Production of monoterpene indole alkaloid compounds in a heterologous host

Abstract

Provided are non-naturally occurring nucleic acids comprising a sequence encoding an enzyme, regulatory protein, or other functional protein that provides for monoterpene indole alkaloid biosynthesis, as well as modified enzyme and protein sequences encoded by the nucleic acid sequences. Also provided are recombinant microorganisms, including novel strains, that comprise the nucleic acid sequences and / or express the enzyme or regulatory protein that provides for the biosynthesis of monoterpene indole alkaloids. Methods of expressing the enzyme or regulatory protein are provided, as are methods of generating monoterpene indole alkaloid compounds, precursors, intermediates, and analogs.

Description

PRODUCTION OF MONOTERPENE INDOLE ALKALOID COMPOUNDS IN AHETEROLOGOUS HOSTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 459,860, filed April 17, 2023, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] This application contains a listing of sequences presented in a series of tables at the end of the specification. Additionally, a computer readable form of the sequence listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The sequence listing is contained in the file created on April 17, 2024, having the file name ‘‘24-0195-WO.xmr’ and is 692 kilobytes in size.FIELD

[0003] The disclosure relates to polynucleotides, enzymes, biosynthetic methods, and recombinant microorganisms useful in the production of monoterpene indole alkaloid compounds. The monoterpene indole alkaloids that can be synthesized according to the disclosure include plant-derived and / or fungal-derived compounds or metabolites that can exert a physiological effect, and are inclusive of compounds such as geissochizine, stemmadenine, coronaridine, hirsutine, cathenamine, voacangine, ibogaine, as well as precursors, intermediates, metabolites, and derivatives thereof. The monoterpene indole alkaloids herein can be generated from precursor compounds that are produced by upstream monoterpene indole alkaloid biosynthetic pathways, which can feed into the biosynthetic pathways (e g., ibogaine pathways) useful in the production the target compounds described herein.BACKGROUND

[0004] Plants, fungi, and bacteria are biochemical factories that can make complex alkaloids molecules, often to defend against herbivores and protect against pathogens. Many of these plant and fungal natural products have medicinal properties. There are multipleexamples of these alkaloid compounds used to treat a wide range of conditions including, for example, drug addiction, depression, pain, cancer, and diabetes.

[0005] Indole alkaloid compounds occur in nature, typically in very' small quantities in certain plants, fungi, and / or bacteria. Isolating such compounds from natural sources typically requires large amounts of starting material (e.g., biomass) as well as complicated separation techniques, and only provides the target compound(s) at low yield. Chemical synthesis strategies that target monoterpene indole alkaloids, particularly from readily available and low-cost precursors, are often highly complicated, involve toxic solvents, and also are associated with low yields.

[0006] Accordingly, there is a need for compositions and methods that provide for the production of indole alkaloid molecules from a renewable source such as recombinant microorganism(s). Hie disclosure provides polynucleotides, amino acid sequences (i.e., enzymes), recombinant cells and strains, and biosynthetic methods that produce indole alkaloids from low-cost feedstock or precursor compounds, as well as from cell lysates and / or in cell -free reactions. The methods and recombinant cells disclosed herein provide for synthesis of indole alkaloid compounds in good quantity and yield, providing advantages in scale, reliability, reproducibility and environmentally-friendly production, particularly when compared to existing production and isolation and / or purification methods.SUMMARY OF THE DISCLOSURE

[0007] In an aspect the disclosure provides for polynucleotide sequences that encode amino acid sequences (e.g., enzymes, transporters, etc.) that are useful in the biosynthesis of a downstream monoterpene indole alkaloid (e.g., from strictosidine or strictosidine aglycone to ibogaine, and intermediates and metabolites thereof). In embodiments of this aspect the polynucleotide sequence comprises a gene that encodes a polypeptide that is associated with monoterpene indole alkaloid synthesis.

[0008] In one aspect, the disclosure relates to a polynucleotide comprising a sequence as disclosed in any of Tables 1-27, or a sequence having about 90% sequence identity to anyone of the disclosed sequences. In some embodiments, the disclosure relates to a polynucleotide sequence having about 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the polynucleotide sequences disclosed in any of Tables 1-27.

[0009] In an aspect, the disclosure relates to an expression vector comprising a polynucleotide in accordance with disclosure and an operatively-linked promoter sequence that allows for expression of the one or more polynucleotide sequences in a cell.

[0010] In one aspect, the disclosure relates to a recombinant cell comprising a polynucleotide and / or an expression vector in accordance with disclosure. In some embodiments, the recombinant cell comprises a bacterial cell, a fungal cell, or a yeast cell. In some embodiments, the yeast cell comprises Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffersomyces , Blakeslea, Rhodotorula, or Yarrowia. In some embodiments, the filamentous fungus cell comprises Aspergillus or Penici Ilium. In some embodiments, the bacterial cell comprises Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or lactobacillus .

[0011] In yet further embodiments, the recombinant cell comprises at least one copy of the one or more polynucleotide sequences stably integrated into the genome of the cell. In further embodiments, a plurality of copies of the one or more polynucleotide sequences are stably integrated into the genome of the cell. In some further embodiments, the recombinant cell comprises a polynucleotide sequence or polynucleotide sequences in accordance with the disclosure that comprises the same sequence. In some further embodiments, the recombinant cell comprises a polynucleotide sequence or polynucleotide sequences in accordance with the disclosure that comprises different sequences. In yet further embodiments, the recombinant cell comprises a polynucleotide sequence or polynucleotide sequences in accordance with the disclosure that encode the same class of enzyme or amino acid sequence. In some further embodiments, the recombinant cell comprises a polynucleotide sequence or polynucleotide sequences in accordance with the disclosure that encode a different class of enzyme or amino acid sequence.

