Methods for production of strictosidine aglycone and monoterpenoid indole alkaloids

TECHNICAL FIELD

The present invention relates to microbial factories, such as microorganism factories in particular yeast factories and bacterial factories, for production of strictosidine aglycone and optionally other plant-derived compounds. Also provided are methods for producing strictosidine aglycone in a microorganism, as well as useful nucleic acids, vectors and host cells.

BACKGROUND

Plants produce some of the most potent human therapeutics and have been used for millennia to treat illnesses. Despite the large repertoire of plant-derived pharmaceuticals, most of these products do not make it to the market because they are found in minute quantities in plants, they are difficult to extract, and there is limited knowledge about their biosynthetic pathways.

Furthermore, sourcing plant-derived pharmaceuticals based on plant-based extraction threatens to cause species extinction. New regulatory laws seek to create conditions to promote biodiversity conservation and sustainable use of genetic resources, which in the short term are expected to further affect the supply chains of many valuable plant natural products.

Moreover, many plant species are not readily genetically manipulated, and synthetic chemistry holds little promise for bulk production of complex plant-derived therapeutics. Together, supporting a need for refactored biosynthesis of new and existing pharmaceuticals, in genetically tractable and sustainable production hosts.

The monoterpenoid indole alkaloids (MIAs) are plant secondary metabolites that show a remarkable structural diversity and pharmaceutically valuable biological activities, such as anti-cancer and anti-psychosis properties. The productions of these alkaloids occurs through highly complicated pathways.

The common precursors for the different MIAs are strictosidine, and its deglycosylated form, strictosidine aglycone. Strictosidine is formed by the coupling of secologanin to tryptamine in a reaction catalysed by the enzyme strictosidine synthase. Strictosidine alglycone is natively produced from hydrolyzing strictosidine by strictosidine-beta-glucosidase (SGD). Over 2,000 MIAs can be produced from strictosidine aglycone.

To enable a sustainable supply of therapeutic MIAs, researchers have for decades attempted to elucidate the biosynthetic pathways from MIA producing plants, including both the platform biosynthetic route to the common MIA precursor strictosidine and the anti-cancer drug vinblastine. Moreover, the platform biosynthetic route from geraniol to strictosidine, and the seven-step biosynthetic pathway from tabersonine to vindoline, the immediate precursor of vinblastine has also been refactored in yeast cell factories.

Current methods for production of strictosidine aglycone are mostly based on chemical synthesis or plant extraction. Such methods are not cost-effective and also have a significant impact on the environment. Therefore, methods for cost-effective and environmental-friendly production of strictosidine aglycone are required.

SUMMARY

The invention concerns a microorganism capable of producing strictosidine aglycone and methods for strictosidine aglycone and monoterpenoid indole alkaloids (MIAs) production in a microorganism.

In one aspect is provided a microorganism capable of producing strictosidine aglycone, said microorganism expresses

- a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone,

wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,

and/or;

wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

Also provided herein are methods for producing strictosidine aglycone in a microorganism, comprising the steps of:

- a) providing a microorganism, said cell expressing:
  - a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone;
- b) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- c) optionally, recovering the strictosidine aglycone;
- d) optionally, further converting the strictosidine aglycone to monoterpenoid indole alkaloids,

and/or;

wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

Also provided herein are nucleic acid constructs comprising a sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 and/or SEQ ID NO:107.

Also provided are vectors comprising the above nucleic acids, as well as host cells comprising said vectors and/or said nucleic acids.

Also provided is a kit of parts comprising a microorganism as described herein, and/or nucleic acid constructs as described herein, and/or a vector as described herein, and instructions for use.

Also provided is the use of above nucleic acids, vectors or host cells for the production of strictosidine aglycone.

Also provided herein are methods for producing monoterpenoid indole alkaloids (MIAs) in a microorganism, said method comprising the steps of:

- a) providing a microorganism capable of converting strictosidine aglycone to tabersonine and/or catharanthine, said cell expressing:
  - optionally, a strictosidine synthase (STR);
  - a strictosidine-beta-glucosidase (SGD);
  - a NADPH-cytochrome P450 reductase (CPR);
  - a Cytochrome b5 (CYB5);
  - a Geissoschizine synthase (GS);
  - a Geissoschizine oxidase (GO);
  - a Redox1;
  - a Redox2;
  - a Stemmadenine O-acetyltransferase (SAT);
  - a O-acetylstemmadenine oxidase (PAS);
  - a Dehydroprecondylocarpine acetate synthase (DPAS);
  - a Tabersonine synthase (TS); and/or
  - a Catharanthine synthase (CS);
- b) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- c) optionally, recovering the MIAs;
- d) optionally, processing the MIAs into a pharmaceutical compound, wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,

and/or;

wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

Also provided herein are strictosidine aglycone, tetrahydroalstonine, heteroyohimbine, rabersonine and/or catharanthine obtained by the method as described herein.

Also provided herein are methods for treating a disorder such as a cancer, arrhythmia, malaria, psychotic diseases, hypertension, depression, Alzheimer's disease, addiction and/or neuronal diseases, comprising administration of a therapeutic sufficient amount of an MIA or a pharmaceutical compound obtained by the as described herein.

DESCRIPTION OF DRAWINGS

FIG. 1: High-resolution analytical results of tetrahydroalstonine (THA) obtained from LC-MS analysis of yeast cells (Saccharomyces cerevisiae) expressing SGD derived from Catharanthus roseus (CroSGD) alone and in various tagged and CroSGD-fusion versions, as well as SGD from Rauvolfia serpentina (RseSGD).

FIG. 2: Sequence identity among SGD derived from Catharanthus roseus (CroSGD), Rauvolfia serpentina (RseSGD), Rauvolfia verticillata (RveSGD), Gelsemium sempervirens (GseSGD), Camptotheca acuminate (CacSGD), Scedosporium apiospermum (SapSGD), Uncaria tomentosa (UtoSGD) and Glycine soja (GsoSGD). The eight protein sequences were aligned with the t-Coffee web server.

FIG. 3: Biosynthesis of the heteroyohimbine tetrahydroalstonine measured on LC-MS. The production of tetrahydroalstonine (THA) was measured in yeast strains expressing either GsoSGD, CacSGD, CroSGD, UtoSGD, GseSGD, SapSGD, RveSGD or RseSGD The yeast strain GsoSGD was used as a negative control. The p-value represents comparison between the negative control (GsoSGD) and CacSGD, CroSGD or UtoSGD, respectively.

FIG. 4: GFP-tagged CroSGD and RseSGD localization in yeast. A) A yeast cell expressing GFP-CroSGD. B) A yeast cell expressing GFP-RseSGD. The arrows mark the localization of SGD in the yeast cells.

FIG. 5: The biosynthesis of the heteroyohimbine alstonine in yeast cell factories, expressing RseSGD, CroTHAS and GseSBE, is shown in triplicates in FIG. 5. Alastonine was measured by Orbitrap Fusion™ Tribrid™ MS.

FIG. 6: The yeast strain MIA-DC was feed with 0.1 mM of secologanine and 1 mM of tryptamine and the production of tabersonine and catharanthine were measured by LC-MS. A) Catharanthine production, B) Tabersonine production, C) Catharanthine standard, and D) Tabersonine standard.

FIG. 7: The yeast strain MIA-DC was feed with 0.1 mM of secologanine and 1 mM of tryptamine and the concentration levels of tabersonine and catharanthine in MIA-DC and MIA-DA (control) were measured by LC-MS.

FIG. 8: Biosynthesis of the heteroyohimbine tetrahydroalstonine measured on LC-MS. The production of tetrahydroalstonine (THA) was measured in yeast strains expressing either CroSGD, VmiSGD1, AhuSGD, HimSGD2, SinSGD, TelSGD, VunSGD, NsiSGD1, LprSGD, AchSGD1, HsuSGD, MroSGD, RseSGD2, PgrSGD, OpuSGD, HpiSGD, HanSGD1, AchSGD2, HimSGD1, IpeSGD, LsaSGD1, CarSGD, OeuSGD, AchSGD3, CmaSGD, MmySGD, VmiSGD3, IniSGD, or NsiSGD2. The p-value represents a comparison between the negative control (CroSGD) and OeuSGD, AchSGD3, CmaSGD, MmySGD, VmiSGD3, IniSGD, and NsiSGD2.

FIG. 9: Biosynthesis of the heteroyohimbine tetrahydroalstonine measured on LC-MS. The production of tetrahydroalstonine (THA) was measured in yeast strains expressing one of the mosaic SGDs: RRCC-SGD, RCCC-SGD, CCCC-SGD, CRCC-SGD, CRCR-SGD, RRCR-SGD, CCCR-SGD, RCCR-SGD, CRRC-SGD, RRRC-SGD, RCRC-SGD, CCRC-RGD, RCRR-SGD, CRRR-SGD, RRRR-SGD, and CCRR-SGD.

CCCC-SGD and RRRR-SGD are identical to the two wild type sequences CroSGD and RseSGD. The p-value represents comparisons between the negative control (CCCC-SGD/CroSGD) and all SGDs containing CroSGD domain 3: RRCC-SGD, RCCC-SGD, CRCC-SGD, CRCR-SGD, RRCR-SGD, CCCR-SGD and RCCR-SGD. The color indicates the identity of domain 3 and 4: Light grey—RseSGD domain 3 & 4, medium grey—RseSGD domain 3 & CroSGD domain 4, dark grey—CroSGD domain 3 & CroSGD/RseSGD domain 4.

FIG. 10: Biosynthesis of the heteroyohimbine tetrahydroalstonine measured on LC-MS. The production of tetrahydroalstonine (THA) was measured in yeast strains expressing one of the wild type SGDs (UtoSGD, GseSGD, CroSGD, or RveSGD) or one of the engineered SGDs (UURR-SGD, GGRR-SGD, CCRR-SGD, or VVRR-SGD).

FIG. 11: Biosynthesis of the common MIA precursor strictosidine (A) and heteroyohimbine tetrahydroalstonine (B) in E. coli measures by LC-MS. The production of strictosidine and tetrahydroalstonine were measures in bacterial strains expressing either CroSGD or RseSGD. A strain with an empty expression vector was included as a negative control.

FIG. 12: Multiple sequence alignment of SGDs proteins derived from Catharanthus roseus (CroSGD), Rauvolfia serpentina (RseSGD and RseSGD2), Rauvolfia verticillata (RveSGD), Gelsemium sempervirens (GseSGD), Camptotheca acuminate (CacSGD), Scedosporium apiospermum (SapSGD), Uncaria tomentosa (UtoSGD), Glycine soja (GsoSGD), Vinca minor (VmiSGD1 and VmiSGD3), Tabernaemontana elegans (TeISGD), Amsonia hubrichtii (AhuSGD), Ophiorrhiza pumila, (OpuSGD), Nyssa sinensis, (NsiSGD1 and NsiSGD2), Coffea arabica (CarSGD), Carapichea ipecacuanha (IpeSGD), Handroanthus impetiginosus (HimSGD2 and HimSGD1), Sesamum indicum (SinSGD), Olea europaea (OeuSGD), Actinidia chinensis var. chinensis (AchSGD1, AchSGD2 and AchSGD3), Helianthus annuus (HanSGD), Lactuca sativa (LseSGD), Ipomoea nil (IniSGD), Chelidonium majus (CmaSGD), Vigna unguiculata (VunSGD), Heliocybe sulcate (HsuSGD), Pyricularia grisea (PgrSGD), Lomentospora prolificans (LprSGD), Hydnomerulius pinastri MD-312 (HpiSGD), Madurella mycetomatis (MmySGD), and Moniliophthora roreri MCA 2997 (MroSGD). The protein sequences were aligned with the t-Coffee web server.

FIG. 13: Pairwise sequence identities among the 36 SGD protein sequences aligned in FIG. 8. The pairwise sequence identities were calculated from the alignment with CLC Main Workbench 8.

DETAILED DESCRIPTION

The present disclosure relates to microorganisms and method for production of strictosidine aglycone and monoterpenoid indole alkaloids (MIA). The microorganism may be any non-natural or natural microorganism. By non-natural is meant an engineered microorganism, which comprises one or more genes which are not native to the microorganism. In some aspects of the present invention the microorganism expresses a heterologous SGD, mosaic SGD or variants thereof.

Microorganisms are microscopic organisms that exist as unicellular, multicellular, or cell clusters. Microorganism may be divided into different types such as bacteria, archaea, yeasts, fungi, protozoa, algae, and viruses. Thus, in one embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, protozoa, algae, and viruses. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, protozoa and algae. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, and algae. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea yeasts and fungi. In another embodiment, the microorganism is selected from bacteria, yeasts and fungi. In another embodiment, the microorganism is selected from bacteria or yeasts. In a preferred embodiment, the microorganism is a bacteria or a yeast.

In some embodiments, the microorganism is a bacteria. In one embodiment, the genus of said bacteria is selected from Escherichia, Corynebacterium, Pseudomonas, Bacillus, Lactococcus, Lactobacillus, Halomonas, Bifidobacterium and Enterococcus. In preferred embodiments, the genus of said bacteria is Escherichia. In another embodiment, the microorganism may be selected from the group consisting of Escherichia, Corynebacterium glutamicum, Pseudomonas putida, Bacillus subtilis, Lactococcus bacillus, Halomonas elongate, Bifidobacterium infantis and Enterococcus faecali. In preferred embodiments, the micororganims is an Escherichia. In some embodiments the bacteria is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, Bacillus subtilis, Lactococcus bacillus, Halomonas elongate, Bifidobacterium infantis and Enterococcus faecal

In some embodiments, the microorganism is a yeast. In some embodiments, the microorganism is a cell from a GRAS (Generally Recognized As Safe) organism or a non-pathogenic organism or strain. In some embodiments, the genus of said yeast is selected from Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. In preferred embodiments, the genus of said yeast is Saccharomyces.

The microorganism may be selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica. In preferred embodiments, the microorganism is a Saccharomyces cerevisiae cell.

Microorganism

Herein is thus provided a microorganism capable of producing strictosidine aglycone, said microorganism expresses

- a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone,
- wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,
- and/or;
- wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

- wherein D₁is a first amino acid sequence from a first SGD,
- wherein D₂is a second amino acid sequence from a second SGD,
- wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,
- wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

The microorganismsdisclosed herein are thus all capable of converting strictosidine to strictosidine aglycone, when strictosidine is provided to the microorganism. In some embodiments, strictosidine is provided to the microorganism, for example by feeding strictosidine to the microorganism in the medium. In other embodiments, the microorganism is capable of synthesising strictosidine, for example the microorganism is further engineered as described below.

In another embodiment said microorganism further expresses a strictosidine synthase (STR), capable of converting secologanin and tryptamine to strictosidine. Thus, microorganisms further expressing STR are capable of converting secologanin and tryptamine to strictosidine aglycone, when secologanin and tryptamine are provided to the microorganism. Secologanin and tryptamine may be provided e.g. in the medium. However, in some embodiments the microorganism is capable of synthesising secologanin and/or tryptamine, for example the microorganismis further engineered to synthesis secologanin and/or tryptamine.

Strictosidine-O-beta-D-glucosidase (SGD)

The first heterologous enzyme expressed in the microorganism is capable of converting strictosidine to strictosidine aglycone. The first heterologous enzyme is not natively expressed in the microorganism. It may be derived from a eukaryote or a prokaryote, as detailed below, preferably a eukaryotic cell such as a plant cell.

In some embodiments, the first heterologous enzyme is a strictosidine-O-beta-D-glucosidase, herein also termed SGD, and having an EC number EC 3.2.1.105. This enzyme catalyses the following reaction:

Strictosidine+H₂O<=>D-glucose+strictosidine aglycone.

Heterologous SGD or Variants Thereof

Thus the microorganism expressing the first heterologous enzyme is capable of converting strictosidine to strictosidine aglycone by the action of the first heterologous enzyme.

The conversion of strictosidine to strictosidine aglycone, may be measured directly by the amount of strictosidine aglycone as known in the art, or surrogate measure of the conversion of strictosidine to strictosidine aglycone may be measured as known in the art. Because strictosidine aglycone is highgly reactive, indirect determination of strictosidine aglycone may be preferred. For example, colorimetric assays to follow strictosidine consumption as described in Geerlings et al., 2000, may be used. The disappearance of strictosidine may also be monitored by UV, as described in Guirimand et al., 2010, or the general p-glucosidase activity in the cells may be measured, e.g. by UV detection of a synthetic substrate such as 4-methylumbelliferyl-β-D-glucoside (Guirimand et al., 2010).

Thus, to determine whether a SGD is capable of converting strictosidine to strictosidine aglycone, the person skilled in the art could use any of said methods, or could use high-precision mass spectrometry to detect the accurate mass of strictosidine aglycone after cultivation of a strain expressing an SGD or an enzyme suspected of having SGD activity in a medium; the cell is either provided with strictosidine in the medium or it has been engineered and can synthesise strictosidine. The strictosidine aglycone can be detected directly in the medium or in a pellet, after centrifugation of the culture broth. Alternatively, the appearance of other products, downstream of strictosidine aglycone, for example tetrahydroalstonine, can be monitored; such products will only form in the presence of a functional SGD, strictosidine, and an enzyme capable of using strictosidine aglycone, as described in e.g. Stavrinides et al., 2015.

In some embodiments, the first heterologous enzyme is an SGD which is native to Rauvolfia serpentina, Gelsemium sempervirens, Scedosporium apiospermum or Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 2997 or a functional variant thereof.

