Heterologous biosynthesis of indole diterpenes

Abstract

Disclosed herein are isolated genes from Aspergillus fresenii, that mediate the production of 14,15- epoxy-3´-GGI, radarin C, radarin A, and the use of these genes to direct heterologous expression of these compounds in isolated host cells. Also disclosed is RosB from Aspergillus fresenii, a gene involved in the production of a new cyclic IDT derived from 14,15-epoxy-3´-GGI; roseyrin. Further disclosed are isolated genes from the Aspergillus muricatus, that mediate the production of 14,15- epoxy-3´-GGI and petromindole. Methods of making and use are provided.

Description

[0001] Heterologous Biosynthesis of Indole Diterpenes

[0002] FIELD OF THE INVENTION

[0003] This invention generally relates to novel polypeptides that catalyze a biochemical reaction leading to the production of radarin compounds and related indole diterpenes, methods of making such polypeptides and polynucleotides, and methods of using such polypeptides and polynucleotides to produce a radarin compound and / or a related indole diterpene derived from C14-C15 epoxidation of 3'-geranylgeranylindole by expression in a recombinant host cell.

[0004] BACKGROUND

[0005] Filamentous fungi produce a diverse repertoire of interesting and useful chemical compounds.

[0006] Meroterpenoids are an abundant class of fungal secondary metabolites (SMs) that have been pursued in various biosynthesis studies due to their impressive structural complexity and potential therapeutic and agricultural applications.1The indole diterpenoids (IDTs) are a large meroterpenoid class, where an indole ring is fused to a cyclised geranylgeranyl (C20) chain (Figure 1A). The IDTs are known to possess bioactivities including anti-MRSA,2anti-cancer,3-5anti-HINl,6insecticidal7'8and tremorgen ic9-11activities, which differ based on the cyclic core or by how that core is chemically decorated. Radarins (Figure IB) are a group of IDTs produced by Aspergillus fresenii, = A. suiphureus nom. illeg.} CBS 550.65 which produces diverse groups of indole diterpenes (IDT), also including the sulpinines, penitrem derivatives and aflavinine compounds. Radarin A is of particular significance because it has been determined to be effective for controlling lepidopteran insects, and possesses significant activity towards cancer cell lines.3A related IDT, namely petromindole, which is produced by Aspergillus muricatus (Petromyces muricatus) CBS 112808,12has structural similarities to the radarins (Figure IB). The unusual skeletons of radarins and petromindole are representative of a branch of IDTs for which the biosynthetic machinery is not yet characterised. Their structures imply that A. fresenii and A. muricatus have the biosynthetic machinery to catalyse regiospecific epoxidation of 3 geranylgeranylindole (GGI) to form predicted precursor 14,15-epoxy-3'-GGI, and subsequent cyclisation to form these IDT architectures (Figure 2).

[0007] Radarins are not easily isolated from the natural producer, Aspergillus fresenii (A. fresenii). The only reported radarin production method is in their initial discovery, and requires A. fresenii to be grown for 21 days at 28 °C in darkness on a layer of corn kernels, followed by a separation of these corn kernels from fungal biomass.3Additionally, obtaining three milligrams of petromindole required 480 Petri dish plates with A. muricatus for 21 days at 35 °C.12These methods of fermentation would not be effective for obtaining useful quantities of the radarins or petromindole, as production of commercial quantities of the radarins / petromindole would be costly and laborious. Additionally, wildtype A. fresenii has multiple competing biosynthetic pathways as it produces a myriad of other IDTs, making efficient production of radarins even more challenging (Figure 3).3A13There are no reported attempts of total chemical synthesis of the radarins due to their complex structure and stereochemistry. Consequently, there is a need in the art for new methods of radarin synthesis and / or biosynthesis that will provide the radarins and biosynthetically-related indole diterpene compounds efficiently, and in useful quantities.

[0008] It is an object of the present invention to provide a polynucleotide encoding a polypeptide that catalyzes the conversion of 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI and / or a polynucleotide encoding a polypeptide that catalyzes the conversion of 14,15-epoxy-3'-GGI to an indole diterpene selected from the group consisting of radarin C, roseyrin or petromindole and / or a polynucleotide encoding a polypeptide that catalyzes the conversion of radarin C to radarin A and / or a method of using such a polynucleotide to make 14,15-epoxy-3'-GGI, radarin C, roseyrin, petromindole and / or radarin A by heterologously expressing one or more such polynucleotides in an isolated host cell and / or to at least provide the public with a useful choice.

[0009] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect the invention relates to an isolated RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO:12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0012] In another aspect the invention relates to an isolated polynucleotide or functional variant thereof encoding a RadB, RadM, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0013] In another aspect the invention relates to an isolated radM, radB, radX, rosB, petMor petB genomic polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively.

[0014] In another aspect the invention relates to an isolated radM, radB, radX, rosB, petMor efFcDNA polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 17 respectively. In another aspect the invention relates to a nucleic acid construct comprising an isolated polynucleotide or functional variant thereof according to the invention.

[0015] In another aspect the invention relates to a transcription unit (TU) comprising an isolated polynucleotide or functional variant thereof according to the invention.

[0016] In another aspect the invention relates to a vector that encodes an isolated polypeptide or functional variant thereof according to the invention.

[0017] In another aspect the invention relates to a vector comprising an isolated polynucleotide or functional variant thereof, nucleic acid construct or a TU according to the invention.

[0018] In another aspect the invention relates to an isolated host cell comprising an isolated polypeptide or functional variant thereof, isolated polynucleotide or functional variant thereof, nucleic acid construct, TU and / or vector according to the invention.

[0019] In another aspect the invention relates to a method of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof comprising expressing a polypeptide or functional variant thereof, isolated nucleic acid sequence or functional variant thereof, TU or vector according to the invention in an isolated host cell.

[0020] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI comprising expressing a) in an isolated recombinant host cell, wherein

[0021] a) is a RadM or PetM polypeptide or functional variant thereof according to the invention. In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to radarin C comprising expressing a) and b) in a recombinant isolated host cell, wherein

[0022] a) is a RadM or PetM polypeptide or functional variant thereof according to the invention, and b) is a RadB polypeptide or functional variant thereof according to the invention.

[0023] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to roseyrin comprising expressing a) and c) in an isolated recombinant host cell, wherein

[0024] a) is a RadM or PetM polypeptide or functional variant according to the invention, and c) is a RosB polypeptide or functional variant thereof according to the invention.

[0025] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to petromindole comprising heterologously expressing a) and d) in an isolated host cell, wherein a) is a RadM or PetM polypeptide or functional variant thereof according to the invention, and

[0026] b) is a PetB polypeptide or functional variant thereof according to the invention.

[0027] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to radarin A comprising expressing a), b) and e) in an isolated recombinant host cell, wherein

[0028] a) is a RadM or PetM polypeptide or functional variant thereof according to the invention, b) is a RadB polypeptide or functional variant thereof according to the invention, and e) is a RadX polypeptide or functional variant thereof according to the invention.

[0029] In another aspect the invention relates to 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole made by a method of the invention.

[0030] In another aspect the invention relates to a compound of Formula VI,

[0031]

[0032] In another aspect the invention relates to a method of making an Aspergillus

[0033]

[0034] polypeptide or functional variant thereof comprising expressing an isolated polynucleotide or functional variant thereof, nucleic acid construct, TU or vector as described herein in an isolated host cell.

[0035] In another aspect the invention relates to an isolated host cell that expresses a polypeptide or functional variant thereof that catalyzes the transformation of 3'-GGI to 14,15-epoxy-3'-GGI.

[0036] In another aspect the invention relates to an isolated host cell that expresses at least three polypeptides or functional variants thereof that together, mediate the transformation of 3'-GGI to radarin A.

[0037] In another aspect the invention relates to an isolated strain of Penicillium paxilli, Aspergillus niger, Aspergillus oryzae, Saccharomyces cerevisiae or Escherichia coii h comprises at least one heterologous polynucleotide or functional variant thereof that encodes an enzyme in the biosynthetic pathway leading from 3'-GGI to radarin A, roseyrin or petromindole.

[0038] In another aspect the invention relates to an isolated strain of Aspergillus s. or Penicillium spp. that comprises at least one genetic modification that leads to an increased biosynthesis of radarin C, roseyrin, petromindole or radarin A. In another aspect the invention relates to a method of making radarin C, radarin A, roseyrin or petromindole comprising expressing a heterologous nucleic acid sequence in Penicillium spp. or Aspergillus spp. wherein a heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway leading from 3'-GGI to radarin C, radarin A, roseyrin or petromindole.

[0039] Various embodiments of the different aspects of the invention as discussed above are also set out below in the detailed description of the invention, but the invention is not limited thereto.

[0040] Other aspects of the invention may become apparent from the following description which is given by way of example only and with reference to the accompanying drawings.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The invention will now be described with reference to the figures in the accompanying drawings. Figure 1: A) Representative IDT structures that biosynthesis has been elucidated. B) Chemical structures of radarins and petromindole.

[0043] Figure 2: Proposed biosynthetic scheme for core biosynthesis of IDTs, showing that radarins and petromindole must derive from the 14,15-epoxy-3'-GGI precursor.

[0044] Figure 3: Chemical structures of all IDTs known to be isolated from Aspergillus fresenii.

[0045] Figure 4: A) Depiction of IDT genes identified in A. fresenii and A. muricatus for biosynthesis of IDTs derived from 14,15-epoxy-3'-GGI. B) Novel steps to 14,15-epoxy-3'-GGI derived IDTs, elucidated by heterologous expression in P. paxiiii.

[0046] Figure 5: Protein sequence identity matrix for experimentally characterised FAD-dependent epoxidases from meroterpenoid biosynthetic pathways.

[0047] Figure 6: Protein sequence identity matrix for experimentally characterised cyclases from meroterpenoid biosynthetic pathways.

[0048] Figure 7: Protein sequence identity matrix for experimentally characterised cytochrome P450 monooxygenases that are from IDT biosynthetic pathways or share >40% sequence homology with RadX.

[0049] Figure 8: Proposed mechanism for catalysed cyclisations of 14,15-epoxy-3'-GGI by RadB, RosB and PetB.

[0050] Figure 9: LC-MS analysis of ten P. paxi / ii 0(2) strains transformed with pRM297 (paxG, paxCand radM expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), / 77 / z406.3 (14,15-epoxy-3'-GGI) and / 77 / z424.3 (14,15-dihydroxy-3'-GGI).

[0051] Figure 10: LC-MS analysis of ten P. paxilli (CY2) strains transformed with pRM318 paxG, paxC and petM expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), / 77 / z406.3 (14,15-epoxy-3'-GGI) and / 77 / 424.3 (14,15-dihydroxy-3'-GGI).

[0052] Figure 11: LC-MS analysis of ten P. paxilli (CY2) strains transformed with pRM298 paxG, paxC, radM and radB expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), m / z 406.3 (radarin C or 14,15-epoxy-3'-GGI).

[0053] Figure 12: LC-MS analysis of ten P. paxilli (CY2) strains transformed with pRM301 paxG, paxC, radM, radB and radX expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), m / z 146.1 (hydroxyindole fragment ion) / 77 / z406.3 (radarin C or 14,15-epoxy-3'-GGI or roseyrin) m / z 422.3 (radarin A).

[0054] Figure 13: LC-MS analysis of six P. paxilli (CY2) strains transformed with pRM308 paxG, paxC and radM and rosB expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), m / z 406.3 (roseyrin).

[0055] Figure 14: LC-MS analysis of ten P. paxilli (CY2) strains transformed with pRM319 paxG, paxC and petM and petB expression construct). Extracted Ion Chromatogram traces shown for each transformant from top to bottom: [M + H]+at m / z 130.1 (indole fragment ion), m / z 406.3 (petromindole).

[0056] Figure 15: Comparison ofXH and13C NMR data for 14,15-dihydroxy-3'-GGI in CDCb

[0057] Figure 16: Summary of13C (150 MHz),XH (600 MHz) and 2D NMR data of 14,15-dihydroxy-3'-GGI in CDCb

[0058] Figure 17: Comparison ofXH and13C NMR data for radarin C in CDCb

[0059] Figure 18: Summary of13C (150 MHz),XH (600 MHz) and 2D NMR data of radarin C in CDCb Figure 19: Comparison ofXH and13C NMR data for radarin A in CDCb

[0060] Figure 20: Summary of13C (150 MHz),XH (600 MHz) and 2D NMR data of radarin A in CDCb Figure 21: Summary of13C (150 MHz),XH (600 MHz) and 2D NMR data of roseyrin in CDCb Figure 22: Comparison ofXH NMR data for petromindole in CDCb

[0061] DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0062] The term "comprising" as used in this specification and claims means "consisting at least in part of"; that is to say when interpreting statements in this specification and claims which include "comprising", the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

[0063] The term "consisting essentially of as used herein means the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0064] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0065] The term "3'-GGI" means a compound of Formula I.

[0066]

[0067] The term "Radarin A" means a compound of Formula III.

[0068]

[0069] The term "Radarin C" means a compound of Formula IV.

[0070]

[0071] The term "petromindole" means a compound of Formula V.

[0072]

[0073] The term "roseyrin" means a compound of Formula VI.

[0074]

[0075] The term "genetic construct" refers to an artificially assembled polynucleotide molecule, where a first polynucleotide, usually double-stranded DNA, but not limited thereto, has been conjugated to another polynucleotide molecule. Preferably a genetic construct is a recombinant nucleic acid molecule. A genetic construct may comprise a single polynucleotide of interest, or multiple polynucleotides of interest. In one non-limiting example a genetic construct is made by inserting a first polynucleotide molecule into a second polynucleotide molecule, for example by restriction / ligation as known in the art. In some embodiments, a genetic construct comprises a single polynucleotide module, at least two polynucleotide modules, or a series of multiple polynucleotide modules assembled into a single contiguous polynucleotide molecule (also referred to herein as a "multigene construct"), but not limited thereto.

[0076] A genetic construct may contain the necessary elements that permit transcription of a polynucleotide molecule, and, optionally, for translating the transcript into a polypeptide. A polynucleotide molecule comprised in and / or by the gene construct may be derived from the host cell or may be derived from a different cell or organism and / or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector. The term "transcription unit" (TU) as used herein refers to a polynucleotide comprising a sequence of nucleotides that code for a single RNA molecule including all the nucleotide sequences necessary for transcription of the single RNA molecule, including a promoter, an RNA-coding sequence, and a terminator, but not limited thereto.

[0077] The term "transcription unit module" (TUM) as used herein refers to a polynucleotide comprising a sequence of nucleotides that encode a single RNA molecule, or parts thereof; or that encode a protein coding sequence (CDS), or parts thereof; or that encode sequence elements, or parts thereof, that control transcription of that RNA molecule; or that encode sequence elements or parts thereof that control translation of the CDS. Such sequence elements may include, but are not limited to, promoters, untranslated regions (UTRs), terminators, polyadenylation signals, ribosome binding sites, transcriptional enhancers and translational enhancers.

[0078] The term "multigene construct" as used herein means a genetic construct that is a polynucleotide comprising at least two TUs.

[0079] The term "marker" as used herein means a nucleic acid sequence in a polynucleotide that encodes a selectable marker or scorable marker.

[0080] The term "selectable marker" as used herein refers to a TU, which when introduced into a cell, confers a trait on the cell that allows the cell to be selected based on the presence or absence of that trait. In one embodiment the cell is selected based on survival under conditions that kill cells not comprising the selectable marker.

[0081] The term "scorable marker" as used herein refers to a TU, which when introduced into a cell, confers a trait on the cell that allows the cell to be scored based on the presence or absence of that trait. In one embodiment the cell comprising the TU is scored by identifying the cell phenotypically from a plurality of cells.

[0082] The term "genetic element" as used herein refers to any polynucleotide sequence that is not a TU or does not form part of a TU. Such polynucleotide sequences may include but are not limited to origins of replication for plasmids and viruses, centromeres, telomeres, repeat sequences, sequences used for homologous recombination, site-specific recombination sequences, and sequences controlling DNA transfer between organisms.

[0083] The term "vector" as used herein refers to any type of polynucleotide molecule that may be used to manipulate genetic material so that it can be amplified, replicated, manipulated, partially replicated, modified and / or expressed, but not limited thereto. In some embodiments a vector may be used to transport a polynucleotide comprised in that vector into a cell or organism. The term "source vector" as used herein refers to a vector into which polynucleotide sequences of interest can be cloned. In some embodiments the polynucleotide sequences are TUs and TUMs as described herein. In some embodiments a source vector is selected from the group consisting of plasmids, bacterial artificial chromosomes (BACs), phage artificial chromosomes (PACs), yeast artificial chromosomes (YACs), bacteriophage, phagemids, and cosmids. In some embodiments, a source vector comprising a polynucleotide sequence of interest is termed an entry clone. In some embodiments the entry clone can serve as a shuttle or destination vector for receiving further polynucleotide sequences.

[0084] The term "shuttle vector" as used herein refers to a vector into which polynucleotide sequences of interest can be cloned and from which they can be manipulated. In some embodiments the polynucleotide sequences are TUs and TUMs as described herein. In some embodiments a shuttle vector is selected from the group consisting of plasmids, BACs, PACs, YACs, bacteriophage, phagemids, and cosmids. In some embodiments, a shuttle vector comprising a polynucleotide sequence of interest can serve as a destination vector for receiving further polynucleotide sequences. The term "destination vector" as used herein refers to a vector into which polynucleotide sequences of interest can be cloned. In some embodiments the polynucleotide sequences are TUs and TUMs as described herein. In some embodiments a destination vector is selected from the group consisting of plasmids, BACs, PACs, YACs, bacteriophage, phagemids, and cosmids. In some embodiments, a destination vector comprising a polynucleotide sequence of interest is an entry clone. In some embodiments the entry clone can serve as a destination vector for receiving further polynucleotide sequences.

[0085] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, CDS, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors and modified polynucleotides. Reference to nucleic acids, nucleic acid molecules, nucleotide sequences and polynucleotide sequences is to be similarly understood.

[0086] The term "gene" as used herein refers to gene the biologic unit of heredity, self-reproducing and located at a definite position (locus) on a particular chromosome. In one embodiment the particular chromosome is a eukaryotic or bacterial chromosome. The term bacterial chromosome is used interchangeably herein with the term bacterial genome.

