Heterologous biosynthesis

Abstract

The present disclosure relates to cyclases from filamentous fungi that mediate the production of indole diterpenes bearing a tetrahydropyran (THP) ring and to isolated host cells that heterologously express such cyclases. Also disclosed are biosynthetic gene clusters encoding PaxA and PaxA equivalent cyclases that direct formation of IDTs comprising THP rings in isolated host cells, preferably isolated fungal cells, and methods of making and use.

Description

[0001] Heterologous Biosynthesis

[0002] FIELD OF THE INVENTION

[0003] This invention generally relates to novel polypeptides that catalyze at least one biochemical reaction leading to the production of indole diterpenes, polynucleotides encoding such polypeptides, methods of making such polypeptides and polynucleotides, and methods of using such polypeptides and polynucleotides to produce at least one indole diterpene compound by heterologous expression in a permissive host.

[0004] BACKGROUND

[0005] Filamentous fungi produce a diverse repertoire of interesting and useful chemical compounds. Indole diterpenes (IDTs) are a complex and structurally diverse class of filamentous fungal natural products that are comprised of an indole ring connected to a variably cyclised diterpenoid moiety.1-3Additional tailoring steps on the core scaffold then massively amplify the structural diversity and associated bioactivity of IDTs. To date, several hundred IDTs have been identified, the majority of which possess a hexacyclic paspa line-type IDT core, including a tetra hydropyran (THP) ring derived from the geranylgeranyl component situated at the opposite end of the molecule from the indole group (Figure 1). These THP-containing IDTs include the archetypical IDT paxilline, as well as the lolitrems, penitrems, shearinines, janthitrems and aflatrems. There are only a few known examples that lack this THP ring, such as the nodulisporic acids (Figure 1A). A number of IDTs are of particular interest due to their chemical diversity and concomitant bioactivities, which include anti-MRSA, anti-cancer, anti-HINl, insecticidal and tremorgenic activities.2

[0006] Many bioactive IDTs are not easily biosynthesized by their natural producers. Additionally, chemical synthesis of bioactive IDTs is long and complicated due to the complex stereochemistry of these compounds, and is therefore not commercially viable.4Accordingly, obtaining commercially useful quantities of the IDTs is challenging, and production of commercial quantities of many IDTs would be costly and laborious. There is a need in the art for new methods of IDT synthesis and / or biosynthesis that will provide useful quantities of the compounds.

[0007] It is an object of the present invention to provide a method of making an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polypeptide that catlayzes the conversion of an expoxidized IDT to an IDT bearing a THP ring and / or a method of converting an epoxidized IDT to an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polypeptide that catalyzes the converstion of an epoxidized IDT to an IDT bearing a THP ring and / or a method of making paspaline comprising expressing in an isolated host cell, a polypeptide that converts an epoxidized IDT to paspaline and / or a method of making emindole DB comprising expressing in an isolated host cell, a polypeptide that converts an epoxidized IDT to paspaline and / or to at least provide the public with a useful choice 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.

[0008] SUMMARY OF THE INVENTION

[0009] In a first aspect the invention relates to a method of converting an epoxidized indole diterpene (IDT) to an IDT bearing a tetra hydropyran (THP) ring comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to an IDT bearing a THP ring.

[0010] In a second aspect the present invention relates to a method of making an indole diterpene (IDT) bearing a tetra hydropyran (THP) ring comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of the IDT bearing a THP ring from an expoxidized IDT.

[0011] In a third aspect the invention relates to a method of converting an epoxidized IDT to an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to an IDT bearing a THP ring.

[0012] In a fourth aspect the invention relates to a method of making an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of the IDT bearing a THP ring from an expoxidized IDT.

[0013] In a fifth aspect the invention relates to a method of converting an epoxidized IDT to paspaline comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to paspaline.

[0014] In a sixth aspect the invention relates to a method of making paspaline comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0015] In a seventh aspect the invention relates to a method of converting an epoxidized IDT to paspaline comprising expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to paspaline. In an eighth aspect the invention relates to a method of making paspaline comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0016] In a ninth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to paspaline.

[0017] In a tenth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0018] In an eleventh aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide that catalyzes the conversion of an epoxidized IDT to paspaline.

[0019] In a twelfth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0020] In a thirteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus s. polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a TH P ring.

[0021] In a fourteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus spp. polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0022] In a fifteenth aspect the invention relates to an isolated recombinant host cell that comprises a heterologous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0023] In an sixteenth aspect the invention relates to an isolated recombinant host cell that comprises a heterologous polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT. In a seventeenth aspect the invention relates to an isolated recombinant host cell comprising a heterologous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring. In an eighteenth aspect the invention relates to an isolated recombinant host cell comprising a heterologous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT. In a ninteenth aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0024] In a twentieth aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0025] In a twenty-first aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0026] In a twenty-second aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0027] In a twenty-third aspect the invention relates to a method of converting an epoxidized IDT to emindole DB comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to emindole DB.

[0028] In a twenty-fourth aspect the invention relates to a method of making emindole DB comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0029] In a twenty-fifth aspect the invention relates to a method of converting an epoxidized IDT to emindole DB comprising expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to emindole DB.

[0030] In an twenty-sixth aspect the invention relates to a method of making emindole DB comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0031] In a twenty-seventh aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to emindole DB.

[0032] In a twenty-eighth aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0033] In an twenty-ninth aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide that catalyzes the conversion of an epoxidized IDT to emindole DB.

[0034] In a thirtyith aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0035] In a thirtyfirst aspect the invention relates to at least one IDT bearing a THP ring made according to a method of the invention.

[0036] In an thirtysecond aspect the invention relates to paspaline made according to a method of the invention.

[0037] In a thirtythird aspect the invention relates to emindole DB made according to a method of the invention.

[0038] 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.

[0039] 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.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The invention will now be described with reference to the figures in the accompanying drawings.

[0042] Figure 1: A) Representative IDTs with the THP ring labelled. B) The biosynthetic pathway for paxilline. Figure 2: A) General reaction showing the conversion of an epoxidized IDT to an IDT bearing a THP ring, R group indicates all positions of the indole would be modifiable, brackets could include any diterpenoid derived structure. B) Conversion of the epoxidized IDT 3', 4'-epoxyemindole SB to the IDT bearing a THP ring paspaline. C) Conversion of the epoxidized IDT 21, 22-epoxyemindole DA to the IDT bearing a THP ring emindole DB.

[0043] Figure 3: IDT biosynthetic gene clusters from Penicillium paxilli {PAX), Penicillium janthinellum {JAN), Penicillium crustosum {PTM) and Aspergillus desertorum {DES). Letters above the / ’A cluster correspond to the gene name and function of the encoded enzyme, - as shown in Figure IB.

[0044] Figure 4: Extracted Ion Chromatogram (EIC) traces showing IDT production at m / z 422 (LHS) and 436 (RHS). Maximum ion intensity counts are shown on the y-axis. A). IDT production of wildtype P. paxilli, ApaxA and a paxA complemented knockout strain. B). Complementation of P. paxilli ApaxA with alternative idtA homologs {janA, ptmA and desA).

[0045] Figure 5: Combined EIC traces showing IDT production at m / z 406 and 422 in P. paxilli strains fed with emindole SB. Ion intensity counts are shown on the y-axis (y-axis range: 0-1 x 106).

[0046] Chromatograms are annotated with the genes present in each strain.

[0047] Figure 6: EIC traces for RC337 {ApaxA and PN2013 (wildtype) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: RC337: undiluted, PN2013: 1 / 50.

[0048] Figure 7: EIC traces for RC337 {ApaxA and PN2013 (wildtype) at m / z 436 showing production of paxilline. Dilutions: RC337: undiluted, PN2013: 1 / 50.

[0049] Figure 8: EIC traces for RC356 {ApaxA: paxA) and PN2013 (wildtype) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: RC356 and PN2013: 1 / 5 (1 pl injection).

[0050] Figure 9: EIC traces for RC356 {ApaxA: paxA) and PN2013 (wildtype) at m / z 436 showing production of paxilline. Dilutions: RC356 and PN2013: 1 / 5 (1 pl injection).

[0051] Figure 10: EIC traces for MH17 paxG, paxB, paxC and paxM) and PN2258 at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: 1 / 20.

[0052] Figure 11: EIC traces for MH45 paxG, paxB, paxC, paxM and paxA) and PN2258 at m / z 422 showing production of paspaline. Dilutions: 1 / 20.

[0053] Figure 12: EIC traces for LS293 {APAX), RC370 {paxM), RC79 paxM, paxB), and RC380 {paxM, paxA) at m / z 406 and 422 showing production emindole SB, 3'4'-epoxyemindole SB and paspaline. Strains were fed with emindole SB. Figure 13: EIC traces for RC389 ApaxA:ptmA) and PN2013 (wildtype) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: RC389 and PN2013: 1 / 5 (1 pl injection).

[0054] Figure 14: EIC traces for RC389 ApaxA:ptmA) and PN2013 (wildtype) at m / z 436 showing production of paxilline. Dilutions: RC389 and PN2013: 1 / 5 (1 pl injection).

[0055] Figure 15: EIC traces for RC383 ApaxA:janA and PN2013 (wildtype) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: RC383 and PN2013: 1 / 5 (1 pl injection).

[0056] Figure 16: EIC traces for RC383 ApaxA:janA and PN2013 (wildtype) at m / z 436 showing production of paxilline. Dilutions: RC383 and PN2013: 1 / 5 (1 pl injection).

[0057] Figure 17: EIC traces for RC391 ApaxA:desA and PN2013 (wildtype) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline. Dilutions: RC391 and PN2013: 1 / 5 (1 pl injection).

[0058] Figure 18: EIC traces for RC391 ApaxA:desA and PN2013 (wildtype) at m / z 436 showing production of paxilline. Dilutions: RC391 and PN2013: 1 / 5 (1 pl injection).

[0059] Figure 19:XH NMR spectrum of 3'4'-epoxyemindole SB

[0060] Figure 20: COSY spectrum of 3'4'-epoxyemindole SB

[0061] Figure 21: HSQC spectrum of 3'4'-epoxyemindole SB

[0062] Figure 22: HMBC spectrum of 3'4'-epoxyemindole SB

[0063] Figure 23: Schematic of paxA knockout (ApaxA) strain design.

[0064] Figure 24: PCR of paxA and? M?from ApaxA transformants. A 1.5% agarose gel showing PCR amplicons for paxA (1131 bp) and paxQ (2087 bp) from DNA of ten ApaxA transformants as well as a wildtype control. A no DNA control (NC) was also included. Figure 25: PCR from ApaxA transformants. 1.5% agarose gel showing PCR amplicons that span the integration site in ApaxA strains and wildtype. A no DNA control (NC) was also included.

[0065] Figure 26: PCR of paxM, paxBan paxA from strains for feeding. 1.5% agarose gel to confirm the presence of target genes in strains for feeding with emindole SB. Strains selected for feeding are indicated with boxes. Expected amplicon sizes are shown in brackets for each gene. A no DNA control (NC) was also included. DETAILED DESCRIPTION OF THE INVENTION

[0066] DEFINITIONS

[0067] 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.

[0068] 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.

[0069] 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.

[0070] The term "epoxidized IDT" as used herein refers to any compound that is covered by the "expoxidized IDT" structure shown in Figure 2A.

[0071] An indole diterpene bearing a THP ring has the structure of an "IDT containing an oxygen bearing heterocycle" as shown in Figure 2A.

[0072] As used herein the term "PaxA equivalent cyclase" refers / means a cyclase that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT. In one embodiment the IDT bearing a THP ring is paspaline and the epoxidized IDT is 3', 4'-epoxy-emindole SB. In one embodiment the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22 epoxy-emindole DA.

[0073] The abbreviation "eeSB" as used herein means 3', 4'-epoxy-emindole SB.

[0074] The abbreviation "eeDA" as used herein means 21, 22 epoxy-emindole DA.

[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. A genetic construct may also be a polynucleotide molecule that has been produced synthetically. 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. A genetic construct may also be a polynucleotide molecule that has been produced wholly or partially by chemical synthesis.

[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.

[0077] 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.

[0078] 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.

[0079] The term "multigene construct" as used herein means a genetic construct that is a polynucleotide comprising at least two TUs.

[0080] 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.

[0081] 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,

[0082] 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, 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] The term "endogenous" as used herein refers to a constituent of a cell, tissue or organism that originates or naturally occurs 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.

[0087] 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.

[0088] "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. A 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. "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.

[0089] "Naturally occurring" as used herein with reference to a polypeptide or polynucleotide sequence as described herein refers to a primary polynucleotide sequence that is found in nature. A synthetic polypeptide or polynucleotide sequence that is identical to a wild type polypeptide or polynucleotide sequence is, for the purposes of this disclosure, considered a naturally occurring sequence. What is important for a naturally occurring polypeptide or polynucleotide sequence is that the actual sequence of amino acid or nucleotide bases that comprise the polypeptide or polynucleotide is found or known from nature.

[0090] For example, a wild type polypeptide or polynucleotide sequence is a naturally occurring amino acid or nucleic acid sequence, but not limited thereto. A naturally occurring polypeptide or polynucleotide sequence also refers to variant polypeptide or polynucleotide sequences as found in nature that differ from wild type. For example, allelic variants and naturally occurring recombinant polypeptide or polynucleotide sequences due to hybridization or horizontal gene transfer, but not limited thereto. "Non-naturally occurring" as used herein with reference to a polypeptide or polynucleotide sequence as described herein refers to a polypeptide or polynucleotide sequence that is not found in nature. A non-naturally occurring polypeptide or polynucleotide sequence is an artificial polypeptide or polynucleotide sequence. Examples of non-naturally occurring polypeptide or polynucleotide sequences include artificially produced mutant and variant polypeptide or polynucleotide sequences, made for example by point mutation, insertion, or deletion, but not limited thereto. Non-naturally occurring polypeptide or polynucleotide sequences also include chemically evolved sequences. What is important for a non-naturally occurring polypeptide or polynucleotide sequence as described herein is that the actual sequence of nucleotide bases that comprise the polypeptide or polynucleotide is not found or known from nature.