[0012] In another aspect, the disclosure provides a method for the biosynthesis of an upstream monoterpene indole alkaloid compound, or a precursor or metabolite thereof, wherein the method comprises culturing a recombinant cell in accordance with any of the aspects and embodiments of the disclosure under conditions that allow for the biosynthesis of the compound. In some embodiments, the method further comprises contacting the recombinant cell with a feedstock comprising one or more exogenous substrate compounds useful in the biosynthesis of one or more MIAs. In some further embodiments, the one or more exogenous substrate compounds may comprise an upstream precursor MIA compound such as, for example, nepetalactol, loganic acid, loganin, secologanin, L-tryptophan, tryptamine, serotonin, 5 -methoxytryptamine, 4-methoxytiyptamine, or 4-hydroxytryptamine,or the feed can comprise a plant extract that includes the one or more exogenous substrate compounds. In some further embodiments, the method can comprise one or more exogenous substrate compounds that are produced by a recombinant cell that can produce one or more upstream precursor MIA compounds, and which are fed into a recombinant cell according to any one of the aspects and embodiments disclosed herein.

[0013] In some embodiments, the biosynthetic method generates one or more of the following compounds: cathenamine, 4,21-dehydrogeissoschizine, 19E-geissoschizine, geissoschizine methyl ether, isositsirikine, tetrahydroalstonine, ajmalicine, 19-epi-ajmalicine, dehydropreakuammicine, preakuammicine, akuammicine, deshydroxymethylstemmadenine, stemmadenine, stemmadenine acetate, precondylocarpine acetate, dihydroprecondylocarpine acetate, secodine, dehydrosecodine, vincadifformine, tabersonine, catharanthine, coronaridine, 10-hydroxycoronaridine (norvoacangine), voacangine, de-ester- voacangine, ibogamine, noribogaine, and ibogaine, among others.

[0014] In a further aspect, the disclosure provides an amino acid sequence comprising a sequence as disclosed in any of Tables 28-46, or a sequence having about 90% sequence identity to any one of the disclosed sequences. In some embodiments, the disclosure relates to an amino acid sequence having about 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequences disclosed in any of Tables 28-46, or a sequence having about 90% sequence identity to any one of the sequences. The disclosure also provides a polynucleotide encoding the amino acid sequences of Tables 28-46 or those with 90%, 95%, 96%, 97%, 98% or 99% sequence identity to those amino acid sequences.

[0015] In some further embodiments, the polynucleotide that encodes a polypeptide associated with monoterpene indole alkaloid synthesis (e.g., arising from MIA precursors such as strictosidine, hydroxystrictosidine, methoxystrictosidine, and / or strictosidine aglycone, etc.) comprises a sequence that is a modified gene. In some embodiments, the polynucleotide sequence comprises a geissoschizine synthase (GS) gene; a geissoschizine oxidase (GO) gene; a Redox 1 (CAD) dehydrogenase gene; a Redox2 (AKR) dehydrogenase gene; a stemmadenine O-acetyltransferase (SAT) gene; a precondylocarpine acetate synthase (PAS) gene; a dihydroprecondylocarpine acetate synthase (DPAS) gene; a coronaridine synthase (CorS) gene; a polyneuridine Aldehyde Esterase (PNAE) gene; an O- methyltransferase (OMT) gene; or a hydroxylase (Ox) gene; an enol-O-methyltransferase (enolMT); or a combination of any two or more the genes.

[0016] In some further embodiments, the polynucleotide encodes a polypeptide that is useful in the biosynthesis of a monoterpene indole alkaloid that encodes for a polypeptide having a wide variety' of biological activity including, for example, a polypeptide associated with electron transport, a polypeptide associated with metabolic processes, transporter protein (importer protein, efflux protein, etc.), an oxidase, a reductase, a dehydrogenase, a peptide synthetase, a halogenase, a transferase (e.g., glycosyltransferase), a synthetase (e.g., tryptophan synthetase) or other polypeptides having functional utility' in the biosynthesis of a monoterpene indole alkaloid. In some embodiments the polynucleotide sequence encodes a cytochrome P450 (CYP), a cytochrome reductase (CPR), a cytochrome B (CYB), a try ptophan synthetase (TRPS), or combinations thereof. In such further embodiments, one or more of the polynucleotides can be used in combination with one or more polynucleotides, as described herein.

[0017] In embodiments, the polynucleotide sequence comprises a GS gene comprising any one or more of the GS sequences disclosed in Table 1 , or a sequence having at least 90% sequence identity to any one of sequences GS_ln-13n.

[0018] In embodiments, the polynucleotide sequence comprises a GO gene comprising any one or more of the GO sequences disclosed in Table 2, or a sequence having at least 90% sequence identity to any one of sequences GO_ln-l In.

[0019] In embodiments, the polynucleotide sequence comprises a Redox 1 (CAD) gene comprising any one or more of the Redox 1 (CAD) sequences disclosed in Table 3, or a sequence having at least 90% sequence identity' to any one of sequences Redoxl_ln-10n.

[0020] In embodiments, the polynucleotide sequence comprises an Redox2 (AKR) gene comprising any one or more of the Redox2 (AKR) sequences disclosed in Table 4, or a sequence having at least 90% sequence identity to any one of sequences Redox2_ln-9n.

[0021] In embodiments, the polynucleotide sequence comprises a SAT gene comprising any one or more of the SAT sequences disclosed in Table 5, or a sequence having at least 90% sequence identity' to any one of sequences SAT_ln-9n.

[0022] In embodiments, the polynucleotide sequence comprises a PAS gene comprising any one or more of the PAS sequences disclosed in Table 6, or a sequence having at least 90% sequence identity' to any one of sequences PAS_ln-14n.

[0023] In embodiments, the polynucleotide sequence comprises a DP AS gene comprising any one or more of the DPAS sequences disclosed in Table 7, or a sequence having at least 90% sequence identity to any one of sequences DPAS_ln-23n.

[0024] In embodiments, the polynucleotide sequence comprises a CorS gene comprising any one or more of the CorS sequences disclosed in Table 8, or a sequence having at least 90% sequence identity to any one of sequences CorS_ln-l 8n.

[0025] In embodiments, the polynucleotide sequence comprises an PNAE gene comprising any one or more of the PNAE sequences disclosed in Table 9, or a sequence having at least 90% sequence identity to any one of sequences PNAE_ln-9n.

[0026] In embodiments, the polynucleotide sequence comprises an OMT gene comprising any one or more of the OMT sequences disclosed in Table 10, or a sequence having at least 90% sequence identity' to any one of sequences 0MT_ln-l In.

