WO2024133899A2 - Production of plant alkaloids - Google Patents

Production of plant alkaloids Download PDF

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WO2024133899A2
WO2024133899A2 PCT/EP2023/087642 EP2023087642W WO2024133899A2 WO 2024133899 A2 WO2024133899 A2 WO 2024133899A2 EP 2023087642 W EP2023087642 W EP 2023087642W WO 2024133899 A2 WO2024133899 A2 WO 2024133899A2
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seq
sequence identity
gene encoding
functional homologue
host cell
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PCT/EP2023/087642
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French (fr)
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Davide MANCINOTTI
Ting Yang
Fernando Geu FLORES
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University Of Copenhagen
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  • the present invention relates to the field of plant alkaloid synthesis, in particular in recombinant host cells. More specifically, the invention relates to production of (-)- sparteine or precursors thereof as well as to enzymes useful for such production.
  • Plant alkaloids are a large family of bioactive, nitrogen-containing compounds commonly used as medical drugs or stimulants. In food and feed crops, however, the accumulation of plant alkaloids is undesirable, as they typically confer toxicity or act as anti-nutritional compounds. Such is the case of the quinolizidine alkaloids (QAs), which are anti-cholinergic compounds produced by the protein crops collectively called lupins (Lupinus spp.). Lupins are the legume crops with the highest seed protein content (up to 44% protein for L. mutabilis). However, their accumulation of QAs limits their use as food and feed crops. Low-alkaloid varieties (sweet varieties) have been produced via traditional breeding, but remaining QA levels are variable and often surpass the thresholds established by the food and feed industries.
  • QAs quinolizidine alkaloids
  • sparteine One of the most versatile QA is the chiral diamine sparteine. Specifically, complexes between sparteine and lithium have proven unrivaled for asymmetric deprotonations, substitutions, carbometalations, and directed orto-metalations. As chemists continue to find new uses for sparteine-lithium complexes, their versatility continues to grow, as exemplified by the development of assembly-line chiral synthesis. Originally, only (-)- sparteine was commercially available, enabling the asymmetric synthesis of just one of two possible products in each case.
  • (+)-sparteine which became known as the (+)-sparteine surrogate.
  • Commercial (-)-sparteine remained cheap throughout the 2000s. However, for reasons that remain unclear, it later became expensive and, at times, fully unavailable.
  • the QAs that accumulate in lupins are mostly of the tetracyclic type, the simplest of which is sparteine.
  • the majority of QAs in lupins share the common tetracyclic backbone of (-)-sparteine and are thought to derive from it.
  • the present invention provides the metabolic pathway for production of (-)-sparteine in Lupinus spp., as well as materials and methods for establishing a metabolic pathway for production of (-)-sparteine or precursors thereof in host cells.
  • QAs are derived from the amino acid L-lysine.
  • the first step of the pathway is decarboxylation of L-lysine by LDC to give cadaverine.
  • CAO oxidizes one end of cadaverine giving an aminoaldehyde that cyclizes readily into A1- piperideine.
  • A1-piperideine exists in equilibrium with its dimer tetahydroanabasine, and NCS facilitates said equilibrium.
  • Tetrahydroanabasine is /V-acetylated by AT to form N- acetylated tetrahydroanabasine.
  • G8HO catalyzes the hydroxylation of /V-acetylated tetrahydroanabasine allowing the formation of a didehydrolusitanine species containing a quinolizidine core.
  • CHYD and NCS1 are subsequently involved in deacetylation coupled to addition of a third A1-piperideine unit to give a tetradehydrosparteine species.
  • the tetradehydrosparteine species is reduced to (-)-sparteine by either VRED1 or VRED2.
  • the invention further shows that expression of CHYD, NCS1 , NCS2, VRED1 or VRED2, LDC, CAO, AT and G8HO is sufficient for production of (-)-sparteine while the additional expression of NCS3 and/or NCS4 leads to more efficient production of (-)- sparteine, whereas additional expression of ARED leads to improved stereoselectivity.
  • the invention provides a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S- norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11, S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the invention provides aforementioned enzymes per se and nucleic acids encoding same.
  • the invention also provides methods for producing (-)-sparteine or a precursor thereof, comprising the steps of: a. Providing a host cell of the invention; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating (-)-sparteine or a precursor thereof.
  • Figure 1 Schematic representation of the proposed biosynthetic pathway of (-)- sparteine.
  • Asterisks indicate that the intermediate exists as a complex equilibrium of different compounds. Shown here are representative forms of the intermediates. A more comprehensive, yet not exhaustive, overview of the forms taken on by the intermediates is presented in the section “Precursors of sparteine”.
  • NCS2 of SEQ ID NO 10 increases the production of ammodendrine in Nicotiana benthamiana.
  • FIG. 3 G8HO of SEQ ID NO 4 catalyzes the hydroxylation of /V-acetylated tetrahydroanabasine allowing the formation of a quinolizidine core.
  • Peaks a, b, and c correspond to the new compounds observed when co-expressing G8HO of SEQ ID NO 4; peak d corresponds to ammodendrine. Traces are combined extracted ion chromatograms (EICs) of m/z 207.15 ⁇ 0.01 ([M+H] + for peak a and [M-H2O+H] + for peaks b and c) and m/z 209.16 ⁇ 0.01 ([M+H] + for peak d). B) comparison of the ESI + CID MS 2 mass spectra of peak a and peak b showing key fragmentations of the apparent products of G8HO of SEQ ID NO 4.
  • the proposed structures are embedded and correspond to a didehydrolusitanine species (m/z 207.15, [M+H] + ) and 2- hydroxyammodendrine (m/z 225.16, [M+H] + ).
  • Parent molecular ions are marked in bold.
  • Neutral losses are marked in grey. Bonds that are cleaved upon fragmentation are marked by dashed lines and the m/z of the resulting fragment ions are indicated at the arrow tips.
  • Figure 4 Production of sparteine in N. benthamiana by co-expression of at least 9 genes.
  • Traces are extracted ion chromatograms (EICs) of the singly and doubly charged molecular ions of sparteine combined (m/z 235.22 ⁇ 0.01 and m/z 118.11 ⁇ 0.01 , respectively).
  • Figure 5 Accumulation of intermediates during the production of sparteine in N. benthamiana.
  • B) purity of sparteine versus two detected sparteine isomers (likely a-isosparteine and p-isosparteine), as determined by LC-MS. Box plots are standard ⁇ n 6 or 7). Letters indicate significant differences (one-way ANOVA with Tukey post hoc test P ⁇ 0.05)
  • BADH acetyltransferase and “AT” are used interchangeably throughout the description.
  • the term “cultivating” as used herein refers maintaining host cells under culture conditions which allow the cells to grow.
  • said culture conditions allow expression of the enzyme(s) encoded by the heterologous gene(s) contained in said host cells.
  • the host cells are cultivated under culture conditions allowing said host cells to produce (-)sparteine or a precursor thereof.
  • “cultivating” refers to maintaining said multicellular organisms under conditions allowing said multicellular organism to grow.
  • “cultivating” refers to maintaining said unicellular organism under conditions allowing said unicellular organism to grow and/or multiply.
  • , where Fraction ((-) isomer) + Fraction ((+) isomer) 1.
  • a mixture of 50% (-) isomer and 50% (+) isomer has an enantiomeric excess of 0%.
  • the mole fractions can be determined in a number of ways known in the art, including but not limited to chromatography using a chiral support, polarimetric measurement of the rotation of polarized light, nuclear magnetic resonance spectroscopy using chiral shift reagents which include but are not limited to lanthanide containing chiral complexes or the Pirkle alcohol, or derivatization of a compounds using a chiral compound such as Mosher's acid followed by chromatography or nuclear magnetic resonance spectroscopy.
  • the enantiomeric purity of (-)-sparteine is determined as described in Example 5 herein below.
  • enzyme refers to proteins or polypeptides, which are capable of catalyzing biochemical reactions. Further, unless context dictates otherwise, as used herein "enzyme” includes protein fragments that retain the relevant catalytic activity, and may include artificial enzymes synthesized to retain the relevant catalytic activity.
  • a functional homologue of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion.
  • a functional homologue shares at least at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence.
  • heterologous gene refers to a gene, which has been inserted into a host cell or into a progenitor of the host cell, e.g. by recombinant or transgenic methods.
  • the respective protein or RNA encoded by a heterologous gene is also referred to as "heterologous”.
  • the heterologous gene may be part of a nonintegrated nucleic acid, e.g. a vector, including but not limited to a plasmid.
  • the heterologous gene(s) are integrated into the host genome.
  • host cell refers to a cell, which comprises one or more heterologous genes.
  • lysine decarboxylase and “LDC” are used interchangeably throughout the description
  • polypeptide refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length. As is known to those skilled in the art, polypeptides may be processed and/or modified, and the term polypeptide may refer to both unmodified or modified polypeptides.
  • sequence identity describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment.
  • sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.
  • the Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.
  • the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • S-norcoclaurine synthase-like protein and “NCS” are used interchangeably throughout the description.
  • sparteine refers to (+)-sparteine, (-)-sparteine, or a mixture of both, “(-)-sparteine” is also known as (7S,7aR,14S,14aS)-dodecahydro-2H,6H-7,14- methanodipyrido[1,2-a:1',2'-e][1,5]diazocine, 7,14-Methano-2H,6H-dipyrido[1,2-a:1',2'- e][1,5]diazocine, dodecahydro-, [7S-(7a,7aa,14a,14aP)], CAS number 90-39-1 , Lupinidine, 6-p,7-a,9-a,11-a-Pachycarpine, l-Sparteine , (-)-Esparteina, (-)-Spartein, or (-)-Sparteinium.
  • “(+)-sparteine” is also known as (7R,7aR,14R,14aS)-dodecahydro- 2H,6H-7,14-methanodipyrido[1,2-a:1',2'-e][1 ,5]diazocine, 7,14-Methano-2H,6H- dipyrido[1,2-a:1',2'-e][1,5]diazocine, dodecahydro-, [7R-(7a,7aa,14a,14aP)], CAS number 492-08-0, Pachycarpine, d-Sparteine, (+)-Esparteina, (+)-Spartein, or (+)- Sparteinium.
  • the present disclosure concerns a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3, geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S- norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S-norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11 , S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or functional homologues of
  • Said enzymes may preferably have the enzyme activity described herein below in the section “Enzyme activity”.
  • the present disclosure concerns a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8- hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO
  • NCS1 S-norcoclaurine synthase-like protein 1
  • NCS2 S-norcoclaurine synthase-like protein 2
  • NCS3 S-norcoclaurine synthase-like protein 3
  • NCS4 S- norcoclaurine synthase-like protein 4
  • a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell in addition comprises one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3 or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.
  • LDC lysine decarboxylase
  • CAO copper amine oxidase
  • AT acetyltransferase
  • the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least
  • the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9 of the following genes: d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; g.
  • the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
  • heterologous genes a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity there
  • the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e.
  • a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto,; and e.
  • a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto f. a gene encoding VRED1 SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof
  • the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue
  • the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue
  • the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue
  • genes encoding enzymes described in this section may encode an enzyme of a specific sequence or a functional homologue thereof sharing at least 70% sequence identity thereto, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity thereto.
  • the present invention provides a biosynthetic pathway for production of alkaloids, notably (-)-sparteine.
  • the pathway comprises a number of enzymes, which for example may be combined as disclosed herein above in the section “Combinations of enzymes”.
  • the host cell comprises a heterologous gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof.
  • LDC of SEQ ID NO 1 or a functional homologue thereof preferably has lysine decarboxylase activity.
  • LDC of SEQ ID NO 1 or functional homologues thereof preferably have the ability to convert L-Lysine to cadaverine.
  • LDC of SEQ ID NO 1 or functional homologues thereof preferably speed up the rate of the decarboxylation of L-Lysine.
  • a functional homologue of LDC of SEQ ID NO 1 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme CAO of SEQ ID NO 2 or a functional homologue thereof.
  • CAO of SEQ ID NO 2 or a functional homologue thereof preferably has amine oxidase activity.
  • CAO of SEQ ID NO 2 or functional homologues thereof preferably have the ability to convert cadaverine to an aminoaldehyde. Furthermore, CAO of SEQ ID NO 2 or functional homologues thereof preferably speed up the rate of the oxidative cleavage of alkylamines into aldehydes and ammonia.
  • a functional homologue of CAO of SEQ ID NO 2 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme AT of SEQ ID NO 3 or a functional homologue thereof.
  • AT of SEQ ID NO 3 or a functional homologue thereof is preferably capable of catalysing /V-acetylation of tetrahydroanabasine.
  • AT of SEQ ID NO 3 or functional homologues thereof preferably have the ability to convert tetrahydrobasine to /V-acetylated tetrahydroanabasine. Furthermore, AT of SEQ ID NO 3 or functional homologues thereof preferably transfer acylated moieties (RC(O)R’) of an acyl-activated CoA thioester donor to an acceptor molecule.
  • acylated moieties R(O)R’
  • a functional homologue of AT of SEQ ID NO 3 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme G8HO of SEQ ID NO 4 or a functional homologue thereof.
  • G8HO of SEQ ID NO 4 or a functional homologue thereof preferably has hydroxylase activity against /V-acetylated tetrahydroanabasine.
  • G8HO of SEQ ID NO 4 or functional homologues thereof preferably have the ability to convert /V-acetylated tetrahydroanabasine to 2-hydroxyammodendrine. Furthermore, G8HO of SEQ ID NO 4 or functional homologues thereof preferably speed up the rate of the hydroxylation of /V-acetylated tetrahydroanabasine.
  • a functional homologue of G8HO of SEQ ID NO 4 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme CHYD of SEQ ID NO 5 or a functional homologue thereof.
  • CHYD of SEQ ID NO 5 or a functional homologue thereof preferably has deacetylase activity against a didehydrolusitanine species in the presence of NCS1 of SEQ ID NO 9.
  • a functional homologue of CHYD of SEQ ID NO 5 or functional homologues thereof preferably have the ability to convert didehydrolusitanine to its deacetylated form in the presence of NCS1 of SEQ ID NO 9.
  • a functional homologue of CHYD of SEQ ID NO 5 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme VRED1 of SEQ ID NO 6 or a functional homologue thereof.
  • VRED1 of SEQ ID NO 6 or a functional homologue thereof preferably is capable of catalysing reduction of a tetradehydrosparteine species.
  • VRED1 of SEQ ID NO 6 or functional homologues thereof preferably have the ability to convert tetradehydrosparteine to (-)-sparteine. Furthermore, VRED1 of SEQ ID NO 6 or functional homologues thereof preferably have increased ability to convert tetradehydrosparteine to (-)-sparteine in the presence of NCS3 of SEQ ID NO 11.
  • a functional homologue of VRED1 of SEQ ID NO 6 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme VRED2 of SEQ ID NO 7 or a functional homologue thereof.
  • VRED2 of SEQ ID NO 7 or a functional homologue thereof preferably is capable of catalysing reduction of di-iminium cations.
  • VRED2 of SEQ ID NO 7 or functional homologues thereof preferably have the ability to convert tetradehydrosparteine to (-)-sparteine. Furthermore, VRED2 of SEQ ID NO 7 or a functional homologue thereof preferably has increased ability to convert tetradehydrosparteine to (-)-sparteine in the presence of NCS3 of SEQ ID NO 11.
  • a functional homologue of VRED2 of SEQ ID NO 7 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme ARED of SEQ ID NO 8 or a functional homologue thereof.
  • ARED of SEQ ID NO 8 or a functional homologue is preferably an enzyme annotated as an oxidoreductase, for example, it may be annotated as an oxidoreductase based on its sequence.
  • ARED of SEQ ID NO 8 or a functional homologue thereof when expressed in N. benthamiana it preferably increases the enantiomeric excess of (-)-sparteine produced in said N. benthamiana to at least 65%, such as preferably to at least 70%, such as preferably to at least 75%, such as preferably to at least 80%, such as preferably to at least 85%, such as preferably to at least 90%, such as preferably to at least 95%, such as most preferably to at least 97% when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5 and VRED1-2 of SEQ ID NO 6-7.
  • ARED of SEQ ID NO 8 or a functional homologue thereof when expressed in N. benthamiana it preferably increases the enantiomeric excess of (-)-sparteine produced in said N. benthamiana to at least 65%, such as preferably to at least 70%, such as preferably to at least 75%, such as preferably to at least 80%, such as preferably to at least 85%, such as preferably to at least 90%, such as preferably to at least 95%, such as most preferably to at least 97% when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-2 of SEQ ID NO 9-10, CHYD of SEQ ID NO 5 and at least one of VRED1 of SEQ ID NO 6 or VRED2 of SEQ ID NO 7.
  • a functional homologue of ARED of SEQ ID NO 8 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme NCS1 of SEQ ID NO 9 or a functional homologue thereof.
  • NCS1 of SEQ ID NO 9 or a functional homologue thereof is preferably capable of catalysing the addition of a A1-piperideine unit to a didehydrolusitanine species in the presence of CHYD of SEQ ID NO 5.
  • a functional homologue of NCS1 of SEQ ID NO 9 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding the enzyme NCS2 of SEQ ID NO 10 or a functional homologue thereof.
  • NCS2 of SEQ ID NO 10 or a functional homologue thereof is preferably capable of catalysing dimerization of A1-piperideine to tetrahydroanabasine.
  • a functional homologue of NCS2 of SEQ ID NO 10 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof.
  • NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of aiding the reduction of a di-iminium cation intermediate by e.g. double-bond isomerization.
  • NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of aiding the reduction of a tetradehydrosparteine species.
  • NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of catalysing formation of (-)-sparteine from a tetradehydrosparteine species in the presence of VRED1 of SEQ ID NO 6 and/or VRED2 of SEQ ID NO 7.
  • a functional homologue of NCS3 of SEQ ID NO 11 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof.
  • NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of aiding the equilibrium between hydroxylated ammodendrine and a quinolizidine-containing compound.
  • NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of aiding the rearrangement of 2-hydroxyammodendrine.
  • NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of catalysing generation of a didehydrolusitanine species from 2- hydroxyammodendrine.
  • a functional homologue of NCS4 of SEQ ID NO 12 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the present invention relates to host cells comprising one or more heterologous genes encoding enzymes of the biosynthetic pathway to (-)-sparteine, notably any of the enzymes or combinations thereof described in the sections “Combinations of enzymes” and “Enzyme activity”.
  • the host cell or a progenitor thereof may be prepared by any useful method available to the skilled person.
  • the heterologous gene(s) may be inserted into a cell by direct uptake, transduction, f-mating, transfection, transformation, bacterial infiltration or any other methods known in the art useful for creating recombinant host cells.
  • the host cell is selected from the group of plant cells, yeast cells, bacteria cells and fungal cells. In some embodiments the host cell is comprised within a multicellular organism. In such embodiments, only some of the cells of said multicellular organism may comprise heterologous gene(s). It is however preferred that all cells of said multicellular organism are host cells that comprise the same heterologous gene(s).