[0091] 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.

[0092] The term "coding sequence" 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 elements. The 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 "coding sequence" (CDS) is capable of being expressed when it is operably linked to a promoter sequence and / or other regulatory element(s).

[0093] "Operably-linked" means that the sequence to be expressed is placed under the control of one or more regulatory elements.

[0094] "Regulatory element(s)" 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. The term "regulatory sequence(s) means the same thing.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] "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.

[0099] "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. Such 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.

[0100] "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.

[0101] The terms "heterologously expressing" and "heterologous expression" refer to the expression of a heterologous polypeptide in a host cell. In some embodiments the host cell is an isolated host cell. In some embodiments the host cell is an isolated recombinant host cell.

[0102] A "functional variant thereof" of a polypeptide is a variant 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 refers to a polypeptide variants including polypeptides comprising variations in primary amino acid sequence as compared to the polypeptide, an aggregate of the polypeptide such as a dimer or other multimer, a fusion polypeptide comprising the polypeptide, or a polypeptide fragment of the polypeptide that comprises a subsequence of the polypeptide, wherein the "functional variant thereof" is capable of performing the polypeptide function required for the biological activity or binding of that polypeptide and / or provides the three-dimensional structure of the polypeptide. "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 at least one purification step.

[0103] "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.

[0104] 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. In some embodiments the isolated host cell is a recombinant cell. For the absence of doubt, an isolated host cell comprising a heterologous or introduced endogenous polynucleotide or polypeptide is a recombinant host cell.

[0105] The term "recombinant" in the context of a polynucleotide sequence is a polynucleotide sequence that is removed from sequences that surround it in its natural context and / or is recombined with polynucleotide sequences that are not present in its natural context. A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

[0106] 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.

[0107] 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. In these embodiments the "variant' is a "functional variant" within the meaning of the term as used herein.

[0108] 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. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

[0109] 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 nucleotide positions, preferably at least 15, 20, 27, 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.

[0110] 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.

[0111] 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. Variant polynucleotides that comprise such "silent variation" 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).

[0112] 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%, and 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. 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.

[0113] 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. Such a variant polypeptide is termed a "functional variant' herein.

[0114] 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.

[0115] 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.

[0116] Other variants include polypeptides with modifications which influence polypeptide stability. Such variants may be considered analogs and 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.

[0117] 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 etal, 1990, Science 247, 1306.

[0118] 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.

[0119] 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.

[0120] DETAILED DESCRIPTION

[0121] Disclosed herein is the inventor's work demonstrating that tetra hydropyran (THP) ring formation is catalyzed by a discrete cluster-encoded cyclase. This demonstration is contrary to all previously published biosynthetic routes that propose THP ring formation is catalysed by IdtB-type terpene cyclases.5-13The first biosynthetic gene cluster (BGC) specifying production of the paspaline-type IDT paxilline was identified in 2001 from Penicillium paxilli. Through a series of gene deletion, complementation, and heterologous reconstruction experiments four genes paxG, paxC, paxMan paxB) were identified as being sufficient to deliver paspaline (Figure IB).13Paspaline biosynthesis is initiated with the formation of geranylgeranyl pyrophosphate (GGPP) catalysed by the cluster-encoded GGPP synthase, PaxG. An indole prenyl transferase (PaxC) then catalyses the first committed step of IDT biosynthesis, producing 3’-geranylgeranylindole (3’-GGI). Construction of hexacyclic paspaline from this acyclic precursor then requires iterative cycles of activation and cyclisation.

[0122] Without wishing to be bound by theory the inventors believe the iterative cycles proceed as follows. In the first of these, the third olefinic moiety of the diterpene tail is activated through epoxidation by the flavin-dependent monooxygenase PaxM. A precise regio- and stereospecific cyclisation cascade is then catalysed by the unusual helical integral membrane terpene cyclase PaxB, producing the first stable cyclised IDT, emindole SB. Generation of the THP ring of paspaline is then primed through a second PaxM -catalysed epoxidation, this time on emindole SB, followed by an additional cyclisation step, which has been attributed by previous studies to the cyclase PaxB.5-13Two P450 monooxygenases, PaxP and PaxQ, then convert paspaline to paxilline.11-13Three additional genes are also present in the paxilline BGC: xZ?and paxO encode enzymes that further decorate the indole moiety of paxilline, and paxA, which encodes a predicted helical transmembrane protein of unknown function.

[0123] Intriguingly, homologs of paxA termed idtAsare. found in BGCs from a number of different fungi known to synthesize THP-containing IDTs (Figure 3).6'7'10'15Based on the work disclosed herein, the inventors have determined, that PaxA, rather than PaxB, is the specific cyclase that catalyzes the formation of the THP ring found in paspaline. Additionally, the inventors have further shown that a number of "PaxA equivalent cyclases" catalyze THP ring formation in the biosynthesis of IDTs that bear a THP ring, preferably paspaline or emindole DB, through heterologous expression in Penicillum paxilli. Without wishing to be bound by theory, the inventors believe that based on the work disclosed herein, PaxA equivalent cyclases catalyze the formation of an IDT bearing a THP ring from an epoxidized IDT for all IDTs that bear a THP ring. To investigate the function of PaxA the inventors created paxA deletion strains ApaxA) in P. paxilli (Figure 24). In contrast to wild type, ApaxA strains were attenuated for the production of paxilline (Figure 4, Figure 7 and accumulated an IDT not observed in wild type (Figure 4, Figure 8). This compound, with a molecular weight of 421 Da, was isolated and characterised as 3',4'-epoxyemindole SB, the proposed substrate for THP ring formation (Figure IB).11

[0124] Reintroduction of paxA into paxA strains complemented this chemotype, attenuating the 3', 4'-epoxyemindole SB peak and restoring paxilline biosynthesis (Figure 4, Figure 8, Figure 9). In a parallel study, the inventors used a biomimetic approach to construct synthetic BGCs containing paxG, paxC, paxM, paxB, and optionally paxA, which were then transformed into the PAX locus of a P. paxilli strain from which the entire / H BGC was deleted (A / H BGC). By employing this methodology, the inventors have shown that the synthetic BGC containing paxA allows a A / H BGC strain to synthesise paspaline without appreciable accumulation of 3',4'-epoxyemindole SB, whereas AZ’ IZBGC strains transformed with the synthetic BGC lacking paxA accumulated 3',4'-epoxyemindole SB (Figure 10, Figure 11). Without wishing to be bound by theory the inventors believe that these results are consistent with the hypothesis that PaxA converts 3',4'-epoxyemindole SB to paspaline. To interrogate the role of PaxA in the absence of other paxilline biosynthetic genes, the inventors carried out a series of feeding experiments using PAX strains into which paxM, paxM+ paxA, or paxM+ paxB a been reintroduced. These strains were fed with purified emindole SB, and while strains expressing paxM+ paxA efficiently converted this emindole SB to paspaline with no accumulation of 3',4'-epoxyemindole SB, strains expressing xA / alone or paxM + xFaccumulated 3',4'-epoxyemindole SB (Figure 5, Figure 12). Collectively, these results demonstrate that PaxA catalyses the conversion of 3',4'-epoxyemindole SB to paspaline in vivo, a role that had previously been incorrectly assigned to the terpene cyclase PaxB.11'13

[0125] As noted above, PaxA homologs are also encoded within IDT BGCs from related Penicillium and Aspergillus species that biosynthesise IDTs bearing a THP ring (Figure 3). Accordingly, the inventors tested the function of the PaxA homologs (also termed PaxA equivalent cyclases herein) JanA and PtmA, encoded within IDT BGCs that mediate the production of paspaline-derived janthitremane and penitrem IDTs, respectively (Figure 1A).10'15Introduction of the janA or ptmA genes into a paxA strain eliminated 3',4'-epoxyemindole SB accumulation and restored paxilline production (Figure 4, Figures 13-16), demonstrating that JanA and PtmA are functional orthologs of PaxA. Additionally, the inventors have shown that desA, which is a paxA homolog located within the emindole DA-specifying DES cluster from Aspergillus desortum, also complemented these chemotypes when added to a paxA strain (Figure 4, Figures 17-18).6This is notable as while emindole DB also contains a THP ring, it is derived from Markovnikov chemistry instead of the anti-Markovnikov chemistry that delivers paspaline, implying that DesA, and likely the other IdtAs, exhibit substrate promiscuity and may catalyse formation of a THP ring on both paspa line-type and emindole DB-type skeletons.6 While the previously proposed biosynthetic pathways for the paspaline derived IDTs included a dual role for unusual terpene cyclase PaxB in constructing both emindole SB and the full paspaline skeleton, the inventor's work disclosed herein shows that this paradigm is incorrect; a distinct cluster-encoded cyclase delivers the THP functionality.5-13

[0126] Due to their bioactive properties, there are a number of IDTs produced by various filamentous fungi that are of commercial interest. However, as described herein, the production of IDTs by chemical synthesis is difficult and costly.4The production of IDTs via heterologous biosynthesis represents a method of production that can generate commercially viable yields.

[0127] Accordingly, the present invention relates generally to the cyclase PaxA from P. paxilli n to PaxA equivalent cyclases from other filamentous fungi, respectively, that mediate the production of IDTs bearing a THP ring, to isolated host cells that heterologously express such cyclases, and to the BGC's encoding PaxA and PaxA equivalent cyclases to direct formation of IDTs comprising THP rings in an isolated host cell, preferably an isolated fungal cell. Using a recently developed technique for manipulating gene sequences termed the Modular Idempotent DNA Assembly System (MIDAS), the inventors have reconstituted the biosynthetic pathways for recombinant, heterologous or introduced endogenous expression of the cyclase, PaxA from P. paxilli. The inventors have also reconstituted the biosynthetic pathways for recombinant, heterologous expression of a representative number of PaxA equivalent cyclases from various fungi in Penicillium paxilli. The 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.

[0128] Using the efficient gene reassembly of MIDAS and heterologous expression in P. paxiiHVne inventors have demonstrated that P. paxiiH s a suitable host for heterologous expression of PaxA and / or PaxA equivalent cyclases that act to convert epoxidized IDTs to IDTs bearing a THP ring. In some embodiments the epoxidized IDTs are IDTs bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0129] In a first aspect the invention relates to a method of converting an epoxidized indole diterpene (IDT) to an IDT bearing a tetra hydropyran (THP) ring comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to an IDT bearing a THP ring.

[0130] In a second aspect the present invention relates to a method of making an indole diterpene (IDT) bearing a tetra hydropyran (THP) ring comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an expoxidized IDT. The following embodiments are specifically contemplated as embodiments of the first and second aspects of the invention.

[0131] In one embodiment the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0132] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of an amino acid sequence having at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0133] In one embodiment the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0134] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:1 (PaxA).

[0135] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:4 (JanA).

[0136] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:7 (PtmA).

[0137] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO: 10 (DesA).

[0138] In one embodiment the polypeptide or functional variant thereof comprises an amino acid motif, [SAJXEXGXsFLXWDYXgW, wherein [SA] is either a Serine (S) or Alanine (A) is present in the first position, E is Glutamic acid, G is Glycine, F is Phenylalanine, L is Leucine, W is Tryptophan, D is Aspartic, and Y is Tyrosine, X represents a single instance of any amino acid and Xnrepresents a series of n instances of any amino acid.

[0139] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an endogenous cyclase.

[0140] In one embodiment the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0141] In one embodiment the epoxidized IDT is 3', 4'-epoxyemindole SB or 21, 22-epoxyemindole DA. In one embodiment the THP ring is comprised in a hexacyclic paspaline-type indole diterpene core. In one embodiment the THP ring is derived from the geranylgeranyl component of the epoxidized IDT and is situated at the opposite end of the molecule from the indole group.

[0142] In one embodiment the IDT bearing a THP ring is paspaline or emindole DB, preferably paspaline, preferably emindole DB.

[0143] In one embodiment the IDT bearing a THP ring is paspaline and the epoxidized IDT is 3', 4'-epoxyemindole SB.

[0144] In one embodiment the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22-epoxyemindole DA.

[0145] In one embodiment the cyclase catalyzes the conversion of 3', 4'-epoxyemindole SB to paspaline. In one embodiment the cyclase is a Penicilliums. or Aspergillus s p- cyclase.

[0146] In one embodiment the cyclase that catalyzes the conversion of 3', 4'-epoxyemindole SB to paspaline is from Penicillum paxilli PK, P. janithrem (JAN), P. crustosum (PTM), or Aspergillus desertorum (DES).

[0147] In one embodiment the cyclase catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB.

[0148] In one embodiment the cyclase that catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB is from Penicillum paxilli PK, P. janithrem (JAN), P. crustosum (PTM), or Aspergillus desertorum (DES).

[0149] In one embodiment the cyclase is from Penicillum paxilli PK and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA).

[0150] In one embodiment the cyclase is from P. janithrem (JAN) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:4 (JanA).

[0151] In one embodiment the cyclase is from P. crustosum (PTM) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:7 (PtmA).

[0152] In one embodiment the cyclase is from Aspergillus desertorum (DES) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:10 (DesA).

[0153] In one embodiment the isolated host cell is a recombinant host cell. 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 contemplated herein include Pseudomonas, Bacillus, Serratia, Klebsiella, Streptomyces, Listeria, Salmonella and Mycobacteria but are not limited thereto.

[0154] In one embodiment the eukaryotic cell is an animal cell, a plant cell, a fungal cell, hyphae, or mycelia, 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.

[0155] In one embodiment the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: Penicillium, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, 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, Volvariella, Flammulina, Lentinula, Auricularia, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably Penicillium, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, Saccharomyces, Pichia, Tricopiusia, or Spondoptera.

