Heterologous biosynthesis

Abstract

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. Specifically, the invention relates to a method of converting an epoxidized indole diterpene (IDT) to an IDT bearing a tetrahydropyran (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.

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 to at least provide the public with a useful choice 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.

[0008] 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.

[0009] SUMMARY OF THE INVENTION

[0010] 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.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] 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. 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In a thirteenth aspect the present invention relates to method of making an Epichioe or Microsera spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Epichloe o Microsera spp. polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0022] In a fourteenth aspect the present invention relates to method of making an Epichloe ox Microsera spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Epichioe ox Microsera spp. polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0023] 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. 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In a thirtyfirst aspect the invention relates to at least one IDT bearing a THP ring made according to a method of the invention.

[0039] In an thirtysecond aspect the invention relates to paspaline made according to a method of the invention.

[0040] In a thirtythird aspect the invention relates to emindole DB made according to a method of the invention.

[0041] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth. 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.

[0042] 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.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The invention will now be described with reference to the figures in the accompanying drawings.

[0045] Figure 1: A) Representative IDTs with the THP ring labelled. B) The lolitrem biosynthetic pathway.

[0046] 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.

[0047] Figure 3: Alignment of biosynthetic gene clusters (BGCs) anchored around / Zt / Sgenes, highlighting synteny and gene similarity across different idtS containing clusters using Clinker.5BGCs are from Tolyplocadium album ( TER), Microcera sp. (MIC) and Epichloe festucae (LTM). Letters below the LTM cluster correspond to gene names and functions of the encoded enzyme.

[0048] Figure 4: A) Schematic representation of the wild-type and Zl / mS'loci. In the wild-type locus, the regions designated as PltmS and TltmS were used as homology arms for the construction of the AltmS plasmid (pYL413). The AltmS locus has the selection cassette integrated in place of the ItmS CDS. The positions of the primers used for PCR screening (805, 806, 807, 808, 823, and 824) are also indicated. B) PCR screening of twenty potential AltmS strains. Primer pair 805 and 806 produce a 1,345 bp band for the wild-type locus and a 2,205 bp band for AltmS. Primer pair 807 and 808 produce a 417 bp band when the / mSCDS is present and no band for an AltmS strain. C) Further PCR screening of strains one and three using the 823 / 824 primer pair. A 3,277 band is produced for wild-type and a 3,650 bp band is produced when the ItmS CDS has been removed.

[0049] Figure 5: EIC traces for YL413 (AltmS) at m / z 22 showing production of 3'4'-epoxyemindole SB and paspaline.

[0050] Figure 6: EIC traces for wild-type at m / z 22 showing production of 3'4'-epoxyemindole SB and paspaline.

[0051] Figure 7: EIC traces for YL413 (AltmS) at / 77 / z686 showing production of lolitrem B.

[0052] Figure 8: EIC traces for wild-type at m / zSSS showing production of lolitrem B. Figure 9: EIC traces for RC358 (ApaxA::ltmS) at m / z 422 showing production of 3'4'-epoxyemindole SB and paspaline.

[0053] Figure 10: EIC traces for RC358 ( paxA ltmS) at m / z 436 showing production of paxilline.

[0054] Figure 11: EIC traces for KS17 (paxG, paxC, micM and micB) and KS31 (paxG, paxC, micM, micB and micS) at m / z 22 showing production of 3'4'-epoxyemindole SB and paspaline.

[0055] Figure 12:XH NMR spectrum of 3'4'-epoxyemindole SB

[0056] Figure 13: COSY spectrum of 3'4'-epoxyemindole SB

[0057] Figure 14: HSQC spectrum of 3'4'-epoxyemindole SB

[0058] Figure 15: HMBC spectrum of 3'4'-epoxyemindole SB

[0059] DETAILED DESCRIPTION OF THE INVENTION

[0060] DEFINITIONS

[0061] 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.

[0062] 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.

[0063] 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.

[0064] The term "epoxidized IDT" as used herein refers to any compound that is covered by the "expoxidized IDT" structure shown in Figure 2A.

[0065] An indole diterpene bearing a THP ring has the structure of an "IDT containing an oxygen bearing heterocycle" as shown in Figure 2A.

[0066] As used herein the term "LtmS 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 the IDT bearing a THP ring is paspaline and the epoxidized IDT is 3', 4'-epoxy-emindole SB. In one embodiment the the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22 epoxy-emindole DA. The abbreviation "eeSB" as used herein means 3', 4'-epoxy-emindole SB.

[0067] The abbreviation "eeDA" as used herein means 21, 22 epoxy-emindole DA.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The term "multigene construct" as used herein means a genetic construct that is a polynucleotide comprising at least two TUs. 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] The terms "under conditions wherein the ... enzyme is active" and "under conditions wherein the ... enzymes are active", and grammatical variations thereof when used in reference to enzyme activity mean that the enzyme will perform it's expected function; e.g., a restriction endonuclease will cleave a nucleic acid at an appropriate restriction site, and a DNA ligase will covalently join two polynucleotides together. 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.

[0079] 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.

[0080] "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.

[0081] "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.

[0082] "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.

[0083] 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.

[0084] "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.

[0085] 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.

[0086] 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.

[0087] 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 sequences. 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 elements.

[0088] "Operably-linked" means that the sequence to be expressed is placed under the control of regulatory elements.

[0089] "Regulatory elements" as used herein refers to any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector, genetic construct or expression cassette and includes promoters, transcription control sequences, translation control sequences, origins of replication, tissue-specific regulatory elements, temporal regulatory elements, enhancers, polyadenylation signals, repressors and terminators. Regulatory elements can be "homologous" or "heterologous" to the polynucleotide insert to be expressed from a genetic construct, expression cassete or vector as described herein. When a genetic construct, expression cassete 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] "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.

[0094] "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 cassete 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.

[0095] "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.

[0096] 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.

[0097] 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.

[0098] "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.

[0099] "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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

[0106] 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.

[0107] 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.

[0108] Polynucleotide sequence identity and similarity can be determined readily by those of skill in the art.

[0109] 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"

[0110] 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 eta / ., 1990, Science 247, 1306).

[0111] 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.

[0112] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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 et al., 1990, Science 247, 1306.

[0119] 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.

[0120] 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.