  • the host cell is plant cells comprised within a plant, within a part of a plant or within the seeds of said plant.
  • all cells of said plant or part thereof are host cells comprising the same heterologous gene(s).
  • the host cells are plant cells, e.g. plant cells from a species of Nicotiana such as Nicotiana benthamiana or Nicotiana tabacum.
  • the host cell is a yeast cell, e.g. a yeast cell of the species Saccharomyces cerevisiae or Saccharomyces pombe.
  • a "plant cell” as used within the present invention refers to a structural and physiological unit of a plant, e.g. a tobacco plant.
  • the plant cell may be in form of a protoplast without a cell wall, an isolated single cell or a cultured cell, or as a part of higher organized unit such as but not limited to, plant tissue, a plant organ, or a whole plant.
  • a "yeast cell” is herein defined to include the group consisting of small, unicellular organisms capable of growth and reproduction through budding or direct division (fission), or by growth as simple irregular filaments (mycelium).
  • the yeast cell may be transformed or transfected with a heterologous vector for expression of a nucleic acid sequence inserted into the heterologous vector.
  • a yeast cell includes Saccharomyces cerevisiae or Saccharomyces pombe, commonly used for transfection and expression of heterologous proteins.
  • a fungi or fungal cell(s) as used herein refers to any cell present within or derived from an organism belonging to the Kingdom Fungi. The methods are applicable to all fungi and fungal cells that are susceptible of genetic modifications.
  • a "bacterial cell” includes prokaryotic cells that may be propagated in culture.
  • the bacterial cell may act as a host cell for the recombinant expression of heterologous proteins.
  • the bacterial cell may be transformed, transfected or infected with a vector for expression of a protein sequence inserted into the vector.
  • suitable bacterial cells include, but are not limited to E. coli, B. megaterium, B. subtilis and B. brevis and various species of Caulobacter, Staphylococcus, and Streptomyces.
  • the host cell is capable of producing lysine, such as L-lysine.
  • the host cell produces lysine, such as L-lysine.
  • the host cell is capable of producing A 1 -piperideine.
  • the host cell produces A 1 -piperideine.
  • the host cell is capable of producing a compound of the structure:
  • the cell is capable of producing (-)- sparteine or a precursor thereof.
  • Said precursor may for example be tetrahydroababasine, /V-acetylated tetrahydroanabasine, 2-hydroxyammodendrine, didehydrolusitanine or tetrahydrosparteine.
  • the host cell is capable of producing tetrahydroanabasine or a precursor thereof.
  • the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.
  • the host cell does not comprise a heterologous gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the method of producing tetrahydroanabasine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating tetrahydroanabasine.
  • Tetrahydroanabasine as used herein include, but is not limited to compounds of the formula: tetrahydroanabasine tetrahydroanabasine tetrahydroanabasine
  • the host cell is capable of producing /V-acetylated tetrahydroanabasine or a precursor thereof.
  • the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
  • the host cell does not comprise a heterologous gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the method of producing /V-acetylated tetrahydroanabasine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating /V-acetylated tetrahydroanabasine.
  • V-acetylated tetrahydroanabasine as used herein include, but is not limited to compounds of the formula:
  • the produced /V-acetylated tetrahydroanabasine may dehydrate to give the dipiperidine alkaloid ammodendrine.
  • the host cell is capable of producing didehydrolusitanine.
  • the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. optionally a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
  • the host cell does not comprise heterologous gene(s) encoding CHYD of SEQ ID NO 5 and/or NCS1 of SEQ ID NO 9 or a functional homologues having at least 70% sequence identity to the aforementioned.
  • the method of producing didehydrolusitanine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding
  • “Didehydrolusitanine” as used herein include, but is not limited to compounds of any one of the formulas:
  • the cell is capable of producing 2- hydroxyammodendrine.
  • the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. optionally a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
  • the host cell does not comprise a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the method of producing 2- hydroxyammodendrine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the hydroxyammodendrine species.
  • 2-hydroxyammodendrine as used herein include, but is not limited to a compound of any one of the formulas:
  • the cell is capable of producing tetradehydrosparteine.
  • the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • the host cell does not comprise a heterologous gene encoding VRED1 of SEQ ID NO 6 or VRED2 if SEQ ID NO:7 or a functional homologue having at least 70% sequence identity to any of the aforementioned.
  • the method of producing tetradehydrosparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto;
  • Tetradehydrosparteine may for example be hydrated (carbinolamine), fully hydrolysed (aminoaldehyde) or tautomerized (iminium and/or enamine) forms of tetradehydrosparteine.
  • tetradehydrosparteine include, but is not limited to compounds of any one of the formulas:
  • the present invention relates to methods of producing (-)-sparteine or a precursor thereof, as well as to enzymes and host cells useful for producing (-)-sparteine.
  • the host cell is capable of producing (-)-sparteine, and more preferably (-)-sparteine of high enantiomeric purity as outlined in detail below.
  • the host cell preferably comprises the heterologous genes outlined below, and it is further preferred that the host cell does not comprise heterologous gene(s) encoding enzymes catalysing conversion of (-)- sparteine to other compounds.
  • the host cell is capable of producing (- )-sparteine and comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene en
  • the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a
  • the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto,; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c
  • a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c.
  • the host cell is capable of producing (- )-sparteine, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a
  • the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a
  • the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a
  • the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a
  • the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a
  • the host cell is capable of producing (-)- sparteine of high enantiomeric purity. Accordingly, it is preferred that the host cell is capable of producing (-)-sparteine with an enantiomeric excess (ee) of at least 60%, such as preferably at least 65%, such as preferably at least 70%, such as preferably at least 75%, such as preferably at least 80%, such as preferably at least 85%, such as preferably at least 90%, such as preferably at least 95%, such as most preferably of at least 97%.
  • the enantiomeric purity of (-)-sparteine may preferably be determined as described herein below in Example 5.
  • the host cell is preferably capable of producing (-)-sparteine with a high yield.
  • the exact yield may be dependent on the type of host cell and/or the cultivation method.
  • the host cell is capable of producing (-)-sparteine at a level of at least 0.005%, such as preferably 0.01%, such as preferably 0.025%, such as preferably 0.4%, such as most preferably 0.05%, such as in the range of 0.005 to 5%, wherein the % is indicated as w/w % of the total weight of the host cell.
  • the invention provides methods of producing (-)-sparteine or precursors thereof, e.g. any of the methods described herein above in the sections “Precursor of sparteine” and “Sparteine”.
  • the methods may comprise a step of cultivating the host cell under conditions enabling growth, and further optionally a step of isolating the (-)-sparteine or the precursor thereof. These steps may for example be performed as described herein in this section.
  • (-)-sparteine or the precursors thereof may be isolated through any useful method known to the skilled person.
  • (-)-sparteine may be isolated by a method comprising one or more of the following:
  • the step of isolating (-)-sparteine or the precursor thereof comprises extraction, e.g. extraction with a solvent, e.g. a solvent comprising an alcohol, such as methanol and/or acid-base extraction.
  • a solvent e.g. a solvent comprising an alcohol, such as methanol and/or acid-base extraction.
  • solvent includes any liquid that can dissolve or substantially disperse another substance.
  • the acid-base extraction may be any procedure using sequential liquid-liquid extractions to purify compounds from mixtures at differential pH.
  • the acid-base extraction may be performed as follows. The mixture is dissolved in a suitable solvent and poured into a separating funnel. An aqueous solution of the acid or base is added, and the pH of the aqueous phase is adjusted to bring the compound of interest into its required form. After shaking and allowing for phase separation, the phase containing the compound of interest is collected. The procedure is then repeated with this phase at the opposite pH range. The order of the steps is not important and the process can be repeated to increase the separation.
  • the method of isolating (-)-sparteine comprises a step of supercritical fluid extraction.
  • supercritical fluid e.g. supercritical carbon dioxide
  • a co-solvent e.g. ethanol
  • the method of isolating (-)-sparteine comprises a step of chromatography, for example cation exchange chromatography or adsorption chromatography.
  • the flour may be extracted with an aqueous solvent (e.g. pure water), and the extract may be run through a cation exchange resin (e.g. AG 50W-X2).
  • the cation exchange resin may be eluted using a basic solvent (e.g. 10% NaOH in ethanol/water 70:30).
  • Adsorption chromatography may involve use of a polymeric resin.
  • the flour may be extracted in basic aqueous solvent (e.g. water basified with 1 M NaOH to pH 11), the extract may be run through a polymeric resin (e.g. Amberlite XAD-16), and the column may be eluted in an organic solvent (e.g. ethanol).
  • basic aqueous solvent e.g. water basified with 1 M NaOH to pH 11
  • the extract may be run through a polymeric resin (e.g. Amberlite XAD-16), and the column may be eluted in an organic solvent (e.g. ethanol).
  • an organic solvent e.g. ethanol
  • the step of isolating (-)-sparteine or the precursor comprises the use of HPLC.
  • HPLC high performance liquid chromatography
  • the step of cultivating the host cell under conditions enabling growth may be performed by any method known to the skilled person.
  • the term "growth" of a host cell or a multicellular organism comprising a host cell should be understood as a reference to proliferation, multiplication, differentiation and/or maintenance of viability of the subject host cell, or multicellular organism. If the host cell is comprised in a multicellular organism, said conditions are usually conditions enabling maintenance of viability and/or growth of the multicellular organism. Thus, if the host cells are plant cells comprised in a plant, the cultivation conditions are conditions suitable for maintenance and/or growth of said plant. That could e.g. sowing seeds or other regenerative parts of the said plant in a field or in a green house. Cultivation may further comprise watering and/or fertilising.
  • cultivation may be incubation in a medium comprising at least a carbon source and a nitrogen source at a temperature suitable for growth of said unicellular organism.
  • the carbon source may e.g. be a carbohydrate, such as as sugars or polysaccharides.
  • the nitrogen source may for example be amino acids or polypeptides.
  • the medium comprises lysine, such as L- Lysine. The skilled person is well able of selecting a suitable cultivation medium based on the particular host cell.
  • the invention also provides enzymes useful in the production of (-)-sparteine or precursors thereof.
  • the invention provides a polypeptide, such as an isolated polypeptide selected from the group of polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • a polypeptide such as an isolated polypeptide selected from the group of polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least
  • the invention further provides nucleic acid encoding any of the polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • the nucleic acid may comprise or consist of a coding sequence (e.g. a cDNA sequence) from wild type lupin, e.g. a cDNA sequence derived from wild type NLL.
  • the nucleic acid sequence may be codon optimised for improved expression in the host cell.
  • the nucleic acid sequence comprises or consists of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, functional homologues thereof encoding the same polypeptide or a polypeptide having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto or codon optimised versions of any of the aforementioned.
  • NCS2 increases the production of N-acetylated tetrahydroanabasine in Nicotiana benthamiana
  • NCSs of SEQ ID NO 9-12 are involved in the dimerization of A 1 - piperideine to tetrahydroanabasine.
  • the full-length coding regions of LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and of the gene candidates NCS1-4 of SEQ ID NO 9-12, were amplified from leaf cDNA of the bitter narrow-leafed lupin (Lupinus angustifolius) cultivar Oskar.
  • the genes were cloned into the plant expression vector pEAQ-USER (Luo et al., 2016) by USER cloning and transformed into E. coli strain Top10. Clones were transformed into electrocompetent Agrobacterium tumefaciens strain AGL-1.
  • AGL-1 strains were grown on YEP liquid medium supplemented with 50 pg/mL kanamycin, 25 pg/mL rifampicin and 50 pg/mL carbenicillin at 28 °C and 220 rpm until ODeoo « 3-4.
  • the diluted strains were mixed in equal portions to obtain the desired gene combination for co-expression in leaves of N. benthamiana. After a 1-3 h incubation at room temperature, the mixtures were infiltrated into the abaxial side of young leaves of 4 week-old N. benthamiana plants.
  • the agroinfiltarted plants were allowed to recover overnight in the dark before being brought back to the greenhouse.
  • Agroinfiltrated leaves were harvested 7 days after agroinfiltration.
  • Two 1-cm leaf discs were punched out of the agroinfiltrated (discolored) portion of the leaves, collected in 1.5 ml screw-cap microcentrifuge tubes, and dried for 4 days at 40 °C.
  • the leaf discs were then pulverized using a steel bead and a TissueLyzerll bead beater (Qiagen) and extracted with 250 pl of extractant (60% methanol, 0.06% formic acid, and 5 ppm caffeine in water) for 3 hours at 400 rpm.
  • the extracts were briefly centrifuged to remove large particulates, diluted 5x with ultrapure water, and filtered through a 0.22 pm filter.
  • Analytical LC-MS was carried out on a Thermo Fisher Dionex 3000 RS HPLC/UPLC system interfaced to a Bruker compact QqTOF mass spectrometer through an ESI source. Analytes were separated on a Luna C18(2) column (150 x 2 mm, 3 pm, 100 A, Phenomenex) kept at 40 °C. Mobile phases A and B consisted of, respectively, 0.05% formic acid in water and 0.05% formic acid in acetonitrile.
  • N2 was used as desolvation, nebulizer and collision cell gas. Values are relative to the plants expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO3, and GFP (mean set to 1), and correspond to the normalized, integrated peak areas from extracted ion chromatograms at m/z 209.16 ⁇ 0.01 ([M+H]+).
  • NCS2 of SEQ ID NO 10 leads to a 3.5-fold increase in accumulation of /V-acetylated tetrahydroanabasine (measured as ammodendrine) in Nicotiana benthamiana when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2 and AT of SEQ ID NO 3 (Fig 2).
  • NCS2 of SEQ ID NO 10 is involved in the dimerization step from A 1 -piperideine to tetrahydroanabasine and that NCS2 of SEQ ID NO 2 increases the production of /V-acetylated tetrahydroanabasine (measured as ammodendrine) in Nicotiana benthamiana when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2 and AT of SEQ ID NO 3.
  • Example 2 G8HO hydroxylates N-acetylated tetrahydroanabasine allowing the formation of a quinolizidine core
  • the ESI + CID mass spectra of the products of G8HO of SEQ ID NO 4 in Nicotiana benthamiana were acquired as described in example 1.
  • the two products appeared to be a hydroxylated form and a dehydrogenated form of ammodendrine ([M+H] + of m/z 225.1596 and m/z 207.1498, respectively).
  • the MS 2 spectrum of the hydroxylated product (peak b) revealed the presence of an alcohol group (neutral loss of H2O) and of a secondary amide group (neutral loss of C2H2O) in the structure.
  • Position 2 is the most likely site of hydroxylation, since it enables the ring-opening and subsequent rearrangement that is essential to proceed in the pathway.
  • the dehydrogenated product (peak a) instead, contained a primary amide group (prominent neutral loss of C2H5NO in the MS 2 spectrum), which is consistent with ring opening and rearrangement leading to a didehydrolusitanine species.
  • G8HO of SEQ ID NO 4 hydroxylates the direct product of AT of SEQ ID NO 3, which can either lose water to become 2-hydroxyammodendrine or undergo rearrangement to give a didehydrolusitanine species, from which subsequent QA biosynthesis can continue.
  • Example 3 Production of sparteine in N. benthamiana by co-expression of at least 9 genes Aim:
  • NCS1 of SEQ ID NO 9 NCS2 of SEQ ID NO 10
  • NCS3 of SEQ ID NO 11 NCS4 of SEQ ID NO 12
  • VRED1 of SEQ ID NO 6 VRED2 of SEQ ID NO 7
  • LDC of SEQ ID NO 1 CAO of SEQ ID NO 2
  • AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana leads to the efficient production of (-)-sparteine.
  • each of the selected candidate genes was replaced by GFP consecutively. In the case of suspected redundancy between two genes, both of them were also replaced simultaneously by GFP.
  • the inventors of the present disclosure have realized that the expression of CHYD of SEQ ID NO 5, NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, VRED1 of SEQ ID NO 6 or VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana leads to the production of (-)- sparteine while the additional expression of NCS3 of SEQ ID NO 11 and NCS4 of SEQ ID NO 12 leads to efficient production of (-)-sparteine.
  • Example 4 Determination of the profile of accumulation of intermediates during the production of sparteine in N. benthamiana.
  • NCS1 of SEQ ID NO 9 NCS2 of SEQ ID NO 10, NCS3 of SEQ ID NO 11 , NCS4 of SEQ ID NO 12, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, NCS2 of SEQ ID NO 10, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana.
  • NCS2 of SEQ ID NO 10 virtually eliminated the production of any of the measured intermediates/shunt products (as well as the end product).
  • absence of NCS4 of SEQ ID NO 12 led to significantly larger amounts of 2- hydroxyammodendrine relative to didehydrolusitanine.
  • CHYD of SEQ ID NO 5 led to the virtual absence of both tetradehydrosparteine and sparteine, a profile that closely resembled that of omitting NCS1 of SEQ ID NO 9.
  • NCS3 of SEQ ID NO 11 led to a much unfavorable ratio of tetradehydrosparteine to (-)- sparteine.
  • This profile was similar to the one observed when omitting both VRED1 of SEQ ID NO 6 and VRED2 of SEQ ID NO 7, except that no final product could be observed in the latter case (Fig. 5).
  • NCS4 suggest a role for NCS4 in aiding the conversion of 2-hydroxyammodendrine to didehydrolusitanine.
  • CHYD indicates that CHYD and NCS1 are responsible for deacetylation coupled to the addition of the third A 1 -piperideine unit.
  • NCS3 suggest a role for NCS3 in the reduction of tetradehydrosparteine to sparteine.
  • VREDS The results regarding the VREDS suggest that the VREDs are directly responsible for the reduction of tetradehydrosparteine to sparteine.
  • Example 5 Co-expression of ARED increases the purity of ( ⁇ )-sparteine produced in N. benthamiana.
  • ARED of SEQ ID NO 8 was cloned into a plant expression vector as described for other narrow-leafed lupin genes in example 1.
  • Agroinfiltrated leaves of N. benthamiana transiently co-expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5, and VRED1-2 of SEQ ID NO 6-7 together with either GFP or ARED of SEQ ID NO 8 were harvested 7 days after agroinfiltration.
  • the remaining agroinfiltrated (discolored) portions of the leaves were clipped out and pooled in 50 ml centrifuge tubes.
  • the leaf discs and the leaf clippings were dried for 4 days at 40 °C after which they were pulverized using steel beads and a TissueLyzerll (Qiagen) bead beater (in the case of the leaf discs) or a benchtop vortex (in the case of the leaf clippings).
  • the pulverized leaf discs were extracted with 250 pl extractant (60% methanol, 0.06% formic acid, and 5 ppm caffeine in water) for 3 hours at 400 rpm.
  • Leaf debris was separated by centrifugation (1 min at 21000xg) and the extracts were diluted 5x in water, filtered through a 0.22-pm filter and analyzed by LC-MS as described in example 1.
