Insect repellent

Abstract

This disclosure concerns a method for the conversion of nepetalactol and / or nepetalactone to dihydronepetalactone catalysed by nepetalactol-related short-chain dehydrogenase enzymes (NEPS) polypeptides from Nepeta mussinii (Nepeta racemosa) and modified NEPS polypeptides with increased activity; and including microbial cells, plant cells and plants transformed to express said modified NEPS polypeptides

Description

[0001] Insect Repellent

[0002] Field of the Invention

[0003] This disclosure concerns a method for the conversion of nepetalactol and / or nepetalactone to dihydronepetalactone catalysed by nepetalactol-related short-chain dehydrogenase enzymes (NEPS). Nucleic acid molecules encoding modified NEPS enzymes, modified NEPS enzymes with increased enzymic activity; microbial cells expressing said modified NEPS enzymes; microbial expression systems comprising said microbial cells for the conversion of nepetalactol and / or nepetalactone to dihydronepetalactone and plants modified to overexpress wild type and mutant NEPS enzymes.

[0004] Background to the Invention

[0005] Infectious diseases transmitted by mosquitoes, flies and ticks cause millions of deaths and infections each year, disproportionally impacting the poorest on the globe. Annually an estimated half a million people alone are killed by Malaria, Yellow fever and the Dengue virus.

[0006] Dihydronepetalactone (DHN) has insect repellent properties and is being developed as an alternative to N, N-Diethyl-meta-toluamide (DEET) which although highly effective has a poor perception in society. Natural insect repellents are sought after and although natural compounds with insect repellent properties exist such as, for example the plant-based citronella oil, the performances are typically poor.

[0007] DHN can be chemically synthesised by the hydrogenation of its precursor nepetalactone, either when purified or through the direct hydrogenation of catmint oil, a plant essential oil rich in nepetalactone. However, the hydrogenation process to produce DHN requires precious metal catalysts, solvents, high pressures and energy, and is expensive. Therefore cleaner, more environmentally friendly methods are sought after.

[0008] Nepetalactones are volatile natural products produced by plants of the genus Nepeta such Nepeta mussinii syn racemosa or N. cataria, also commonly known as catmint or catnip. Catmint or catnip are most commonly known due to their euphoric effect on house cats with the active compound causing the euphoric effects being nepetalactone.

[0009] Nepetalactone synthesis is known in the art and starts with the transformation of geranyl pyrophosphate to produce nepetalactone via several intermediates. US2021207183 discloses three cyclases (NEPS1-3) isolated from N. mussinii that are responsible for the stereoselective cyclisation and / or subsequent oxidation of activated 8-oxocitronellyl enol / enolate into distinct nepetalactone and nepetalactol diastereomers. Nepetalactol-related short-chain dehydrogenases (NEPS) are described in Lichman et al. 2019, Lichman et al 2020 and Lozada et al 2022.

[0010] US2022 / 0356497 discloses a recombinant microbial cell comprising a heterologous nepetalactol synthase for the production of nepetalactol from the 8-oxocitronellyl enol intermediate. Further, recombinant microbial cells encoding a heterologous dihydronepetalactone dehydrogenase (DND) to produce dihydronepetalactone from nepetalactone, are also disclosed, however, the sequence of this hypothetic enzyme is not described.

[0011] We disclose that DHN can be obtained under reducing conditions using NEPS and nepetalactone or nepetalactol as a substrate. NEPS modified enzymes improving the yield of DHN and plants or microbial cells transformed and adapted to express said modified enzymes are also disclosed.

[0012] Statement of the invention

[0013] According to an aspect of the invention there is provided a method to produce dihydronepetalactone (DHN) comprising the steps:

[0014] (i) providing a substrate selected from the group: nepetalactone, nepetalactol, iridodial or catmint oil comprising nepetalactone,

[0015] (ii) providing at least one nepetalactol-related short chain reductase (NEPS) polypeptide,

[0016] (iii) forming a preparation comprising said substrate with said NEPS polypeptide in the presence of a reducing co-substrate to form a reaction mixture under conditions to convert said substrate to DHN.

[0017] Nepetalactone is conveniently provided in oily extracts from Nepeta species such as N.mussinii syn racemosa or N. cataria. The oily extracts are commonly known as catmint or catnip oil. In the context of this invention catmint oil and catnip oil are used interchangeably.

[0018] In a preferred method of the invention said substrate is iridodial or nepetalactone.

[0019] In a further preferred method said substrate is catmint oil comprising nepetalactone.

[0020] In a further preferred method said substrate is nepetalactone.

[0021] In a preferred method of the invention said NEPS polypeptide is provided as an isolated polypeptide.

[0022] In a preferred method of the invention said method is an in vitro method. As known in the art there are eight stereoisomers of nepetalactone such as the 7R- and 7S forms of cis, cis-, cis, trans-, trans, cis- and trans, trans-nepetalactone with 7S-cis-trans, 7S-cis- cis and 7S-trans-cis the preferred forms found in Nepeta cataria or Nepeta mussinii. More formally these are known as 7S,4aS,7a / ?-nepetalactone, 7S,4a / ?,7aS-nepetalactone and 7S,4aS,7aS-nepetalactone, respectively. Also known to the skilled artisan is that DHN has sixteen possible stereochemical forms.

[0023] Preferably, nepetalactone is cis-trans nepetalactone, preferably 7S-cis-trans nepetalactone.

[0024] In an alternative method nepetalactone is trans-cis nepetalactone, preferably 7S-trans-cis- nepetalactone.

[0025] Preferably DHN is selected from the group consisting of: (4 / ?,7S,4aS,7aS)- dihydronepetalactone (DHN1), (4S,7S,4aS,7a / ?)-dihydronepetalactone (DHN2) and (4 / ?,7S,4aS,7a / ?)-dihydronepetalactone (DHN3).

[0026] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide comprises or consists of an amino acid sequence with at least 80% at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the full-length amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and has retained or enhanced NEPS polypeptide activity.

[0027] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide has at least 80% at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the full-length amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and has retained or enhanced NEPS polypeptide activity.

[0028] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide has at least 91%, at least 92%, at least 93%, at least 94% identity to the full-length amino acid sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and has retained or enhanced NEPS polypeptide activity.

[0029] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a combination thereof. In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide is selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a combination thereof.

[0030] Preferably, the nepetalactol-related short chain reductase (NEPS) polypeptide set forth in SEQ ID NO: 1 is combined with one or more NEPS polypeptides selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, preferably SEQ ID NO 1 and SEQ ID NO: 4; or NEPS set forth in SEQ ID NO 2 is combined with NEPS selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 4, preferably SEQ ID NO: 2 and SEQ ID NO: 3. Even more preferably said NEPS polypeptide set forth in SEQ ID NO:1 is combined with NEPS set forth in SEQ ID NO: 3 and SEQ ID NO: 4 or SEQ ID NO: 2 and SEQ ID NO: 4.