[0087] The term "gene cluster" as used herein refers to a group of genes located closely together on the same chromosome whose products play a coordinated role in a specific aspect of cellular primary or secondary metabolism. In one example a gene cluster comprises a group of CDSs the products of which all participate in a series of biochemical reactions that comprise the biosynthetic pathway or array that produces a given metabolite, particularly a secondary metabolite.

[0088] The term "secondary metabolite" as used herein refers to compounds that are not involved in primary metabolism, and therefore differ from the more prevalent macromolecules such as proteins and nucleic acids that make up the basic machinery of life.

[0089] The terms "under conditions wherein the... enzyme is active" and "under conditions wherein the... enzymes are active", and grammatical variations thereof when used in reference to enzyme activity mean that the enzyme will perform it's expected function; e.g., a restriction endonuclease will cleave a nucleic acid at an appropriate restriction site, and a DNA ligase will covalently join two polynucleotides together.

[0090] The phrase "produces 3'-GGI endogenously" and grammatical variations thereof refers to a host cell that naturally produces endogenous 3 '-GGI.

[0091] The phrase "produces 3'-GGI heterologously" and grammatical variations thereof refers to a host cell that is a recombinant host cell that has been transformed with at least one polynucleotide encoding a at least one polypeptide that catalyzes the formation of 3 '-GGI from "a 3 '-GGI precursor substrate molecule". The "3 '-GGI precursor substrate molecule" may be a substrate molecule that is produced endogenously by the host cell or that is produced heterologously by a recombinant host cell.

[0092] The term "endogenous" as used herein refers to a constituent of a cell, tissue or organism that originates or is produced naturally within that cell, tissue or organism. An "endogenous" constituent may be any constituent including but not limited to a polynucleotide, a polypeptide including a non-ribosomal polypeptide, a fatty acid or a polyketide, but not limited thereto.

[0093] The term "exogenous" as used herein refers to any constituent of a cell, tissue or organism that does not originate or is not naturally occurring within that cell, tissue or organism. An exogenous constituent may be, for example, a polynucleotide sequence that has been introduced into a cell, tissue or organism, or a polypeptide expressed in that cell, tissue or organism from that polynucleotide sequence, but is not limited thereto. In some embodiments an exogenous constituent is an introduced endogenous polynucleotide sequence that has been introduced into a cell, tissue or organism to create a recombinant cell, tissue or organism, wherein the polynucleotide encodes an introduced endogenous polypeptide.

[0094] "Endogenous" as used herein with reference to a polynucleotide or polypeptide in a host organism means that the polynucleotide or polypeptide is a native and naturally-occurring polynucleotide or polynucleotide within that host organism. An endogenous polynucleotide may be operably linked to a homologous or heterologous regulatory element so that an endogenous polypeptide may be expressed from a TU, genetic element or vector comprising the endogenous polynucleotide as described herein.

[0095] "Introduced endogenous" as used herein with reference to polynucleotide or polypeptide in a host organism means that the polynucleotide or polypeptide is a native and naturally-occurring polynucleotide or polynucleotide within that host organism that has been introduced into the organism by experimental techniques. In one embodiment the experimental technique is transformation and the host organism is a recombinant host organism. An introduced endogenous polynucleotide may be operably linked to a homologous or heterologous regulatory element so that an introduced endogenous polypeptide may be expressed from a TU, genetic element or vector comprising a homologous polynucleotide as described herein.

[0096] "Naturally occurring" as used herein with reference to a polynucleotide sequence according to the invention refers to a primary polynucleotide sequence that is found in nature. A synthetic polynucleotide sequence that is identical to a wild polynucleotide sequence is, for the purposes of this disclosure, considered a naturally occurring sequence. What is important for a naturally occurring polynucleotide sequence is that the actual sequence of nucleotide bases that comprise the polynucleotide is found or known from nature.

[0097] For example, a wild-type polynucleotide sequence is a naturally occurring polynucleotide sequence, but not limited thereto. A naturally occurring polynucleotide sequence also refers to variant polynucleotide sequences as found in nature that differ from wild type. For example, allelic variants and naturally occurring recombinant polynucleotide sequences due to hybridization or horizontal gene transfer, but not limited thereto.

[0098] "Non-naturally occurring" as used herein with reference to a polynucleotide sequence as described herein refers to a polynucleotide sequence that is not found in nature. A non-naturally occurring polynucleotide sequence is an artificial polynucleotide sequence. Examples of non-naturally occurring polynucleotide sequences include artificially produced mutant and variant polynucleotide sequences, made for example by point mutation, insertion, or deletion, but not limited thereto. Non-naturally occurring polynucleotide sequences also include chemically evolved sequences. What is important for a non-naturally occurring polynucleotide sequence according to the invention is that the actual sequence of nucleotide bases that comprise the polynucleotide is not found or known from nature. The term, "wild type" when used herein with reference to a polynucleotide refers to a naturally occurring; non-mutant form of a polynucleotide. A mutant polynucleotide means a polynucleotide that has sustained a mutation as known in the art, such as point mutation, insertion, deletion, substitution, amplification or translocation, but not limited thereto. The term, "wild type" when used herein with reference to a polypeptide refers to a naturally occurring, non-mutant form of a polypeptide. A wild type polypeptide is a polypeptide that is capable of being expressed from a wild type polynucleotide.

[0099] The term "coding sequence" (CDS) or "open reading frame" (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and / or a polypeptide under the control of appropriate regulatory sequences. A CDS is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct or an expression cassette, a CDS is capable of being expressed when it is operably linked to a promoter sequence and / or other regulatory elements.

[0100] "Operably-linked" means that the sequence to be expressed is placed under the control of regulatory elements.

[0101] "Regulatory elements" as used herein refers to any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector, genetic construct or expression cassette and includes promoters, transcription control sequences, translation control sequences, origins of replication, tissue-specific regulatory elements, temporal regulatory elements, enhancers, polyadenylation signals, repressors and terminators. Regulatory elements can be "homologous" or "heterologous" to the polynucleotide insert to be expressed from a genetic construct, expression cassette or vector as described herein. When a genetic construct, expression cassette or vector as described herein is present in a cell, a regulatory element can be "endogenous", "exogenous", "naturally occurring" and / or "non-naturally occurring" with respect to cell.

[0102] The term "noncoding region" refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.

[0103] Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.

[0104] The term "promoter" refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate the transcription of a polynucleotide sequence. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes. In one non-limiting example, bacterial promoters may comprise a "Pribnow box" (also known as the -10 region), and other motifs that are bound by transcription factors and promote transcription. Promoters can be homologous or heterologous with respect to polynucleotide sequence to be expressed. When the polynucleotide sequence is to be expressed in a cell, a promoter may be an endogenous or exogenous promoter. Promoters can be constitutive promoters, inducible promoters or regulatable promoters as known in the art.

[0105] "Homologous" as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is a native and naturally-occurring polynucleotide regulatory element. A homologous polynucleotide regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a TU, genetic element or vector according to the invention.

[0106] "Homologous" as used herein with reference to polynucleotide or polypeptide in a host organism means that the polynucleotide or polypeptide is a native and naturally-occurring polynucleotide or polynucleotide within that host organism. A homologous polynucleotide may be operably linked to a homologous or heterologous regulatory element so that a homologous polypeptide may be expressed from a TU, genetic element or vector comprising the homologous polynucleotide as described herein. "Heterologous" as used herein with reference to polynucleotide regulatory elements, means a polynucleotide regulatory element that is not a native and naturally-occurring polynucleotide regulatory element. A heterologous polynucleotide regulatory element is not normally associated with the CDS to which it is operably linked. A heterologous regulatory element may be operably linked to a polynucleotide of interest such that the polynucleotide of interest can be expressed from a vector, genetic construct or expression cassette according to the invention. Non-limiting examples of heterologous regulatory elements include promoters, enhancers and / or terminators. In one nonlimiting example, "promoters" may include promoters normally associated with other genes, ORFs or coding regions, and / or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cell.

[0107] "Heterologous" as used herein with reference to a polynucleotide or polypeptide in a host organism means a polynucleotide or polypeptide that is not a native and naturally-occurring polynucleotide or polypeptide in that host organism. A heterologous polynucleotide may be operably linked to a heterologous or homologous regulatory element so that a heterologous polypeptide may be expressed from a TU, genetic element or vector comprising the heterologous polynucleotide as described herein. The terms "heterologously expressing" and "heterologous expression" mean the expression of a heterologous polynucleotide or heterologous polypeptide in a host cell.

[0108] A "biochemical reaction in the biosynthetic pathway leading from 3'-GGI to radarins or indole diterpenes derived from C14-C15 epoxidation of 3'-GGI" means a biochemical reaction catalyzed by RadM or PetM to transform the substrate molecule 3'-GGI to 14,15-epoxy-3'-GGI, a biochemical reaction catalyzed by RadB to transform the substrate molecule 14,15-epoxy-3'-GGI to radarin C, a biochemical reaction catalyzed by RosB to transform the substrate molecule 14,15-epoxy-3'-GGI to roseyrin, a biochemical reaction catalyzed by PetB to transform the substrate molecule 14,15-epoxy- 3'-GGI to petromindole, or a biochemical reaction catalyzed by RadX to transform the substrate molecule radarin C to radarin A, and does not include similar enzymes within a host cell that may have similar functions but that do not act on the particular named intermediates above.

[0109] A "functional variant thereof" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity or binding of that polypeptide and / or provides the three-dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or functional polypeptide derivative thereof that is capable of performing the polypeptide activity.

[0110] "Isolated" as used herein with reference to polynucleotide or polypeptide sequences describes a sequence that has been removed from its natural cellular environment. An isolated molecule may be obtained by any method or combination of methods as known and used in the art, including biochemical, recombinant, and synthetic techniques. The polynucleotide or polypeptide sequences may be prepared by a purification step.

[0111] "Isolated" when used herein in reference to a cell or host cell describes to a cell or host cell that has been obtained or removed from an organism or from its natural environment and is subsequently maintained in a laboratory environment as known in the art. The term encompasses single cells, per se, as well as cells or host cells comprised in a cell culture and can include a single cell or single host cell.

[0112] The term "isolated host cell" as used herein with reference to a fungal host cell encompasses single cells of unicellular fungi and the hyphae and mycelia of filamentous fungi including septate and non-septate forms.

[0113] The term "recombinant" refers to a polynucleotide sequence that is removed from sequences that surround it in its natural context and / or is recombined with sequences that are not present in its natural context. A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0114] As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities that are the same or similar to those of a corresponding wild type molecule; i.e., the parent polypeptides or polynucleotides. In certain embodiments, variants of the polypeptides described herein have biological activities that are similar, or that are substantially similar to their corresponding wild type molecules. In certain embodiments the similarities are similar activity and / or binding specificity.

[0115] In certain embodiments, variants of polypeptides described herein have biological activities that differ from their corresponding wild type molecules. In certain embodiments the differences are altered activity and / or binding specificity.

[0116] The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

[0117] Variant polynucleotide sequences preferably exhibit at least 50%, at least 60%, preferably at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 8 nucleotide positions, preferably at least 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1200, preferably at least 1400 nucleotide positions, preferably over the entire length of a polynucleotide as described herein.

[0118] Polynucleotide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance.

[0119] Polynucleotide sequence identity and similarity can be determined readily by those of skill in the art. Variant polynucleotides also encompass polynucleotides that differ from the polynucleotide sequences described herein but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[0120] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie etaL, 1990, Science 247, 1306).

[0121] The term "variant" with reference to polypeptides also encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 35%, preferably at least 40%, 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 2 amino acid positions, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, preferably at least 500 amino acid positions, preferably over the entire length of a polypeptide used in or identified according to a method of the invention.

[0122] Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, and which could not reasonably be expected to have occurred by random chance.

[0123] Polypeptide sequence identity and similarity can be determined readily by those of skill in the art. A variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid or non-conservative substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide.

[0124] Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0125] Analysis of evolved biological sequences has shown that not all sequence changes are equally likely, reflecting at least in part the differences in conservative versus non-conservative substitutions at a biological level. For example, certain amino acid substitutions may occur frequently, whereas others are very rare. Evolutionary changes or substitutions in amino acid residues can be modelled by a scoring matrix also referred to as a substitution matrix. Such matrices are used in bioinformatics analysis to identify relationships between sequences and are known to the skilled worker.

[0126] Other variants include peptides with modifications which influence peptide stability. Such analogs may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids, e.g. beta or gamma amino acids and cyclic analogs.

[0127] Substitutions, deletions, additions or insertions may be made by mutagenesis methods known in the art. A skilled worker will be aware of methods for making phenotypically silent amino acid substitutions. See for example Bowie eta!., 1990, Science 247, 1306. A polypeptide as used herein can also refer to a polypeptide that has been modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, by derivatization using blocking / protecting groups and the like. Such modifications may increase stability or activity of the polypeptide.

[0128] The terms "modulate(s) expression", "modulated expression" and "modulating expression" of a polynucleotide or polypeptide, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide to be expressed according to the invention is modified thus leading to modulated expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "modulated expression" can be related to an increase or decrease in the amount of messenger RNA and / or polypeptide produced and may also result in an increase or decrease in the activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

[0129] The terms "modulate(s) activity", "modulated activity" and "modulating activity" of a polynucleotide or polypeptide, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide to be expressed according to the invention is modified thus leading to modulated expression of a polynucleotide or modulated expression or activity of polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The "modulated activity" can be related to an increase or decrease in the amount of messenger RNA and / or polypeptide produced and may also result in an increase or decrease in the functional activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.

[0130] As used herein, the nomenclature RadM, RadB, RadX, RosB, PetM, and PetB designates polypeptides. As used herein the nomenclature radM, radB, radX, rosB, petMand petB designates polynucleotides. It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0131] DETAILED DESCRIPTION

[0132] Disclosed herein is the inventor's work demonstrating the biosynthetic machinery to deliver IDTs derived from 14,15-epoxy-3 '-GGI. This work provides a method to deliver the subset of IDTs that are accessed biosynthetically by regiospecific epoxidation at C14-C15 of 3'-GGI, and subsequent enzyme-catalysed cyclisation. Generally, the early steps in IDT biosynthesis are well established. The first dedicated step is formation of geranylgeranyl pyrophosphate (GGPP), typically by a GGPP synthase (IdtG) that is encoded within an IDT biosynthetic gene cluster (Figure 2). A prenyltransferase (IdtC) then catalyses condensation of indole-3 '-glycerol phosphate with GGPP to yield 3'-geranylgeranylindole (3'-GGI). 3'-GGI is epoxidised by an FAD-dependent monooxygenase (IdtM) to prime the scaffold for protonation-initiated cyclisation by a transmembrane terpene cyclase (IdtB), delivering a variety of cyclized skeletons (Figure 2).14'15These scaffolds are often substrates for further derivatization by oxidases,16prenyltransferases,17'18and other decorative enzymes.19

[0133] To date, all described IDT biosynthetic pathways include an IdtM epoxidase that regioselectively oxidizes the C10-C11 or "internal" olefin of 3 '-GGI (Figure 1A). The resulting 10,ll-epoxy-3 '-GGI product is then cyclized by an IdtB cyclase, most frequently to form emindole SB,20-22but also to give an array of other IDT architectures.14'15While the route that leads to IDTs via epoxidation of the internal olefin have been well described, there are currently no reported examples of IdtMs that catalyse the initial epoxidation of the terminal olefin, nor IdtBs that cyclise 14,15-epoxy-3'-GGI (Figure 2). However, the isolated radarin3and petromindole12IDTs have unusual skeletons that imply such machinery for primary C14-C15 epoxidation of 3'-GGI and subsequent cyclisation must exist in nature (Figure IB).23-26

[0134] Radarins are bioactive IDTs produced by Aspergillus fresenii, with radarin A being of particular significance due to proven insecticidal activity against lepidopterans and anti-cancer activity3, whereas petromindole is not yet known to have any associated bioactivity. However, as described herein, the production of radarins by direct synthesis has not been achieved, and only one study has been able to generate partial synthesis of the radarin cyclic core.27. Further, the biosynthetic production of the radarins (by fermentation of the native host) in quantities that would be useful at even a small-scale commercial level would be difficult to achieve, if at all. Fermentation processes for petromindole would also not be feasible on a commercial level,12. Enzymatic synthesis of petromindole has been achieved by a lupeol synthase (LUPI) from Arabidopsis thaliana, but only provided the synthetic 14,15-epoxy-3'-GGI, which has limitations on scalability.28

[0135] Accordingly, disclosed herein are a series of isolated genes from the fungus, Aspergillus fresenii, that mediate the production of 14,15-epoxy-3'-GGI, radarin C, radarin A, and the use of these genes to direct the heterologous and / or introduced endogenous expression of these compounds in an isolated host cell, preferably an isolated fungal cell. Also disclosed is an additional isolated gene, RosB, from Aspergillus fresenii, which when combined with one other gene, RadM, mediates the production of a new cyclic IDT derived from 14,15-epoxy-3'-GGI; roseyrin. Further disclosed herein are a series of isolated genes from the fungus, Aspergillus muricatus, that mediate the production of 14,15-epoxy-3'-GGI and petromindole. Using the alternate host Penicillium paxilli, the inventors have reconstituted the biosynthetic pathways for 14,15-epoxy-3'-GGI, radarin C, radarin A and roseyrin from A. fresenii, and for 14,15-epoxy-3'-GGI and petromindole from A. muricatus. A variety of multi-gene constructs were generated using the Modular Idempotent DNA Assembly System (MIDAS).29The MIDAS platform and method of using the MIDAS platform are described herein in US 11,655,476, the entirety of which is hereby incorporated by reference.

[0136] The inventors analysed the genomic sequence of, fresenii n identified a three-gene cluster (the RAD cluster) and a single unclustered gene comprising the following genomic and predicted coding sequences (CDSs): / aoWgDNA (SEQ ID NO: 1), / a< WcDNA(SEQ ID NO: 2), / ao^gDNA (SEQ ID NO:4), r t / FcDNA (SEQ ID NO: 5), r tZ gDNA (SEQ ID NO: 7), and / a XcDNA (SEQ ID NO: 8), rosB gDNA (SEQ ID NO: 10), and rosFcDNA (SEQ ID NO: 11) that are expected to encode enzymes necessary for the biosynthesis of 14,15-epoxy-3'-GGI, radarin C, radarin A or roseyrin (Figure 4A). The inventors also analysed the genomic sequence of, muricatusand identified two clustered genes comprising the following genomic and predicted CDSs: efA / gDNA (SEQ ID NO: 13), efA / cDNA (SEQ ID NO: 14), efFgDNA (SEQ ID NO: 16), efFcDNA (SEQ ID NO: 17) that are expected to encode enzymes necessary for the biosynthesis of 14,15-epoxy-3'-GGI and petromindole (Figure 4A).