  • Extracted ion chromatograms of combined m/z ratios of 235.22 ⁇ 0.01 and 118.11 ⁇ 0.01 were produced, which corresponded to the [M+H] + and [M+2H] 2+ ions, respectively.
  • Three peaks were detected and integrated from the extracted ion chromatograms and added together to give a total peak area.
  • the % purity of sparteine was evaluated by comparing the area of the sparteine peak to the total peak area.
  • the dichloromethane extracts were transferred to clean 12-ml glass vials and dried with anhydrous MgSC The dried extracts were transferred to clean vials and evaporated under reduced pressure. The residues were reconstituted in 100 pl ethyl acetate and analyzed by chiral GC-MS.
  • Chiral GC-MS was carried out on a Shimadzu Nexis GC-2030 gas chromatograph equipped with a Shimadzu AGC-6000 autosampler and coupled to a Shimadzu GCMS- QP2020 NX single quadrupole mass spectrometer. Analytes were separated on an Agilent J&W CycloSil-B capillary column (30 m x 0.25 mm x 0.25 pm) using He as carrier gas. Ethyl acetate extracts were injected in splitless mode (1 pl) at an inlet temperature of 250 °C.
  • Analytes were separated using the following column temperature program at a constant carrier gas pressure of 51 .0 kPa: initial 80 °C, hold for 3 min; ramp to 125 °C at 30 °C/min; ramp to 240 °C at 2 °C/min.
  • the separated analytes were ionized using an electron impact ion source at 250 °C.
  • MS spectra were acquired in Scan mode (m/z 10- 300) at an energy of 70 eV. (+)- and (-)-sparteine were identified by comparison with commercial standards (Sigma-Aldrich), (-)-sparteine and (+)-sparteine were identified based on comparison with commercial preparations (Sigma-Aldrich). The enantiomeric excess was calculated from extracted ion chromatograms of m/z 137.
  • benthamiana in the presence of ARED of SEQ ID NO 8 was -5.7 pg in two leaf discs of 1-cm diameter. Assuming -12 mg fresh weight for the two leaf discs, this amounts to -0.05% yield on a fresh weight basis (Fig. 5).
  • the reductase ARED of SEQ ID NO 8 is able to improve the stereoselectivity of the (-)-sparteine pathway engineered in N. benthamiana, leading to a higher purity of sparteine (97% based on non-chiral LC-MS peak areas) as well as a higher enantiomeric purity of (-)-sparteine (97% enantiomeric excess based on chiral GC-MS).
  • NCS1 Enzyme S-norcoclaurine synthase-like 1
  • NCS2 Enzyme S-norcoclaurine synthase-like 2
  • NCS3 Enzyme S-norcoclaurine synthase-like 3
  • NCS4 Enzyme S-norcoclaurine synthase-like 4
  • SEQ ID NO 20 Nucleotide sequence of the enzyme ARED from narrow-leafed lupin cultivar Oskar:
  • NCS1 S-norcoclaurine synthase-like 1
  • SEQ ID NO 22 Nucleotide sequence of the enzyme S-norcoclaurine synthase-like 2 (NCS2) from narrow-leafed lupin cultivar Oskar:
  • NCS3 S-norcoclaurine synthase-like 3
  • NCS4 S-norcoclaurine synthase-like 4
  • a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S- norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11, S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • G8HO ger
  • the host cell according to item 1 wherein the host cell further comprises one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3 or a functional homologue of any one of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
  • LDC lysine decarboxylase
  • CAO copper amine oxidase
  • AT BADH acetyltransferase
  • the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as 12 of the following genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d.
  • a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the host cell comprises a heterologous gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof, wherein said functional homologue has lysine decarboxylase activity.
  • the host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme CAO of SEQ ID NO 2 or a functional homologue thereof, wherein said functional homologue has amine oxidase activity.
  • the host cell comprises a heterologous gene encoding the enzyme AT of SEQ ID NO 3 or a functional homologue thereof, wherein said functional homologue is capable of catalysing /V-acetylation of tetrahydroanabasine.
  • the host cell comprises a heterologous gene encoding the enzyme G8HO of SEQ ID NO 4 or a functional homologue thereof, wherein said functional homologue has hydroxylase activity against /V-acetylated tetrahydroanabasine.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme CHYD of SEQ ID NO 5 or a functional homologue thereof, wherein said functional homologue has deacetylase activity against a didehydrolusitanine species in the presence of NCS1 or functional homologues thereof.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme VRED1 of SEQ ID NO 6 or a functional homologue thereof, wherein said functional homologue is capable of catalysing reduction of a tetradehydrosparteine species.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme VRED2 of SEQ ID NO 7 or a functional homologue thereof, wherein the functional homologue is capable of catalysing reduction of di-iminium cations.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme ARED of SEQ ID NO 8 or a functional homologue thereof, wherein the functional homologue is annotated as an oxidoreductase.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme NCS1 of SEQ ID NO 9 or a functional homologue thereof, wherein the functional homologue is capable of catalysing the addition of a A1-piperideine unit to didehydrolusitanine species in the presence of CHYD or functional homologues thereof.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding the enzyme NCS2 of SEQ ID NO 10 or a functional homologue thereof, wherein the functional homologue is capable of catalysing dimerization of A1-piperideine to tetrahydroanabasine.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding NCS3 of SEQ ID NO 11 or a functional homologue enzyme, wherein the functional homologue is capable of aiding the reduction of a tetradehydrosparteine species in the presence of VRED1 or VRED2 or functional homologues thereof.
  • the host cell according to any one of the preceding items wherein the host cell comprises a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof, wherein the functional homologue is capable of catalysing the equilibration between a 2-hydroxyammodendrine species and a didehydrolusitanine species.
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene
  • a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d.
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous genes a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a.
  • a gene encoding VRED1 SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
  • the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b.
  • the host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene
  • the host cell according to any one of the preceding items, wherein the host cell is selected from the group of plant cells, yeast cells, bacteria cells and fungal cells.
  • the host cell according to any one of the preceding items, wherein the host cell is plant cells comprised within a plant, within a part of a plant or within the seeds of said plant.
  • the host cell according to any one of the preceding items, wherein the host cell is capable of producing tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.
  • the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing /V-acetylated tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing a didehydrolusitanine species and/or a 2- hydroxyammodendrine species, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of
  • a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto e. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and f. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
  • the recombinant host cell according to any one of the preceding items, wherein the cell is capable of producing a tetradehydrosparteine species, including hydrated (carbinolamine), fully hydrolysed (aminoaldehyde), iminium, or enamine forms of tetradehydrosparteine, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c.
  • a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d.
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c.
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b.
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e.
  • heterologous gene(s) a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional
  • the host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c.
  • a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
  • a method of producing (-)-sparteine or a precursor thereof comprising the steps of: a. Providing a host cell according to any of items 1 to 37; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating (-)-sparteine or a precursor thereof.
  • the method according to any one of items 40 to 41 , wherein the method of isolating (-)-sparteine comprises a step of extraction, for example extraction with a solvent comprising an alcohol, acid-base extraction and/or supercritical fluid extraction.
  • the method according to any one of items 40 to 42, wherein the method of isolating (-)-sparteine comprises a step of chromatography, for example cation exchange chromatography, adsorption chromatography using a polymeric resin or HPLC.

Abstract

The present invention provides a pathway for production of (-)-sparteine. In particular, enzymes involved in the pathway are provided as well as nucleic acids encoding same. The invention also provides host cells capable of producing (-)-sparteine or precursors thereof, as well methods of producing (-)-sparteine or precursors thereof.

Description

P6112PC00
Production of plant alkaloids
Technical field
The present invention relates to the field of plant alkaloid synthesis, in particular in recombinant host cells. More specifically, the invention relates to production of (-)- sparteine or precursors thereof as well as to enzymes useful for such production.
Background
Plant alkaloids are a large family of bioactive, nitrogen-containing compounds commonly used as medical drugs or stimulants. In food and feed crops, however, the accumulation of plant alkaloids is undesirable, as they typically confer toxicity or act as anti-nutritional compounds. Such is the case of the quinolizidine alkaloids (QAs), which are anti-cholinergic compounds produced by the protein crops collectively called lupins (Lupinus spp.). Lupins are the legume crops with the highest seed protein content (up to 44% protein for L. mutabilis). However, their accumulation of QAs limits their use as food and feed crops. Low-alkaloid varieties (sweet varieties) have been produced via traditional breeding, but remaining QA levels are variable and often surpass the thresholds established by the food and feed industries.
One of the most versatile QA is the chiral diamine sparteine. Specifically, complexes between sparteine and lithium have proven unrivaled for asymmetric deprotonations, substitutions, carbometalations, and directed orto-metalations. As chemists continue to find new uses for sparteine-lithium complexes, their versatility continues to grow, as exemplified by the development of assembly-line chiral synthesis. Originally, only (-)- sparteine was commercially available, enabling the asymmetric synthesis of just one of two possible products in each case. This led to the development of a synthetically accessible molecule with the expected functionality of (+)-sparteine, which became known as the (+)-sparteine surrogate. Commercial (-)-sparteine remained cheap throughout the 2000s. However, for reasons that remain unclear, it later became expensive and, at times, fully unavailable.
Summary
Hence there is an unmet need for cheap and stable methods for production of (-)- sparteine. The QAs that accumulate in lupins are mostly of the tetracyclic type, the simplest of which is sparteine. The majority of QAs in lupins share the common tetracyclic backbone of (-)-sparteine and are thought to derive from it.
The present invention provides the metabolic pathway for production of (-)-sparteine in Lupinus spp., as well as materials and methods for establishing a metabolic pathway for production of (-)-sparteine or precursors thereof in host cells.
QAs are derived from the amino acid L-lysine. The first step of the pathway is decarboxylation of L-lysine by LDC to give cadaverine. In the next step CAO oxidizes one end of cadaverine giving an aminoaldehyde that cyclizes readily into A1- piperideine. A1-piperideine exists in equilibrium with its dimer tetahydroanabasine, and NCS facilitates said equilibrium. Tetrahydroanabasine is /V-acetylated by AT to form N- acetylated tetrahydroanabasine. G8HO catalyzes the hydroxylation of /V-acetylated tetrahydroanabasine allowing the formation of a didehydrolusitanine species containing a quinolizidine core. CHYD and NCS1 are subsequently involved in deacetylation coupled to addition of a third A1-piperideine unit to give a tetradehydrosparteine species. The tetradehydrosparteine species is reduced to (-)-sparteine by either VRED1 or VRED2.
The invention further shows that expression of CHYD, NCS1 , NCS2, VRED1 or VRED2, LDC, CAO, AT and G8HO is sufficient for production of (-)-sparteine while the additional expression of NCS3 and/or NCS4 leads to more efficient production of (-)- sparteine, whereas additional expression of ARED leads to improved stereoselectivity.
An overview of the proposed pathway for biosynthesis of (-)-sparteine provided by the present invention is shown herein in figure 1.
In one aspect the invention provides a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S- norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11, S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In addition, the invention provides aforementioned enzymes per se and nucleic acids encoding same.
The invention also provides methods for producing (-)-sparteine or a precursor thereof, comprising the steps of: a. Providing a host cell of the invention; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating (-)-sparteine or a precursor thereof.
Description of Drawings
Figure 1. Schematic representation of the proposed biosynthetic pathway of (-)- sparteine. A) Early part of the pathway leading to formation of tetrahydroanabasine. B) Middle part of the pathway progressing through acetylated intermediates and leading to didehydrolusitanine. C) Late part of the pathway including formation of the tetracyclic core of sparteine. Asterisks indicate that the intermediate exists as a complex equilibrium of different compounds. Shown here are representative forms of the intermediates. A more comprehensive, yet not exhaustive, overview of the forms taken on by the intermediates is presented in the section “Precursors of sparteine".
Figure 2. NCS2 of SEQ ID NO 10 increases the production of ammodendrine in Nicotiana benthamiana. A) accumulation of ammodendrine in the leaves of Nicotiana benthamiana upon transient co-expression of LDC of SEQ ID NO 1, CAO of SEQ ID NO 2 and AT of SEQ ID NO 3 with GFP (negative control) or with each of the four NCS candidates. Leaf samples were harvested at 7 days post agroinfiltration. Values are relative to the plants expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, and GFP (mean set to 1), and correspond to the normalized, integrated peak areas from extracted ion chromatograms at m/z 209.16 ± 0.01 ([M+H]+). Box plots are standard (n = 6 or 7). Letters indicate significant differences (one-way ANOVA with Tukey post hoc test P<0.05).
Figure 3. G8HO of SEQ ID NO 4 catalyzes the hydroxylation of /V-acetylated tetrahydroanabasine allowing the formation of a quinolizidine core. A) representative LC-MS chromatograms of leaf extracts of N. benthamiana expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, NCS2 of SEQ ID NO 10 and three oxidase candidates (G8HO of SEQ ID NO 4 and two other cytochrome P450s referred to as CYP450_1 and CYP450_2, respectively) at 5 days post agroinfiltration. Peaks a, b, and c correspond to the new compounds observed when co-expressing G8HO of SEQ ID NO 4; peak d corresponds to ammodendrine. Traces are combined extracted ion chromatograms (EICs) of m/z 207.15 ± 0.01 ([M+H]+ for peak a and [M-H2O+H]+ for peaks b and c) and m/z 209.16 ± 0.01 ([M+H]+for peak d). B) comparison of the ESI+ CID MS2 mass spectra of peak a and peak b showing key fragmentations of the apparent products of G8HO of SEQ ID NO 4. The proposed structures are embedded and correspond to a didehydrolusitanine species (m/z 207.15, [M+H]+) and 2- hydroxyammodendrine (m/z 225.16, [M+H]+). Parent molecular ions are marked in bold. Neutral losses are marked in grey. Bonds that are cleaved upon fragmentation are marked by dashed lines and the m/z of the resulting fragment ions are indicated at the arrow tips.
Figure 4. Production of sparteine in N. benthamiana by co-expression of at least 9 genes. A) representative LC-MS chromatograms of N. benthamiana leaves showing production of sparteine (peak a) and its isomers (peak b, p-isosparteine; peak c, a- isosparteine) upon transient expression of 12 biosynthetic gene candidates (7 days post agroinfiltration). Traces are extracted ion chromatograms (EICs) of the singly and doubly charged molecular ions of sparteine combined (m/z 235.22 ± 0.01 and m/z 118.11 ± 0.01 , respectively). B) comparison of the ESP CID MS2 mass spectra of the singly charged molecular ion of sparteine (24.1 eV) from a commercial standard or from transiently transformed N. benthamiana (tobacco) leaves. C) comparison of the ESP CID MS2 mass spectra of the doubly-charged molecular ion of sparteine (18.8 eV) from a commercial standard or from transiently transformed N. benthamiana (tobacco) leaves. D) determination of the essential genes for the production of sparteine in N. benthamiana. The production of sparteine in leaves expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5, and VRED1-2 of SEQ ID NO 6-7 was compared to leaves in which selected genes (or pairs of) were replaced by GFP. Leaves were harvested 5 days post agroinfiltration. Bars represent mean normalized peak areas of sparteine (m/z 118.11 ± 0.01 , [M+2H]2+) and error bars are 1.5 standard deviations (n = 3 or 4). Data are relative to the mean peak area of sparteine in the samples where none of the genes was replaced by GFP. Letters indicate significant differences (oneway ANOVA with Tukey post hoc test P<0.05).
Figure 5. Accumulation of intermediates during the production of sparteine in N. benthamiana. The gene combinations used are the same as in Figure 3D. Bars represent mean peak areas of /V-acetylated tetrahydroanabasine (quantified as ammodendrine, m/z 209.16 ± 0.01 , [M+H]+), 2-hydroxyammodendrine (m/z 207.15 ± 0.01 , [M-H2O+H]+), didehydrolusitanine m/z 207.15 ± 0.01 , [M+H]+), tetradehydrosparteine {m/z 116.10 ± 0.01 , [M]2+), and sparteine {m/z 118.11 ± 0.01 , [M+2H]2+). Error bars represent 1.5 standard deviations {n = 3 or 4). Comparisons between the accumulation of selected intermediates within a given gene combination are show as fold-changes on top of dotted, curved arrows.
Figure 6. Co-expression of ARED of SEQ ID NO 8 increases the purity of (-)-sparteine produced in N. benthamiana. All data refer to agroinfiltrated N. benthamiana leaves transiently expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5, VRED1-2 of SEQ ID NO 6-7 and either GFP or ARED of SEQ ID NO 8 at 7 days post agroinfiltration. A) yield of sparteine (pg) in two leaf discs of 1 cm diameter, as determined by LC-MS in comparison to a commercial standard. Box plots are standard {n = 6 or 7). Yield differences were not significant based on one-way ANOVA with Tukey post hoc test at P<0.05. B) purity of sparteine versus two detected sparteine isomers (likely a-isosparteine and p-isosparteine), as determined by LC-MS. Box plots are standard {n = 6 or 7). Letters indicate significant differences (one-way ANOVA with Tukey post hoc test P<0.05) C) chiral GC-MS chromatograms of sparteine extracted from agroinfiltrated N. benthamiana leaves compared to enantiomerically pure, commercial standards. Chromatograms are extracted ion chromatograms at m/z 137 (El base peak of sparteine). Detailed description
Definitions
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.
The terms “BADH acetyltransferase” and “AT” are used interchangeably throughout the description.
The terms “copper amine oxidase” and “CAO” are used interchangeably throughout the description.
The term “cultivating" as used herein refers maintaining host cells under culture conditions which allow the cells to grow. Preferably, said culture conditions allow expression of the enzyme(s) encoded by the heterologous gene(s) contained in said host cells. Preferably, the host cells are cultivated under culture conditions allowing said host cells to produce (-)sparteine or a precursor thereof. In embodiments where the host cell is contained within a multicellular organism (e.g. a plant), “cultivating” refers to maintaining said multicellular organisms under conditions allowing said multicellular organism to grow. In embodiments where the host cell is a unicellular organism, “cultivating” refers to maintaining said unicellular organism under conditions allowing said unicellular organism to grow and/or multiply.
The term “some embodiments” can include one, or more than one embodiment.
"Enantiomeric purity" as used herein refers to the purity of one enantiomer compared to the other enantiomer. Enantiomeric purity is herein measured as “enantiomeric excess” (ee). “Enantiomeric excess” (ee) reflects the degree to which a sample contains one enantiomer in greater amounts than the other. The enantiomeric excess is defined as the absolute difference between the mole fraction of each enantiomer: ee = | Fraction ((-) isomer) - Fraction ((+) isomer) |, where Fraction ((-) isomer) + Fraction ((+) isomer) = 1. As an example, a mixture of 50% (-) isomer and 50% (+) isomer has an enantiomeric excess of 0%. A mixture of 98.5% and 1.5% of the two isomers, has an enantiomeric excess of 97%. The mole fractions can be determined in a number of ways known in the art, including but not limited to chromatography using a chiral support, polarimetric measurement of the rotation of polarized light, nuclear magnetic resonance spectroscopy using chiral shift reagents which include but are not limited to lanthanide containing chiral complexes or the Pirkle alcohol, or derivatization of a compounds using a chiral compound such as Mosher's acid followed by chromatography or nuclear magnetic resonance spectroscopy. Preferably, the enantiomeric purity of (-)-sparteine is determined as described in Example 5 herein below.