[0031] In a preferred method of the invention the NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1 is combined with one or more NEPS polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, preferably with SEQ ID NO: 4.

[0032] In a further preferred method of the invention the NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2 is combined with one or more NEPS polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 4, preferably with SEQ ID NO: 3.

[0033] In a further preferred method of the invention the NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO 1 is combined with one or more NEPS polypeptides comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 4; or set forth in SEQ ID NO: 2 and SEQ ID NO: 4.

[0034] Preferably, when the NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1 is combined with NEPS polypeptide comprising or consisting of an amino acid sequence set forth in i) SEQ ID NO: 4, or ii) SEQ ID NO 2 and SEQ ID NO:4, or iii) SEQ ID NO 3 and SEQ ID NO: 4; said substrate is cis-trans nepetalactone.

[0035] Alternatively, when the NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO:2 is combined with SEQ ID NO: 3, said substrate is cis-trans nepetalactone. Alternatively, when NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO 1 is combined with NEPS polypeptide comprising or consisting of an amino acid sequence set forth in i) SEQ ID NO 4, or ii) SEQ ID NO 2 and SEQ ID NO: 4, or iii) SEQ ID NO 3 and SEQ ID NO: 4, said substrate is trans-cis nepetalactone.

[0036] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, is modified and has at least one amino acid substitution.

[0037] In a preferred method of the invention said nepetalactol-related short chain reductase polypeptide sequence set forth in SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 is modified and has at least one amino acid substitution.

[0038] Preferably said amino acid substitutions are conservative amino acid substitutions. Conservative amino acid substitutions, as known by the skilled artisan, replace one amino acid with an amino acid having similar biochemical properties.

[0039] Preferably said amino acid substitutions are non-conservative amino acid substitutions. Nonconservative amino acid substitutions, as known by the skilled artisan, replace one amino acid with an amino acid having different biochemical properties.

[0040] In a further preferred embodiment of the invention said amino acid substitution is at amino residue 49, 163 and / or 208 of the sequence set forth in SEQ ID NO: 1 and wherein said modified polypeptide has retained or enhanced nepetalactol-related short chain reductase activity.

[0041] In a preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0042] In a further preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to glycine (G) or alanine (A) substitution.

[0043] In a preferred embodiment of the invention said modification at amino acid residue 163 is a serine (S) to aspartic acid (D) substitution.

[0044] In a preferred embodiment of the invention said modification at amino acid residue 208 is a Methionine (M) to Glutamic acid (E) substitution. In a preferred embodiment of the invention said modified nepetalactol-related short chain reductase polypeptide is selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO;

[0045] 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or a combination thereof.

[0046] In a preferred embodiment of the invention said modified nepetalactol-related short chain reductase polypeptide comprises or consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, or a combination thereof.

[0047] In a further preferred embodiment of the invention said amino acid substitution is at amino residue 41 , 159 and / or 204 of the sequence set forth in SEQ ID NO: 2 and wherein said modified polypeptide has retained or enhanced nepetalactol-related short chain reductase activity.

[0048] In a preferred embodiment of the invention said modification at amino acid residue 41 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0049] In a preferred embodiment of the invention said modification at amino acid residue 159 is an isoleucine (I) to aspartic acid (D) substitution.

[0050] In a preferred embodiment of the invention said modification at amino acid residue 204 is a methionine (M) to glutamic acid (E) substitution.

[0051] In a further preferred embodiment of the invention said amino acid substitution is at amino residue 49, 164 and / or 209 of the sequence set forth in SEQ ID NO 3 and wherein said modified polypeptide has retained or enhanced nepetalactol-related short chain reductase activity.

[0052] In a preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0053] In a preferred embodiment of the invention said modification at amino acid residue 164 is an asparagine (N) to aspartic Acid (D) substitution.

[0054] In a preferred embodiment of the invention said modification at amino acid residue 209 is a methionine (M) to glutamic acid (E) substitution. In a further preferred embodiment of the invention said amino acid substitution is at amino residue 49, 163 and / or 208 of the sequence set forth in SEQ ID NO: 4 and wherein said modified polypeptide has retained or enhanced nepetalactol-related short chain reductase activity.

[0055] In a preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0056] In a preferred embodiment of the invention said modification at amino acid residue 163 is an asparagine (N) to aspartic acid (D) substitution.

[0057] In a preferred embodiment of the invention said modification at amino acid residue 208 is a methionine (M) to glutamic Acid (E) substitution.

[0058] In a preferred method of the invention said reduced co-substrate is NADPH or NADH.

[0059] Due to the high costs of cofactors such as NADH and NADPH the skilled artisan knows that commercially available enzymes such as alcohol dehydrogenase could be used to reduce the cofactors and therefore providing a recycling system.

[0060] In a preferred method of the invention said reaction mixture further comprises stabilisers such as organic solvent.

[0061] Organic solvents can be categorised into polar and non-polar organic solvents including nonpolar hydrocarbon solvents such as benzene or toluene, nonpolar ether solvent such as diethyl ether or tetra hydrofuran, nonpolar chlorocarbon solvents such as chloroform, polar aprotic solvents such as dichloromethane or acetonitrile, polar aprotic solvents such as n- butanol or ammonia.

[0062] In a preferred method of the invention said organic solvent is acetonitrile.

[0063] According to an aspect of the invention there is provided an isolated nucleic acid molecule that encodes a modified nepetalactol-related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence set forth in SEQ ID NO; 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i) and retains nepetalactol- related short chain reductase activity, iii) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO; 8, SEQ ID NO; 9 and SEQ ID NO: 10, iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue in the amino acid sequence as represented in iii) above and which has retained or enhanced nepetalactol-related short chain reductase activity.

[0064] In a preferred embodiment of the invention said modified amino acid sequence has at least 90, 91 , 92, 93 or 94% identity to the full-length amino acid sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10 and has retained or enhanced NEPS activity.

[0065] In a further preferred embodiment of the invention said modified amino acid sequence has at least 95, 96, 97, 98, 99 % identity to the full-length amino acid sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO: 10 and has retained or enhanced NEPS activity.

[0066] In a preferred embodiment said modified amino acid sequence is not SEQ ID NO: 1.

[0067] According to an aspect of the invention there is provided an isolated polypeptide selected from the group consisting of: i) a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; ii) a polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition, deletion or substitution or at least one amino acid residue of the sequence represented in SEQ ID NO: 1 which has retained or enhanced nepetalactol-related short chain reductase activity; iii) a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO:1 wherein said amino acid sequence is modified at amino acid residue 49, 163 and / or 208 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; and iv) a polypeptide comprising or consisting of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity to the full length sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 which has retained or enhanced nepetalactol-related short chain reductase activity but does not comprise or consist the sequence set forth in SEQ ID NO 1.

[0068] In a preferred embodiment of the invention said modification at amino acid residue 49, 163 and / or 208 is a substitution.

[0069] Preferably said amino acid substitution is a conservative amino acid substitution.