[0156] In one embodiment, the fungal cell, hyphae or mycelia is from Penicillium paxilli, P. janthineiium, P. simpicissimum, P. crustosum, Aspergillus aiiiaceus, A. desertorum, A. fiavus, A. nominus, Epichloe festucae, Periglandula ipomoeae, Claviceps purpurea, or Aciculosporium take.

[0157] In one embodiment the fungal cell, hyphae or mycelia is from Penicillium paxilli, P. janthineiium, P. crustosum, Aspergillus fiavus, A. desertorum, Epichloe festucae, Periglandula ipomoeae, Claviceps purpurea, or Aciculosporium take.

[0158] In one embodiment the fungal cell, hyphae or mycelia is from Penicillium paxilli, P. janthineiium, P. crustosum, or Aspergillus desertorum.

[0159] In one embodiment the isolated host cell further comprises an endogenous polypeptide having the same activity as the heterologously expressed polypeptide.

[0160] In one embodiment the endogenous polypeptide is a cyclase.

[0161] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0162] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0163] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline. In one embodiment the endogenous cyclase catalyses the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0164] In one embodiment the polypeptide or functional variant thereof is expressed from an isolated polynucleotide or functional variant thereof.

[0165] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 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, or SEQ ID NO:11.

[0166] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.

[0167] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO: 12.

[0168] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12.

[0169] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:2. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 3.

[0170] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:5. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 6.

[0171] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:8. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 9.

[0172] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:11. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 12.

[0173] In one embodiment the polynucleotide or functional variant therof is a heterologous polynucleotide or functional variant thereof. In one embodiment the polynucleotide or functional variant thereof is an introduced endogenous polynucleotide or functional variant thereof.

[0174] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0175] In one embodiment the polynucleotide or functional variant thereof, nucleic acid construct or transcription unit is comprised in a vector.

[0176] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct.

[0177] In one embodiment the polynucleotide or functional variant thereof is comprised in a transcription unit (TU) or transcription unit module (TUM).

[0178] In one embodiment the polynucleotide or functional variant thereof is comprised in a vector.

[0179] In one embodiment the nucleic acid construct, TU or TUM is comprised in a vector.

[0180] In some embodiments the vector is 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.

[0181] Polynucleotides, nucleic acid constructs, TUs and TUMs 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 vitro or 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, the entirety of which is hereby incorporated by reference) and the like.

[0182] Suitable viral vectors include but are not limited to vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdovi ruses, 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. 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 at least one TU or TUM.

[0183] 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 nucleic acid construct comprising at least one TU or TUM.

[0184] In one embodiment the nucleic acid construct is a multigene construct comprising at least two TUs or TUMs.

[0185] In one embodiment, the polynucleotide or functional variant thereof is operatively linked to 5' or 3' untranslated regulatory elements in the nucleic acid construct, TU, TUM or vector. The design of a particular nucleic acid construct, TU, TUM or vector will depend on various factors including the host cells in which the operatively linked polynucleotide or functional variant thereof is to be expressed and the desired level of polynucleotide expression.

[0186] Likewise, the selection of various untranslated regulatory elements including promoters, enhancers and / or other genetic elements for a nucleic acid construct, TU, TUM or vector will depend on various factors including the host cells and expression levels discussed above. In one embodiment, the nucleic acid construct, TU, TUM or vector comprises a heterologous or a homologous promoter operatively linked to the polynucleotide or functional variant thereof. In one embodiment, the homologous promoter is an introduced endogenous promoter.

[0187] In one embodiment, the 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 the polynucleotide functional variant thereof. Many such elements are described in the literature and are available through commercial suppliers.

[0188] By way of example only, a promoter useful in a nucleic acid construct, TU, TUM or vector as described herein can be any suitable eukaryotic or prokaryotic promoter.

[0189] 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 elements (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.

[0190] Additional promoters contemplated 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.

[0191] Prokaryotic promoters contemplated 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.

[0192] Enhancers contemplated herein include SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin and the like.

[0193] In one embodiment, in the nucleic acid construct, TU, TUM or vector is driven by a T3, T7 or SP6 cytoplasmic expression system.

[0194] 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).

[0195] A functional variant of a polynucleotide (including orthologues) may be identified by the methods described. Functionally variant polynucleotides may be identified using PCR-based methods as known in the art. Typically, the polynucleotide sequence of a primer, useful to amplify functional variants of polynucleotide molecules by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.

[0196] Further methods for identifying functionlally 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.

[0197] In one embodiment the method comprises expressing at least two isolated polypeptides that catalyze the conversion of an epoxidized IDT to an IDT bearing a THP ring. Specifically contemplated here are the embodiments related to an epoxidized IDT and an IDT bearing a THP ring as set forth above. In one embodiment the at least two isolated polypeptides are cyclases.

[0198] In one embodiment one of the at least two polypeptides is an exogenous cyclase and one of the at least two polypeptides is an endogenous cyclase.

[0199] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0200] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline. In one embodiment the exogenous cyclase catalyzes the conversion of eeDA to emindole DB.

[0201] In one embodiment the endogenous cyclase catalyzes the conversion of eeDA to emindole DB. In a third aspect the invention relates to a method of converting an epoxidized IDT to an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to an IDT bearing a THP ring.

[0202] In a fourth aspect the invention relates to a method of making an IDT bearing a THP ring comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of the IDT bearing a THP ring from an expoxidized IDT.

[0203] The following embodiments are specifically contemplated as embodiments of the third or fourth aspects of the invention.

[0204] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 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, or SEQ ID NO:11.

[0205] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.

[0206] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO: 12.

[0207] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12.

[0208] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:2. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 3.

[0209] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:5. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 6. In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:8. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 9.

[0210] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:11. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO: 12.

[0211] In one embodiment the polynucleotide or functional variant therof is a heterologous polynucleotide or functional variant thereof.

[0212] In one embodiment the polynucleotide or functional variant thereof is an introduced endogenous polynucleotide or functional variant thereof.

[0213] Additionally, specifically contemplated as embodiments of the third and fourth aspects of the invention are all of the embodiments set forth in the first and second aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0214] In a fifth aspect the invention relates to a method of converting an epoxidized IDT to paspaline comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxizied IDT to paspaline.

[0215] In a sixth aspect the invention relates to a method of making paspaline comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0216] Additionally, specifically contemplated as embodiments of the fifth and sixth aspects of the invention are all of the embodiments set forth in the first and second aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing the above polypeptides including nucleic acid sequences, nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0217] In a seventh aspect the invention relates to a method of converting an epoxidized IDT to paspaline comprising expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to paspaline.

[0218] In an eighth aspect the invention relates to a method of making paspaline comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0219] Additionally, specifically contemplated as embodiments of the seventh and eighth aspects of the invention are all of the embodiments set forth in the third and fourth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0220] In a ninth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to paspaline.

[0221] In a tenth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0222] The following embodiments are specifically contemplated as embodiemnts of the ninth and tenth aspects of the invention.

[0223] In one embodiment the isolated host cell comprises an endogenous polypeptide having the same activity as the expressed introduced endogenous polypeptide.

[0224] In one embodiment the endogenous polypeptide is a cyclase.

[0225] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to paspaline. In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0226] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0227] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0228] In one embodiment the method comprises expressing at least two polypeptides or functional variants thereof that catalyze the conversion of the epoxidized IDT to paspaline.

[0229] In one embodiment the at least two polypeptides or functional variants thereof are cyclases.

[0230] In one embodiment one of the at least two polypeptides or functional variants thereof is an exogenous cyclase and one of the at least two polypeptides or functional variants thereof is an endogenous cyclase.

[0231] In one embodiment the exogenous cyclase is an introduced endogenous polypeptide or functional variant thereof.

[0232] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0233] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0234] Additionally, specifically contemplated as embodiments of the ninth and tenth aspects of the invention are all of the embodiments set forth in the first, second, fifth and sixth aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0235] In an eleventh aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide that catalyzes the conversion of an epoxidized IDT to paspaline.

[0236] In a twelfth aspect the invention relates to a method of making paspaline comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

[0237] Additionally, specifically contemplated as embodiments of the eleventh and twelfth aspects of the invention are all of the embodiments set forth in the third, fourth, seventh and eighth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0238] In a thirteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus s. polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0239] In a fourteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus spp. polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0240] The following embodiments are specifically contemplated as embodiments of the thirteenth and fourteenth aspects of the invention.

[0241] In one embodiment the isolated host cell is a recombinant host cell.

[0242] In one embodiment the isolated host cell does not comprise an endogenous polypeptide having the same activity as the expressed polypeptide.

[0243] In one embodiment the isolated host cell heterologously expresses the Penicillium or Aspergillus spp. polypeptide or functional variant thereof.

[0244] In one embodiment the isolated host cell comprises an endogenous polypeptide having the same activity as the expressed polypeptide.

[0245] In one embodiment the endogenous polypeptide is a cyclase.

[0246] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring. In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0247] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0248] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0249] In one embodiment the expressed polypeptide is an introduced endogenous polypeptide or functional variant thereof.

[0250] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0251] In one embodiment the method comprises expressing at least two polypeptides or functional variants thereof that catalyze the conversion of the epoxidized IDT to an IDT bearing a THP ring.

[0252] In one embodiment the at least two polypeptides or functional variants thereof are cyclases.

[0253] In one embodiment one of the at least two polypeptides or functional variants thereof is an exogenous cyclase and one of the at least two polypeptides or functional variants thereof is an endogenous cyclase.

[0254] In one embodiment the exogenous cyclase is an introduced endogenous polypeptide or functional variant thereof.

[0255] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0256] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0257] In one embodiment the exogenous or endogenous cyclase, or both, that catalyzes the conversion of 3', 4'-epoxyemindole SB to paspaline is from Penicillum paxilli PK, P. janithrem (JAN), P. crustosum (PTM), or Aspergillus desertorum (DES).

[0258] In one embodiment the exogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0259] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0260] In one embodiment the exogenous or endogenous cyclase, or both, that catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB is from Penicillum paxilli PPX, P. janithrem (JAN), P. crustosum (PTM), or Aspergillus desertorum (DES). Additionally, specifically contemplated as embodiments of the thirteenth and fourteenth aspects of the invention are all of the embodiments set forth in the first, second, fifth, sixth, ninth and tenth aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0261] In a fifteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus s. polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0262] In a sixteenth aspect the present invention relates to method of making an Penicillium or Aspergillus spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Penicillium or Aspergillus spp. polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0263] Additionally, specifically contemplated as embodiments of the fifteenth and sixteenth aspects of the invention are all of the embodiments set forth in the third, fourth, seventh, eighth, eleventh and twelfth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0264] In a seventeenth aspect the invention relates to an isolated recombinant host cell that comprises a heterologous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring. In an eighteenth aspect the invention relates to an isolated recombinant host cell that comprises a heterologous polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0265] In an embodiment of the seventeenth and eighteenth aspects, the heterologous polypeptide or functional variant thereof is expressed in the host cell.

[0266] Additionally, specifically contemplated as embodiments of the seventeenth and eighteenth aspects of the invention are all of the embodiments set forth in the first, second, fifth, sixth, ninth, tenth, thirteenth and fourtheenth aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0267] In a ninteenth aspect the invention relates to an isolated recombinant host cell comprising a heterologous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring. In an twentith aspect the invention relates to an isolated recombinant host cell comprising a heterologous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT. In one embodiment of the ninteenth and twentith aspects of the invention, the heterologous polynucleotide or functional variant thereof is expressed in the host cell.

[0268] Additionally, specifically contemplated as embodiments of the nineteenth and twentith aspects of the invention are all of the embodiments set forth in the third, fourth, seventh, eighth, eleventh, twelfth, fifteenth and sixteenth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells. In a twenty-first aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0269] In a twenty-second aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0270] In one embodiment of the twenty-first and twenty-second aspects of the invention, the introduced endogenous polypeptide or functional variant thereof is expressed in the host cell.

[0271] Additionally, specifically contemplated as embodiments of the twenty-first and twenty-second aspects of the invention are all of the embodiments set forth in the first, second, fifth, sixth, ninth, tenth, thirteenth, fourtheenth, seventeenth and eighteenth aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells. In a twenty-third aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0272] In a twenty-fourth aspect the invention relates to an isolated recombinant host cell that comprises an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0273] In one embodiment of the twenty-thirdand twenty-fourth aspects of the invention, the introduced endogenous polynucleotide or functional variant thereof is expressed in the host cell.

[0274] Additionally, specifically contemplated as embodiments of the twenty-third and twenty-fourth aspects of the invention are all of the embodiments set forth in the third, fourth, seventh, eighth, eleventh, twelfth, fifteenth, sixteenth, nineteenth and twentith aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA) or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0275] In a twenty-fifth aspect the invention relates to a method of converting an epoxidized IDT to emindole DB comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to emindole DB.

[0276] In a twenty-sixth aspect the invention relates to a method of making emindole DB comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0277] The following embodiments are specifically contemplated as embodiments of the twenty-fifth and twenty-sixth aspects of the invention.

[0278] In one embodiment the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0279] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of an amino acid sequence having at least 75%, preferably at least 80%, 85%, 90%, 95%, preferably at least 99% amino acid sequence identity to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0280] In one embodiment the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0281] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0282] In one embodiment the polypeptide or functional variant thereof comprises an amino acid motif, [SAJXEXGXsFLXWDYXgW, wherein [SA] is either a Serine (S) or Alanine (A) is present in the first position, E is Glutamic acid, G is Glycine, F is Phenylalanine, L is Leucine, W is Tryptophan, D is Aspartic, and Y is Tyrosine, X represents a single instance of any amino acid and Xnrepresents a series of n instances of any amino acid.

[0283] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an endogenous cyclase. In one embodiment the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0284] In one embodiment the epoxidized IDT is 21, 22-epoxyemindole DA.

[0285] In one embodiment the THP ring is derived from the geranylgeranyl component of the epoxidized IDT and is situated at the opposite end of the molecule from the indole group.