In other words, in some embodiments the SGD is derived from Rauvolfia serpentina, Gelsemium sempervirens, Scedosporium apiospermum, Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 2997 or a functional variant thereof. Functional variants of SGD are modified enzymes which retain the capability to convert strictosidine to strictosidine aglycone. In some embodiments, the SGD is RseSGD as set forth in SEQ ID NO: 24 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24. In other embodiments, the SGD is GseSGD as set forth in SEQ ID NO: 25 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 25. In other embodiments, the SGD is SapSGD as set forth in SEQ ID NO: 26 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 26. In other embodiments, the SGD is RveSGD as set forth in SEQ ID NO: 27 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 27. In other embodiments, the SGD is VmiSGD1 as set forth in SEQ ID NO: 47 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 47. In other embodiments, the SGD is AhuSGD as set forth in SEQ ID NO: 48 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 48. In other embodiments, the SGD is HimSGD2 as set forth in SEQ ID NO: 49 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 49. In other embodiments, the SGD is SinSGD as set forth in SEQ ID NO: 50 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 50. In other embodiments, the SGD is TelSGD as set forth in SEQ ID NO: 51 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 51. In other embodiments, the SGD is VunSGD as set forth in SEQ ID NO: 52 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 52. In other embodiments, the SGD is NsiSGD1 as set forth in SEQ ID NO: 53 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 53. In other embodiments, the SGD is LprSGD as set forth in SEQ ID NO: 54 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 54. In other embodiments, the SGD is AchSGD1 as set forth in SEQ ID NO: 55 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 55. In other embodiments, the SGD is HsuSGD as set forth in SEQ ID NO: 56 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 56. In other embodiments, the SGD is MroSGD as set forth in SEQ ID NO: 57 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 57. In other embodiments, the SGD is RseSGD2 as set forth in SEQ ID NO: 58 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 58. In other embodiments, the SGD is PgrSGD as set forth in SEQ ID NO: 59 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 59. In other embodiments, the SGD is OpuSGD as set forth in SEQ ID NO: 60 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 60. In other embodiments, the SGD is HpiSGD as set forth in SEQ ID NO: 61 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 61. In other embodiments, the SGD is HanSGD1 as set forth in SEQ ID NO: 62 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 62. In other embodiments, the SGD is AchSGD2 as set forth in SEQ ID NO: 63 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 63. In other embodiments, the SGD is HimSGD as set forth in SEQ ID NO: 64 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 64. In other embodiments, the SGD is IpeSGD as set forth in SEQ ID NO: 65 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 65. In other embodiments, the SGD is LsaSGD as set forth in SEQ ID NO: 66 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 66. In other embodiments, the SGD is CarSGD as set forth in SEQ ID NO: 67 or a functional variant thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 67.

Preferably, the SGD is RseSGD or a functional variant thereof.

In some embodiments, the SGD originates from a MIA producing plant species, wherein said SGD shares at least 65% sequence identity to RseSGD. Thus, in some embodiments, the SGD is selected from the group consisting of RseSGD, RveSGD, TelSGD, or VmiSGD or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 51 or SEQ ID NO: 47.

In some embodiments, the SGD originates from a MIA producing plant species, wherein said SGD shares at the most 65% sequence identity to RseSGD. Thus, in some embodiments, the SGD is selected from the group consisting of GseSGD, NsiSGD, OpuSGD, AhuSGD, or RseSGD2 or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 25, SEQ ID NO: 53 SEQ ID NO: 60, SEQ ID NO: 48 or SEQ ID NO: 58.

A person skilled in the art would know how to determine sequence identity between two species by using known methods in the art.

In some embodiments, the SGD originates from a non-MIA producing plant species. Thus, in some embodiments, the SGD is selected from the group consisting of AchSGD1, AchSGD2, CarSGD, HanSGD, HimSGD1, HimSGD2, LsaSGD1, SinSGD, VunSGD or IpeSGD or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 55, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 49, SEQ ID NO: 66, SEQ ID NO: 50, SEQ ID NO: 52 or SEQ ID NO: 65.

In some embodiments, the SGD originates from a non-MIA producing fungi species. Thus, in some embodiments, the SGD is selected from the group consisting of HpiSGD, HsuSGD, LprSGD, MroSGD, PgrSGD, or SapSGD or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 61, SEQ ID NO: 56, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 59 or SEQ ID NO: 26.

In other embodiments, said microorganism, such as the yeast cell or the bacteria cell, is capable of producing at least 1 μM tetrahydroalstonine. Thus, in some embodiments, the SGD is selected from the group consisting of RseSGD, VmiSGD or AhuSGD, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24, SEQ ID NO: 47 or SEQ ID NO: 48.

In other embodiments the SGD is selected from the group consisting of RseSGD, GseSGD, SapSGD or RveSGD, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID NO: 27.

In other embodiments the SGD is selected from the group consisting of RseSGD, GseSGD, SapSG, RveSGD, VmiSGD, AhuSGD or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 47 or SEQ ID NO: 48.

In other embodiments the SGD is selected from the group consisting of RseSGD, RveSGD, VmiSGD, AhuSGD, HimSGD, SinSGD or TelSGD, or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), or LsaSGD1 (SEQ ID NO: 66), or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from RseSGD (SEQ ID NO: 24), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

In some embodiments, said SGD is selected from GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

Thus, in some embodiments the microorganism according to the present invention may express a SGD as described herein above. In other embodiments, the microorganism according to the present invention may express a mosaic SGD. The microorganism may be a yeast cell or a bacteria cell, as described herein.

Mosaic SGD or Variants Thereof

The inventors have engineered new and active mosaic SGDs capable of converting strictosidine into strictosidine aglycone. Said mosaic SGDs are useful in microorganism factories, such as yeast factories and bacteria factories, for production of strictosidine aglycone, tetrahydroalstonine and/or other MIA products.

Thus, the present invention also relates to a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

The mosaic SGD thus comprises at least one domain of RseSGD, namely the third domain D₃, and at least one other domain as defined above which is not a domain of RseSGD.

The inventors found that a SGD can be divided into four domains:

- Domain 1 (D₁)
- Domain 2 (D₂)
- Domain 3 (D₃)
- Domain 4 (D₄)

Examples hereof are described in Examples 8 and 9 herein below.

Each of domain 1-4 consists of a consecutive sequence of amino acids. Domain 1 is the most N-terminal amino acid sequence in the SGD. The first amino acid residue in domain 1 is typically methionine, as this is the first amino acid which is translated from a start codon, however it may occur that the first domain actually starts with another residue in embodiments where part of the domain would be cleaved off, thereby removing the methionine. Being the first domain in SGD, domain 1 is followed by domain 2, which is followed by domain 3, which is followed by domain 4. Domain 4 is the most C-terminal amino acid sequence in the SGD. The last amino acid residue in domain 4 is the last amino acid residue in the consecutive sequence of the SGD.

The positions of the amino acids in each domain 1-4 of a SGD may be defined by aligning the SGD amino acid sequence to the amino acid sequence RseSGD of SEQ ID NO:24, hereby using RseSGD as a reference sequence. Thus, is it to be understood that following alignment between a SGD amino acid sequence and the reference amino acid sequence of SEQ ID NO:24, an amino acid corresponds to position X of SEQ ID NO:24 if it aligns to the same position.

For example, the domains can be defined as follows. Starting from an SGD which is not RseSGD, and which hereinafter is termed XxxSGD, a pairwise alignment of the two amino acid sequences of RseSGD and XxxSGD is performed to determine the boundaries of the domains in XxxSGC.

Domain 1 in XxxSGD can thus be defined as follows. Domain 1 of RseSGD (as set forth in SEQ ID NO: 89) is used to align XxxSGD. The first domain is then defined as the region of XxxSGD starting with the amino acid that aligns with the first residue of SEQ ID NO: 89 and finishing with the amino acid that aligns with the last residue of SEQ ID NO: 89. In embodiments where this amino acid is not a methionine, the introduction of a methionine immediately upstream of this first domain may be necessary in order to ensure proper translation of the protein, as is known in the art.

The same procedure can be repeated for domains 2 and 3, as needed. Domain 2 in XxxSGD can thus be defined as follows. Domain 2 of RseSGD (as set forth in SEQ ID NO: 90) is used to align XxxSGD. The second domain is then defined as the region of XxxSGD starting with the amino acid that aligns with the first residue of SEQ ID NO: 90 and finishing with the amino acid that aligns with the last residue of SEQ ID NO: 90. Domain 3 in XxxSGD can thus be defined as follows. Domain 3 of RseSGD (as set forth in SEQ ID NO: 91) is used to align XxxSGD. The third domain is then defined as the region of XxxSGD starting with the amino acid that aligns with the first residue of SEQ ID NO: 91 and finishing with the amino acid that aligns with the last residue of SEQ ID NO: 91. The third domain of the mosaic SGD is domain D₃of RseSGD as set forth in SEQ ID NO: 91, but it may still be useful to determine the position of domain 3 in XxxSGD, particularly in order to determine the position of domain 4 in XxxSGD.

Domain 4 in XxxSGD preferably corresponds to the region starting with the first amino acid immediately downstream of domain 3 of the same XxxSGD and finishing with the last amino acid of XxxSGD. In other words, if domain 3 of XxxSGD ends with residue number n, then domain 4 starts with residue n+1, where n is an integer.

The term “domain 1” as used herein refers to one or more sequential groups of amino acids corresponding to amino acids from position 1 to 115 of SEQ ID NO:24.

The term “domain 2” as used herein refers to one or more sequential groups of amino acids corresponding to amino acids from position 116 to 266 of SEQ ID NO:24.

The term “domain 3” as used herein refers to one or more sequential groups of amino acids corresponding to amino acids from position 267 to 456 of SEQ ID NO:24.

The term “domain 4” as used herein refers to one or more sequential groups of amino acids corresponding to amino acids from position 457 to 532 of SEQ ID NO:24.

The four domains of the mosaic SGD may be linked by, or separated by, small sequences, for example amino acid linkers, as is known in the art. It will thus be understood that the mosaic SGD may comprise additional amino acids which can be added to each of the four domains, as is known in the art.

In some embodiments, the mosaic SGD may be further modified, for example by the introduction of additional domains which may increase the stability or longevity or half-life of the protein, or localidation domains targeting the mosaic SGD to specific cellular localisations. Relevant additional domains are known in the art.

A non-functional SGD as used herein referes to a SGD which is not capable of converting strictosidine to strictosidine aglycone, whereas in contrast, a functional SGD is capable of converting strictosidine to strictosidine aglycone. By introducing some domains of RseSGD into a non-functional SGD however, it may be possible to restore function of a non-functional SGD, as shown in the examples, thus obtaining a functional mosaic SGD.

In some embodiments, D₁is a first amino acid sequence from a first SGD. Said first SGD may be any SGD, such as a functional or a non-functional SGD. It is preferred that said first SGD has at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to RseSGD of SEQ ID NO: 24.

In some embodiments, D₂is a second amino acid sequence from a second SGD. Said second SGD may be any SGD, such as a functional or a non-functional SGD. It is preferred that said second SGD has at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to RseSGD of SEQ ID NO: 24.

Interestingly, the inventors found that domain 3 (D₃) of RseSGD consisting of an amino acid sequence of SEQ ID NO:91 is capable of rescuing the inability of a non-functional SGDs of converting strictosidine to strictosidine aglycone (see FIGS. 9 and 10). Thus in preferred embodiments, the mosaic SGD comprises 4 domains, of which at least one comprises or consists of domain 3 of RseSGD; this domain is set forth in SEQ ID NO: 91.

Thus, in some embodiments of the present invention, the mosaic SGD comprises a D_3,wherein said D₃is a third amino acid sequence consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% identity to SEQ ID NO: 91. In other words, said D₃is an amio acid sequence of domain 3 of RseSGD.

In some embodiments, D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% identity to SEQ ID NO: 92. Said fourth SGD may be any SGD, such as a functional or a non-functional SGD. It is preferred that said fourth SGD has at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% identity to RseSGD of SEQ ID NO: 24.

In a preferred embodiment, said mosaic SGD comprises a D_4,wherein said D₄is a fourth amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof.

Said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD. In other words, said mosaic SGD may not be an RseSGD of SEQ ID NO: 24. Thus, said first first SGD, second SGD and fourth SGD, may be of the same species or different species, however said first first SGD, second SGD and fourth SGD may not all be native to Rauvolfia serpentina.

The third domain of the mosaic SGD comprises or consists of the third domain of RseSGD as detailed above, and at least one of the first domain, the second domain and the fourth domain is from a second organism which is not Rauvolfia serpentina, for example at least one of D₁, D₂or D₄is from an SGD native to an organism selected from Gelsemium sempervirens, Scedosporium apiospermum or Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 2997 or a variant thereof—as explained above, the variant here does not need to be functional to begin with, as its activity may be rescued by the D₃domain of RseSGD.

In some embodiments, each of D₁, D₂and D₄are from different SGDs, and are derived from different organisms independently selected from the group consisting of Scedosporium apiospermum, Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 299. In such embodiments, one of D₁, D₂and D₄may be D₁, D₂or D₄from RseSGD as set forth in SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 92, respectively, or variants thereof having at least 70% identity or homology thereto.

In some embodiments, two of D₁, D₂and D₄are from the same SGD, and are derived from one organism and the remaining domain is from another SGD. Relevant organisms and SGDs have been described above in the section “ Strictosidine-O-beta-D-glucosidase”. For example, D₁and D₂are from one SGD from a first organism, and

D₄is from another SGD from another organism; or D₁and D₄are from one SGD from a first organism, and D₂is from another SGD from another organism; or D₂and D₄are from one SGD from a first organism, and D₁is from another SGD from another organism, which may be Rauvolfia serpentina. The first organism and the other organism may be different organisms which are independently selected from the group consisting of Scedosporium apiospermum, Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 299.

In some embodiments, all of D₁, D₂and D₄are from the same SGD of the same organism, which is not Rauvolfia serpentina. D₁, D₂and D₄may be of an SGD native to an organism selected from the group consisting of Scedosporium apiospermum, Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 299.

Thus in some embodiments, the first, second and fourth SGD are all from the same SGD, which is not RseSGD. In other embodiments, the first and second SGD are from the same SGD and the fourth SGD is from another SGD; at least one said two SGDs is not RseSGD. In other embodiments, the first and third SGD are from the same SGD and the fourth SGD is from another SGD; at least one said two SGDs is not RseSGD. In other embodiments, the fourth and second SGD are from the same SGD and the fourth SGD is from another SGD; at least one said two SGDs is not RseSGD. In some embodiments, the first, second and fourth SGD are all from different SGDs, one of which may be RseSGD.

In one embodiment, the mosaic SGD comprises or consists of an amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 108, or variants thereof having at least 90% identity or homology thereto, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity or homology thereto.

The SGD may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a SGD. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 1, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1. Thus, the microorganism of the invention or the microorganism used in the methods of the invention preferably comprises at least a nucleic acid sequence identical to or having at least 90% identity to SEQ ID NO: 1.

In other embodiments, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107 such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88 SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107.

As is known in the art, in the event that the first domain of XxxSGD used in the mosaic SGD is not a methionine, the skilled person will readily be able to introduce a start codon in the nucleic acid sequence encoding the mosaic SGD in order to ensure proper translation of the mosaic SGD. The skilled person will also know how to introduce short nucleic acid sequences corresponding to linkers separating the different domains in the mosaic SGD.

The microorganism according to the present invention, expressing a heterologous SGD or variant thereof, and/or a mosaic SGD or variant thereof, is capable of converting strictosidine to strictosidine aglycone.

The conversion of strictosidine to strictosidine aglycone, may be measured directly by the amount of strictosidine aglycone as known in the art, or surrogate measure of the conversion of strictosidine to strictosidine aglycone may be measured as known in the art. Because strictosidine aglycone is highgly reactive, indirect determination of strictosidine aglycone may be preferred. For example, colorimetric assays to follow strictosidine consumption as described in Geerlings et al., 2000, may be used. The disappearance of strictosidine may also be monitored by UV, as described in Guirimand et al., 2010, or the general 8-glucosidase activity in the cells may be measured, e.g. by UV detection of a synthetic substrate such as 4-methylumbelliferyl-β-D-glucoside (Guirimand et al., 2010).

Strictosidine Synthase (STR)

Strictosidine may be provided to the microorganism, for example as part of the medium the cell is incubated in. In some embodiments, however, the microorganism is engineered and is capable of synthesising strictosidine from secologanin and tryptamine.

Thus in some embodiments the microorganism expresses a heterologous strictosidine synthase having an EC number EC 4.3.3.2. Such enzymes catalyse a Pictet-Spengler reaction between the aldehyde group of secologanin and the amino group of tryptamine to yield strictosidine.

Thus microorganisms expressing a heterologous STR are capable of converting secologanin and tryptamine to strictosidine.

In some embodiments, the STR is the STR native to Catharanthus roseus or a functional variant thereof which retains the ability to convert secologanin and tryptamine to strictosidine. Thus in some embodiments, the STR is CroSTR as set forth in SEQ ID NO: 30 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 30.

Thus, in some embodiments, the microorganism expresses RseSGD as set forth in SEQ ID NO: 24 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto. In some embodiments, the microorganism expresses GseSGD as set forth in SEQ ID NO: 25 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto. In some embodiments, the microorganism expresses SapSGD as set forth in SEQ ID NO: 26 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto. In some embodiments, the microorganism expresses RveSGD as set forth in SEQ ID NO: 27 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto.

The STR may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes an STR. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 7, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 7.

Tetrahydroalstonine Synthase, Heteroyohimbine Synthase

In addition to the above, the microorganism may be further engineered so that it can produce tetrahydroalstonine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, and further expresses a heterologous tetrahydroalstonine synthase (THAS), which is not natively present in the cell. Tetrahydroalstonine synthase has an EC number EC 1.-.-.- and catalyses conversion of strictosidine aglycone to tetrahydroalstonine. The microorganism when expressing a THAS is thus able to convert strictosidine aglycone to tetrahydroalstonine, thus producing tetrahydroalstonine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, and further expresses a heteroyohimbine synthase (HYS), which is not natively present in the cell. Heteroyohimbine synthase has an EC number EC 1.-.-.- and catalyses conversion of strictosidine aglycone to tetrahydroalstonine, ajmalicine, or mayumbine.