[0070] Alternatively said amino acid substitution is a non-conservative amino acid substitution.

[0071] In a preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0072] In a further preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to glycine (G) or alanine (A) substitution.

[0073] In a preferred embodiment of the invention said modification at amino acid residue 163 is a serine (S) to aspartic acid (D) substitution.

[0074] In a preferred embodiment of the invention said modification at amino acid residue 208 is a methionine (M) to glutamic acid (E) substitution.

[0075] According to an aspect of the invention there is provided an isolated polypeptide selected from the group consisting of: i) a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2 wherein said amino acid sequence is modified at amino acid residue 41 , 159 and / or 204 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; ii) a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3 wherein said amino acid sequence is modified at amino acid residue 49, 164 and / or 209 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; and iii) a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4 wherein said amino acid sequence is modified at amino acid residue 49, 163 and / or 208 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity.

[0076] In a preferred embodiment of the invention said modification at amino acid residue 41 , 49, 159, 163, 164, 204, 208 and / or 209 is a substitution.

[0077] Preferably, said amino acid substitution is a conservative amino acid substitution. Alternatively, said amino acid substitution is a non-conservative amino acid substitution.

[0078] In a preferred embodiment of the invention said modification at amino acid residue 41 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0079] In a preferred embodiment of the invention said modification at amino acid residue 159 is an isoleucine (I) to aspartic acid (D) substitution.

[0080] In a preferred embodiment of the invention said modification at amino acid residue 204 is a methionine (M) to glutamic acid (E) substitution.

[0081] In a preferred embodiment of the invention said modification at amino acid residue 49 is an aspartic acid (D) to serine (S), asparagine (N), glycine (G) or alanine (A) substitution.

[0082] In a preferred embodiment of the invention said modification at amino acid residue 164 is an asparagine (N) ) to aspartic acid (D) substitution.

[0083] In a preferred embodiment of the invention said modification at amino acid residue 209 is a methionine (M) to glutamic acid (E) substitution.

[0084] In a preferred embodiment of the invention said modification at amino acid residue 163 is anasparagine (N) to aspartic Acid (D) substitution.

[0085] In a preferred embodiment of the invention said modification at amino acid residue 208 is a methionine (M) to glutamic acid (E) substitution.

[0086] According to an aspect of the invention there is provided an isolated nucleic acid molecule that encodes a modified nepetalactol-related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence that encodes a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 2 wherein said amino acid sequence is modified at amino acid residue 41 , 159 and / or 204 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; ii) a nucleotide that encodes a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 3 wherein said amino acid sequence is modified at amino acid residue 49, 164 and / or 209 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; iii) a nucleotide sequence that encodes a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 4 wherein said amino acid sequence is modified at amino acid residue 49, 163 and / or 208 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity, and iv) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i)-iii) and retains nepetalactol-related short chain reductase activity,

[0087] According to an aspect of the invention there is provided a transcription cassette comprising a nucleic acid molecule according to the invention.

[0088] Preferably, transcription from said transcription cassette is regulated by a heterologous promoter sequence.

[0089] According to a further aspect of the invention there is provided a vector comprising a nucleic acid molecule encoding a polypeptide according to the invention wherein said nucleic acid molecule is operably linked to a nucleic acid molecule comprising a promoter sequence.

[0090] In a preferred embodiment of the invention said promoter sequence is a heterologous promoter sequence.

[0091] In a preferred embodiment said promoter sequence is adapted for expression in the trichome cells of a plant.

[0092] In a preferred embodiment of the invention said vector is adapted for expression in a plant cell.

[0093] According to a further aspect of the invention there is provided a vector comprising a transcription cassette according to the invention encoding a polypeptide according to the invention.

[0094] In a preferred embodiment of the invention said transcription cassette comprises at least two nucleic acid molecule encoding one or more polypeptides according to the invention.

[0095] In a preferred embodiment said promoter sequence is adapted for expression in the trichome cells of a plant.

[0096] T richome specific promoters are know and can be specific for expression of genes in glandular or non-glandular trichomes. Examples of trichome specific promoters are for example GaMYB2, NtCYP71 D16 or NtCPS2. In a preferred embodiment of the invention said vector is adapted for expression in a microbial host cell.

[0097] In a preferred embodiment of the invention there is provided a microbial host cell comprising the vector according to the invention.

[0098] In a preferred embodiment of the invention said microbial host cell is a eukaryotic microbial cell.

[0099] Preferably, said eukaryotic microbial host cell is a fungal cell, for example a yeast cell such as Saccharomyces cerevisae.

[0100] In an alternative preferred embodiment of the invention said microbial host cell is a prokaryotic host cell. Preferably, said prokaryotic host cell is a bacterial cell, for example an Escherichia coli (E. coli) cell.

[0101] If microbial cells are used as organisms in the process method according to the invention they are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulphate, trace elements such as salts of iron, copper, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0 °C and 100 °C, preferably between 10 °C and 60 °C, while gassing in oxygen.

[0102] The pH of the liquid medium can either be kept constant and regulated during the culturing period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. To this end, the organisms can advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

[0103] The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).

[0104] As described above, these media which can be employed in accordance with the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements. Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats.

[0105] Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids, or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract, and others. The nitrogen sources can be used individually or as a mixture.

[0106] Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulphate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, and iron.

[0107] Inorganic sulphur-containing compounds such as, for example, sulphates, sulphites, dithionites, tetrathionates, thiosulfates, sulphides, or else organic sulphur compounds such as mercaptans and thiols may be used as sources of sulphur for the production of sulphur- containing fine chemicals and pathway intermediates, in particular of methionine.

[0108] Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

[0109] Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

[0110] The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, panthothenate, and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Editors PM. Rhodes, PF. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

[0111] All media components are sterilized, either by heat (20 min at 1.5 bar and 121 °C) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

[0112] The culture temperature is normally between 15 °C and 45 °C, preferably at from 25 °C to 40 °C and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of vectors it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20 °C to 45 °C and preferably 25 °C to 40 °C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 10 to 160 hours.

[0113] The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It is advantageous to process the biomass after its separation.

[0114] In a preferred embodiment of the invention said vector is adapted for expression in a plant cell.

[0115] According to a further aspect of the invention there is provided a cell culture comprising a microbial cell according to the invention.

[0116] According to a further aspect of the invention there is provided a fermenter comprising a microbial cell culture according to the invention.

[0117] According to a further aspect of the invention there is provided a plant cell modified by transformation or transfection of a nucleic acid molecule or vector according to the invention. In a preferred embodiment of the invention said nucleic acid molecule or vector comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO 10.

[0118] In a further preferred embodiment of the invention said nucleic acid molecule or vector comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO 9.

[0119] In a further preferred embodiment of the invention said nucleic acid molecule or vector comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO 8.

[0120] In a further preferred embodiment of the invention said nucleic acid molecule or vector comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO 5.

[0121] In a further preferred embodiment of the invention said nucleic acid molecule or vector comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO 6.