[0286] In one embodiment the IDT bearing a THP ring is emindole DB.

[0287] In one embodiment the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22-epoxyemindole DA.

[0288] In one embodiment the cyclase is an Aspergillus sp. or Penicillium spp. cyclase.

[0289] In one embodiment the cyclase catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB.

[0290] In one embodiment the cyclase is from Penicillum paxilli PK, P. janithrem (JAN), P. crustosum (PTM), or Aspergillus desertorum (DES) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

[0291] In one embodiment the isolated host cell is a recombinant host cell.

[0292] In one embodiment the isolated host cell further comprises an endogenous polypeptide having the same activity as the heterologously expressed polypeptide.

[0293] In one embodiment the endogenous polypeptide is a cyclase.

[0294] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0295] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0296] In one embodiment the endogenous cyclase catalyses the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0297] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 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, or SEQ ID NO:11. In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.

[0298] In one embodiment the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12..

[0299] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12..

[0300] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:11.. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12.

[0301] In one embodiment the method comprises expressing at least two isolated polypeptides that catalyze the conversion of an epoxidized IDT to emindole DB. Specifically contemplated here are the embodiments related to an epoxidized IDT and an IDT bearing a THP ring, preferably emindole DB, as set forth above.

[0302] In one embodiment the at least two isolated polypeptides are cyclases.

[0303] In one embodiment one of the at least two polypeptides is an exogenous cyclase and one of the at least two polypeptides is an endogenous cyclase.

[0304] In one embodiment the exogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0305] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0306] Additionally, specifically contemplated as embodiments of the twenty-fifth and twenty-sixth aspects of the invention are all of the embodiments set forth in the first, second, fifth, sixth, ninth, tenth, thirteenth, fourtheenth, seventeenth, eighteenth, twenty-first and twenty-second aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0307] In a twenty-seventh aspect the invention relates to a method of converting an epoxidized IDT to emindole DB comprising expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the conversion of the epoxidized IDT to emindole DB.

[0308] In an twenty-eighth aspect the invention relates to a method of making emindole DB comprising heterologously expressing in an isolated host cell, a polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0309] Additionally, specifically contemplated as embodiments of the twenty-seventh and twenty-eighth aspects of the invention are all of the embodiments set forth in the third, fourth, seventh, eighth, eleventh, twelfth, fifteenth, sixteenth, nineteenth, twentieth, twenty-third and twenty-fourth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0310] In a twenty-ninth aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to emindole DB.

[0311] In a thirtieth aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0312] Additionally, specifically contemplated as embodiments of the twenty-ninth and thirtieth aspects of the invention are all of the embodiments set forth in the first, second, fifth, sixth, ninth, tenth, thirteenth, fourtheenth, seventeenth, eighteenth, twenty-first, twenty-second, twenty-fifth and twenty-sixth aspects of the invention that are directed to polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0313] In an thirty-first aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide that catalyzes the conversion of an epoxidized IDT to emindole DB.

[0314] In a thirty-second aspect the invention relates to a method of making emindole DB comprising expressing in an isolated host cell, an introduced endogenous polynucleotide or functional variant thereof that encodes a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

[0315] Additionally, specifically contemplated as embodiments of the thirtyfirst and thirtysecond aspects of the invention are all of the embodiments set forth in the third, fourth, seventh, eighth, eleventh, twelfth, fifteenth, sixteenth, nineteenth, twentieth, twenty-third, twenty-fourth, twenty-seventh and twenty-eighth aspects of the invention that are directed to polynucleotides encoding and / or expressing various polypeptides including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory elements and functional variants; polypeptides comprising, consisting or consisting essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA) (including those encoded by polynucleotides) including primary amino acid sequences, % amino acid sequence identity, amino acid motifs, specified amino acid residues and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0316] In a thirty-third aspect the invention relates to at least one IDT bearing a THP ring made according to a method of the invention.

[0317] In one embodiment the IDT bearing a THP ring is paspaline or emindole DB.

[0318] In a thirty-fourth aspect the invention relates to paspaline made according to a method of the invention.

[0319] In a thirty-fifth aspect the invention relates to emindole DB made according to a method of the invention. Additionally, specifically contemplated as embodiments of the thirty-third, thirty-fourth and thirty-fifth aspects of the invention are all of the embodiments set forth above in the method aspects of the invention.

[0320] 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.

[0321] The invention will now be illustrated in a non-limiting way by reference to the following examples. EXAMPLES

[0322] Example 1 - Generation and analysis of ApaxA and reconstruction strains

[0323] Generation of AoaxA strains using CRISPR-Cas9

[0324] To investigate the function of paxA in P. paxilli, paxA knockout (ApaxA) strains were engineered using a plasmid (pRC337) designed to facilitate homologous recombination leading to the replacement of the paxA coding sequence (CDS) with a nourseoth ricin selection cassette. RC337 was created using a MIDAS level 2 reaction by combining level 1 plasmids containing homology arms (HAs) that spanned 542 bp upstream of the paxA start codon (HA1), 752 downstream of the paxA stop codon (HA2) and a nourseoth ricin selection cassette (Figure 23). To increase the efficiency of homologous recombination at the paxA locus Cas9 was added along with two sgRNAs (RCC7 and RCC8), to generate cuts at both ends of the paxA locus. RCC7 and RCC8 RNPs were transformed into wildtype (PN2013) P. paxilli protoplasts with 100 ng of RC337 and Cas9 protein. Ten transformants were selected and grown up following standard methods.

[0325] Confirmation of o3%4 by PCR and sequencing

[0326] Ten nourseoth ricin resistant transformants were screened for the presence of the paxA CDS using PCR. The complete paxA CDS was amplified with paxA_F and paxA_R primers. The axCCDS was also amplified with paxQ_fragl_F and paxQ_frag4_R primers as a control. A paxA amplicon was only observed in the wildtype sample with no amplification of paxA in any of the ten transformants (Figure 24). The paxQ control band was present in all transformants as expected.

[0327] Further PCR screening was performed to confirm RC337 had integrated as expected within the paxA locus. A 6952 bp fragment that included 173 bp upstream of the left homology arm and 2876 bp downstream of the right homology arm was amplified with the paxG_fragl_R and paxB_F primers.

[0328] 10 / 10 transformants had a band of the expected size (Figure 25). Amplicons were sequenced from RC337-6, 8 and 10 confirming integration of RC337 into the paxA locus as expected. A band of the expected size (5488 bp) was present in the wildtype sample.

[0329] LC-MS analysis of ApaxA transformants

[0330] Extracts were obtained from ten ApaxA (RC337) strains and analysed by LC-MS. All ten RC337 transformants had a 422.3 / 77 / zpeak at 9.4 min that corresponds to 3'4'-epoxyemindole SB, this peak is absent in wildtype (PN2013) (Figure 6). A peak at 12.8 min that corresponds to paspaline is also present in all transformants. A paxilline peak (436.3 m / z, 7.2 min) was not detected in 9 / 10 RC337 transformants and RC337-9 had a greatly reduced paxilline peak compared to wildtype (Figure 7). The accumulation of 3'4'-epoxyemindole SB and reduction of paxilline in ApaxA strains indicates that PaxA is important in the conversion of 3'4'-epoxyemindole SB to paspaline and that without it paxilline biosynthesis is inefficient.

[0331] LC-MS analysis of ApaxA::paxA transformants

[0332] To confirm that the presence of the 3'4'-epoxyemindole SB peak and reduction in paxilline production was due to the loss of PaxA, a ApaxA strain (RC337-6) was complemented by random integration with a plasmid containing the paxA CDS driven by the paxA promoter (RC356). Extracts were obtained from ten RC356 strains and analysed by LC-MS. Extracted ion chromatograms are shown for 422.3 / 77 / z(3'4'-epoxyemindole SB and paspaline) (Figure 8) and 436.3 m / z (paxilline) below (Figure 9). Eight out of ten RC356 transformants had no observable 3'4'-epoxyemindole SB peak. Instead, a paxilline peak was present in these transformants at 7.2 min (436.3 m / z, suggesting a restoration of a wildtype phenotype.

[0333] These experiments show that PaxA has a role in THP-ring formation and functions to convert 3', 4'-epoxyemindole SB to paspaline. This role was previously attributed to PaxB.11'13Through these experiments we have defined the role of PaxA in IDT biosynthesis.

[0334] Example 2 - Complementation with other IdtAs

[0335] Generation of ApaxA complementation strains

[0336] To confirm that other known idtAs are orthologs of paxA we complemented the ApaxA strain; RC337-6 with idtAs from IDT biosynthetic gene clusters known to produce THP-ring containing compounds. Plasmids were engineered to contain either ptmA (pRC389), janA (pRC383) or desA (pRC391) from the biosynthetic gene clusters that produce penitrems, shearinines, and emindole DB respectively. Genes were introduced by random integration and transformants selected following standard methods.

[0337] LC-MS analysis of 403x4 complementation strains

[0338] IDTs were extracted from ten strains for each idtA gene and analysed by LC-MS using standard methods. Extracted ion chromatograms are shown for 422.3 / 77 / z(3'4'-epoxyemindole SB and paspaline) (Figures 13, 15, 17) and 436.3 / 77 / z (paxilline) (Figures 14, 16, 18). Eight out of nine ApaxA::ptmA (RC389) strains lacked the 422.3 m / z peak at 9.4 min that corresponds to 3'4'-epoxyemindole SB (Figure 13). This peak is present in the ApaxA strain (RC337-6). A paxilline peak was also observed in seven of the ten RC389 strains (Figure 14), indicating that PtmA is able to complement the function of PaxA and restore paxilline production. A similar result was observed for ApaxA::janA (RC383) with all strains lacking an 3'4'-epoxyemindole SB peak and seven out of ten having a paxilline peak (Figures 15-16). A similar result was also obtained for ApaxA::desA (RC391) with no observable 3'4'-epoxyemindole SB peak in any of the strains (Figure 17) and a paxilline peak present in eight out of ten strains (Figure 18).

[0339] These experiments show that other IdtAs, including PtmA, JanA, and DesA perform the same function as PaxA in IDT biosynthesis.

[0340] Example 3 - testing of PaxA using a biomimetic strain

[0341] Generation of reconstruction strains

[0342] To further investigate the role of PaxA, plasmids were constructed containing either paxG, paxB, paxC, and A7(pMH17) or paxG, paxB, paxC, paxM, and paxA (pMH45). These plasmids were then integrated into a Z’ IXcl uster knockout (APAX) strain (LS293), which has the entire paxilline biosynthetic gene cluster replaced with a hygromycin resistance cassette and the thymine kinase gene. The plasmids were engineered with HAs designed to flank the hygromycin and thymine kinase cassette, facilitating their targeted integration into the genomic region normally occupied by the paxilline biosynthetic gene cluster. HA3 included a 999 bp fragment located between 423 bp and 1422 bp upstream of the zCstop codon, while HA4 contained a 1014 bp fragment spanning from 22 bp to 1031 bp upstream of the hypothetical protein PP122 (Gen Bank accession no. HM171111.1). pMH17 and pMH45 were transformed into LS293 protoplasts along with RNPs generated from two sgRNAs (sgRNA_LJSl and sgRNA_LJS2). Transformants were selected using both fdu for negative selection and nourseoth ricin for positive selection.

[0343] LC-MS analysis of reconstruction strains

[0344] Extracts were obtained from thirteen MH17 and MH45 strains and analysed by LC-MS. Extracted ion chromatograms are shown for 422.3 m / z (3'4'-epoxyemindole SB and paspaline) (Figures 10-11). The retention times differ in this experiment from previous experiments due to the use of the 50 x 2.1 mm column rather than the 3.0 x 100 mm column. Twelve of the thirteen MH17 transformants had a 422.3 m / z peak at 8.8 min that corresponded to 3'4'-epoxyemindole SB (Error! Reference source not found. 10). The 3'4'-epoxyemindole SB peak was not observed in the ApaxP strain (PN2258), which carries a mutation in paxP, resulting in an accumulation of paspaline (422.3 m / z, 11.9 min). In contrast, all thirteen MH45 transformants lacked an 3'4'-epoxyemindole SB peak and twelve of the thirteen transformants had a paspaline peak at 11.9 min (Figure 11). This provides further evidence that PaxA has a role in the conversion of 3'4'-epoxyemindole SB to paspaline and without PaxA an accumulation of 3'4'-epoxyemindole SB is observed.

[0345] Example 4 - Feeding studies

[0346] Generation of strains for feeding

[0347] To further interrogate the role of PaxA a feeding study was performed. All strains for feeding were built from LS239 ( / ’ ). Plasmids were engineered using the same HAs as described in example 1 and contained either axA7(pRC370) alone, xA / and xF(pRC379) or xA / and paxA (pRC380). RNPs were generated from sgRNA_LJSl and sgRNA_LJS2 to facilitate replacement of the hygromycin and thymidine kinase cassette with / H genes. PCR screening was used to ensure strains contained the target genes (Figure 26).

[0348] In iz / i / o Feeding

[0349] Three strains of RC370 (3,4 and 7), RC379 (4,5 and 6), (1,2 and 3) and LS293 were grown under standard conditions. Cultures were fed with 100 pl of 1.4 mg / ml emindole SB in methanol on days zero and four. Emindole SB was selected for feeding instead of 3'4'-epoxyemindole SB to prevent issues with compound stability.