The microorganism when expressing an HYS is thus able to convert strictosidine aglycone to tetrahydroalstonine, ajmalicine, or mayumbine, thus producing tetrahydroalstonine.

In some embodiments, the microorganism expresses a SGD and optionally an STR and further expresses a THAS and an HYS.

In preferred embodiments, the THAS is the THAS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert strictosidine aglycone to tetrahydroalstonine. Thus in some embodiments, the THAS is CroTHAS as set forth in SEQ ID NO: 28 or a functional variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 28.

The THAS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a THAS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 5, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5.

In other preferred embodiments, the HYS is the HYS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert strictosidine aglycone to tetrahydroalstonine, ajmalicine, or mayumbine. Thus in some embodiments, the HYS is CroHYS as set forth in SEQ ID NO: 46 or variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 46.

The HYS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes an HYS. In particular, the nucleic acid sequence is identical to or has at least 90% to SEQ ID NO: 23, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 23.

In some embodiments, the microorganism expresses CroHYS and/or CroTHAS or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 46 and/or SEQ ID NO: 28.

The microorganism expressing THAS and/or HYS further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

The cell may also further express an STR as described herein, in particular CroSTR as set forth in SEQ ID NO: 30, or a functional variant thereof having at least 90% identity thereto. In some embodiments, the microorganism thus also expresses RseSGD as set forth in SEQ ID NO: 24 and CroSTR as set forth in SEQ ID NO: 30; GseSGD as set forth in SEQ ID NO: 25 and CroSTR as set forth in SEQ ID NO: 30; SapSGD as set forth in SEQ ID NO: 26 and CroSTR as set forth in SEQ ID NO: 30; or RveSGD as set forth in SEQ ID NO: 27 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90% identity thereto.

Sarpargan Bridge Enzyme (SBE)

In addition to the above, the microorganism may be further engineered so that it can produce a heteroyohimbine, in particular alstonine and serpentine. Heteroyohimbines are a prevalent subclass of the monoterpene indole alkaloids, which are found in many plant species, primarily from the Apocynaceae and Rubiaceae families. Examples of heteroyohimbines include the al-adrenergic receptor antagonist ajmalicine, and the benzodiazepine receptor ligand mayumbine (19-epi-ajmalicine). Oxidized β-carboline heteroyohimbines also exhibit potent pharmacological activity: serpentine has shown topoisomerase inhibition activity and alstonine has been shown to interact with 5-HT2A/C receptors and may act as an anti-psychotic agent. In addition, heteroyohimbines are biosynthetic precursors of many oxindole alkaloids, which also display a wide range of biological activities.

In some embodiments, the microorganism expresses an SGD and optionally an STR, and further expresses a heterologous sarpargan bridge enzyme (SBE), which is not natively present in the cell. This enzyme has an EC number EC 1.14.14.- and catalyses conversion of tetrahydroalstonine and ajmalicine to the corresponding alstonine and serpentine, respectively, or converts by cyclization the strictosidine-derived geissoschizine to the sarpagan alkaloid polyneuridine aldehyde. The microorganism when expressing an SBE is thus able to convert tetrahydroalstonine to alstonine and serpentine. In embodiments where the cell is capable of producing ajmalicine, the microorganism when expressing an SBE is able to convert tetrahydroalstonine and ajmalicine to alstonine and serpentine.

In preferred embodiments, the SBE is the SBE native to Gelsemium sempervirens or a functional variant thereof which retains the ability to convert tetrahydroalstonine and ajmalicine to alstonine and serpentine. Thus in some embodiments, the SBE is GseSBE as set forth in SEQ ID NO: 29 or a functional variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 29.

The SBE may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes an SBE. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 6, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 6.

The microorganism also expresses a SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

The microorganism may also express a THAS and/or an HYS as described herein, in particular the microorganism expresses CroHYS and/or CroTHAS or functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 46 and SEQ ID NO: 28.

NADPH-Cytochrome P450 Reductase, Cytochrome b5 and Geissoschizine Synthase

The microorganism may be further engineered so that it can produce 19E-geissoschizine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, and further expresses a heterologous NADPH-cytochrome P450 reductase (CPR), a heterologous Cytochrome b5 (CYB5) and a heterologous Geissoschizine synthase (GS) which are not natively present in the microorganism. NADPH-cytochrome P450 reductase has an EC number EC 1.6.2.4 and is required for electron transfer from NADP to cytochrome P450. Cytochrome b5 has an EC number EC 1.6.2.2 and is a membrane bound hemoprotein which function as an electron carrier. Geissoschizine synthase has an EC number EC 1.3.1.36 and catalyzes the reduction of strictosidine aglycone to 19E-geissoschizine. The microorganism when expressing CPR, CYB5 and GS is thus able to convert strictosidine aglycone to 19E-geissoschizine, thus producing 19E-geissoschizine.

In some embodiments, the microorganism expresses an SGD and optionally an STR and further expresses CPR, CYB5 and GS.

In preferred embodiments, the CPR is the CPR native to Catharanthus roseus or a functional variant thereof which retains the ability to transfer electrons from NADP to cytochrome P450. Thus in some embodiments, the CPR is CroCPR as set forth in SEQ ID NO: 31 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 31.

The CPR may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a CPR. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 8, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 8.

In preferred embodiments, the CYB5 is the CYB5 native to Catharanthus roseus or a functional variant thereof which retains the ability to function as an electron carrier. Thus in some embodiments, the CYB5 is CroCYB5as set forth in SEQ ID NO: 32 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 32.

The CYB5 may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a CYB5. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 9, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 9.

In preferred embodiments, the GS is the GS native to Catharanthus roseus or a functional variant thereof which retains the ability to catalyze the reduction of strictosidine aglycone to 19E-geissoschizine. Thus in some embodiments, the GS is CroGS as set forth in SEQ ID NO: 33 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 33.

The GS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a GS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 10, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 10.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25,SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

Geissoschizine Oxidase, Redox1 and Redox2

The microorganism may be further engineered so that it can produce stemmadenine.

The microorganism may be as described herein above. In some embodiments, the microorganism is a yeast cell. In other embodiments the microorganism is a bacterial cell.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5 and GS and further expresses a Geissoschizine oxidase (GO), a Redox1 and a Redox2, which are not natively present in the cell. Geissoschizine oxidase has an EC number EC 1.14.14.—and catalyzes the oxidation of 19E-geissoschizine to produce a short-lived MIA unstable intermediate which can be oxidized either by Redox1 and Redox2 to produce stemmadenine and 16S/R-deshydroxymethylstemmadenine (16S/R-DHS) or by spontaneous conversion to akuammicine. Redox1 has a EC number EC 1.14.14.—and catalyses the first of two oxidation steps that the converts the unstable product resulting from oxidation of 19E-geissoschizine by geissoschizine oxidase (GO) to stemmadenine. Redox2 has an EC number EC 1.7.1.—and catalyses the second of two oxidation steps that the converts the unstable product resulting from oxidation of 19E-geissoschizine by geissoschizine oxidase (GO) to stemmadenine. The microorganism when expressing GO, Redox1 and Redox2 is thus able to convert 19E-geissoschizine to stemmadenine, thus producing 19E-stemmadenine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5 and GS and further expresses GO, Redox1 and Redox2.

In preferred embodiments, the GO is the GO native to Catharanthus roseus or a functional variant thereof which retains the ability to catalyze the oxidation of 19E-geissoschizine to produce a short-lived MIA unstable intermediate which can be oxidized either by Redox1 and Redox2 to produce stemmadenine. Thus in some embodiments, the GO is CroGO as set forth in SEQ ID NO: 34 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 34.

The GO may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a GO. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 11, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 11.

In preferred embodiments, the Redox1 is the Redox1 native to Catharanthus roseus or a functional variant thereof which retains the ability to catalyse the first of two oxidation steps that the converts the unstable product resulting from oxidation of 19E-geissoschizine by geissoschizine oxidase (GO) to stemmadenine. Thus in some embodiments, the Redox1 is CroRedox1 as set forth in SEQ ID NO: 35 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 35.

The Redox1 may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a Redox1. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 12, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 12.

In preferred embodiments, the Redox2 is the Redox2 native to Catharanthus roseus or a functional variant thereof which retains the ability to catalyse the second of two oxidation steps that the converts the unstable product resulting from oxidation of 19E-geissoschizine by geissoschizine oxidase (GO) to stemmadenine. Thus in some embodiments, the Redox2 is CroRedox2 as set forth in SEQ ID NO: 36 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 36.

The Redox2 may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a Redox2. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 13, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 13.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

Stemmadenine O-Acetyltransferase

The microorganism may be further engineered so that it can produce O-acetylstemmadenine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS, GO, Redox1 and Redox2, and further expresses Stemmadenine O-acetyltransferase which is not natively present in the cell. Stemmadenine O-acetyltransferase has an EC number EC 1.7.1.—and catalyzes the acetylation of stemmadenine to O-acetylstemmadenine. The microorganism when expressing SAT is thus able to convert stemmadenine to O-acetylstemmadenine, thus producing O-acetylstemmadenine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS GO, Redox1 and Redox2 and further expresses SAT.

In preferred embodiments, the SAT is the SAT native to Catharanthus roseus or a functional variant thereof which retains the ability to convert stemmadenine to O-acetylstemmadenine. Thus in some embodiments, the SAT is CroSAT as set forth in SEQ ID NO: 37 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity identityto SEQ ID NO: 37.

The SAT may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a SAT. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 14, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 14.

The microorganism further expresses an SGD as described herein, in particular

RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

O-Acetylstemmadenine Oxidase

The microorganism may be further engineered so that it can produce dihydroprecondylocarpine acetate.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS, GO, Redox1, Redox2 and SAT, and further expresses O-acetylstemmadenine oxidase (PAS) which is not natively present in the cell. O-acetylstemmadenine oxidase has an EC number EC 1.21.3.—and converts O-acetylstemmadenine to precondylocarpine acetate. The microorganism when expressing PAS is thus able to convert O-acetylstemmadenine to precondylocarpine acetate, thus producing precondylocarpine acetate.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS GO, Redox1, Redox2, and SAT and further expresses PAS.

In preferred embodiments, the PAS is the PAS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert O-acetylstemmadenine to precondylocarpine acetate. Thus in some embodiments, the PAS is CroPAS as set forth in SEQ ID NO: 38 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 38.

The PAS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a PAS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 15, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 15.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

Dehydroprecondylocarpine Acetate Synthase

The microorganism may be further engineered so that it can produce dihydroprecondylocarpine acetate.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS, GO, Redox1, Redox2, SAT and PAS, and further expresses dihydroprecondylocarpine acetate synthase (DPAS) which is not natively present in the cell. Dihydroprecondylocarpine acetate synthase has an EC number EC 1.1.1.—and converts precondylocarpine acetate to dihydroprecondylocarpine acetate. The microorganism when expressing DPAS is thus able to convert precondylocarpine acetate to dihydroprecondylocarpine acetate, thus producing dihydroprecondylocarpine acetate.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS GO, Redox1, Redox2, SAT and PAS and further expresses DPAS.

In preferred embodiments, the DPAS is the DPAS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert precondylocarpine acetate to dihydroprecondylocarpine acetate. Thus in some embodiments, the DPAS is CroDPAS as set forth in SEQ ID NO: 39 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 39.

The DPAS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a DPAS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 16, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 16.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

Tabersonine Synthase

The microorganism may be further engineered so that it can produce tabersonine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS, GO, Redox1, Redox2, SAT, PAS and DPAS, and further expresses Tabersonine synthase (TS) which is not natively present in the cell. Tabersonine synthase has an EC number EC 4.-.-.- and converts dihydroprecondylocarpine acetate to tabersonine. The microorganism when expressing TS is thus able to convert dihydroprecondylocarpine acetate to tabersonine, thus producing tabersonine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS GO, Redox1, Redox2, SAT, PAS and DPAS, and further expresses TS.

In preferred embodiments, the TS is the TS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert dihydroprecondylocarpine acetate to tabersonine. Thus in some embodiments, the TS is CroTS as set forth in SEQ ID NO: 40 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 40.

The TS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a TS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 17, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 17.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

The cell may also further express an STD as described herein, in particular CroSTR as set forth in SEQ ID NO: 30, or a functional variant thereof having at least 90% identity thereto. In some embodiments, the microorganism thus also expresses RseSGD as set forth in SEQ ID NO: 24 and CroSTR as set forth in SEQ ID NO: 30; GseSGD as set forth in SEQ ID NO: 25 and CroSTR as set forth in SEQ ID NO: 30; SapSGD as set forth in SEQ ID NO: 26 and CroSTR as set forth in SEQ ID NO: 30; or RveSGD as set forth in SEQ ID NO: 27 and CroSTR as set forth in SEQ ID NO: 30, or functional variants thereof having at least 90% identity thereto.

Catharanthine Synthase

The microorganism may be further engineered so that it can produce catharanthine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS, GO, Redox1, Redox2, SAT, PAS and DPAS, and further expresses Catharanthine synthase (CS) which is not natively present in the cell. Catharanthine synthase has an EC number EC 4.-.-.- and converts dihydroprecondylocarpine acetate to catharanthine. The microorganism when expressing CS is thus able to convert dihydroprecondylocarpine acetate to catharanthine, thus producing catharanthine.

In some embodiments, the microorganism expresses an SGD and optionally an STR, CPR, CYB5, GS GO, Redox1, Redox2, SAT, PAS and DPAS, and further expresses CS. Optionally the microorganism also expresses TS.

In preferred embodiments, the CS is the CS native to Catharanthus roseus or a functional variant thereof which retains the ability to convert dihydroprecondylocarpine acetate to catharanthine. Thus in some embodiments, the CS is CroCS as set forth in SEQ ID NO: 41 or a variant thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 41.

The CS may be expressed in the microorganism by introducing a nucleic acid sequence as detailed further below, which encodes a CS. In particular, the nucleic acid sequence is identical to or has at least 90% identity to SEQ ID NO: 18, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 18.

The microorganism further expresses an SGD as described herein, in particular RseSGD as set forth in SEQ ID NO: 24, GseSGD as set forth in SEQ ID NO: 25, SapSGD as set forth in SEQ ID NO: 26, or RveSGD as set forth in SEQ ID NO: 27, or functional variants thereof having at least 90% identity thereto.

Methods for producing strictosidine aglycone and monoterpenoid indole alkaloids The microorganisms described herein are useful as platform for producing plant compounds, in particular strictosidine aglycone and monoterpenoid indole alkaloids (MIAs).

Herein is provided a method of producing strictosidine aglycone in a microorganism, said method comprising the steps of:

- a) providing a microorganism, said cell expressing:
  - a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone;
- b) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- c) optionally, recovering the strictosidine aglycone;
- d) optionally, further converting the strictosidine aglycone to monoterpenoid indole alkaloids.

The microorganism may be as described herein above. Thus, the microorganism may be any microorganism.

Thus, in one embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, protozoa, algae, and viruses. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, protozoa and algae. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea, yeasts, fungi, and algae. In another embodiment, the microorganism is selected from the group consisting of bacteria, archaea yeasts and fungi. In another embodiment, the microorganism is selected from bacteria, yeasts and fungi. In another embodiment, the microorganism is selected from bacteria or yeasts. In a preferred embodiment, the microorganism is a bacteria or a yeast.

The strictosidine aglycone produced in the cell may in some embodiments of the methods be further converted into monoterpenoid indole alkaloids. The term “further conversion” herein simply means that the produced strictosidine aglycone is transformed or converted into another compound which is a monoterpenoid indole alkaloid. The conversion may happen in vivo, i.e. within the cell, which may be capable of catalysing further conversion of the strictosidine aglycone into other compounds. The methods however may also comprise the steps of recovering the strictosidine aglycone from the microorganism or from the medium by methods known in the art, and thereafter converting the strictosidine aglycone into monoterpenoid indole alkaloids, i.e. the further conversion may be an ex vivo conversion.

Preferably, the microorganism expresses an SGD as described herein; the SGD may be a heterologous SGD or a mosaic SGD as described herein above. In preferred embodiments, the SGD is selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO:

62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) and functional variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity hereto.

The microorganism may be any of the microorganisms described herein. Thus, the microorganism in some embodiments expresses an SGD as described in the section “Strictosidine-O-beta-glucosidase (SGD)” and is capable of converting strictosidine to strictosidine aglycone. In some embodiments the SGD is a heterologous SGD as described in the section “Heterologous SGD or variants thereof”. In some embodiments, the SGD is a mosaic SGD as described in the section “Mosaic SGD or variants thereof”. The mosaic SGD is as described above and comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

The microorganism may also express an STR as described in the section “Strictosidine synthase (STR)” and may thus be capable of synthesising strictosidine from secologanin and tryptamine. Preferably, secologanin and tryptamine are provided to the cell, e.g. in the medium; in such embodiments, the medium need not comprise strictosidine. In other embodiments, particularly where the microorganism cannot synthesise strictosidine, strictosidine is provided to the microorganism as part of the medium.

The microorganism may be further engineered to produce tetrahydroalstonine as described in the section “Tetrahydroalstonine synthases, heteroyohimbine synthase”. For example, the microorganism may express a heterologous THAS and/or a heterologous HYS.

The microorganism may be further engineered to produce a heteroyohimbine, in particular alstonine and serpentine, as described in the section “Sarpargan bridge enzyme (SBE)”. For example, the microorganism may express a heterologous sarpargan bridge enzyme (SBE).