[0122] In a further preferred embodiment said plant cell is further modified by the transformation or transfection with a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol-related short chain reductase polypeptide or a vector comprising a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol-related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence comprising or consisting of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19 or SEQ ID NO 20, ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i) and retains nepetalactol-related short chain reductase activity, iii) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue in the amino acid sequence as represented in iii) above and which has retained or enhanced nepetalactol-related short chain reductase activity. In a preferred embodiment of the invention said modified amino acid sequence has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 % identity to the full-length amino acid sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and has retained or enhanced NEPS activity.

[0123] According to a further aspect of the invention there is provided a plant cell modified by transformation or transfection with a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol-related short chain reductase polypeptide or a vector comprising a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol- related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence comprising or consisting of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19 or SEQ ID NO 20, ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i) and retains nepetalactol-related short chain reductase activity, iii) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4. iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue in the amino acid sequence as represented in iii) above and which has retained or enhanced nepetalactol-related short chain reductase activity.

[0124] In a preferred embodiment of the invention said modified amino acid sequence has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 % identity to the full-length amino acid sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and has retained or enhanced NEPS activity.

[0125] In a preferred embodiment said transcription cassette is regulated by a heterologous promoter sequence

[0126] According to an aspect of the invention there is provided a plant comprising a modified plant cell according to the invention.

[0127] In a preferred embodiment of the invention said plant is of the genus Nepeta. Preferably, said plant is the Nepeta species: N. mussinii syn racemosa or N. cataria.

[0128] In an alternative preferred embodiment of the invention said plant is selected from: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerate), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana spp.), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citrus tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus spp.), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

[0129] Preferably, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops. Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum.

[0130] Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava been, lentils, chickpea, etc.

[0131] In a preferred embodiment of the invention said modified plant comprises enhanced levels of DHN when compared to wild type plants.

[0132] According to a further aspect of the invention there is provided the use of a nucleic acid molecule that encodes a polypeptide according to the invention in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

[0133] Preferably said conversion is the conversion of nepetalactone to dihydronepetalactone. According to a further aspect of the invention there is provided the use of a polypeptide according to the invention in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

[0134] Preferably said conversion is the conversion of nepetalactone to dihydronepetalactone.

[0135] According to a further aspect of the invention there is provided the use of a cell according to the invention in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

[0136] Preferably said conversion is the conversion of nepetalactone to dihydronepetalactone.

[0137] According to a further aspect of the invention there is provided the use of a plant according to the invention in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

[0138] Preferably said conversion is the conversion of nepetalactone to dihydronepetalactone.

[0139] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers.

[0140] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0141] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0142] An embodiment of the invention will now be described by example only and with reference to the following figures:

[0143] Figure 1. Chemicals of interest. A. Nepetalactone and dihydronepetalactone (DHN) isomers. B. Proposed route to DHN from nepetalactone. C. Nepetalactone biosynthesis pathway in Nepeta. ISY = iridoid synthase, NEPS = nepetalactolrelated short chain reductases; Figure 2. NEPS enzymes. A. Multiple alignment of NEPS used. B. Protein sequence identity of the alignment. C. SDS -page showing purification of NEPS2 as example process;

[0144] Figure 3. Enzymatic conversion of nepetalactone to DHN. A. GC-MS analysis of enzyme reaction containing: 0.5 mM cis-trans-nepetalactone, 5 pM NEPS5, 4 mM NADH, 0.5 M MOPS pH 7.5, 3h, 30 °C. Control is missing enzyme. Small peak observed in enzyme reactions, coeluting with DHN2 standard. C. El spectrum of DHN2 (A1) and enzyme peak co-eluting with DHN2 standard (B1);

[0145] Figure 4. Formation of DHN2 from cis-trans-nepetalactone with NEPS. DHN2 peak area from three replicates (individual reactions dots, averages bars). A. Single NEPS. B. Mixture of NEPS. Conditions: GC-MS analysis of enzyme reaction containing: 0.5 mM cis-trans- nepetalactone, 5 pM total enzyme, 4 mM NADH, 0.5 M MOPS pH 7.5, 3h, 30 °C (N1 = SEQ ID NO: 1 , N2 = SEQ ID NO: 2, N4 = SEQ ID NO: 3, N5 = SEQ ID NO: 4);

[0146] Figure 5. Formation of putative DHN isomers from trans-cis-nepetalactone with NEPS5. A. GC-MS analysis of enzyme reaction containing: 0.5 mM cis-trans-nepetalactone, 5 pM total enzyme, 4 mM NADH, 0.5 M MOPS pH 7.5, 3h, 30 °C. Control is missing enzyme. Peaks observed in enzyme reactions, but not co-eluting with standard. B. El spectrum of putative DHN isomers (RT = 20.7 (top) and 21.5 (bottom) respectively). C. Products (RT = 20.7 and 21.5) peak areas from single enzyme reactions. D. Mixtures of NEPS showing RT 21.4 product;

[0147] Figure 6. Formation of DHN from crude Passmore oil. A. GC-MS analysis of enzyme reaction containing: MOPS buffer 0.5 M pH 7.5, Passmore oil 0.5 mM, NADH 4 mM, NEPS 100 pM. Formation of DHN2 clear in NEPS1 (shown here) and NEPS5. Nepetalactone (NL) content decreases. B. Time course showing DHN2 formation in the reaction over 48 h;

[0148] Figure 7. Optimisation of reaction with crude Passmore oil and NEPS. A. Formation of DHN2 after 24 h at different pHs. B. Nepetalactone content at 0 h and 24 h across different pHs. Conditions: NEPS1 75 pM, 4 mM NADH, 0.1 mg / mL CPO and bis-tris propane buffer at 100 mM and a pH range from 6.5-9.0 and tracked this over 24 h;

[0149] Figure 8. Buffer optimisation for passmore oil. A. DHN2 output with different buffers (Conditions: buffer 0.5 M, Passmore oil 0.5 mM, NADH 4 mM, NEPS2 enzyme 20 pM, incubated at 30°C, then quenched at24 h). B. DHN2 output with different additives (conditions: MES 100 mM pH 6.5, Passmore oil 1 mg / mL, NADH 4 mM, NEPS1 75 pM, with additives (1 mM DTT, 1 mM EDTA, 5% v / v MeOH, 5% v / v DMSO, 5% v / v IPA, 250 mM NaCI or 4 uM MLP);

[0150] Figure 9. A. Nepetalactone content in reactions without enzyme. 3 h and 24 h samples run as duplicates. In buffer nepetalactone content drops to near zero at 24 h, with more nepetalactone in the neat water sample and then MeCN containing sample. B. NMR spectrum of nepetalactone incubated in buffer or water. In water the nepetalactone is stable but in buffer it breaks down to nepetalic acid; and C Impact of MeCN on DHN formation. Left: Nepetalactone content after 24 h with 0, 10 and 20% v / v MeCN, Right: DHN2 formation after 24 h with 0, 10 and 20% v / v MeCN, Y-axis shows peak area relative to internal standard; and B. Nepetalactone stability in water and buffer.

[0151] Figure 10. Formation of DHN from NEPS variants. A. Testing different cofactors. Conditions: 100 mM Bis-Tris pH 8, 0.1 mg / mL Passmore oil, NADH 4 mM, NEPS1 variants 35 pM, 24 h. B. Formation of DHN2 in NEPS variants: 100 mM Bis-Tris pH 8; 0.1 mg I mL Passmore oil; NADH 4 mM; NEPS1 35 pM; 24 h, RT.

[0152] Figure 11 : Sequences of the various nepetalactol-related short chain reductases disclosed herein.

[0153] Figure 12: illustrates cofactor switching showing the ratio of product production comparing NADPH / NADH.

[0154] Figure 13. A. Increase in DHN with variant expression. B. El spectrum showing peak match to DHN isomer.

[0155] Figure 14: Sequences of the nepetalactol-related short chain reductases set forth in SEQ ID NO 17- 20.

[0156] Materials and Methods

[0157] NEPS genes and expression constructs

[0158] Full length genes for Nepeta racemosa NEPS1 , 2, 4 and 5 (Lichman et al. 2019, 2020) were expressed in pOPINF vectors to generate N-terminal hexahistidine fusions under an IPTG inducible promoter. Mutations of NEPS1 (D49A, D49G, D49N, D49S, S163D and M208E) were ordered as synthetic gene constructs from TWIST BioScience and subcloned into the pHREAC vector (Peyret, Brown, and Lomonossoff 2019) through In-Fusion cloning, followed by heat shock transformation into Stellar competent cells and grown overnight on LB agar containing 50 pg / mL kanamycin. Positive colonies were identified by colony PCR whereafter 5 mL of an overnight LB broth culture was miniprepped using Qiagen kit. Two positive clones were verified through whole plasmid sequencing with Plasmidsaurus. A single clone was than transformed into Agrobacterium tumefaciens GV3101 and grown for 3-5 days on LB plates containing 25 pg / ml gentamycin and 50 pL / ml kanamycin. Positive transformants were verified through colony PCR whereafter an overnight culture was prepared. Cryogenic stocks were prepared at final glycerol concentration of 25% v / v.

[0159] Enzyme purification

[0160] E. coli SoluBL21 strains expressing NEPS genes were grown in 2XYT broth containing 100 pg / mL carbenicillin and induced with 0.5 mM IPTG at an QD600 between 0.5 and 0.8. Induction took place at 18 °C overnight. Cells were pelleted and resuspended in lysis buffer (0.2 mg / mL lysozyme, 50 mM Tris-HCL, 50 mM glycine, 5% v / v glycerol, 0.5 M NaCI, 20 mM imidazole, pH 8) with Complete Mini EDTA-free Protease Inhibitor Cocktail (Roche Diagnostics) at 1 tablet per 50 mL buffer and 250U / mL Benzonase nuclease. After 30 min of incubation at 4°C the lysis solution was passed through a cell press at 25 kPSI followed by high-speed centrifugation (35000 xg, 20 min). The supernatant was passed through a Minisart Glass fibre syringe filter (Sartorius, Germany) prior to affinity purification using either Co-NTA agarose resin (BioServUK, UK) or a 1mL HisTrap HP column coupled to an AKTA. Elution buffers were exchanged with storage buffer (20 mM HEPES pH 7.5,10% glycerol) using PD10 columns (Merck Life Sciences, UK) and where necessary concentrated through Vivaspin 10 kDA centrifugation columns (Sartorius, Germany). Protein concentrations were estimated through UV absorbance with a Nanodrop and the size verified through SDS-PAGE gel electrophoresis.

[0161] General assay conditions

[0162] Assays were performed at 30 °C in 1.5 mL tubes at a final volume of 100 pL. Assays were quenched by the addition of 100 pL EtOAc containing 0.1 mM carvone, followed by 5 min of vortex mixing and 10 min of centrifugation at 10,000 xg. The solvent layer was transferred into glass GCMS vials and crimp sealed. The carvone internal standard was prepared in MeCN as a 10X stock. The absence of enzyme served as a negative control for all assay conditions tested.

[0163] NEPS activity time course.

[0164] Assay conditions were set up with 0.5 M MOPS pH 7.5, 0.5 mM Passmore oil (assuming 100% nepetalactone content), and 4 mM NADH. NEPS1 was added at 0, 25, 50, 75 and 100 pM, and the reactions ran for 24 h and 48 h at 30 °C. NEPS activity in different buffers

[0165] The activity of NEPS1 was tested in MOPS, PBS and HEPES buffers at a final concentration of 0.5 M and pH 7. Reactions contained 0.5 mM Passmore oil (assuming 100% nepetalactone content), 4 mM NADH and 20 pM of enzyme.

[0166] Activity of NEPS over a pH range

[0167] Passmore oil (0.1 mg / mL) was used as substrate with 75 pM NEPS1 and 4 mM NADH. Activity was tested for a pH range from 6.5 to 9.0 at 0.5 increments in 100 mM Bis-Tris propane buffer.

[0168] Sample preparation

[0169] For tissue extracts, tissue (100 mg) was extracted with hexane (600 pL, containing 30 ng / pL nonyl acetate). For in vitro reactions, reactions (100 pL) were extracted with ethyl acetate (100 pL, containing 100 mM carvone). In both cases the samples were vortexed and centrifuged and the top layer removed into a sealed vial for GC-MS analysis.

[0170] GCMS of in vitro assays

[0171] Samples (2 pL) were analysed using a 2:1 split through GC-MS on an Agilent 7890A gas chromatograph (Agilent Technologies) interfaced to an Agilent 5975C MSD (Agilent Technologies). Separation was performed on a Zebron ZB5-HT-INFERNO column (5% phenyl methylsiloxane; length: 30 m; diameter: 250 pm; Phenomenex) interfaced with auxiliary transfer line (1.5 m, 150 pm) and helium as mobile phase at a constant flow of 1.2 mL / min. The inlet temperature was 250 °C with column temperatures as follow: 5 min at 80 °C, increased to 110 °C at 2.5 °C / min and then to 280 °C at 10 °C / min, where it was held for 4 min. A 5 min solvent delayed was used with a mass scan range of 50-400 at a threshold of 150. The MS source and quad was set to 230 °C and 150 °C, respectively.

[0172] NMR Assays

[0173] Cis-trans-nepetalactone was prepared at 50 mg / mL in MeCN and 60 pL aliquoted into 1.5 mL tubes. The solvent was dried off using an EZvac for 45 min with a maximum temperature of 50 °C using the low boiling point setting. Assays were performed in 20% D2O at a total volume of 600 pL. Final concentrations were 5 mg / mL nepetalactone, 60 pM NEPS1 , 12 mM NADH and 0.1 M phosphate buffer. Reference samples contained 0.1 M buffer, no additives (water only) or 20% v / v MeCN. The assay mixture was vortexed briefly to mix and transferred into NMR tubes using a glass pasteur pipette. 1 H NMR spectra were recorded on a Bruker 700 MHz NEO with a TCI Prodigy Cryoprobe, operating at 700 MHz. All spectral data was acquired at 295 K. Standards were analysed at ~5 mg / mL in neat D2O. Spectra were aligned using solvent peaks.