The term "enzyme" as used herein refers to proteins or polypeptides, which are capable of catalyzing biochemical reactions. Further, unless context dictates otherwise, as used herein "enzyme" includes protein fragments that retain the relevant catalytic activity, and may include artificial enzymes synthesized to retain the relevant catalytic activity.
The term "functional homologue" of an amino acid sequence, refers to a polypeptide comprising said amino acid sequence with the proviso that one or more amino acids are substituted, deleted, added, and/or inserted, and which polypeptide has (qualitatively) the same enzymatic functionality for substrate conversion. Preferably, a functional homologue shares at least at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity to said amino acid sequence.
The terms “geraniol-8-hydroxylase” and “G8HO” are used interchangeably throughout the description.
The term “heterologous gene” refers to a gene, which has been inserted into a host cell or into a progenitor of the host cell, e.g. by recombinant or transgenic methods. The respective protein or RNA encoded by a heterologous gene is also referred to as "heterologous”. The heterologous gene may be part of a nonintegrated nucleic acid, e.g. a vector, including but not limited to a plasmid. Preferably the heterologous gene(s) are integrated into the host genome.
The terms “high performance liquid chromatography” and “HPLC” are used interchangeably throughout the description. The term "host cell" refers to a cell, which comprises one or more heterologous genes.
The terms “lysine decarboxylase” and “LDC” are used interchangeably throughout the description
The term "polypeptide" as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length. As is known to those skilled in the art, polypeptides may be processed and/or modified, and the term polypeptide may refer to both unmodified or modified polypeptides.
The term “sequence identity” as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e. a candidate sequence (e.g. a mutant sequence) and a reference sequence (such as a wild type sequence) based on their pairwise alignment. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443- 453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss_needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
The Needleman-Wunsch algorithm is also used to determine whether a given amino acid in a sequence other than the reference sequence corresponds to a given position in a reference sequence.
For purposes of the present invention, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment).
The terms “S-norcoclaurine synthase-like protein” and “NCS” are used interchangeably throughout the description.
The term “sparteine” as used herein refers to (+)-sparteine, (-)-sparteine, or a mixture of both, “(-)-sparteine” is also known as (7S,7aR,14S,14aS)-dodecahydro-2H,6H-7,14- methanodipyrido[1,2-a:1',2'-e][1,5]diazocine, 7,14-Methano-2H,6H-dipyrido[1,2-a:1',2'- e][1,5]diazocine, dodecahydro-, [7S-(7a,7aa,14a,14aP)], CAS number 90-39-1 , Lupinidine, 6-p,7-a,9-a,11-a-Pachycarpine, l-Sparteine , (-)-Esparteina, (-)-Spartein, or (-)-Sparteinium. “(+)-sparteine” is also known as (7R,7aR,14R,14aS)-dodecahydro- 2H,6H-7,14-methanodipyrido[1,2-a:1',2'-e][1 ,5]diazocine, 7,14-Methano-2H,6H- dipyrido[1,2-a:1',2'-e][1,5]diazocine, dodecahydro-, [7R-(7a,7aa,14a,14aP)], CAS number 492-08-0, Pachycarpine, d-Sparteine, (+)-Esparteina, (+)-Spartein, or (+)- Sparteinium.
Combinations of enzymes
The present disclosure concerns a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3, geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S- norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S-norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11 , S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or functional homologues of any of the aforementioned. Said enzymes may preferably have the enzyme activity described herein below in the section “Enzyme activity”. In a main aspect, the present disclosure concerns a host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8- hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO
6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S-norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11 , S- norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure the host cell, in addition comprises one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3 or a functional homologue of any one of the aforementioned having at least 70% sequence identity thereto.
In some embodiments of the present disclosure the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least
7, such as at least 8, such as at least 9, such as at least 10, such as at least 11, such as 12 of the following genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and
2.1. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9 of the following genes: d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and/or l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto and optionally in addition, at least one, such as at least two, for example at least 3 of the following genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and/or c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto,; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto, f. a gene encoding VRED1 SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. In some embodiments of the present disclosure, the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and i.. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
All of the genes encoding enzymes described in this section may encode an enzyme of a specific sequence or a functional homologue thereof sharing at least 70% sequence identity thereto, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferably at least 98% sequence identity thereto.
Enzyme activity
The present invention provides a biosynthetic pathway for production of alkaloids, notably (-)-sparteine. The pathway comprises a number of enzymes, which for example may be combined as disclosed herein above in the section “Combinations of enzymes”.
The enzymes preferably have the enzyme activity described in this section. In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof. LDC of SEQ ID NO 1 or a functional homologue thereof preferably has lysine decarboxylase activity. The person skilled in the art will appreciate that LDC of SEQ ID NO 1 or functional homologues thereof preferably have the ability to convert L-Lysine to cadaverine. Furthermore, LDC of SEQ ID NO 1 or functional homologues thereof preferably speed up the rate of the decarboxylation of L-Lysine. A functional homologue of LDC of SEQ ID NO 1 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme CAO of SEQ ID NO 2 or a functional homologue thereof. CAO of SEQ ID NO 2 or a functional homologue thereof preferably has amine oxidase activity.
CAO of SEQ ID NO 2 or functional homologues thereof preferably have the ability to convert cadaverine to an aminoaldehyde. Furthermore, CAO of SEQ ID NO 2 or functional homologues thereof preferably speed up the rate of the oxidative cleavage of alkylamines into aldehydes and ammonia. A functional homologue of CAO of SEQ ID NO 2 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme AT of SEQ ID NO 3 or a functional homologue thereof. AT of SEQ ID NO 3 or a functional homologue thereof is preferably capable of catalysing /V-acetylation of tetrahydroanabasine.
AT of SEQ ID NO 3 or functional homologues thereof preferably have the ability to convert tetrahydrobasine to /V-acetylated tetrahydroanabasine. Furthermore, AT of SEQ ID NO 3 or functional homologues thereof preferably transfer acylated moieties (RC(O)R’) of an acyl-activated CoA thioester donor to an acceptor molecule.
A functional homologue of AT of SEQ ID NO 3 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto. In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme G8HO of SEQ ID NO 4 or a functional homologue thereof. G8HO of SEQ ID NO 4 or a functional homologue thereof preferably has hydroxylase activity against /V-acetylated tetrahydroanabasine.
G8HO of SEQ ID NO 4 or functional homologues thereof preferably have the ability to convert /V-acetylated tetrahydroanabasine to 2-hydroxyammodendrine. Furthermore, G8HO of SEQ ID NO 4 or functional homologues thereof preferably speed up the rate of the hydroxylation of /V-acetylated tetrahydroanabasine. A functional homologue of G8HO of SEQ ID NO 4 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure the host cell comprises a heterologous gene encoding the enzyme CHYD of SEQ ID NO 5 or a functional homologue thereof. CHYD of SEQ ID NO 5 or a functional homologue thereof preferably has deacetylase activity against a didehydrolusitanine species in the presence of NCS1 of SEQ ID NO 9.
The person skilled in the art will appreciate that functional homologues of CHYD of SEQ ID NO 5 or functional homologues thereof preferably have the ability to convert didehydrolusitanine to its deacetylated form in the presence of NCS1 of SEQ ID NO 9. A functional homologue of CHYD of SEQ ID NO 5 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme VRED1 of SEQ ID NO 6 or a functional homologue thereof. VRED1 of SEQ ID NO 6 or a functional homologue thereof preferably is capable of catalysing reduction of a tetradehydrosparteine species.
VRED1 of SEQ ID NO 6 or functional homologues thereof preferably have the ability to convert tetradehydrosparteine to (-)-sparteine. Furthermore, VRED1 of SEQ ID NO 6 or functional homologues thereof preferably have increased ability to convert tetradehydrosparteine to (-)-sparteine in the presence of NCS3 of SEQ ID NO 11. A functional homologue of VRED1 of SEQ ID NO 6 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme VRED2 of SEQ ID NO 7 or a functional homologue thereof. VRED2 of SEQ ID NO 7 or a functional homologue thereof preferably is capable of catalysing reduction of di-iminium cations.
VRED2 of SEQ ID NO 7 or functional homologues thereof preferably have the ability to convert tetradehydrosparteine to (-)-sparteine. Furthermore, VRED2 of SEQ ID NO 7 or a functional homologue thereof preferably has increased ability to convert tetradehydrosparteine to (-)-sparteine in the presence of NCS3 of SEQ ID NO 11. A functional homologue of VRED2 of SEQ ID NO 7 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme ARED of SEQ ID NO 8 or a functional homologue thereof. ARED of SEQ ID NO 8 or a functional homologue is preferably an enzyme annotated as an oxidoreductase, for example, it may be annotated as an oxidoreductase based on its sequence.
When ARED of SEQ ID NO 8 or a functional homologue thereof is expressed in N. benthamiana it preferably increases the enantiomeric excess of (-)-sparteine produced in said N. benthamiana to at least 65%, such as preferably to at least 70%, such as preferably to at least 75%, such as preferably to at least 80%, such as preferably to at least 85%, such as preferably to at least 90%, such as preferably to at least 95%, such as most preferably to at least 97% when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5 and VRED1-2 of SEQ ID NO 6-7. In another embodiment, when ARED of SEQ ID NO 8 or a functional homologue thereof is expressed in N. benthamiana it preferably increases the enantiomeric excess of (-)-sparteine produced in said N. benthamiana to at least 65%, such as preferably to at least 70%, such as preferably to at least 75%, such as preferably to at least 80%, such as preferably to at least 85%, such as preferably to at least 90%, such as preferably to at least 95%, such as most preferably to at least 97% when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-2 of SEQ ID NO 9-10, CHYD of SEQ ID NO 5 and at least one of VRED1 of SEQ ID NO 6 or VRED2 of SEQ ID NO 7. A functional homologue of ARED of SEQ ID NO 8 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme NCS1 of SEQ ID NO 9 or a functional homologue thereof. NCS1 of SEQ ID NO 9 or a functional homologue thereof is preferably capable of catalysing the addition of a A1-piperideine unit to a didehydrolusitanine species in the presence of CHYD of SEQ ID NO 5. A functional homologue of NCS1 of SEQ ID NO 9 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding the enzyme NCS2 of SEQ ID NO 10 or a functional homologue thereof. NCS2 of SEQ ID NO 10 or a functional homologue thereof is preferably capable of catalysing dimerization of A1-piperideine to tetrahydroanabasine. A functional homologue of NCS2 of SEQ ID NO 10 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof. NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of aiding the reduction of a di-iminium cation intermediate by e.g. double-bond isomerization. For example, NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of aiding the reduction of a tetradehydrosparteine species. In one embodiment NCS3 of SEQ ID NO 11 or a functional homologue thereof is preferably capable of catalysing formation of (-)-sparteine from a tetradehydrosparteine species in the presence of VRED1 of SEQ ID NO 6 and/or VRED2 of SEQ ID NO 7. A functional homologue of NCS3 of SEQ ID NO 11 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
In some embodiments of the present disclosure, the host cell comprises a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof. NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of aiding the equilibrium between hydroxylated ammodendrine and a quinolizidine-containing compound. For example, NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of aiding the rearrangement of 2-hydroxyammodendrine. In one embodiment, NCS4 of SEQ ID NO 12 or a functional homologue thereof is preferably capable of catalysing generation of a didehydrolusitanine species from 2- hydroxyammodendrine. A functional homologue of NCS4 of SEQ ID NO 12 has at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
Host cells
The present invention relates to host cells comprising one or more heterologous genes encoding enzymes of the biosynthetic pathway to (-)-sparteine, notably any of the enzymes or combinations thereof described in the sections “Combinations of enzymes” and “Enzyme activity”.
The host cell or a progenitor thereof may be prepared by any useful method available to the skilled person. For example, the heterologous gene(s) may be inserted into a cell by direct uptake, transduction, f-mating, transfection, transformation, bacterial infiltration or any other methods known in the art useful for creating recombinant host cells.
In some embodiments of the present disclosure, the host cell is selected from the group of plant cells, yeast cells, bacteria cells and fungal cells. In some embodiments the host cell is comprised within a multicellular organism. In such embodiments, only some of the cells of said multicellular organism may comprise heterologous gene(s). It is however preferred that all cells of said multicellular organism are host cells that comprise the same heterologous gene(s).
In some embodiments of the present disclosure, the host cell is plant cells comprised within a plant, within a part of a plant or within the seeds of said plant. Preferably, all cells of said plant or part thereof are host cells comprising the same heterologous gene(s).
In some embodiments of the present disclosure, the host cells are plant cells, e.g. plant cells from a species of Nicotiana such as Nicotiana benthamiana or Nicotiana tabacum.
In some embodiments of the present disclosure, the host cell is a yeast cell, e.g. a yeast cell of the species Saccharomyces cerevisiae or Saccharomyces pombe.
The person skilled in the art will appreciate that a "plant cell" as used within the present invention refers to a structural and physiological unit of a plant, e.g. a tobacco plant. The plant cell may be in form of a protoplast without a cell wall, an isolated single cell or a cultured cell, or as a part of higher organized unit such as but not limited to, plant tissue, a plant organ, or a whole plant. Furthermore, the person skilled in the art will appreciate that a "yeast cell" is herein defined to include the group consisting of small, unicellular organisms capable of growth and reproduction through budding or direct division (fission), or by growth as simple irregular filaments (mycelium). The yeast cell may be transformed or transfected with a heterologous vector for expression of a nucleic acid sequence inserted into the heterologous vector. An example of a yeast cell includes Saccharomyces cerevisiae or Saccharomyces pombe, commonly used for transfection and expression of heterologous proteins. The person skilled in the art will appreciate that a fungi or fungal cell(s) as used herein refers to any cell present within or derived from an organism belonging to the Kingdom Fungi. The methods are applicable to all fungi and fungal cells that are susceptible of genetic modifications. The person skilled in the art will appreciate that a "bacterial cell" includes prokaryotic cells that may be propagated in culture. The bacterial cell may act as a host cell for the recombinant expression of heterologous proteins. The bacterial cell may be transformed, transfected or infected with a vector for expression of a protein sequence inserted into the vector. Examples of suitable bacterial cells include, but are not limited to E. coli, B. megaterium, B. subtilis and B. brevis and various species of Caulobacter, Staphylococcus, and Streptomyces.
In some embodiments, the host cell is capable of producing lysine, such as L-lysine. Thus, in other embodiments, the host cell produces lysine, such as L-lysine. In some embodiments, the host cell is capable of producing A1-piperideine. Hence, in other embodiments, the host cell produces A1-piperideine. In some embodiments, the host cell is capable of producing a compound of the structure:
Figure imgf000025_0001
Precursors of sparteine
In some embodiments of the present disclosure, the cell is capable of producing (-)- sparteine or a precursor thereof. Said precursor may for example be tetrahydroababasine, /V-acetylated tetrahydroanabasine, 2-hydroxyammodendrine, didehydrolusitanine or tetrahydrosparteine.
In some embodiments of the present disclosure, the host cell is capable of producing tetrahydroanabasine or a precursor thereof. In such embodiments it is preferred that the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.
Even more preferably, in such embodiments the host cell does not comprise a heterologous gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the method of producing tetrahydroanabasine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating tetrahydroanabasine.
’’Tetrahydroanabasine” as used herein include, but is not limited to compounds of the formula:
Figure imgf000026_0001
tetrahydroanabasine tetrahydroanabasine tetrahydroanabasine
(carbinolamine form) (imine form) (enamine form)
Figure imgf000026_0002
Figure imgf000026_0003
tetrahydroanabasine (aminoaldehyde form)
The person skilled in the art will appreciate that the wavy single bonds are the standard way to represent unknown or unspecified stereochemistry or a mixture of isomers (as with tetrahedral stereocenters).
In some embodiments of the present disclosure, the host cell is capable of producing /V-acetylated tetrahydroanabasine or a precursor thereof. In such embodiments, the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
Even more preferably, in such embodiments the host cell does not comprise a heterologous gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the method of producing /V-acetylated tetrahydroanabasine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating /V-acetylated tetrahydroanabasine.
“/V-acetylated tetrahydroanabasine” as used herein include, but is not limited to compounds of the formula:
Figure imgf000028_0001
/V-acetylated /V-acetylated tetrahydroanabasine tetrahydroanabasine (carbinolamide form) (/V-acetyl-iminium form)
Figure imgf000028_0002
y tetrahydroanabasine /V-acetylated
(/V-acetyl-enamine form) tetrahydroanabasine
(ammodendrine)
Figure imgf000028_0003
(/V-acetyl-aminoaldehyde form)
The produced /V-acetylated tetrahydroanabasine may dehydrate to give the dipiperidine alkaloid ammodendrine.
In some embodiments of the present disclosure, the host cell is capable of producing didehydrolusitanine. In such embodiments the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. optionally a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
Even more preferably, in such embodiments the host cell does not comprise heterologous gene(s) encoding CHYD of SEQ ID NO 5 and/or NCS1 of SEQ ID NO 9 or a functional homologues having at least 70% sequence identity to the aforementioned.
In some embodiments of the present disclosure, the method of producing didehydrolusitanine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding
G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the didehydrolusitanine species.
“Didehydrolusitanine” as used herein include, but is not limited to compounds of any one of the formulas:
Figure imgf000030_0001
In some embodiments of the present disclosure, the cell is capable of producing 2- hydroxyammodendrine. In such embodiments, the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. optionally a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
Even more preferably, in such embodiments, the host cell does not comprise a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the method of producing 2- hydroxyammodendrine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the hydroxyammodendrine species.
“2-hydroxyammodendrine” as used herein include, but is not limited to a compound of any one of the formulas:
Figure imgf000032_0001
In some embodiments of the present disclosure, the cell is capable of producing tetradehydrosparteine. In such embodiments, the host cell preferably comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
Even more preferably, in such embodiments, the host cell does not comprise a heterologous gene encoding VRED1 of SEQ ID NO 6 or VRED2 if SEQ ID NO:7 or a functional homologue having at least 70% sequence identity to any of the aforementioned.