[0137] The RAD cluster was identified by searching for the / . fresenii genome for regions that share homology with known IDT epoxidase and cyclase encoding genes from other fungi. Details of these predicted genes in the cluster and their proposed function are shown in Table 2. Two of the cluster genes share some homology to those found in other IDT or meroterpenoid biosynthetic gene clusters (Table 2). The protein product of the two predicted genes that share homology to IDT epoxidase and cyclase encoding genes from other fungi share less than 45% amino acid identity (Figure 5 and Figure 6). These are the FAD-dependent oxidase (RadM (SEQ ID NO: 3) and an IDT cyclase (RadB (SEQ ID NO: 6)). The other putative ORF was predicted to encode a cytochrome P450 oxygenase (RadX (SEQ ID NO: 9), the protein product is highly dissimilar to other known IDT cytochrome P450 oxygenases (Figure 7). The RAD cluster does not contain a specific gene encoding a secondary geranylgeranyl pyrophosphate synthase, nor a specific gene encoding an indole prenyltransferase, required to provide the 3'-GGI substrate, however these are found elsewhere in the genome. The inventors also identified an additional gene named / os£(SEQ ID NO: 10, SEQ ID NO: 11), that was predicted to encode an IDT cyclase (SEQ ID NO: 12), that shared high similarity with RadB (SEQ ID NO: 6) (Figure 6).

[0138] The two genes identified in / . muricatus (petM S Q ID NO: 13, SEQ ID NO: 14) efF(SEQ ID NO: 16, SEQ ID NO: 17) were identified by searching the genome for homologues of RadM (SEQ ID NO: 3) and RadB (SEQ ID NO: 6). The details of these predicted genes in the PET cluster (Figure 4A) and their proposed function are shown in Table 3. These are an FAD-dependent oxidase PetM (SEQ ID NO: 15) and an IDT cyclase PetB (SEQ ID NO: 18). To determine the roles of the RAD cluster encoded protein machinery, and directly establish their respective roles in radarin biosynthesis the inventors constructed a series of plasmids harbouring various combinations of these genes, which they then transformed into an appropriate P. paxilli host for heterologous production of the radarins. Accordingly, the A. fresenii genes of interest were ordered pre-cloned into a MIDAS Level-1 vector, pMLl (Table 4). At MIDAS Level-2, the cloned genes were placed under the control of heterologous promoter and transcriptional terminator modules to generate full-length transcription units (Table 5), which were then used to generate the multi-gene plasmids (Table 6). The inventors used P. x / / / / knockout strain devoid of its native / ’AYcluster (such that no IDTs are produced by this strain) to determine the roles of the RAD cluster genes and reconstruct the pathway to produce radarin A. Following transformation of P. paxi / ii hosts with multigene plasmids, the inventors determined the chemical phenotypes of the transformants initially reversed-phase liquid chromatography-mass spectrometry (LC-MS) analysis of fungal extracts. The inventors purified the expressed metabolites, as determined by liquid chromatography mass spectrometry (LC-MS), by normal phase silica gel chromatography and semi-preparative reversed-phase high-performance liquid chromatography (HPLC). The structures of the compounds were confirmed by Nuclear Magnetic Resonance (NMR) (1H,13C, and HSQC, HMBC, COSY and NOESY). An equivalent experimental process was used to determine the roles of PetM and PetB protein machinery from A. muricatusto establish their roles in petromindole biosynthesis. The same applies for determining the role of RosB protein machinery from A. fresenii o determine if there is a new indole diterpene compound it specifies for.

[0139] Using this methodology, and by expression alongside xCand xC(HM171111.1), the inventors identified that / aW(SEQ ID NO: 1 and SEQ ID NO: 2) encodes a FAD-dependent oxidases RadM (SEQ ID NO: 3) that mediates the regiospecific epoxidation of 3'-GGI, to provide 14,15-epoxy-3'-GGI, and its corresponding diol (14,15-dihydroxy-3'-GGI) anticipated from spontaneous hydrolysis of any singly epoxidized form of 3'-GGI (Figure 9). Up-scaled growth of this strain enabled isolation of 14,15-dihydroxy-3'-GGI which was confirmed to be the C14-C15 isomer using NMR spectroscopy (Figure 15 and Figure 16), demonstrating that RadM catalyzes singular epoxidation of the terminal olefin, providing the substrate required for radarin biosynthesis. Notably the inventors have identified the first epoxidase (RadM) in IDT biosynthesis to catalyze regioselective epoxidation of 3'-GGI at the C14-C15 position. The inventors also identified that rar#? (SEQ ID NO: 4 and SEQ ID NO: 5), encodes the IDT cyclase RadB (SEQ ID NO: 6) that catalyses the cyclisation of 14,15-epoxy-3'-GGI to form radarin C (Figure 11). Isolation and structural characterization by NMR confirmed the structure to be radarin C (Figure 17 and Figure 18). Importantly the inventors have uncovered the genetic basis for biosynthesis of radarin C, of which the cyclisation requires a unique and highly complex backbone rearrangement via formation of a ketone (Figure 8). The inventors further confirmed that radX (SEQ ID NO: 7 and SEQ ID NO: 8) encodes a cytochrome P450 monooxygenases RadX (SEQ ID NO: 9) that catalyzes hydroxylation of the indole ring of radarin C at C-7' to form radarin A (Figure 12, Figure 19 and Figure 20). Importantly the RadX sequence (SEQ ID NO: 9) shares little similarity to known IDT cytochrome P450 monooxygenases (Figure 7), and in IDT biosynthesis, the regiospecificity of this hydroxylation has not previously been observed.

[0140] The additional gene, / osF(SEQ ID NO: 10 and SEQ ID NO: 11), was expressed alongside / aW(SEQ ID NO:1), xCand xC(HM171111.1), to provide it with 14,15-epoxy-3'-GGI substrate and confirm that it encodes an IDT cyclase RosB (SEQ ID NO: 12) by detection of an alternate compound with [M + H]+m / z 406.3 (Figure 13). Up-scaled growth of this strain enabled isolation of a new compound, named roseyrin, and its chemical structure was confirmed using NMR spectroscopy (Figure 21), demonstrating that RosB catalyzes a cyclization of 14,15-epoxy-3'-GGI, that contains a trans-decalin scaffold not previously observed in the IDT class. The cyclisation by RosB (SEQ ID NO: 12) appears to be an early termination of radarin C cyclisation, by deprotonation of intermediate X to form its distinctive exocyclic alkene (Figure 8). Importantly, the discovery of roseyrin shows that there may be other cyclic variations of IDTs that derive from 14,15-epoxy-3'-GGI that remain to be discovered. To determine the roles of the PET cluster protein machinery from A. muricatus, the inventors again used the same methodology. By expressing efA7(SEQ ID NO: 13 and SEQ ID NO: 14) alongside paxG and xC(HM171111.1), the inventors show that petM encodes a FAD-dependent oxidase PetM (SEQ ID NO: 15) that mediates the regiospecific epoxidation of 3'-GGI, to provide 14,15-epoxy-3'-GGI, and its corresponding diol as the major product (14,15-dihydroxy-3'-GGI) confirming that PetM is a functional orthologue of RadM (Figure 10). The inventors also identified that efF(SEQ ID NO: 16 and SEQ ID NO: 17), encodes the IDT cyclase PetB (SEQ ID NO: 18) that catalyzes the cyclisation of 14,15-epoxy-3'-GGI to form petromindole (Figure 14). Partial isolation and NMR analysis confirmed production of petromindole (Figure 22). Importantly the inventors have uncovered the genetic basis for biosynthesis of petromindole, which is the only other known IDT structure derived from 14,15-epoxy-3'-GGI.

[0141] Using the efficient gene reassembly of MIDAS and heterologous expression in P. paxilliVne inventors have demonstrated that P. paxilli\s a suitable host for heterologous expression studies of radarins, roseyrin and petromindole, enabling the inventors to confirm the function of radM, radB, radX n rosB genes from A. fresenii, and efA / and efFfrom A. muricatus.

[0142] Polypeptides

[0143] Accordingly, in one aspect the invention relates to an isolated RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0144] In one embodiment the isolated RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprises at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0145] In one embodiment the isolated RadM polypeptide comprises, consists or consists essentially of the amino acid sequence of SEQ ID NO: 3. In one embodiment the isolated RadB polypeptide comprises, consists or consists essentially of SEQ ID NO: 6. In one embodiment the isolated RadX polypeptide comprises, consists or consists essentially of SEQ ID NO: 9. In one embodiment the isolated RosB polypeptide comprises, consists or consists essentially of the amino acid sequence of SEQ ID NO: 12. In one embodiment the isolated PetM polypeptide comprises, consists or consists essentially of SEQ ID NO: 15. In one embodiment the isolated PetB polypeptide comprises, consists or consists essentially of SEQ ID NO: 18.

[0146] In one embodiment the isolated RadM polypeptide or functional variant thereof has epoxidase activity, preferably terminal epoxidase activity. Preferably the epoxidase activity is epoxidation at the terminal C14-C15 olefin of 3 '-geranylgeranyl indole (3'-GGI) (Figure 4B).

[0147] In one embodiment the isolated RadB polypeptide or functional variant thereof has cyclase activity, preferably IDT cyclase activity. Preferably the cyclase activity is cyclisation of 14,15-epoxy-3'-GGI to form radarin C (Figure 4B).

[0148] In one embodiment the isolated RadX polypeptide or functional variant thereof has oxygenase activity, preferably cytochrome P450 oxygenase activity. Preferably the oxygenase activity is oxidation of C7' of radarin C to form radarin A (Figure 4B).

[0149] In one embodiment the isolated RosB polypeptide or functional variant thereof has cyclase activity, preferably IDT cyclase activity. Preferably the cyclase activity is cyclisation of 14,15-epoxy-3 '-GGI to form roseyrin (Figure 4B).

[0150] In one embodiment the isolated PetM polypeptide or functional variant thereof has epoxidase activity, preferably terminal epoxidase activity. Preferably the epoxidase activity is epoxidation at the terminal C14-C15 olefin of 3 '-geranylgeranyl indole (3'-GGI) (Figure 4B).

[0151] In one embodiment the isolated PetB polypeptide or functional variant thereof has cyclase activity, preferably IDT cyclase activity. Preferably the cyclase activity is cyclisation of 14,15-epoxy-3 '-GGI to form radarin C (Figure 4B).

[0152] In one embodiment the isolated polypeptide or functional variant thereof is a recombinant polypeptide or functional variant thereof.

[0153] Polynucleotides In another aspect the invention relates to an isolated polynucleotide or functional variant thereof encoding a RadB, RadM, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0154] In one embodiment the RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprises at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0155] In one embodiment the RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprise, consist of or consist essentially of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

[0156] Specifically contemplated as separate embodiments of this aspect of the invention are each of the isolated polynucleotides or functional variants thereof encoding a RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof as set forth above.

[0157] In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadM and a RadB polypeptide or a functional variant of either or both as defined in the first aspect of the invention.

[0158] In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadM and a RadX polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadB and a RadX polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadM, a RadB and a RadX polypeptide or functional variant of any one or all as defined in the first aspect of the invention.

[0159] In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadM and a RosB polypeptide or a functional variant of either or both as defined in the first aspect of the invention.

[0160] In one embodiment the isolated polynucleotide or functional variant thereof encodes a RadM and a PetB polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a PetM and a RadB polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a PetM and a PetB polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a PetM and a RosB polypeptide or a functional variant of either or both as defined in the first aspect of the invention.

[0161] In one embodiment the isolated polynucleotide or functional variant thereof encodes a PetM and a RadX polypeptide or functional variant of either or both as defined in the first aspect of the invention. In one embodiment the isolated polynucleotide or functional variant thereof encodes a PetM, a RadB and a RadX polypeptide or functional variant of any one or all as defined in the first aspect of the invention.

[0162] In another aspect the invention relates to an isolated radM, radB, radX, rosB, petM or petB genomic polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively.

[0163] In one embodiment the isolated radM, radB, radX, rosB, petM or petB genomic polynucleotide sequence or functional variant thereof comprises at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively. In one embodiment the isolated radM, radB, radX, rosB, petM or petB genomic polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively.

[0164] In one embodiment the isolated genomic polynucleotide sequence or functional variant thereof is comprised in an isolated polynucleotide that comprises, consists of, or consists essentially of at least two of i) - vi) wherein i) - vi) are defined as:

[0165] i) a radM genomic polynucleotide sequence or functional variant thereof as set forth above, ii) a radB genomic polynucleotide sequence or functional variant thereof as set forth above, iii) a radX genomic polynucleotide sequence or functional variant thereof as set forth above, iv) a rosB genomic polynucleotide sequence or functional variant thereof as set forth above, v) a petM genomic polynucleotide sequence or functional variant thereof as set forth above, and

[0166] vi) a petB genomic polynucleotide sequence or functional variant thereof as set forth above. In one embodiment the isolated cDNA polynucleotide sequence or functional variant thereof comprises i) and ii); i) and iii); ii) and iii); i), ii) and iii); i) and iv); i) and vi); v) and vi); v) and ii); v) and iv); v) and iii) and v), ii) and iii). Each of the combinations listed are also specifically contemplated as separate embodiments.

[0167] Specifically contemplated as separate embodiments of this aspect of the invention are each of the isolated radM, radB, radX, rosB, petMor petB genomic polynucleotide sequences or functional variants thereof as set forth above.

[0168] In another aspect the invention relates to an isolated radM, radB, radX, rosB, petMor efFcDNA polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 17 respectively.

[0169] In one embodiment the isolated radM, radB, radX, rosB, petMor efFcDNA polynucleotide sequence or functional variant thereof comprises at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 17 respectively. In one embodiment the isolated radM, radB, radX, rosB, petMor petB XMP polynucleotide sequence comprises, consists of or consists essentially of SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, or SEQ ID NO: 17 respectively.

[0170] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists of, or consists essentially of at least two of vii) - xii) wherein vii) - xii) are defined as:

[0171] vii) a radMcDNP polynucleotide sequence or functional variant thereof as set forth above, viii) a / ao^cDNA polynucleotide sequence or functional variant thereof as set forth above, ix) a radX cDNA polynucleotide sequence or functional variant thereof as set forth above, x) a rosFcDNA polynucleotide sequence or functional variant thereof as set forth above, xi) a efA / cDNA polynucleotide sequence or functional variant thereof as set forth above, and

[0172] xii) a efFcDNA polynucleotide sequence or functional variant thereof as set forth above. In one embodiment the isolated polynucleotide or functional variant thereof comprises vii) and viii); vii) and ix); viii) and ix); vii), viii) and ix); vii) and x); vii) and xii); xi) and xii); xii) and viii); xi) and x); xii) and ix); and xi), viii) and ix). Each of the combinations listed are also specifically contemplated as separate embodiments. Specifically contemplated as separate embodiments of this aspect of the invention are each of the isolated radM, radB, radX, rosB, petMor petB P polynucleotide sequences or functional variants thereof as set forth above.

[0173] In one embodiment i), v), vii) and xi) each encode a polypeptide having epoxidase activity, preferably terminal epoxidase activity. Preferably the epoxidase activity is epoxidation at the terminal C14-C15 olefin of 3' -geranylgeranyl indole (3'-GGI) (Figure 4B).

[0174] In one embodiment ii), iv), vi), viii), x) and xii) each encode a polypeptide having cyclase activity, preferably IDT cyclase activity. Preferably the cyclase activity is cyclisation of 14,15-epoxy-3'-GGI to form radarin C (Figure 4B).

[0175] In one embodiment iii) and ix) each encode a polypeptide having oxygenase activity, preferably cytochrome P450 oxygenase activity.

[0176] In one embodiment, an isolated polynucleotide of any of the embodiments set forth above is a recombinant polynucleotide. In one embodiment an isolated polynucleotide of any of the embodiments set forth above comprises at least one heterologous regulatory sequence, preferably a heterologous promoter, enhancer or terminator or any combination thereof.

[0177] Nucleic acid molecules contemplated herein may be isolated. They can be isolated from a biological sample, chemically synthesized, or produced using synthetic biology, employing a variety of techniques known in the art. By way of example, such polynucleotides can be isolated through use of the polymerase chain reaction (PCR), amplified using primers, such as defined herein, and / or would be derived from the polynucleotide sequences contemplated herein.

[0178] Further methods for isolating polynucleotides include use of all, or portions of, a polynucleotide as described herein as hybridization probes. For technique of hybridizing labeled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen genomic or cDNA libraries. Similarly, probes may be coupled to beads and hybridized to the target sequence. Isolation can be carried out using known art protocols such as magnetic separation. The choice of appropriately stringent hybridization and wash conditions is believed to be within the skill of those in the art.

[0179] Polynucleotide fragments may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.

[0180] A partial polynucleotide sequence may be used as a probe, in methods well-known in the art to identify the corresponding full length polynucleotide sequence in a sample. Such methods include PCR-based methods, 5'RACE and hybridization-based method, computer / database-based methods as known in the art. Detectable labels such as radioisotopes, fluorescent, chemiluminescent and bioluminescent labels may be used to facilitate detection. Inverse PCR also permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region as known and used in the art. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized as known in the art. Primers and primer pairs which allow amplification of polynucleotides of the invention, also form a further aspect of this invention.

[0181] Variants (including orthologues) may be identified by the methods described. Variant polynucleotides may be identified using PCR-based methods as known in the art. Typically, the polynucleotide sequence of a primer, useful to amplify variants of polynucleotide molecules by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

[0182] Further methods for identifying variant polynucleotides include use of all, or portions of the specified polynucleotides as hybridization probes to screen genomic or cDNA libraries as described above. Typically probes based on a sequence encoding a conserved region of the corresponding amino acid sequence may be used. Hybridization conditions may also be less stringent than those used when screening for sequences identical to the probe.

[0183] In another aspect the invention relates to a nucleic acid construct comprising an isolated polynucleotide or functional variant thereof as described herein.