[0174] Transient expression in Nepeta cataria

[0175] Genes encoding enzymes (NEPS and NEPS variants) able to produce DHN in vitro were selected for transient expression in Nepeta cataria. Nepeta cataria seedlings were germinated on F2+S soil. One week old seedlings were transferred to P40 plant pots and grown under greenhouse conditions for another three to four weeks. Plants received no water for 24h prior to infiltration. Agrobacterium tumefaciens GV3101 expressing genes of interest in pHREAC plasmids was grown overnight in YEB broth supplemented with MgCI2. Cells were harvested by centrifugation at 4000g for 15 min and resuspended in an infiltration buffer (10 mM MES and 10 mM MgCI2). Acetosyringone was added to a final concentration of 100 uM, followed by 3h of incubation at room temperature and gentle shaking. The infiltration mixture was diluted to an GD600 of 0.4 in infiltration buffer containing silwet at 0.015% v / v. Plant leaves were briefly and gently massaged to perforate the trichomes whereafter they were submerged upside down in the infiltration mixture. The pot was covered with foil to prevent soil from falling into the infiltration mixture. Plants were placed in a vacuum chamber at -0.98 Bar for 30 seconds. If more than 50% of the leaf area was translucent we tagged the leaf as infiltrated. Leaves were inspected under a UV light 5-7 days post infiltration for GFP signal. Fluorescent leaves from a single plant were pooled as a biological replicate with 3-6 plants infiltrated per expression construct. Pooled samples were flash frozen in liquid nitrogen and stored at -70C.

[0176] GCMS of leaves

[0177] Frozen leaf samples were homogenised to a fine powder using a tissue lyser. Roughly 100mg of powder was weighed into a 2mL Eppendorf tube and extracted into 600uL of hexane containing 30ng / uL of nonyl acetate as internal standard. Samples were analysed by GCMS and compounds of interest identified using authentic standards for isomers of nepetalactol, nepetalactone and dihydronepetalactone.

[0178] Stable transformation of Nepeta

[0179] Genes encoding enzymes (NEPS and NEPS variants) able to produce DHN in vitro and via transient expression in Nepeta cataria were selected for stable transformation. Standard parts for Goldengate assembly of expression constructs were used in combination with a trichomespecific N. cataria promoter for controlled expression. A. tumefaciens GV3101 was transformed via heat shock and verified by colony PCR. Overnight cultures of bacteria grown in YEB broth containing antibiotics were harvested by centrifugation, washed and diluted in MMA media at an OD600 of 0.8. Leaves from 30 day old plants were cut into ~1cm squares, avoiding the main vein, and sterilised by 30 sec submersion in 1.2% NaOCI (+ 0.01% v / v Tween). Sterile leaves were washed twice in sterile dFW (+ 0.01 % v / v Tween). Leaves were suspended in the transformation mix for 30min, dried on sterile water-wetted Whatman paper and sealed with micropore tape. Plates were wrapped in foil and kept in the dark for two days at room temperature. The Whatman paper was used to transfer the leaf disc onto a plate containing a wash solution (100mg / L Kanamycin and 250 mg / L Cefotaxime). Discs were washed for 3min under gentle shaking. Discs were dried on sterile Whatman paper and placed on plates containing shoot induction medium. Plates were incubated in a light cabinet under constant light and high humidity at 23C. Once shoots started developing from the calli we excised the shoot with a sterile blade and transferred to a polypot containing rooting media. Once established we removed the media and transferred the plant to soil, maintaining high humidity for 2-3 weeks. After growth of explants, PCR and sequencing were used to verify the gene integration and its location in the genome. Plants were grown to maturity and propagated clonally via cuttings. The content of DHN was measured using GC-MS as described above. Alternative routes to stable transformation may also be considered such as via cell cultures and using Cas9 for targeted mutations.

[0180] Example 1

[0181] The molecule dihydronepetalactone (DHN) is a reduced form of nepetalactone (NL) (Figure 1A). Although it is a known natural product the pathway is unknown. Figure 1 C shows the natural NL biosynthesis pathway. DHN is being explored as an insect repellent as it is efficacious and stable (compared to NL). Industrially it is generated from NL using catalytic hydrogenation. We propose using reductase enzymes for make DHN from NL (Figure 1 B). However, as the double bond in NL is not in the typical form for enzymatic reduction and would not typically be converted by a reductase, this reaction would be surprising.

[0182] Example 2

[0183] Demonstration of NEPS activity with NL

[0184] NEPS were selected to convert NL to DHN. We were able to express and purify NEPS enzymes from N. racemosa using standard E. coli expression system and nickel affinity purification (Figure 2). We incubated NEPS enzymes with 0.5 mM c / s,trans-nepetalactone (ctNLone), 4 mM NADH and 0.5 M MOPS buffer pH 7.5 for 3 h at 30 °C. After analysis of the reactions through GC-MS analysis, we were able to observe a peak co-eluting with DHN2 peak of the mixed standard (Figure 3A). The El spectrum matched the between the enzyme product and compound standard (Figure 3B) confirming its identity. The evidence of DHN2 formation with enzymes from ctNLone is clear based on standards. There was limited accumulation of possible intermediates or shunt products in this pathway. Formation of DHN2 from ctNLone was seen in NEPS1 and NEPS5 but not NEPS2 or 4 (Figure 4A). We also attempted combined different enzymes and observed DHN2 production from ctNLone with all combinations attempted (NEPS1+2+5, NEPS1+4+5, NEPS1+5, NEPS2+4) (Figure 4B).

[0185] We then incubated NEPS enzymes with 0.5 mM trans, c / s-nepetalactone, 4 mM NADH and 0.5 M MOPS buffer pH 7.5 for 3 h at 30 °C. After analysis of the reactions through GC-MS analysis, we did not observe peaks co-eluting with DHN2 or DHN3 of our mixed standard. (Figure 5A). With NEPS5 we did see peaks emerge that had similar retention time to the standards and had El spectra that matched previously described trans-fused DHN isomers (e.g. DHN1 , where the 4a and 7a hydrogens are on opposite sides of the molecule) (Zimmermann et al. 2012) (Figure 5B), indicating DHN isomers were formed in the reaction. We also observed other new broad peaks that may represent alcohol or aldehyde intermediates or shunt products. We attempted enzyme combinations here too and saw the putative DHN isomer forming with NEPS1+2+5, NEPS1+4+5, NEPS1+5 but not NEPS2+4 (Figure 5C).