The microorganism may be further engineered to produce tabersonine and/or caranthine as described herein. In particular, the microorganism may be further engineered to synthesise 19E-geissoschizine as described in the section “NADPH-cytochrome P450 reductase, Cytochrome b5 and Geissoschizine synthase”. For example, the microorganism may express a heterologous NADPH-cytochrome P450 reductase (CPR), a heterologous Cytochrome b5 (CYB5) and a heterologous Geissoschizine synthase (GS). The microorganism may be further engineered so that it can synthesise stemmadenine, as described in the section “Geissoschizine oxidase, Redox1 and Redox2”. For example, the microorganism may express a GO, a Redox1 and a Redox2. The microorganism may be further engineered so that it can synthesise O-acetylstemmadenine as described in section “Stemmadenine O-acetyltransferase”. For example, the microorganism may express SAT. The microorganism may be further engineered so that it can synthesise dihydroprecondylocarpine acetate as described in section “O-acetylstemmadenine oxidase”. For example, the microorganism may express a PAS. The microorganism may be further engineered so that it can produce dihydroprecondylocarpine acetate, as described in the section “Dehydroprecondylocarpine acetate synthase”. For example, the microorganism may express a DPAS. The microorganism may be further engineered so that it can produce tabersonine, as described in the section “Tabersonine synthase”. For example, the microorganism expresses TS. The microorganism may be further engineered so that it can produce catharanthine, as described in the section “Catharanthine synthase”. For example, the microorganism may express a CS.

Thus, the microorganism may be as described above, and may produce one or more of:

- strictosidine
- strictosidine aglycone
- tetrahydroalstonine
- alstonine
- tabersonine
- catharanthine

The necessary substrates for each product may be provided to the cell as part of the medium used to grow the cells. Alternatively, the substrates for each of the above products may be synthesised by the cell itself. In all cases, the microorganism is capable of synthesising strictosidine aglycone.

Each of the above products may be recovered from the medium by methods known in the art if desirable. Accordingly, the method may comprise the step of recovering one or more of:

- strictosidine
- strictosidine aglycone
- tetrahydroalstonine
- alstonine
- tabersonine
- catharanthine

In some embodiments, the medium comprises a substrate which is strictosidine. The microorganism can convert said strictosidine to strictosidine aglycone as described in detail herein above.

In some embodiments, the medium comprises strictosidine, at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.5 mM, such as at least 1 mM.

In other embodiments, the medium comprises tryptamine and secologanin, preferably at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.5 mM, such as at least 1 mM.

The present invention also related to a method of producing indole alkaloids (MIAs) in a microorganism.

Thus, herein is provided a method of producing monoterpenoid indole alkaloids (MIAs) in a microorganism, said method comprising the steps of:

- i) providing a microorganism capable of converting strictosidine to tabersonine and/or catharanthine, said cell expressing:
  - a strictosidine-beta-glucosidase (SGD);
  - a NADPH-cytochrome P450 reductase (CPR);
  - a Cytochrome b5 (CYB5);
  - a Geissoschizine synthase (GS);
  - a Geissoschizine oxidase (GO);
  - a Redox1;
  - a Redox2;
  - a Stemmadenine O-acetyltransferase (SAT);
  - a O-acetylstemmadenine oxidase (PAS);
  - a Dehydroprecondylocarpine acetate synthase (DPAS);
  - a Tabersonine synthase (TS); and/or
  - a Catharanthine synthase (CS);
- ii) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- iii) optionally, recovering the MIAs;
- iv) optionally, processing the MIAs into a pharmaceutical compound,

and/or;

wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

wherein D₁is a first amino acid sequence from a first SGD,

wherein D₂is a second amino acid sequence from a second SGD,

wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,

wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

The microorganism may optionally further express a strictosidine synthase (STR).

The microorganism capable of producing monoterpenoid indole alkaloids (MIAs) may be any microorgsnims as described herein under section “Deteiled description”.

Titers

The microorganisms and methods disclosed herein can be used to produce different plant-derived compounds at high titers. Strictosidine aglycone may thus be obtained with a total titer of at least 0.1 λM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM, such as at least 25 μM, such as at least 30 μM, such as at least 35 μM, such as at least 40 μM, such as at least 50 μM, or more, wherein the total titer is the sum of the intracellular strictosidine aglycone titer and the extracellular strictosidine aglycone. Indeed, the produced strictosidine aglycone may be secreted from the cell—extracellular strictosidine aglycone—or it may be retained in the cell—intracellular strictosidine aglycone.

The microorganism may be capable of producing extracellular strictosidine aglycone with a titer of at least 0.1 μM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM, such as at least 25 μM, such as at least 30 μM, such as at least 35 μM, such as at least 40 μM, such as at least 50 μM, or more.

The microorganism may be capable of producing intracellular strictosidine aglycone with a titer of at least 0.1 μM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM, such as at least 25 μM, such as at least 30 μM, such as at least 35 μM, such as at least 40 μM, such as at least 50 μM, or more.

Methods for determining the strictosidine aglycone titer are known in the art. For example, the cells can be lysed and the titers determined by Orbitrap Fusion Tribid MS (see example 5) to determine the intracellular or secreted strictosidine aglycone titers. The titers can also be determined by Orbitrap Fusion Tribid MS in supernatant fractions from which the cells have been removed.

The microorganism may be capable of producing tetrahydroalstonine with a titre of at least 1 μM, such as at least 2 μM, such as at least 4 μM, such as at least 6 μM, such as at least 8 μM such as at least 10 μM or more.

The microorganism may be capable of producing alstonine with a titre of at least 0.1 μM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM or more.

The microorganism may be capable of producing tabersonine with a titre of at least 0.01 μM, such as at least 0.02 μM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM or more.

The microorganism may be capable of producing catharanthine with a titre of at least 0.01 μM, such as at least 0.02 μM, such as at least 0.5 μM, such as at least 1 μM, such as at least 2 μM, such as at least 3 μM, such as at least 4 μM, such as at least 5 μM, such as at least 6 μM, such as at least 7 μM L, such as at least 8 μM, such as at least 9 μM, such as at least 10 μM, such as at least 11 μM, such as at least 12 μM, such as at least 13 μM, such as at least 14 μM, such as at least 15 μM, such as at least 20 μM or more.

Nucleic Acids, Vectors and Host Cells

Also disclosed herein are useful nucleic acid constructs for constructing a microorganism as described above, or useful in general in the methods described herein. Such nucleic acid constructs encode the heterologous enzymes useful for constructing the microorganisms of the invention.

It will be understood that the term “nucleic acid constructs” may refer to one nucleic acid molecule, or to a plurality of nucleic acid molecules, comprising the relevant nucleic acid sequences. The nucleic acid construct may thus be one nucleic acid molecule, which may encode several enzymes, or it may be several nucleic acid molecules, each comprising one sequence encoding an enzyme. The relevant nucleic acid sequences may thus be comprised on one vector, or on several vectors. They may also be integrated in the genome, on one chromosome or even together in one location, or they may be integrated on different chromosomes. It is also possible to have some sequences on one or more vectors, and some integrated in the genome.

Also provided herein are nucleic acid constructs comprising a nucleic acid sequence identical to or having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107. Thus, the microorganism of the invention or the microorganism used in the methods of the invention preferably comprises at least a nucleic acid sequence identical to or having at least 90% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 or SEQ ID NO:107. Preferably the nucleic acid is identical to or has at least 90% identity to SEQ ID NO: 1.

The nucleic acid construct may further comprise a nucleic acid sequence identical to or having at 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 7.

The nucleic acid construct may further comprise a sequence identical to or having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5 and/or SEQ ID NO: 23.

The nucleic acid construct may further comprise a nucleic acid sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and/or SEQ ID NO: 18.

All nucleic acid sequences may have been codon-optimised for expression in the microorganism, as is known in the art.

It may be of interest to take advantage of inducible promoters. Thus in some embodiments, the nucleic acid constructs comprises one or more of the above nucleic acid sequences under the control of an inducible promoter. This allows more control of when the enzyme encoded by the sequence is actually expressed, and can be advantageous for example if production of one of the plant compounds negatively affects cell growth. The skilled person will have no difficulty in identifying suitable inducible promoters.

In some embodiments, the nucleic acid construct is one or more vectors, for examples an integrative or a replicative vector. Suitable vectors are known in the art and readily available to the skilled person.

Also provided herein is a vector comprising one of more of the nucleic acid sequences above, in particular SEQ ID NO: 1 or a sequence having at least 90% identity thereto. The vector may further comprise any of SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 23, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and/or SEQ ID NO: 18 or a sequence having at least 90% identity thereto.

Also provided herein is a host cell comprising one or more nucleic acid sequence or vector as defined herein above, in particular SEQ ID NO: 1 or a sequence having at least 90% identity thereto, or a vector comprising SEQ ID NO: 1 or a sequence having at least 90% identity thereto, and one or more of SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 23, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17 and/or SEQ ID NO: 18 or a sequence having at least 90% identity thereto.

The host cell may be any host cell, such as a primary cell or a cell from a cell line. In preferred embodiments, the host cell is from a mammalian or human cell line. The host cell may be a prokaryote or a eukaryote. In a preferred embodiment, the cell is a eukaryote.

A host cell according to the present invention may be comprised within a host organism, such as an animal.

Also provided herein is the use of the nucleic acid constructs, the microorganisms, the vectors or the host cells described herein for producing strictosidine aglycone and/or tetrahydroalstonine, alstonine, tabersonine and/or catharanthine in a microorganism. In some embodiments, the nucleic acid constructs, the microorganisms, the vectors or the host cells described herein are used in a method for producing strictosidine aglycone and/or tetrahydroalstonine, alstonine, tabersonine and/or catharanthine in a microorganism as described herein.

Pharmaceutical Compounds

The plant compounds obtainable by the present methods may be useful for manufacturing pharmaceutical compounds. Thus, the methods may further comprise a step of producing a pharmaceutical compound from any of the compounds, in particular monoterpenoid indole alkaloids, produced by the microorganism of the present invention.

Thus is also provided a method of treating a disorder such as a cancer, arrhythmia, malaria, psychotic diseases, hypertension, depression, Alzheimer's disease, addiction and/or neuronal diseases, comprising administration of a therapeutic sufficient amount of an MIA or a pharmaceutical compound obtained by the methods described herein.

Sequences

TABLE 1

Sequence

ID NO:
Description
Details

1
DNA
Strictosidine-O-beta-D-glucosidase

RseSGD
EC 3.2.1.105

from Rauvolfia
Hydrolyses strictosidine to strictosidine

serpentina

aglycone

2
DNA
strictosidine glucosidase

GseSGD
EC 3.2.1.-

from Gelsemium
Putative function: Hydrolyses O-

sempervirens

glycosyl compounds

3
DNA
3-alpha-(S)-strictosidine beta-

SapSGD
glucosidase

from Scedosporium
EC 3.2.1.105

apiospermum

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

4
DNA
Strictosidine-beta-D-glucosidase

RveSGD
EC 3.2.1.105

from Rauvolfia
Putative function: Hydrolyses

verticillata

strictosidine to strictosidine aglycone

5
DNA
Tetrahydroalstonine synthase

CroTHAS
EC.1.-.-.-

from Chatharanthus
Converts strictosidine aglycone to

roseus

tetrahydroalstonine

6
DNA
Sarpagan bridge enzyme (CYP71AY5)

GseSBE
EC 1.14.14.-

from Gelsemium
Converts by aromatization the

sempervirens

tetrahydroalstonine and ajmalicine to

the corresponding alstonine and

serpentine, respectively or converts by

cyclization the strictosidine-derived

geissoschizine to the sarpagan alkaloid

polyneuridine aldehyde

7
DNA
Strictosidine synthase

CroSTR from
EC 4.3.3.2

Catharanthus roseus

Converts secologanin and tryptamine to

strictosidine by stereospecific

condensation.

8
DNA
NADPH-cytochrome P450 reductase

CroCPR from
EC 1.6.2.4

Catharanthus roseus
This enzyme is required for electron

transfer from NADP to cytochrome

P450

9
DNA
Cytochrome b5

CroCYB5 from
EC 1.6.2.2

Catharanthus roseus

Membrane bound hemoprotein which

function as an electron carrier

10
DNA
Geissoschizine synthase (CrADH14)

CroGS from Catharanthus
EC 1.3.1.36

roseus

Catalyzes the reduction of strictosidine

aglycone to 19E-geissoschizine

11
DNA
Geissoschizine oxidase (CYP71AY2)

CroGO from Catharanthus
EC 1.14.14.-

roseus

Catalyzes the oxidation of 19E-

geissoschizine to produce a short-lived

MIA unstable intermediate which can

be oxidized either by Redox1 and

Redox2 to produce stemmadenine and

16S/R-

deshydroxymethylstemmadenine

(16S/R-DHS) or by spontaneous

conversion to akuammicine

12
DNA
Redox 1

CroRedox1 from
EC 1.14.14.-

Catharanthus roseus

Catalyzes the first of two oxidation

steps that the converts the unstable

product resulting from oxidation of 19E-

geissoschizine by geissoschizine

oxidase (GO) to stemmadenine

biosynthesis

13
DNA
Redox 2

CroRedox2 from
EC 1.7.1.-

Catharanthus roseus

Catalyzes the second of two oxidation

steps that the converts the unstable

product resulting from oxidation of 19E-

geissoschizine by geissoschizine

oxidase (GO) to stemmadenine

biosynthesis

14
DNA
Stemmadenine O-acetyltransferase

CroSAT from
EC 1.7.1.-

Catharanthus roseus

Catalyzes the acetylation of

stemmadenine to O-acetylstemmadenine

15
DNA
O-acetylstemmadenine oxidase

CroPAS from
(precondylocarpine acetate synthase)

Catharanthus roseus

EC 1.21.3.-

Converts O-acetylstemmadenine to

dihydroprecondylocarpine acetate

16
DNA
Dehydroprecondylocarpine acetate

CroDPAS from
synthase

Catharanthus roseus

EC 1.1.1.-

Converts precondylocarpine acetate to

dihydroprecondylocarpine acetate

17
DNA
tabersonine synthase (Hydrolyase 2)

CroTS from Catharanthus
EC 4.-.-.-

roseus
Catalyzes the conversion of

dihydroprecondylocarpine acetate to

tabersonine

18
DNA
Catharanthine synthase (Hydrolase 1)

CroCS from Catharanthus
EC 4.-.-.-

roseus

Catalyzes the conversion of

dihydroprecondylocarpine acetate to

catharanthine

19
DNA
Putative strictosidine beta-D-

UtoSGD from Uncaria
glucosidase

tomentosa

EC 3.2.1.105

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

20
DNA
Strictosidine-O-beta-D-glucosidase

CroSGD from
EC 3.2.1.105

Catharanthus roseus

Hydrolyses strictosidine to strictosidine

aglycone

21
DNA
Putative strictosidine beta-D-

CacSGD from
glucosidase

Camptotheca acuminata

EC 3.2.1.105

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

22
DNA
Uncharacterized protein

GsoSGD from Glycine
EC 3.2.-.-

soja

Putative function: Hydrolyses O-

glycosyl compounds

23
DNA
Heteroyohimbine synthase

CroHYS
EC.1.-.-.-

Converts strictosidine aglycone to

tetrahydroalstonine, ajmalicine, or

mayumbine

24
Protein
Strictosidine-O-beta-D-glucosidase

RseSGD
EC 3.2.1.105

from Rauvolfia
Q8GU20

serpentina

Hydrolyses strictosidine to strictosidine

aglycone

25
Protein
strictosidine glucosidase

GseSGD
EC 3.2.1.-

from Gelsemium
AXK92564.1

sempervirens

Putative function: Hydrolyses O-

glycosyl compounds

26
Protein
3-alpha-(S)-strictosidine beta-

SapSGD
glucosidase

from Scedosporium
EC 3.2.1.105

apiospermum

A0A084GBX6

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

27
Protein
Strictosidine-beta-D-glucosidase

RveSGD
EC 3.2.1.105

from Rauvolfia
M9NGS2

verticillata

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

28
Protein
Tetrahydroalstonine synthase

CroTHAS
EC.1.-.-.-

from Chatharanthus
A0A0F6SD02

roseus

Converts strictosidine aglycone to

tetrahydroalstonine

29
Protein
Sarpagan bridge enzyme (CYP71AY5)

GseSBE
EC 1.14.14.-

from Gelsemium
P0DO14

sempervirens

Converts by aromatization the

tetrahydroalstonine and ajmalicine to

the corresponding alstonine and

serpentine, respectively or converts by

cyclization the strictosidine-derived

geissoschizine to the sarpagan alkaloid

polyneuridine aldehyde

30
Protein
Strictosidine synthase

CroSTR from
EC 4.3.3.2

Catharanthus roseus

P18417

Converts secologanin and tryptamine to

strictosidine by stereospecific

condensation.

31
Protein
NADPH-cytochrome P450 reductase

CroCPR from
EC 1.6.2.4

Catharanthus roseus

Q05001

This enzyme is required for electron

transfer from NADP to cytochrome

P450

32
Protein
Cytochrome b5

CroCYB5 from
EC 1.6.2.2

Catharanthus roseus

A0A0C5DKP2

Membrane bound hemoprotein which

function as an electron carrier

33
Protein
Geissoschizine synthase (CrADH14)

CroGS from Catharanthus
EC 1.3.1.36

roseus

W8JWW7

Catalyzes the reduction of strictosidine

aglycone to 19E-geissoschizine

34
Protein
Geissoschizine oxidase (CYP71AY2)

CroGO from Catharanthus
EC 1.14.14.-

roseus

I1TEM0

Catalyzes the oxidation of 19E-

geissoschizine to produce a short-lived

MIA unstable intermediate which can

be oxidized either by Redox1 and

Redox2 to produce stemmadenine and

16S/R-

deshydroxymethylstemmadenine

(16S/R-DHS) or by spontaneous

conversion to akuammicine

35
Protein
Redox 1

CroRedox1 from
EC 1.14.14.-

Catharanthus roseus

A0A2P1GIW4

Catalyzes the first of two oxidation

steps that the converts the unstable

product resulting from oxidation of 19E-

geissoschizine by geissoschizine

oxidase (GO) to stemmadenine

biosynthesis

36
Protein
Redox 2

CroRedox2 from
EC 1.7.1.-

Catharanthus roseus

A0A2P1GIY9

Catalyzes the second of two oxidation

steps that the converts the unstable

product resulting from oxidation of 19E-

geissoschizine by geissoschizine

oxidase (GO) to stemmadenine

biosynthesis

37
Protein
Stemmadenine O-acetyltransferase

CroSAT from
EC 1.7.1.-

Catharanthus roseus

A0A2P1GIW7

Catalyzes the acetylation of

stemmadenine to O-

acetylstemmadenine

38
Protein
O-acetylstemmadenine oxidase

CroPAS from
(precondylocarpine acetate synthase)

Catharanthus roseus

EC 1.21.3.-

MH213134.1

Converts O-acetylstemmadenine to

dihydroprecondylocarpine acetate

39
Protein
Dehydroprecondylocarpine acetate

CroDPAS from
synthase

Catharanthus roseus

EC 1.1.1.-

A0A1B1FHP3

Converts precondylocarpine acetate to

dihydroprecondylocarpine acetate

40
Protein
tabersonine synthase (Hydrolyase 2)

CroTS from Catharanthus
EC 4.-.-.-

roseus

A0A2P1GIW3

Catalyzes the conversion of

dihydroprecondylocarpine acetate to

tabersonine

41
Protein
Catharanthine synthase (Hydrolase 1)

CroCS from Catharanthus
EC 4.-.-.-

roseus

A0A2P1GIW2

Catalyzes the conversion of

dihydroprecondylocarpine acetate to

catharanthine

42
Protein
Putative strictosidine beta-D-

UtoSGD from Uncaria
glucosidase

tomentosa

EC 3.2.1.105

I6ZQ42

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

43
Protein
Strictosidine-O-beta-D-glucosidase

CroSGD from
EC 3.2.1.105

Catharanthus roseus
B8PRP4

Hydrolyses strictosidine to strictosidine

aglycone

44
Protein
Putative strictosidine beta-D-

CacSGD from
glucosidase

Camptotheca acuminata

EC 3.2.1.105

G8E0P8

Putative function: Hydrolyses

strictosidine to strictosidine aglycone

45
Protein
Uncharacterized protein

GsoSGD from Glycine
EC 3.2.-.-

soja

A0A0R0H2R3

Putative function: Hydrolyses O-

glycosyl compounds

46
Protein
Heteroyohimbine synthase

CroHYS from
EC.1.-.-.-

Catharanthus roseus

A0A1B1FHP5

Converts strictosidine aglycone to

tetrahydroalstonine, ajmalicine, or

mayumbine

47
Protein VmiSGD1 from
Uncharacterized protein

Vinca minor

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

48
Protein AhuSGD from
Uncharacterized protein

Amsonia hubrichtii

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

49
Protein HimSGD2 from
Uncharacterized protein

Handroanthus

EC 3.2.-.-

impetiginosus

PIN06789.1

Putative function: Hydrolyses O-

glycosyl compounds

50
Protein SinSGD from
Uncharacterized protein

Sesamum indicum

EC 3.2.-.-

XP_011094151.1

Putative function: Hydrolyses O-

glycosyl compounds

51
Protein TelSGD from
Uncharacterized protein

Tabernaemontana

EC 3.2.-.-

elegans

Putative function: Hydrolyses O-

glycosyl compounds

52
Protein VunSGD from
Uncharacterized protein

Vigna unguiculata

EC 3.2.-.-

XP_027910736.1

Putative function: Hydrolyses O-

glycosyl compounds

53
Protein NsiSGD1 from
Uncharacterized protein

Nyssa sinensis

EC 3.2.-.-

KAA8549635.1

Putative function: Hydrolyses O-

glycosyl compounds

54
Protein LprSGD from
Uncharacterized protein

Lomentospora prolificans

EC 3.2.-.-

PKS11920.1

Putative function: Hydrolyses O-

glycosyl compounds

55
Protein AchSGD1 from
Uncharacterized protein

Actinidia chinensis var.
EC 3.2.-.-

chinensis

PSS10019.1

Putative function: Hydrolyses O-

glycosyl compounds

56
Protein HsuSGD from
Uncharacterized protein

Heliocybe sulcata

EC 3.2.-.-

TFK52902.1

Putative function: Hydrolyses O-

glycosyl compounds

57
Protein MroSGD from
Uncharacterized protein

Moniliophthora roreri

EC 3.2.-.-

MCA 2997
ESK96275.1

Putative function: Hydrolyses O-

glycosyl compounds

58
Protein RseSGD2 from
Raucaffricine-O-beta-D-glucosidase

Rauvolfia serpentina

EC 3.2.1.125

AAF03675.1

Function: Hydrolyses the MIA

raucaffricine

59
Protein PgrSGD from
Uncharacterized protein

Pyricularia grisea
EC 3.2.-.-

AAX07701.1

Putative function: Hydrolyses O-

glycosyl compounds

60
Protein OpuSGD from
Uncharacterized protein

Ophiorrhiza pumila

EC 3.2.-.-

BAP90523.1

Putative function: Hydrolyses O-

glycosyl compounds

61
Protein HpiSGD from
Uncharacterized protein

Hydnomerulius pinastri

EC 3.2.-.-

MD-312
KIJ63193.1

Putative function: Hydrolyses O-

glycosyl compounds

62
Protein HanSGD1 from
Uncharacterized protein

Helianthus annuus

EC 3.2.-.-

XP_022015317.1

Putative function: Hydrolyses O-

glycosyl compounds

63
Protein AchSGD2 from
Uncharacterized protein

Actinidia chinensis var.
EC 3.2.-.-

chinensis

PSR88404.1

Putative function: Hydrolyses O-

glycosyl compounds

64
Protein HimSGD1 from
Uncharacterized protein

Handroanthus

EC 3.2.-.-

impetiginosus

PIN07435.1

Putative function: Hydrolyses O-

glycosyl compounds

65
Protein IpeSGD from
beta-glucosidase

Carapichea ipecacuanha

EC 3.2.1.21

BAH02544.1

function: hydrolyses glucosidic Ipecac

alkaloids

66
Protein LsaSGD1 from
Uncharacterized protein

Lactuca sativa

EC 3.2.-.-

XP_023770227.1

Putative function: Hydrolyses O-

glycosyl compounds

67
Protein CarSGD from
Uncharacterized protein

Coffea arabica

EC 3.2.-.-

XP_027073002.1

Putative function: Hydrolyses O-

glycosyl compounds

68
DNA VmiSGD1 from Vinca
Uncharacterized protein

minor

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

69
DNA AhuSGD from
Uncharacterized protein

Amsonia hubrichtii
EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

70
DNA HimSGD2 from
Uncharacterized protein

Handroanthus

EC 3.2.-.-

impetiginosus

Putative function: Hydrolyses O-

glycosyl compounds

71
DNA SinSGD from
Uncharacterized protein

Sesamum indicum

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

72
DNA TelSGD from
Uncharacterized protein

Tabernaemontana

EC 3.2.-.-

elegans

Putative function: Hydrolyses O-

glycosyl compounds

73
DNA VunSGD from Vigna
Uncharacterized protein

unguiculata

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

74
DNA NsiSGD1 from Nyssa
Uncharacterized protein

sinensis

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

75
DNA LprSGD from
Uncharacterized protein

Lomentospora prolificans

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

76
DNA AchSGD1 from
Uncharacterized protein

Actinidia chinensis var.
EC 3.2.-.-

chinensis

Putative function: Hydrolyses O-

glycosyl compounds

77
DNA HsuSGD from
Uncharacterized protein

Heliocybe sulcata

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

78
DNA MroSGD from
Uncharacterized protein

Moniliophthora roreri

EC 3.2.-.-

MCA 2997
Putative function: Hydrolyses O-

glycosyl compounds

79
DNA RseSGD2 from
Raucaffricine-O-beta-D-glucosidase

Rauvolfia serpentina

EC 3.2.1.125

Function: Hydrolyses the MIA

raucaffricine

80
DNA PgrSGD from
Uncharacterized protein

Pyricularia grisea

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

81
DNA OpuSGD from
Uncharacterized protein

Ophiorrhiza pumila

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

82
DNA HpiSGD from
Uncharacterized protein

Hydnomerulius pinastri

EC 3.2.-.-

MD-312
Putative function: Hydrolyses O-

glycosyl compounds

83
DNA HanSGD1 from
Uncharacterized protein

Helianthus annuus

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

84
DNA AchSGD2 from
Uncharacterized protein

Actinidia chinensis var.
EC 3.2.-.-

chinensis

Putative function: Hydrolyses O-

glycosyl compounds

85
DNA HimSGD1 from
Uncharacterized protein

Handroanthus

EC 3.2.-.-

impetiginosus

Putative function: Hydrolyses O-

glycosyl compounds

86
DNA IpeSGD from
Beta-glucosidase

Carapichea ipecacuanha

EC 3.2.1.21

Function: hydrolyses glucosidic Ipecac

alkaloids

87
DNA LsaSGD1 from
Uncharacterized protein

Lactuca sativa

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

88
DNA CarSGD from Coffea
Uncharacterized protein

arabica

EC 3.2.-.-

Putative function: Hydrolyses O-

glycosyl compounds

89
Domain 1 of RseSGD from
M1-R115

Rauvolfia serpentina

MDNTQAEPLVVAIVPKPNASTEHTNS

HLIPVTRSKIVVHRRDFPQDFIFGAGG

SAYQCEGAYNEGNRGPSIWDTFTQR

SPAKISDGSNGNQAINCYHMYKEDIKI

MKQTGLESYR

90
Domain 2 of RseSGD from
F116-G266

Rauvolfia serpentina

FSISWSRVLPGGRLAAGVNKDGVKFY

HDFIDELLANGIKPSVTLFHWDLPQAL

EDEYGGFLSHRIVDDFCEYAEFCFWE

FGDKIKYWTTFNEPHTFAVNGYALGE

FAPGRGGKGDEGDPAIEPYVVTHNIL

LAHKAAVEEYRNKFQKCQEG

91
Domain 3 of RseSGD from
E267-G456

Rauvolfia serpentina
IGIVLNSMWMEPLSDVQADIDAQKRA

LDFMLGWFLEPLTTGDYPKSMRELVK

GRLPKFSADDSEKLKGCYDFIGMNYY

TATYVTNAVKSNSEKLSYETDDQVTK

TFERNQKPIGHALYGGWQHVVPWGL

YKLLVYTKETYHVPVLYVTESGMVEE

NKTKILLSEARRDAERTDYHQKHLAS

VRDAIDDG

92
Domain 4 of RseSGD from
V457-T532

Rauvolfia serpentina

VNVKGYFVWSFFDNFEWNLGYICRY

GIIHVDYKSFERYPKESAIWYKNFIAG

KSTTSPAKRRREEAQVELVKRQKT

93
Protein sequence of
Mosaic SGD

CCRR

94
Protein sequence of
Mosaic SGD

CRRR

95
Protein sequence of
Mosaic SGD

RCRR

96
Protein sequence of
Mosaic SGD

RRRC

97
Protein sequence of
Mosaic SGD

RCRC

98
Protein sequence of
Mosaic SGD

CCRC

99
Protein sequence of
Mosaic SGD

VVRR

100
DNA of CCRR
Mosaic SGD

101
DNA of CRRR
Mosaic SGD

102
DNA of RCRR
Mosaic SGD

103
DNA of CRRC
Mosaic SGD

104
DNA of RRRC
Mosaic SGD

105
DNA of RCRC
Mosaic SGD

106
DNA of CCRC
Mosaic SGD

107
DNA of VVRR
Mosaic SGD

108
Protein sequence of
Mosaic SGD

CRRC

EXAMPLES

Strains

Different strains were developed to validate the functionalization of RseSGD in the production of strictosidine aglycone and selected MIAs.

TABLE 2

Strain
Genotype
Substrate → Product

MIA-BJ
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
OR

@XII-2, [CroSTR-CroSLS] @X-4,
Geraniol + tryptamine →

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
strictosidine

CroADH2] @XII-4

MIA-CA-1
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or tetrahydroalstonine if

@XII-2, [CroSTR-CroSLS] @X-4,
the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [CroSGD-

CroHYS]@XII-5

MIA-CA-2
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [RseSGD-

CroHYS]@XII-5

MIA-CA-3
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [RveSGD-

CroHYS]@XII-5

MIA-CA-4
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [GseSGD-

CroHYS]@XII-5

MIA-CA-5
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or tetrahydroalstonine if

@XII-2, [CroSTR-CroSLS] @X-4,
the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [CacSGD-

CroHYS]@XII-5

MIA-CA-6
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanine + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [SapSGD-

CroHYS]@XII-5

MIA-CA-7
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or tetrahydroalstonine if

@XII-2, [CroSTR-CroSLS] @X-4,
the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [UtoSGD-

CroHYS]@XII-5

MIA-CA-8
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or tetrahydroalstonine if

@XII-2, [CroSTR-CroSLS] @X-4,
the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [GsoSGD-

CroHYS]@XII-5

MIA-BZ-1
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or strictosidine aglycone

@XII-2, [CroSTR-CroSLS] @X-4,
if the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [CroSGD]@XII-5

MIA-BZ-2
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine aglycone

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [RseSGD]@XII-5

MIA-BZ-3
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]
* or tetrahydroalstonine if

@XII-2, [CroSTR-CroSLS] @X-4,
the candidate SGD does

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-
function

CroADH2] @XII-4 [CroSGD-

CroTHAS]@XII-5

MIA-BZ-4
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR-Cro8HGO] @XI-3, [CroIS-CroIO]

@XII-2, [CroSTR-CroSLS] @X-4,

[Cro7DLGT-Cro7DLH] @XI-1, [CroLAMT-

CroADH2] @XII-4 [RseSGD-

CroTHAS]@XII-5

MIA-DA
Cas9@XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
No production

adh6Δ, [CroCPR-CroCYB5]@XI-3

MIA-DC
Cas9@XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroCPR-CroCYB5]@XI-3,
→ tabersonine +

[CroSTR-CroGS-RseSGD-CroGO-
catharanthine

CroRedoxI -CroRedox2]@XII-5, [CroSAT-

CroPAS-CroDPAS-CroTS-CroCG]@XI-5

MIA-DE
Cas9@XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
tabersonine → Vindoline

adh6Δ, [CroCPR-CroCYB5]@XI-3,
OR

[CroNMT-CroD4H-CroDAT-CroPER-
Tabersonine +

CroT16H1]@X-4, [CroT16H2-Cro16OMT-
catharanthine →

CroT3O-CroT3R]@XII-4
vinblastine

OR

Vindoline + catharanthine

→ vinblastine

MIA-FA
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
OR

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
Geraniol + tryptamine →

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
strictosidine*

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-
*or tetrahydroalstonine if

NcISY] @XII-5, [CroHYS] @IV-1
functional SGD is co-

expressed

MIA-FC-1
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[CroSGD] @IV-2

MIA-FC-2
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[VmiSGDI] @IV-2

MIA-FC-3
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[AhuSGD] @IV-2

MIA-FC-4
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[HimSGD2] @IV-2

MIA-FC-5
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[SinSGD] @IV-2

MIA-FC-6
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[TelSGD] @IV-2

MIA-FC-7
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[VunSGD] @IV-2

MIA-FC-8
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[NsiSGD1] @IV-2

MIA-FC-9
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[LprSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

10
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[AchSGD1] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

11
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[HsuSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

12
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[MroSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

13
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[RseSGD2] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

14
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[PgrSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

15
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[OpuSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

16
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[HpiSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

17
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[HanSGD1] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

18
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[AchSGD2] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

19
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[HimSGD1] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

20
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[IpeSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

21
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[LsaSGD1] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secologanin + tryptamine

22
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ tetrahydroalstonine

[CroCPR] @XI-3, [CroIO] @XII-2,

[CroSTR-CroSLS] @X-4, [Cro7DLGT-

Cro7DLH] @XI-1, [CroLAMT-CroADH2]

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[CarSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

23
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[OeuSGD2] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

24
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[AchSGD3] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

25
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[CmaSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

26
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[MmySGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

27
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[VmiSGD3] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

28
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[IniSGD] @IV-2

MIA-FC-
Cas9 @ XII-1, atf1Δ oye2Δ, oye3Δ ari1Δ
Secolocanin + tryptamine

29
adh6Δ, [CroG8H-CroCYB5] @X-3,
→ strictosidine*

[CroCPR] @XI-3, [CroIO] @XII-2,
* or tetrahydroalstonine if

[CroSTR-CroSLS] @X-4, [Cro7DLGT-
the candidate SGD does

Cro7DLH] @XI-1, [CroLAMT-CroADH2]
function

@XII-4, [Vmi8HGO-A] @X-2, [NcMLP-

NcISY] @XII-5, [CroHYS] @IV-1,

[NsiSGD2] @IV-2

Example 1

Construction of USER Backbones

All USER vectors were constructed based on pCfB2315 (pRS413-HIS), linearized by restriction enzymes Xhol and Sac! (Thermo-Fisher FastDigest™). All terminators were amplified from CEN.PK113-7D genome using primers flanked with Xhol and Sac! restriction sites. A DNA cassette containing the ccdB counter-selection marker (Steyaert J. et al. 1993) was inserted into all USER vectors to ensure high cloning efficiency.