[0027] In embodiments, the polynucleotide sequence comprises an Ox gene comprising any one or more of the Ox sequences disclosed in Table 1 1 , or a sequence having at least 90% sequence identity to any one of sequences Ox_ln-20n.

[0028] In embodiments, the polynucleotide sequence comprises, a tabersonine synthase (TS or TabS), sequence disclosed in Table 47, or a sequence having at least 90% sequence identity to sequence TS ln.

[0029] In embodiments, the polynucleotide sequence comprises a CYB5 gene comprising any one or more of the CYB5 sequences disclosed in Table 12, or a sequence having at least 90% sequence identity to any one of sequences CYB5_ln-2n.

[0030] In embodiments, the polynucleotide sequence comprises a CPR gene comprising any one or more of the CPR sequences disclosed in Table 13, or a sequence having at least 90% sequence identity' to any one of sequences CPR_ln-4n.

[0031] In embodiments, the polynucleotide sequence comprises a transporter gene comprising any one or more of the sequences disclosed in Table 14, or a sequence having at least 90% sequence identity to any one of sequences trxporter_ln-6n.

[0032] In embodiments, the polynucleotide sequence comprises an enolMT gene comprising any one or more of the sequences disclosed in Table 15, or a sequence having at least 90% sequence identity' to any one of sequences enolMT_ln-7n.

[0033] In embodiments, the polynucleotide sequence comprises a fusion gene or a fusion 2A gene, comprising any two or more of the polynucleotide sequences disclosed herein. In some particular embodiments, the polynucleotide sequence comprises a fusion gene comprising GO-2A-GS, RED1-2A-RED2, PAS-2A-SAT, DPAS1-2A-DPAS2, OX (I10H)- 2A-OMT (I0MT), CPR-2A-TDC, STR-2A-SGD, CORS-DPAS1, CORS-DPAS2, DPAS1- CORS, DPAS2-CORS as disclosed in the Tables herein, or a sequence having at least 90% sequence identity to any one of the disclosed sequences.

[0034] In another aspect the disclosure provides for amino acid sequences (e g., enzymes, transporters, etc.) that are useful in the biosynthesis of a downstream monoterpene indole alkaloid. In some embodiments, amino acid sequence comprises a modified sequence of a polypeptide (e.g., protein, enzyme, etc.) sequence that comprises a geissoschizine synthase (GS); a geissoschizine oxidase (GO); a Redox 1 (CAD) dehydrogenase; a Redox2 (AKR) dehydrogenase; a stemmadenine O-acetyltransferasc (SAT); a precondylocarpine acetate synthase (PAS); a dihydroprecondylocarpine acetate synthase (DPAS); a coronaridine synthase (CorS); a polyneuridine Aldehyde Esterase (PNAE); an O-methyltransferase (OMT); an enol-O-methyltransferase (enolMT); or a hydroxylase (Ox); or a combination of any two or more of such polypeptides.

[0035] In some further embodiments the amino acid sequence useful in the biosynthesis comprising a polypeptide having a wide variety of biological activity including, for example, a polypeptide associated with electron transport, a polypeptide associated with metabolic processes, transporter protein (importer protein, efflux protein, etc.), an oxidase, a reductase, a dehydrogenase, a peptide synthetase, a halogenase, a transferase (e.g., glycosyltransferase), a synthetase (e.g., tiyptophan synthetase) or other polypeptides having functional utility in the biosynthesis of a monoterpene indole alkaloid. In some embodiments the polypeptide comprises a sequence of a cytochrome P450 (CYP), a cytochrome reductase (CPR), a cytochrome B (CYB), a tryptophan synthetase (TRPS), or combinations thereof. In such further embodiments, one or more of the polypeptides can be used in combination.

[0036] In some embodiments, the amino acid sequence comprises a GS sequence disclosed in Table 28, or a sequence having at least 90% sequence identity to any one of sequences GS_lp-13p.

[0037] In some embodiments, the amino acid sequence comprises a GO sequence disclosed in Table 29, or a sequence having at least 90% sequence identity' to any one of sequences G8H_lp-l Ip.

[0038] In embodiments, the amino acid sequence comprises a Redoxl / CAD sequence disclosed in Table 30, or a sequence having at least 90% sequence identity to any one of sequences Redoxl_lp-10p.

[0039] In embodiments, the amino acid sequence comprises a Redox2 / AKR sequence disclosed in Table 31, or a sequence having at least 90% sequence identity to any one of sequences Redox2_lp-9p.

[0040] In embodiments, the amino acid sequence comprises a SAT sequence disclosed in Table 32, or a sequence having at least 90% sequence identity to any one of sequences SAT_lp-14p.

[0041] In embodiments, the amino acid sequence comprises a PAS sequence disclosed in Table 33, or a sequence having at least 90% sequence identity to any one of sequences PAS_lp-14p.

[0042] In embodiments, the amino acid sequence comprises a DPAS sequence disclosed in Table 34, or a sequence having at least 90% sequence identity to any one of sequences DPAS_lp-23p.

[0043] In embodiments, the amino acid sequence comprises a CorS sequence disclosed in Table 35, or a sequence having at least 90% sequence identity to any one of sequences CorS_lp-18p.

[0044] In embodiments, the amino acid sequence comprises a PNAE sequence disclosed in Table 36, or a sequence having at least 90% sequence identity' to any one of sequences PNAE_lp-9p.

[0045] In embodiments, the amino acid sequence comprises an OMT sequence disclosed in Table 37, or a sequence having at least 90% sequence identity to any one of sequences OMT_lp-l lp.

[0046] In embodiments, the amino acid sequence comprises an Ox sequence disclosed in Table 38, or a sequence having at least 90% sequence identity to any one of sequences Ox_lp-20p.

[0047] In embodiments, the amino acid sequence comprises a CYB5 sequence disclosed in Table 39, or a sequence having at least 90% sequence identity to any one of sequences CYB5_lp-2p.

[0048] In embodiments, the amino acid sequence comprises a CPR sequence disclosed in Table 40, or a sequence having at least 90% sequence identity to any one of sequences CPR_lp-4p.

[0049] In embodiments, the amino acid sequence comprises a transporter protein comprising any one or more of the sequences disclosed in Table 41, or a sequence having at least 90% sequence identity' to any one of sequences trxporter_lp-6p.