[0186] DHN2 was preferably produced from the c / s,trans-nepetalactone substrate. Moreover, the reaction with this substrate showed a lack of other peaks that are likely enzyme derived. In contrast, when using trans, c / s-nepetalactone as a substrate only NEPS5 showed the production of DHN isomers (that were not DHN2 or DHN3).

[0187] Example 3

[0188] Crude oil

[0189] Selected cultivars (chemotypes) of catmint are available which produce an oil rich in cis,trans- nepetalactone (and low in trans, c / s-nepetalactone), eg N. cataria cultivar CR9. Such oils would be a more realistic feedstock for an industrial process compared to using a pure chemical as substrate. Therefore, we elected to test NEPS enzymes using crude CR9 oil instead of a purified substrate. This reaction was performed with NEPS1 or NEPS5 (100 pM) and with 0.5 M MOPS buffer pH 7.5 and 4 mM NADH. CR9 oil was added at 0.5 mM, this concentration calculated assuming that it was 100% nepetalactone. We identified DHN2 accumulating in the reaction by GC-MS (Figure 5A). There was also a hint that the nepetalactone content was decreasing in both the control and enzyme reaction (Figure 5A). We showed that 48 h the product was still increasing but the rate of increase had slowed down (Figure 5B).

[0190] Example 4

[0191] Optimisation To optimise the reaction conditions to maximise conversion we performed it with NEPS1 75 pM, 4 mM NADH, 0.1 mg / mL CR9 oil and bis-tris propane buffer at 100 mM and a pH range from 6.5-9.0 and tracked this over 24 h. We saw largest accumulation of DHN2 at pH 8 (Figure 6A). Surprisingly we saw large decrease in nepetalactone in all conditions, but greater consumption at higher pHs (Figure 6B). Even the zero-hour reactions which are quenched immediate after formation clearly showed nepetalactone degradation. Using a standard curve to quantify the data, it was evident that DHN2 production was a maximum 10% at pH 8 yet there was nearly a complete consumption of nepetalactone.

[0192] Other optimisation was attempted including testing different buffers and pHs (HEPES, MOPS, PBS pH 6, 7, 8). Here we saw greatest DHN formation in PBS pH 7 and 8 and MOPS pH 8 (Figure 7A). IPA addition with MOPS did not increase DHN formation further. Later we examined NEPS1 activity with CR9 oil with these additives: 1 mM DTT, 1 mM EDTA, 5% v / v MeOH, 5% v / v DMSO, 5% v / v IPA, 250 mM NaCI or 4 uM MLP. None of these increase DHN significantly beyond the non-additive control (Figure 7B).

[0193] Example 5

[0194] Nepetalactone stabilisation

[0195] To identify the cause of non-enzymatic nepetalactone breakdown and potential for preventing this we investigated its stability without enzymes or cofactors present. We incubated CR9 oil in buffer, water and water with 20% v / v MeCN. We followed the content of nepetalactone at zero, three and 24 hours. We observed that in buffer the nepetalactone content halved after 3 h and was near completely depleted after 24 h (Figure 8A). In neat water, nepetalactone breakdown was negligible at three hours but observed after 24 h. Addition of MeCN appeared to protect nepetalactone. To determine what nepetalactone was being converted into we incubated it in an NMR tube and followed the reaction over 24 h and analysed the proton spectrum (Figure 8B). In the reaction containing just water, nepetalactone was stable after 24 h, we could see a clean sample with the expected proton shifts for nepetalactone. In contrast the nepetalactone incubated in buffer (phosphate buffer) gained new peaks. These corresponded with the open form of nepetalic acid, as we could observed shifts characteristic of this compound, most notably the aldehyde at 9.5 ppm. This indicates that the buffer is acting as a general acid to break open the nepetalactone in aqueous solution. However, even if we were to not add buffer to the reaction, the enzyme is stored in buffer so nepetalactone degradation may still occur. We tested NEPS1 with 0, 10, 20 % v / v MeCN additive and CR9 oil over 24 h and did see protection of NLone. But MeCN causes DHN formation to drop at 10% and be eliminated at 20% indicating that MeCN is detrimental to the enzyme activity (Figure 9).

[0196] Example 6

[0197] Mutants

[0198] The first NEPS1 mutant altered the selectivity of the cofactor from NADH to NADPH. This would be more suitable for a reductase in planta as NADPH is typically used for reductases, and NAD+ used for oxidases.

[0199] The NEPS1 variant mutant enzymes were ordered as synthetic genes from Twist Bioscience prepared in pET28a vectors for E. coli expression.

[0200] The mutants were expressed and purified from E. coli and assayed with CR9 oil. We observed no formation of DHN with NADH for D49 mutant but for D49A and D49G formation of DHN was observed with NADPH (Figure 10A). This suggests that the co-factor selectivity has been modified. For WT, M208E and S163D we saw DHN formation with both NADH and NADPH, interestingly M208E and S163D appeared to show enhanced formation of DHN with NADH compared to WT. This was verified in a subsequent experiment (Figure 10B).

[0201] Example 7

[0202] We have successfully developed a transient expression system in Nepeta cataria, verified by GFP fluorescence and qRT-PCR. We use Agrobacterium mediated transient expression with a vector encoding NEPS1 variants. The Agrobacterium media is syringe or vacuum infiltrated into leaves of Nepeta cataria, and the gene of interest is expressed and metabolite content can be modified. We infiltrated NEPS variants into Nepeta cataria using transient expression and analysed the metabolites by GC-MS. We identified a peak that increased in concentration with the infiltration of NEPS1 D49S, a variant that has NADPH co-factor selectivity (Figure 13A). The mass spectrum of this peak matched literature data for trans-trans- dihydronepetalactone (Figure 13B). This result demonstrates that the transient expression of NEPS variants in with enhanced NADPH selectivity in Nepeta cataria can boost formation of DHN isomers.

[0203] References

[0204] Lichman, Benjamin R., Grant T. Godden, John P. Hamilton, Lira Palmer, Mohamed O.

[0205] Kamileen, Dongyan Zhao, Brieanne Vaillancourt, et al. 2020. “The Evolutionary Origins of the Cat Attractant Nepetalactone in Catnip.” Science Advances 6 (20): eaba0721.

[0206] Lichman, Benjamin R., Mohamed O. Kamileen, Gabriel R. Titchiner, Gerhard Saalbach, Clare E. M. Stevenson, David M. Lawson, and Sarah E. O’Connor. 2019. “Uncoupled Activation and Cyclization in Catmint Reductive Terpenoid Biosynthesis.” Nature Chemical Biology 5 (1): 71-79.

[0207] Peyret, Hadrien, James K. M. Brown, and George P. Lomonossoff. 2019. “Improving Plant Transient Expression through the Rational Design of Synthetic 5’ and 3’ Untranslated Regions.” Plant Methods 15 (September): 108.