In some embodiments of the present disclosure, the method of producing tetradehydrosparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the tetradehydrosparteine species. Tetradehydrosparteine may for example be hydrated (carbinolamine), fully hydrolysed (aminoaldehyde) or tautomerized (iminium and/or enamine) forms of tetradehydrosparteine. In particular, tetradehydrosparteine include, but is not limited to compounds of any one of the formulas:
Figure imgf000034_0001
Figure imgf000035_0001
Sparteine
The present invention relates to methods of producing (-)-sparteine or a precursor thereof, as well as to enzymes and host cells useful for producing (-)-sparteine. In preferred embodiments of the present disclosure, the host cell is capable of producing (-)-sparteine, and more preferably (-)-sparteine of high enantiomeric purity as outlined in detail below. In such embodiments the host cell preferably comprises the heterologous genes outlined below, and it is further preferred that the host cell does not comprise heterologous gene(s) encoding enzymes catalysing conversion of (-)- sparteine to other compounds.
In some embodiments of the present disclosure, the host cell is capable of producing (- )-sparteine and comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto and g. a encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto,; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto, f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell is capable of producing (- )-sparteine, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the host cell is capable of producing (- )-sparteine, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
In some embodiments of the present disclosure, the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the (-)-sparteine.
In some embodiments of the present disclosure, the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto and a gene encoding NCS3 of SEQ ID NO 11 , or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the (-)-sparteine.
In some embodiments of the present disclosure, the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto and a gene encoding NCS4 of SEQ ID NO 12, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the (-)-sparteine.
In some embodiments of the present disclosure, the method of producing (-)-sparteine comprises the steps of: a. Providing a host cell comprising a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding VRED1 or VRED 2 of SEQ ID NO 6-7 or a functional homologue thereof having at least 70% sequence identity thereto a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto and a gene encoding NCS3 of SEQ ID NO 11 , or a functional homologue having at least 70% sequence identity thereto and a gene encoding NCS4 of SEQ ID NO 12, or a functional homologue having at least 70% sequence identity thereto; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating the (-)-sparteine.
(-)-sparteine” as used herein refers to compounds of the formula:
Figure imgf000041_0001
(-)-sparteine
As mentioned above, it is preferred that the host cell is capable of producing (-)- sparteine of high enantiomeric purity. Accordingly, it is preferred that the host cell is capable of producing (-)-sparteine with an enantiomeric excess (ee) of at least 60%, such as preferably at least 65%, such as preferably at least 70%, such as preferably at least 75%, such as preferably at least 80%, such as preferably at least 85%, such as preferably at least 90%, such as preferably at least 95%, such as most preferably of at least 97%. The enantiomeric purity of (-)-sparteine may preferably be determined as described herein below in Example 5.
The host cell is preferably capable of producing (-)-sparteine with a high yield. The exact yield may be dependent on the type of host cell and/or the cultivation method. In some embodiments the host cell is capable of producing (-)-sparteine at a level of at least 0.005%, such as preferably 0.01%, such as preferably 0.025%, such as preferably 0.4%, such as most preferably 0.05%, such as in the range of 0.005 to 5%, wherein the % is indicated as w/w % of the total weight of the host cell.
Method
The invention provides methods of producing (-)-sparteine or precursors thereof, e.g. any of the methods described herein above in the sections “Precursor of sparteine” and “Sparteine”. The methods may comprise a step of cultivating the host cell under conditions enabling growth, and further optionally a step of isolating the (-)-sparteine or the precursor thereof. These steps may for example be performed as described herein in this section.
The (-)-sparteine or the precursors thereof may be isolated through any useful method known to the skilled person. For example, (-)-sparteine may be isolated by a method comprising one or more of the following:
• Extraction, e.g. acid/base extraction and/or supercritical fluid extraction
• Precipitation
• Crystallization and/or recrystallization
• Chromatography, e.g. cation exchange chromatography, adsorption chromatography and/or HPLC
In some embodiments of the present disclosure, the step of isolating (-)-sparteine or the precursor thereof comprises extraction, e.g. extraction with a solvent, e.g. a solvent comprising an alcohol, such as methanol and/or acid-base extraction.
The “solvent” as used herein includes any liquid that can dissolve or substantially disperse another substance.
The acid-base extraction may be any procedure using sequential liquid-liquid extractions to purify compounds from mixtures at differential pH. In one embodiment, the acid-base extraction may be performed as follows. The mixture is dissolved in a suitable solvent and poured into a separating funnel. An aqueous solution of the acid or base is added, and the pH of the aqueous phase is adjusted to bring the compound of interest into its required form. After shaking and allowing for phase separation, the phase containing the compound of interest is collected. The procedure is then repeated with this phase at the opposite pH range. The order of the steps is not important and the process can be repeated to increase the separation.
In some embodiments of the present disclosure, the method of isolating (-)-sparteine comprises a step of supercritical fluid extraction. In superfcritical fluid extraction, supercritical fluid (e.g. supercritical carbon dioxide) may be used to extract the flour at high pressure (e.g. 25 MPa) in the absence or presence of a co-solvent (e.g. ethanol). In some embodiments of the present disclosure, the method of isolating (-)-sparteine comprises a step of chromatography, for example cation exchange chromatography or adsorption chromatography. For cation exchange, the flour may be extracted with an aqueous solvent (e.g. pure water), and the extract may be run through a cation exchange resin (e.g. AG 50W-X2). The cation exchange resin may be eluted using a basic solvent (e.g. 10% NaOH in ethanol/water 70:30).
Adsorption chromatography may involve use of a polymeric resin. For such chromatography, the flour may be extracted in basic aqueous solvent (e.g. water basified with 1 M NaOH to pH 11), the extract may be run through a polymeric resin (e.g. Amberlite XAD-16), and the column may be eluted in an organic solvent (e.g. ethanol).
In some embodiments of the present disclosure, the step of isolating (-)-sparteine or the precursor, comprises the use of HPLC.
The person skilled in the art will appreciate that "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase on a support matrix, typically a densely packed column.
The step of cultivating the host cell under conditions enabling growth may be performed by any method known to the skilled person.
The term "growth" of a host cell or a multicellular organism comprising a host cell should be understood as a reference to proliferation, multiplication, differentiation and/or maintenance of viability of the subject host cell, or multicellular organism. If the host cell is comprised in a multicellular organism, said conditions are usually conditions enabling maintenance of viability and/or growth of the multicellular organism. Thus, if the host cells are plant cells comprised in a plant, the cultivation conditions are conditions suitable for maintenance and/or growth of said plant. That could e.g. sowing seeds or other regenerative parts of the said plant in a field or in a green house. Cultivation may further comprise watering and/or fertilising.
If the host cell is a unicellular organism, cultivation may be incubation in a medium comprising at least a carbon source and a nitrogen source at a temperature suitable for growth of said unicellular organism. The carbon source may e.g. be a carbohydrate, such as as sugars or polysaccharides. The nitrogen source may for example be amino acids or polypeptides. In some embodiments, the medium comprises lysine, such as L- Lysine. The skilled person is well able of selecting a suitable cultivation medium based on the particular host cell.
Polypeptides and nucleic acids
In addition to the host cells, the invention also provides enzymes useful in the production of (-)-sparteine or precursors thereof.
In some embodiments of the present disclosure, the invention provides a polypeptide, such as an isolated polypeptide selected from the group of polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11 , SEQ ID NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
The invention further provides nucleic acid encoding any of the polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto. The skilled person will appreciate that many different nucleic acid sequences may encode the same polypeptide. In some embodiment, the nucleic acid may comprise or consist of a coding sequence (e.g. a cDNA sequence) from wild type lupin, e.g. a cDNA sequence derived from wild type NLL. In other embodiments, the nucleic acid sequence may be codon optimised for improved expression in the host cell.
In one embodiment the nucleic acid sequence comprises or consists of any one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, functional homologues thereof encoding the same polypeptide or a polypeptide having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto or codon optimised versions of any of the aforementioned..
Examples
Example 1: NCS2 increases the production of N-acetylated tetrahydroanabasine in Nicotiana benthamiana
Aim:
To investigate whether NCSs of SEQ ID NO 9-12 are involved in the dimerization of A1- piperideine to tetrahydroanabasine.
Material and Methods:
The full-length coding regions of LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and of the gene candidates NCS1-4 of SEQ ID NO 9-12, were amplified from leaf cDNA of the bitter narrow-leafed lupin (Lupinus angustifolius) cultivar Oskar. The genes were cloned into the plant expression vector pEAQ-USER (Luo et al., 2016) by USER cloning and transformed into E. coli strain Top10. Clones were transformed into electrocompetent Agrobacterium tumefaciens strain AGL-1. For agroinfiltration, AGL-1 strains were grown on YEP liquid medium supplemented with 50 pg/mL kanamycin, 25 pg/mL rifampicin and 50 pg/mL carbenicillin at 28 °C and 220 rpm until ODeoo « 3-4. The bacteria were collected by centrifugation and resuspended in ultrapure H2O to ODeoo = 1. The diluted strains were mixed in equal portions to obtain the desired gene combination for co-expression in leaves of N. benthamiana. After a 1-3 h incubation at room temperature, the mixtures were infiltrated into the abaxial side of young leaves of 4 week-old N. benthamiana plants. The agroinfiltarted plants were allowed to recover overnight in the dark before being brought back to the greenhouse. Agroinfiltrated leaves were harvested 7 days after agroinfiltration. Two 1-cm leaf discs were punched out of the agroinfiltrated (discolored) portion of the leaves, collected in 1.5 ml screw-cap microcentrifuge tubes, and dried for 4 days at 40 °C. The leaf discs were then pulverized using a steel bead and a TissueLyzerll bead beater (Qiagen) and extracted with 250 pl of extractant (60% methanol, 0.06% formic acid, and 5 ppm caffeine in water) for 3 hours at 400 rpm. The extracts were briefly centrifuged to remove large particulates, diluted 5x with ultrapure water, and filtered through a 0.22 pm filter.
The extracts were then analyzed by analytical LC-MS. Analytical LC-MS was carried out on a Thermo Fisher Dionex 3000 RS HPLC/UPLC system interfaced to a Bruker compact QqTOF mass spectrometer through an ESI source. Analytes were separated on a Luna C18(2) column (150 x 2 mm, 3 pm, 100 A, Phenomenex) kept at 40 °C. Mobile phases A and B consisted of, respectively, 0.05% formic acid in water and 0.05% formic acid in acetonitrile. Analytes were eluted using the following gradient at a constant flow rate of 0.3 mL/min: 0-0.5 min, 2% B (constant); 0.5-2.375 min, 2-6% B (linear); 2.375-7 min, 6-25% B (linear), 7-13 min, 25-100% B (linear); 13-14 min, 100% B (constant); 14- 14.5 min, 100-2% B (linear); and 14.5-20 min, 2% B (constant). ESI mass spectra (m/z 50-1000) were acquired in positive ionization mode with automatic MS2 acquisition using the following parameters: capillary voltage 4500 V; end plate offset -500 V; source temperature 250 °C; desolvation gas flow 8.0 L/min; and nebulizer pressure 2.5 bar. N2 was used as desolvation, nebulizer and collision cell gas. Values are relative to the plants expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO3, and GFP (mean set to 1), and correspond to the normalized, integrated peak areas from extracted ion chromatograms at m/z 209.16 ± 0.01 ([M+H]+).
Results:
Co-expression of NCS2 of SEQ ID NO 10 leads to a 3.5-fold increase in accumulation of /V-acetylated tetrahydroanabasine (measured as ammodendrine) in Nicotiana benthamiana when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2 and AT of SEQ ID NO 3 (Fig 2).
Conclusion:
The inventors of the present disclosure have realized that NCS2 of SEQ ID NO 10 is involved in the dimerization step from A1-piperideine to tetrahydroanabasine and that NCS2 of SEQ ID NO 2 increases the production of /V-acetylated tetrahydroanabasine (measured as ammodendrine) in Nicotiana benthamiana when co-expressed with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2 and AT of SEQ ID NO 3.
Example 2: G8HO hydroxylates N-acetylated tetrahydroanabasine allowing the formation of a quinolizidine core
Aim:
To investigate which enzyme oxidises /V-acetylated tetrahydroanabasine to yield a dihydrolusitanine species Material and Methods:
The full-length coding regions of G8H0 of SEQ ID NO 4, CYP450_1 , and CYP450_2 were cloned into a plant expression vector as described for other narrow-leafed lupin genes in example 1. These candidate genes were individually co-expressed in N. benthamiana with LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, and NCS2 of SEQ ID NO 10 as described in example 1. The LC-MS chromatograms of leaf extracts were obtained at 5 days post agroinfiltration as described in example 1.
The ESI+ CID mass spectra of the products of G8HO of SEQ ID NO 4 in Nicotiana benthamiana were acquired as described in example 1. The two products appeared to be a hydroxylated form and a dehydrogenated form of ammodendrine ([M+H]+ of m/z 225.1596 and m/z 207.1498, respectively). The MS2 spectrum of the hydroxylated product (peak b) revealed the presence of an alcohol group (neutral loss of H2O) and of a secondary amide group (neutral loss of C2H2O) in the structure. These features are consistent with a hydroxylated ammodendrine. Position 2 is the most likely site of hydroxylation, since it enables the ring-opening and subsequent rearrangement that is essential to proceed in the pathway. The dehydrogenated product (peak a), instead, contained a primary amide group (prominent neutral loss of C2H5NO in the MS2 spectrum), which is consistent with ring opening and rearrangement leading to a didehydrolusitanine species.
Results:
The co-expression of G8HO of SEQ ID NO 4 with LDC of SEQ ID NO1 , CAO of SEQ ID NO 2, NCS2 of SEQ ID NO 10 and AT of SEQ ID NO 3 leads to a decrease in ammodendrine accumulation as well as appearance of a hydroxylated ammodendrine (2-hydroxyammodendrine) and didehydrolusitanine (Fig. 3).
Conclusions:
The inventors of the present disclosure have realized that G8HO of SEQ ID NO 4 hydroxylates the direct product of AT of SEQ ID NO 3, which can either lose water to become 2-hydroxyammodendrine or undergo rearrangement to give a didehydrolusitanine species, from which subsequent QA biosynthesis can continue.
Example 3: Production of sparteine in N. benthamiana by co-expression of at least 9 genes Aim:
To investigate whether the expression of CHYD of SEQ ID NO 5, NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, NCS3 of SEQ ID NO 11 , NCS4 of SEQ ID NO 12, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana leads to the efficient production of (-)-sparteine.
Material and Methods:
The full-length coding regions of CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, and VRED2 of SEQ ID NO 7, were cloned into a plant expression vector as described for other narrow-leafed lupin genes in example 1. These candidate genes were coexpressed in N. benthamiana with NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, NCS3 of SEQ ID NO 11 , NCS4 of SEQ ID NO 12, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 as described in example 1. The LC-MS chromatograms of leaf extracts were obtained at 5 days post agroinfiltration as described in example 1. To analyse (-)-sparteine, extracted ion chromatograms (EICs) of the singly and doubly charged molecular ions of (-)-sparteine combined (m/z 235.22 ± 0.01 and m/z 118.11 ± 0.01 , respectively) were monitored.
To determine whether each of the selected candidate genes was necessary for the efficient production of sparteine, each of the genes were replaced by GFP consecutively. In the case of suspected redundancy between two genes, both of them were also replaced simultaneously by GFP.
Results:
The combination of CHYD of SEQ ID NO 5, NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, NCS3 of SEQ ID NO 11 , NCS4 of SEQ ID NO 12, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 led to (-)-sparteine production in N. benthamiana. The individual absence of NCS2 of SEQ ID NO 10, CHYD of SEQ ID NO 5, and NCS1 of SEQ ID NO 9 led to the complete absence of (-)-sparteine accumulation. The absence of the other NCSs, namely NCS3 of SEQ ID NO 11 and NCS4 of SEQ ID NO 12, did not lead to statistically significant changes in (-)-sparteine accumulation but led to noticeably lower average values. The combined absence of VRED1 of SEQ ID NO 6 and VRED2 of SEQ ID NO 7 led to a complete loss of sparteine accumulation (Fig. 4)
Conclusions:
The inventors of the present disclosure have realized that the expression of CHYD of SEQ ID NO 5, NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, VRED1 of SEQ ID NO 6 or VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana leads to the production of (-)- sparteine while the additional expression of NCS3 of SEQ ID NO 11 and NCS4 of SEQ ID NO 12 leads to efficient production of (-)-sparteine.
Example 4: Determination of the profile of accumulation of intermediates during the production of sparteine in N. benthamiana.
Aim:
To gain insights into the activity of CHYD of SEQ ID NO 5, NCS1 of SEQ ID NO 9, NCS2 of SEQ ID NO 10, NCS3 of SEQ ID NO 11 , NCS4 of SEQ ID NO 12, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, NCS2 of SEQ ID NO 10, AT of SEQ ID NO 3 and G8HO of SEQ ID NO 4 in N. benthamiana.
Material and Methods:
In addition to (-)-sparteine, relative levels of the pathway intermediates/shunt products ammodendrine, 2-hydroxyammodendrine, and didehydrolusitanine in N. benthamiana leaves expressing different gene combinations were quantified. Apart from these, also a tetradehydrosparteine species (possibly the di-iminium cation intermediate, m/z 116.10 ± 0.01 , [M]2+) that accumulated with particular gene combinations was quantified. The gene combinations used are the same ones as in Example 3.
The LC-MS chromatography of N. benthamiana leaves 7 days post agrifiltration was performed as described in example 1. For (-)-sparteine, the extracted ion chromatograms (EICs) of the singly and doubly charged molecular ions of sparteine combined (m/z 235.22 ± 0.01 and m/z 118.11 ± 0.01 , respectively) were monitored. For the mentioned pathway intermediates/shunt products, the following molecular ions were monitored: ammodendrine [M+H]+ at m/z 209.16 ± 0.01 ; 2-hydroxyammodendrine [M-H2O+H]+ at m/z 207.15 ± 0.01 ; didehydrolusitanine [M+H]+ at m/z 207.15 ± 0.01 ; and tetradehydrosparteine [M]2+ at m/z 116.10 ± 0.01.
Results:
The absence of NCS2 of SEQ ID NO 10 virtually eliminated the production of any of the measured intermediates/shunt products (as well as the end product). Compared to the control, absence of NCS4 of SEQ ID NO 12 led to significantly larger amounts of 2- hydroxyammodendrine relative to didehydrolusitanine. Omitting CHYD of SEQ ID NO 5 led to the virtual absence of both tetradehydrosparteine and sparteine, a profile that closely resembled that of omitting NCS1 of SEQ ID NO 9. In turn, the absence of NCS3 of SEQ ID NO 11 led to a much unfavorable ratio of tetradehydrosparteine to (-)- sparteine. This profile was similar to the one observed when omitting both VRED1 of SEQ ID NO 6 and VRED2 of SEQ ID NO 7, except that no final product could be observed in the latter case (Fig. 5).