[0121] DETAILED DESCRIPTION

[0122] 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.6-14The first biosynthetic gene cluster (BGC) specifying production of a paspaline-type IDT was identified in 2001 from Penicillium pax i.13Through a series of gene deletion, complementation, and heterologous reconstruction experiments four genes paxG, paxC, paxMand paxB) were identified as being sufficient to deliver paspaline (Figure IB).14An equivalent set of genes were later identified in Epichloe festucae as part of a BGC split across three loci specifying production of the paspaline derived lolitrems ItmG, ItmC, ItmM and Paspaline biosynthesis is initiated with the formation of geranylgeranyl pyrophosphate (GGPP) catalysed by the cluster-encoded GGPP synthase, IdtG. An indole prenyl transferase (IdtC) 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.

[0123] 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 IdtM. A precise regio- and stereospecific cyclisation cascade is then catalysed by the unusual helical integral membrane terpene cyclase IdtB, producing the first stable cyclised IDT, emindole SB. Generation of the THP ring of paspaline is then primed through a second IdtM -catalysed epoxidation, this time on emindole SB, followed by an additional cyclisation step, which has been attributed by previous studies to the cyclase IdtB.6-14Six additional cluster encoded products are required to convert paspaline to lolitrem B, this includes four P450 monoxygenases (LtmP, LtmQ, LtmK and LtmJ) and two prenyl transferases (LtmF and LtmE). The Z.77 cluster also contains another gene, ItmS, that encodes a predicted helical transmembrane protein of unknown function.

[0124] Intriguingly, homologs of ItmS termed idtSsare. found in BGCs from a number of different fungi known to synthesize THP-containing IDTs including the lolitrems, terpendoles, and epoxy-janthitrems (Figure 3).16'17Based on the work disclosed herein, the inventors have determined, that LtmS, rather than LtmB, is the specific cyclase that catalyzes the formation of the THP ring found in paspaline, an intermediate of lolitrem biosynthesis. Additionally, the inventors have further shown that a number of "LtmS 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 Epichloe festucae anA PeniciHum paxilli. Without wishing to be bound by theory, the inventors believe that based on the work disclosed herein, LtmS equivalent cyclases catalyze the formation of an IDT bearing a THP ring from an epoxidized IDT for all IDTs that bear a THP ring.

[0125] To investigate the function of LtmS the inventors generated ItmS deletion strains ( ltmS) in E. festucae. Lolium perenne seeds were inoculated with AltmS strains to look at IDT production. (Figure 4). In contrast to wild type, plant samples containing AltmS strains were attenuated for the production of lolitrem B (Figures 7-8) and accumulated an IDT not observed in wild type (Figures 5- 6). 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).12Without wishing to be bound by theory it is considered that these results are consistent with LtmS being able to convert 3',4'-epoxyemindole SB to paspaline and that reintroduction of / mSinto AltmS strains would restore the wildtype chemotype by attenuating 3',4'-epoxyemindole SB production and restoring lolitrem B production.

[0126] To further interrogate the role of LtmS in THP ring formation, and assess its function in an alternative host, ItmS was transformed into a strain of P.paxilliVna accumulates 3',4'-epoxyemindole SB.Introduction of ItmS to this strain attenuated the accumulation of 3',4'-epoxyemindole SB and restored paxilline production (Figures 9-10). Without wishing to be bound by theory the results are consistent with LtmS also being able to convert 3',4'-epoxyemindole SB to paspaline in alternative host organisms.

[0127] As noted above, LtmS orthologs (also termed IdtSs or LtmS equivalent cyclases herein) are also encoded within IDT BGCs from related filamentous fungi that biosynthesise IDTs bearing a THP ring (Figure 3).17Accordingly, the inventors tested the function of MicS, an idtS found in a BGC that encodes for production of terpendoles in Microcera sp. (Figure 3). When an idtG, idtC, idtM, and idtB were transformed into a P. paxilli strain from which the entire / H BGC was deleted, accumulation of 3',4'-epoxyemindole SB was observed (Figure 11). When micS was transformed alongside these genes then 3',4'-epoxyemindole SB production was attenuated and paspaline production increased (Figure 11). Without wishing to be bound by theory these results are consistent with MicS, and all other LtmS equivalent cyclases, converting 3',4'-epoxyemindole SB to paspaline.

[0128] While the previously proposed biosynthetic pathways for the paspaline derived IDTs included a dual role for unusual terpene cyclase LtmB 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.6-14Without wishing to be bound by theory it is considered that the results presented here are also consistent with LtmS, and all other LtmS equivalent cyclases, converting 21',22'-epoxyemindole DA to emindole DB.

[0129] 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.

[0130] Accordingly, the present invention generally relates to the cyclase LtmS from Epichloe festucae and to LtmS 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 LtmS and LtmS equivalent cyclases to direct formation of IDTs comprising THP rings in an isolated host cell, preferably an isolated fungal cell. Using a 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 homologous expression of the cyclase, LtmS from E. festucae 'm alternate fungal hosts. The inventors have also reconstituted the biosynthetic pathways for recombinant, heterologous expression of an LtmS equivalent cyclase from alternate fungi in an alternate fungal host, 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. Using the efficient gene reassembly of MIDAS and heterologous expression in P. paxHHV e. inventors have demonstrated that P. paxilli\s a suitable host for heterologous expression of LtmS and / or LtmS 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.

[0131] 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.

[0132] In a second aspect the present invention relates to a method of making an indole diterpene (IDT) bearing a tetrahydropyran (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.

[0133] The following embodiments are specifically contemplated as embodiments of the first and second aspects of the invention.

[0134] 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 (LtmS) or SEQ ID NO:4 (MicS).

[0135] 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 (LtmS) or SEQ ID NO:4 (MicS).

[0136] In one embodiment the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (LtmS), SEQ ID NO:4 (MicS).

[0137] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:1 (LtmS).

[0138] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:4 (MicS).

[0139] In one embodiment the polypeptide or functional variant thereof comprises an amino acid motif, ARLRQINEX2GTS[SG]GFFX[AT]XGL[FL], wherein A is Alanine, R is Arginine, L is Leucine, Q is Glutamine, I is isoleucine, N is Asparagine, E is Glutamic acid, G is glycine, T is Threonine, S is Serine, [SG] is either a Serine (S) or Glycine (G), F is Phenylalanine, [AT] is either Alanine (A) or Threonine (T), [FL] is Phenylalanine (F) or Leucine (L), X represents a single instance of any amino acid and Xn represents a series of n instances of any amino acid.

