN-2224WOClt Ref: S24-220Table 1 : Enzymes for Taxol (paclitaxel) BiosynthesisTable 2: Enzymes used in synthesis of taxanes other than TaxolBFF Ref: STAN-2224WOClt Ref: S24-220BFF Ref: STAN-2224WOClt Ref: S24-220INFORMAL SEQUENCE LISTINGSEQ ID NO: 1 FoT01 facilitator of taxane oxidation (gene id: ctg24251_gene.1 )MAETMNEKVGADEKLIEDSRNTGQEENDYSKRLGPLSRNIIPHLINIYTCIATPRDLEIY HPDATFEDFFVRAFGIKEIKSIHYSFPKLVYDGKILEYSVEENETSPGCGELLFNIKQQYKVLYLGKEVNITTLMVLQIENGKIIKHEDRINQHPVRGRHDISVPLVGRAREGIRRLTML MLHVRMGFGKDPTPPSEQ ID NO: 2 T9aH1 (T9aH-750C) taxane 9-alpha hydroxylase 1 (gene id: ctg10747_gene.1)MAFSRLIEAAAAEAPTIVTVLLLSFIFYLWRRNSSSSSTRLPPGPFQWPVIGNLHQFGRLPHLSIQHLANKYGPIMWLRLGYYPVVVVSTTEMAKEFLKIHDLAFSSRPKSGVGEHLVYNYKSMGFSPYGDYWRHIRRVWMTELMTPKRLSSFRSIREEEVCSAMRSIWEKSEQGRVAVNVSKAIDWISSSIVWRTVAGKKCSEDRDGKDLCDMVKRLMLTVKEVNGREIIPCIGWFDLQGVTRRMKETHRIFDGVAQNIIDQHINGRKREQSSDVKDIVDVLLEMADTGAINIQLDSIKAIIFDVLTGGIETATSSLEWTMTEMVRNPDIARKLQQEIESVVGKHRTVTE SDLPNIEYLQCVVQESLRLHPPAPLIFPRESTEACTVGAEGYVIPPKTRLMINVWAIGRDPAVWEDSLTFKPERFMGKDMDIKGRSDFRMLPFGGGRRGCPGAQMAIGNMELILAQ LMHCFDWRAEGDPSELDMSEALGTSLSRKHNLFAVPTLKLLNCIBFF Ref: STAN-2224WO Clt Ref: S24-220SEQ ID NO: 3 T7AT taxane 7p-O-acetyltransferase (gene id: ctg3030_gene.2)MENPSSTDFLVKKFDPVVVAPSLPLPKTTLQLSPIDNQIGFRGFFNSLSVYNAPDDISADPVKIIREALSKVLVHYFPLAGRFRNKENGELEVDCTGEGALFVEAMVEDNISVLRDFDDLNPSFQQLVFWPPMGANIEDLHLLVVQVTRFTCGDITIGVTVCHSIFDGCGAAQFVTALADMARGEVKPLLEPIWNRELLKPEDPLHLQLYQFDSLCPPPILFEELGQASLIINSNTIKYMKQCIMEECKVFCSTFEVMAALVWVARTKAFQIPHTETVKLLFAMDMRRSFNPPFPNGYYGNAIGTAYAMDNVEDLLNGSLSRVVMIIKKSKVSLRDNYLRSNKVKDPYSLDVNKKDNNVLALSDWRRLGFHEANFGWGDPVNVTAPQLLGKGLPLLSYYLFLQPSKNQPDGIKILMSCMHPSAVKSIKMEMEAMINKFVNKSSEQ ID NO: 4 T1 pH-686 taxane 1 -beta hydroxylase 686 (gene id: ctg5594_gene.1 )MASSAQNGVELSYIDLSQFSFDSEGLKNLQNHPGVATVREACKEFGCFGVLNTGIPDDVVQKLESVSHELCAMPSEMKDRAITSNPYDSYNRIPYRESFWFPTTWDSDSVLAYFNKLWPEKDNLNLCETVMAYALGMAELQRKISFIIIASLGLDVETFYHSDFEKATSYMRVHHHYSEGKFAAGEEALFGHYDPNCFTMLYQDIGGGLQIESKEGKWVDAKPGSLVINVAESLKAWSNGRYSSAKHRVVYKDWMHRLSVGWVMQFPDKEICAPAELVDEQHPQLYRPFPYPPFLDASMKYRITIDKYAGISPIYSEQ ID NO: 5 DeAc898 taxane C9 deacetylase (gene id: ctg19840_gene.3)MEAATAEPRVMQDMHGFIKVYSDGSVVRAGEPYFPAAISEENNDKLGPYKDVVYNAELGLWARIYLPPPPHKKTRLPVLLFFHASGFCILSPATPVVHRLCLLWAAKTGVIIVSVKYRLAPEHRLPAAYDDSIAALQWLLAMKSTEPGAVAVDPWLHSHADLSNIFVAGESAGGNIAHYLGCWVAAQDGEIQAQVKGLILVCPFFGGEDRTPSEEGNLAVMSEADIIWKYALPVGSNRDHPFCNPVGEGRESAISSLALPPILFVIAGLDVLRDKELQYCELLKKCGKQLEVVMFDEENHGFTLFNAEDQKSLEVIRCISDFVWSKSSEQ ID NO: 6 T9ox taxane C9 oxidase (gene id: ctg4166_gene.5)MEQNNMRNEVDLPFIDLSQFSFDSEGIKNLQNHPGVATVMESCQEWGFFRIMNTGIPBFF Ref: STAN-2224WOClt Ref: S24-220NDVFQKVESVSHELFAMPQEMKDRAITSSPHDTYINHPYRESFWFPTPPHSDSVLAFCNKLWPEKDNLKLCETIGTYILSMEDLKRKISSIIIASLGLDKVETFYHSDFENGTSVFRIHHYYSDGKFAAGEEALFAHTDPHCFTILYQDNGGGLQIQSKEGNWVHVKNIPNSLIINVADSLKAWSNGRYRSVNHRVVYKDWTNRISLGWFMMFPDKEIRVPAEFIDDQHPQCYRPFTYLQFRDAFMKDRIDIDGYAGIIPTYSEQ ID NO: 7 DeAc1023 taxane C7 deacetylase (gene id: ctg1975_gene.2)MADNSEPRVVENLYDGVIKLYSDGSIVRGDQQSPPPPTDDYNCVPFKDIVFDHTLGLWARVYLPPQTAKTRVPVLVYYHGGGFCCEFPPSTAILDCMCHKWAATLGVIIVSAEYRLAPEHRLPAAYHDAISALHWIDSMKSGAVEVDPWFRSHADFCKVFVAGDSAGGNIANHVGIWAAGAHGDGDLQIQIKGIILGCPFFGGEERTPSGSHNSPVFNLEISDTMWRLSLPLGSNRDHPFCNPVGVGDLKEADLPPMLFVIAGQDILKDKQLQYCEFLKGCGKQVEVHVFEEEDHGFTALKMENRSAVEALRCISHFINLTNBFF Ref: STAN-2224WOClt Ref: S24-220SEQ ID NO: 8 POL phenylalanine-CoA ligase (gene id: ctg867_gene.5)MDAEVVKSVRELGVDDVVQAGLPRHRAEIFYGQLQRAIADIGGSQTSLWHRVSKELLAPHHPHALHQLMYYSIYKNWDTSENGPPLYWFPTQESARLTNLGWMMETYGPQLLGSSYYNPITSFQSFQQFTVDHPEVYWSLVLKELSVVFHESPRCILDTSDKSRSGGVWLPGSVLNVAESCLSAKESINKTDNSIAIVWREEGRNEYPVNKMTLGELRAKVMRIANALDVVFTKGDAIAIDMPMTVNAVAIYLALILAGYVVVSIADSFVPKEIATRVRVTKAKGIFTQDFILRGGKRIPLYSRVVESGAPKAIVIPAEEELGTQLREIDVAWSKFLSFSDHLRSPEYYSAVRQPVDARTNILFSSGTSGEPKAIPWTHSPPIRCGSECWSHLDVKAGDIFCWPTNLGWVMGPVLVYSCFLSGATAAIYEGSPLDRGFGKFVQDARVTVLGTVPSMVKTWKSTGCMEGLDWSHIRTFASTGEASSIDDDLWLSSKGWYKPVIELCGGTELSACFVHGSLLQPQALGMFSTPTMTTGFVLFDDQQIPYPNDQPCIGEIGLFPRFFGSSYTLLNADHDAVYFKGMPMYKGMRLRRHGDMIERTVGGYYKAHGRSDDTMNLGGIKTSAIEIERVCNRAHEQVLETAAISISSSEGGPELLAILTVLKDGPTVSMDTLKLAFSKAIQSNLNPLFKVSFVKVISDFPRTASNKIMRRVLRDQIKQEFSLHKSRLSEQ ID NO: 9 Ti pH-184 taxane 1 -beta hydroxylase 184 (gene id: ctg13625_gene.1 )MASSLQNEDDLPIISLSQFSFESEGLKNLQNYPGLAKVREACKKWGFFRIVNTGIPNDVFRKMESVSHELCVMPQEMKDRAITSDPYDSYSQTPSRESFWFPTPSHSVSVQDFCNKLWPEKDNLKLCQTIGAYMFGMKELQRKISVIILASLGLDLETFYHSDFEKGTSIFRIHHHYSDGKFAVGEEALFGHTDPNCFTILYQDNGGGLQIQSKEGNWVDVKPVPNSLVINIADSLKAWSNGRYRSAKHRVVYKDWTNRISFVWMLMFPDKEIRAPTELIDEQHPQHYRPFTYHPFREATMKDHVNIDGYAGIFPTYBFF Ref: STAN-2224WOClt Ref: S24-220SEQ ID NO: 10. BCL benzoyl-CoA ligaseMEELKRCPANYPPLTPIGFIERAATVYRDCTSIVYNTTRFTWSQTFNRCRKLASALSSRNISRGDVVSVVAPNVPAIYEMHFAVPMAGAVLNNVNIRLDARTMAAQLTHCEPKFVFVDYQFLPLVREALSEIGNKPCVVVIEELDYGREIATSAWLTYEGLIGEGDPEFEIRWPEDEWQAAVLSYTSGTTSAPKGVVHCHRGLYTIAMDNVVMWGMKAQPVFLWTLPMFHTNGWCFPWAIAAVGGTNICLRKFDAKVIFDAITDHRVTHLCAAPVVLSMMANAHPSERKLLPGKVEILTGGAPPPAAVLLKAEELGFSVTHAYGLTETAGVVVICSLKGEWDKLSGQERARMKARQGVRSLSTAHVEVKDPATMASVARDGVEMGELMIRGASVMKGYLKNERVTAQTMQDGWLRTGDVAVVHPDGYLEIKDRSKDVIISGGENISSVEVESVLYSHPLIMEAAVVARPDPFWGETPCAFVSINNNSKEVLSEAQVISFCREHMAHFMAPKSVIFMKELPK TSTGKIQKFVLREMARALPLPSSRLSEQ ID NO: 1 1 ABC transporter Putative transporter-2MGVLSVDGTCHANMLSRQRPFSCVSVGHSNGKAKYSCRKKFSVARASATGDVLLEVKGLKAVVAESRMEILQGIDLVIRQGEVHAIMGKNGSGKSTFAKVLVGHPDYEVTGGSVIFKGENLLDKEPEERSRAGCFMSFQSPVEIPGVSNMDFLRMACNARRATLGLPELGPLEFYGFLSPKLAILNMDPKFLNRNVNEGFSGGEKKRNEILQFAVLEADLAILDEIDSGLDVDALQDVAKAVNGLLTPKNAVLMITHYQRLLDFIKPIYVHIIENGRIIKTGNALLAKQLEEG GYRAIASVSEQ ID NO: 12 lipid transfer protein Putative transporter-3MGLRNETSSISCCNYMLLCVVVLLSSQVVAAADNEDTCINSIVPCAPYLNATTRPPSNCCEPLLNVISTQQQCLCNLLTSDVISQFNVNITKALEIPTLCGAKNVSTDTCTKAGTTVGTPPASGTPSGSESSNKSSAEAASVEILLPLLAFVFLGVSVBFF Ref: STAN-2224WO Clt Ref: S24-220SEQ ID NO: 13 T1 pH-246 taxane 1 -beta hydroxylase 246MAEIDLPVIDIAQFPKEFDGGYFHHPDVVKLREACEEWGFFRVVNHGFPPDLLQKVLSVSRDLLSRPLEYKEKVPTYNALKYNPLHDEVIDAYGVLISDLAITITKFLIAALNLDPRAFYQSNFGKCTADLVISSFNFLEHDKCVRDEALVSHADTCVVTILYNDDKEGLEVRSKQGQWVNVKPTPDSFIVNVGDSMKVWSNGRYRSAHHRVLLKGWKNRLSLPLFFNFPLEEPIYAPEELVDKDNPRCYKPCTFSELLIEIKNKKSGGELYDAVERVASIM