Conclusion:
The elimination of the production of any of these intermediates in the absence of NCS2 confirms a role in the early QA pathway. The results in regards to NCS4 suggest a role for NCS4 in aiding the conversion of 2-hydroxyammodendrine to didehydrolusitanine. The results in regards to CHYD indicate that CHYD and NCS1 are responsible for deacetylation coupled to the addition of the third A1-piperideine unit. The results in regards to NCS3 suggest a role for NCS3 in the reduction of tetradehydrosparteine to sparteine. The results regarding the VREDS suggest that the VREDs are directly responsible for the reduction of tetradehydrosparteine to sparteine.
Based on the results it is proposed that the pathway for production of (-)- sparteine in NLL is as shown in Fig. 1.
Example 5: Co-expression of ARED increases the purity of (~)-sparteine produced in N. benthamiana.
Aim: To investigate if the putative reductase ARED of SEQ ID NO 8 leads to an increased amount of sparteine compared to a-isosparteine and p-isosparteine.
Material and Methods:
The full-length coding regions of ARED of SEQ ID NO 8 was cloned into a plant expression vector as described for other narrow-leafed lupin genes in example 1. Agroinfiltrated leaves of N. benthamiana transiently co-expressing LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5, and VRED1-2 of SEQ ID NO 6-7 together with either GFP or ARED of SEQ ID NO 8 were harvested 7 days after agroinfiltration. Two leaf discs of 1-cm diameter were punched out of each infiltrated leaf (n = 6 or 7) and collected in 1.5 ml screw-cap microcentrifuge tubes. The remaining agroinfiltrated (discolored) portions of the leaves were clipped out and pooled in 50 ml centrifuge tubes. The leaf discs and the leaf clippings were dried for 4 days at 40 °C after which they were pulverized using steel beads and a TissueLyzerll (Qiagen) bead beater (in the case of the leaf discs) or a benchtop vortex (in the case of the leaf clippings).
To determine the purity of sparteine compared to the isosparteines, the pulverized leaf discs were extracted with 250 pl extractant (60% methanol, 0.06% formic acid, and 5 ppm caffeine in water) for 3 hours at 400 rpm. Leaf debris was separated by centrifugation (1 min at 21000xg) and the extracts were diluted 5x in water, filtered through a 0.22-pm filter and analyzed by LC-MS as described in example 1. Extracted ion chromatograms of combined m/z ratios of 235.22 ± 0.01 and 118.11 ± 0.01 were produced, which corresponded to the [M+H]+ and [M+2H]2+ ions, respectively. Three peaks were detected and integrated from the extracted ion chromatograms and added together to give a total peak area. The % purity of sparteine was evaluated by comparing the area of the sparteine peak to the total peak area.
To determine the enantiomeric purity of sparteine, 50 mg of the pulverized leaf clippings were transferred to 12-ml glass vials and extracted with 3 ml of 1 M HCI(aq) by incubating for 1 hour at 400 rpm. Leaf debris was separated by centrifugation and the cleared extracts were transferred to clean 12 ml glass vials. The extracts were defatted with n- hexane (3x 1.5-ml portions), and the aqueous extracts were transferred to clean 12-ml glass vials. The extracts were basified with 2 ml of 2 M NaOH(aq) and the alkaloids were extracted with dichloromethane (3x 1.5-ml portions). The dichloromethane extracts were transferred to clean 12-ml glass vials and dried with anhydrous MgSC The dried extracts were transferred to clean vials and evaporated under reduced pressure. The residues were reconstituted in 100 pl ethyl acetate and analyzed by chiral GC-MS.
Chiral GC-MS was carried out on a Shimadzu Nexis GC-2030 gas chromatograph equipped with a Shimadzu AGC-6000 autosampler and coupled to a Shimadzu GCMS- QP2020 NX single quadrupole mass spectrometer. Analytes were separated on an Agilent J&W CycloSil-B capillary column (30 m x 0.25 mm x 0.25 pm) using He as carrier gas. Ethyl acetate extracts were injected in splitless mode (1 pl) at an inlet temperature of 250 °C. Analytes were separated using the following column temperature program at a constant carrier gas pressure of 51 .0 kPa: initial 80 °C, hold for 3 min; ramp to 125 °C at 30 °C/min; ramp to 240 °C at 2 °C/min. The separated analytes were ionized using an electron impact ion source at 250 °C. MS spectra were acquired in Scan mode (m/z 10- 300) at an energy of 70 eV. (+)- and (-)-sparteine were identified by comparison with commercial standards (Sigma-Aldrich), (-)-sparteine and (+)-sparteine were identified based on comparison with commercial preparations (Sigma-Aldrich). The enantiomeric excess was calculated from extracted ion chromatograms of m/z 137.
Results:
Co-expression of LDC of SEQ ID NO 1 , CAO of SEQ ID NO 2, AT of SEQ ID NO 3, G8HO of SEQ ID NO 4, NCS1-4 of SEQ ID NO 9-12, CHYD of SEQ ID NO 5 and VRED1-2 of SEQ ID NO 6-7 genes with ARED of SEQ ID NO 8 in N. benthamiana leads to a significantly improved relative amount of sparteine vs. a-isosparteine and - isosparteine from -86% to -95%. ARED of SEQ ID NO 8 was also able to improve the enantiomeric excess of (-)-sparteine from -60% to -97%. The total yield of (-)- sparteine produced in N. benthamiana in the presence of ARED of SEQ ID NO 8 was -5.7 pg in two leaf discs of 1-cm diameter. Assuming -12 mg fresh weight for the two leaf discs, this amounts to -0.05% yield on a fresh weight basis (Fig. 5).
Conclusions:
The reductase ARED of SEQ ID NO 8 is able to improve the stereoselectivity of the (-)-sparteine pathway engineered in N. benthamiana, leading to a higher purity of sparteine (97% based on non-chiral LC-MS peak areas) as well as a higher enantiomeric purity of (-)-sparteine (97% enantiomeric excess based on chiral GC-MS). Sequence overview
SEQ ID NO 1 :
Enzyme lysine decarboxylase (LDC) from narrow-leafed lupin cultivar Oskar:
MPSLLLSEGLYQAKGATTPLSLKAIYNASGVKGKRVTPLLANEQEGGISHFIQSIIRNTP
DIDSPFLVLDLGVIMHLMEKWTTNLPTVQSYYAVKCNPNPSLLGVLAALGSSFDCASR
AEIESVLSLGVSPDRIIYANPCKSESHIKYAATVGVNVTTFDSKEEIHKIKKWHPKCELLI
RIKPPQDSGARNALGLKYGALPEEVKPLLQAAKDAELKWGVSFHIGSGGADSRTYH
GAIAAAKSVFDMASNELGMPRMKIVDIGGGFTCGNQFDAASFHVNEALEDNFGKEEG
VWIGEPGRYFAESPFTLASKVIGKRVRGEVREYWIDDGIYGSLNCIMYDFATVTCTPL
ACSSKPEDPECKNAKMYPSTVFGPTCDSLDTVLTDYLLPELELDDWVVFPNMGAYTT
SSGTNFNGFNTTAISTYLAYSTPIAMEKSMF
SEQ ID NO 2:
Enzyme copper amine oxidase (CAO) from narrow-leafed lupin cultivar Oskar:
MASASEKMVPPSCACCSAGNDSAIIPHIAAAAAPSADWTANVPDDGRLNKMTIVHPV
DSLPQPSINAKGIITLPRPQPSHPLDPLSPAEISLAVATVRAAGKTPELKDGLRFMEIAL
LEPDKHVVALADAYFFPPFQPSLLPKGGFVIPTKLPPRCARLLVYNRKTNETSLWIVEL
SQVHAVTRGGNHLGKVISSQWPDVQPPMDAVEYAECEAAVKSYPPFIEAMKKRGIE
NMELVMVDPWCAGYFSEADDPNRRLAKPIIFCKCESDCPMENGYARPVEGIFVLVDM
QKMEVIQFEDRKLVPLPPVDPLRNYTHAATRGGTDRSDLKPLKIVQPEGPSFSVNGYY
VEWQKWNFRIGFTPKEGLVIYSVAYVDGSQGLRPVAHRLSFVEMWPYGDPNDPHY
RKNAFDAGEDGLGRNAHSLKKGCDCSGIVKYFDAHFTNFTGGVETIENCVCLHEEDH
GILWKHQDWRTGLSEVRRSRRLSVSFICTVANYEYGFFWHFYQDGKMEAEVKLTGIL
SMGALMPGEYRKYGTVIAPGLYAPVHQHFFVARMNMAVDSRPGEALNQWEVNVKA
EEPGDHNVHNNAFYAEETLLRSEMEAMRDCDPMTARSWIVRNTRSTNRTGHLTGYK
LVPGSNCLPFAHSDAKFLRRGAFLKHNLWVTAYSPDELFPGGEFPNQNPRIGDGLPT
WVTQNRSLEESDIVLWYVFGVTHVPRLEDWPVMPVEHIGFMLMPHGFFNCSPAIDVP
PSKCELEAKEKDIKDNGVLKPIENSLASKL
SEQ ID NO 3:
Enzyme BADH acetyltransferase (AT) from narrow-leafed lupin cultivar Oskar:
MAYQMASLKLEMNEVVHVKPSTPTPSIVLPLSTLDHRPYPDSIWPIVHVYRSASNGKL
DPAFVLKQALSKALVYYYPLAGKLVKQPDGKVAINCNNDGVPFLEAIANCNLSSLNYL
DDHDILIAKQLVFDLHVQDENEYPHPVSFKLTKFQCGGFTIGMSTSHIVCDGWGACQF
FRAIVELASGKSEPFVKPVWERERLIGSITTQPMPNPMDEATAAVSPFLPATDVMYEL FKVDKESIRRLKMSLMKEISGNETMEQGFTSFESLAAYVWRSRARALNLNNEGKTLLV
FSVQVRQHMSPPLSDGYYGTAITEGQVVLTMKELNEKPLSDIVKLVKESKNIAFTGDFI
KNTIDTLESNPENFNVEEGPGATLALSDWKHLGFMPNVDFGWKEPINMVPAPCNMFE
YEGLCIFLSPSKYDPSMEGGVRVFISLPSVAMPKFREEMEALKVTTP
SEQ ID NO 4:
Enzyme geraniol-8-hydroxylase (G8HO) from narrow-leafed lupin cultivar Oskar:
MDYLTLFLLISFVWTSIYVLFSKLGIKTSKYAPGPYPLPIIGNIFELGKLPHQTLSKLSQTY
GPIMTLKFGSVTAIVISSPQVAKEALQKNDQVFSFRPTPDTLRAHDHHIYSVAWMQPS
ADWRALRKACAIKVFSSRMLDSTQFLRQKKVQELMDYVKESCKKGEALDIGKATFKT
VLNSISNTLFSMDLAHYTSDKFQEFKDIICGITEEAGKPNYVDYFPILSFLDPQGAHGR
MKGYFGKLIKFFDDLIEERLQLRATQKESKACKDVLDSVLELMLEDNSQITRLHVSHLF
VDLFVAGIDTTSITIEWAMAELLRNPEKLKKVRKELQQVTSKGEQLEETHISKLPFLEAV
IKETFRLHPPAAFLVPRMSGDNVELCGYMVPKNAQIMINAWAMGRDSSVWANPNEFI
PERFLNNEIDFKGQYFELIPFGAGRRICPGLPLASKTVHTVLASLLCGYDWKLVDGGE
GENMDMSEEYGLTLHKAQPLLVIPIKA
SEQ ID NO 5:
Enzyme CHYD from narrow-leafed lupin cultivar Oskar:
MSNKASSQSQPFDPYEYLQIVQSPDGTLTCSIEYPKAPPTSDPNLLIPVLTKDVTINES
TKTWVRLFLPRRTLLSSHGSNSNHKLPIIVFFHSGGFICASAATIVVHDFCVDMADNVE
AIVVSVDYRLAPKHRLPAQYDDAMDALYWIRSSQDEWLTKYADISNCYLMGNSAGANI
SYHTGLRVAEDVDHFKPLKIQGFIFRQPFFGGIKRTDSELRLENDPVIPVSTTDLMWEL
ALPIGANREHEYCNLRVGNGPKKLDEFRKLGWRALVSWTGGDQLGDRGKELVQLLD
EKGVQVVSDFHEEGCHEVEYNEPLKAKQLLGLVKGFISS
SEQ ID NO 6:
ENZYME VRED1 from narrow-leafed lupin cultivar Oskar:
MEEGKGRVCVTGGGGFIASMIIKRLLLEGYSVNTTVRPGKSKKDVSFLTNLPGASRKL
QVFNADLNNPESFIPAIEGCVGVFHTATPYDLQKDEDEHTLTKRAIGGALGILQASISSK
TVKRVVYTASGAAIINSGKEVEDLDESYWSDIDFMYKTKPFAWTIAISQTLTEKAVLEFA
AQHENELDVVTLILPYVIGPFICSKLPESVESAFAWLFGKYQFGVFLRFPLTHVDDVAR
AQIFLLEHPNPKGRFNCSLSGTVTFEEIGDILMAKYPEFQIPTRESLKEIKGWTIPSINSK
KLRDAGFKFNYGTKEIIEETIQCCKENGYL SEQ ID NO 7:
Enzyme VRED2 from narrow-leafed lupin cultivar Oskar:
MEEGKGRVCVTGGGGFIASMIIRRLLLEGYSVNTTVRPGKSKKDLSFLTNLPGASRTL
QVFNADLNNPESFIPAIEGCIGVFHTATPYDMQKDEDEKILTKRAIGGALGILNASISSK
TVKRVVYTASAAAIINSGKEVEELDESHWSDIDFMYKTKPFAWTIAISQTLTEKAVLEFA AQHENELDVVTLILPYVIGPFICSKLPDSVQFAFPWLFGDYQLGIFLRFPLVHVDDVAR AHIFLLEHPNPKGRFNCSLSGTVTFEEIADILRSKYPEFQMPTLESLKEIAGWTIPSINSK
KLKDAGFKYNYGTKEIIEETIQCCKENGYL
SEQ ID NO 8:
Enzyme ARED from narrow-leafed lupin cultivar Oskar:
MEKRCKVCVTGGSSYIGSYLIKKLLEKGYIVHTTLRNLNDEAKIGILRSFPEANTRLVLF
KADIYKPHEFEPAIKGCEFVFHIATPYEHQMDSQFKSTSEAAVAAVKSIASYCIESGTV
KRLIYTASLLAYSPYKDDGTGFKDYIDETCWTSLNLLNRTIDDDLTNYINSKTQAEKELL SYENGENGSEMEVVSLACGIVGGDTVLSYISESIAVLISQVKDDETTYQTLKFIEDLDG
KIPWHIDDVCEALIFCTKKPSMHGRFLVAAAYVSSSDVANYYFQTYPEFKLKRKYLEG
PKREIKWASTKLKDKGFAYNHDLNKILDDCITCARRIGDL
SEQ ID NO 9:
Enzyme S-norcoclaurine synthase-like 1 (NCS1) from narrow-leafed lupin cultivar
Oskar:
MALSGILSSEIGIKAPASKWFNLFTKQLQRIPIIVDGVEKVTLLQGDWHTIGSVKQWSEL VDGKVATFKEKI EAI DEKN KWI RYN I FDGEM NQH YKVYI LTIQVI EKDDGSASI KWTI EYE KVN ESLEPPYHYM DSITKGCKDI DAELLKN
SEQ ID NO 10:
Enzyme S-norcoclaurine synthase-like 2 (NCS2) from narrow-leafed lupin cultivar
Oskar:
MALKGILSSEVGVHASASKWFNLFAKELHNVQNTTDRVHKTKLLEGDDWHSIGSVKQ WTDIVDGKESHYKERLDAIDEENKTIVYTLFDGDFSKDYNVFKLLFQVIEKNNGAFIKW Tl EYEKVN EKVEPPYGFM DH FTKSTKEI DVFLLKA
SEQ ID NO 11 :
Enzyme S-norcoclaurine synthase-like 3 (NCS3) from narrow-leafed lupin cultivar
Oskar: MPIRGKLEGGFEAKSSADKFWGALRNWYTFFPEAFPSVYKAVEVVEGDGKAVGSVF
RVSISEDSPFAKSIREKIEAVDDVKRTLILDVAGIDGNVFHIYKKYVLHVSVTPKGDGSV
VKVAVEYENPTVKDPEPTEFIDVEVQGFQDLDAYLQNK
SEQ ID NO 12:
Enzyme S-norcoclaurine synthase-like 4 (NCS4) from narrow-leafed lupin cultivar
Oskar:
MATLEKI EAI I El KTNADKYWRTFRDYRTVFPKVFSKYRSI El LEGDGKSVGSVLRH ITFE
GSLKSATEKIEAVDDEKRTLTYAVIDADILQDYKNYKGHISVTPKGNGSEVKWIAEYEK
ASQEVPDPISIKDYLVDTFLKLDAYIQKA
SEQ ID NO 13:
Nucleotide sequence of the enzyme lysine decarboxylase (LDC) from narrow-leafed lupin cultivar Oskar:
ATGCCTTCACTACTACTGAGCGAGGGATTATACCAGGCCAAGGGTGCAACAACAC
CTTTGAGCCTGAAGGCCATTTATAATGCTTCTGGGGTTAAGGGTAAGCGAGTCAC
TCCATTACTCGCTAATGAACAAGAAGGTGGCATCTCTCATTTCATTCAATCCATCAT
TCGAAACACACCAGATATTGATTCACCATTCTTAGTACTTGATCTTGGCGTAATCAT
GCACCTCATGGAGAAATGGACCACTAATCTTCCCACGGTTCAATCTTATTATGCAG
TCAAGTGCAACCCTAACCCATCCTTGTTAGGTGTACTCGCAGCACTTGGTTCGAG
CTTCGACTGCGCCAGCCGAGCCGAAATCGAATCGGTTTTATCACTTGGAGTCTCA
CCGGACCGGATCATTTATGCGAACCCATGCAAATCAGAGAGTCACATTAAATATG
CAGCTACTGTTGGTGTCAACGTCACAACATTCGATTCTAAAGAAGAGATCCATAAG
ATTAAAAAGTGGCACCCAAAATGTGAGTTACTTATTCGTATCAAGCCACCACAAGA
CAGTGGAGCAAGAAATGCTTTGGGTCTCAAATACGGTGCGCTTCCTGAAGAAGTT
AAGCCACTCTTACAAGCCGCTAAAGACGCGGAATTGAAAGTTGTTGGTGTTTCGTT
CCACATAGGAAGTGGTGGTGCTGATTCTAGAACCTACCATGGAGCAATTGCTGCT
GCTAAAAGTGTTTTCGACATGGCTTCTAACGAACTAGGCATGCCAAGAATGAAAAT
AGTGGACATTGGTGGCGGTTTCACTTGTGGGAACCAATTTGATGCAGCTTCTTTTC
ACGTGAATGAGGCTCTTGAGGACAATTTTGGAAAAGAGGAAGGTGTTGTGGTAAT
TGGAGAACCTGGTCGTTATTTTGCTGAGTCACCTTTTACTTTGGCTAGTAAAGTTA
TTGGGAAGCGCGTGAGGGGAGAAGTGAGGGAGTATTGGATTGATGATGGGATTT
ACGGTTCTCTTAATTGCATAATGTATGACTTTGCAACTGTTACTTGCACGCCACTC
GCGTGTAGCTCAAAACCAGAGGATCCAGAATGCAAAAACGCCAAAATGTACCCTT CAACTGTGTTTGGACCCACTTGTGATTCGTTAGATACTGTTCTAACAGATTACTTGT TACCGGAACTGGAACTTGATGATTGGGTTGTGTTCCCAAATATGGGTGCTTATACC
ACATCGTCAGGGACTAATTTCAATGGGTTTAACACAACAGCTATTTCTACTTACCTA
GCATATTCCACCCCAATTGCGATGGAAAAATCTATGTTCTAA
SEQ ID NO 14:
Nucleotide sequence of the enzyme copper amine oxidase (CAO) from narrow-leafed lupin cultivar Oskar:
ATGGCTTCAGCTTCTGAAAAAATGGTACCTCCTTCTTGTGCTTGTTGTTCAGCCGG
TAATGACTCTGCCATCATTCCCCACATTGCGGCTGCCGCCGCTCCCTCTGCTGAC
TGGACTGCCAATGTCCCCGATGATGGCCGCCTAAATAAGATGACCATCGTTCATC CTGTCGACTCCCTACCGCAACCTTCCATCAATGCCAAAGGAATCATTACACTGCCA
AGGCCTCAACCAAGCCACCCTTTGGACCCTTTATCTCCTGCTGAAATCTCTCTGG
CAGTAGCTACTGTGAGGGCTGCTGGAAAAACTCCTGAGCTTAAAGACGGTTTGCG
ATTCATGGAAATAGCTTTGCTCGAACCGGATAAACATGTCGTTGCACTAGCAGATG
CTTATTTTTTTCCACCTTTCCAGCCATCATTGCTTCCTAAAGGAGGGTTTGTGATCC
CAACTAAACTCCCTCCAAGATGTGCTAGACTTCTTGTTTACAATAGGAAGACAAAT
GAGACTAGTCTTTGGATCGTCGAGTTATCGCAAGTTCATGCTGTAACTCGAGGTG
GAAATCATTTAGGAAAAGTAATTTCATCACAAGTTGTACCTGATGTTCAGCCTCCA
ATGGATGCTGTGGAGTATGCAGAATGTGAGGCTGCTGTTAAAAGTTATCCTCCATT
TATAGAGGCTATGAAGAAAAGGGGTATTGAAAACATGGAGCTTGTGATGGTAGAT
CCCTGGTGTGCTGGTTACTTCAGTGAAGCTGATGATCCGAACCGAAGACTTGCTA
AACCAATAATATTTTGCAAGTGTGAGAGTGATTGCCCTATGGAAAATGGCTATGCT
CGCCCGGTCGAGGGAATCTTTGTTCTTGTTGATATGCAAAAGATGGAGGTGATAC
AGTTCGAAGACCGCAAACTTGTTCCTCTGCCTCCTGTAGATCCCTTAAGGAACTAT
ACACATGCTGCAACTAGAGGTGGCACTGATAGAAGTGACTTAAAACCATTGAAAAT