[0140] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXAXGLF (SEQ ID NO: 9).

[0141] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXAXGLL (SEQ ID NO: 10).

[0142] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXTXGLF (SEQ ID NO: 11).

[0143] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXTXGLL (SEQ ID NO: 12).

[0144] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXAXGLF (SEQ ID NO: 13).

[0145] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXAXGLL (SEQ ID NO: 14).

[0146] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXTXGLF (SEQ ID NO: 15).

[0147] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXTXGLL (SEQ ID NO: 16).

[0148] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an endogenous cyclase.

[0149] In one embodiment the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0150] In one embodiment the epoxidized IDT is 3', 4'-epoxyemindole SB or 21, 22-epoxyemindole DA.

[0151] In one embodiment the THP ring is comprised in a hexacyclic paspaline-type indole diterpene core.

[0152] 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.

[0153] In one embodiment the IDT bearing a THP ring is paspaline or emindole DB, preferably paspaline, preferably emindole DB. In one embodiment the IDT bearing a THP ring is paspaline and the epoxidized IDT is 3', 4'- epoxyemindole SB.

[0154] In one embodiment the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22- epoxyemindole DA.

[0155] In one embodiment the cyclase catalyzes the conversion of 3', 4'-epoxyemindole SB to paspaline.

[0156] In one embodiment the cyclase is a Epichloe spp. or Microcera spp. cyclase.

[0157] In one embodiment the cyclase is from Epichloe festucae (LTM) or Microcera sp. (MIC) .

[0158] In one embodiment the cyclase catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB.

[0159] In one embodiment the cyclase that catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB is from Epichloe festucae (LTM) or Microcera sp. (MIC) .

[0160] In one embodiment the cyclase is from Epichloe festucae (LTM) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (LtmS).

[0161] In one embodiment the cyclase is from Microcera sp. (MIC) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:4 (MicS). In one embodiment the isolated host cell is a recombinant host cell.

[0162] 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.

[0163] 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.

[0164] In one embodiment the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: PeniciiHum, Aspergillus, Epichloe, Periglandula, Ciaviceps, Acicuiosporium, Trichoderma, Neurospora, Fusarium, Mortiereiia, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichopiusia, Ashbya, Hansenuia, Pichia, Kluveromyces, Schizzosaccharomyces, Monascus, Taiaromyces, Cryptonectria, Endothia, Toiypociadium, Hypocrea, Gibbereiia, Acremonium, Agaricus, Pieurotus, Voivarieiia, Flammulina, Lentinuia, Auricuiaria, Ganoderma, (Rhizo)mucor, Riopus, or Saccharomyces, preferably PeniciiHum, Aspergillus, Epichloe, Periglandula, Ciaviceps, Acicuiosporium, Saccharomyces, Pichia, Tricopiusia, or Spondoptera. In one embodiment, the fungal cell, hyphae or mycelia is from PeniciHium paxilli, P. janthinellum, P. simplcissimum, P. crustosum, Aspergillus alliaceus, A. desertorum, A. flavus, A. nominus, Epich / oe festucae, Periglandula ipomoeae, Claviceps purpurea, or Aciculosporium take.

[0165] In one embodiment the fungal cell, hyphae or mycelia is from PeniciHium paxilli, P. janthinellum, P. crustosum, Aspergillus flavus, A. desertorum, Epich / oe festucae, Periglandula ipomoeae, Claviceps purpurea, or Aciculosporium take.

[0166] In one embodiment the fungal cell, hyphae or mycelia is from Epich / oe festucae (LTM) or Microcera sp. (MIC) .

[0167] In one embodiment the isolated host cell further comprises an endogenous polypeptide having the same activity as the heterologously expressed polypeptide.

[0168] In one embodiment the endogenous polypeptide is a cyclase.

[0169] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0170] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0171] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0172] In one embodiment the endogenous cyclase catalyses the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0173] In one embodiment the polypeptide or functional variant thereof is expressed from an isolated polynucleotide or functional variant thereof.

[0174] 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 or SEQ ID NO:5

[0175] 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 or SEQ ID NO:5

[0176] 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 or SEQ ID NO:6

[0177] 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 or SEQ ID NO:6 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.

[0178] 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.

[0179] In one embodiment the polynucleotide or functional variant therof is a heterologous polynucleotide or functional variant thereof.

[0180] In one embodiment the polynucleotide or functional variant thereof is an introduced endogenous polynucleotide or functional variant thereof.

[0181] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0182] In one embodiment the polynucleotide or functional variant thereof, nucleic acid construct or transcription unit is comprised in a vector.

[0183] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct.

[0184] In one embodiment the polynucleotide or functional variant thereof is comprised in a transcription unit (TU) or transcription unit module (TUM).

[0185] In one embodiment the polynucleotide or functional variant thereof is comprised in a vector.

[0186] In one embodiment the nucleic acid construct, TU or TUM is comprised in a vector.

[0187] 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.

[0188] 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) and the like.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] In one embodiment the nucleic acid construct is a multigene construct comprising at least two TUs or TUMs.

[0193] In one embodiment, the polynucleotide or functional variant thereof is operatively linked to 5' or 3' untranslated regulatory sequences 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.

[0194] Likewise, the selection of various untranslated regulatory sequences 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.

[0195] 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.

[0196] 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. In one embodiment, the eukaryotic promoter can be a eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Expression levels of an operably linked polynucleotide in a particular cell type will be determined by the nearby presence (or absence) of specific gene regulatory sequences (e.g., enhancers, silencers and the like). Any suitable promoter / enhancer combination (see: Eukaryotic Promoter Data Base EPDB) can be used to drive expression of a polynucleotide as described herein.

[0197] 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.

[0198] 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.

[0199] Enhancers contemplated herein include SV40 enhancer, cytomegalovirus early promoter enhancer, globin, albumin, insulin and the like.

[0200] In one embodiment, in the nucleic acid construct, TU, TUM or vector is driven by a T3, T7 or SP6 cytoplasmic expression system.

[0201] 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).

[0202] 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.

[0203] 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. 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.

[0204] In one embodiment the at least two isolated polypeptides are cyclases.

[0205] 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.

[0206] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0207] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0208] In one embodiment the exogenous cyclase catalyzes the conversion of eeDA to emindole DB.

[0209] In one embodiment the endogenous cyclase catalyzes the conversion of eeDA to emindole DB.

[0210] 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.

[0211] 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.