[0350] LC-MS analysis of feeding strains

[0351] Extracts were obtained from three RC370, RC379, RC380, and LS293 strains and analysed by LC-MS. Extracted ion chromatograms for 406.3 m / z (emindole SB) and 422.3 / 77 / z(3'4'-epoxyemindole SB and paspaline) were combined and shown below (Figure 12). All chromatograms have the same y-axis range (0 - l 106). Residual emindole SB was seen in all strains with LS293 strains having the largest peaks, as expected due to a lack of enzymes to create any downstream products. RC380-2 also contained a large 3'4'-epoxyemindole SB peak, but this is likely due to a problem with gene expression in that strain as DNA from the target genes was shown to be present (Figure 26). The 3'4'-epoxyemindole SB peak was only present in RC370 and RC379 with RC380 having no evidence of 3'4'-epoxyemindole SB (Figure 12). This indicates that efficient conversion of 3'4'-epoxyemindole SB to paspaline only occurs when paxA is present. This study provides additional evidence that PaxA is required for the efficient conversion of 3'4'-epoxyemindole SB to paspaline. This experiment also shows that adding PaxB (the cyclase previously thought to catalyse THP ring formation), does not have any effect on the IDT profile.

[0352] General methods:

[0353] Bacterial protocols

[0354] Routine growth of Escherichia coii as performed at 37 °C in Luria Broth (LB). Chemically competent E. coli HST08 Stellar cells (Clontech Laboratories, Inc., 636763) and in-house DH5o cells (original stock - NEB® 5-alpha, C2988J) were used for transformation and maintenance of plasmids. Bacterial transformations were performed using standard protocols, 1 pl of plasmid was added to 25 pl of competent cells and incubated on ice for 30 min. Heat shock at 42 °C for 45 seconds was followed by immediate transfer to ice for 2 min. Then, 400 pl of SOC medium was added, and the cells were incubated at 37 °C for 1 hour with shaking. Afterward, 50-100 pl of the transformation mixture was spread onto LB agar plates containing appropriate selection agents and incubated overnight at 37 °C. Individual colonies were selected using a sterile pipette tip. Colonies were transferred to a 14 ml sterile tube containing 4 ml of LB (and appropriate selection agents) using a sterile pipette tip. The cultures were then incubated overnight at 37 °C with shaking (200 rpm).

[0355] Media and reagents used for bacterial protocols

[0356] Luria Broth:

[0357] Luria Broth was prepared by adding 25 g of Miller's LB base (Invitrogen, 12795-084) to 1 1 of deionised water and autoclaving. LB agar (LBA) was prepared by dissolving 25 g of Miller's LB base (Invitrogen, 12795-084) and 20 g of select agar (SigmaAldrich, A5054) in 1 I of deionised water and autoclaving.

[0358] SOC medium:

[0359] Super Optimal Broth with Catabolite Repression (SOC) medium was prepared as follows: 20g of Tryptone (ThermoFisher, LP0042B), 5 g of yeast extract (Oxoid, LP0021), 2 ml of 5 M sodium chloride (Sigma, S9888), and 1.25 ml of 2 M potassium chloride (Sigma Aldrich, P9333) were added to 1 I of deionised water and autoclaved. After the medium cooled, 10 ml of a 2 M filter-sterilized glucose solution (20 mM working concentration) was added, along with 5 ml each of filter-sterilized 2 M magnesium chloride (10 nM working concentration) (Sigma, M8266) and 2 M MgSO4.7H2O (10 mM working concentration) (Aaron Chemicals, AR0002CV).

[0360] Selection:

[0361] MIDAS level 1 agar plates: LBA was supplemented with Isopropyl 0-d-l-thiogalactopyranoside (IPTG) (PureScience, N1038215) at a working concentration 0.26 pM of and 5-bromo-4-chloro-3-indolyl-0-d-ga lactopyranoside (X-Gal) (PureScience, N1016210-1), at a working concentration of 53.3 pg / ml. MIDAS level 2 agar plates: LBA was supplemented with 4-chloro-dl-phenylalanine (4CP) at a working concentration of 0.25 g / l (Sigma, C6506-5G) and kanamycin (ThermoFisher, 11815032) at a working concentration of 50 pg / ml.

[0362] MIDAS level 3 agar plates: LBA was supplemented with IPTG at a working concentration of 0.26 mM and X-Gal at a working concentration of 53.3 pg / ml. Either spectinomycin (Sigma, S4014-5G) or carbenicillin (Aaron Chemicals, AR00I9G0) were also added at working concentrations of 120 pg / ml and 100 pg / ml respectively. Fungal protocols - Penicillium oaxilli protoplast preparation

[0363] The preparation of fungal protoplasts for transformation followed the protocol outlined by Yelton et al. with modifications.16Protoplasts were generated by combining 1 x 10A7 spores with 50 ml of CYDE medium in 250 ml Erlenmeyer flasks and incubating at 28 °C with shaking (200 rpm) for 31 hours. The resulting mycelia were collected and filtered through a sterile blue cloth, followed by three rinses with sterile water and one rinse with OM buffer (10 mM Na2HPO4 (SigmaAldrich, S9390-1KG) and 1.2 M MgSO4.7H2O (Aaron Chemicals, AR0002CV), adjusted to pH 5.8 with 100 mM NaH2PO4.2H2O (Vetec, V800376-500G)). The mycelia were then resuspended in 50 ml of filter-sterilized Lysing Enzyme solution (prepared by resuspending VinoTaste® Pro (Novozymes) 2 mg / ml in 50 ml OM buffer) and incubated for 18 hours at 30 °C, 80 rpm. Protoplasts were filtered through a sterile blue cloth into a 250 ml Erlenmeyer flask and further filtered through a sterile 40 pM cell strainer to remove any residual mycelia. The protoplasts were divided between two sterile 50 ml centrifuge tubes and overlaid with 10 ml of ST buffer (0.6 M sorbitol (Sigma Aldrich, S1876-1KG) and O.1 M Tris-HCI (Invitrogen, 1550420) at pH 8.0) to create a gradient, followed by centrifugation at 4 °C, 4300 rpm for 15 min. Protoplasts were then washed with STC buffer (1 M sorbitol (Sigma Aldrich, S1876-1KG), 50 mM Tris-HCI at pH 8.0, and 50 mM CaCI2 (Sigma, C3306-500G) and pelleted by centrifugation. After pooling into a single tube, the protoplasts were resuspended in STC buffer and their concentration was estimated using a hemocytometer. The protoplast stock was diluted to a final concentration of 1.25 x 10A8 protoplasts per ml of STC buffer. Aliquots of protoplasts (100 pl) were either used immediately or preserved in 8% PEG solution (80 pl of protoplasts added to 20 pl of 40% w / v PEG 4000 (Sigma, 81240) in STC buffer) in 1.7 ml micro-centrifuge tubes. Protoplasts were slowly cooled to -80 °C at 1 °C / min using a Mr. Frosty freezing container (Nalgene, C1562-1).

[0364] Fungal protocols - transformation of P. oaxilli

[0365] P. paxilli protoplasts were transformed following a method modified from Vollmer and Yanofsky and Oliver et al.17'18Transformations were conducted in 1.7 ml micro-centrifuge tubes using 100 pl (1.25xlOA7) of P. paxilli protoplasts. In a separate 1.7 ml micro-centrifuge tube, 2 pl of spermidine (50 mM in H2O) (Sigma, S2626) and 5 pl heparin (Sigma, H3393-100KU) (5 mg / mL in STC buffer) were combined with 5-15 pg of plasmid, for random integration, or 100-500 ng of plasmid for CRISPR-Cas9 transformations, with the amount of plasmid adjusted according to plasmid size. The contents of the 1.7 ml micro-centrifuge tube were then combined with the 100 pl of protoplasts and incubated on ice for 30 min. Subsequently, 900 pl of 40% PEG solution (40% w / v PEG 4000 in STC buffer) was added, and protoplasts were further incubated for 20 min. Protoplasts were then transferred to 20 ml of 0.8% RGA medium (prewarmed to 50 °C) in sterile 50 ml tubes, mixed by inversion, and 3.5 ml aliquots were dispensed onto 5 x 1.5% RGA plates. Following overnight incubation at 28 °C, 5 ml of 0.8% RGA supplemented with either nourseothricin, G418, or hygromycin was overlaid onto each plate, with selection agent concentrations adjusted to account for the agar volume of the entire plate. Plates were further incubated for 4 days at 28 °C, and spores were picked from individual colonies and streaked onto CDYE agar plates supplemented with the appropriate selection. Streaked plates were incubated at 28 °C for an additional 4 days. Spores from individual colonies were suspended in 50 pl of 0.01% v / v triton X-100 (Sigma, T8787-100ML) and spread onto 35 mm cell culture dishes containing 3 ml of RGA (containing appropriate selection). Spore plates were then incubated at 28 °C for 3-4 days, and spore stocks were prepared as follows: the top layer, containing the mycelia and spores, was removed from the 35 mm dishes and added to 3 ml of 0.01% v / v triton X-100 in glass vials. Spores were separated from the mycelia by shaking, and 800 pl of the spore suspension was mixed with 200 pl of 50% w / v glycerol in a 1.7 ml micro-centrifuge tube. Spore concentration was estimated by measuring the optical density at 600 nm of a 1 / 10 dilution, and spore stocks were adjusted to a concentration of 1 x 10A8 spores / ml. Spore stocks were either used immediately for growth in liquid culture or stored at -80 °C

[0366] Fungal protocols - Growth of P. paxilliin liquid culture (small scale)

[0367] Fungal liquid cultures were grown in 125 ml Erlenmeyer flasks sealed with cotton wool, each filled with 25 ml of CDYE medium. The flasks were inoculated with 25 pl (2.5xlOA6 spores) of spore stock and incubated at 28°C with shaking at 200 rpm for 7 days.

[0368] Media and reagents used for fungal protocols

[0369] CDYE (Czapex-Dox / Yeast extract) + trace elements medium:

[0370] Made with deionised water and contained 3.34% (w / v) Czapex-Dox (Duchefa Biochemie, C1714), 0.5% (w / v) yeast extract (Oxoid, LP0021), and 0.5% (v / v) trace element solution. For agar plates, select agar (SigmaAldrich, A5054-1KG) was added to 1.5% (w / v). Trace element solution was made in deionised water and contained 0.004% (w / v) cobalt(II) chloride hexahydrate (ThermoFisher, 1697492), 0.005% (w / v) copper(II) sulfate pentahydrate (ThermoFisher, BSPCL942.500), 0.05% (w / v) iron(II) sulfate heptahydrate (Sigma, F7002), 0.014% (w / v) manganese(II) sulfate tetrahydrate (ThermoFisher, 1683385) and 0.05% (w / v) zinc sulfate heptahydrate (Acros Organics, 205980010). The solution was preserved with 1 drop of 12 M hydrochloric acid.

[0371] Regeneration (RG) medium

[0372] Made with deionised water and contained 2% (w / v) malt extract (ThermoFisher, LP0039B), 2% (w / v) D(+)-glucose anhydrous (Scharlau, SCARGL01251000), 1% (w / v) mycological peptone (ThermoFisher, LP0040B), and 27.6% sucrose (BayStyle Crown Brands, BSWS3). Depending on whether the media was to be used for plates (1.5% RGA) or overlays (0.8% RGA), Select agar (SigmaAldrich, A5054-1KG) was added to 1.5% or 0.8% (w / v), respectively. Potato dextrose medium

[0373] Potato dextrose broth was prepared by adding 24 g of potato dextrose broth powder (Bio-strategy, SCAR02-483-500) to 1 1 of deionised water. Potato dextrose agar (PDA) was prepared by dissolving 39 g of potato dextrose agar mix (Oxoid, CM0139B) in 1 1 of deionised water.

[0374] Agar plate Selection

[0375] Agar plates were supplemented with the following selection agents: Geneticin (G418) (PureScience, G-418-10G) at a working concentration of 150 pg / ml, nourseoth ricin (Pure Science, N-500-1) at a working concentration of 100 pg / ml and hygromycin (Invitrogen, 10687010) at a working concentration of 150 pg / ml. For negative selection, 5-Fluoro-2' -deoxyuridine (fdu) (AK Scientific, J50306) was used at a working concentration of 50 pM

[0376] Chemical extraction from small scale P. paxilli cultures

[0377] Mycelia were isolated from fermentation broths by filtration through a blue cloth and washed with sterile water. Indole diterpenes (IDTs) were extracted from 850 mg of wet weight mycelia or 50 mg of freeze-dried mycelia via homogenisation with 500 pl EtOAc (ThermoFisher, E-0900-17) in a bead beating apparatus (MPBio FastPrep24 5G bead beater grinder and lysis system, 40 s, 8 m / s). The extract was recovered by centrifuging the homogenised sample at 13,000 rpm for 10 minutes and solvent removed in vacuo using a speedvac (Labconco Centrivap DNA concentrator). Extracts were resuspended in MeCN (150 pl) (ThermoFisher, M / 4000 / PC17), filtered using 0.2 pm syringe filters prior to LC-MS analysis.

[0378] Liquid Chromatography - Mass Spectrometry (LC-MS)

[0379] LC-MS was performed on an Agilent 1260 Infinity II LC-MS system with DAD and electrospray ionisation. Either a Phenomenex C18 Kinetex column (2.6 p, 100 A, 50 x 2.1 mm) equipped with a Phenomenex C18 guard cartridge or a Phenomenex C18 Kinetex column (2.6 p, 100 A, 3.0 x 100 mm) with a Phenomenex C18 guard cartridge was used. The columns were maintained at 40 °C and eluted with a mobile phase of A: H2O and B: MeCN, both containing 0.1% formic acid (ThermoFisher, Al 17-50). For the 50 x 2.1 mm column an injection volume of 10 pl and flow rate of 0.4 ml / min were used. The gradient was as follows: 0-1 min 40% B, 1-5 min 40-60% B, 5-24 min 60-90% B, 24-25 min 90-100% B, 25-27 min 100% B. For the 3.0 x 100 mm column an injection volume of 10 pl and flow rate of 0.6 ml / min were used. The gradient was as follows: 1-5 min 40-60% B, 5-24 min 60-90% B, 24-24.10 min 90-100% B, 24.10-27 min 100% B.

[0380] MIDAS protocols

[0381] MIDAS cloning was performed as described in van Dolleweerd et al (WO2019064242 - the entirety of which is hereby incorporated by reference) with the following modifications: • Protocols for MIDAS Level-1 module cloning - Esp3I (NEB, R0734L) was used as alternative to BsmBI in some reactions. All reactions were incubated overnight at 37 °C.