[0184] In another aspect the invention relates to a TU comprising an isolated polynucleotide or functional variant thereof as described herein.

[0185] In one embodiment the nucleic acid construct or TU is comprised in vector, preferably an expression vector. In one embodiment the vector is selected from the group consisting of plasmids, BACs, (PACs), YACs, bacteriophage, phagemids, and cosmids. Preferably the vector is a plasmid.

[0186] In another aspect the invention relates to a vector that encodes an isolated polypeptide or functional variant thereof as described herein.

[0187] In another aspect the invention relates to a vector comprising an isolated polynucleotide or functional fragment thereof, nucleic acid construct or TU as described herein.

[0188] In one embodiment the isolated polynucleotide or nucleic acid sequence is comprised in a nucleic acid construct or a TU.

[0189] In one embodiment the vector is selected from the group consisting of plasmids, BACs, PACs, YACs, bacteriophage, phagemids, and cosmids. Preferably the vector is a plasmid. In one embodiment the vector is an expression vector. Nucleic acid sequences, polynucleotides, nucleic acid constructs and TUs as described herein can be incorporated into any suitable vector capable of expressing that polynucleotide or, where applicable, an encoded polypeptide as described herein in vitroor in a host cell. Preferably the vector is an expression vector. Examples of suitable expression vectors include, but not limited to, plasmid DNA vectors, viral DNA vectors (such as adenovirus and adeno-associated virus), or viral RNA vectors (such as a retroviral vectors). In some embodiments the plasmid and / or phage vectors may be selected from the following vectors or variants thereof including pUC18, pU19, Mpl8, Mpl9, ColEl, PCR1 and pKRC; lambda gtlO and M13 plasmids such as pBR322, pACYC184, pT127, RP4, plJlOl, SV40 and BPV. Also included are vectors such as, but not limited to, cosmids, YACS, BACs shuttle vectors such as pSA3, PAT28 transposons (such as described in US 5,792,294) and the like.

[0190] Suitable viral vectors include but are not limited to vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. Viral vectors employed herein can be appropriately modified by pseudotyping with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as known and used in the art.

[0191] In one embodiment the expression vector comprises an, preferably at least two, preferably at least three isolated polynucleotides as described herein.

[0192] In one embodiment the expression vector comprises an, preferably at least two, preferably at least three TUs as described herein.

[0193] In one embodiment the vector is a component in a cloning system. In one embodiment the cloning system is useful for making a gene construct comprising a TU, preferably at least two, preferably at least three TUs.

[0194] In one embodiment the vector is comprised in a vector set, the vector set being part of a cloning system. In one embodiment the cloning system is useful for making a gene construct comprising a TU, preferably at least two, preferably at least three TUs.

[0195] In one embodiment the cloning system is useful for making a gene construct comprising a TU, preferably at least two, preferably at least three TUs.

[0196] In one embodiment the gene construct is a multigene construct comprising at least two, preferably at least three TUs.

[0197] The nucleic acid constructs and TUs described herein may comprise one or more of the isolated polynucleotide or nucleic acid sequences described herein and / or one or more of the isolated polynucleotides or nucleic acid sequences encoding the isolated polypeptides described herein. In one embodiment a TU, nucleic acid construct or vector is constructed to drive expression of at least one of, preferably at least two of, at least three of, preferably all four of 14,15-epoxy-3'-GGI, radarin C, roseyrin, petromindole and radarin A, either in vitro or in vivo. In one embodiment, the TU, nucleic acid construct or vector comprises a polynucleotide as described herein operatively linked to 5' or 3' untranslated regulatory sequences. The design of a particular TU, nucleic acid construct or vector will depend on various factors including the host cells in which the operatively linked polynucleotide is to be expressed and the desired level of polynucleotide expression.

[0198] Likewise, the selection of various promoters, enhancers and / or other genetic elements for a TU, nucleic acid construct or vector will depend on various factors including the host cells and expression levels discussed above. In one embodiment, the TU, nucleic acid construct or vector comprises a homologous promoter operatively linked to a polynucleotide as described herein. In another embodiment, the TU, nucleic acid construct or vector comprises a heterologous promoter operatively linked to a polynucleotide of the invention. In one embodiment, the homologous or heterologous promoter is an inducible, repressible or regulatable promoter. A suitable promoter may be chosen and used under the appropriate conditions to direct high-level expression of a polynucleotide of the invention. Many such elements are described in the literature and are available through commercial suppliers.

[0199] By way of example only, a promoter useful in the TUs, nucleic acid constructs or vectors described herein can be any suitable eukaryotic or prokaryotic promoter. In one embodiment, the eukaryotic promoter can be a eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Expression levels of an operably linked polynucleotide in a particular cell type will be determined by the nearby presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers, and the like). Any suitable promoter / enhancer combination (see: Eukaryotic Promoter Data Base EPDB) can be used to drive expression of a polynucleotide as described herein. Additional promoters useful in a TU, nucleic acid construct or vector as described herein include 0-lactamase, alkaline phosphatase, tryptophan, and tac promoter systems which are all well known in the art. Yeast promoters include 3-phosphoglycerate kinase, enolase, hexokinase, pyruvate decarboxylase, glucokinase, and glyceraldehydrate-3-phosphanate dehydrogenase but are not limited thereto.

[0200] Prokaryotic promoters useful in in a TU, nucleic acid construct or vector as described herein include constitutive promoters as known in the art (such as the int promoter of bacteriophage lamda and the bla promoter of the beta-lactamase gene sequence of pBR322) and regulatable promoters (such as lacZ, recA and gal). A ribosome binding site upstream of the CDS may also be required for expression.

[0201] Enhancers useful in in a TU, nucleic acid construct or vector as described herein include SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin and the like. In one embodiment, in a TU, nucleic acid construct or vector as described herein may be driven by a T3, T7 or SP6 cytoplasmic expression system.

[0202] The choice of a particular promoter / enhancer / cell type combination for protein expression is within the ordinary skill of those in the art of molecular biology (see, for example, Sambrook et al. (1989) which is incorporated herein by reference).

[0203] In another aspect the invention relates to an isolated host cell comprising an isolated polypeptide or functional variant thereof, isolated polynucleotide or functional variant thereof, TU, nucleic acid construct or vector as described herein.

[0204] In one embodiment the isolated host cell is a prokaryotic or eukaryotic cell. Prokaryotes most commonly employed as host cells are strains of Escherichia coii (E. coii). Other prokaryotic hosts include Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria but are not limited thereto.

[0205] In one embodiment the eukaryotic cell is an animal cell, a plant cell, a fungal cell or a protist cell. In one embodiment the animal cell is an insect cell or a mammalian cell. In one embodiment the fungal cell is a single cell of a unicellular fungal host strain. In one embodiment the fungal cell comprises fungal hyphae or the mycelia of a fungal host strain.

[0206] In one embodiment the fungal cell, hyphae or mycelia of the fungal host strain is from a species of fungi in one of the following genera: Aspergillus, Trichoderma, Neurospora, Fusarium, Mortierella, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichoplusia, Ashbya, Hansenula, Pichia, Kluveromyces, Schizzosaccharomyces, Monascus, Talaromyces, Cryptonectria, Endothia, Tolypocladium, Hypocrea, Gibberella, Acremonium, Agaricus, Pleurotus, PeniciHium, Volvariella, Flammulina, Lentinula, Auricularia, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably PeniciHium, Aspergillus, Saccharomyces, Pichia, Tricopiusia, and Spondoptera. Preferably the fungal cell is from Saccharomyces. Preferably the fungal hyphae or mycelia is from Aspergillus, preferably A. fresenii. Preferably the fungal hyphae or mycelia is from PeniciHium, preferably P. paxiHi.

[0207] In another aspect the invention relates to a method of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof comprising expressing an isolated polypeptide or functional variant thereof, isolated polynucleotide or functional variant thereof, TU, nucleic acid construct or vector as described herein in an isolated host cell.

[0208] In some embodiments, the method comprises making 14,15-epoxy-3'-GGI and radarin C comprising expressing at least two isolated polypeptides, isolated polynucleotides, TUs, or nucleic acid constructs as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadM and RadB or PetM and RadB. In one embodiment the at least two polynucleotides include radM and radB or petMand radB. In some embodiments, the method comprises making 14,15-epoxy-3'-GGI and radarin A comprising expressing at least two isolated polypeptides, isolated polynucleotides, TUs, or nucleic acid constructs as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadM, RadB and RadX or PetM, RadB and RadX. In one embodiment the at least two polynucleotides include radM, radBax\d radX x petM, radBax\d radX.

[0209] In some embodiments, the method comprises making radarin C and radarin A comprising expressing at least two isolated polypeptides, isolated polynucleotides, TUs, or nucleic acid constructs as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadB and RadX. In one embodiment the at least two polynucleotides include r r / Fand radX.

[0210] In some embodiments, the method comprises making 14,15-epoxy-3'-GGI, radarin C and radarin A comprising expressing an isolated polypeptide, isolated polynucleotide, TU, nucleic acid construct or vector as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadM, RadB and RadX or PetM, RadB and RadX. In one embodiment the at least two polynucleotides include radM, radBax\d radX x petM, radB and radX.

[0211] In some embodiments, the method comprises making 14,15-epoxy-3'-GGI and roseyrin comprising expressing an isolated polypeptide, isolated polynucleotide, TU, nucleic acid construct or vector as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadM and RosB or PetM and RosB. In one embodiment the at least two polynucleotides include radM and rosB ox petM and rosB.

[0212] In some embodiments, the method comprises making 14,15-epoxy-3'-GGI and petromindole comprising expressing an isolated polypeptide, isolated polynucleotide, TU, nucleic acid construct or vector as described herein in an isolated host cell. In one embodiment the at least two polypeptides include RadM and PetB or PetM and PetB. In one embodiment the at least two polynucleotides include radM and petB ox petM and petB.

[0213] In one embodiment the at least two isolated polypeptides, isolated polynucleotides, TUs, or nucleic acid constructs are expressed from a single vector. In one embodiment the at least two isolated polypeptides, isolated polynucleotides, TUs, or nucleic acid constructs are expressed from at least two vectors.

[0214] In one embodiment 14,15-epoxy-3'-GGI, radarin C, roseyrin, petromindole and radarin A are as depicted in Figure 4B.

[0215] In one embodiment expressing is heterologous expression or introduced endogenous expression. In one embodiment, heterologous expression or introduced endogenous expression comprises expression of an isolated polypeptide as described herein from an isolated polynucleotide, nucleic acid construct, TU or vector as described herein, in an isolated fungal host cell or in the hyphae or mycelia of an isolated fungal strain as described herein. In one embodiment the hyphae or mycelia are from Aspergillus, preferably, freseniior Penicillium, preferably P. paxilli.

[0216] In one embodiment the nucleic acid construct or TU is comprised in a multigene construct comprising at least two, preferably at least three isolated polynucleotides encoding at least two, preferably at least three isolated polypeptides as described herein. Based on the description provided herein the skilled person can determine which isolated polynucleotides to include in the multigene construct to make any one, combination of, or all of 14,15-epoxy-3'-GGI, radarin C, roseyrin, petromindole and radarin A.

[0217] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and isolated host cells. In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI comprising expressing a) in an isolated recombinant host cell, wherein

[0218] a) is a RadM or PetM polypeptide or functional variant thereof as described herein.

[0219] In one embodiment a) catalyzes the conversion of 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI.

[0220] In one embodiment the isolated recombinant host cell produces 3'-GGI endogenously. In one embodiment the isolated recombinant host cell produces 3' -GGI heterologously.

[0221] In one embodiment the method further comprises converting 14,15-epoxy-3'-GGI to radarin C comprising expressing b) in the isolated host cell, wherein

[0222] b) is a RadB polypeptide or functional variant thereof as described herein.

[0223] In one embodiment b) catalyzes the conversion of 14,15-epoxy-3'-GGI to radarin C.

[0224] In one embodiment the method further comprises converting 14,15-epoxy-3'-GGI to roseyrin comprising expressing c) in the isolated host cell, wherein

[0225] c) is a RosB polypeptide or functional variant thereof as described herein.

[0226] In one embodiment c) catalyzes the conversion of 14,15-epoxy-3'-GGI to roseyrin.

[0227] In one embodiment the method further comprises converting 14,15-epoxy-3'-GGI to petromindole comprising heterologously expressing d) in the isolated host cell, wherein d) is a PetB polypeptide or functional variant thereof as described herein.

[0228] In one embodiment d) catalyzes the conversion of 14,15-epoxy-3'-GGI to petromindole.

[0229] In one embodiment the method further comprises converting radarin C to radarin A comprising heterologously expressing e) in the isolated host cell, wherein

[0230] e) is a RadX polypeptide or functional variant thereof as described herein.

[0231] In one embodiment e) catalyzes the conversion of radarin C to radarin A.

[0232] In one embodiment expressing comprises heterologous expression of a) - d) or e) or any combination thereof. In one embodiment expressing is introduced homologous expression of a) - d) or e) or any combination thereof.

[0233] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and host cells.

[0234] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to radarin C comprising expressing a) and b) in a recombinant isolated host cell, wherein

[0235] a) is a RadM or PetM polypeptide or functional variant thereof as described herein, and b) is a RadB polypeptide or functional variant thereof as described herein.

[0236] In one embodiment the isolated host cell produces 3'-GGI endogenously. In one embodiment the isolated host cell produces 3'-GGI heterologously.

[0237] In one embodiment a) catalyzes the conversion of 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI.

[0238] In one embodiment b) catalyzes the conversion of 14,15-epoxy-3'-GGI to radarin C.

[0239] In one embodiment expressing comprises heterologous expression of a) or b) or both. In one embodiment expressing comprises introduced endogenous expression of a) or b) or both.

[0240] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and host cells. In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to roseyrin comprising expressing a) and c) in an isolated recombinant host cell, wherein

[0241] a) is a RadM or PetM polypeptide or functional variant as described herein, and

[0242] c) is a RosB polypeptide or functional variant thereof as described herein.

[0243] In one embodiment the isolated host cell produces 3'-GGI endogenously. In one embodiment the isolated host cell produces 3'-GGI heterologously.

[0244] In one embodiment a) catalyzes the conversion of 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI.

[0245] In one embodiment c) catalyzes the conversion of 14,15-epoxy-3'-GGI to roseyrin.

[0246] In one embodiment expressing comprises heterologous expression of a) or c) or both. In one embodiment expressing comprises introduced endogenous expression of a) or c) or both.

[0247] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and host cells.

[0248] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to petromindole comprising heterologously expressing a) and d) in an isolated host cell, wherein

[0249] c) is a RadM or PetM polypeptide or functional variant thereof as described herein, and d) is a PetB polypeptide or functional variant thereof as described herein.

[0250] In one embodiment the isolated host cell produces 3'-GGI endogenously. In one embodiment the isolated host cell is a recombinant host cell that produces 3'-GGI heterologously.

[0251] In one embodiment a) catalyzes the conversion of 3'-geranylgeranylindole (3'-GGI) to 14,15-epoxy-3'-GGI.

[0252] In one embodiment d) catalyzes the conversion of 14,15-epoxy-3'-GGI to petromindole.

[0253] In one embodiment expressing comprises heterologous expression of a) or d) or both. In one embodiment expressing comprises introduced endogenous expression of a) or d) or both.

[0254] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and host cells.

[0255] In another aspect the invention relates to a method of converting 3'-geranylgeranylindole (3'-GGI) to radarin A comprising expressing a), b) and e) in an isolated recombinant host cell, wherein

[0256] a) is a RadM or PetM polypeptide or functional variant thereof as described herein, b) is a RadB polypeptide or functional variant thereof as described herein, and

[0257] e) is a RadX polypeptide or functional variant thereof as described herein.

[0258] In one embodiment expression is heterologous expression of a), b) or e) or any combination thereof. In one embodiment expression is introduced endogenous expression of a), b) or e), or any combination thereof.

[0259] Specifically contemplated as embodiments are all combinations of a), b) and e) wherein expression of any of a), b) and / or e) may be heterologous or introduced endogenous expression or any combination of both. In one example non-limiting example, expression of a) is heterologous expression and expression of b) and e) is introduced endogenous expression. In another non-limiting example, expression of a) and b) is heterologous expression and expression of e) is introduced endogenous expression.

[0260] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and host cells.

[0261] In another aspect the invention relates to 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof, made by a method as described herein.

[0262] In another aspect the invention relates to a compound of Formula VI,

[0263]

[0264] The compound of Formula VI is named herein as "roseyrin".

[0265] In another aspect the invention relates to a method of making an Aspergillus s. polypeptide or functional variant thereof comprising expressing an isolated polynucleotide or functional variant thereof, nucleic acid construct, TU or vector as described herein in an isolated host cell. In one embodiment the Aspergillus s - is A. fresenii. In this embodiment the polypeptide or functional variant thereof is a RadM, RadB, RadX or RosB polypeptide or functional variant thereof as described herein. In this embodiment, the isolated polynucleotide or functional variant thereof is a radM, radB, radX or rosB polynucleotide or functional variant thereof as described herein.

[0266] In one embodiment the Aspergillus s. is A. muricatus. In this embodiment the isolated polypeptide or functional variant thereof is a PetM or PetB polypeptide or functional variant thereof. In this embodiment the isolated polynucleotide or functional variant thereof is a petM or petB polynucleotide or functional variant thereof.

[0267] In one embodiment expressing is heterologous expression. In one embodiment expressing is introduced homologous expression.

[0268] In one embodiment the isolated host cell is a recombinant host cell. In one embodiment the isolated host cell comprises fungal hyphae or mycelia of an Aspergillus s. or Peniciffiums. host cell, preferably A. fresenii, preferably A. muricatus, preferably P. paxiiii.

[0269] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and methods of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof. In another aspect the invention relates to an isolated host cell that expresses a polypeptide or functional variant thereof that catalyzes the transformation of 3'-GGI to 14,15-epoxy-3'-GGI.

[0270] In one embodiment the polypeptide or functional variant thereof is an epoxidase, preferably a terminal epoxidase. Preferably, the epoxidase catalyzes the epoxidation of the terminal C14-C15 olefin of 3'- GGI (Figure 4B). Preferably the epoxidase is a RadM or PetM polypeptide as described herein. Preferably the RadM or PetM polypeptide is expressed respectively from an isolated radM <yc petM polynucleotide or functional variant thereof as described herein.