[0212] The following embodiments are specifically contemplated as embodiments of the third or fourth aspects of the invention.

[0213] 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 or SEQ ID NO:5.

[0214] 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 or SEQ ID NO:5.

[0215] 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 or SEQ ID NO:6.

[0216] 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 or SEQ ID NO:6. 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.

[0217] 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.

[0218] In one embodiment the polynucleotide or functional variant therof is a heterologous polynucleotide or functional variant thereof.

[0219] In one embodiment the polynucleotide or functional variant thereof is an introduced endogenous polynucleotide or functional variant thereof.

[0220] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0221] 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.

[0222] 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.

[0223] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid sequences, nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells. 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.

[0224] 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.

[0225] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0226] 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.

[0227] 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.

[0228] The following embodiments are specifically contemplated as embodiemnts of the ninth and tenth aspects of the invention.

[0229] In one embodiment the isolated host cell comprises an endogenous polypeptide having the same activity as the expressed introduced endogenous polypeptide.

[0230] In one embodiment the endogenous polypeptide is a cyclase.

[0231] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to paspaline.

[0232] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0233] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline. In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0234] 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.

[0235] In one embodiment the at least two polypeptides or functional variants thereof are cyclases.

[0236] 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.

[0237] In one embodiment the exogenous cyclase is an introduced endogenous polypeptide or functional variant thereof.

[0238] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0239] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0240] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0241] 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.

[0242] 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.

[0243] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0244] In a thirteenth aspect the present invention relates to method of making an Epichioe or Microsera spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Epichloe o Microsera spp. polypeptide or functional variant thereof that catalyzes the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0245] In a fourteenth aspect the present invention relates to method of making an Epichloe ox Microsera spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Epichioe ox Microsera spp. polypeptide or functional variant thereof that catalyzes the formation of an IDT bearing a THP ring from an epoxidized IDT.

[0246] The following embodiments are specifically contemplated as embodiments of the thirteenth and fourteenth aspects of the invention.

[0247] In one embodiment the isolated host cell is a recombinant host cell.

[0248] In one embodiment the isolated host cell does not comprise an endogenous polypeptide having the same activity as the expressed polypeptide.

[0249] In one embodiment the isolated host cell heterologously expresses the Epichioe ox Microsera spp. polypeptide or functional variant thereof.

[0250] In one embodiment the isolated host cell comprises an endogenous polypeptide having the same activity as the expressed polypeptide.

[0251] In one embodiment the endogenous polypeptide is a cyclase.

[0252] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0253] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0254] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0255] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB. In one embodiment the expressed polypeptide is an introduced endogenous polypeptide or functional variant thereof.

[0256] In one embodiment the polynucleotide or functional variant thereof is comprised in a nucleic acid construct or transcription unit (TU).

[0257] 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.

[0258] In one embodiment the at least two polypeptides or functional variants thereof are cyclases.

[0259] 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.

[0260] In one embodiment the exogenous cyclase is an introduced endogenous polypeptide or functional variant thereof.

[0261] In one embodiment the exogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0262] In one embodiment the endogenous cyclase catalyzes the conversion of eeSB to paspaline.

[0263] In one embodiment the exogenous or endogenous cyclase, or both, that catalyzes the conversion of 3', 4'-epoxyemindole SB to paspaline is from Epichloe festucae (LTM) or Microsera (MIC).

[0264] In one embodiment the exogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0265] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0266] In one embodiment the exogenous or endogenous cyclase, or both, that catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB is from Epichloe festucae (LTM) or Microsera (MIC).

[0267] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0268] In a fifteenth aspect the present invention relates to method of making an Epichloe or Microsera spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, a Epichloe or Microsera spp. 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.

[0269] In a sixteenth aspect the present invention relates to method of making an Epichloe or Microsera spp. spp. polypeptide or functional variant thereof comprising expressing in an isolated host cell, an Epichloe or Microsera 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.

[0270] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0271] 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.

[0272] 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.

[0273] In an embodiment of the seventeenth and eighteenth aspects, the heterologous polypeptide or functional variant thereof is expressed in the host cell.

[0274] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0275] 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.

[0276] 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.

[0277] 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.

[0278] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO: 1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0279] 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.

[0280] 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.

[0281] 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.

[0282] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0283] 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.

[0284] In a twenty-fourthaspect 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.

[0285] 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.

[0286] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0287] 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.

[0288] 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. The following embodiments are specifically contemplated as embodiments of the twenty-fifth and twenty-sixth aspects of the invention.

[0289] 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 (LtmS) or SEQ ID NO:4 (MicS).

[0290] 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 (LtmS) or SEQ ID NO:4 (MicS).

[0291] In one embodiment the polypeptide or functional variant thereof comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS).

[0292] In one embodiment the polypeptide or functional variant thereof comprises, consists or consists essentially of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS).

[0293] In one embodiment the polypeptide or functional variant thereof comprises an amino acid motif, ARLRQINEX2GTS[SG]GFFX[AT]XGL[FL], wherein A is Alanine, R is Arginine, L is Leucine, Q is Glutamine, I is isoleucine, N is Asparagine, E is Glutamic acid, G is glycine, T is Threonine, S is Serine, [SG] is either a Serine (S) or Glycine (G), F is Phenylalanine, [AT] is either Alanine (A) or Threonine (T), [FL] is Phenylalanine (F) or Leucine (L), X represents a single instance of any amino acid and Xn represents a series of n instances of any amino acid.

[0294] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXAXGLF (SEQ ID NO: 9).

[0295] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXAXGLL (SEQ ID NO: 10).

[0296] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXTXGLF (SEQ ID NO: 11).

[0297] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSSGFFXTXGLL (SEQ ID NO: 12).

[0298] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXAXGLF (SEQ ID NO: 13).

[0299] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXAXGLL (SEQ ID NO: 14). In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXTXGLF (SEQ ID NO: 15).

[0300] In one embodiment the amino acid motif comprises, consists or consists essentially of ARLRQINEXXGTSGGFFXTXGLL (SEQ ID NO: 16).

[0301] In one embodiment the polypeptide or functional variant thereof is a cyclase, preferably an exogenous cyclase, preferably an endogenous cyclase.

[0302] In one embodiment the epoxidized IDT bears an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule.

[0303] In one embodiment the epoxidized IDT is 21, 22-epoxyemindole DA.

[0304] 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.

[0305] In one embodiment the IDT bearing a THP ring is emindole DB.