[0050] In embodiments, the amino acid sequence comprises an enol-O-methyltransferase (enolMT) sequence disclosed in Table 42, or a sequence having at least 90% sequence identity to any one of sequences enolMT_lp-7p.

[0051] In embodiments, the amino acid sequence comprises, a tabersonine synthase (TS or TabS), sequence disclosed in Table 50, or a sequence having at least 90% sequence identity to sequence TS lp.

[0052] In embodiments, the amino sequence comprises a fusion or a 2A fusion, comprising any two or more of the enzyme sequences disclosed herein. In some particular embodiments, the amino acid sequence comprises a fusion comprising GO-2A-GS, RED1- 2A-RED2, PAS-2A-SAT, DPAS1-2A-DPAS2, OX (I10H)-2A-OMT (I0MT), CPR-2A- TDC, STR-2 A-SGD, CORS-DPAS1, CORS-DPAS2, DPAS1-CORS, DPAS2-CORS as disclosed in the Tables herein, or a sequence having at least 90% sequence identity to any one of the disclosed sequences.

[0053]

[0054] In another aspect the disclosure provides for vectors (e.g., expression cassettes, episomes) that comprise one or more of the polynucleotide sequences, including multiple copies of the same sequence or a sequence encoding the same or similar type of functional amino acid sequence, as described above.

[0055] In another aspect the disclosure provides recombinant cells (or "host cells") that are engineered to produce an upstream monoterpene indole alkaloid, as discussed herein. In some embodiments, the recombinant cell comprises one or a plurality of the polynucleotide sequences of the disclosure. In some embodiments, the recombinant cell comprises one or a plurality of the polypeptide sequences of the disclosure. In some embodiments, the recombinant cell is transformed with a vector, or otherwise genetically manipulated, to express one or a plurality of the polynucleotide sequences of the disclosure. In some embodiments, the recombinant cell comprises a modification to one or more naturally- occurring genes in the cell. In some further embodiments, the genetic modification comprises a deletion of an endogenous gene. In some embodiments, the genetic modification comprises an addition of an exogenous gene. In yet further embodiments, the exogenous gene encodes a polypeptide that is different from the polypeptides of the disclosure. In some embodiments, the genetic modification comprises a combination of exogenous genes comprising a sequence that encodes a polypeptide of the disclosure and a sequence that encodes a polypeptide that is different from the polypeptides of the disclosure.

[0056] In any of the above aspects and embodiments relating to a recombinant cell, the cell can be fungal cell or a microbial cell. In some embodiments, a microbial cell can be a bacterial cell (e.g., E. colt, or the like) or a yeast cell (e.g., Saccharomyces or Pichia, or the like). In some embodiments, the recombinant cell comprises a further genetic modificationthat improves the cells ability to produce a monoterpene indole alkaloid, or a precursor or metabolite thereof.

[0057] In another aspect, the disclosure provides a method for the biosynthesis of a downstream monoterpene indole alkaloid, or a precursor or metabolite thereof, the method comprising culturing a recombinant cell in accordance with the aspects and embodiments of the disclosure under conditions that allow for the biosynthesis.

[0058] In some embodiments, the method provides for the biosynthesis of one or more compounds including 4,21-dehydrogeissoschizine geissoschizine methyl ether, isositsirikine, dehydropreakuammicine, preakuammicine, akuammicine, stemmadenine, stemmadenine acetate, precondylocarpine acetate, dihydroprecondylocarpine acetate, condylocarpine, tubotaiwine, dehydrosecodine, angryline, secodine, vincadifformine, coronaridine, de-ester coronaridine, norvoacangine, voacangine, de-ester voacangine, ibogamine, noribogaine, and / or ibogaine, among others.

[0059] In some embodiments, the method provides for the biosynthesis of noribogaine or ibogaine.

[0060] In some embodiments, the method provides for the biosynthesis of ibogamine.

[0061] In some embodiments, the method provides for the biosynthesis of stemmadenine.

[0062] In some embodiments, the method provides for the biosynthesis of coronaridine.

[0063] In some embodiments, the method provides for the biosynthesis of norvoacangine, de-ester voacangine, or voacangine.

[0064] In some embodiments, the method provides for the biosynthesis of a downstream monoterpene indole alkaloid that is fed precursors generated by an upstream MIA biosynthetic pathway providing compounds such as loganin, loganic acid, strictosidine, or strictosidine aglycone, etc.

[0065] Other aspects and embodiments falling within the scope and spirit of the disclosure will be apparent to those of skill in the art in light of the following description and illustrative examples.BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 depicts the chemical structures of example monoterpene indole alkaloid (MIA) compounds, including (A) the orientation of the monoterpene and indole structures in an illustrative MIA compound, strictosidine; (B) an example of a downstream MIA compound, ibogaine reflecting ring system designations and position numbering; (C) an example of a substituted MIA compound, ibogaine reflecting a particular stereoisomer, wherethe substituted moiety ‘R’ comprises an additional substituent attached to an atom on the indole portion of the ring, and Z comprising a carbon or heteroatom substituted on the indole ring of the compound and in accordance with the description; (D) specific examples of substituted MIA compounds, oxa-ibogaine and 1-methyl-ibogaine; and (E) a structural genus of contemplated derivative compounds based on downstream MIA compounds (e.g., ibogaine).

[0067] FIG. 2 depicts an illustrative biosynthetic pathway to form the 5-ring MIA (A) coronaridine, which acts as an intermediate to form the MIA ibogaine via (B) hydroxylation to voacangine to ibogaine or (C) de-esterification to ibogamine to ibogaine.

[0068] FIG. 3 depicts an illustrative diagram of a recombinant host cell expressing combinations of genes for downstream MIA synthesis in accordance with embodiments of the disclosure that can produce MIAs such as stemmadenine, coronaridine, voacangine, ibogaine, and related MIA analogs.

[0069] FIG. 4 depicts the bioconversion of MIA precursors into successive later (downstream) target MIAs, such as (A) MIA precursors, such as loganin, from a plant source fed to a cell to generate strictosidine or strictosidine aglycone, and / or (B) a co-culture, mixed lysate, or cell-free conversion of MIA precursor compounds (e.g., strictosidine, etc.) generated by or sourced from an exogenous recombinant cell, and reacted with (i.e., fed to) a recombinant cell or cell lysate from a cell expressing downstream pathway enzymes to generate ibogaine.