[0271] In one embodiment the isolated host cell also expresses a polypeptide or functional variant thereof that catalyzes a transformation of 14,15-epoxy-3'-GGI to radarin C.

[0272] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an indole diterpene (IDT) cyclase. Preferably the cyclase catalyzes the cyclisation of 14,15-epoxy-3'-GGI to form radarin C (Figure 4B). Preferably the cyclase is a RadB polypeptide as described herein.

[0273] Preferably the RadB polypeptide is expressed from an isolated radB polynucleotide or functional variant thereof as described herein. In one embodiment the isolated host cell also expresses a polypeptide or functional variant thereof that catalyzes a transformation of 14,15-epoxy-3'-GGI to roseyrin.

[0274] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an IDT cyclase. Preferably the cyclase catalyzes the cyclisation of 14,15-epoxy-3'-GGI to form roseyrin (Fig.

[0275] 1). As noted previously herein roseyrin is a compound of Formula VI,

[0276]

[0277] In one embodiment the cyclase is a RosB polypeptide or functional variant thereof as described herein. Preferably the RosB polypeptide is expressed from an isolated rosB polynucleotide or functional variant thereof as described herein.

[0278] In one embodiment the isolated host cell further expresses a polypeptide or functional variant thereof that catalyzes the transformation of 14,15-epoxy-3'-GGI to petromindole.

[0279] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an IDT cyclase. Preferably the cyclase catalyzes the cyclisation of 14,15-epoxy-3'-GGI to form petromindole (Fig. 1). In one embodiment the cyclase is a PetB polypeptide or functional variant thereof as described herein. Preferably the PetB polypeptide is expressed from an isolated petB polynucleotide or functional variant thereof as described herein.

[0280] In one embodiment the isolated host cell also expresses a polypeptide or functional variant thereof that catalyzes the transformation of radarin C to radarin A.

[0281] In one embodiment the isolated polypeptide or functional variant thereof is an oxygenase, preferably a cytochrome P450 oxygenase. Preferably the oxygenase is a RadX polypeptide or functional variant thereof as described herein. Preferably the RadX polypeptide is expressed from an isolated radX polynucleotide or functional variant thereof as described herein.

[0282] In one embodiment expression is heterologous expression. In one embodiment expression is introduced endogenous expression.

[0283] In one embodiment the isolated host cell is a prokaryotic or eukaryotic cell. Prokaryotes most commonly employed as host cells are strains of Escherichia coii (E. coii). Other prokaryotic hosts include Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria but are not limited thereto. In one embodiment the eukaryotic cell is an animal cell, a plant cell, a fungal cell or a protist cell. In one embodiment the animal cell is an insect cell or a mammalian cell. In one embodiment the fungal cell is a single cell of a unicellular fungal host strain. In one embodiment the fungal cell comprises fungal hyphae or the mycelia of a fungal host strain.

[0284] In one embodiment the fungal cell, hyphae or mycelia of the fungal host strain is from a species of fungi in one of the following genera: Aspergillus, Trichoderma, Neurospora, Fusarium, Mortiereiia, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichopiusia, Ashbya, Hansenuia, Pichia, Kiuveromyces, Schizzosaccharomyces, Monascus, Taiaromyces, Cryptonectria, Endothia, Toiypociadium, Hypocrea, Gibbereiia, Acremonium, Agaricus, Pieurotus, Peniciiiium, Voivarieiia, Fiammuiina, Lentinuia, Auricuiaria, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably Peniciiiium, Aspergillus, Saccharomyces, Pichia, Tricopiusia, and Spondoptera. Preferably the fungal cell is from Saccharomyces. Preferably the fungal hyphae or mycelia is from Aspergillus, preferably A. fresenii. Preferably the fungal hyphae or mycelia is from Peniciiiium, preferably P. paxilli.

[0285] In one embodiment the isolated host cell is a recombinant host cell.

[0286] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and methods of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof. In another aspect the invention relates to an isolated host cell that expresses at least three polypeptides or functional variants thereof that together, mediate the transformation of 3 '-GGI to radarin A.

[0287] In one embodiment at least one of the polypeptides or functional variants thereof is an epoxidase, preferably a terminal epoxidase. Preferably, the epoxidase catalyzes the epoxidation of the terminal C14-C15 olefin of 3'-GGI (Figure 4B). Preferably the epoxidase is a RadM or PetM polypeptide as described herein. Preferably the RadM or PetM polypeptide is expressed respectively from an isolated radMor petM polynucleotide or functional variant thereof as described herein.

[0288] In one embodiment at least one of the polypeptides or functional variants thereof is a cyclase, preferably an indole diterpene (IDT) cyclase. Preferably the cyclase catalyzes the cyclisation of 14,15-epoxy-3'-GGI to form radarin C (Figure 4B). Preferably the cyclase is a RadB polypeptide as described herein. Preferably the RadB polypeptide is expressed from an isolated radB polynucleotide or functional variant thereof as described herein.

[0289] In one embodiment at least one of the isolated polypeptides or functional variants thereof is an oxygenase, preferably a cytochrome P450 oxygenase. Preferably the oxygenase is a RadX polypeptide or functional variant thereof as described herein. Preferably the RadX polypeptide is expressed from an isolated radX polynucleotide or functional variant thereof as described herein. In one embodiment the isolated host cell expresses RadM, RadB and RadX polypeptides or functional variants thereof as described herein. In one embodiment the RadM, RadB and RadX polypeptides or functional variants thereof are expressed from radM, radB n radX polynucleotides or functional variants thereof as described herein.

[0290] In one embodiment the isolated host cell expresses PetM, RadB and RadX polypeptides or functional variants thereof as described herein. In one embodiment the RadM, RadB and RadX polypeptides or functional variants thereof are expressed from petM, radBanA radX polynucleotides or functional variants thereof as described herein.

[0291] In one embodiment expression is heterologous expression. In one embodiment expression is introduced endogenous expression. In some embodiments, expression is a combination of heterologous expression and introduced homologous expression.

[0292] In one embodiment the isolated host cell is a prokaryotic or eukaryotic cell. Prokaryotes most commonly employed as host cells are strains of Escherichia coii (E. coii). Other prokaryotic hosts include Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria but are not limited thereto.

[0293] In one embodiment the eukaryotic cell is an animal cell, a plant cell, a fungal cell or a protist cell. In one embodiment the animal cell is an insect cell or a mammalian cell. In one embodiment the fungal cell is a single cell of a unicellular fungal host strain. In one embodiment the fungal cell comprises fungal hyphae or the mycelia of a fungal host strain.

[0294] In one embodiment the fungal cell, hyphae or mycelia of the fungal host strain is from a species of fungi in one of the following genera: Aspergillus, Trichoderma, Neurospora, Fusarium, Mortierella, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichoplusia, Ashbya, Hansenula, Pichia, Kluveromyces, Schizzosaccharomyces, Monascus, Talaromyces, Cryptonectria, Endothia, Tolypocladium, Hypocrea, Gibberella, Acremonium, Agaricus, Pleurotus, PeniciHium, Volvariella, Flammulina, Lentinula, Auricularia, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably PeniciHium, Aspergillus, Saccharomyces, Pichia, Tricopiusia, and Spondoptera. Preferably the fungal cell is from Saccharomyces. Preferably the fungal hyphae or mycelia is from Aspergillus, preferably A. fresenii. Preferably the fungal hyphae or mycelia is from PeniciHium, preferably P. paxiHi.

[0295] In one embodiment the isolated host cell is a recombinant host cell.

[0296] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements) and methods of making 14,15-epoxy-3'-GGI, radarin C, and radarin A or any combination thereof.

[0297] In another aspect the invention relates to an isolated strain of Penicillium paxilli, Aspergillus niger, Aspergillus oryzae, Saccharomyces cerevisiae or Escherichia colit at. comprises at least one heterologous polynucleotide or functional variant thereof that encodes an enzyme in the biosynthetic pathway leading from 3'-GGI to radarin A, roseyrin or petromindole.

[0298] In one embodiment the at least one heterologous polynucleotide or functional variant thereof is a radM, radB, radX, rosB, petM or petB polynucleotide or a functional variant thereof as described herein.

[0299] In one embodiment the isolated strain comprises at least two heterologous polynucleotides or functional variants thereof as described herein that are radM an radB polynucleotides, radM and rosB polynucleotides, radM and petB polynucleotides, efA / and rosB polynucleotides, efA / and radB polynucleotides, efA / and petB polynucleotides, / ao^and radX polynucleotides, radM and radX polynucleotides, or petM and radX polynucleotides.

[0300] In one embodiment the isolated strain comprises at least three heterologous polynucleotides or functional variants thereof that are radM, radB and radX polynucleotides as described herein.

[0301] In one embodiment the at least one heterologous polynucleotide is an isolated polynucleotide. In one embodiment the at least one heterologous polynucleotide is expressed in the isolated strain.

[0302] In one embodiment the enzyme is a RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof as described herein that is encoded by the radM, radB, radX, rosB, petM or petB polynucleotide or a functional variant thereof respectively.

[0303] In one embodiment the isolated strain is an isolated host cell as described herein.

[0304] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements), host cells, and methods of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof.

[0305] In another aspect the invention relates to an isolated strain of Aspergillus s p. or Penicillium spp. that comprises at least one genetic modification that leads to an increased biosynthesis of radarin C, roseyrin, petromindole or radarin A. In one embodiment the at least one genetic modification is transformation of the isolated strain with at least one heterologous or introduced endogenous polynucleotide or functional variant thereof as described herein. In some embodiments transformation comprises transformation with at least two, preferably at least three heterologous or introduced endogenous polynucleotides or functional variants thereof as described herein.

[0306] In one embodiment the at least one genetic modification leads to increased biosynthesis of radarin C. In this embodiment the at least one genetic modification is transformation with a radM, petMor radB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a radM an a radB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a efA / and a radB polynucleotide or functional variant thereof as described herein.

[0307] In one embodiment any or all of the radM, petMor radB polynucleotides or functional variants thereof are heterologous polynucleotides or introduced endogenous polynucleotides.

[0308] In one embodiment the at least one genetic modification leads to increased biosynthesis of roseyrin. In this embodiment the at least one genetic modification is transformation with a radM, petMor rosB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a radM and a rosB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a efA / and a rosB polynucleotide or functional variant thereof as described herein.

[0309] In one embodiment any or all of the radM, petMor rosB polynucleotides or functional variants thereof are heterologous polynucleotides or introduced endogenous polynucleotides.

[0310] In one embodiment the at least one genetic modification leads to increased biosynthesis of petromindole.

[0311] In this embodiment the at least one genetic modification is transformation with a radM, petMor petB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a radM and a petB polynucleotide or functional variant thereof as described herein. In one embodiment transformation is with a efA / and a petB polynucleotide or functional variant thereof as described herein.

[0312] In one embodiment any or all of the radM, petMor petB polynucleotides or functional variants thereof are heterologous polynucleotides or introduced endogenous polynucleotides.

[0313] In one embodiment the at least one genetic modification leads to increased biosynthesis of radarin A. In this embodiment the at least one genetic modification is transformation with a radM, petM, radB ox radX polynucleotide or functional variant thereof as described herein. In various embodiments transformation is with radM and radB ox radMax\6 radX ox radBax\6 radX ox radM, radB and radX polynucleotides or functional variants thereof as described herein.

[0314] In one embodiment any or all of the radM, radB and radX polynucleotides or functional variants thereof are heterologous polynucleotides or functional variants thereof or introduced endogenous polynucleotides or functional variants thereof.

[0315] In one embodiment the heterologous polynucleotides or introduced endogenous polynucleotides are isolated. In one embodiment heterologous polynucleotides or functional variants thereof set out in this aspect of the invention are expressed in the isolated strain.

[0316] In one embodiment the isolated strain is an isolated host cell as described herein.

[0317] In another aspect the invention relates to a method of making radarin C, radarin A, roseyrin or petromindole comprising expressing a heterologous nucleic acid sequence in Penicillium spp. or Aspergillus spp. wherein a heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway leading from 3'-GGI to radarin C, radarin A, roseyrin or petromindole.

[0318] Specifically contemplated as embodiments of this aspect of the invention are the embodiments set out in the previous aspects of the invention that relate to isolated polypeptides and polynucleotides (including identified amino acid and nucleic acid sequences and % sequence identities), TUs, nucleic acid constructs, vectors (including choice of appropriate regulatory elements), host cells, enzymes and methods of making 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin or petromindole or any combination thereof.

[0319] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents; or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0320] The invention will now be illustrated in a non-limiting way by reference to the following examples. EXAMPLES

[0321] Example 1 - Heterologous biosynthesis of 14,15-epoxy-3'-GGI

[0322] To determine the function of radMax\6 petM, plasmids were constructed containing xCand paxC for making the 3'-GGI precursor, and either / a< W(pRM297) or efA7(pRM318). These plasmids were each transformed (by random integration in the genome) into a P. paxilli strain (CY2) that lacks the native paxilline IDT gene cluster ( / H cIuster). Ten transformants were selected by positive selection with geneticin (plasmids contain the t / gene) and processed as described in materials and methods.

[0323] The ten strains containing / 3< YA7(SEQ ID NO: 1) were grown in liquid cultures, extracted, and analysed by LC-MS. All ten strains showed detection of 14,15-epoxy-3'-GGI (m / z 406.3 [M + H]+) and the corresponding diol (14,15-dihydroxy-3'-GGI) as the major product (m / z 424.3 [M + H]+), based on extracted ion chromatograms as shown in Figure 9. The equivalent was carried out for the ten strains containing efA7(SEQ ID NO: 13), and the extracted ion chromatograms for m / z 406.3 [M + H]+and / 77 / Z424.3 [M + H]+also confirmed detection of 14,15-epoxy-3'-GGI and 14,15-dihydroxy-3'-GGI (Figure 10). Up-scaled growth of a radM containing strain (RM297-2), followed by chromatography enabled isolation of 14,15-dihydroxy-3'-GGI, where NMR spectroscopy demonstrated the hydroxyl groups are at the C14 and C15 position (Figure 15 and Figure 16).

[0324] These experiments confirm that RadM (SEQ ID NO: 3) and PetM (SEQ ID NO: 15) are FAD-dependent monooxygenases that catalyze a singular regiospecific epoxidation at the terminal C14-C15 olefin of 3'-GGI, defining the biosynthetic machinery that is central to accessing all IDTs derived from 14,15-epoxy-3'-GGI substrate.

[0325] Example 2 - Heterologous biosynthesis of radarin C

[0326] To determine the function of radB, a plasmid was constructed containing paxG, paxC, radM an radB (pRM298). This plasmid was transformed (by random integration in the genome) into a P. paxilli strain (CY2) that lacks the native paxilline IDT gene cluster ( / HYcluster). Ten transformants were selected by positive selection with geneticin (plasmids contain the nptllgene) and processed as described in materials and methods.

[0327] The ten strains containing radB SEQ ID NO: 4) were grown in liquid cultures, extracted, and analysed by LC-MS. Nine of ten strains showed detection of a mass consistent with radarin C (m / z 406.3 [M + H]+) based on extracted ion chromatograms as shown in Figure 11. Up-scaled growth of a radB containing strain (RM298-10), enabled isolation and structural characterization by NMR spectroscopy, confirming the structure to be radarin C (Figure 17 and Figure 18).

[0328] These experiments confirm that RadB (SEQ ID NO: 6) is an indole diterpene cyclase that catalyzes a complex rearrangement / cyclisation of 14,15-epoxy-3'-GGI to form the unique terpene skeleton of radarin C.

[0329] Example 3 - Heterologous biosynthesis of petromindole

[0330] To determine the function of petB, a plasmid was constructed containing the paxG, paxC, radM and efF(pRM319). This plasmid was transformed (by random integration in the genome) into a P. paxilli strain (CY2) that lacks the native paxilline IDT gene cluster ( / HYcluster). Ten transformants were selected by positive selection with geneticin (plasmids contain the tZZgene) and processed as described in materials and methods.

[0331] The ten strains containing efF(SEQ ID NO: 16) were grown in liquid cultures, extracted, and analysed by LC-MS. Seven of ten strains showed detection of a mass consistent with petromindole ( / 77 / z406.3 [M + H]+) along with 14,15-dihydroxy-3'-GGI {m / z 406.3 [M + H]+) based on extracted ion chromatograms as shown in Figure 14. Up-scaled growth of a petB containing strain (RM319-7), enabled isolation of petromindole to approximately 50% purity as confirmed by NMR spectroscopy (Figure 22).

[0332] These experiments confirm that PetB (SEQ ID NO: 18) is an indole diterpene cyclase that catalyzes a cyclisation of 14,15-epoxy-3'-GGI to form petromindole.

[0333] Example 4 - Heterologous biosynthesis of roseyrin

[0334] To determine the function of rosB, a plasmid was constructed containing the paxG, paxC, radMan / osF(pRM308). This plasmid was transformed (by random integration in the genome) into a P. paxilli strain (CY2) that lacks the native paxilline IDT gene cluster ( / ’AYcluster). Six transformants were selected by positive selection with geneticin (plasmids contain the nptllgene) and processed as described in materials and methods.

[0335] The six strains containing / os£(SEQ ID NO: 10) were grown in liquid cultures, extracted, and analysed by LC-MS. Four of six strains showed detection of a mass consistent with roseyrin {m / z 406.3 [M + H]+) based on extracted ion chromatograms as shown in Figure 13. Up-scaled growth of a rosB containing strain (RM308-2), enabled isolation and structural characterization by NMR spectroscopy, confirming the structure to be a new compound, named roseyrin (Figure 21).

[0336] These experiments confirm that RosB (SEQ ID NO: 12) is an indole diterpene cyclase that catalyzes a cyclisation of 14,15-epoxy-3'-GGI to form the new IDT skeleton of roseyrin.

[0337] Example 5 Heterologous biosynthesis of radarin A

[0338] To determine the function of radX, a plasmid was constructed containing the paxG, paxC, radM, radB and radX (pRM301). This plasmid was transformed (by random integration in the genome) into a P. paxilli xa\r\ (CY2) that lacks the native paxilline IDT gene cluster ( / ’AY cluster). Ten transformants were selected by positive selection with geneticin (plasmids contain the nptllgene) and processed as described in materials and methods.