[0306] In one embodiment the IDT bearing a THP ring is emindole DB and the epoxidized IDT is 21, 22- epoxyemindole DA.

[0307] In one embodiment the cyclase is an Epichloe spp. or Microsera spp. cyclase.

[0308] In one embodiment the cyclase catalyzes the conversion of 21, 22-epoxyemindole DA to emindole DB.

[0309] In one embodiment the cyclase is from Epichloe festucae (LTM) or Microsera (MIC) and comprises, consists, or consists essentially of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS).

[0310] In one embodiment the isolated host cell is a recombinant host cell.

[0311] In one embodiment the isolated host cell further comprises an endogenous polypeptide having the same activity as the heterologously expressed polypeptide.

[0312] In one embodiment the endogenous polypeptide is a cyclase.

[0313] In one embodiment the same activity is catalyzing the conversion of an epoxidized IDT to an IDT bearing a THP ring.

[0314] In one embodiment the epoxidized IDT is an IDT bearing an epoxide on the terminal end of the geranylgeranyl derived portion of the molecule. In one embodiment the endogenous cyclase catalyses the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0315] 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 or SEQ ID NO:5

[0316] 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 or SEQ ID NO:5.

[0317] 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 or SEQ ID NO:6.

[0318] 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 or SEQ ID NO:6.

[0319] In one embodiment the isolated polynucleotide or functional variant thereof comprises, consists, or consists essentially of SEQ ID NO:2 or SEQ ID NO:5. In one embodiment the isolated polynucleotide comprises, consists, or consists essentially of SEQ ID NO:3 or SEQ ID NO:6.

[0320] 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.

[0321] In one embodiment the at least two isolated polypeptides are cyclases.

[0322] 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.

[0323] In one embodiment the exogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0324] In one embodiment the endogenous cyclase catalyzes the conversion of 21, 22 epoxyemindole DA to emindole DB.

[0325] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0326] 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.

[0327] 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.

[0328] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0329] 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.

[0330] 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.

[0331] 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 of the amino acid sequence of SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS) (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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; cyclases, epoxidized IDTs, IDTs bearing a THP ring, and isolated host cells.

[0332] 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.

[0333] 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.

[0334] 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 polypeptide embodiments including nucleic acid constructs, transcription units, transcripton unit modules, vectors, multigene constructs, untranslated regulatory sequences and functional variants; polypeptides comprising, consisting or consisting essentially of of the amino acid sequence of SEQ ID NO: 1 (LtmS) or SEQ ID NO:4 (MicS) (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.

[0335] 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.

[0336] In one embodiment the IDT bearing a THP ring is paspaline or emindole DB.

[0337] In a thirty-fourth aspect the invention relates to paspaline made according to a method of the invention.

[0338] 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.

[0339] 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.

[0340] The invention will now be illustrated in a non-limiting way by reference to the following examples.

[0341] EXAMPLES

[0342] Example 1 - Generation and analysis of A / tmS strains

[0343] Generation of Zl / mS strains in Eoichloe festucae

[0344] To investigate the function of ItmS in E. festucae, ItmS knockout (AltmS) strains were engineered using a plasmid (pYL413) designed to facilitate homologous recombination leading to the replacement of the ItmS coding sequence (CDS) with a neomycin selection cassette. YL413 was created using a MIDAS level 2 reaction by combining level 1 plasmids containing the ItmS promoter, geneticin selection cassette under the TrpC promoter and terminator, and the ItmS terminator with a level 2 +BF backbone vector.18The ItmS promoter sequence consisted of 999 bp starting immediately upstream of the start codon. The terminator consisted of an 853 bp sequence that began immediately downstream of the stop codon. The ItmS promoter and terminator sequences were designed to act as homology arms (HAs) to facilitate integration of the neomycin selection cassette into the / f / nS locus through homologous recombination (Figure 4).

[0345] To increase the efficiency of homologous recombination, Cas9 was added along with two sgRNAs (YLsgRNAl and YsgRNA2) to generate cuts at both ends of the ItmS locus. YLsgRNAl and YLsgRNA2 RNPs were transformed into wildtype E. festucae protoplasts with pYL413.

[0346] Confirmation of / fr Sbv PCR and sequencing:

[0347] Twenty Geneticin resistant transformants were screened for the presence of the ItmS CVS using PCR. A multiplex PCR was performed to amplify a region between the ItmS promoter and terminator (807 / 808), and an internal region within the ItmS CDS (805 / 806). Successful integration of the Geneticin cassete should produce a larger 2,205 bp band with primer set 805 / 806 compared to the 1,345 bp band for wildtype. This was observed for all the transformants (Figure 4A,B). Eleven of the twenty transformants (1, 3, 7, 10, 11, 12, 13, 16, 18, 19 and 20) also lacked the 417 bp internal PCR product for the / f / nSCDS that would be expected from primer set 805 / 806 when the CDS is present (Figure 4B). All of these eleven transformants therefore have expected pattern of bands for ItmS knockouts. Transformants one and three were further screened by PCR with primers that amplified the region surrounding the ItmS locus (823 / 824). Both transformants one and three have the longer band (3,650 bp) that would expected when the selection cassette had correctly integrated into the ItmS locus (Figure 4C). Strain one was used to inoculate L perenne seeds for analysis of IDT production.

[0348] LC-MS analysis of / frnS transformants:

[0349] Extracts were obtained from fourteen AltmS infected plants and eleven wild-type plants and analysed by LC-MS using standard methods. Thirteen of the fourteen AltmS samples had a large 422.3 m / z peak at 9.4 min that corresponds to 3'4'-epoxyemindole SB, this peak was also oberserved in the wild-type samples but it was greatly reduced (Figures 5-6). A peak at 12.8 min that corresponds to paspaline is also present in both the Zl / fmS'and wildtype samples. A lolitrem B peak (686.3 m / z, 12 min) was observed in all wild-type samples (Figure 8) . AltmS samples had a greatly reduced lolitrem peak and some samples had no observable lolitrem B peak (Figure 7). The accumulation of 3'4'- epoxyemindole SB and reduction of lolitrem B in AltmS samples indicates that LtmS is important in the conversion of 3'4'-epoxyemindole SB to paspaline and that without it lolitrem biosynthesis is inefficient.

[0350] Example 2 - Complementation of ApaxA with ItmS o show function in an alternative host.