USER Assembly of Plasmids

All plasmids were constructed using the USER method (Jensen NB et al. 2013). Biobrick for plant genes were amplified from synthetic gBlocks (Integrated DNA Technologies and Twist Biosciences), codon optimized for expression in yeast host. Biobrick for promoters were amplified from yeast CEN.PK113-7D genome.

Construction of Strains

All strains were constructed using the CRISPR-Cas9 method described in Jakoc̆iūnas T. et al. 2015.

Example 2

Showing that CroSGD does not Function in Yeast

Geerlings et al. (Geerlings, A., 2000 and WO 00/42200) originally isolated a full-length cDNA clone from a Catharanthus roseus cDNA library giving rise to SGD activity in an in vitro assay.

To confirm if CroSGD could be validated and functionalized in yeast, CroSGD was expressed according to Geerlings et al. by using the strong glycolytic and constitutive active promoters TDH3 and TEF1, respectively.

The following yeast strains were produced, containing SGD and tetrahydroalstonine (THA) synthase both from Catharantus roseus, i.e. CroSGD and CroTHAS.

Strain MIA-BJ (EZ-Swap, full CroSTR) expressing:

- P1-TDH3-CroSGD_nls-P2_TEF1-CroTHAS_nls
- P1-TDH3-CroSGD_cyt-P2_TEF1-CroTHAS_cyt
- P2-TEF1-CroSGD-5xGS-CroTHAS_nls
- P2-TEF1-CroTHAS-5xGS-CroSGD_nls
- P2-TEF1-CroSGD-5xGS-CroTHAS_cyt
- P2-TEF1-CroTHAS-5xGS-CroSGD_cyt
- P1-TEF1-CroSGD_nls-P2_PGK1-CroTHAS_nls
- P1-TEF1-CroSGD_cyt-P2_PGK1-CroTHAS_cyt
- P1-TEF1-CroSGD_nls-P2_PGK1-CroTHAS_cyt
- P1-TEF1-CroSGD_cyt-P2_PGK1-CroTHAS_nls

The high-resolution analytical results obtained from LC-MS analysis expressing CroSGD alone and in various tagged and CroSGD-fusion versions contradicts the results presented by Geerlings et al. are not valid.

FIG. 1 shows the LC-MS analysis of tetrahydroalstonine (THA). From FIG. 1 it can be seen that none of the strains expressing CroSGD could produce detectable amount of tetrahydroalstonine.

As a positive control, the following strains were created, strain MIA-BJ (EZ-Swap, full CroSTR) expressing:

- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_nls
- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_cyt

Surprisingly, and in contrast to the strains expressing CroSGD, the yeast stain expressing RseSGD (P1-TEF1-RseSGD-P2_PGK1-CroTHAS_nls) was able to produce tetrahydroalstonine, thus showing that RseSGD is functional in yeast (FIG. 1). Tetrahydroalstonine was detected in both samples from supernatant (filtered medium) and cell pellet.

Example 3

SGD Homology Search

To further investigate, and ultimately enable, functionalization of the critical SGD node in yeast, a homology-search for SGDs against the NCBI database and using the CroSGD protein sequence as a query was performed. From this search, eight different SGD homologs from Catharanthus roseus (CroSGD), Rauvolfia serpentina (RseSGD), Rauvolfia verticillata (RveSGD), Gelsemium sempervirens (GseSGD), Camptotheca acuminate (CacSGD), Scedosporium apiospermum (SapSGD), Uncaria tomentosa (UtoSGD) and Glycine soja (GsoSGD) were selected.

The eight protein sequences were aligned with the t-Coffee web server (FIG. 2).

Among the eight SGDs selected for this test, two (Catharanthus roseus and Rauvolfia serpentina) are known to have SGD activity in vitro, four are putative SGD from MIA producing plants (Rauvolfia verticillata, Gelsemium sempervirens, Camptotheca acuminate and Uncaria tomentosa). Scedosporium apiospermum is a fungus known to produce other alkaloids. Glycine soja, which is unlikely to have SGD activity, was chosen as a negative control. See table 3 below.

TABLE 3

MIA production

in the origin

Abbreviation
Function
Species
Family
organism

RseSGD
In vitro

Rauvolfia serpentina

Apocyanaceae
Yes

verified

SGD

RveSGD
Putative

Rauvolfia verticillate

Apocyanaceae
Yes

SGD

CroSGD
In vitro

Catharanthus roseus

apocyanaceae
Yes

verified

SGD

GseSGD
Putative

Gelsemium

Gelsemiacea
Yes

SGD

sempervirens

UtoSGD
Putative

Uncaria tomentosa

Rubiaceae
Yes

SGD

CacSGD
Putative

Camptotheca

Nyssaseae
Yes

SGD

acuminata

SapSGD
Putative

Scedosporium

Microascaceae
Yes

SGD

apiospermum

(fungi)

GsoSGD
Putative

Glycine soja

Phaseoleae
No

GH1 beta-

gucosidase

Each one of the eight SGD together with the CroHYS (capable of converting strictosidine aglycone to tetrahydroalsoinine) gene were integrated into a MIA-BJ strain expressing CroG8H+CroCYB5+CroCPR+Cro8HGO+CrolS+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2, resulting in strains MIA-CA-1 to MIA-CA-8

MIA-CA-1: MIA-BJ strain+CroSGD+CroHYS

MIA-CA-2: MIA-BJ strain+RseSGD+CroHYS

MIA-CA-3: MIA-BJ strain+RveSGD+CroHYS

MIA-CA-4: MIA-BJ strain+GseSGD+CroHYS

MIA-CA-5: MIA-BJ strain+CacSGD+CroHYS

MIA-CA-6: MIA-BJ strain+SapSGD+CroHYS

MIA-CA-7: MIA-BJ strain+UtoSGD+CroHYS

MIA-CA-8: MIA-BJ strain+GsoSGD+CroHYS

First, all strains were grown (in triplicates) in 150 uL of YPD for overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of synthetic complete (SC) medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL supernatant was filtered through a 0.2 pm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure secologanin, strictosidine and tetrahydroalstonine concentrations.

Yeast strains expressing GseSGD, SapSGD, RveSGD and RseSGD were able to produce tetrahydroalstonine (FIG. 3). Whereas, CacSGD, CroSGD and UtoSGD, as well as their control GsSGD were not able to produce tetrahydroalstonine. The p-value represents comparison between the negative control (GsoSGD) and each of CacSGD, CroSGD and UtoSGD.

The yeast strain expressing RseSGD was able to produce at least 10 μM tetrahydroalstonine.

Example 4

Cellular Localisation and Expression

In order to understand the functional discrepancy between CroSGD and RseSGD in yeast, the two enzymes were GFP-tagged and their subcellular localization was studied. A clear difference in both level of expression and localization was observed for CroSGD and RseSGD.

The yeast cells expressing GFP-linker-CroSGD showed weak expression of CroSGD, as well as a nuclear localization of the CroSGD, whereas the yeast cells expressing GFP-linker-RseSGD showed higher RseSGD expression and a supramolecular localization pattern (FIG. 4) resembling CroSGD localization in planta.

Example 5

Production of Strictosidine Aglycone and Heteroyohimbines

Strictosidine Aglycone and Tetrahydroalstonine

CroSGD or RseSGD alone or in combination with the CroTHAS were inserted into the MIA-BJ strain (CroG8H+CroCYB5+CroCPR+Cro8HGO+CrolS+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2), resulting in strains MIA-BZ-1 to MIA-BZ-4:

- MIA-BZ-1: MIA-BJ strain+pTEF1->CroSGD-tADH1
- MIA-BZ-2: MIA-BJ strain+p TEF1->RseSGD-tADH 1
- MIA-BZ-3: MIA-BJ strain+tCYC1-CroTHAS<-pPGK1-pTEF1->CroSGD-tADH1
- MIA-BZ-4: MIA-BJ strain+tCYC1-CroTHAS<-pPGK1-pTEF1->RseSGD-tADH1

The yeast strains MIA-BZ-1 to MIA-BZ-4 as well as their control (MIA-BJ strain), were tested in batch fermentation using 96-well deep plate as the following.

After 6 days, 200 uL supernatant was filtered through a 0.2 μm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as an internal standard before analysis on the LC-MS.

Strictosidine aglycone was measured by Orbitrap Fusion™ Tribrid™ MS.

Analysis of strictosidine aglycone peaks on the Orbitrap Fusion™ Tribrid™ MS (positive mode, mass 351.1703 Da) is shown in table 4.

TABLE 4

Mass pos mode, 351.1703 Da

Strictosidine aglycone production

4.08 min
4.40 min
4.52 min

MIA-BJ (EZ-Swap, full CroSTR)
N.D.
N.D.
N.D.

MIA-BJ + CroSGD
N.D.
N.D.
N.D.

MIA-BJ + RseSGD
3.90E+06
7.31E+06
4.31E+06

MIA-BJ + CroSGD + CroTHAS
N.D.
N.D.
N.D.

MIA-BJ + RseSGD + CroTHAS
1.56E+06
2.14E+06
1.18E+06

These results show that yeast strains expressing RseSGD are able to convert secologanin and tryptamine into strictosidine aglycone. Whereas the yeast strains expressing CroSGD, alone or in combination with CroTHAS, do not produce strictosidine aglycone. This shows that RseSGD is functional in yeast, while CroSGD is not functional in yeast.

Alstonine

To further explore if yeast could be used as a microbial platform for MIA biosynthesis RseSGD and CroTHAS were co-expressed with a sapargan bridge enzymes (SBE) from either Gelsemium sempervirens (GseSBE), Catharantus roseus (CroSBE) or Rauvolfia serpentina (RseSBE), thereby enabling production of a second heteroyohimbine, alstonine.

Strain MIA-BJ (EZ-Swap, full CroSTR) expressing:

- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_empty vector
- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_P1-FET1-CroSBE
- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_P1-FET1-RseSBE
- P1-TEF1-RseSGD-P2_PGK1-CroTHAS_P1-FET1-GseSBE

The sample caffeine mixtures were analysed on LC-MS to measure secologanin, strictosidine and tetrahydroalstonine concentrations.

The biosynthesis of the heteroyohimbine alstonine in yeast cell factories is shown in triplicates in FIG. 5. Alastonine was measured by Orbitrap Fusion™ Tribrid™ MS.

The yeast cells expressing RseSGD, CroTHAS and GseSBE were capable of converting secologanin and tryptamine to strictosidine aglycone and further capable of converting strictosidine aglycone to tetrahydroalstonine and further capable of converting tetrahydroalstonine to alstonine. This example confirms that RseSGD is functional in yeast.

Example 6

Production of Tabersonine and Catharanthine

To further demonstrate functionalized RseSGD in yeast, the biosynthetic pathway steps from strictosidine aglycone to tabersonine and catharanthine (MIA-DC) were engineered.

Strain MIA-DC:

CroCPR+CroCYB5+CroCPR+CroCYB5+CroSTR+CroGS+RseSGD+CroGO+CroRedoc1+CroRedox2+CroSAT+CroPAS+CroCPAS+CroTS+CroCS

The MIA-DC and MIA-DA (control) strains were tested in batch fermentation using 96-well deep plate as the following.

First, all strains were grown (in triplicates) in 150 uL YPD for overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of synthetic complete (SC) medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL of supernatant was filtered through a 0.2 pm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The production of tabersonine and catharanthine were measured by LC-MS.

Yeast-based production of tabersonine and catharanthine were detected, based on precursor feeding of 0.1 mM of secologanine and 1 mM of tryptamine upstream the RseSGD in strain MIA-DC (FIGS. 6A-D and 7).

Example 7

Expanded SGD Homology Search

To further investigate, and ultimately enable, functionalization of the critical SGD node in yeast, a homology-search for SGDs against the NCO database and the PhytoMetaSyn database was performed using the RseSGD and SapSGD protein sequences as queries. From this search, 28 different SGD homologs were selected from Rauvolfia serpentina (RseSGD2), Vinca minor (VmiSGD1 and VmiSGD3), Tabernaemontana elegans (TeISGD), Amsonia hubrichtii (AhuSGD), Ophiorrhiza pumila, (OpuSGD), Nyssa sinensis, (NsiSGD1 and NsiSGD2), Coffea arabica (CarSGD), Carapichea ipecacuanha (IpeSGD), Handroanthus impetiginosus (HimSGD2 and HimSGD1), Sesamum indicum (SinSGD), Olea europaea (OeuSGD), Actinidia chinensis var. chinensis (AchSGD1, AchSGD2 and AchSGD3), Helianthus annuus (HanSGD), Lactuca sativa (LseSGD), Ipomoea nil (IniSGD), Chelidonium majus (CmaSGD), Vigna unguiculata (VunSGD), Heliocybe sulcate (HsuSGD), Pyricularia grisea (PgrSGD), Lomentospora prolificans (LprSGD), Hydnomerulius pinastri MD-312 (HpiSGD), Madurella mycetomatis (MmySGD), and Moniliophthora roreri MCA 2997 (MroSGD).

The 28 protein sequences together with RseSGD, RveSGD, CroSGD, GseSGD, CacSGD, UtoSGD, GsoSGD, and SapSGD were aligned using the t-coffee server (FIG. 12). Pairwise sequence identities were calculated from this alignment with CLC Main Workbench 8.0. (FIG. 13)

Among the 28 selected sequences for this test two (RseSGD2 and I peSGD) are known to have low SGD activity in vitro, seven are putative beta-glucosidases or hypothetical proteins from MIA producing plants (Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis), one (OeuSGD) is a oleuropein beta-glucosidase from Olea europaea, and 12 are putative beta-glucosidases with various putative activities from plants that do not produce MIAs but a range on different glycosylated natural products (Coffea arabica, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Chelidonium majus, and Vigna unguiculata). Six of the selected sequences are putative beta-glucosidases and hypothetical proteins from fungi (Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, Madurella mycetomatis, and Moniliophthora roreri MCA 2997). Nothing has been reported on glycosylated natural products produced by any of these fungi.

TABLE 5

MIA production

in the origin

Abbreviation
Function
Species
Family
organism

RseSGD2
raucaffricine-

Rauvolfia

Apocynaceae
Yes

O-beta-D-

serpentina

glucosidase

VmiSGD1
Putative

Vinca minor

Apocynaceae
Yes

beta-

glucosidase

VmiSGD3
Putative

Vinca minor

Apocynaceae
Yes

Beta-

glucosidase

TelSGD
Putative

Tabernaemontana

Apocynaceae
Yes

beta-

elegans

glucosidase

AhuSGD
Putative

Amsonia

Apocynaceae
Yes

beta-

hubrichtii

glucosidase

OpuSGD
Putative

Ophiorrhiza

Rubiaceae
Yes

beta-

pumila

glucosidase

NsiSGD1
Hypothetical

Nyssa sinensis

Nyssaceae
Yes

protein

NsiSGD2
Hypothetical

Nyssa sinensis

Nyssaceae
Yes

protein

CarSGD
Putative

Coffea arabica

Rubiaceae
No

raucaffricine-

O-beta-D-

glucosidase

IpeSGD
Beta-

Carapichea

Rubiaceae
No

glucosidase

ipecacuanha

HimSGD1
Putative

Handroanthus

Bignoniaceae
No

beta-

impetiginosus

glucosidase

HimSGD2
Putative

Handroanthus

Bignoniaceae
No

beta-

impetiginosus

glucosidase

SinSGD
Putative

Sesamum

Pedaliaceae
No

beta-

indicum

glucosidase

OeuSGD
Oleuropein

Olea europaea

Oleaceae
No

beta-

glucosidase

AchSGD1
Putative

Actinidia

Actinidiaceae
No

beta-

chinensis var.

glucosidase

chinensis

AchSGD2
Putative

Actinidia

Actinidiaceae
No

beta-

chinensis var.

glucosidase

chinensis

AchSGD3
Putative

Actinidia

Actinidiaceae
No

beta-

chinensis var.

glucosidase

chinensis

HanSGD
Putative SGD

Helianthus

Asteraceae
No

annuus

LsaSGD
Putative

Lactuca sativa

Asteraceae
No

beta-

glucosidase

IniSGD
Putative

Ipomoea nil

Convolvulaceae
No

raucaffricine-

O-beta-D-

glucosidase

CmaSGD
Putative

Chelidonium

Papaveraceae
No

beta-

majus

glucosidase

VunSGD
Putative

Vigna

Fabaceae
No

cyanogenic

unguiculata

beta-

glucosidase

HsuSGD
Putative

Heliocybe

Gloeophyllaceae
No

beta-

sulcata

(fungi)

glucosidase

PgrSGD
Putative

Pyricularia

Magnaporthaceae
No

lactase-

grisea

(fungi)

phlorizin

hydrolase

LprSGD
Hypothetical

Lomentospora

Microascaceae
No

protein

prolificans

(fungi)

HpiSGD
Putative GH1

Hydnomerulius

(fungi)
No

family beta-

pinastri MD-312

glucosidase

MmySGD
Putative

Madurella

(fungi)
No

Beta-

mycetomatis

glucosidase

MroSGD
Putative

Moniliophthora

(fungi)
No

beta-

roreri MCA 2997

glucosidase

Each one of the 28 SGD and CroSGD together with the CroHYS (capable of converting strictosidine aglycone to tetrahydroalsoinine) gene were integrated into a MIA-FA strain expressing CroG8H+Vmi8HGO-A+NcMLP+NcISY+CroCYB5+CroCPR+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2+CroHYS , resulting in strains MIA-FC-1 to MIA-FC-29. CroSGD was included as a negative control since it was already shown in example 2 to be unable to convert strictosidine to strictosidine aglycone in yeast.