[0339] The ten strains containing r o (SEQ ID NO: 7) were grown in liquid cultures, extracted, and analysed by LC-MS. Five of ten strains showed detection of a mass consistent with radarin A {m / z 422.3 [M + H]+) based on extracted ion chromatograms as shown in Figure 12. Presence of the m / z 146.1 fragment ion was diagnostic of hydroxylation of the indole ring of radarin A (Figure 12). Up-scaled growth of a radX containing strain (RM301-9), enabled isolation and structural characterization by NMR spectroscopy, confirming the structure to be radarin A (Figure 19 and Figure 20).

[0340] These experiments confirm that RadX (SEQ ID NO: 9) is cytochrome P450 monooxygenase that catalyzes a regiospecific hydroxylation of C-7' of radarin C to form radarin A. Hydroxylation of the indole ring of indole diterpenes has not been previously observed and may be significant for the concomitant bioactive properties of radarin A.

[0341] Materials and Methods

[0342] Identification and annotation of A. fresenii IDT genes {RAD cluster scaffold 214, and rosB scaffold 92)

[0343] The genome of A. fresenii CBS 550.65 (Joint Genome Institute)30'31was searched for regions where putative / oWand / k / fFgenes are clustered together, using PaxM and PaxB protein sequences as queries. This revealed the RAD cluster on scaffold 214 of, fresenii, which contained homologous sequences to PaxM and PaxB (r oA / and radB respectively), along with a gene predicted to encode a cytochrome P450 radX}. Additionally, an unclustered idtBgene that shared some homology to the PaxB protein sequence, was identified on scaffold 92 {rosB). Each gene was subjected to NCBI BLAST analyses that were carried out using default algorithm parameters. Introns were validated manually by comparison of sequence alignments of known IdtBs with FgeneSH32(based on Aspergillus niduians} used as a supporting tool to confirm intron / exon boundaries and overall gene structure.

[0344] Identification and annotation of A. muricatus DT genes {PET cluster scaffold 8)

[0345] The genome of A. muricatus CBS 112808 (Joint Genome Institute)30'31was searched for regions where putative / bWand idtB genes are clustered together, using RadM and RadB protein sequences as queries. This revealed the PET cluster on scaffold 8 of, muricatus, which contained homologous sequences to RadM and RadB ( efA / and petB respectively). Each gene was subjected to NCBI BLAST analyses that were carried out using default algorithm parameters. Introns were validated manually by comparison of sequence alignments of known IdtBs with FgeneSH32(based on Aspergillus niduians used as a supporting tool to confirm intron / exon boundaries and overall gene structure.

[0346] Protein sequence alignment generation

[0347] Protein sequences were aligned using Clustal Omega 1.2.3.

[0348] Synthetic genes

[0349] Synthetic genes were domesticated in siiico to remove restriction enzyme recognition sites that are incompatible with MIDAS cloning (BsmBI, Aarl, Bsal, BtgZI and associated schizoisomers). These synthetic genes {radM, radB, radX and rosB) were purchased from Twist Bioscience, who carried out DNA synthesis and cloning into the MIDAS Level-1 custom vector pMLl_CA0543 (MIDAS Level-1 vector). MIDAS cloning

[0350] Cloning reactions and protocols were carried out as described in van Dolleweerd etal. 201829Media / reagents for fungal protocols

[0351] All media was made up in in Milli-Q® Water and sterilised by autoclaving. Antibiotics and supplements were added to cool media post sterilisation. Media and reagents were made up as outlined in McLellan et al. 2022.19

[0352] Protoplast preparation and transformation of P. paxilli

[0353] The preparation of fungal protoplasts for transformation was according to Yelton et al. 1984 with modifications.33Method of transformation of P. paxilli as adapted from van Dolleweerd et. al. 201829, which was originally modified from Vollmer and Yanosfsky 198634and Oliver et al. 198735. All additional modifications are outlined in McLellan etal. 2022.19

[0354] LC-MS screening of transformant metabolites

[0355] The EtOAc supernatant from extracted mycelia was concentrated using a vacuum concentrator. The dried extracts were resuspended in 150 pL MeCN and syringe filtered through a Millex®-LG, 4mm, 0.2 urn PTFE membrane into amber LC-MS vials (with glass inserts). LC-MS analyses of transformants was performed on a Agilent 1260 Infinity II LC-MS system with a Diode Array Detector and electrospray ionisation, coupled with a Phenomenex Kinetex®, 2.6 pm C18 (50 x 2.1 mm) column or a Phenomenex Kinetex®, 2.6 pm C18 (100 x 3.0 mm) column equipped with a Phenomenex C18 guard cartridge and maintained at 40 °C using positive ion mode with gradients of MeCN-FW (containing 0.1% formic acid) with a flow rate of 0.4 mL / min or 0.6 mL / min. Data was exported from Agilent OpenLab ChemStation software and processed with Mestrenova.

[0356] HPLC

[0357] Reverse-phase (RP) HPLC was performed using an Agilent 1260 Infinity II LC system equipped with diode array detection. Semipreparative RP-HPLC utilized a Phenomenex Luna® 5 pm C18 100 A column (250 mm x 10 mm) and preparative RP HPLC utilized a Phenomenex Luna® 5 pm C18 100 A column (250 mm x 15 mm). Columns were eluted using gradients of MeCN: H2O (each containing 0.1% formic acid).

[0358] HR-MS

[0359] HR-MS Spectra were collected using a Shimadzu QTOF 9030 mass spectrometer with ESI in positive ion mode and a collision energy of 30 eV. Samples were delivered using a Shimadzu LC40Bx3 UHPLC with an isocratic solvent system consisting of 50:50 acetonitrile:water containing 0.1% formic acid.

[0360] Compound isolation from P. paxilli strains and structural elucidation

[0361] General growth conditions and metabolite extraction

[0362] Cultures of CDYE + TE liquid medium in Erlenmeyer flasks capped with cotton wool were inoculated with ~107spores per 50 mL and incubated at 28 °C with shaking at 200 RPM for 7 days. Mycelia from fungal cultures were separated from the media by filtration through nappy liners and squeezed. Mycelia was then extracted twice with EtOAc (300-400 mL) overnight. Organic extracts were separated from the aqueous layer, washed with brine, and concentrated in-vacuo.

[0363] Isolation of 14,15-dihydroxy-3'-GGI

[0364] 2.0 L (4 x 400 mL, 2 x 200 mL) cultures containing P. paxilli strain RM297-2 spores (CY2 / paxGCradM) were grown, and the mycelia (188.5 g wet weight) was extracted and concentrated according to General growth conditions and metabolite extraction. The crude extract (600 mg) was fractionated on a Grace Reveleris flash chromatography system using a Reveleris HP 40 g silica flash cartridge and a CHCb: MeOH gradient (liquid injection dissolved in CHCb; mobile phase gradient: 6 CV at 1% MeOH, 12 CV gradient to 10% MeOH, 2 CV gradient to 100% MeOH, 3 CV at 100% MeOH). Fractions of interest, as determined by TLC, were combined, and concentrated in vacuo. Fraction A (~385 mg) was determined via LC-MS to contain 14,15-dihydroxy-3'-GGI, and then subjected to preparative RP-HPLC (C18 250x15 mm column; flow-rate = 15 mL / min; I = 230 nm, CH3CN / H2O (with 0.1% formic acid) = 80 / 20) to afford 14,15-dihydroxy-3'-GGI (fe = 9.8-10.5 min (major), 1.1 mg).

[0365] 14,15-dihydroxy-3'-GGI: colourless oil; HRMS (ESI) m / z. [M + H]+calcd for C28H42NO2, 424.32101; found, 424.32171; NMR data tabulated in Figure 15 and Figure 16

[0366] Isolation of radarin C

[0367] 1.6 L (6 x 200 mL, 8 x 50 mL) cultures containing P. paxilli strain RM298-10 spores (CY2 / paxGCradMB) were grown, and the mycelia (161.6 g wet weight) was extracted and concentrated according to General growth conditions and metabolite extraction. The crude extract (682 mg) was fractionated on a Grace Reveleris flash chromatography system using a Reveleris HP 40 g silica flash cartridge and a CHCb: MeOH gradient (liquid injection dissolved in CHCb; mobile phase gradient: 5 CV at 1% MeOH, 12 CV gradient to 10% MeOH, 2 CV gradient to 100% MeOH, 3 CV at 100% MeOH). Fractions of interest, as determined by TLC, were combined and concentrated in vacuo. Fractions A and B were confirmed by LC-MS to contain radarin C and combined. Combined fraction RM298-10_AB (~304 mg) was then subjected to semi-preparative RP-HPLC (C18250x10 mm column; flow-rate = 5 mL / min; I = 230 nm, CH3CN / H2O (with 0.1% formic acid) = 90 / 10) to afford radarin C (fe = 16.0-17.0 min (major), 26.1 mg).

[0368] Radarin C: white film; HRMS (ESI) m / z. [M + H]+calcd for C28H40NO, 406.31044; found, 406.31084; NMR data tabulated in Figure 17 and Figure 18.

[0369] Isolation of radarin A

[0370] 1.6 L (4 x 200 mL, 4 x 100 mL, 8 x 50 mL) cultures containing P. paxilli strain RM301-9: CY2 spores (CY2 / paxGCradMBX) were grown, and the mycelia (161.1 g wet weight) was extracted and concentrated according to General growth conditions and metabolite extraction. The crude extract (599 mg) was fractionated on a Grace Reveleris flash chromatography system using a Reveleris HP 40 g silica flash cartridge and a CHCh: MeOH gradient (liquid injection dissolved in CHCh; mobile phase gradient: 5 CV at 1% MeOH, 11 CV gradient to 10% MeOH, 2 CV gradient to 100% MeOH, 3 CV at 100% MeOH). Fractions of interest, as determined by TLC, were combined, and concentrated in vacuo. Fraction C (~17 mg) was confirmed by LC-MS to contain radarin A and was then subjected to semi-preparative RP-HPLC (C18 250x10 mm column; flow-rate = 5 mL / min; I = 230 nm, CH3CN / H2O (with 0.1% formic acid) = 90 / 10) to afford radarin A (fe = 13.9-15.4 min (major), 9.8 mg).

[0371] Radarin A: white film; HRMS (ESI) m / z. [M + H]+calcd for C28H40NO2, 422.30536; found, 422.30611, NMR data tabulated in Figure 19 and Figure 20.

[0372] Isolation of roseyrin

[0373] 2.0 L (1 x 600 mL, 5 x 200 mL, 4 x 100 mL) cultures containing P. paxi / H strain RM308-2 spores (CY2 / paxGCradMrosB) were grown, and the mycelia (204.8 g wet weight) was extracted and concentrated according to General growth conditions and metabolite extraction. The crude extract (561 mg) was fractionated on a Grace Reveleris flash chromatography system using a Reveleris HP 40 g silica flash cartridge and a CHCh: MeOH gradient (liquid injection dissolved in CHCh; mobile phase gradient: 5 CV at 1% MeOH, 11 CV gradient to 10% MeOH, 2 CV gradient to 100% MeOH, 3 CV at 100% MeOH). Fractions of interest, as determined by TLC, were combined, and concentrated in vacuo. Fraction A (~271 mg) was confirmed by LC-MS to contain roseyrin and was then subjected to semi-preparative RP-HPLC (C18 250x15 mm column; flow-rate = 15 mL / min; I = 230 nm, CH3CN / H2O (with 0.1% formic acid) = 78 / 22) to afford roseyrin (fe = 19.3-20.0 min (major), 6.8 mg).

[0374] Roseyrin: white film; HRMS (ESI) m / z. [[M + H]+calcd for C28H40NO, 406.31044; found, 406.31079., NMR data tabulated in Figure 21.

[0375] Isolation of petromindole

[0376] 1.6 L (4 x 400 mL) cultures containing P. paxiiii strain RM319-7 spores (CY2 / paxGCpetMpetB) were grown, and the mycelia (181.5 g wet weight) was extracted and concentrated according to General growth conditions and metabolite extraction. The crude extract (1271 mg) was fractionated on a Grace Reveleris flash chromatography system using a Reveleris HP 40 g silica flash cartridge and a CHCh: MeOH gradient (liquid injection dissolved in CHCh; mobile phase gradient: 6 CV at 1% MeOH, 11 CV gradient to 10% MeOH, 2 CV gradient to 100% MeOH, 3 CV at 100% MeOH). Fractions of interest, as determined by TLC, were combined, and concentrated in vacuo. Fraction B (~271 mg) was confirmed by LC-MS to contain petromindole and was then subjected to semi-preparative RP-HPLC (C18 250x15 mm column; flow-rate = 15 mL / min; I = 230 nm, CH3CN / H2O (with 0.1% formic acid) = 78 / 22) to afford petromindole (fe = 11.4-11.8 min (50% purity), 3.9 mg).

[0377] Petromindole: white film; NMR data tabulated in Figure 22. Tables 1-6 referenced in this specification are set out below:

[0378] Table 1. Fungal strains used in this study

[0379]

[0380] Table 2. Gene prediction of the RAD duster genes and rosB gene from Aspergillus fresenii NRRL4077

[0381]

[0382] [a]Proposed function based on homologues or analysis of NCBI Conserved Domain Search.

[0383] [b]If available, closest characterised homologue is shown. Uncharacterised homologues referred to by locus tag. Table 3. Gene prediction of the PET cluster genes from Aspergillus muricatus NRRL4077

[0384]

[0385] [a]Proposed function based on homologues or analysis of NCBI Conserved Domain Search.

[0386] [b]If available, closest characterised homologue is shown. Uncharacterised homologues referred to by locus tag.

[0387] Table 4. MIDAS Level 1 plasmids

[0388]

[0389] aAII MIDAS Level 1 plasmids assembled in pMLl vector.29b"P" at the start indicates promoter region, T at the start indicates terminator region Table 5. MIDAS Level 2 plasmids[1]

[0390]

[0391] [1]MIDAS Level 2 plasmids assembled in pML2(+)BF, pML2(+)BR, pML2(+)WF or pML2(+)WR vectors.29 Table 6. MIDAS Level 3 plasmids[1]

[0392]

[0393] SEQ ID NO: 1 - radM genomic DNA ATGGAGTCAAAATTCAGAGTCATCATCGTGGGCGGATCCATTGCAGGTCTAACGCTCGCGCACTGCTTGGACC ACCTAGGGGTTGATTATGTTGTACTGGAGAAGCGAAAAGAAATCGCACCTCAGGAGGGTGCGTCAATTGTTGT CATGCCCCATGCTGGACGAATTCTAGACCAGCTGGGATTACTTAGCCATCTAGGAGAATTTGTCGAGCCTCTGC ACACAGCGCACGTATCCTATCCCGACGGCTTTAAGCTAACTGACCGATCGCCGAATGTGCTTCTTGAGCGGTGC GTTGTTAmCTGAATCTCTCCTCTGCACTTTATGGCCAGTTGGCTAACAACATACTTTGCTGCGCACTAGGTT TGGGCTCCCTCTAACCTTCCTTGAACGGCGTAAGCTGCTGCAAGTTCTATACAATTCGCTACGAGATCCGTCGC GCGTGTTAGTTGACAGTACTGTTGTCTCTGTACTGCAGAACGAGGGCGACATCGGCGCTGTGCAAGTACAGGA TGGATCGATTTATCGGGGCGATCTTATCGTCGGAGCCGATGGTATACACAGTCGGATTCGGCGTGAAATGTGG AGACTGGCAGGATTGAATCATCCGGGCAGAATATCAGAGAAAGAGAAGAACGGTGAATCCGCGTCCCAATTGC ATACATGTGATTCCAACGAGGGTAAGCTGATGTATGGGATCCACTTAGCTATGAAGATCGACTACGCATGTGT ATTCGGTATATCGGATGCCGTGCCGGACCTCCGCCCCGGTGAGCAAATAACAGGCTTTCAGTATGGGAGGTCC TTGCTCATATTCCCTGGAAAGAACGGCCGGGTTTTCTGGTTCATTTTCAAAAAGCTTGATCGGCAATATTCTTA TTCCTCGGCTCCTCGGTGGTCTAAATGTGATGCGGACAAAATAGCGGAGGAGTTTGGGCTGGACTACGTTTGG GCTGGTCTCCGATTCAAGCATGTATGGAATACCAGACAAGTCGTTGGTATAACCAATCrCGAGGAAAACGTCTT TTCTACCTGGTACTCTGGCCGCGTGGTCTTCGTCGGAGACAGCATGCACAAGGTATGTTAGGCCGAACTGTCA TGGAATGCACTCrCACAGAAAATCTGGGTAGTTCGCrCCCAATACAGGACAAGGGGCAAACrGTGCCATCGAA GACGCCGCAGTGTTGGCCAATTATATCAGCAAAGCACTCGCCACCGAAGAGGATGTGGCTCGTCCTTCCTACG ATACGCTCCAGAAACTTCTTCATTCCTTCAGTCAAAGTCGGTCCCAGCGCGCCCAGGAGATATCTAAAAGCGCC AGGTTCGCGATAAGGCTTTCTACATGCGATAGTCTGTTCCTGCGCCTTCTGGGTAGGTTTGTCTTGCCATATAT GGGTGACTTTCGAGCAGAGGTTGCGTCTAGAATAATCGGCCGCGGAGTGCAGTTGTCGTmTGGCACCCTCG CGGTGCTCTGCATCTGGATGGCAAACCTTTCACCCTGATCGGGAGTCTAGATCTACCTACACGGCTATAATTAT GGTTTTGGGTmTTAATGTGCTTGGCTGCTTGTAGAGTATACGGATTCGGGCTTTAA