[0351] ItmS complementation of ApaxA strain ( ApaxA ::!tmS)

[0352] To show that ItmS is also functional in an alternative host we complemented a P. paxilli ApaxA strain with a plasmid containing / f / nScDNA (pRC358). Genes were introduced by random integration and transformants selected following standard P. paxilli methods.

[0353] LC-MS analysis of 1 x4.-.7fr77S complementation strains:

[0354] Extracts from ten ApaxA: :!tmS (RC358) strains were analysed by LC-MS using standard methods. No observable 3'4'-epoxyemindole SB peak (422.3 m / z, 9.4 min) was present in eight out of ten RC358 strains and five out of ten had a strong paxilline peak (436.3 m / z, 7.2 min) with a further two out of ten showing some evidence of paxilline production (Figures 9-10). The restoration of paxilline production when ItmS was introduced to an ApaxA strain confirms that LtmS is able to perform the function of PaxA and catalyse the conversion of 3'4'-epoxyemindole SB to paspaline in an alternative host (P. paxilli). Example 3 - pathway reconstruction in P.paxilli with micS gene

[0355] Generation of micS reconstruction strains

[0356] To investigate the role of LtmS orthologs, a plasmid (pKS31) was constructed with an / tf / Sfrom a Microcera sp. micS) in combination with paxG, paxCand micM and micB, (homologs of idtMand idtB). A second plasmid was also constructed that was identical but lacked the m / cSgene (pKS17). These plasmids were randomly integrated into the heterologous host that lacked the PAX BGC to determine the function of MicS.

[0357] LC-MS analysis of reconstruction strains

[0358] Extracts were obtained from nine pKS17 and four pKS31 strains and analysed by LC-MS. Extracted ion chromatograms are shown for 422.3 m / z (3'4'-epoxyemindole SB and paspaline) (Figure 11). Three of the nine pKS17 transformants had a large 422.3 m / z peak at 8.8 min that corresponded to 3'4'- epoxyemindole SB. These transformants also contained a paspaline peak at 11.9 min. The remaining six tranformants did not have either of these peaks. In contrast three of the four pKS31 transformants contained only a 422.3 m / z peak at 11.9 min that corresponded to paspaline and no 3'4'-epoxyemindole SB peak was observed at 8.8 min. This provides evidence that MicS is a functional homolog of ItmS and has a role in the conversion of 3'4'-epoxyemindole SB to paspaline and without MicS an accumulation of 3'4'-epoxyemindole SB is observed.

[0359] Fungal protocols - Ejo / cA / oe esf / cae transformation:

[0360] Epich / oe festucae strains were grown at 22 °C on 2.4% (w / v) PDA. E. festucae protoplasts were prepared and transformed as described previously19'20with the addition of CRIPSR-Cas9 ribonucleoproteins (RNPs). 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.

[0361] Ta ble 1 : Components of CRISPR-Cas9 reaction

[0362] Four or five Lolium perenne seeds were surface sterilised and artificially inoculated using a method adapted from Latch and Christensen.21Seeds were soaked in 50% (v / v) H2SO4 for 30 min, rinsed in sterile H2O and soaked in 50% (v / v) commercial bleach (NaOCI 21.5 g / l), rinsed thoroughly with sterile H2O and air-dried in a laminar flow cabinet. Inoculation was performed by making a shallow 2- 3 mm long incision between the mesocotyl and coleoptile regions of L. perenne seedlings (7 d old germinated on 1.5% PDA) and a small amount of E. festucae mycelia were placed into this cut. Inoculated seedlings were maintained on PDA for 7 days in darkness followed by 7 days with a 16 h photoperiod, then were transferred to root trainers containing fungicide-free organic seed mix (Daltons®) and maintained in a Conviron GEN1000 plant growth chamber with a photoperiod of 16 h of light (25% intensity) and 75% humidity.

[0363] Chemical extraction from E. festuca

[0364] Five-day old mycelial cultures (25 ml) were freeze-dried and resuspended in 5 ml 1% HCI. Freeze- dried plant pseudostem samples (50-200 mg dry weight) were ground into fine powder using MPBio FastPrep24 5G bead beater grinder and lysis system (40 s, 6m / s) and resuspended in 1 ml 1% HCI. Metabolites were extracted with 3 ml (for 25 ml mycelial cultures) or 0.5 ml (for plant pseudostem samples) dichloromethane (DCM) and resuspended in acetonitrile (MeCN).

[0365] Isolation and characterisation of 3'4'-eDOxyemindole SB

[0366] A strain of Penicillium paxilli was grown using standard growth conditions. 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 C18 250 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 12-15).

[0367] High-resolution mass spectrometric data and MS / MS spectra

[0368] 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.

[0369] NMR spectroscopy

[0370] 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.

[0371] Protein sequence information

[0372] >LtmS

[0373] MSRSDWI FI SLQGFFCLAGVIWKSREGYPI IDFPCPLQFIDSSDATLSYGTTSPWFGFRAVASMQSIWDNGPWFW LHLMLYIAQLVGLILI ILHETVPHGAFLRKFESLAALGYLSYTVGLSTAFPVFSLWILNQYRAEKLVTAWPRRQE KAFLRTI FWCTGI SHIGI FMVAIVATLLHRDATAPFHIGNSLLGVPDCSQFPCSEIAARHARLRQINEMTGTSSG FFLTVGLFSQALEAENKHLSLRVMVRMFFVSLIAGPAAGSADVLLLRDSITRSKKDCG ( SEQ ID NO : 1 )

[0374] >MicS

[0375] MAPNDWLFSLLQMFFGLAGMAWKIREGYPIMDSPGLLWHFDSSDRALGHTELSSWVELCLGTSVLLFRNNWPWFS LHLILYIGQLVGFMLTILHEI PPYGPLGKLGYAALGCLSLCFGFSTAMPAVSLWILHKNRAKSFVPAQSRRRAKA FLGAI FWFSGITHVGAFLVAFTTTLMPQQVAPFHVMNSLLGVPDCSQLPCSEVAQRQARLRQINEMVGTSSGFFL TVGLFCQVLDAKNKHMSSRLLIRMFLVSLVI GPAAGGADVLLLKDALMRFGAA ( SEQ ID NO : 4 )

[0376] Nucleic acid Sequence information

[0377] >l tmS ( gDNA)

[0378] ( SEQ ID NO : 2 )