MIA-FC-1: MIA-FA+CroSGD

MIA-FC-2: MIA-FA+VmiSGD1

MIA-FC-3: MIA-FA+AhuSGD

MIA-FC-4: MIA-FA+HimSGD2

MIA-FC-5: MIA-FA+SinSGD

MIA-FC-6: MIA-FA+TelSGD

MIA-FC-7: MIA-FA+VunSGD

MIA-FC-8: MIA-FA+NsiSGD1

MIA-FC-9: MIA-FA+LprSGD

MIA-FC-10: MIA-FA+AchSGD1

MIA-FC-11: MIA-FA+HsuSGD

MIA-FC-12: MIA-FA+MroSGD

MIA-FC-13: MIA-FA+RseSGD2

MIA-FC-14: MIA-FA+PgrSGD

MIA-FC-15: MIA-FA+OpuSGD

MIA-FC-16: MIA-FA+HpiSGD

MIA-FC-17: MIA-FA+HanSGD1

MIA-FC-18: MIA-FA+AchSGD2

MIA-FC-19: MIA-FA+HimSGD1

MIA-FC-20: MIA-FA+IpeSGD

MIA-FC-21: MIA-FA+LsaSGD1

MIA-FC-22: MIA-FA+CarSGD

MIA-FC-23: MIA-FA+OeuSGD

MIA-FC-24: MIA-FA+AchSGD3

MIA-FC-25: MIA-FA+CmaSGD

MIA-FC-26: MIA-FA+MmySGD

MIA-FC-27: MIA-FA+VmiSGD3

MIA-FC-28: MIA-FA+IniSGD

MIA-FC-29: MIA-FA+NsiSGD2

First, all strains were grown (in triplicates) in 150 uL of YPD overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of synthetic complete (SC) medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL supernatant was filtered through a 0.2 pm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure secologanin and tetrahydroalstonine concentrations.

Yeast strains expressing VmiSGD1, AhuSGD, HimSGD2, SinSGD, TelSGD, VunSGD, NsiSGD1, LprSGD, AchSGD1, HsuSGD, MroSGD, RseSGD2, PgrSGD, OpuSGD, HpiSGD, HanSGD1, AchSGD2, HimSGD1, IpeSGD, LsaSGD1, and CarSGD were able to produce tetrahydroalstonine and hereby also strictosidine aglycone (FIG. 8) whereas yeast strains expressing OeuSGD, AchSGD3, CmaSGD, MmySGD, VmiSGD3, IniSGD, and NsiSGD2, as well as the negative control CroSGD were not able to produce tetrahydroalstonine. The p-value represents comparison between the negative control (CroSGD) and each of OeuSGD, AchSGD3, CmaSGD, MmySGD, VmiSGD3, IniSGD, and NsiSGD2. More homologs from MIA and non-MIA producing plants were tested, but none were able to produce tetrahydroalstonine.

Example 8

8.1 Characterization of SGD Domains

To investigate which sequence domains are critical for SGD functionalization in yeast the protein sequences of a functional SGD (RseSGD) and a non-functional SGD (CroSGD) were aligned and divided into four domains which were then reassembled in all 16 possible combinations. The domains of RseSGD are termed R and the domains of CroSGD are termend C in this Example. Two combinations (RRRR-SGD and CCCC-SGD) corresponds to the two wild type protein sequences (RseSGD and CroSGD). The four domains are 76 to 203 amino acids long with varying sequence identity (table 6).

TABLE 6

Domain 1
Domain 2
Domain 3
Domain 4

start
stop
start
stop
start
stop
start
stop

RseSGD
M1
R115
F116
G266
E267
G456
V457
stop

115
152
190
76

CroSGD
M1
R123
F124
G274
E275
G477
V478
stop

123
151
203
78

Seq_ID
63.80%
79.60%
64.20%
77.60%

Each of the 16 shuffled SGDs were cloned with USER fusion (Geu-Flores F et al. 2007) on a plasmid and transformed into a MIA-FA strain capable of expressingCroG8H+Vmi8HGO-A+NcMLP+NcISY+CroCYB5+CroCPR+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2+CroHYS, resulting in strains MIA-FD-1 to MIA-FD-16 (table 7). The MIA-FA strain is capable of synthesizing strictosidine when fed tryptamine and secologanin, or other precursors in the secologanin biosynthetic pathway from geraniol, and is also capable of converting strictosidine aclycone to tetrahydroalstonine if a functional SGD capable of converting strictosidine to strictosidine aglycone is coexpressed.

TABLE 7

Strain
Domain 1
Domain 2
Domain 3
Domain 4

MIA-FD-1: MIA-FA +
CroSGD
CroSGD
CroSGD
CroSGD

pRS413U_pTEF1_CCCC-SGD

MIA-FD-2: MIA-FA +
CroSGD
RseSGD
CroSGD
CroSGD

pRS413U_pTEF1_CRCC-SGD

MIA-FD-3: MIA-FA +
CroSGD
RseSGD
CroSGD
RseSGD

pRS413U_pTEF1_CRCR-SGD

MIA-FD-4: MIA-FA +
CroSGD
CroSGD
CroSGD
RseSGD

pRS413U_pTEF1_CCCR-SGD

MIA-FD-5: MIA-FA +
CroSGD
RseSGD
RseSGD
CroSGD

pRS413U_pTEF1_CRRC-SGD

MIA-FD-6: MIA-FA +
CroSGD
CroSGD
RseSGD
RseSGD

pRS413U_pTEF1_CCRC-SGD

MIA-FD-7: MIA-FA +
CroSGD
RseSGD
RseSGD
RseSGD

pRS413U_pTEF1_CRRR-SGD

MIA-FD-8: MIA-FA +
CroSGD
CroSGD
RseSGD
RseSGD

pRS413U_pTEF1_CCRR-SGD

MIA-FD-9: MIA-FA +
RseSGD
RseSGD
CroSGD
CroSGD

pRS413U_pTEF1_RRCC-SGD

MIA-FD-10: MIA-FA +
RseSGD
CroSGD
CroSGD
CroSGD

pRS413U_pTEF1_RCCC-SGD

MIA-FD-11: MIA-FA +
RseSGD
RseSGD
CroSGD
RseSGD

pRS413U_pTEF1_RRCR-SGD

MIA-FD-12: MIA-FA +
RseSGD
CroSGD
CroSGD
RseSGD

pRS413U_pTEF1_RCCR-SGD

MIA-FD-13: MIA-FA +
RseSGD
RseSGD
RseSGD
CroSGD

pRS413U_pTEF1_RRRC-SGD

MIA-FD-14: MIA-FA +
RseSGD
CroSGD
RseSGD
CroSGD

pRS413U_pTEF1_RCRC-SGD

MIA-FD-15: MIA-FA +
RseSGD
CroSGD
RseSGD
RseSGD

pRS413U_pTEF1_RCRR-SGD

MIA-FD-16: MIA-FA +
RseSGD
RseSGD
RseSGD
RseSGD

pRS413U_pTEF1_RRRR-SGD

First, all strains were grown (in triplicates) in 150 uL of synthetic complete without histidine (SC-HIS) overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of SC-HIS medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL supernatant was filtered through a 0.2 pm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure secologanin tetrahydroalstonine concentrations.

Results

Yeast strains expressing CRRC-SGD, RRRC-SGD, RCRC-SGD, CCRC-SGD, CRRR-SGD, CCRR-SGD, RCRR-SGD, and RRRR-SGD were able to produce tetrahydroalstonine (FIG. 9). All functional SGD variants have RseSGD domain 3. All SGD variants with CroSGD domain 3 were not able to produce tetrahydroalstonine. The identity of domain 1 and 2 has low or no effect. Of the functional SGD variants, the four sequences with RseSGD domain 3 and domain 4 (CRRR-SGD, CCRR-SGD,

RCRR-SGD, and RRRR-SGD) are able to produce the highest amount of tetrahydroalstonine. CCRR-SGD is the best variant capable of producing more tetrahydroalstonine than the wild type RseSGD (RRRR-SGD)

8.2 Production of Tetrahydroalstonine in a Yeast Strain Expressing CCRR_SGD

The best SGD variant (CCRR-SGD) were integrated in the MIA-FA strain MIA-FA capable of strain expressing CroG8H+Vmi8HGO-A+NcMLP+NcISY+CroCYB5+CroCPR+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2+CroHYS, resulting in the strain MIA-FE:

MIA-FE: MIA-FA+CCRR-SGD

First, MIA-FE was grown (in triplicates) in 150 uL of YPD overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of synthetic complete (SC) medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL supernatant was filtered through t a 0.2 μm filter membrane suitable for aquaeus solutions such as he AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure tetrahydroalstonine concentrations.

Results

The yeast strain expressing CCRR-SGD was able to produce 13.30 μM (±1.29 μM) tetrahydroalstonine.

Example 9

Rescuing the function of other SGD homologs with RseSGD domain 3 and 4

Encouraged by the capability of RseSGD domain 3 and 4 to rescue the non-functional CroSGD in yeast three more SGD variants were cloned swapping domain 3 and 4 between RseSGD and UtoSGD (U), GseSGD (G), and RveSGD (V) respectively. Even though swapping domain 3 alone was able to make CroSGD functional swapping both domain 3 and domain 4 gave the largest improvement and therefor this swapping strategy was expanded to other SGD sequences.

The sequences of the four domains of UtoSGD, GseSGD and RveSGD were determined from a multiple sequence alignment (FIG. 12). The first residue in domain 1 is always the start methionine and the last residue in domain 4 is always the last residue in the sequence. The remaining first and last residues are defined as the residues aligning with the first and last residues in the four RseSGD domains. Table 8 summarizes the four domains of RseSGD, CroSGD, UtoSGD, GseSGD, and RveSGD.

TABLE 8

Domain 1
Domain 2
Domain 3
Domain 4
Seq_ID to

start
stop
start
stop
start
stop
start
stop
RseSGD

RseSGD
M1
R115
F116
G266
E267
G456
V457
stop

UtoSGD
M1
R88
F89
G277
K278
G459
V460
stop
40.70%

GseSGD
M1
R92
F93
G265
Q266
G456
V457
stop
53.90%

CroSGD
M1
R123
F124
G274
E275
G477
V478
stop
70.30%

RveSGD
M1
R115
F116
G265
E266
G459
V460
stop
89.90%

Three domain-swap SGD variants and the three wild type SGDs were cloned with USER fusion. The plasmids were transformed into a MIA-FA strain capable of expressing CroG8H+Vmi8HGO-A+NcMLP+NcISY+CroCYB5+CroCPR+CrolO+CroSTR+CroSLS+Cro7DLGT+Cro7DLH+CroLAMT+CroADH2+CroHYS, resulting in strains MIA-FD-17 to MIA-FD-22 (table 9). The MIA-FA strain is capable of synthesizing strictosidine when fed tryptamine and secologanin, or other precursors in the secologanin biosynthetic pathway from geraniol, and is also capable of converting strictosidine aclycone to tetrahydroalstonine if a functional SGD capable of converting strictosidine to strictosidine aglycone is coexpressed

TABLE 9

MIA-FD-17: MIA-FA +
UtoSGD
UtoSGD
UtoSGD
UtoSGD

pRS413U_pTEF1_UtoSGD-SGD

MIA-FD-18: MIA-FA +
UtoSGD
UtoSGD
RseSGD
RseSGD

pRS413U_pTEF1_UURR-SGD

MIA-FD-19: MIA-FA +
GseSGD
GseSGD
GseSGD
GseSGD

pRS413U_pTEF1_GseSGD-SGD

MIA-FD-20: MIA-FA +
GseSGD
GseSGD
RseSGD
RseSGD

pRS413U_pTEF1_GGRR-SGD

MIA-FD-21: MIA-FA +
RveSGD
RveSGD
RveSGD
RveSGD

pRS413U_pTEF1_RveSGD-SGD

MIA-FD-22: MIA-FA +
RveSGD
RveSGD
RseSGD
RseSGD

pRS413U_pTEF1_VVRR-SGD

First, all six strains plus two control strains (MIA-FD-1 and 8) were grown (in triplicates) in 150 uL of synthetic complete without histidine (SC-HIS) overnight to saturation. Then, 10 ul preculture was transferred into 500 uL of SC-HIS medium with 2% glucose, supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 6 days, 200 uL supernatant was filtered through a 0.2 pm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure tetrahydroalstonine concentrations.

As already shown in example 9, swapping in RseSGD domain 3 and 4 rescued the function of the non-functional CroSGD (FIG. 9). Wild type RveSGD is capable of producing tetrahydroalstonine. Swapping in RseSGD domain 3 and 4 improved the tetrahydroalstonine production about seven fold. GseSGD and UtoSGD have lower sequence identity to RseSGD (53.9% and 40.7% respectively) than CroSGD and RveSGD (70.3% and 89.9%). GseSGD can produce tetrahydroalstonine in low concentrations whereas UtoSGD is incapable of tetrahydroalstonine production. Swapping in RseSGD domain 3 and 4 into these two SGDs did not rescue the function of UtoSGD and abolished the low tetrahydroalstonine production of GseSGD.

Example 10

Minimum Strictosidine Aglycone Production in Yeast

Strictosidine aglycone is chemically unstable and was impossible to either purchase or purify to use as a standard for quantification. The minimum strictosidine aglycone produced by the tested SGD homologs was calculated from the measured tetrahydroalstonine produced by the yeast strains and the measured secologanin left in the media. It is possible that not all produced strictosidine aglycone is converted to tetrahydroalstonine, and therefore the true strictosidine aglycone titres might in some cases be higher than the estimated minimum production.

Strictosidine Aqlycone Production in μM:

Since strictosidine aglycone is converted to tetrahydroalstonine in equimolar amounts, the minimum strictosidine aglycone titre equals the tetrahydroalstonine titre.

c(strictosidine aglycone)=c(tetrahydroalstonine)

Strictosidine Alycone Yields:

The minimum strictosidine algycone yield can be estimated from the strictosidine aglycone titre and the theoretical strictosidine titre. It is assumed that all secologanin taken up by the yeast strain is converted to strictosidine.

Strictosidine_aglycone_%=c(strictosidine aglycone)/(c(secologanin supplemented in media)−c(secologanin left after cultivation))

Example 11

Production of THA in Escherichia coli

To test if RseSGD or CroSGD could be used for production of strictosidine aglycone and MIAs in prokaryotic microorganisms an expression system was established in the gram-negative bacterium Escherichia coli for in vivo conversion of secologanin and tryptamine to strictosidine by CroSTR, conversion of strictosidine to strictosidine aglycone by RseSGD or CroSGD and conversion of strictosidine aglycone to tetrahydroalstonine by CroHYS. Two low-copy plasmids were cloned for co-expression of the three genes from a polycistronic mRNA under control of a medium strength constitutive promoter. The plasmids were based on pCfB3510(p15A_P2BCD2GFP).

The two plasmids and an empty plasmid were transformed into the strain DH5-α giving the three strains MIA-ECO-1 to MIA-ECO-3.

MIA-ECO-1: D H5-α+p15A-AmpR-CroSTR-CroHYS-CroSGD

MIA-ECO-2: DH5-α+p15A-AmpR-CroSTR-CroHYS-RseSGD

MIA-ECO-3: DH5-α+p15A-AmpR

First, all three strains were grown (in triplicates) in 150 uL of Lysogeny broth (LB) medium with 100 μg/mL ampicillin overnight to saturation. Then, 10 ul preculture was transferred into 500 uL LB medium with 100 μg/mL ampicillin and supplemented with 0.1 mM of secologanin and 1 mM of tryptamine. After 48 hours, 200 uL supernatant was filtered through a 0.2 μm filter membrane suitable for aquaeus solutions such as the AcroPrep™ Advance, 350 uL, 0.2 micron Supor® membrane for media/water. Next, 20 uL of 250 mg/L caffeine was added to each sample as internal standard before analysis on the LC-MS.

The sample caffeine mixtures were analysed on LC-MS to measure secologanin, strictosidine, and tetrahydroalstonine concentrations.

Results

The E. coli strain MIA-ECO-2 expressing RseSGD, CroSTR, and CroHYS was able to produce tetrahydroalstonine (FIG. 11-B). No strictosidine was detected in the media of the E. coli expressing RseSGD. MIA-ECO-1 expressing CroSGD, CroSTR, and CroHYS produced strictosidine (FIG. 11-A) but no tetrahydroalstonine, indicating that like in yeast RseSGD is functional and CroSGD is non-functional.

REFERENCES

Geerlings, A., Ibanez, M. M., Memelink, J., van Der Heijden, R. & Verpoorte, R. Molecular cloning and analysis of strictosidine beta-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus. J. Biol. Chem. 275, 3051-3056 (2000).

Fernando Geu-Flores, Hussam H. Nour-Eldin, Morten T. Nielsen and Barbara A. Halkier 2007. USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Research, 2007, Vol. 35, No. 7 e55. doi:10.1093/nar/gkm106

Guirimand G., Courdavault V., Lanoue A., Mahroug S., Guihur A., Blanc N., Giglioli-Guivarc'h N., St-Pierre B., Burlat V. Strictosidine activation in Apocynaceae: towards a “nuclear time bomb”? BMC Plant Biology 2010, 10:182

Jakoc̆iūnas T, Rajkumar A S, Zhang J, Arsovska D, Rodriguez A, Jendresen C B, Skjodt M L, Nielsen A T, Borodina I, Jensen M K, Keasling J D. CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. ACS Synth Biol. 2015 Nov. 20; 4(11):1226-34. doi: 0.1021/acssynbio.5b00007. Epub 2015 Mar. 26.