[0070] FIG. 5 depicts LC-MS extracted ion counts (EIC) and detection of fermentation- derived voacangine (EIC = 369) resulting from a recombinant cell expressing OMT enzyme in accordance with example embodiments of the disclosure, compared to standard and negative control.

[0071] FIG. 6 depicts HPLC chromatograms and detection of fermentation-derived MIA pathway product, ibogaine, from a recombinant cell expressing PNAE enzymes in accordance with example embodiments of the disclosure.

[0072] FIG. 7 depicts LC-MS / MS fragmentation of yeast fermentation-derived ibogaine from a recombinant cell expressing PNAE enzymes in accordance with example embodiments of the disclosure.

[0073] FIG. 8 depicts the methylation of geissoschizine by enol-O-methyltransferase (enolMT) activity to generate geissoschizine methyl ether along with LC-MS extracted ion counts (EIC) of yeast fermentation-derived geissoschizine methyl ether from a recombinantcell expressing GS and enolMT enzymes in accordance with example embodiments of the disclosure.

[0074] FIG. 9 depicts the oxidation of geissoschizine by geissoschizine oxidase (GO) activity to generate akuammicine along with LC-MS and LC-MS / MS fragmentation of yeast fermentation-derived akuammicine from a recombinant cell expressing GS and GO enzymes in accordance with example embodiments of the disclosure.

[0075] FIG. 10 depicts LC-MS data for tabersonine, which shares common precursor molecules with coronaridine, produced from recombinant cells expressing enzymes in accordance with the example embodiments of the disclosure.

[0076] FIG. 11 depicts LC-MS / MS EIC and fragmentation pattern data identifying ibogamine produced from recombinant cells expressing enzymes in accordance with the example embodiments of the disclosure.

[0077] FIG. 12 and FIG. 13 depict LC-MS data identifying norvoacangine (hydroxycoronaridine) produced by recombinant cells (FIG. 12) and cell lysate (FIG. 13) that include or express enzymes in accordance with the example embodiments of the disclosure.

[0078] FIG. 14 depicts LC-MS data identify ing norvoacangine (EIC 355 m / z) and voacangine (EIC 369 m / z) produced by a co-culture of recombinant cells expressing the enzymes in accordance with the example embodiments of the disclosure.

[0079] FIG. 15 depicts recombinant production of coronaridine. FIG. 15A depicts LC- MS and LC-MS / MS data relating to coronaridine (standard, fermentation-derived, with negative controls), with detailed coronaridine MS data (FIG. 15B) as produced by recombinant cells expressing enzymes in accordance with the example embodiments of the disclosure.DETAILED DESCRIPTION

[0080] In a general sense, the disclosure provides for novel sequences (polynucleotide, proteins, and enzymes) that are useful in the production of recombinant cells and biosynthetic processes for the synthesis of indole alkaloids. In various aspects and embodiments, the disclosure provides a biosynthetic method that is sufficient for the production of at least one monoterpene indole alkaloid (MIA) (e.g., a downstream MIA in the ibogaine pathway), synthetic intermediates, metabolites, or precursor species thereof. In various aspects and embodiments, the disclosure provides sequences, recombinant cells, and methods comprisingthe same for the biosynthesis of monoterpene indole alkaloids, synthetic intermediates, metabolites, or precursor species thereof. In some aspects and embodiments, the disclosure provides a sequence comprising one or more polynucleotide sequences, including polynucleotide sequences that encode for enzymes, transporter proteins, localization proteins, and / or regulatory protein sequences, associated with the biosynthetic pathway for the production of indole alkaloids including ibogaine, that comprise any one or combination of a tryptophan decarboxylase (TDC), a geissoschizine synthase (GS), a geissoschizine oxidase (GO), (Redox 1 / CAD) and (Redox2 / AKR) dehydrogenases, a stemmadenine O- acetyltransferase (SAT), a precondylocarpine acetate synthase (PAS), a dihydroprecondylocarpine acetate synthase (DPAS), a coronaridine synthase (CorS), a polyneuridine Aldehyde Esterase (PNAE), an O-methyltransferase (OMT), a hydroxylase (Ox), an enol-O-methyltransfers (enolMT), a tabersonine synthase (TS or TabS), a cytochrome bS (CYB5), a cytochrome P450 reductase (CPR), a tryptophan importer (TAT2), a SAMe importer (SAM3), a transporter (trxporter), a methionine importer (MUP1), a mRNA stabilizer (IME4), a P450 / CYP activity enhancer (ICE2 and / or INO2), and / or a SAMe pathway enhancer (ADK1 and / or SAM2).Abbreviations and Definitions

[0081] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. A number of terms and abbreviations appear throughout the disclosure and, unless otherwise indicated, should be understood to have the definitions that follow.

[0082] A "nucleic acid" or "polynucleotide" sequence are used herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form. The terms encompass nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

[0083] The terms "nucleic acid primer," "nucleic acid probe," and "oligonucleotide" are all used herein to refer to a short nucleic acid sequence, which may comprise or consist of a fragment of a longer polynucleotide sequence. Oligonucleotides, nucleic acid primers, and / ornucleic acid probes can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof and are typically single-stranded. However, they can also be doublestranded having two complementing strands that can be separated (e.g., melted) under denaturating conditions (e.g., stringent, moderately stringent, or highly stringent conditions). In some embodiments an oligonucleotide, primer, and / or probe has a length of from about 8 nucleotides to about 200 nucleotides, or from about 12 nucleotides to about 100 nucleotides, or from about 18 to about 50 nucleotides. In embodiments, oligonucleotides, primers, and / or probes can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications (e.g., to inhibit or prevent degradation).

[0084] The terms "amino acid sequence," "polypeptide," "protein," and "peptide" as used herein all refer to a sequence of amino acid residues linked by peptide bonds or modified peptide bonds. The amino acid sequence can be of any length of greater than two amino acids. Polypeptides can include modified forms of the sequence, such as naturally occurring or synthetically generated post-translational modifications, or modifications to the chemical structure of one or more amino acid residues. Non-limiting examples of modified forms include glycosylated sequences, phosphorylated sequences, myristoylated sequences, palmitoylated sequences, ribosylated sequences, acetylated sequences, and the like. Modifications can also include intra- or inter-molecular crosslinking or covalent attachments to moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the naturally-encoded twenty amino acids may also be included in a polypeptide.