[0394] SEQ ID NO: 2 - / aoWcDNA ATGGAGTCAAAATTCAGAGTCATCATCGTGGGCGGATCCATTGCAGGTCTAACGCTCGCGCACTGCTTGGACC ACCTAGGGGTTGATTATGTTGTACTGGAGAAGCGAAAAGAAATCGCACCTCAGGAGGGTGCGTCAATTGTTGT CATGCCCCATGCTGGACGAATTCTAGACCAGCTGGGATTACTTAGCCATCTAGGAGAATTTGTCGAGCCTCTGC ACACAGCGCACGTATCCTATCCCGACGGCTTTAAGCTAACTGACCGATCGCCGAATGTGCTTCTTGAGCGGTTT GGGCTCCCTCTAACCTTCCTTGAACGGCGTAAGCTGCTGCAAGTTCTATACAATTCGCTACGAGATCCGTCGCG CGTGTTAGTTGACAGTACTGTTGTCTCTGTACTGCAGAACGAGGGCGACATCGGCGCTGTGCAAGTACAGGAT GGATCGATTTATCGGGGCGATCTTATCGTCGGAGCCGATGGTATACACAGTCGGATTCGGCGTGAAATGTGGA GACTGGCAGGATTGAATCATCCGGGCAGAATATCAGAGAAAGAGAAGAACGCTATGAAGATCGACTACGCATG TGTATTCGGTATATCGGATGCCGTGCCGGACCTCCGCCCCGGTGAGCAAATAACAGGCTTTCAGTATGGGAGG TCCTTGCTCATATTCCCTGGAAAGAACGGCCGGGTTTTCTGGTTCATTTTCAAAAAGCTTGATCGGCAATATTC TTATTCCTCGGCTCCTCGGTGGTCTAAATGTGATGCGGACAAAATAGCGGAGGAGTTTGGGCTGGACTACGTT TGGGCTGGTCTCCGATTCAAGCATGTATGGAATACCAGACAAGTCGTTGGTATAACCAATCTCGAGGAAAACG TCmTCTACCTGGTACTCTGGCCGCGTGGTCTTCGTCGGAGACAGCATGCACAAGTTCGCTCCCAATACAGGA CAAGGGGCAAACTGTGCCATCGAAGACGCCGCAGTGTTGGCCAATTATATCAGCAAAGCACTCGCCACCGAAG AGGATGTGGCTCGTCCTTCCTACGATACGCTCCAGAAACTTCTTCATTCCTTCAGTCAAAGTCGGTCCCAGCGC GCCCAGGAGATATCTAAAAGCGCCAGGTTCGCGATAAGGCTTTCTACATGCGATAGTCTGTTCCTGCGCCTTCT GGGTAGGTTTGTCTTGCCATATATGGGTGACTTTCGAGCAGAGGTTGCGTCTAGAATAATCGGCCGCGGAGTG CAGTTGTCGTTTTTGGCACCCTCGCGGTGCTCTGCATCTGGATGGCAAACCTTTCACCCTGATCGGGAGTCTA GATCTACCTACACGGCTATAATTATGGTTTTGGGTCTTTTAATGTGCTTGGCTGCTTGTAGAGTATACGGATTC GGGCTTTAA SEQ ID NO: 3 - RadM MESKFRVIIVGGSIAGLTLAHCLDHLGVDYWLEKRKEIAPQEGASIWMPHAGRILDQLGLLSHLGEFVEPLHTAHV SYPDGFKLTDRSPNVLLERFGLPLTFLERRKLLQVLYNSLRDPSRVLVDSTWSVLQNEGDIGAVQVQDGSIYRGDL IVGADGIHSRIRREMWRLAGLNHPGRISEKEKNAMKIDYACVFGISDAVPDLRPGEQITGFQYGRSLLIFPGKNGR VFWFIFKKLDRQYSYSSAPRWSKCDADKIAEEFGLDYVWAGLRFKHVWNTRQVVGITNLEENVFSTWYSGRVVF VGDSMHKFAPNTGQGANCAIEDAAVLANYISKALATEEDVARPSYDTLQKLLHSFSQSRSQRAQEISKSARFAIRLS TCDSLFLRLLGRFVLPYMGDFRAEVASRIIGRGVQLSFLAPSRCSASGWQTFHPDRESRSTYTAIIMVLGLLMCLAA CRVYGFGL*

[0395] SEQ ID NO: 4 - radB genomic DNA ATGGAAGGATTCGACTTTGATACAGGGCCTCCCGAGTTCCAACGCGTGAGATCACTGTCGCAGGGCATGTTCT TCCTCGCCGGTCTAGGATGGATAATATACTACTTTCTCGCGATCCGAACAGCATTTCGCGATAACACAGCGGGC TTGCCCCTGGCTGGACTCTGTAACCACATATCATGGGAGATTACATATGCTGTnTTCACTGTCCACCAGACTG GGTTAACCAAGTTATACTCCGTGTCTGGTTATCCATTAGCATATTTGTTCTCTACGCTATGGTCAAGAATACAG CCAAGACGCATTCAGATTCCCrCTTTCrACCACGCrATACrCCCCrATTTACGATGCTCTACATTGGATTTTTCG TTTCCGCTCATTGCACTCTCGCTGCACATCTGGGGGATATTGAGGCCCTCTATTGGGGCGGAATGGCATCTCA GGTCTTTATGAATGCTAGTGCACTCAGTAGGCTCATCCAACGGGGAAGCACGCGTGGATGCACCTACGGAATG TGGTAAGTCTCTATCCATGCCACAGTGGCGAATATATTCCTAACGGGTTTTCATATCTACTAGGTTGTCAGAAT TTATGGGTTTAATATTTACGCTAATTGGAGTTTACCTGCGAGCGTGTTCCTGGCCGGCGAAATGGGGGTGGTC TAATAGTATTTTGATGGCGTGGTTTGCTTTCTGGACrGTTATTTTGGAAATTGCGTACGTGTGTTTTTTCTGGC TAACTCGGCGAGCTGAGCATGCGAGACTGCCGTCAAAAGAAGAATGA SEQ ID NO: 5- / ao^cDNA ATGGAAGGATTCGACTTTGATACAGGGCCTCCCGAGTTCCAACGCGTGAGATCACTGTCGCAGGGCATGTTCT TCCTCGCCGGTCTAGGATGGATAATATACTACTTTCTCGCGATCCGAACAGCATTTCGCGATAACACAGCGGGC TTGCCCCTGGCTGGACTCTGTAACCACATATCATGGGAGATTACATATGCTGTnTTCACTGTCCACCAGACTG GGTTAACCAAGTTATACTCCGTGTCTGGTTATCCATTAGCATATTTGTTCTCTACGCTATGGTCAAGAATACAG CCAAGACGCATTCAGATTCCCrCTTTCrACCACGCrATACrCCCCTATTTACGATGCTCTACATTGGATTTTTCG TTTCCGCTCATTGCACTCTCGCTGCACATCTGGGGGATATTGAGGCCCTCTATTGGGGCGGAATGGCATCTCA GGTCTTTATGAATGCTAGTGCACTCAGTAGGCTCATCCAACGGGGAAGCACGCGTGGATGCACCTACGGAATG TGAATTTATGGGTTTAATATTTACGCTAATTGGAGTTTACCTGCGAGCGTGTTCCTGGCCGGCGAAATGGGGG TGGTCTAATAGTATTTTGATGGCGTGGTTTGCTTTCTGGACrGTTATTTTGGAAATTGCGTACGTGTGTTTTTT CTGGCTAACTCGGCGAGCTGAGCATGCGAGACTGCCGTCAAAAGAAGAATGA SEQ ID NO: 6 - RadB MEGFDFDTGPPEFQRVRSLSQGMFFLAGLGWIIYYFLAIRTAFRDNTAGLPLAGLCNHISWEITYAVFHCPPDWVN QVILRVWLSISIFVLYAMVKNTAKTHSDSLFLPRYTPLFTMLYIGFFVSAHCTLAAHLGDIEALYWGGMASQVFMNA SALSRLIQRGSTRGCTYGMWLSEFMGLIFTLIGVYLRACSWPAKWGWSNSILMAWFAFWTVILEIAYVCFFWLTR RAEHARLPSKEE*

[0396] SEQ ID NO: 7- / a Xgenomic DNA ATGCACTCCTCTACAGCTGTCTCTTTCGTGGCAGGGGTCCTGTCACACCTAGCGTACTTTAACAAAGGCGAGCA CCATCTATATGGCATGGATTATCTGCAGGTTTTCCTCGCAGCGATTGCATCAAGTGCCATCTGTCTGCAATATC AACAAGACTTGCCATGGAGCACAGCCCTGGCCAACACrCrGCATTTCGCCGCGTTGTATCrCACCGGCGTCTAT ACCAGCCTTATCACCTATCGCCTTTTTTTCCATCCGCTTCGACGATTCCCCGGCCCCATAGGCGCGCGCATCTC TAGCTTATGGTTCTGCACGCAGGTCTCTAAACACGACACCCATCGCCAGCTCCTTGCTCTTCACAAGAAATACG GTCCGATCGTGCGCATCGGCTCATCTGACCTGTCAATCATTCATCCAAGCGGCATCGGCGCCCTCTATGGCCA GAATACCCGCTGCACCAAGGGCTCCTGGTACGACATGTCATACCCCGCGATTTCTATGCAGACGATGCGCGAT CCGGTAGAGCATAGAATCAGACGACGAGCTTGGAGCCCTGCGTTCGGGGATAACCAACTGCGCGGGTACGAG GTGCGCCTGCGCCCCTATCGACAAAGGCTGCTCGATCGGTTTACCGGGATGGTTGACGAGCCTGTCGATATTA GGAAATGGTTCAATCTCTACACGTTCGACGTGATGGGCGACCTGACGTTCGGAGAGGGCTTTGGGGGTATCGA GCGGGGGCAGCTCCTCGCrCCGCTTGAGCrGGTGGGCACCATGGTATACTTCATCGGGTTGTTTATGCCGGTG TGGGTGTTGTGTCTTCrCrCCCGTCrCCCGCrGGCATCGAAAGGCrGGTTTTTGTTTTTGGATTGGGCCGCTG AGCTACTTAATAAGCGCATCAAGGTGTTTCTCTCTCACCTTCTAGGATATTCTTCAGGTTGAATCAACTTGGCT AATCATCATAGCAAGAACCCTCCTTCGATTCCCGACATCAGCGCCGCGCTTATCGCCCACCTCGACGGTCGCGA GCCCACCAAAAAAGACCAGCTGCTGCTAGGAGGCGACTCGCGCCTCATCATGTTCGCCGGTAGCGATACCACC TCGACGGCACTGACAGCGATTGTGTACGAACTGCTGCATAACCCTGGAGAGCTTTCCAAATTGCGCAGCGAGC TAGCCCCGCACATCGAAGCAGACGGAGATTTCGTGCACCTCAAAATCCAGCATCTCGAGCACCTAAATGGGATC ATCAACGAAGCCTTGCGCCTGCACCCCGTCGTGCCGGACAACATGCAGCGGAAGACACCGCCGGAAGGGATTC GTGTCGACGGGGTGTGGATTCCTGGAGACATGACGGTGATGTGTCCGCAATATGTGCTGGGGAGGTGTCAGT CTTTCTCCCTCTAAGATGCTAGAATAATTTCTCGATGCGGACTAACGCAATCAATAGCGGAGCGATGCTATGTT AAGCCCAACTCGTTCATCCCCGAGCGATGGTACAGTTCCCCCCAGCTGGTCCGCGATAAATCGGCGTTTGCTCC ATTTTCAATTGGTTGGTCTTCTTCATCGCCTCTTATGGTTGTTCCCTTGTAACCAAGATATATCGGTAATCACA GGACCAGCTAGCTGCATCGGCAAACCGCTAGCGCTGATGAACGTTCGGGCGACTATCGCTCGATTCGTCATGG AATTCGATTTTGAATTTGCGCCAGGGACGGATCGACGACAATTTGAGGACGAGGCCATGGATAATTTTATCCTA GTTCCGGGGAAGTTGACGGTTGTTTTGAAAAGAAGAGAGAAGGGAGTTTGA SEQ ID NO: 8- / 30 cDNA ATGCACTCCTCTACAGCTGTCTCTTTCGTGGCAGGGGTCCTGTCACACCTAGCGTACTTTAACAAAGGCGAGCA CCATCTATATGGCATGGATTATCTGCAGGTTTTCCTCGCAGCGATTGCATCAAGTGCCATCrGTCrGCAATATC AACAAGACTTGCCATGGAGCACAGCCCTGGCCAACACTCTGCATTTCGCCGCGTTGTATCTCACCGGCGTCTAT ACCAGCCTTATCACCTATCGCCTTTTTTTCCATCCGCTTCGACGATTCCCCGGCCCCATAGGCGCGCGCATCTC TAGCTTATGGTTCTGCACGCAGGTCTCTAAACACGACACCCATCGCCAGCTCCTTGCTCTTCACAAGAAATACG GTCCGATCGTGCGCATCGGCTCATCrGACCTGTCAATCATTCATCCAAGCGGCATCGGCGCCCTCTATGGCCA GAATACCCGCTGCACCAAGGGCTCCTGGTACGACATGTCATACCCCGCGATTTCTATGCAGACGATGCGCGAT CCGGTAGAGCATAGAATCAGACGACGAGCTTGGAGCCCTGCGTTCGGGGATAACCAACTGCGCGGGTACGAG GTGCGCCTGCGCCCCTATCGACAAAGGCTGCTCGATCGGTTTACCGGGATGGTTGACGAGCCTGTCGATATTA GGAAATGGTTCAATCTCTACACGTTCGACGTGATGGGCGACCTGACGTTCGGAGAGGGCTTTGGGGGTATCGA GCGGGGGCAGCTCCTCGCrCCGCTTGAGCrGGTGGGCACCATGGTATACTTCATCGGGTTGTTTATGCCGGTG TGGGTGTTGTGTCTTCrCrCCCGTCrCCCGCrGGCATCGAAAGGCrGGTTTTTGTTTTTGGATTGGGCCGCTG AGCTACTTAATAAGCGCATCAAGAACCCTCCTTCGATTCCCGACATCAGCGCCGCGCTTATCGCCCACCTCGAC GGTCGCGAGCCCACCAAAAAAGACCAGCTGCTGCTAGGAGGCGACTCGCGCCTCATCATGTTCGCCGGTAGCG ATACCACCTCGACGGCACTGACAGCGATTGTGTACGAACTGCTGCATAACCCTGGAGAGCTTTCCAAATTGCGC AGCGAGCTAGCCCCGCACATCGAAGCAGACGGAGATTTCGTGCACCTCAAAATCCAGCATCTCGAGCACCTAAA TGGGATCATCAACGAAGCCTTGCGCCTGCACCCCGTCGTGCCGGACAACATGCAGCGGAAGACACCGCCGGAA GGGATTCGTGTCGACGGGGTGTGGATTCCTGGAGACATGACGGTGATGTGTCCGCAATATGTGCTGGGGAGG TCGGAGCGATGCTATGTTAAGCCCAACTCGTTCATCCCCGAGCGATGGTACAGTTCCCCCCAGCTGGTCCGCG ATAAATCGGCGTTTGCTCCATTTTCAATTGCTAGCTGCATCGGCAAACCGCTAGCGCTGATGAACGTTCGGGC GACTATCGCTCGATTCGTCATGGAATTCGATTTTGAATTTGCGCCAGGGACGGATCGACGACAATTTGAGGAC GAGGCCATGGATAATTTTATCCTAGTTCCGGGGAAGTTGACGGTTGTTTTGAAAAGAAGAGAGAAGGGAGTTT GA SEQ ID NO: 9- RadX

[0397] M HSSTAVSFVAGVLSH LAYFN KGEH H LYGM DYLQVFLAAIASSAICLQYQQDLPWSTALANTLH FAALYLTGVYTS LITYRLFFHPLRRFPGPIGARISSLWFCTQVSKHDTHRQLLALHKKYGPIVRIGSSDLSIIHPSGIGALYGQNTRCTK GSWYDMSYPAISMQTMRDPVEHRIRRRAWSPAFGDNQLRGYEVRLRPYRQRLLDRFTGMVDEPVDIRKWFNLY TFDVMGDLTFGEGFGGIERGQLLAPLELVGTMVYFIGLFMPVWVLCLLSRLPLASKGWFLFLDWAAELLNKRIKNP PSIPDISAALIAHLDGREPTKKDQLLLGGDSRLIMFAGSDTTSTALTAIVYELLHNPGELSKLRSELAPHIEADGDFV HLKIQHLEHLNGIINEALRLHPWPDNMQRKTPPEGIRVDGVWIPGDMTVMCPQYVLGRSERCYVKPNSFIPERW YSSPQLVRDKSAFAPFSIASCIGKPLALMNVRATIARFVMEFDFEFAPGTDRRQFEDEAMDNFILVPGKLTWLKRR EKGV*

[0398] SEQ ID NO: 10— rosB genomic DNA ATGGAAGGATTCGACTTTGACGCAGGTCCTCCCGAGTTCCAACGCGTGAGATCACTGTCCCAGGCTATGTTCT TCCTCGCTGGCCTATTATTCATAATAAACTACTTTCTTGCAATCCGAACAGCATTTCGCCATAACGCAGCGGGT GTGCCCCTCGCTTCGCTCAGTTGTAATATATCCTGGGAGGTTACATACGCCCTmTTACTGTCCACCAGACTG GGTTAGCCAAGTCATACTCCGTGTCTGGCTGTTCATTAACATCTTTGTTCTCTACGCCATGATCACGAATACGG GAAAGGCGCAGCCGGATTCCCTCTTCCCACCACGCTATGTCCCCTTATTCACGACGTTCTACATCGTATTCTTC CTTTCTGCTCATTACACTCTCGCTACGCATCTGGGGGGTGTTAAGGCCCTCTACTGGGGTGGAATGGCATCTC AAGTCTTTATGAATGCCAGTGCGCTTAGTGGGCTTATCCAACGGGGAAGCACGCGTGGATGCACCTACGGGAT GTGGTATGTCTCTAATCATACGAACGTGGCTAATATATTCATTCCTAACGGGTTGTCATGTCTACTAGGCTGTC AGAATTTATGGGGTTAGTGTTTACGCTAATTGGAGTTTACCTGCGAGCGTGGTCCTGGCCGGAGAAATGGGGG TGGTCrAGTGGTATTTTGATGGCGTGGTTTGCGACCTGGAGTGTTATTTTGAATATTGCGTACGCAGTTTGTT TCTGGTTAACTCGGCGAGCTGAGCAGGTGAGACAACCGTCAAAGCAAGAATGA SEQ ID NO: 11- / osFcDNA ATGGAAGGATTCGACTTTGACGCAGGTCCTCCCGAGTTCCAACGCGTGAGATCACTGTCCCAGGCTATGTTCT TCCTCGCTGGCCTATTATTCATAATAAACTACTTTCTTGCAATCCGAACAGCATTTCGCCATAACGCAGCGGGT GTGCCCCTCGCTTCGCTCAGTTGTAATATATCCTGGGAGGTTACATACGCCCTmTTACTGTCCACCAGACTG GGTTAGCCAAGTCATACTCCGTGTCTGGCTGTTCATTAACATCTTTGTTCTCTACGCCATGATCACGAATACGG GAAAGGCGCAGCCGGATTCCCTCTTCCCACCACGCTATGTCCCCTTATTCACGACGTTCTACATCGTATTCTTC CTTTCTGCTCATTACACTCTCGCTACGCATCTGGGGGGTGTTAAGGCCCTCTACTGGGGTGGAATGGCATCTC AAGTCTTTATGAATGCCAGTGCGCTTAGTGGGCTTATCCAACGGGGAAGCACGCGTGGATGCACCTACGGGAT GTGGCTGTCAGAATTTATGGGGTTAGTGTTTACGCTAATTGGAGTTTACCTGCGAGCGTGGTCCTGGCCGGAG AAATGGGGGTGGTCrAGTGGTATTTTGATGGCGTGGTTTGCGACCTGGAGTGTTATTTTGAATATTGCGTACG CAGTTTGTTTCTGGTTAACTCGGCGAGCTGAGCAAGTGAGACAACCGTCAAAGCAAGAATGA SEQ ID NO: 12 - RosB MEGFDFDAGPPEFQRVRSLSQAMFFLAGLLFIINYFLAIRTAFRHNAAGVPLASLSCNISWEVTYALFYCPPDWVSQ VILRVWLFINIFVLYAMITNTGKAQPDSLFPPRYVPLFTTFYIVFFLSAHYTLATHLGGVKALYWGGMASQVFMNAS ALSGLIQRGSTRGCTYGMWLSEFMGLVFTLIGVYLRAWSWPEKWGWSSGILMAWFATWSVILNIAYAVCFWLTR RAEQVRQPSKQE*