Jensen N B, Strucko T, Kildegaard K R, David F, Maury J, Mortensen U H, Forster J, Nielsen J, Borodina I. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 2014 March; 14(2):238-48. doi: 10.1111/1567-1364.12118. Epub 2013 Nov. 18.

Luijendick T. J. C., Stenvens, L. H., Verpoorte R. Reaction for the Localization of Strictosidine Glucosidase Activity on Polyacrylamide gels. Phytochemical analysis (1996). doi:3.0.00;2-H″>10.1002/(SICI)1099-1565(199601)7:1<16::AID-PCA280>3.0.CO; 2-H.

Stavrinides A., Tatsis E. C., Foureau E., Caputi L., Kellner F., Courdavault V., O'Connor S. E. Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism. Chemistry & Biology 22, 336-341, Mar. 19, 2015

Steyaert J, Van Melderen L, Bernard P, Thi M H, Loris R, Wyns L, Couturier M. J Mol Purification, circular dichroism analysis, crystallization and preliminary X-ray diffraction analysis of the F plasmid CcdB killer protein Biol. 1993 May 20; 231(2):513-5.

WO 00/4220: Verpoorte, R., Van Der Heijden, R., Memelink, J. & Geerlings, A. Strictosidine glucosidase from Catharanthus roseus and its use in alkaloid production. World Patent (2000).

Items

1. A microorganism capable of producing strictosidine aglycone, said microorganism expresses
- a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone,
- wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,
- and/or;
- wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

- wherein D₁is a first amino acid sequence from a first SGD,
- wherein D₂is a second amino acid sequence from a second SGD,
- wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,
- wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,
2. wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD. The microorganism according to item 1, further expressing
- a strictosidine synthase (STR), capable of converting secologanin and tryptamine to strictosidine, whereby the microorganism is capable of synthesising strictosidine,
- wherein said STR is preferably CroSTR or variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 30.
3. The microorganism according to any one of the preceding items, wherein D₁comprises or consists of an amino acid sequence corresponding to amino acids M1 to R115 of SEQ ID NO:24.
4. The microorganism according to any one of the preceding items, wherein D₂comprises or consists of an amino acid sequence corresponding to amino acids F116 to G266 of SEQ ID NO:24.
5. The microorganism according to any one of the preceding items, wherein D₄comprises or consists of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92.
6. The microorganism according to any one of the preceding items, wherein at least one of D₁, D₂or D₄is from an SGD which is native to a first organism selected from Gelsemium sempervirens, Scedosporium apiospermum or Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 2997.
7. The microorgagnism according to any one of the preceding items, wherein the first SGD, the second SGD and the fourth SGD are identical or different.
8. The microorganism according to any one of the preceding items, wherein two of the first SGD, the second SGD and the fourth SGD are identical, or wherein the first SGD, the second SGD and the fourth SGD are different, or wherein the first SGD, the second SGD and the fourth SGD are identical.
9. The microorganism according to any one of the preceding items, wherein said mosaic SGD comprises or consists of an amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 108, or variants thereof having at least 90% identity or homology thereto, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity or homology thereto.
10. The microorganism according to any one the preceding items, further expressing
- a tetrahydroalstonine synthase (THAS) and/or a heteroyohimbine synthase (HYS), capable of converting strictosidine aglycone to tetrahydroalstonine, whereby the microorganism is capable of synthesising tetrahydroalstonine,
- wherein said THAS is preferably CroTHAS and/or HYS is CroHYS or variants thereof, having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 28 and/or SEQ ID NO: 46.
11. The microorganism according to any of the preceding items, further expressing
- a sarpargan bridge enzymes (SBE), capable of converting tetrahydroalstonine and ajmalicine to a heteroyohimbine selected from the group consisting of alstonine and serpentine, whereby the microorganism is capable of synthesising alstonine and serpentine,
- wherein said SBE is preferably GseSBE or variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 29.
12. The microorganism according to any one of the preceding items, further expressing
- a NADPH-cytochrome P450 reductase (CPR);
- a Cytochrome b5 (CYB5);
- a Geissoschizine synthase (GS);
- a Geissoschizine oxidase (GO);
- a Redox1;
- a Redox2;
- a Stemmadenine O-acetyltransferase (SAT);
- a O-acetylstemmadenine oxidase (PAS);
- a Dehydroprecondylocarpine acetate synthase (DPAS);
- a Tabersonine synthase (TS); and/or
- a Catharanthine synthase (CS),
- whereby the microorganism is capable of synthesising tabersonine and/or catharanthine,
- wherein preferably said CPR is CroCPR, said CYB5 is CroCYB5, said GS is CroSG, said GO is CroGO, said Redox1 is CroRedox1, said Redox2 is CroRedox2, said SAT is CroSAT, said PAS is CroPAS, said DPAS is CroDPAS, said TS is CroTS and/or said CS is CroCS or variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and/or SEQ ID NO: 41, respectively.
13. The microorganism according to any one of the preceding items, capable of producing strictosidine aglycone with a titre of at least 1 μM, such as at least 2 μM, such as at least 4 μM, such as at least 6 μM, such as at least 8 μM such as at least 10 μM or more.
14. The microorganism according to item 10, capable of producing tetrahydroalstonine with a titre of at least 1 μM, such as at least 2 μM, such as at least 4 μM, such as at least 6 μM, such as at least 8 μM such as at least 10 μM or more.
15. The microorganism according to item 11, capable of producing alstonine with a titre of at least 1 μM, such as at least 2 μM, such as at least 4 μM, such as at least 6 μM, such as at least 8 μM such as at least 10 μM or more.
16. The microorganism according to item 12, capable of producing tabersonine with a titre of at least 0.01 μM, such as at least 0.02 μM.
17. The microorganism according to item 12, capable of producing catharanthine with a titre of at least 0.01 μM, such as at least 0.02 μM.
18. The microorganism according to any of the preceding items, wherein the microorganism is selected from the group consisting of yeasts, bacteria, archaea, fungi, protozoa, algae, and viruses, preferably the microorganism is a yeast or a bacteria.
19. The microorganism according to any one of the preceding items, wherein the microorganism is a bacteria.
20. The microorganism according to item 19, wherein the genus of said bacteria is selected from the groups consisting of Escherichia, Corynebacterium, Pseudomonas, Bacillus, Lactococcus, Lactobacillus, Halomonas, Bifidobacterium and Enterococcus.
21. The microorganism according to any one of items 19 to 20, wherein the bacteria is selected from the group consisting of Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, Bacillus subtilis, Lactococcus bacillus, Halomonas elongate, Bifidobacterium infantis and Enterococcus faecal.
22. The microorganism according to any one of items 19 to 21, wherein the bacteria is Escherichia coli.
23. The microorganism according to any one of the preceding items, wherein the microorganism is a yeast.
24. The microorganism according to item 23, wherein the genus of said yeast cell is selected from the group consisting of Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
25. The microorganism according to any one of items 23 to 24, wherein the yeast is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan and Yarrowia lipolytica
26. The microorganism according to any one of items 23 to 25, wherein the yeast is Saccharomyces cerevisiae.
27. The microorganism according to any of the preceding items, wherein the microorganism comprises a nucleic acid encoding SGD, said nucleic acid having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1.
28. A method of producing strictosidine aglycone in a microorganism, said method comprising the steps of:
- a) providing a microorganism, said cell expressing:
  - a strictosidine-beta-glucosidase (SGD), capable of converting strictosidine to strictosidine aglycone;
- b) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- c) optionally, recovering the strictosidine aglycone;
- d) optionally, further converting the strictosidine aglycone to monoterpenoid indole alkaloids,
- wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,
- and/or;
- wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

- wherein D₁is a first amino acid sequence from a first SGD,
- wherein D₂is a second amino acid sequence from a second SGD,
- wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,
- wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,
- wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.
29. The microorganism according to item 28, wherein the SGD, the heterologous SGD and/or the mosaic SGD is as defined in any one of the preceding items.
30. The microorganism according to any one of items 28 to 29, wherein D₁comprises or consists of an amino acid sequence corresponding to amino acids M1 to R115 of SEQ ID NO:24.
31. The microorganism according to any one of items 28 to 30, wherein D2 comprises or consists of an amino acid sequence corresponding to amino acids F116 to G266 of SEQ ID N0:24.
32. The microorganism according to any one of items 28 to 31, wherein D₄comprises or consists of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92.
33. The microorganism according to any one of items 28 to 32, wherein at least one of D₁, D₂or D₄is from an SGD which is native to a first organism selected from Gelsemium sempervirens, Scedosporium apiospermum or Rauvolfia verticillata, Vinca minor, Tabernaemontana elegans, Amsonia hubrichtii, Ophiorrhiza pumila, Nyssa sinensis, Coffea arabica, Carapichea ipecacuanha, Handroanthus impetiginosus, Sesamum indicum, Actinidia chinensis var. chinensis, Helianthus annuus, Lactuca sativa, Ipomoea nil, Vigna unguiculata, Heliocybe sulcate, Pyricularia grisea, Lomentospora prolificans, Hydnomerulius pinastri MD-312, and Moniliophthora roreri MCA 2997.
34. The microorgagnism according to any one of items 28 to 33, wherein the first SGD, the second SGD and the fourth SGD are identical or different.
35. The microorganism according to any one of items 28 to 34, wherein two of the first SGD, the second SGD and the fourth SGD are identical, or wherein the first SGD, the second SGD and the fourth SGD are different, or wherein the first SGD, the second SGD and the fourth SGD are identical.
36. The microorganism according to items 28 to 35, wherein said mosaic SGD comprises or consists of an amino acid sequence of SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 108, or variants thereof having at least 90% identity or homology thereto, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity or homology thereto.
37. The method according to any one of items 28 to 36, wherein the substrate is secologanin and/or tryptamine, and wherein said microorganism further expresses:
- a strictosidine synthase (STR), capable of converting secologanin and tryptamine to strictosidine;
- wherein said STR is preferably CroSTR or variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 30.
38. The method according to any one of items 28 to 37, wherein the method comprising step d) and wherein said microorganism further expresses:
- a tetrahydroalstonine synthase (THAS) and/or or a heteroyohimbine synthase (HSY), capable of converting strictosidine aglycone to tetrahydroalstonine;
- wherein preferably said THAS is identical to or has at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 28 and/or HYS is identical to or has at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 46.
39. The method according to items 28 to 38, wherein said method further comprises the step of recover tetrahydroalstonine.
40. The method according to any one of items 28 to 39, wherein the method comprising step d) and wherein said microorganism further expresses:
- a sapargan bridge enzyme (SBE), capable of converting tetrahydroalstonine to alstonine;
- wherein preferably said SBE is identical to or has at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 29.
41. The method according to item 40, wherein said method further comprises the step of recovering alstonine.
42. The method according to any one of items 28 to 41, wherein the method comprises step d) and wherein said microorganism further expresses:
- a NADPH-cytochrome P450 reductase (CPR);
- a Cytochrome b5 (CYB5);
- a Geissoschizine synthase (GS);
- a Geissoschizine oxidase (GO);
- a Redox1;
- a Redox2;
- a Stemmadenine O-acetyltransferase (SAT);
- a O-acetylstemmadenine oxidase (PAS);
- a Dehydroprecondylocarpine acetate synthase (DPAS);
- a Tabersonine synthase (TS); and/or
- a Catharanthine synthase (CS),
- wherein preferably said CPR is CroCPR, said CYB5is CroCYB5, said GS is CroSG, said GO is CroGO, said Redox1 is CroRedox1, said Redox2 is CroRedox2, said SAT is CroSAT, said PAS is CroPAS, said DPAS is CroDPAS, said TS is CroTS and/or said CS is CroCS or variants thereof having at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and/or SEQ ID NO: 41, respectively.
- wherein the microorganism is capable of producing tabersonine and/or catharanthine, optionally wherein said method further comprises the step of recovering tabersonine and/or catharanthine.
43. The method according to any one of items 28 to 42, wherein the medium comprises at least strictosidine, preferably at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.5 mM, such as at least 1 mM.
44. The method according to any one of items 288 to 43, wherein the medium comprises at least tryptamine and secologanin, preferably at a concentration of at least 0.05 mM, such as at least 0.1 mM, such as at least 0.5 mM, such as at least 1 mM.
45. A nucleic acid construct comprising a sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:72, SEQ ID NO: 73, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106 and/or SEQ ID NO:107.
46. The nucleic acid construct according to item 45, further comprising a sequence identical to or having at 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 7.
47. The nucleic acid construct according to any of items 45 to 46, further comprising a sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 5 and/or SEQ ID NO: 23.
48. The nucleic acid construct according to any of items 45 to 47, further comprising a nucleic acid sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 6.
49. The nucleic acid construct according to any one of items 45 to 48, further comprising a nucleic acid sequence identical to or having at least 90% identity, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and/or SEQ ID NO: 18.
50. The nucleic acid construct according to any of items 45 to 49, wherein at least one of the one or more nucleic acid sequences are under the control of an inducible promoter.
51. The nucleic acid construct according to any of items 45 to 50, wherein the nucleic acid construct is a vector such as an integrative vector or a replicative vector.
52. A vector comprising a nucleic acid sequence as defined in any one of items 45 to 50.
53. A host cell comprising one or more nucleic acid sequence as defined in any of items 45 to 50, or the vector according to item 52.
54. A kit of parts comprising a microorganism according to any one of items 1 to 36, and/or nucleic acid constructs according to any one of items 45 to 50, and/or a vector according to item 52, and instructions for use.
55. Use of the nucleic acid construct according to any one of items 45 to 50, of the microorganism according to any of items 1 to 36, the vector according to item 52, or the host cell according to item 53, for the production of strictosidine aglycone and/or tetrahydroalstonine, alstonine, tabersonine and/or catharanthine in a microorganism.
56. The use according to item 55 in the method according to items 37 to 44.
57. Strictosidine aglycone obtained by the method according to any of items 37 to 44.
58. Tetrahydroalstonine obtained by the method according to any of items 39 to 44.
59. Heteroyohimbine obtained by the method according to any of items 41 to 44.
60. Tabersonine and/or catharanthine obtained by the method according item 42 to 44.

61. A method of producing monoterpenoid indole alkaloids (MIAs) in a microorganism, said method comprising the steps of:

- a) providing a microorganism capable of converting strictosidine to tabersonine and/or catharanthine, said cell expressing:
  - a strictosidine-beta-glucosidase (SGD);
  - a NADPH-cytochrome P450 reductase (CPR);
  - a Cytochrome b5 (CYB5);
  - a Geissoschizine synthase (GS);
  - a Geissoschizine oxidase (GO);
  - a Redox1;
  - a Redox2;
  - a Stemmadenine O-acetyltransferase (SAT);
  - a O-acetylstemmadenine oxidase (PAS);
  - a Dehydroprecondylocarpine acetate synthase (DPAS);
  - a Tabersonine synthase (TS); and/or
  - a Catharanthine synthase (CS);
  - optionally, a strictosidine synthase (STR);
- b) incubating said microorganism in a medium comprising strictosidine or a substrate which can be converted to strictosidine by said microorganism;
- c) optionally, recovering the MIAs;
- d) optionally, processing the MIAs into a pharmaceutical compound,
- wherein said SGD is a heterologous SGD selected from RseSGD (SEQ ID NO: 24), GseSGD (SEQ ID NO: 25), SapSGD (SEQ ID NO: 26), RveSGD (SEQ ID NO: 27), VmiSGD1 (SEQ ID NO: 47), AhuSGD (SEQ ID NO: 48), HimSGD2 (SEQ ID NO: 49), SinSGD (SEQ ID NO: 50), TelSGD (SEQ ID NO: 51), VunSGD (SEQ ID NO: 52), NsiSGD1 (SEQ ID NO: 53), LprSGD (SEQ ID NO: 54), AchSGD1 (SEQ ID NO: 55), HsuSGD (SEQ ID NO: 56), MroSGD (SEQ ID NO: 57), RseSGD2 (SEQ ID NO: 58), PgrSGD (SEQ ID NO: 59), OpuSGD (SEQ ID NO: 60), HpiSGD (SEQ ID NO: 61), HanSGD1 (SEQ ID NO: 62), AchSGD2 (SEQ ID NO: 63), HimSGD1 (SEQ ID NO: 64), IpeSGD (SEQ ID NO: 65), LsaSGD1 (SEQ ID NO: 66), or CarSGD (SEQ ID NO: 67) or variants thereof having at least 70%, such as at least 80%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity thereto,
- and/or;
- wherein said SGD is a mosaic SGD, wherein said mosaic SGD comprises an amino acid sequence having the general formula

D₁-D₂-D₃-D₄

- wherein D₁is a first amino acid sequence from a first SGD,
- wherein D₂is a second amino acid sequence from a second SGD,
- wherein D₃is a third amino acid sequence comprising or consisting of amino acids of SEQ ID NO:91 or a variant thereof having at least 90% identity to SEQ ID NO: 91,
- wherein D₄is a fourth amino acid sequence from a fourth SGD or an amino acid sequence consisting of amino acids of SEQ ID NO:92 or a variant thereof having at least 90% identity to SEQ ID NO: 92,

wherein said first SGD, second SGD and fourth SGD can be the same or different, with the proviso that said first SGD, second SGD and fourth SGD are not all RseSGD.

62. The method according to item 61, wherein the microorganism is as defined in any one of the preceding items.
63. A method of treating a disorder such as a cancer, arrhythmia, malaria, psychotic diseases, hypertension, depression, Alzheimer's disease, addiction and/or neuronal diseases, comprising administration of a therapeutic sufficient amount of an MIA or a pharmaceutical compound obtained by the method according to any of items 24 to 30, 47 or 61 to 62.

Methods for production of strictosidine aglycone and monoterpenoid indole alkaloids

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

Priority Claims (1)

PCT Information

Provisional Applications (1)