[0085] The polypeptide sequences or polynucleotide sequences can be isolated and / or purified, both of which refer to a polypeptide (or a polynucleotide) that is substantially separated from other cellular components (i.e., proteins, DNA, RNA, lipids, membranes, cell debris) of the organism in which the sequence is produced (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0086] As used herein, a "conservative amino acid substitution" refers to an amino acid substitution in a polypeptide sequence wherein the substituted amino acid(s) has similar characteristics to the amino acid in the native sequence, for example charge, hydrophobicity and / or hydrophilicity profile, polarity, size, and the like. Non-limiting examples of conservative amino acid substitutions are Ser for Ala, Thr, or Cys; Lys for Aig; Gin for Asn, His, or Lys; His for Asn; Glu for Asp or Lys; Asn for His or Gin; Asp for Glu; Pro for Gly;Leu for lie. Phe, Met, or Vai; Vai for He or Leu; lie for Leu, Met, or Vai; Arg for Lys; Met for Phe; Tyr for Phe or Trp; Thr for Ser; Tip for Tyr; and Phe for Tyr. Non-natural amino acids can also serve as a conservative amino acid substitution for a naturally occurring amino acid.

[0087] The term "functional variant" refers to a recombinant biological sequence that is structurally different from a naturally occurring sequence and capable of performing the same function as the naturally occurring sequence. For example, a functional variant of a pathway gene or enzyme comprises a nucleotide and / or amino acid sequence that is altered by one or more nucleotides and / or amino acids compared to the native indole alkaloid pathway gene or enzyme sequences, and is capable of performing the function of the native or parent indole alkaloid pathway gene or enzyme (e.g., an STR enzyme capable of generating strictosidine). In embodiments, a modification to the native or parent sequence may provide for the same or improved functional activity and reaction parameters without altering the underlying function of the native biological sequence. Functional variants may comprise conservative sequence substitutions, sequence additions, and sequence deletions. The sequence modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids, and / or analogs thereof. In some embodiments, recombinant biological sequences, including functional variants, comprise amino acid analogs (e.g. amino acids other than the 20 amino acids encoded by DNA or RNA) and / or labeled amino acids and amino acid analogs comprising, for example, fluorescent dyes, radioisotopes, electron dense agents, and the like.

[0088] A "recombinant" nucleic acid or amino acid sequence is a nucleic acid or polypeptide produced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012). The terms "recombinant," "heterologous," and "exogenous," can be used interchangeably herein and, when referring to polynucleotides, mean a polynucleotide (e.g., a DNA or RNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found. Similarly, the terms when referring topolypeptides, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. As such, recombinant DNA molecules can be expressed in a host cell to produce a recombinant polypeptide.[0089J The terms "transformed," "transgenic," and "recombinant," when used with reference to host cells typically refer to an isolated cell or a cell in culture, such as a plant, fungal, or microbial (e.g. bacterial or yeast) cell, into which a heterologous polynucleotide has been introduced or a heterologous amino acid sequence is expressed. The polynucleotide can be integrated into the genome of the host cell, or it can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating, as discussed herein. Transformed cells, tissues, or subjects are understood to encompass not only the end-product of a transformation process, but also transgenic progeny thereof.[0090[ The terms "plasmid," "vector,” and "cassette" (e.g., transformation cassette, expression vector, or expression cassette) generally refer to an extra-chromosomal element that comprises nucleic acid sequences (e.g., genes, promoters, regulatory elements (inducers, repressors, etc.) and the like) which are not part of the central metabolism of the cell, and can be circular, double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences (linear or circular) of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. Merely for purposes of clarity, a "transformation cassette" refers to a vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. An "expression cassette" or "expression vector" refers to a vector containing a foreign gene and having elements in addition to the foreign gene that allow for expression and / or enhanced expression of that gene in a foreign host. Thus, the terms can refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. A nonlimiting example of an expression vector includes a gene encoding an enzyme with a promoter that is functional in yeast, where the promoter and gene are oriented such that the promoter drives expression of the enzyme in the yeast cell. A non-limiting example of a vector capable of extra-chromosomal replication is an episome.

[0091] A "linker" refers to a short amino acid sequence that separates multiple domains of a polypeptide. In some embodiments, the linker prohibits eneigetically or structurally unfavorable interactions between the discrete domains.

[0092] A recombinant gene can be "codon optimized" when its nucleotide sequence is modified to accommodate codon bias of the host oiganism, typically to improve gene expression and increase translational efficiency of the gene.

[0093] As used herein a "coding sequence" generally refers to a DNA sequence that encodes for a specific amino acid sequence.

[0094] A "regulatory sequence" is generally used to refer to a polynucleotide sequence located upstream (5* non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0095] A "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Commonly, a coding sequence is oriented or located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different natural promoters, or comprise synthetic DNA segments. Different promoters may direct the expression of a gene in different cell types, or at different stages of development or cell growth / cycle, or in response to different environmental conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters."

[0096] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0097] The term "expression" generally refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid sequence. "Over-expression" refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non-transformed organisms.

[0098] "Transformation" is used according to its ordinary and customary meaning as understood by a person of ordinary skill in the art, and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide can beincorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

[0099] Compounds that fall within the scope of the disclosure comprise compounds that can be used in or generated by at least one step or part of the downstream indole alkaloid biosynthetic pathway described herein, including indole alkaloids, synthetic intermediates, metabolites, or precursor species thereof (which may also be referred to as "target" compounds), as generally described herein. Non-limiting examples of such compounds include cathenamine, 4,21 -dehydrogeissoschizine, 19E-geissoschizine, geissoschizine methyl ether, isositsirikine, tetrahydroalstonine, ajmalicine, 19-epi-ajmalicine, dehydropreakuammicine, preakuammicine, akuammicine, deshydroxymethylstemmadenine, stemmadenine, stemmadenine acetate, precondylocarpine acetate, dihydroprecondyiocarpine acetate, secodine, dehydrosecodine, vincadifformine, tabersonine, catharanthine, coronaridine, 10-hydroxycoronaridine (norvoacangine), voacangine, de-ester-voacangine, ibogamine, noribogaine, and ibogaine, among others.