[0379] ATGTCGCGAAGTGATTGGATCTTTATCTCTCTGCAGGGTTTCTTTTGTTTAGCCGGCGTAATATGGAAGTCACGG

[0380] GAAGGATATCCGATCATTGACTTTCCCTGCCCATTACAGTTCATTGATTCTTCGGATGCTACATTAAGCTATGGA

[0381] ACCACATCACCATGGTTCGGGTTTCGCGCTGTTGCGTCGATGCAGTCCATTTGGGATAATGGGCCTTGGTTTTGG

[0382] CTTCACCTAATGCTTTACATTGCTCAGCTGGTTGGGCTCATTTTGATAATTCTGCATGAGACCGTACCGCATGGC

[0383] GCATTCCTCCGCAAGTTTGAGTATGTGATTCCCTGTTAACTCTGACTCACTGACTATGCTTACTTTTTGAAGAAG

[0384] TTTGGCCGCACTAGGCTATCTCTCTTACACAGTTGGACTTTCCACTGCCTTTCCCGTCTTTTCACTGTGGATCCT

[0385] AAATCAATATCGTGCCGAGAAATTAGTAACAGCGTGGCCGAGGAGGCAAGAGAAGGCTTTCCTAAGGACTATTTT

[0386] CTGGTGCACTGGTATAAGCCACATTGGCATTTTTATGGTAGCGATCGTGGCGACTCTCCTTCACAGAGACGCCAC

[0387] TGCTCCCTTTCATATAGGGAATAGCCTCCTTGGTGTACCCGATTGCTCGCAGTTTCCGTGTTCCGAGATCGCTGC

[0388] ACGACATGCTAGACTCAGACAGATTAATGAGATGACTGGTACATCAAGTGGATTTTTCTTGACCGTGGGGCTATT

[0389] TTCTCAAGCACTTGAAGCAGAAAATAAACACTTGAGCTTACGAGTTATGGTAAGGATGTTTTTTGTCAGTCTCAT

[0390] AGCTGGTCCAGCTGCAGGTAGCGCGGATGTGCTACTTCTACGAGACTCAATAACAAGATCTAAAAAGGACTGCGG GTAG >l tmS ( cDNA)

[0391] ( SEQ ID NO : 3 )

[0392] ATGTCGCGAAGTGATTGGATCTTTATCTCTCTGCAGGGTTTCTTTTGTTTAGCCGGCGTAATATGGAAGTCACGG GAAGGATATCCGATCATTGACTTTCCCTGCCCATTACAGTTCATTGATTCTTCGGATGCTACATTAAGCTATGGA ACCACATCACCATGGTTCGGGTTTCGCGCTGTTGCGTCGATGCAGTCCATTTGGGATAATGGGCCTTGGTTTTGG CTTCACCTAATGCTTTACATTGCTCAGCTGGTTGGGCTCATTTTGATAATTCTGCATGAGACTGTACCGCATGGC GCATTCCTCCGCAAGTTTGAAAGTTTGGCCGCACTAGGCTATCTCTCTTACACAGTTGGACTTTCCACTGCCTTT CCCGTCTTTTCACTGTGGATCCTAAATCAATATCGTGCCGAGAAATTAGTAACAGCGTGGCCGAGGAGGCAAGAG AAGGCTTTCCTAAGGACTATTTTCTGGTGCACTGGTATAAGCCACATTGGCATTTTTATGGTAGCGATCGTGGCG ACTCTCCTTCACAGAGATGCCACTGCTCCCTTTCATATAGGGAATAGCCTCCTTGGTGTACCCGATTGCTCGCAG TTTCCGTGTTCCGAGATCGCTGCACGACATGCTAGACTCAGACAGATTAATGAGATGACTGGTACATCAAGTGGA TTTTTCTTGACCGTGGGGCTATTTTCTCAAGCACTTGAAGCAGAAAATAAACACTTGAGCTTACGAGTTATGGTA AGGATGTTTTTTGTCAGTCTCATAGCTGGTCCAGCTGCAGGTAGCGCGGATGTGCTACTTCTACGAGACTCAATA ACAAGAT CTAAAAAGGACT GCGGGTAG

[0393] >micS ( gDNA)

[0394] ATGGCGCCAAACGACTGGCTGTTCAGCCTCCTGCAAATGTTTTTCGGTCTCGCCGGCATGGCATGGAAGATACGA GAAGGATATCCAATCATGGATTCACCAGGCCTGCTATGGCATTTTGATTCTTCAGACCGTGCATTGGGCCACACA GAACTATCTTCATGGGTTGAGCTTTGCTTGGGCACGTCAGTGCTACTCTTTCGGAACAATTGGCCCTGGTTCTCG CTTCATCTTATCCTCTACATTGGCCAACTAGTGGGGTTTATGTTGACAATACTGCATGAGATCCCACCGTATGGA CCGCTTGGCAAGCTCGGGTATGTCAAACTGTTCTACATGTTTCCAACGCGCTGACCATGCCCCCGGCTCGAAGGA CTTTTGCCGCACTAGGCTGTCTCTCTCTTTGCTTTGGGTTTTCAACTGCTATGCCTGCCGTATCGTTGTGGATCC TTCATAAGAACCGTGCCAAGAGTTTTGTACCAGCGCAGTCAAGAAGGAGAGCGAAAGCGTTCTTGGGGGCCATCT TCTGGTTCTCTGGGATCACGCACGTTGGTGCTTTCTTGGTGGCATTCACAACAACTCTGATGCCTCAGCAAGTTG CCCCGTTTCACGTAATGAACAGTCTTCTTGGTGTACCTGATTGTTCACAACTTCCATGTTCGGAGGTGGCTCAGA GACAGGCGAGACTGAGACAGATCAATGAGATGGTTGGCACTTCAAGCGGGTTTTTCTTGACCGTGGGGCTCTTTT GCCAAGTTCTTGATGCAAAAAACAAGCACATGAGCTCACGACTTCTGATAAGGATGTTTCTTGTCAGTTTGGTGA TCGGCCCCGCTGCAGGTGGTGCTGACGTACTCCTGCTGAAGGATGCTTTGATGAGGTTCGGAGCGGCCTGA ( SEQ ID NO : 5 )

[0395] >micS ( cDNA)