[0208] Zimmermann, Nicole, Robert Hilgraf, Lutz Lehmann, Daniel Ibarra, and Wittko Francke. 2012. “Stereoselective Synthesis of Trans-Fused Iridoid Lactones and Their Identification in the Parasitoid Wasp Alloxysta Victrix, Part I: Dihydronepetalactones.” Beilstein Journal of Organic Chemistry 8 (August): 1246-55.

[0209] Nestor J. Hernandez Lozada, Benke Hong, Joshua C. Wood, Lorenzo Caputi, Jerome Basquin, Ling Chuang, Maritta Kunert, Carlos E. Rodriguez Lopez, Chloe Langley, Dongyan Zhao, C. Robin Buell, Benjamin R. Lichman & Sarah E. O’Connor. 2022.

[0210] “Biocatalytic routes to stereo-divergent iridoids”, Nature Communications, volume 13, Article number: 4718

Claims

Claims1. A method to produce dihydronepetalactone (DHN) comprising the steps:(i) providing a substrate selected from the group: nepetalactone, nepetalactol, iridodial or catmint oil comprising nepetalactone,(ii) providing at least one nepetalactol-related short chain reductase polypeptide (NEPS)(iii) forming a preparation comprising said substrate with said NEPS in the presence of a reducing co-substrate to form a reaction mixture under conditions to convert said substrate to DHN.

2. The method according to claim 1 wherein said substrate is nepetalactone or catmint oil comprising nepetalactone.

3. The method according to claims 1 or 2 wherein said NEPS is provided as an isolated polypeptide.

4. The method according to any one of claims 1 to 3 wherein said nepetalactone is cis, trans nepetalactone.

5. The method according to any one of claims 1 to 4 wherein said nepetalactone is trans, cis nepetalactone.

6. The method according to any one of claims 1 to 5 wherein said nepetalactol-related short chain reductase polypeptide comprises or consists of an amino acid sequence with at least 80% at least 85% at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the full-length sequence set forth in SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 and has retained or enhanced NEPS activity.

7. The method according to any one of claims 1 to 5 wherein said nepetalactol-related short chain reductase polypeptide comprising or consisting of an amino acid sequence selected from the group: SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4,8. The method according to any one of claims 1 to 7 wherein said NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO 1 is combined with one or more NEPS polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID: NO 3 and SEQ ID NO:4, preferably with SEQ ID NO 4.

9. The method according to any one of claims 1 to 7 wherein said NEPS polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO 2 is combinedwith one or more NEPS polypeptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO: 3 and SEQ ID NO: 4, preferably with SEQ ID NO 3.

10. The method according to any one of claims 1 to 7 wherein said NEPS polypeptide comprising or consisting of an amino acid sequence set forth with SEQ ID NO 1 is combined with one or more NEPS polypeptides comprising or consisting of an amino acid sequence set forth in SEQ 3 and SEQ ID NO: 4, or set forth in SEQ ID NO 2 and SEQ ID NO: 4.

11. The method according to any one of claims 6 to 10 wherein said sequence set forth in SEQ ID NO 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 is modified and has at least one amino acid substitution.

12. The method according to claim 11 wherein said modified sequence is selected from the group consisting of: SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, or a combination thereof13. The method according to any one of claims 1 to 12 wherein said reduced co-substrate is NADPH or NADH.

14. The method according to any one of claims 1 to 13 wherein said reaction mixture further comprises stabilisers, preferably an organic solvent such for example acetonitrile.

15. An isolated nucleic acid molecule that encodes a modified nepetalactol-related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence set forth in SEQ ID NO 11 , SEQ ID NO 12 SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15 or SEQ ID NO 16 ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i) and retains nepetalactol-related short chain reductase activity, iii) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10, iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion, or substitution of at least one amino acid residue as represented in iii) above and which has retained or enhanced nepetalactol-related short chain reductase activity.

16. An isolated polypeptide selected from the group consisting of: i) a polypeptide comprising or consisting of an amino acid sequence selected from the group consisting of: SEQ ID NO 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10; ii) a polypeptide comprising or consisting of a modified amino acid sequence wherein said polypeptide is modified by addition, deletion or substitution or at least one amino acid residue of the sequence represented in SEQ ID NO: 1 which has retained or enhanced nepetalactol-related short chain reductase activity; iii) a polypeptide comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 1 wherein said amino acid sequence is modified at amino acid residue 49, 163 and / or 208 and wherein said polypeptide has retained or enhanced nepetalactol-related short chain reductase activity; and iv) a polypeptide comprising or consisting of an amino acid sequence that has at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the full length sequence set forth in SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, which has retained or enhanced nepetalactol-related short chain reductase activity but does not comprises or consist of the sequence set forth in SEQ ID NO 1 .

17. The isolated polypeptide according to claim 16 wherein said modification at amino acid residue 49 is a aspartic Acid (D) to serine (S,) asparagine (N), glycine (G) or alanine (A) substitution, said modification at amino acid residue 163 is a aerine (S) to aspartic Acid (D) substitution and said modification at amino acid residue 208 is a methionine (M) to glutamic Acid (E) substitution.

18. A transcription cassette comprising a nucleic acid molecule according to claim 15.

19. The transcription cassette according to claim 18 wherein said transcription cassette is regulated by a heterologous promoter sequence.

20. A vector comprising the transcription cassette according to claims 18 or 1921. The vector according to claim 20 wherein said vector is adapted for expression in a microbial host cell.

22. A microbial cell comprising the vector according to claim 21 .

23. A cell culture comprising a microbial cell according to claim 22.

24. A plant cell modified by transformation or transfection with a nucleic acid molecule according to claim 15, transcription cassette according to claims 18 or 19 or vector according to claim 20.

25. A plant cell modified by transformation or transfection with a nucleic acid molecule that encodes a nepetaloctol-related short chain reductase polypeptide, a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol-related short chain reductase polypeptide ora vector comprising a transcription cassette comprising a nucleic acid molecule that encodes a nepetalactol-related short chain reductase polypeptide wherein said nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group: i) a nucleotide sequence comprising or consisting of SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19 or SEQ ID NO 20, ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence as defined in i) and retains nepetalactol-related short chain reductase activity, iii) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO 1 , SEQ ID NO 2, SEQ ID NO 3 or SEQ ID NO 4. iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition, deletion or substitution of at least one amino acid residue in the amino acid sequence as represented in iii) above and which has retained or enhanced nepetalactol-related short chain reductase activity.

26. The plant cell according to claim 25 wherein said modified amino acid sequence has at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 % identity to the full-length amino acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 and has retained or enhanced NEPS activity.

27. A plant comprising a modified plant cell according to any one of claims 24 to 26.

28. The plant according to claim 27 wherein said plant is of the genus Nepeta.

29. The use of a nucleic acid molecule according to claim 15 in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

30. The use of a polypeptide according to claims 16 or 17 in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.31 . The use of a cell according to claim 22 in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

30. The use of a plant according to claim 27 for use in the conversion of nepetalactone, nepetalactol or iridodial to dihydronepetalactone.

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