[0100] Sequences, including polynucleotide and amino acid sequences that fall within the scope of the disclosure include sequences that comprise a protein or enzyme (or sequences encoding the same) involved in at least a portion of the indole alkaloid biosynthetic pathway for ibogaine, including sequences that may facilitate the expression or activity of one or more other sequences of the biosynthetic pathway. Non-limiting examples of such sequences include enzymes and transport and regulatory proteins, as well as polynucleotides that encode for such enzymes and proteins comprising:, a geissoschizine synthase (GS), a geissoschizine oxidase (GO), (Redox 1 / CAD) and (Redox2 / AKR) dehydrogenases, a stemmadenine O- acetyltransferase (SAT), a precondylocarpine acetate synthase (PAS), a dihydroprecondyiocarpine acetate synthase (DPAS), a coronaridine synthase (CorS), an O- methyltransferase (OMT), a hydroxylase (Ox), an enol-O-methyltransferase (enolMT), a tabersonine synthase (TS or TabS), a polyneuridine Aldehyde Esterase (PNAE), among others.

[0101] Other non-limiting examples of such sequences associated with the biosynthetic pathways include enzymes and regulatory' proteins, as well as polynucleotides that encode for such enzymes and regulatory proteins comprising: a cytochrome bS (CYB5), a cytochrome P450 reductase (CPR), a tryptophan importer (TAT2), a SAMe importer (SAM3), a transporter (trxporter), a methionine importer (MUP1), a mRNA stabilizer (IME4), aP450 / CYP activity enhancer (ICE2 and / or INO2), and / or a SAMe pathway enhancer (ADK 1 and / or SAM2), among others.Polynucleotides and related Enzymes and Proteins

[0102] In various aspects the disclosure provides for polynucleotide sequences that encode amino acid sequences (e.g., enzymes, regulatory proteins, transporters, etc.) that are useful in the biosynthesis of an indole alkaloid. In various aspects the disclosure provides for polynucleotide sequences that encode amino acid sequences (e.g., enzymes, transporters, etc.) that are useful in the biosynthesis of an indole alkaloid such as, for example, a compound in the biosynthetic pathway for ibogaine production. In embodiments of this aspect, the polynucleotide sequence comprises a gene that encodes a polypeptide that is associated with downstream indole alkaloid synthesis (i.e., compounds in a biosynthetic pathway from strictosidine or strictosidine aglycone through to ibogaine, as well as precursors and metabolites thereof).

[0103] Thus, in embodiments, the polynucleotide sequence comprises a modified gene sequence that encodes an enzyme or regulatory protein associated with the indole alkaloid biosynthetic pathway (i.e., from strictosidine or strictosidine aglycone to ibogaine), comprising one or more of a geissoschizine synthase (GS), a geissoschizine oxidase (GO), (Redoxl / CAD) and (Redox2 / AKR) dehydrogenases, a stemmadenine O-acetyltransferase (SAT), a precondylocarpine acetate synthase (PAS), a dihydroprecondylocarpine acetate synthase (DPAS), a coronaridine synthase (CorS), a polyneuridine Aldehyde Esterase (PNAE), an O-methyltransferase (OMT), a hydroxylase (Ox), an enol-methyltransferase (enolMT), a tabersonine synthase (TS or TabS), a cytochrome bS (CYB5), a cytochrome P450 reductase (CPR), a tryptophan importer (TAT2), a SAMe importer (SAM3), a transporter (trxporter), a methionine importer (MUP 1 ), a mRNA stabilizer (IME4), a P450 / CYP activity enhancer (ICE2 and / or INO2), and / or a SAMe pathway enhancer (ADK1 and / or SAM2).

[0104] In various embodiments of the biosynthetic methods and recombinant organisms disclosed herein the disclosure provides a combination of one or more of the polynucleotide sequences each encoding a functional amino acid sequence (e.g., enzyme, regulatoiy protein, transport protein, etc ), as described herein, that participates in the biosynthetic process.

[0105] In some embodiments, the sequences comprise a geissoschizine synthase (GS) involved in NADPH-dependent iminium reduction on 4,21 -dehydrogeissoschizine to produce 19E-geissoschizine. In some embodiments, the polynucleotide comprises a geissoschizinesynthase (GS) gene comprising any of the sequences in Table 1, including GS ln - GS_13n (SEQ ID Nos. 1-13), or a sequence having about 90% sequence identity to any of the GS sequences. In some embodiments, the polynucleotide comprises a geissoschizine synthase (GS) gene sequence that encodes a geissoschizine synthase (GS) amino acid sequence in Table 28, including GS lp - GS_13p (SEQ ID Nos. 211-223), or a sequence having about 90% sequence identity' to any of the GS amino acid sequences.

[0106] In some embodiments, the sequences comprise a geissoschizine oxidase (GO), a p450 monooxygenase that catalyzes the oxidation of, for example, 19E-geissoschizine, to produce a series of short-lived MIA unstable intermediates. These intermediates can be spontaneously converted into akuammicine. In some embodiments, the polynucleotide comprises a geissoschizine oxidase (GO) gene comprising any of the sequences in Table 2, including GO ln - GO_1 In (SEQ ID Nos. 14-24) or a sequence having about 90% sequence identity' to any of the GO sequences. In some embodiments, the polynucleotide comprises a geissoschizine oxidase (GO) gene sequence that encodes a geissoschizine oxidase (GO) amino acid sequence in Table 29, including GO lp - GO_1 Ip (SEQ ID Nos. 224-234), or a sequence having about 90% sequence identity to any of the GO amino acid sequences.