Claims

BFF Ref: STAN-2224WOClt Ref: S24-220WHAT IS CLAIMED IS:1 . A method of producing a taxane, comprising: obtaining a cell comprising an expression cassette, wherein the expression cassette comprises a sequence encoding FoT01 operably linked to a promoter; and harvesting a taxane from the cell.

2. The method of claim 1 , wherein FoTO1 has at least 75% sequence identity to SEQ ID NO: 1.

3. The method of claim 1 or 2, wherein FoTO1 has at least 95% sequence identity to SEQ ID NO: 1.

4. The method of any one of claims 1 -3, wherein the sequence encoding FoT01 is from a Taxus species.

5. The method of any one of claims 1 -4, wherein the sequence encoding FoTO1 is from T. chinensis or T. media.

6. The method of any one of claims 1 -5, wherein the sequence encoding FoTO1 is codon optimized for the cell.

7. The method of any one of claims 1 -6, wherein the promoter is heterologous to FoTO1 .

8. The method of any one of claims 1 -7, wherein the promoter is selected from an inducible promoter, a constitutive promoter.

9. The method of any one of claims 1 -8, wherein the promoter is an inducible promoter, and the method further comprises providing a triggering agent to induce expression of the sequence encoding FoTO1 .

10. The method of any one of claims 1-9, wherein the expression cassette further comprises a sequence encoding one or more of: T9oH, T7AT, Ti pH, DeAc898,BFF Ref: STAN-2224WO Clt Ref: S24-220 DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.11 . The method of claim 10, further wherein the expression cassette further comprises a sequence encoding one or more of: TS, T5aH, TAT, T10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

12. The method of any one of claims 1 -11 , wherein obtaining the cell comprises transfecting the cell with vector comprising the expression cassette.

13. The method of claim 12, wherein the vector integrates the expression cassette into the genome of the cell.

14. The method of claim 2, wherein the vector is non-integrating.

15. The method of any one of claims 1 -9, wherein the cell comprises a second expression cassette, wherein the second expression cassette comprises a sequence encoding one or more of: T9oH, T7AT, Ti pH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

16. The method of claim 15, wherein the second expression cassette comprises a sequence encoding one or more of: TS, T5aH, TAT, T10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

17. The method of claim 15 or 16, wherein obtaining the cell comprises transfecting the cell with a vector comprising the second expression cassette.

18. The method of any one of claim 1 -17, wherein the cell is a plant cell, an animal cell, a bacterial cell, or a yest cell.

19. The method of any one of claims 1 -18, wherein the cell is from Nicotiana benthamiana or Saccharomyces cerevisiae.

20. The method of any one of claims 1 -19, wherein the cell is comprised in a cell culture.BFF Ref: STAN-2224WO Clt Ref: S24-22021 . The method of any one of claims 1-20, wherein the cell is comprised in a tissue.

22. The method of claim 21 , wherein the tissue is an N. benthamiana leaf.

23. The method of any one of claims 1 -22, wherein the cell is comprised within a vessel.

24. The method of claim 23, wherein the vessel is selected from a petri dish, a flask, a bioreactor, and a vat.

25. A cell comprising an expression cassette, wherein the expression cassette comprises a sequence encoding FoTO1 operably linked to a promoter.

26. The cell of claim 25, wherein FoTO1 has at least 75% sequence identity to SEQ ID NO: 1.

27. The cell of claim 25 or 26, wherein FoTO1 has at least 95% sequence identity to SEQ ID NO: 1.

28. The cell of any one of claims 25-27, wherein the sequence encoding FoT01 is from a Taxus species.

29. The cell of any one of claims 25-28, wherein the sequence encoding FoTO1 is from T. chinensis or T. media.

30. The cell of any one of claims 25-29, wherein the sequence encoding FoTO1 is codon optimized for the cell.31 . The cell of any one of claims 25-30, wherein the promoter is heterologous to FoTO1 .

32. The cell of any one of claims 25-31 , wherein the promoter is selected from an inducible promoter, a constitutive promoter.

33. The cell of any one of claims 25-32, wherein the promoter is an inducible promoter,BFF Ref: STAN-2224WO Clt Ref: S24-220 and the method further comprises providing a triggering agent to induce expression of the sequence encoding FoT01.

34. The cell of any one of claims 25-33, wherein the expression cassette further comprises a sequence encoding one or more of: T9oH, T7AT, Ti pH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

35. The cell of claim 34, further wherein the expression cassette further comprises a sequence encoding one or more of: TS, T5aH, TAT, T10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

36. The cell of any one of claims 25-35, wherein the expression cassette is integrated into the genome of the cell.

37. The cell of any one of claims 25-35, wherein the expression cassette is not integrated into the genome of the cell.

38. The cell of any one of claims 25-33, wherein the cell comprises a second expression cassette, wherein the second expression cassette comprises a sequence encoding one or more of: T9aH, T7AT, Ti pH, DeAc898, DeAc1023,T9ox, PCL, BCL, ABC transporter, and lipid transporter protein.

39. The cell of claim 38, wherein the second expression cassette comprises a sequence encoding one or more of: TS, T5aH, TAT, T10PH, DBAT, T13aH, T2aH, TBT, T7pH, and TOT.

40. The cell of any one of claim 25-39, wherein the cell is a plant cell, an animal cell, a bacterial cell, or a yest cell.41 . The cell of any one of claims 25-40, wherein the cell is from Nicotiana benthamiana or Saccharomyces cerevisiae.

42. The cell of any one of claims 25-41 , wherein the cell is comprised in a cell culture.

43. The cell of any one of claims 25-42, wherein the cell is comprised in a tissue.BFF Ref: STAN-2224WO Clt Ref: S24-22044. The cell of claim 43, wherein the tissue is an N. benthamiana leaf.

45. The cell of any one of claims 25-44, wherein the cell is comprised within a vessel.

46. The cell of claim 45, wherein the vessel is selected from a petri dish, a flask, a bioreactor, and a vat.

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