TGTTCAACCTGAAGGTCCAAGCTTTTCCGTCAATGGATATTATGTTGAATGGCAAA
AGTGGAACTTTCGGATTGGATTCACACCCAAAGAAGGTTTAGTTATATATTCTGTT
GCATATGTTGATGGTAGTCAAGGTCTAAGGCCTGTAGCTCATAGGTTGAGTTTTGT
GGAGATGGTTGTACCCTACGGAGATCCAAACGATCCACATTACAGGAAAAATGCT
TTTGATGCTGGGGAAGATGGCCTAGGAAGAAATGCACATTCCTTGAAGAAGGGAT
GTGATTGTTCTGGCATAGTCAAATATTTTGATGCTCACTTCACAAATTTCACTGGTG
GTGTGGAGACAATTGAAAATTGTGTATGTTTGCATGAAGAAGATCATGGAATTCTT
TGGAAGCATCAAGATTGGAGAACTGGCTTATCAGAAGTCCGAAGGTCTAGAAGGC
TTTCAGTTTCATTTATATGTACTGTGGCTAACTATGAGTATGGATTTTTTTGGCACT TTTATCAGGATGGAAAGATGGAAGCTGAAGTTAAGCTAACTGGAATTCTGAGCATG GGAGCCTTAATGCCCGGAGAGTATCGAAAATATGGAACCGTGATTGCCCCAGGTC
TATATGCTCCAGTTCATCAACACTTTTTTGTTGCTCGTATGAACATGGCTGTTGATT
CTAGACCTGGTGAAGCTTTGAATCAGGTTGTGGAAGTCAATGTGAAAGCTGAGGA
ACCTGGTGATCATAATGTTCACAATAATGCATTCTATGCCGAAGAAACTTTGCTCA
GATCTGAAATGGAAGCAATGCGTGATTGCGATCCCATGACTGCTCGATCTTGGAT
TGTAAGGAATACAAGATCAACCAATAGAACTGGACACTTGACAGGCTACAAGCTA
GTACCTGGCTCGAACTGCTTACCATTCGCGCATTCGGATGCCAAGTTTTTAAGAA
GAGGTGCTTTCTTGAAGCATAATCTTTGGGTTACAGCTTACTCACCCGATGAGCTG
TTTCCTGGAGGAGAATTTCCTAATCAAAATCCACGCATTGGCGACGGATTACCTAC
ATGGGTTACGCAGAACCGATCTTTAGAAGAGTCTGATATAGTTCTTTGGTATGTAT
TTGGAGTCACACATGTTCCTCGTTTAGAAGACTGGCCTGTTATGCCAGTAGAGCA
CATTGGTTTTATGCTCATGCCTCATGGATTCTTCAATTGTTCCCCTGCAATAGATGT
TCCACCTAGTAAATGTGAATTGGAGGCTAAAGAAAAAGATATAAAGGATAATGGGG
TTTTGAAGCCAATTGAGAATTCCTTAGCATCAAAGCTCTAA
SEQ ID NO 15:
Nucleotide sequence of the enzyme BADH acetyltransferase (AT) from narrow-leafed lupin cultivar Oskar:
ATGGCATATCAAATGGCATCACTGAAACTTGAGATGAATGAAGTAGTGCATGTCAA
ACCTTCTACACCAACACCTTCCATTGTTCTTCCTCTATCTACCCTTGACCATAGACC
CTATCCTGATAGCATTTGGCCTATAGTTCATGTTTACCGGTCAGCCTCAAATGGGA
AGCTAGATCCTGCTTTTGTGCTCAAACAAGCCCTTTCAAAGGCTTTGGTTTATTATT
ACCCTCTTGCAGGTAAGCTAGTAAAACAACCCGACGGAAAAGTTGCTATCAATTG
CAACAATGATGGAGTTCCATTCCTGGAAGCAATTGCAAATTGTAATCTTTCCTCTC
TTAATTATCTAGATGATCATGACATCCTAATTGCAAAACAATTGGTTTTCGATTTAC
ATGTTCAAGATGAAAATGAATACCCACATCCAGTTTCGTTCAAGTTGACCAAATTC
CAATGTGGAGGTTTCACAATTGGAATGAGCACATCACATATTGTGTGTGATGGTTG
GGGAGCATGTCAGTTCTTCCGAGCCATTGTTGAACTGGCAAGTGGTAAAAGTGAG
CCCTTTGTGAAACCTGTTTGGGAGAGAGAAAGATTAATAGGATCAATCACTACACA
ACCAATGCCAAATCCAATGGATGAGGCTACTGCTGCAGTTTCACCATTTCTTCCAG
CCACTGATGTTATGTATGAGTTGTTTAAGGTTGACAAGGAAAGCATAAGAAGACTC
AAGATGAGTTTAATGAAGGAAATTAGTGGCAATGAAACAATGGAACAAGGCTTCAC
AAGTTTTGAATCTCTTGCTGCATATGTGTGGAGATCAAGAGCAAGGGCCTTAAACC TAAATAATGAAGGGAAAACTTTGCTTGTTTTCTCAGTGCAGGTGAGACAACACATG AGTCCTCCTTTATCTGATGGGTACTATGGAACTGCTATCACAGAAGGGCAAGTTGT
GCTAACCATGAAGGAGCTCAATGAGAAACCACTCTCAGATATAGTGAAGCTTGTC
AAAGAGAGTAAAAATATTGCTTTCACTGGTGATTTTATCAAAAACACAATTGATACA
TTGGAGTCTAATCCAGAGAATTTTAATGTTGAAGAAGGTCCTGGTGCAACATTGGC
TTTATCAGATTGGAAGCATTTGGGTTTCATGCCAAATGTGGATTTTGGATGGAAGG
AACCAATAAATATGGTACCTGCTCCATGCAACATGTTTGAGTATGAGGGTTTGTGC
ATTTTCTTGTCTCCTAGTAAGTATGACCCATCAATGGAAGGAGGAGTTAGGGTTTT
CATATCACTCCCTAGTGTTGCCATGCCTAAGTTTAGAGAGGAGATGGAAGCTCTG
AAGGTTACTACACCCTAG
SEQ ID NO 16:
Nucleotide sequence of the enzyme geraniol-8-hydroxylase (G8HO) from narrow- leafed lupin cultivar Oskar:
>
ATGGATTATCTAACACTTTTTCTACTCATTTCCTTTGTTTGGACAAGCATTTATGTC
CTATTCTCCAAATTAGGAATCAAAACATCCAAATATGCCCCAGGTCCATACCCTTT
ACCTATCATAGGTAACATCTTTGAACTTGGAAAACTCCCACACCAAACACTTTCTAA
GCTCTCTCAAACTTATGGACCTATAATGACCTTAAAGTTTGGTAGTGTTACTGCTAT
AGTTATTTCCTCTCCACAAGTAGCCAAAGAAGCACTTCAAAAAAATGACCAAGTTT
TCTCTTTTAGGCCAACCCCAGATACCCTTAGGGCACATGACCATCATATATACTCA
GTGGCATGGATGCAGCCTTCAGCTGATTGGAGGGCCCTTAGGAAAGCTTGCGCA
ATCAAAGTGTTCTCATCTCGAATGCTCGATTCGACGCAATTTTTACGACAAAAGAA
GGTGCAAGAGTTGATGGATTATGTTAAGGAAAGTTGCAAGAAAGGTGAGGCTTTG
GATATTGGCAAGGCAACTTTTAAGACTGTGCTTAATTCTATATCAAACACTTTGTTC
TCTATGGACTTGGCTCATTATACTTCTGATAAGTTTCAAGAGTTCAAGGACATTATT
TGTGGGATCACTGAAGAAGCTGGAAAGCCTAACTATGTGGATTATTTTCCAATCCT
TAGTTTTCTTGATCCACAAGGTGCCCATGGAAGAATGAAGGGTTATTTTGGAAAGT
TGATTAAATTTTTTGATGATCTTATAGAAGAAAGGCTACAATTAAGAGCTACACAAA
AGGAATCCAAGGCTTGCAAAGATGTTCTAGATTCTGTGCTAGAACTCATGCTGGAA
GACAATTCTCAAATTACTAGGCTCCATGTTTCGCATTTGTTTGTGGATTTATTCGTG
GCTGGAATAGATACCACATCAATCACAATAGAATGGGCAATGGCAGAGTTGCTAC
GTAATCCAGAAAAGCTAAAAAAAGTTAGAAAAGAACTTCAACAAGTTACAAGCAAA
GGTGAACAACTTGAAGAAACACACATATCAAAGCTTCCTTTCTTAGAAGCAGTGAT
TAAAGAAACTTTTCGTTTGCATCCACCAGCAGCATTCTTAGTGCCACGCATGTCAG
GAGATAATGTTGAACTATGTGGTTACATGGTACCTAAAAATGCACAAATTATGATTA
ATGCATGGGCCATGGGAAGAGATTCAAGTGTTTGGGCCAACCCAAATGAATTTAT CCCTGAAAGATTCTTGAATAATGAGATTGATTTTAAAGGTCAATATTTTGAGCTTAT
TCCTTTTGGTGCTGGAAGAAGGATTTGTCCTGGTTTACCATTGGCTTCTAAGACTG
TGCACACTGTCTTGGCCTCACTTTTATGTGGCTATGATTGGAAGCTTGTTGATGGA
GGAGAGGGAGAGAATATGGATATGTCTGAGGAATATGGGCTTACCTTACATAAGG
CACAACCTCTCCTAGTTATTCCTATCAAAGCATAA
SEQ ID NO 17:
Nucleotide sequence of the enzyme CHYD from narrow-leafed lupin cultivar Oskar:
ATGTCGAATAAAGCCTCCTCACAATCTCAACCATTTGATCCCTATGAGTACCTCCA
AATTGTTCAAAGCCCCGATGGCACACTCACTTGCTCAATAGAATACCCTAAAGCCC
CACCCACTTCAGATCCCAACCTTCTAATCCCTGTCCTCACCAAAGATGTCACCATT
AACGAATCAACCAAAACTTGGGTTCGATTATTCCTACCACGAAGAACATTATTATC
AAGTCATGGTTCTAATTCTAACCACAAGCTACCCATCATTGTTTTCTTTCACAGTGG
TGGATTCATCTGTGCAAGTGCAGCTACCATCGTTGTCCATGATTTCTGTGTGGACA
TGGCAGATAACGTAGAAGCTATCGTTGTTTCCGTTGATTATCGCCTTGCTCCTAAG
CACCGATTACCGGCGCAATATGATGATGCCATGGATGCTTTGTATTGGATTAGAA
GCAGCCAAGATGAATGGTTGACAAAATATGCTGACATTTCCAATTGTTACCTAATG
GGGAATAGTGCTGGGGCCAACATTTCCTATCATACAGGTCTACGTGTAGCAGAAG
ATGTGGACCATTTTAAGCCATTGAAAATCCAAGGATTCATATTTCGCCAGCCATTC
TTTGGTGGGATCAAAAGGACTGATTCAGAGTTGAGGCTTGAGAATGATCCAGTGA
TTCCTGTGTCCACTACTGATTTGATGTGGGAGCTAGCACTGCCAATTGGAGCTAA
CCGTGAGCATGAGTATTGCAATCTAAGGGTTGGGAATGGTCCTAAGAAACTAGAT
GAATTCAGAAAACTTGGGTGGAGGGCACTGGTGAGTTGGACTGGTGGGGACCAA
TTAGGGGATCGTGGGAAGGAACTGGTGCAATTGTTGGATGAAAAGGGTGTTCAAG
TGGTGAGTGACTTTCATGAAGAGGGTTGCCATGAGGTTGAATATAATGAGCCCTT
GAAAGCAAAGCAACTCCTTGGGTTGGTGAAAGGTTTCATTTCCTCATAA
SEQ ID NO 18:
Nucleotide sequence of the enzyme VRED1 from narrow-leafed lupin cultivar Oskar:
ATGGAAGAAGGGAAGGGAAGAGTATGTGTCACCGGAGGTGGAGGGTTTATTGCT
TCAATGATTATAAAGAGATTACTCCTAGAAGGTTACTCGGTTAATACGACTGTTAG
ACCAGGTAAAAGTAAGAAAGATGTAAGCTTCCTGACAAATTTACCTGGTGCATCCC
GAAAGTTACAAGTTTTCAATGCGGATCTCAATAATCCAGAAAGTTTTATTCCAGCAA
TTGAAGGATGCGTTGGGGTCTTCCATACTGCTACTCCATATGATTTGCAAAAAGAT
GAAGATGAGCACACATTAACCAAAAGAGCCATTGGTGGAGCCTTAGGGATTTTAC AGGCTTCAATTAGTTCAAAGACTGTGAAGCGAGTTGTTTACACTGCCAGTGGCGC
TGCTATTATCAACAGTGGTAAAGAAGTTGAAGATTTAGATGAGAGTTATTGGAGTG
ATATAGATTTTATGTATAAGACGAAGCCATTTGCTTGGACTATTGCAATTTCTCAGA
CACTGACAGAAAAAGCAGTTCTTGAATTTGCAGCACAACATGAGAATGAGTTGGAT
GTTGTTACTCTTATTCTTCCTTATGTTATTGGACCTTTCATTTGTTCTAAGCTTCCAG
AATCAGTCGAGTCTGCGTTTGCTTGGTTATTTGGTAAATATCAGTTTGGTGTTTTCC
TTCGTTTTCCTCTAACACATGTGGATGATGTGGCAAGAGCACAAATTTTTTTACTTG
AGCATCCAAACCCTAAAGGAAGGTTTAATTGCTCACTAAGTGGTACTGTGACTTTC
GAAGAGATAGGTGATATTCTTATGGCAAAATACCCGGAATTTCAAATCCCAACACG
AGAGTCGCTAAAGGAAATCAAAGGTTGGACAATACCAAGTATAAACTCAAAGAAAC
TCAGGGATGCTGGATTCAAGTTTAACTATGGGACTAAGGAAATAATCGAGGAAAC
AATTCAATGCTGCAAGGAAAATGGTTATCTGTAA
SEQ ID NO 19:
Nucleotide sequence of the enzyme VRED2 from narrow-leafed lupin cultivar Oskar:
ATGGAAGAAGGGAAGGGAAGAGTATGTGTCACCGGAGGTGGAGGGTTTATTGCT
TCAATGATCATACGGAGACTACTCCTAGAAGGTTACTCGGTTAATACCACTGTTAG
ACCAGGTAAAAGCAAGAAAGATCTAAGCTTCCTGACAAATTTACCTGGTGCATCCC
GCACGTTACAAGTTTTTAATGCGGATCTCAACAATCCAGAAAGTTTTATTCCAGCA
ATTGAAGGATGCATTGGGGTTTTCCATACTGCTACTCCATATGACATGCAAAAAGA
TGAAGACGAGAAAATATTAACAAAAAGAGCCATTGGTGGAGCCTTAGGAATTTTAA
ATGCTTCGATTAGTTCAAAGACTGTGAAGCGAGTTGTTTACACTGCCAGTGCCGCT
GCTATTATCAACAGTGGTAAAGAAGTTGAAGAACTAGATGAGAGTCATTGGAGTGA
TATAGATTTTATGTATAAAACGAAGCCATTTGCTTGGACTATTGCAATATCTCAGAC
ACTGACAGAAAAAGCAGTTCTTGAATTTGCAGCACAACATGAGAATGAGTTGGATG
TTGTTACTCTTATTCTTCCTTATGTTATTGGACCTTTCATTTGTTCTAAGCTTCCCGA
TTCAGTCCAGTTTGCGTTTCCTTGGTTATTTGGTGATTATCAGTTAGGTATTTTCCT
TCGTTTTCCTCTAGTACATGTGGATGATGTGGCCAGAGCACATATTTTTTTACTTGA
GCATCCAAACCCTAAAGGAAGATTTAATTGCTCACTAAGTGGTACTGTGACTTTTG
AAGAGATAGCTGATATTCTTCGCTCAAAATACCCAGAATTTCAAATGCCAACACTA
GAGTCGCTAAAGGAAATCGCAGGTTGGACAATACCAAGTATAAACTCAAAGAAAC
TCAAGGATGCTGGATTCAAGTATAACTATGGGACTAAGGAGATAATTGAGGAAACA
ATTCAATGCTGCAAAGAAAATGGTTATCTGTAA
SEQ ID NO 20: Nucleotide sequence of the enzyme ARED from narrow-leafed lupin cultivar Oskar:
ATGGAAAAGAGGTGCAAGGTATGCGTCACTGGCGGTTCAAGCTATATTGGTTCTT
ACCTCATCAAGAAGCTTTTGGAAAAGGGCTACATTGTCCATACAACGCTTAGAAAC
TTAAATGATGAGGCTAAGATTGGTATTTTGAGAAGCTTTCCTGAAGCCAATACTAG
ATTGGTGTTATTTAAAGCAGACATATACAAGCCACATGAGTTTGAGCCTGCAATAA
AAGGCTGTGAGTTTGTCTTTCATATTGCTACTCCCTACGAACATCAAATGGATTCT
CAGTTTAAAAGCACAAGTGAAGCTGCAGTAGCCGCGGTAAAAAGCATTGCTAGTT
ATTGTATTGAATCAGGAACAGTGAAACGACTGATTTACACTGCGTCCTTGCTTGCT
TATTCTCCGTATAAGGATGATGGAACTGGTTTCAAGGATTACATCGATGAAACTTG
TTGGACTTCTCTTAACCTTTTAAATAGAACCATTGATGATGATCTTACGAACTACAT
TAATTCGAAGACACAAGCTGAGAAAGAGTTATTGAGTTATGAAAATGGGGAAAATG
GCAGTGAAATGGAGGTGGTAAGTCTGGCTTGTGGAATAGTGGGAGGTGACACTG
TTCTAAGTTACATATCCGAGAGTATTGCTGTGCTTATATCTCAGGTAAAAGATGAT
GAAACCACATACCAAACCTTAAAGTTCATAGAAGACTTGGATGGAAAGATTCCCGT
TGTCCACATTGATGATGTTTGTGAAGCTCTTATTTTCTGTACGAAGAAACCATCAAT
GCATGGTAGATTCTTGGTTGCTGCTGCATATGTTTCGTCTTCAGACGTAGCAAATT
ACTATTTCCAAACTTACCCAGAGTTCAAATTGAAGAGGAAGTATTTGGAAGGGCCT
AAAAGAGAAATTAAATGGGCATCAACAAAGCTTAAAGACAAAGGATTTGCATACAA
CCATGACCTCAACAAGATATTAGATGATTGTATCACATGCGCAAGAAGGATTGGC
GATCTCTAG
SEQ ID NO 21 :
Nucleotide sequence of the enzyme S-norcoclaurine synthase-like 1 (NCS1) from narrow-leafed lupin cultivar Oskar:
ATGGCTCTTAGTGGTATATTGAGTTCTGAAATTGGGATTAAAGCACCAGCATCTAA
ATGGTTCAACCTCTTCACAAAGCAACTTCAACGCATTCCAATTATTGTTGATGGAG
TAGAAAAAGTTACTTTGCTCCAAGGGGATTGGCATACCATAGGTTCAGTTAAACAA
TGGTCTGAATTAGTAGATGGTAAAGTAGCAACATTTAAGGAGAAAATTGAAGCCAT
TGATGAAAAGAACAAGTGGATCAGATACAACATATTTGATGGTGAAATGAATCAAC
ATTACAAAGTTTACATTCTCACCATTCAAGTGATAGAAAAAGATGATGGAAGTGCTT
CTATTAAATGGACTATTGAATATGAGAAGGTTAATGAGAGTCTTGAACCTCCATAT
CATTACATGGATTCTATAACTAAGGGTTGTAAGGATATTGATGCTGAACTTCTAAA
GAACTAG
SEQ ID NO 22: Nucleotide sequence of the enzyme S-norcoclaurine synthase-like 2 (NCS2) from narrow-leafed lupin cultivar Oskar:
ATGGCTTTGAAAGGTATATTGAGTTCTGAAGTTGGGGTTCATGCATCTGCTTCAAA
GTGGTTCAATCTCTTTGCTAAGGAACTTCACAATGTTCAAAATACAACTGATAGAG
TGCACAAAACTAAGTTGCTTGAAGGTGATGATTGGCATAGCATTGGTTCAGTTAAG
CAATGGACCGACATCGTAGATGGAAAGGAATCACATTATAAGGAGAGACTTGATG
CTATTGATGAAGAGAACAAGACAATTGTATACACACTCTTTGATGGAGATTTCAGT
AAGGATTACAATGTCTTCAAGCTTCTTTTTCAAGTAATTGAAAAGAATAATGGTGCA
TTTATTAAATGGACTATTGAATATGAGAAAGTGAATGAGAAAGTTGAACCACCATAT
GGCTTTATGGATCACTTCACTAAGAGTACTAAAGAAATTGATGTTTTTCTTCTTAAG
GCATAG
SEQ ID NO 23:
Nucleotide sequence of the enzyme S-norcoclaurine synthase-like 3 (NCS3) from narrow-leafed lupin cultivar Oskar:
ATGCCTATCCGTGGAAAGCTTGAGGGAGGTTTTGAGGCCAAGTCTAGTGCAGACA
AGTTCTGGGGAGCTCTTAGGAACTGGTACACATTTTTTCCTGAAGCCTTCCCCAGT
GTTTACAAAGCCGTTGAGGTTGTCGAGGGTGATGGAAAGGCTGTTGGTTCTGTCT
TTCGTGTATCAATTAGTGAAGATTCGCCATTTGCAAAATCAATTAGAGAGAAGATT
GAAGCTGTTGATGATGTAAAGAGGACATTAATCTTGGACGTGGCTGGGATTGATG
GGAACGTCTTCCATATCTACAAAAAATATGTGTTACACGTTAGTGTAACACCTAAG
GGAGATGGAAGCGTGGTGAAAGTCGCAGTTGAGTATGAAAATCCAACCGTAAAAG
ATCCTGAGCCAACTGAGTTCATAGATGTAGAAGTACAGGGCTTCCAAGATTTGGAT
GCCTATCTTCAAAACAAATAG
SEQ ID NO 24:
Nucleotide sequence of the enzyme S-norcoclaurine synthase-like 4 (NCS4) from narrow-leafed lupin cultivar Oskar:
ATGGCAACCCTTGAAAAGATAGAGGCCATCATTGAGATTAAGACTAATGCAGACAA
GTACTGGAGAACCTTTAGGGATTATCGAACTGTATTCCCAAAGGTATTTTCAAAAT
ATAGAAGTATTGAGATTCTTGAAGGTGATGGCAAATCAGTTGGTTCTGTCCTTCGC
CACATTACTTTTGAAGGTTCATTAAAATCAGCCACAGAGAAGATTGAGGCTGTTGA
TGATGAAAAGAGGACACTTACCTATGCTGTAATTGATGCTGATATCCTCCAAGACT
ATAAAAATTACAAGGGACACATTAGTGTAACACCTAAGGGAAATGGAAGTGAGGT GAAATGGATTGCTGAGTATGAAAAAGCTTCTCAAGAAGTTCCTGATCCTATTAGCA TCAAAGATTATTTAGTTGATACCTTCCTTAAATTGGATGCCTATATTCAAAAGGCAT AG References
Luo, D. et al. Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L. Proc. Natl. Acad. Sci. II. S. A. 113, E5082-E5089 (2016).
Items
1. A host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S- norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11, S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
2. The host cell according to item 1, wherein the host cell further comprises one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3 or a functional homologue of any one of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
3. The host cell according to any one of the preceding items, wherein the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as 12 of the following genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
4. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof, wherein said functional homologue has lysine decarboxylase activity.
5. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme CAO of SEQ ID NO 2 or a functional homologue thereof, wherein said functional homologue has amine oxidase activity.
6. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme AT of SEQ ID NO 3 or a functional homologue thereof, wherein said functional homologue is capable of catalysing /V-acetylation of tetrahydroanabasine. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme G8HO of SEQ ID NO 4 or a functional homologue thereof, wherein said functional homologue has hydroxylase activity against /V-acetylated tetrahydroanabasine. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme CHYD of SEQ ID NO 5 or a functional homologue thereof, wherein said functional homologue has deacetylase activity against a didehydrolusitanine species in the presence of NCS1 or functional homologues thereof. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme VRED1 of SEQ ID NO 6 or a functional homologue thereof, wherein said functional homologue is capable of catalysing reduction of a tetradehydrosparteine species. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme VRED2 of SEQ ID NO 7 or a functional homologue thereof, wherein the functional homologue is capable of catalysing reduction of di-iminium cations. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme ARED of SEQ ID NO 8 or a functional homologue thereof, wherein the functional homologue is annotated as an oxidoreductase. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme NCS1 of SEQ ID NO 9 or a functional homologue thereof, wherein the functional homologue is capable of catalysing the addition of a A1-piperideine unit to didehydrolusitanine species in the presence of CHYD or functional homologues thereof. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding the enzyme NCS2 of SEQ ID NO 10 or a functional homologue thereof, wherein the functional homologue is capable of catalysing dimerization of A1-piperideine to tetrahydroanabasine. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding NCS3 of SEQ ID NO 11 or a functional homologue enzyme, wherein the functional homologue is capable of aiding the reduction of a tetradehydrosparteine species in the presence of VRED1 or VRED2 or functional homologues thereof. The host cell according to any one of the preceding items, wherein the host cell comprises a heterologous gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof, wherein the functional homologue is capable of catalysing the equilibration between a 2-hydroxyammodendrine species and a didehydrolusitanine species. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
20. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
24. The host cell according to any one of the preceding items, wherein the host cell is selected from the group of plant cells, yeast cells, bacteria cells and fungal cells.
25. The host cell according to any one of the preceding items, wherein the host cell is plant cells comprised within a plant, within a part of a plant or within the seeds of said plant.
26. The host cell according to any one of the preceding items, wherein the plant cell is from a species of Nicotiana such as Nicotiana benthamiana orNicotiana tabacum.
27. The host cell according to any one of the preceding items, wherein the host cell is a yeast cell of the species Saccharomyces cerevisiae or Saccharomyces pombe.
28. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof.
29. The host cell according to any one of the preceding items, wherein the host cell is capable of producing tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto. 30. The host cell according to any one of the preceding items, wherein the cell is capable of producing /V-acetylated tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
31. The host cell according to any one of the preceding items, wherein the cell is capable of producing a didehydrolusitanine species and/or a 2- hydroxyammodendrine species, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto; and e. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and f. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
32. The recombinant host cell according to any one of the preceding items, wherein the cell is capable of producing a tetradehydrosparteine species, including hydrated (carbinolamine), fully hydrolysed (aminoaldehyde), iminium, or enamine forms of tetradehydrosparteine, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto; and h. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and i. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and i. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and k. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and k. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto. The host cell according to any one of the preceding items, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
38. A polypeptide selected from the group of polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ I D NO 11 , SEQ I D NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
39. A nucleic acid encoding any of the polypeptides according to item 38.
40. A method of producing (-)-sparteine or a precursor thereof, comprising the steps of: a. Providing a host cell according to any of items 1 to 37; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating (-)-sparteine or a precursor thereof.
41. The method according to item 40, wherein the method comprises the step of isolating (-)-sparteine or the precursor, wherein said step comprises extraction and/or chromatography.
42. The method according to any one of items 40 to 41 , wherein the method of isolating (-)-sparteine comprises a step of extraction, for example extraction with a solvent comprising an alcohol, acid-base extraction and/or supercritical fluid extraction. The method according to any one of items 40 to 42, wherein the method of isolating (-)-sparteine, comprises a step of chromatography, for example cation exchange chromatography, adsorption chromatography using a polymeric resin or HPLC. The method according to any one of items 40 to 43, wherein the method is a method of producing tetrahydroanabasine, and wherein the host cell is the host cell according to item 29. The method according to any one of items 40 to 43, wherein the method is a method of producing /V-acetylated tetrahydroanabasine, and wherein the host cell is the host cell according to item 30. The method according to any one of items 40 to 43, wherein the method is a method of producing a didehydrolusitanine species, and wherein the host cell is the host cell according to item 31. The method according to any one of items 40 to 43, wherein the method is a method of producing a tetradehydrosparteine species, and wherein the host cell is the host cell according to item 32. The method according to any one of items 40 to 43, wherein the method is a method of producing (-)-sparteine, and wherein the host cell is the host cell according to any one of items 33 to37.

Claims

Claims
1. A host cell comprising one or more heterologous gene(s) encoding an enzyme selected from the group of geraniol-8-hydroxylase (G8HO) of SEQ ID NO 4, CHYD of SEQ ID NO 5, VRED1 of SEQ ID NO 6, VRED2 of SEQ ID NO 7, ARED of SEQ ID NO 8, S-norcoclaurine synthase-like protein 1 (NCS1) of SEQ ID NO 9, S-norcoclaurine synthase-like protein 2 (NCS2) of SEQ ID NO 10, S- norcoclaurine synthase-like protein 3 (NCS3) of SEQ ID NO 11, S-norcoclaurine synthase-like protein 4 (NCS4) of SEQ ID NO 12, or a functional homologue of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
2. The host cell according to claim 1 , wherein the host cell further comprises one or more heterologous gene(s) encoding an enzyme selected from the group of lysine decarboxylase (LDC) of SEQ ID NO 1, copper amine oxidase (CAO) of SEQ ID NO 2, BADH acetyltransferase (AT) of SEQ ID NO 3 or a functional homologue of any one of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
3. The host cell according to any one of the preceding claims, wherein the host cell comprises at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11 , such as 12 of the following genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto. k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
4. The host cell according to any one of the preceding claims, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
5. The host cell according to any one of the preceding claims, wherein the host cell comprises at least the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto, or a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
6. The host cell according to any one of the preceding claims, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
7. The host cell according to any one of the preceding claims, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
8. The host cell according to any one of the preceding claims, wherein the host cell comprises at least the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto.
9. The host cell according to any one of the preceding claims, wherein the host cell is selected from the group of plant cells, yeast cells, bacteria cells and fungal cells.
10. The host cell according to any one of the preceding claims, wherein the host cell is a plant cell from a species of Nicotiana such as Nicotiana benthamiana or Nicotiana tabacum.
11. The host cell according to any one of the preceding claims, wherein the host cell is a yeast cell of the species Saccharomyces cerevisiae or Saccharomyces pombe.
12. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof.
13. The host cell according to any one of the preceding claims, wherein the host cell is capable of producing tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding NCS2 of SEQ ID NO 10, or a functional homologue having at least 70% sequence identity thereto.
14. The host cell according to any one of the preceding claims, wherein the cell is capable of producing /V-acetylated tetrahydroanabasine or a precursor thereof, wherein the host cell comprises the following heterologous gene(s): a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto.
15. The host cell according to any one of the preceding claims, wherein the cell is capable of producing a didehydrolusitanine species and/or a 2- hydroxyammodendrine species, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto; and e. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and f. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
16. The recombinant host cell according to any one of the preceding claims, wherein the cell is capable of producing a tetradehydrosparteine species, including hydrated (carbinolamine), fully hydrolysed (aminoaldehyde), iminium, or enamine forms of tetradehydrosparteine, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue having at least 70% sequence identity thereto; and h. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
17. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and i. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
18. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and i. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
19. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and k. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
20. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and h. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and j. optionally a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue having at least 70% sequence identity thereto; and k. optionally a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue having at least 70% sequence identity thereto.
21. The host cell according to any one of the preceding claims, wherein the cell is capable of producing (-)-sparteine or a precursor thereof, wherein the host cell comprises the following heterologous genes: a. a gene encoding LDC of SEQ ID NO 1 or a functional homologue thereof having at least 70% sequence identity thereto; and b. a gene encoding CAO of SEQ ID NO 2 or a functional homologue thereof having at least 70% sequence identity thereto; and c. a gene encoding AT of SEQ ID NO 3 or a functional homologue thereof having at least 70% sequence identity thereto; and d. a gene encoding G8HO of SEQ ID NO 4 or a functional homologue thereof having at least 70% sequence identity thereto; and e. a gene encoding CHYD of SEQ ID NO 5 or a functional homologue thereof having at least 70% sequence identity thereto; and f. a gene encoding VRED1 of SEQ ID NO 6 or a functional homologue thereof having at least 70% sequence identity thereto; and g. a gene encoding VRED2 of SEQ ID NO 7 or a functional homologue thereof having at least 70% sequence identity thereto, h. a gene encoding ARED of SEQ ID NO 8 or a functional homologue thereof having at least 70% sequence identity thereto; and i. a gene encoding NCS1 of SEQ ID NO 9 or a functional homologue thereof having at least 70% sequence identity thereto; and j. a gene encoding NCS2 of SEQ ID NO 10 or a functional homologue thereof having at least 70% sequence identity thereto; and k. a gene encoding NCS3 of SEQ ID NO 11 or a functional homologue thereof having at least 70% sequence identity thereto; and l. a gene encoding NCS4 of SEQ ID NO 12 or a functional homologue thereof having at least 70% sequence identity thereto.
22. A polypeptide selected from the group of polypeptides of SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ I D NO 11 , SEQ I D NO 12, and functional homologues of any of the aforementioned having at least 70% sequence identity, preferably at least 80%, preferably at least 85% sequence identity, preferably at least 90% sequence identity, preferably at least 95% sequence identity, more preferred at least 98% sequence identity thereto.
23. A nucleic acid encoding any of the polypeptides according to claim 22.
24. A method of producing (-)-sparteine or a precursor thereof, comprising the steps of: a. Providing a host cell according to any of claims 1 to 21 ; b. Cultivating the host cell under conditions enabling growth; c. Optionally isolating (-)-sparteine or a precursor thereof.
25. The method according to claim 24, wherein: i. the method is a method of producing tetrahydroanabasine, and wherein the host cell is the host cell according to claim 13; ii. the method is a method of producing /V-acetylated tetrahydroanabasine, and wherein the host cell is the host cell according to claim 14; iii. the method is a method of producing a didehydrolusitanine species, and wherein the host cell is the host cell according to claim 15; iv. the method is a method of producing a tetradehydrosparteine species, and wherein the host cell is the host cell according to claim 16; or v. the method is a method of producing (-)-sparteine, and wherein the host cell is the host cell according to any one of claims 17 to 21.
PCT/EP2023/087642 2022-12-22 2023-12-22 Production of plant alkaloids WO2024133899A2 (en)

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