[0396] ATGGCGCCAAACGACTGGCTGTTCAGCCTCCTGCAAATGTTTTTCGGTCTCGCCGGCATGGCATGGAAGATACGA GAAGGATATCCAATCATGGATTCACCAGGCCTGCTATGGCATTTTGATTCTTCAGACCGTGCATTGGGCCACACA GAACTATCTTCATGGGTTGAGCTTTGCTTGGGCACGTCAGTGCTACTCTTTCGGAACAATTGGCCCTGGTTCTCG CTTCATCTTATCCTCTACATTGGCCAACTAGTGGGGTTTATGTTGACAATACTGCATGAGATCCCACCGTATGGA CCGCTTGGCAAGCTCGGGTATGCCGCACTAGGCTGTCTCTCTCTTTGCTTTGGGTTTTCAACTGCTATGCCTGCC GTATCGTTGTGGATCCTTCATAAGAACCGTGCCAAGAGTTTTGTACCAGCGCAGTCAAGAAGGAGAGCGAAAGCG TTCTTGGGGGCCATCTTCTGGTTCTCTGGGATCACGCACGTTGGTGCTTTCTTGGTGGCATTCACAACAACTCTG ATGCCTCAGCAAGTTGCCCCGTTTCACGTAATGAACAGTCTTCTTGGTGTACCTGATTGTTCACAACTTCCATGT TCGGAGGTGGCTCAGAGACAGGCGAGACTGAGACAGATCAATGAGATGGTTGGCACTTCAAGCGGGTTTTTCTTG ACCGTGGGGCTCTTTTGCCAAGTTCTTGATGCAAAAAACAAGCACATGAGCTCACGACTTCTGATAAGGATGTTT CTTGTCAGTTTGGTGATCGGCCCCGCTGCAGGTGGTGCTGACGTACTCCTGCTGAAGGATGCTTTGATGAGGTTC GGAGCGGCCTGA ( SEQ ID NO : 6 ) sqRNAs:

[0397] >YLsgRNAl

[0398] CCGUGACUUCCAUAUUACGCC ( SEQ ID NO : 7 )

[0399] > YLsgRNA2

[0400] ACAAGAUCUAAAAAGGACUG ( SEQ ID NO : 8 ) Table 2: Plasmid components:

[0401] * Richardson et al (2022)

[0402] +GenBank accession : HM 171111.1

[0403] * GenBank accession : ABD64366.1

[0404] * GenBank accession : JN613320.1

[0405] 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.

[0406] 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).

[0407] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0408] 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.

[0409] 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.

[0410] 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).

[0411] 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.

[0412] 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.

[0413] 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.

[0414] INDUSTRIAL APPLICATION

[0415] The invention has industrial application in the production of indole diterpene compounds bearing a THP ring, particularly paspaline and emindole DB.

[0416] REFERENCES

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[0433] 18. van Dolleweerd, C. J.; Kessans, S. A.; Van de Bittner, K. C.; Bustamante, L. Y.; Bundela, R.; Scott, B.; Nicholson, M. J.; Parker, E. J., MIDAS: A Modular DNA Assembly System for Synthetic Biology. ACS Synthetic Biology. 2018, 7 (4), 1018-1029.

[0434] 19. Itoh, Y.; Johnson, R.; Scott, B., Integrative transformation of the mycotoxin-producing fungus, Penicillium paxilli. Current Genetics 1994, 25 (6), 508-13.

[0435] 20. Young, C. A.; Bryant, M. K.; Christensen, M. J.; Tapper, B. A.; Bryan, G. T.; Scott, B., Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Molecular Genetics and Genomics 2005, 274 (1), 13-29.

[0436] 21. Latch, G. C. M.; Christensen, M. J., Artificial infection of grasses with endophytes. Annals of Applied Biology 1985, 107 (1), 17-24.

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 the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS).

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 (LtmS) or SEQ ID NO:4 (MicS).

4. The method of any one of claims 1 to 3 wherein the polypeptide or functional variant thereof comprises an amino acid motif, ARLRQINEX2GTS[SG]GFFX[AT]XGL[FL], wherein A is Alanine, R is Arginine, L is Leucine, Q is Glutamine, I is isoleucine, N is Asparagine, E is Glutamic acid, G is glycine, T is Threonine, S is Serine, [SG] is either a Serine (S) or Glycine (G), F is Phenylalanine, [AT] is either Alanine (A) or Threonine (T), [FL] is Phenylalanine (F) or Leucine (L), X represents a single instance of any amino acid and Xn represents a series of n instances of any amino acid .

5. The method of anyone of claims 1 to 4 wherein the 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 the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: PeniciHium, Aspergillus, Epichioe, Periglandula, Claviceps, Aciculosporium, Trichoderma, Neurospora, Fusarium, Mortierella, Chrysosporium, Candida, Geotrichum, Yarrowia,Eremothecium, Trichop / usia, Ashbya, Hansenu / a, Pichia, K / uveromyces, Schizzosaccharomyces, Monascus, Ta / aromyces, Cryptonectria, Endothia, To / ypodadium, 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, preferably from Penicillium paxilli, 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 or SEQ ID NO:5.

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 or SEQ ID NO:6.

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 the polypeptide or functional variant thereof comprises an amino acid sequence that is at least 70% identical to SEQ ID NO:1 (LtmS) or SEQ ID NO:4 (MicS).

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 (LtmS) or SEQ ID NO:4 (MicS).

16. The method of any one of claims 12 to 15 wherein the polypeptide or functional variant thereof comprises an amino acid motif, ARLRQINEX2GTS[SG]GFFX[AT]XGL[FL], wherein A is Alanine, R is Arginine, L is Leucine, Q is Glutamine, I is isoleucine, N is Asparagine, E is Glutamic acid, G is glycine, T is Threonine, S is Serine, [SG] is either a Serine (S) or Glycine (G), F is Phenylalanine, [AT] is either Alanine (A) or Threonine (T), [FL] is Phenylalanine (F) or Leucine (L), X represents a single instance of any amino acid and Xn represents a series of n instances of any amino acid.

17. The method of anyone of claims 12 to 16 wherein the 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 the fungal cell, hyphae or mycelia is from a species of fungi in one of the following genera: Peniciffum, Aspergillus, Epichloe, Periglandula, Claviceps, Aciculosporium, Trichoderma, Neurospora, Fusarium, Mortierella, Chrysosporium, Candida, Geotrichum, Yarrowia, Eremothecium, Trichoplusia, Ashbya, Hansenula, Pichia, Kiuveromyces, 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, preferably from Penicillium paxilli, 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 or SEQ ID NO:5.

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 or SEQ ID NO:6.

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