[0382] • Protocols for MIDAS Level-2 TU assembly - 80 ng of pML2 was combined with level 1 vectors at 1:2 molar ratio. All reactions were incubated overnight at 37 °C.

[0383] • Protocols for MIDAS Level-3 multigene assembly - Esp3I and PaqCI (NEB, R0745L) were used as alternatives to BsmBI and Aarl in some reactions. When PaqCI was used 0.5 pl of PaqCI activator was added to the reaction. 80 ng of the pML3 destination vector was combined with level 2 plasmids at a 1:2 molar ratio. Reactions were incubated overnight at 37 °C. In certain cases, an alternative level 3 destination vector with ampicillin resistance was utilised instead of the standard level 3 destination vector.

[0384] General Molecular biology - P. oaxilli

[0385] MIDAS cloning reagents including restriction endonucleases and were purchased from New England Biolabs (NEB), except Aarl, which was purchased from Thermo Fisher Scientific. T4 DNA Ligase, 10x T4 DNA Ligase buffer, and 10 mM ATP were from NEB. Primers were synthesized by Macrogen and synthetic MIDAS level 1 constructs ordered from Twist Bioscience. Monarch® Kits (NEB) were used for plasmid purification and PCR products were purified using the NucleoSpin Gel and PCR Clean-up Mini kit for gel extraction and PCR clean up (Machery-Nagel). Genomic DNA from P. paxilli was isolated using the ZR Fungal / Bacterial DNA MicroPrep Kit (Zymo Research). All PCRs for the construction of the MIDAS source, shuttle, and destination vectors and for amplification of MIDAS modules were performed using Phusion® High-Fidelity PCR Master Mix with HF Buffer (NEB). PCRs for fungal strain screening were performed with OneTaq® DNA Polymerase (NEB), LongAmp® Taq DNA Polymerase or Phusion® High-Fidelity PCR Master Mix with HF Buffer. PCR products were sequenced using the linear / Amplicon sequencing service from Plasmidsaurus. CRISPR-Cas9 ribonucleoproteins (RNPs) were generated using the reaction mixture described in Table 1. A separate reaction was performed for each sgRNA. Reactions were incubated for 15 min at room temperature prior to transformation into fungal protoplasts.

[0386] Table 1: Components of CRISPR-Cas9 reaction

[0387]

[0388] Isolation and characterisation of 3'4'-eooxyem indole SB

[0389] A ApaxA strain was grown using standard growth conditions for P. paxilli. Spores were used to inoculate 2 x 2 L shake flasks each containing 500 mL of CDYE liquid media and incubated at 28 °C and 200 rpm for 8 days. Mycelia (approx. 100 g) was separated from media by vacuum filtration and subsequently extracted overnight with EtOAc (approx. 300 mL). The extract was collected by vacuum filtration, aqueous layer removed, and organic layer concentrated in vacuo. The sample was then dissolved in MeCN (4 mL) and purified using an Agilent 1260 Infinity II preparative HPLC system equipped with DAD and a Phenomenex Luna C18250 x 15 mm 100 A 5 pm column. The mobile phase was A: H2O and B: MeCN and followed a linear gradient from 60-98% B over 30 min at 15 mL min-1. A peak eluting at 16.5 min was collected and concentrated in vacuo to give a white powder (2.1 mg). Analysis by 2D NMR spectroscopy confirmed the compound was 3'4'-epoxyemindole SB (Figures 19-22).

[0390] High-resolution mass spectrometric data and MS / MS spectra

[0391] High-resolution mass spectrometric data and MS / MS spectra were obtained with an Agilent 6530 Accurate Mass Q-TOF fitted with an electrospray ion source and equipped with an Agilent 1260 Infinity II LC system. Chromatography was carried out using an Agilent Accucore C18 2.0 pm 50 x 2.1 mm column eluted with a mobile phase of A: H2O and B: MeCN, both containing 0.1% formic acid. The flow rate was 0.3 mL / min and injection volume 5 pL. The gradient used was as follows: 0-1 min 40% B, 1-30 min 40-100% B, 30-35 min 100% B. The mass spec parameters used were: positive ion mode, mass range 100-1000 Da, acquisition rate 2 scans / s, capillary temperature 300 °C, capillary voltage 3500 V, fragmentor voltage 175 V, drying gas flow 8 L / min, sheath gas temp 350 °C, sheath gas flow 11 L / min, and a nebulizer pressure of 35 psi. MS / MS data were acquired for selected masses using CID with an isolation width of M 1.6 and collision energy of 30 eV.

[0392]

[0393] NMR spectroscopy

[0394] NMR spectroscopy was conducted on a JEOL JNM-ECZ600R with a nitrogen cooled 5 mm SuperCOOL cryogenic probe (600 MHz for 1H nuclei and 150 MHz for 13C nuclei). The residual solvent peak was used as an internal reference for 1H [6H 7.26, CDCI3] and 13C [6C 77.16, CDCI3] chemical shifts. Protein sequence information

[0395] > PaxA MTSITTSVLVLHSLLAANFKYYQSFQNGFIDMLSAMADSNSVSGLPGQLCREYTGIRPLDTFLTSCTVFFWPTFQ GEIPGLSLYGIAFASAMIPMWLI IVIDVHRRRQPFGALVELIAFAGPLIQCIGPGLVMPLLLARIHTPSRDSKSA SQFDYRTFIPSMI IGYILPLLLASLPAPLILSYHNKQQFIAIWQGWPLYSSVLMWAFRRRSGHVHCSRHKGLKHA CIFALACSSAGHLVLLSLTWLWSLSYWGYIQSAPWNEPPLASLEAGVLRFLQWDYTLSASATLSWAIAFRHEWQ QKSLRISLLSLLRCLIGIVFLGPCSMVALLYWQTCSLQEEAGQKAPAKDKQLDQEI (SEQ ID NO: 1)

[0396] > JanA MSQTTTAALISLSVFAAYAKYYQSFQNGFIALLSDMADTKSLSGLPGGLHCEYTGFAPLDRFLTACNIFFWPVFQ GEVPNLSLYGVAFASALVPMWLVIVLETHRGRRPVAALMELAFLAGPLVQCLGPGLVIPAILSRLPMSTPGTKLP FGFDIGFYPSSMI IGYILPLILAALPTPRVTAYEAKQQLIAVWQGWPVYTSLIMLI IHYLRPMRASQDWQLKIAC AFAFACSTAGHLAFLWFARAKTASYHVFLPPIPWRELQVASIEAGVLRFLQWDYTLSASAMLVWTVASYCRATGK RIGQSSSVILIFGMAGWFLGPCSVALLLYTSVALQRKGVTNYDCCRSS (SEQ ID NO: 4 )

[0397] > PtmA

[0398] SRVIRSILI ILGSVALYTKYYLSFQNGFIDLLSTMGSQGSLAGLQSGLRSHYTGLDPLDRFLKACNVFFWPIFHG TSPALSLYAIAFAGSMIPMWLILLMHTCVNSSIVEIVMINALAGLLVQGIGPGVIMCVLLAMRNTSMKEFAVTGI PAVSILGPNDLPLSLWCYILPLALSSLPAPASISVPSKQLFIAIWQGWPLYIALAVGIAHSLRNHYRRNRPQQL FRHAYAFALACS I I SHVGLLS I SFLSVSPQSPFLSLHSADLHPRSLL I PRLPWQEVKI TSLESGVLRFLHWDYS I SSTGTLLWCYDVYWKDRMRGRGWI AFFSLSSRLATMSLAFGPCSVALALYWAALSNNLMKSENARKR ( SEQ ID NO: 7)

[0399] > DesA MDVWHKLRIMLACLALVAVYTKYYLSFQNGFFPLLSLLKAQTLLPGNAGLLRTQYTGCTFLDDFFAACILFFWPV FSGSSWLLSLYALAFAGGMVPTCVILGTHAWKSANPLRSLVRNILAGIAIQSIGPGVMVPVLLALQSPTLQRTRY SASGNCISIPIAVFYGLPLFLASGAAPKIVSISMKQQLIAIWQGWSLYVLLSVPILQWVAANVSMSRLSKRPARH DRTHVYAFAFLCSTLTHWTMI CALSLVKSD I I STSCVWNSLTVPRLSWREDQVAS I EEGVQHFLQWDYS I AVSL LMWCVTLYQHHARMAPVSIETFRCLLWVMAAAFIJCGPCSAAVWIJYYEAEITVIJAKEP (SEQ ID NO: 10)

[0400] Nucleic acid Sequence information

[0401] >paxA (gDNA) ATGACATCCATCACCACGAGTGTTCTTGTTTTACACTCGCTACTTGCGGCAAACTTCAAGTATTACCAATCCTTT CAAAATGGCTTCATCGACATGTTATCAGCAATGGCCGACAGCAATTCCGTTTCGGGACTTCCCGGACAACTCTGC CGCGAGTATACAGGAATAAGGCCCCTTGATACATTCTTGACAAGCTGTACTGTATTCTTCTGGCCAACCTTTCAG GGTGAGATTCCCGGATTGTCCCTTTATGGAATCGCTTTCGCTAGTGCCATGATTCCGATGTGGTTGATAATCGTG ATCGATGTACATCGGCGCCGTCAGCCGTTTGGCGCCTTGGTGGAGTAAGCCCGCGTGCAGATCATCGGTATAGCG ACCCAAGCTGACAGGAATCGCTTCGATAGGCTCATAGCTTTTGCCGGACCTTTGATTCAATGTATTGGACCGGGC CTTGTAATGCCACTTCTCTTAGCAAGGATTCATACACCAAGTCGCGACAGCAAATCAGCTTCTCAATTCGATTAT CGCACATTCATCCCCTCAATGATCATCGGATACATCTTACCTTTACTTCTCGCCTCACTCCCAGCTCCACTTATC CTTTCGTATCATI\ATI\AGCAGCI\ATTTATTGCGATCTGGCI\AGGATGGCCCCTGTACAGTTCCGTTCTCATGTGG GCCTTCCGTCGCCGCTCGGGCCACGTACATTGTTCGCGCCATAAGGGGCTAAAGCATGCATGCATATTTGCCTTG GCTTGCTCATCTGCAGGCCACCTGGTTCTTCTGTCATTGACTTGGCTCTGGAGTTTGTCCTATTGGGGTTACATT CAATCTGCCCCATGGAACGAACCTCCATTGGCCAGTCTAGAAGCTGGCGTACTCCGCTTCCTTCAGTGGGACTAC ACGTTATCGGCTTCAGCCACGTTGAGCTGGGCGATTGCTTTCCGACACGAAGTAGTTCAACAAAAGTCCCTCCGC ATTTCGTTACTCTCACTCCTTCGTTGCCTAATTGGGATCGTTTTTCTTGGGCCATGCAGCATGGTGGCCTTGTTG TATTGGCAAACATGCTCACTGCAGGAGGAAGCTGGGCAAAAAGCGCCGGCTAAAGATAAACAACTGGACCAGGAG ATCTAG (SEQ ID NO: 2 )

[0402] >paxA (cDNA) ATGACATCCATCACCACGAGTGTTCTTGTTTTACACTCGCTACTTGCGGCAAACTTCAAGTATTACCAATCCTTT CAAAATGGCTTCATCGACATGTTATCAGCAATGGCCGACAGCAATTCCGTTTCGGGACTTCCCGGACAACTCTGC CGCGAGTATACAGGAATAAGGCCCCTTGATACATTCTTGACAAGCTGTACTGTATTCTTCTGGCCAACCTTTCAG GGTGAGATTCCCGGATTGTCCCTTTATGGAATCGCTTTCGCTAGTGCCATGATTCCGATGTGGTTGATAATCGTG ATCGATGTACATCGGCGCCGTCAGCCGTTTGGCGCCTTGGTGGAGCTCATAGCTTTTGCCGGACCTTTGATTCAA TGTATTGGACCGGGCCTTGTAATGCCACTTCTCTTAGCAAGGATTCATACACCAAGTCGCGACAGCAAATCAGCT TCTCAATTCGATTATCGCACATTCATCCCCTCAATGATCATCGGATACATCTTACCTTTACTTCTCGCCTCACTC CCAGCTCCACTTATCCTTTCGTATCATAATAAGCAGCAATTTATTGCGATCTGGCAAGGATGGCCCCTGTACAGT TCCGTTCTCATGTGGGCCTTCCGTCGCCGCTCGGGCCACGTACATTGTTCGCGCCATAAGGGGCTAAAGCATGCA TGCATATTTGCCTTGGCTTGCTCATCTGCAGGCCACCTGGTTCTTCTGTCATTGACTTGGCTCTGGAGTTTGTCC TATTGGGGTTACATTCAATCTGCCCCATGGAACGAACCTCCATTGGCCAGTCTAGAAGCTGGCGTACTCCGCTTC CTTCAGTGGGACTACACGTTATCGGCTTCAGCCACGTTGAGCTGGGCGATTGCTTTCCGACACGAAGTAGTTCAA CAAAAGTCCCTCCGCATTTCGTTACTCTCACTCCTTCGTTGCCTAATTGGGATCGTTTTTCTTGGGCCATGCAGC ATGGTGGCCTTGTTGTATTGGCAAACATGCTCACTGCAGGAGGAAGCTGGGCAAAAAGCGCCGGCTAAAGATAAA CAACTGGACCAGGAGATCTAG (SEQ ID NO: 3 )