[0399] SEQ ID NO: 13- petM genomic DNA ATGGAGTCAGAGTTCAGAGTCGTCATTGTGGGCGGATCTATTGCAGGTCTAACGCTTGCTCATTGCTTGGATC ACATGGGGGTCGATTATGTCGTGTTGGAGAAACGAAAAGAAATTGCACCTCAGGAGGGTGCATCCATCGTGGT TATGCCTCATGCTGGGCGAATTCTGGACCAGCTGGGATTGATGGACGAGGTTGGGAAGTTCGTCGAGCCGCTA CACACAGCCCATGTGTCCTATCCCGACGGCTTCAAGCAAACAGACCGATCrCCGCAGGTGCrCTTTGAACGGTG CGTGATTACTCCTGCATCCCCTTCGTCTGCCCGCTATGGCTAGTTTGCTGACGATCCATAATGCCATATAATAG GTTTGGACTGCCTTTGGCATTTCTGGAACGACGTAAGCTGCTAGAAATTTTATATACCTCGCTACGGGACCCGT CCCGCGTGCTAGTCGACAAGGCTGTTGTATCTGTACTGCAAGATGAGGGCGACATTGGCGCAGTGCAAGTACA AGATGGATCGATCTATCGAGGTGATCTTATCGTCGGAGCCGACGGAGTACATAGTCGAATAAGACGTGAAATG TGGCGTCTGGATGAATTGAAGCATCCGGGCAGGATATCACAGAAAGAAAAGAATGGTCAGTGCGCGTTCCGGA TGCATGACCGTGATGGCAAAGGTGAACATCTGACCATATAGAATCCATCTAGCCATGAAGATCGACTACGCATG TATATTTGGTATATCGAATGCTGTGCCCAACCTCAATCCTGGTGAGCAAGTAACAGGCTTTCAAAATGGGAGGT CCTTGCrCGTTTTCCCTGGAAAGAATGGTCGGGTATACTGGTTTCTTTTCAAAAAGCTTGACCGACAACATTCA TATTGTTCGGGCCCTCGATGGTCCAACTTGGATGCGGCTGAAATAGCAAAGGAATTTGCGCCGGACAATGTTT GGGCTGGCCTTCAATTTAAGGGCATTTGGGAGACCAGAGAAGTCGTTGGCATAACCAATCTCGAGGAAAATGT CTTTTCTACCTGGCACTCCGGCCGTGTGGTCTGCATTGGGGACAGCGTGCACAAGGTATGGTAGATACTGGAC AGCGAATGGATCTATACTCACAGATAACrGGGGTAGTTGGCCCCAAATACAGGTCAAGGGGCAAATTGTGCAA TCGAAGATGCCGCAGCTTTGGCCAATTGTATCAGCAAAGCAATGGCCACGAAGCCGAATGCTGTTCGCCCTTC CTACGATATGATGCAGCCATTTCTCTGTTCCTTTAGCCGGAGTCAGATCCGGCGTGCCCAAGAGATATCAAAGA GCGCCAGGCTAGTGATCAGGCTTTCCACAGGCGATAGCCTGCmTGCGACTTGTGGGTAGGTTCGTCTTGCC ATATATCGGAGACTTTCGAACAGAGATCGCATCCAAAATAATAGGCGGTGGAATGCAGCTGTCGTmTGCCG CCATCACAGTGCTCTGCATCrGGATGGCAAAGATTTTGCCGGGATCGTGAGTCCAAATTAAGTTATACrGTCGT GATCACGGCTCTAGGGATTTCTATGTGCTTCGCTGCTTGTGTTGGATATGGACTTCTCTGGCCATAA SEQ ID NO: 14- eWcDNA ATGGAGTCAGAGTTCAGAGTCGTCATTGTGGGCGGATCTATTGCAGGTCTAACGCTTGCTCATTGCTTGGATC ACATGGGGGTCGATTATGTCGTGTTGGAGAAACGAAAAGAAATTGCACCTCAGGAGGGTGCATCCATCGTGGT TATGCCTCATGCTGGGCGAATTCTGGACCAGCTGGGATTGATGGACGAGGTTGGGAAGTTCGTCGAGCCGCTA CACACAGCCCATGTGTCCTATCCCGACGGCTTCAAGCAAACAGACCGATCTCCGCAAGTGCTCTTTGAACGTTT GGACTGCCTTTGGCATTTCTGGAACGACGTAAGCTGCTAGAAATTTTATATACCTCGCTACGGGACCCGTCCCG CGTGCTAGTCGACAAGGCTGTTGTATCTGTACTGCAAGATGAGGGCGACATTGGCGCAGTGCAAGTACAAGAT GGATCGATCTATCGAGGTGATCTTATCGTCGGAGCCGACGGAGTACATAGTCGAATAAGACGTGAAATGTGGC GTCTGGATGAATTGAAGCATCCGGGCAGGATATCACAGAAAGAAAAGAATGCCATGAAGATCGACTACGCATG TATATTTGGTATATCGAATGCTGTGCCCAACCTCAATCCTGGTGAGCAAGTAACAGGCTTTCAAAATGGGAGGT CCTTGCrCGTTTTCCCTGGAAAGAATGGTCGGGTATACTGGTTTCTTTTCAAAAAGCTTGACCGACAACATTCA TATTGTTCGGGCCCTCGATGGTCCAACTTGGATGCGGCTGAAATAGCAAAGGAATTTGCGCCGGACAATGTTT GGGCTGGCCTTCAATTTAAGGGCATTTGGGAAACCAGAGAAGTCGTTGGCATAACCAATCTCGAGGAAAATGT CTTTTCTACCTGGCACrCCGGCCGTGTGGTCrGCATTGGGGACAGCGTGCACAAGTTGGCCCCAAATACAGGT CAAGGGGCAAATTGTGCAATCGAAGATGCCGCAGCTTTGGCCAATTGTATCAGCAAAGCAATGGCCACGAAGC CGAATGCTGTTCGCCCTTCCTACGATATGATGCAGCCATTTCTCTGTTCCTTTAGCCGGAGTCAGATCCGGCGT GCCCAAGAGATATCAAAGAGCGCCAGGCTAGTGATCAGGCTTTCCACAGGCGATAGCCTGCmTGCGACTTG TGGGTAGGTTCGTCTTGCCATATATCGGAGACTTTCGAACAGAGATCGCATCCAAAATAATAGGCGGTGGAAT GCAGCTGTCGTTTTTGCCGCCATCACAGTGCTCrGCATCrGGATGGCAAAGATTTTGCCGGGATCGTGAGTCC AAATTAAGTTATACTGTCGTGATCACGGCTCTAGGGATTTCTATGTGCTTCGCTGCTTGTGTTGGATATGGACT TCTCTGGCCATAA SEQ ID NO: 15 - PetM MESEFRWIVGGSIAGLTLAHCLDHMGVDYWLEKRKEIAPQEGASIWMPHAGRILDQLGLMDEVGKFVEPLHTA HVSYPDGFKQTDRSPQVLFERFGLPLAFLERRKLLEILYTSLRDPSRVLVDKAVVSVLQDEGDIGAVQVQDGSIYRG DLIVGADGVHSRIRREMWRLDELKHPGRISQKEKNAMKIDYACIFGISNAVPNLNPGEQVTGFQNGRSLLVFPGKN GRVYWFLFKKLDRQHSYCSGPRWSNLDAAEIAKEFAPDNVWAGLQFKGIWETREWGITNLEENVFSTWHSGRV VCIGDSVHKLAPNTGQGANCAIEDAAALANCISKAMATKPNAVRPSYDMMQPFLCSFSRSQIRRAQEISKSARLVI RLSTGDSLLLRLVGRFVLPYIGDFRTEIASKIIGGGMQLSFLPPSQCSASGWQRFCRDRESKLSYTWITALGISMCF AACVGYGLLWP*

[0400] SEQ ID NO: 16- petB genomic DNA ATGGATGGATTTGATTTTGATGCGGGTCCCCCAGAGTTCCAGCAGGTGAGGTCACTCGCGCAGGCCAACTTTT TCCTCGCCGGCGTGGGATGGCTGGCATACAACTACTTCTTGATCCGGTCAGCTTATCGCGATAGTACAGCCGG CGTGTCCCTGATTCCACTCTGCAATAACTTTGCCTGGGAGCTTACCTTCGCCCTCGTCTATTGCCCGCCAGACT TGGCTACTAAATCTGTAGGCCGGGCCTGGCTGTTTATCAATTGTTTTGTTGTGTACACCATGGTCAGGTTTACA GGGAGGTCGCAATCTGATTCCCTCTTTCTACCACAATATACTCCCCTATTTGTGGTCGTCGGCATCGGGTTCTT CTTTTCGGCCCATTGGACTCTCGCTGCGCATCTGGGGGGCATCAGGGCCCTCTACTGGAGCGGATTAGGAGAC CTGATGATCATGCATATCAGTGCACTTAATCTTCTCGTACAACGAGGGAATACGCTCGGGTCATCGTACGGAAT GTGGTAAGGTTCTATGCTTATCTGTAATCCAGCGACCCTGATGTTTCTAACTTGTTTATCTCGGATAGGCTGTC ACGATTTGCCGGTACCTTGTTTGCGTTTGTGGGAGTGTATCTACGAGCATTTACTTGGCCGGACAAATGGGGA TGGTCCAATAATATAGTGATGACGTGGTTTGTTGCTGCATGGTTGCTTTTGGATATTGTGTACGGGTTCTTCTT CTGGCTCACTCGGCGAGTCGAGCAGCAGGCAAGATTCAAATCAAAGGACGGATGA SEQ ID NO: 17- efFcDNA ATGGATGGATTTGATTTTGATGCGGGTCCCCCAGAGTTCCAGCAAGTGAGGTCACTCGCGCAGGCCAACTTTT TCCTCGCCGGCGTGGGATGGCTGGCATACAACTACTTCTTGATCCGGTCAGCTTATCGCGATAGTACAGCCGG CGTGTCCCTGATTCCACTCTGCAATAACTTTGCCTGGGAGCTTACCTTCGCCCTCGTCTATTGCCCGCCAGACT TGGCTACTAAATCTGTAGGCCGGGCCTGGCTGTTTATCAATTGTTTTGTTGTGTACACCATGGTCAGGTTTACA GGGAGGTCGCAATCTGATTCCCTCTTTCTACCACAATATACTCCCCTATTTGTGGTCGTCGGCATCGGGTTCTT CTTTTCGGCCCATTGGACTCTCGCTGCGCATCTGGGGGGCATCAGGGCCCTCTACTGGAGCGGATTAGGAGAT CTGATGATCATGCATATCAGTGCACTTAATCTTCTCGTACAACGAGGGAATACGCTCGGGTCATCGTACGGAAT GTGGCTGTCACGATTTGCCGGTACCTTGTTTGCGTTTGTGGGAGTGTATCTACGAGCATTTACTTGGCCGGAC AAATGGGGATGGTCCAATAATATAGTGATGACGTGGTTTGTTGCTGCATGGTTGCmTGGATATTGTGTACG GGTTCTTCTTCTGGCTCACrCGGCGAGTCGAGCAGCAGGCAAGATTCAAATCAAAGGACGGATGA

[0401] SEQ ID NO: 18 - PetB MDGFDFDAGPPEFQQVRSLAQANFFLAGVGWLAYNYFLIRSAYRDSTAGVSLIPLCNNFAWELTFALVYCPPDLAT KSVGRAWLFINCFWYTMVRFTGRSQSDSLFLPQYTPLFWVGIGFFFSAHWTLAAHLGGIRALYWSGLGDLMIMH ISALNLLVQRGNTLGSSYGMWLSRFAGTLFAFVGVYLRAFTWPDKWGWSNNIVMTWFVAAWLLLDIVYGFFFWL TRRVEQQARFKSKDG* INDUSTRIAL APPLICATION

[0402] The invention has industrial application in the production of indole diterpene compounds derived from from C14-C15 epoxidation of 3'-geranylgeranylindole, particularly 14,15-epoxy-3'-GGI, radarin C, radarin A, roseyrin and petromindole.

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Claims

What we claim is:

1. A method of converting 3 '-geranylgeranylindole (3 '-GGI) to radarin A comprising expressing a), b) and e) in an isolated recombinant host cell, whereina) is a RadM or PetM polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3 or SEQ ID NO: 15 respectively, b) is a RadB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 6, ande) is a RadX polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 9.

2. The method of claim 1 wherein expression is heterologous expression of a), b) or e) or any combination thereof.

3. The method of claim 1 wherein expression is introduced endogenous expression of a), b) or e), or any combination thereof.

4. The method of claim 1 wherein expression of any of a), b) and / or e) is heterologous or introduced endogenous expression or any combination of both.

5. An isolated RadM, RadB, RadX, RosB, PetM or PetB polypeptide or functional variant thereof comprising at least 70% amino acid sequence identity to SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18 respectively.

6. An isolated polynucleotide or functional variant thereof encoding a RadB, RadM, RadX, RosB, PetM or PetB polypeptide of claim 5.

7. An isolated radM, radB, radX, rosB, petMor petB genomic polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively.

8. A transcription unit (TU) comprising an isolated polynucleotide of claim 7.

9. A vector that encodes an isolated polypeptide of claim 5.

10. A vector comprising an isolated polynucleotide of claim 6 or claim 7, or a TU of claim 8.

11. An isolated host cell comprising an isolated polypeptide of claim 5, an isolated polynucleotide of claim 6 or claim 7, a TU of claim 8 and / or a vector of claim 9 or claim 10.

12. A compound of Formula VI,13. A method of making anpolypeptide or functional variant thereof comprising expressing an isolated polynucleotide or functional variant thereof of claim 6 or 7, a TU of claim 8, or vector of claim 9 or 10 in an isolated host cell.

14. The method of claim 13 wherein the Aspergillus sp. is A. fresenii.

15. The method of claim 14 wherein the polypeptide or functional variant thereof is a RadM, RadB, RadX or RosB polypeptide or functional variant thereof as defined in claim 1.

16. An isolated host cell that heterologously expresses a polypeptide or functional variant thereof that catalyzes the transformation of 3 '-GGI to 14,15-epoxy-3 '-GGI.

17. The isolated host cell of claim 16 wherein the polypeptide or functional variant thereof is an epoxidase, preferably wherein the epoxidase is a RadM or PetM polypeptide or functional variant thereof as defined in claim 5.

18. The isolated host cell of claim 16 or 17 that also expresses a polypeptide or functional variant thereof that catalyzes a transformation of 14,15-epoxy-3'-GGI to radarin C.

19. The isolated host cell of claim 18 wherein the polypeptide or functional variant thereof that catalyzes a transformation of 14,15-epoxy-3'-GGI to radarin C is a cyclase, preferably wherein the cyclase is a RadB polypeptide or functional variant thereof as defined in claim 5.

20. The isolated host cell of claim 18 that also expresses a polypeptide or functional variant thereof that catalyzes the transformation of radarin C to radarin A.

21. The isolated host cell of claim 20 wherein the polypeptide or functional variant thereof that catalyzes the transformation of radarin C to radarin A is an oxygenase, preferably a cytochrome P450 oxygenase, preferably wherein the cytochrome P450 oxygenase is a RadX polypeptide or functional variant thereof as defined in claim 5.

22. An isolated strain of Penicillium paxilli, Aspergillus niger, Aspergillus oryzae, Saccharomyces cerevisiae or Escherichia coii h comprises at least one heterologous polynucleotide or functional variant thereof that encodes an enzyme in the biosynthetic pathway leading from 3'-GGI to radarin A, roseyrin or petromindole.

23. The isolated strain of claim 22 wherein the at least one heterologous polynucleotide or functional variant thereof is a radM, radB, radX, rosB, petMor petB polynucleotide or a functional variant thereof as defined in claim 6 or claim 7.

24. The isolated strain of claim 23 that comprises at least three heterologous polynucleotides or functional variants thereof that are radM, radB n radX polynucleotides or functional variants thereof as defined in claim 6 or claim 7.

25. A method of making radarin C, radarin A, roseyrin or petromindole comprising expressing a heterologous nucleic acid sequence in PeniciHium spp. or Aspergillus spp. wherein a heterologous nucleic acid sequence encodes an enzyme in a biosynthetic pathway leading from 3'-GGI to radarin C, radarin A, roseyrin or petromindole.

26. The method of claim 25 wherein the heterologous nucleic acid sequence is an isolated polynucleotide or functional variant thereof encoding a RadB, RadM, RadX, RosB, PetM or PetB polypeptide.

27. The method of claim 25 or 26 wherein the heterologous nucleic acid sequence is an isolated radM, radB, radX, rosB, petM or petB genomic polynucleotide sequence or functional variant thereof comprising at least 70% nucleic acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, or SEQ ID NO: 16 respectively.

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