[0403] >janA (gDNA) ATGTCGCAAACAACAACAGCCGCTCTGATTTCACTCTCTGTCTTTGCGGCCTATGCCAAGTACTACCAGTCATTC CAAAACGGCTTTATTGCTCTCTTATCTGATATGGCGGACACTAAGTCTCTATCTGGGCTACCTGGTGGACTGCAC TGCGAGTACACGGGATTTGCTCCTCTGGATCGATTCCTGACCGCATGCAATATATTCTTCTGGCCAGTATTCCAG GGAGAAGTTCCTAATTTGTCGCTATACGGAGTGGCATTCGCTAGTGCCCTAGTACCAATGTGGTTGGTGATCGTA CTCGAGACACATCGAGGAAGACGTCCCGTTGCTGCATTGATGGAGTAAGTTTTTGTCCGGTTTGCCGACGCATTG AGGTGTATTGACCGAGTTCTATACGACAGGCTAGCTTTTCTAGCCGGCCCACTAGTCCAATGCCTTGGCCCAGGA CTTGTGATACCTGCGATATTGTCGAGATTGCCTATGTCAACTCCTGGCACCAAGTTGCCTTTTGGATTCGATATA GGCTTCTATCCCTCGTCGATGATCATTGGATACATTCTCCCACTCATCCTCGCTGCATTACCCACCCCGCGTGTC ACTGCATATGAAGCAAAGCAGCAACTGATCGCAGTTTGGCAGGGTTGGCCTGTTTACACGTCACTTATCATGTTG ATCATCCACTACCTACGCCCTATGCGAGCCTCGCAAGATTGGCAGCTGAAGATAGCATGTGCGTTTGCCTTTGCA TGTTCCACTGCTGGACATCTGGCGTTTCTGTGGTTTGCTCGAGCCAAGACTGCCTCGTATCACGTTTTTCTTCCA CCGATTCCCTGGAGGGAACTGCAAGTGGCGAGTATCGAGGCGGGAGTGTTGCGATTTCTCCAATGGGACTATACG TTATCTGCATCGGCTATGCTGGTCTGGACAGTTGCCTCCTACTGTCGGGCAACCGGAAAAAGGATTGGTCAGTCT TCTTCGGTTATACTGATTTTCGGAATGGCCGGCGTGGTCTTTCTAGGGCCTTGCAGTGTGGCTTTGTTGCTGTAC ACTTCGGTTGCGTTGCAGAGAAAGGGCGTCACGAACTATGACTGTTGCAGATCATCATGA (SEQ ID NO: 5)

[0404] >janA (cDNA) ATGTCGCAAACAACAACAGCCGCTCTGATTTCACTCTCTGTCTTTGCGGCCTATGCCAAGTACTACCAGTCATTC CAAAACGGCTTTATTGCTCTCTTATCTGATATGGCGGACACTAAGTCTCTATCTGGGCTACCTGGTGGACTGCAC TGCGAGTACACGGGATTTGCTCCTCTGGATCGATTCCTGACCGCATGCAATATATTCTTCTGGCCAGTATTCCAG GGAGAAGTTCCTAATTTGTCGCTATACGGAGTGGCATTCGCTAGTGCCCTAGTACCAATGTGGTTGGTGATCGTA CTCGAGACACATCGAGGAAGACGTCCCGTTGCTGCATTGATGGAGCTAGCTTTTCTAGCCGGCCCACTAGTCCAA TGCCTTGGCCCAGGACTTGTGATACCTGCGATATTGTCGAGATTGCCTATGTCAACTCCTGGCACCAAGTTGCCT TTTGGATTCGATATAGGCTTCTATCCCTCGTCGATGATCATTGGATACATTCTCCCACTCATCCTCGCTGCATTA CCCACCCCGCGTGTCACTGCATATGAAGCAAAGCAGCAACTGATCGCAGTTTGGCAGGGTTGGCCTGTTTACACG TCACTTATCATGTTGATCATCCACTACCTACGCCCTATGCGAGCCTCGCAAGATTGGCAGCTGAAGATAGCATGT GCGTTTGCCTTTGCATGTTCCACTGCTGGACATCTGGCGTTTCTGTGGTTTGCTCGAGCCAAGACTGCCTCGTAT CACGTTTTTCTTCCACCGATTCCCTGGAGGGAACTGCAAGTGGCGAGTATCGAGGCGGGAGTGTTGCGATTTCTC CAATGGGACTATACGTTATCTGCATCGGCTATGCTGGTCTGGACAGTTGCCTCCTACTGTCGGGCAACCGGAAAA AGGATTGGTCAGTCTTCTTCGGTTATACTGATTTTCGGAATGGCCGGCGTGGTCTTTCTAGGGCCTTGCAGTGTG GCTTTGTTGCTGTACACTTCGGTTGCGTTGCAGAGAAAGGGCGTCACGAACTATGACTGTTGCAGATCATCATGA

[0405] (SEQ ID NO: 6 )

[0406] >ptmA (gDNA) ATGTCGCGTGTCATACGATCTATATTGATCATTCTCGGATCCGTGGCCCTATATACAAAGTATTATTTATCCTTT CAAAATGGCTTCATTGATCTTCTGTCGACCATGGGCTCTCAGGGCTCTCTTGCTGGACTTCAAAGTGGTCTTCGG TCACATTATACTGGCTTGGACCCACTTGATAGATTCCTAAAGGCATGCAACGTTTTCTTCTGGCCAATTTTCCAT GGTACCTCGCCTGCTTTATCCCTCTATGCGATTGCATTCGCGGGCTCTATGATCCCGATGTGGCTGATCCTCCTC ATGCATACGTGCGTGAACAGCTCAATTGTGGAAATAGTCATGATGTACGTATGTACTCACATTGTATAGCGTTAT ACTGATCTTCCAAAATTAGAAATGCCCTCGCCGGTCTGCTGGTGCAAGGAATCGGTCCAGGGGTGATTATGTGTG TTCTACTAGCAATGAGAAATACATCCATGAAAGAGTTTGCCGTCACCGGTATACCAGCGGTATCTATACTGGGGC CAAATGATCTTCCACTGTCACTGGTCGTCTGTTATATACTGCCCTTAGCCTTGAGCAGCCTTCCAGCGCCTGCAA GCATATCTGTTCCGTCTAAGCAATTATTTATTGCCATTTGGCAAGGATGGCCCCTTTATATTGCACTTGCCGTTG GAATCGCACACTCCTTAAGGAATCACTATAGACGAAATCGCCCACAGCAACTGTTCAGGCATGCTTACGCCTTTG CCCTTGCGTGTTCCATCATCAGCCACGTTGGTCTTCTTTCGATCTCATTCCTTTCGGTATCTCCTCAGAGCCCTT TCTTGTCACTGCACTCGGCAGACCTTCACCCACGGAGCCTTTTGATCCCCAGACTTCCATGGCAGGAGGTGAAAA TTACATCTCTAGAGTCTGGAGTCCTTAGGTTTTTGCACTGGGACTATAGTATCTCATCTACGGGAACTCTGCTGT GGTGCTATGATGTGTATTGGAAAGACAGAATGAGAGGCAGGGGGTGGATCGCTTTTTTCTCTCTTTCATCTCGAC TAGCTACAATGAGCCTGGCGTTTGGTCCTTGTAGTGTAGCACTTGCACTCTACTGGGCAGCATTGTCAAATAATT TGATGAAGAGCGAGAATGCAAGGAAGAGATAA (SEQ ID NO: 8 )

[0407] >ptmA (cDNA) ATGTCGCGTGTCATACGATCTATATTGATCATTCTCGGATCCGTGGCCCTATATACAAAGTATTATTTATCCTTT CAAAATGGCTTCATTGATCTTCTGTCGACCATGGGCTCTCAGGGCTCTCTTGCTGGACTTCAAAGTGGTCTTCGG TCACATTATACTGGCTTGGACCCACTTGATAGATTCCTAAAGGCATGCAACGTTTTCTTCTGGCCAATTTTCCAT GGTACCTCGCCTGCTTTATCCCTCTATGCGATTGCATTCGCGGGCTCTATGATCCCGATGTGGCTGATCCTCCTC ATGCATACGTGCGTGAACAGCTCAATTGTGGAAATAGTCATGATAAATGCCCTCGCCGGTCTGCTGGTGCAAGGA ATCGGTCCAGGGGTGATTATGTGTGTTCTACTAGCAATGAGAAATACATCCATGAAAGAGTTTGCCGTCACCGGT ATACCAGCGGTATCTATACTGGGGCCAAATGATCTTCCACTGTCACTGGTCGTCTGTTATATACTGCCCTTAGCC TTGAGCAGCCTTCCAGCGCCTGCAAGCATATCTGTTCCGTCTAAGCAATTATTTATTGCCATTTGGCAAGGATGG CCCCTTTATATTGCACTTGCCGTTGGAATCGCACACTCCTTAAGGAATCACTATAGACGAAATCGCCCACAGCAA CTGTTCAGGCATGCTTACGCCTTTGCCCTTGCGTGTTCCATCATCAGCCACGTTGGTCTTCTTTCGATCTCATTC CTTTCGGTATCTCCTCAGAGCCCTTTCTTGTCACTGCACTCGGCAGACCTTCACCCACGGAGCCTTTTGATCCCC AGACTTCCATGGCAGGAGGTGAAAATTACATCTCTAGAGTCTGGAGTCCTTAGGTTTTTGCACTGGGACTATAGT ATCTCATCTACGGGAACTCTGCTGTGGTGCTATGATGTGTATTGGAAAGACAGAATGAGAGGCAGGGGGTGGATC GCTTTTTTCTCTCTTTC TCTCG CT GCT C TG GCCTGGCGTTTGGTCCTTGT GTGT GC CTTGC CTC TACTGGGCAGCATTGTCAAATAATTTGATGAAGAGCGAGAATGCAAGGAAGAGATAA (SEQ ID NO: 9)

[0408] >desA (gDNA) ATGGATGTCTGGCACAAATTGAGAATCATGCTGGCGTGCCTGGCGCTGGTGGCAGTCTACACCAAGTATTATTTG TCCTTTCAAAACGGTTTTTTTCCATTGCTCTCCTTACTCAAGGCTCAGACATTACTTCCTGGGAACGCAGGGCTG TTAAGAACACAGTACACGGGGTGCACCTTCCTGGACGACTTCTTTGCCGCATGTATCCTCTTCTTTTGGCCGGTC TTCAGTGGAAGTTCATGGTTGTTGTCACTCTACGCCCTCGCCTTTGCGGGTGGCATGGTACCAACTTGTGTCATA CTGGGCACCCATGCCTGGAAGAGCGCGAATCCACTCAGGTCCCTCGTTCGGTACGTCACAACTGACCAAATACTT GATCGGCATGTTGATATGTCAAAACAGAAACATACTCGCGGGCATTGCAATTCAGAGCATAGGCCCTGGAGTAAT GGTCCCCGTCCTCTTGGCCTTGCAGTCGCCGACTTTGCAGCGCACACGTTACAGTGCTTCTGGGAACTGCATATC CATTCCCATTGCCGTATTCTATGGATTGCCATTATTTCTGGCATCGGGTGCCGCTCCCAAAATAGTCTCAATCAG CATGAAGCAACAGCTGATTGCTATTTGGCAAGGGTGGTCATTGTATGTCTTACTCTCGGTGCCCATATTGCAATG GGTCGCAGCCAACGTGTCAATGTCTAGGTTGTCCAAACGACCTGCAAGACATGACAGAACGCATGTATATGCATT TGCGTTTCTCTGCTCCACACTTACCCATTGGACTATGATCTGTGCTCTGTCTCTCGTCAAATCAGACATCATTTC GACCAGTTGTGTTTGGAATTCCCTCACTGTGCCCAGGCTATCCTGGCGAGAGGATCAGGTGGCGAGTATTGAGGA AGGTGTGCAGCATTTTCTCCAATGGGATTATAGCATTGCTGCTGTCTCGCTATTAATGTGGTGCGTGACTCTATA CCAACACCACGCGAGAATGGCTCCGGTGTCCATCGAGACTTTTCGCTGTCTCCTGTGGGTGATGGCAGCGGCGTT CCTTTGTGGGCCCTGTAGTGCGGCCGTATGGCTGTACTACGAAGCCGAGATTACCGTCTTAGCGAAGGAGCCATA

[0409] G (SEQ ID NO: 11)

[0410] >desA (cDNA) ATGGATGTCTGGCACAAATTGAGAATCATGCTGGCGTGCCTGGCGCTGGTGGCAGTCTACACCAAGTATTATTTG TCCTTTCAAAACGGTTTTTTTCCATTGCTCTCCTTACTCAAGGCTCAGACATTACTTCCTGGGAACGCAGGGCTG TTAAGAACACAGTACACGGGGTGCACCTTCCTGGACGACTTCTTTGCCGCATGTATCCTCTTCTTTTGGCCGGTC TTCAGTGGAAGTTCATGGTTGTTGTCACTCTACGCCCTCGCCTTTGCGGGTGGCATGGTACCAACTTGTGTCATA CTGGGCACCCATGCCTGGAAGAGCGCGAATCCACTCAGGTCCCTCGTTCGAAACATACTCGCGGGCATTGCAATT CAGAGCATAGGCCCTGGAGTAATGGTCCCCGTCCTCTTGGCCTTGCAGTCGCCGACTTTGCAGCGCACACGTTAC AGTGCTTCTGGGAACTGCATATCCATTCCCATTGCCGTATTCTATGGATTGCCATTATTTCTGGCATCGGGTGCC GCTCCCAAAATAGTCTCAATCAGCATGAAGCAACAGCTGATTGCTATTTGGCAAGGGTGGTCATTGTATGTCTTA CTCTCGGTGCCCATATTGCAATGGGTCGCAGCCAACGTGTCAATGTCTAGGTTGTCCAAACGACCTGCAAGACAT GACAGAACGCATGTATATGCATTTGCGTTTCTCTGCTCCACACTTACCCATTGGACTATGATCTGTGCTCTGTCT CTCGTCAAATCAGACATCATTTCGACCAGTTGTGTTTGGAATTCCCTCACTGTGCCCAGGCTATCCTGGCGAGAG GATCAGGTGGCGAGTATTGAGGAAGGTGTGCAGCATTTTCTCCAATGGGATTATAGCATTGCTGCTGTCTCGCTA TTAATGTGGTGCGTGACTCTATACCAACACCACGCGAGAATGGCTCCGGTGTCCATCGAGACTTTTCGCTGTCTC CTGTGGGTGATGGCAGCGGCGTTCCTTTGTGGGCCCTGTAGTGCGGCCGTATGGCTGTACTACGAAGCCGAGATT ACCGTCTTAGCGAAGGAGCCATAG (SEQ ID NO: 12 )

[0411] sqRNAs:

[0412] > RCC7

[0413] GUGUAAAACAAGAACACUCG (SEQ ID NO: 13 )

[0414] > RCC8

[0415] UAAAGAUAAACAACUGGACC (SEQ ID NO: 14 )

[0416] >sgRNA_LJSl

[0417] AGCAAACGGUGGUCAAAGGA (SEQ ID NO: 15)

[0418] >sgRNA_LJS2

[0419] CGGCACUGGCAAUAACAAGC (SEQ ID NO: 16 )

[0420] Primers

[0421] >paxG_f ragl_R (8 )

[0422] CTTCTACGTCTCGTACTGTTCTIkATCGTGCTTGGTG (SEQ ID NO: 17)

[0423] >paxB_F (21)

[0424] CGATGTACGTCTCACTCGAATGGACGGTTTTGATGTTTCCCAA (SEQ ID NO: 18 )

[0425] >paxA_F

[0426] CGATGTACGTCTCACTCGAATGACATCCATCACCACGAGTG (SEQ ID NO: 19)

[0427] >paxA_R

[0428] GACCTTTCGTCTCTGTCTCAAAGCCTAGATCTCCTGGTCCAGTTGTTTATCTTTAGCC (SEQ ID NO: 20) > paxQ_fragl_F

[0429] CGATGTACGTCTCACTCGAATGGATTTCGTGTTATCAGCCTTAC (SEQ ID NO: 21)

[0430] > paxQ_frag4_R

[0431] GACCTTTCGTCTCTGTCTCAAAGCTCATGCCGAGACAGACTTTCTG (SEQ ID NO: 22 ) Table 2: Plasmid components:

[0432]

[0433]

[0434]

[0435] All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0436] Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e. g., all exact exemplary values provided with respect to a particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate).

[0437] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0438] The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise indicated. No language in the specification should be construed as indicating any element is essential to the practice of the invention unless as much is explicitly stated.

[0439] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability and / or enforceability of such patent documents.

[0440] The description herein of any aspect or embodiment of the invention using terms such as reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of," "consists essentially of" or "substantially comprises" that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). This invention includes all modifications and equivalents of the subject matter recited in the aspects or claims presented herein to the maximum extent permitted by applicable law. All publications and patent applications cited in this specification are herein incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0441] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0442] INDUSTRIAL APPLICATION

[0443] The invention has industrial application in the production of indole diterpene compounds bearing a THP ring, particularly paspaline and emindole DB.

[0444] REFERENCES

[0445] 1. Ozaki, T.; Minami, A.; Oikawa, H., Biosynthesis of indole diterpenes: a reconstitution approach in a heterologous host. Natural Product Reports 2023, 40(1), 202-213.

[0446] 2. Niu, J.; Qi, J.; Wang, P.; Liu, C.; Gao, J. M., The chemical structures and biological activities of indole diterpenoids. Natural Products and Bioprospecting 2023, 5(1), 3.

[0447] 3. Jiang, M.; Wu, Z.; Liu, L.; Chen, S., The chemistry and biology of fungal meroterpenoids (2009-2019). Organic & Biomoiecuiar Chemistry 2021, 19 (8), 1644-1704.

[0448] 4. Zou, Y.; Smith, A. B., Total synthesis of architecturally complex indole terpenoids: strategic and tactical evolution. The Journal of Antibiotics 2018, 71 (2), 185-204.

[0449] 5. Hibbard, T. R.; McLellan, R. M.; Stevenson, L. J.; Richardson, A. T.; Nicholson, M. J.; Parker, E. J., Functional Crosstalk between Discrete Indole Terpenoid Gene Clusters in Tolypocladium album. Organic Letters 2023, 25 (41), 7470-7475.

[0450] 6. Bundela, R.; Cameron, R. C.; Singh, A. J.; McLellan, R. M.; Richardson, A. T.; Berry, D.; Nicholson, M. J.; Parker, E. J., Generation of Alternate Indole Diterpene Architectures in Two Species of Aspergilli. Journal of the American Chemical Society 2023, 145(5), 2754-2758.

[0451] 7. McLellan, R. M.; Cameron, R. C.; Nicholson, M. J.; Parker, E. J., Aminoacylation of Indole Diterpenes by Cluster-Specific Monomodular NRPS-like Enzymes. Organic Letters 2022, 2 (12), 2332-2337.

[0452] 8. Jiang, Y.; Ozaki, T.; Harada, M.; Miyasaka, T.; Sato, H.; Miyamoto, K.; Kanazawa, J.; Liu, C.; Maruyama, J.-i.; Adachi, M.; Nakazaki, A.; Nishikawa, T.; Uchiyama, M.; Minami, A.; Oikawa, H., Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton. Angewandte Chemie International Edition 2020, 59(41), 17996-18002.

[0453] 9. Tang, M.-C.; Lin, H.-C.; Li, D.; Zou, Y.; Li, J.; Xu, W.; Cacho, R. A.; Hillenmeyer, M. E.; Garg, N. K.; Tang, Y., Discovery of Unclustered Fungal Indole Diterpene Biosynthetic Pathways through Combinatorial Pathway Reassembly in Engineered Yeast. Journal of the American Chemical Society 2015, 757(43), 13724-13727.

[0454] 10. Liu, C.; Tagami, K.; Minami, A.; Matsumoto, T.; Frisvad, 1 C.; Suzuki, H.; Ishikawa, J.; Gomi, K.; Oikawa, H., Reconstitution of biosynthetic machinery for the synthesis of the highly elaborated indole diterpene penitrem. Angewandte Chemie International Edition 2015, 54(19), 5748-52.

[0455] 11. Tagami, K.; Liu, C.; Minami, A.; Noike, M.; Isaka, T.; Fueki, S.; Shichijo, Y.; Toshima, H.; Gomi, K.; Dairi, T.; Oikawa, H., Reconstitution of Biosynthetic Machinery for Indole-Diterpene Paxilline in Aspergillus oryzae. Journal of the American Chemical Society 2013, 755(4), 1260-1263.

[0456] 12. Saikia, S.; Parker, E. J.; Koulman, A.; Scott, B., Defining paxilline biosynthesis in Penicillium paxiiii: functional characterization of two cytochrome P450 monooxygenases. Journal of Biological Chemistry 2007, 252(23), 16829-16837.

[0457] 13. Saikia, S.; Parker, E. J.; Koulman, A.; Scott, B., Four gene products are required for the fungal synthesis of the indole-diterpene, paspaline. FEBS Letters 2006, 555(6), 1625-30.

[0458] 14. Young, C.; McMillan, L.; Telfer, E.; Scott, B., Molecular cloning and genetic analysis of an indole-diterpene gene cluster from Penicillium paxilli. Molecular Microbiology 2001, 55(3), 754-64.

[0459] 15. Nicholson, M. J.; Eaton, C. J.; Starkel, C.; Tapper, B. A.; Cox, M. P.; Scott, B., Molecular Cloning and Functional Analysis of Gene Clusters for the Biosynthesis of Indole-Diterpenes in Penicillium crustosum and P. janthinellum. Toxins 2015, 7(8), 2701-22.

[0460] 16. Yelton, M. M.; Hamer, J. E.; Timberlake, W. E., Transformation of Aspergillus nidulans by using a trpC plasmid. Proceedings of the National Academy of Sciences of the United States of America 1984, 57 (5), 1470-4.

[0461] 17. Oliver, R. P.; Roberts, I. N.; Harling, R.; Kenyon, L.; Punt, P. J.; Dingemanse, M. A.; van den Hondel, C. A. M. J. J., Transformation of Fulvia fulva, a fungal pathogen of tomato, to hygromycin B resistance. Current Genetics 1987, 72(3), 231-233.

[0462] 18. Vollmer, S. J.; Yanofsky, C., Efficient cloning of genes of Neurospora crassa. Proceedings of the National Academy of Sciences of the United States of America 1986, 55(13), 4869-73.

[0463] 19. Richardson, A. T.; Cameron, R. C.; Stevenson, L. J.; Singh, A. J.; Lukito, Y.; Berry, D.; Nicholson, M. J.; Parker, E. J., Biosynthesis of Nodulisporic Acids: A Multifunctional Monooxygenase Delivers a Complex and Highly Branched Array. Angewandte Chemie International Edition 2022.

[0464] 20. Wymelenberg, A. J. V.; Cullen, D.; Spear, R. N.; Schoenike, B.; Andrews, J. H., Expression of Green Fluorescent Protein in Aureobasidium pullulans and Quantification of the Fungus on Leaf Surfaces. BioTechniques 1997, 25(4), 686-690.

[0465] 21. Malonek, S.; Rojas, M. C.; Hedden, P.; Gaskin, P.; Hopkins, P.; Tudzynski, B., The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. Journal of Biological Chemistry 2004, 279 (24), 25075-84.

Claims

What we claim is:

1. A method of making an indole diterpene (IDT) bearing a tetra hydropyran (THP) ring comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an expoxidized IDT.

2. The method of claim 1 wherein the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

3. The method of claim 1 or claim 2 wherein the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

4. The method of any one of claims 1 to 3 wherein the polypeptide or functional variant thereof comprises an amino acid motif, [SAJXEXGXsFLXWDYXgW, wherein [SA] is either a Serine (S) or Alanine (A) is present in the first position, E is Glutamic acid, G is Glycine, F is Phenylalanine, L is Leucine, W is Tryptophan, D is Aspartic, and Y is Tyrosine, X represents a single instance of any amino acid and Xnrepresents a series of n instances of any amino acid.

5. The method of anyone of claims 1 to 4 wherein the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an introduced endogenous cyclase.

6. The method of anyone of claims 1 to 5 wherein the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule, preferably wherein the epoxidized IDT is 3', 4'-epoxyemindole SB or 21, 22-epoxyemindole DA.

7. The method of anyone of claims 1 to 6 wherein the THP ring is derived from the geranylgeranyl component of the epoxidized IDT and is situated at the opposite end of the molecule from the indole group.

8. The method of anyone of claims 1 to 7 wherein the IDT bearing a THP ring is paspaline or emindole DB, preferably paspaline, preferably emindole DB.

9. The method of anyone of claims 1 to 8 wherein the isolated host cell is a prokaryotic or eukaryotic cell, preferably wherein the eukaryotic cell is a fungal cell, hyphae, or mycelia, preferably wherein the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: Penicillium, Aspergillus, Epichioe, Periglandula, Claviceps, Aciculosporium, Trichoderma, Neurospora, Fusarium, Mortiereiia, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichopiusia, Ashbya, Hansenuia, Pichia, Kiuveromyces, Schizzosaccharomyces, Monascus, Taiaromyces, Cryptonectria, Endothia, Toiypociadium, Hypocrea, Gibbereiia,Acremonium, Agaricus, Pleurotus, Volvariella, Flammulina, Lentinula, Auricularia, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably PeniciHium, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, Saccharomyces, Pichia, Tricoptusia, or Spondoptera, preferably from PeniciHium paxiiii, P. janthineiium, P. crustosum, or Aspergillus desertorum.

10. The method of anyone of claims 1 to 9 wherien the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 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, or SEQ ID NO:11.

11. The method of anyone of claims 1 to 9 wherien the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO:12.

12. A method of making paspaline comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of paspaline from an epoxidized IDT.

13. A method of making emindole DB comprising heterologously expressing in an isolated host cell, a polypeptide or functional variant thereof that catalyzes the formation of emindole DB from an epoxidized IDT.

14. The method of claim 12 or claim 13 wherein the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

15. The method of of any one of claims 12 to 14 wherein the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (PaxA), SEQ ID NO:4 (JanA), SEQ ID NO:7 (PtmA), or SEQ ID NO: 10 (DesA).

16. The method of any one of claims 12 to 15 wherein the polypeptide or functional variant thereof comprises an amino acid motif, [SAJXEXGXsFLXWDYXgW, wherein [SA] is either a Serine (S) or Alanine (A) is present in the first position, E is Glutamic acid, G is Glycine, F is Phenylalanine, L is Leucine, W is Tryptophan, D is Aspartic, and Y is Tyrosine, X represents a single instance of any amino acid and Xnrepresents a series of n instances of any amino acid.

17. The method of anyone of claims 12 to 16 wherein the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an introduced endogenous cyclase.

18. The method of anyone of claims 12 to 17 wherein the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule, preferably wherein the epoxidized IDT is 3', 4'-epoxyemindole SB.

19. The method of anyone of claims 12 to 18 wherein the THP ring is derived from the geranylgeranyl component of the epoxidized IDT and is situated at the opposite end of the molecule from the indole group.

20. The method of anyone of claims 12 to 19 wherein the isolated host cell is a prokaryotic or eukaryotic cell, preferably wherein the eukaryotic cell is a fungal cell, hyphae, or mycelia, preferably wherein the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: Penicillium, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, 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, Voivarieiia, Fiammuiina, Lentinuia, Auricuiaria, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably Penicillium, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, Saccharomyces, Pichia, Tricopiusia, or Spondoptera, preferably from Penicillium paxi / ii, P. janthineiium, P. crustosum, or Aspergillus desertorum.

21. The method of anyone of claims 12 to 20 wherien the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 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, or SEQ ID NO:11.

22. The method of anyone of claims 12 to 21 wherien the isolated polynucleotide or functional variant thereof comprises at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, preferably at least 99% nucleic acid sequence identity to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, or SEQ ID NO: 12.

Images