[0107] In some embodiments, the sequences comprise a Redox 1 (CAD) sequence and / or a Redox2 ( AKR) sequence, both medium chain alcohol dehydrogenases that together perform an iminium reduction of two unstable intermediates to form stemmadenine, for example, from preakuammicine. In some embodiments, the polynucleotide comprises a Redox 1 (CAD) gene comprising any of the sequences in Table 3, including Redox l_ln - Redox 1 1 On (SEQ ID Nos. 25-34), or a sequence having about 90% sequence identity' to any of the Redox 1 sequences. In some embodiments, the polynucleotide comprises Redox 1 (CAD) gene sequence that encodes a Redox 1 (CAD) amino acid sequence in Table 30, including Redoxl ip - Redoxl lOp (SEQ ID Nos. 235-244), or a sequence having about 90% sequence identity to any of the Redox 1 amino acid sequences.

[0108] In some embodiments, the polynucleotide comprises a Redox2 (AKR) gene comprising any of the sequences in Table 4, including Redox2_ln - Redox2_9n (SEQ ID Nos. 35-43), or a sequence having about 90% sequence identity to any of the Redox2 sequences. In some embodiments, the polynucleotide comprises Redox2 (AKR) gene sequence that encodes a Redox2 (AKR) amino acid sequence in Table 31, including Redox2_lp - Redox2_9p (SEQ ID Nos. 245-253), or a sequence having about 90% sequence identity to any of the Redox2 amino acid sequences.

[0109] In some embodiments, the sequences comprise a stemmadenine O- acetyltransferase (SAT), an acetyltransferase that uses acetyl-CoA as a donor to acetylate hydroxyl compounds such as stemmadenine to stemmadenine acetate. In some embodiments, the polynucleotide comprises a stemmadenine O-acetyltransferase (SAT) gene comprising any of the sequences in Table 5, including SAT ln - SAT_9n (SEQ ID Nos. 44-52), or a sequence having about 90% sequence identity' to the SAT sequences. In some embodiments, the polynucleotide comprises a stemmadenine O-acetyltransferase (SAT) gene sequence that encodes a stemmadenine O-acetyltransferase (SAT) amino acid sequence in Table 32, including SAT lp - SAT_9p (SEQ ID Nos. 254-262), or a sequence having about 90% sequence identity to any of the SAT amino acid sequences.

[0110] In some embodiments, the sequences comprise a precondylocarpine acetate synthase (PAS) that catalyzes the oxidation of stemmadenine acetate for example to precondylocarpine acetate using FAD as a cofactor. In some embodiments, the polynucleotide comprises a precondylocarpine acetate synthase (PAS) gene comprising any of the sequences in Table 6, including PAS ln - PAS_14n (SEQ ID Nos. 53-66), or a sequence having about 90% sequence identity to the PAS sequences. In some embodiments, the polynucleotide comprises a precondylocarpine acetate synthase (PAS) gene sequence that encodes a precondylocarpine acetate synthase (PAS) amino acid sequence in Table 33, including PAS lp - PAS_14p (SEQ ID Nos. 263-276), or a sequence having about 90% sequence identity to any of the PAS amino acid sequences.

[0111] In some embodiments, the sequences comprise a dihydroprecondylocarpine acetate synthase (DPAS), a medium chain alcohol dehydrogenase that requires NADPH as a cofactor and that catalyzes, for example, the reduction of precondylocarpine acetate to dihydroprecondylocarpine acetate. In some embodiments such reduction can lead to the spontaneous formation of products such as secodine, dehydrosecodine, angryline or vincadifformine. In some embodiments the DPAS over-reduces the precondylocarpine acetate to form secodine or dehydrosecodine. In some embodiments, with excess NADPH, DPAS catalyzes the formation of vincadifformine. In some embodiments, the polynucleotide comprises a dihydroprecondylocarpine acetate synthase (DPAS) gene comprising any of the sequences in Table 7, including DPAS ln - DPAS_23n (SEQ ID Nos. 67-89), or a sequence having about 90% sequence identity' to the DPAS sequences. In some embodiments, the polynucleotide comprises a dihydroprecondylocarpine acetate synthase (DPAS) gene sequence that encodes a dihydroprecondylocarpine acetate synthase (DPAS) amino acidsequence in Table 34, including DPAS lp - DPAS_23p (SEQ ID Nos. 277-299), or a sequence having about 90% sequence identity to any of the DPAS amino acid sequences.

[0112] In some embodiments, the sequences comprise a coronaridine synthase (CorS), which is a combination, cyclase and reductase that generates the unique scaffold, (-)- coronaridine from dehydrosecodine. In some embodiments, the polynucleotide comprises a coronaridine synthase (CorS) gene comprising any of the sequences in Table 8, including CorS ln - CorS_18n (SEQ ID Nos. 90-107), or a sequence having about 90% sequence identity to the CorS sequences. In some embodiments, the polynucleotide comprises a coronaridine synthase (CorS) gene sequence that encodes a coronaridine synthase (CorS) amino acid sequence in Table 35, including CorS lp - CorS ISp (SEQ ID Nos. 300-317), or a sequence having about 90% sequence identity- to any of the CorS amino acid sequences.

[0113] In some embodiments, the sequences comprise a polyneuridine aldehyde esterase (PNAE) that de-esterifies (-)-voacangine, for example, and which is non-enzymatically converted to (-)-ibogaine in a slow, decarboxylation accelerated by heat. In some embodiments, the polynucleotide comprises a polyneuridine Aldehyde Esterase (PNAE) gene comprising any of the sequences in Table 9, including PNAE ln - PNAE_9n (SEQ ID Nos. 108-116), or a sequence having about 90% sequence identity- to the PNAE sequences. In some embodiments, the polynucleotide comprises a polyneuridine Aldehyde Esterase (PNAE) gene sequence that encodes a polyneuridine Aldehyde Esterase (PNAE) amino acid sequence in Table 36, including PNAE lp - PNAE_9p (SEQ ID Nos. 318-326), or a sequence having about 90% sequence identity to any of the PNAE amino acid sequences.

[0114] In some embodiments, the sequences comprise an O-methyltransferase (OMT or I-OMT) that catalyzes, for example, the 10-O-methylation of noribogaine to produce ibogaine and 10-O-methylation of 10-hydroxycoronaridine to voacangine. In some embodiments, the method comprises an O-methyltransferase (OMT) gene of Table 10, including OMT ln - 0MT 1 In (SEQ ID Nos. 1 17-127), or a sequence having about 90% sequence identity to the OMT sequences. In some embodiments, the polynucleotide comprises an O-methyltransferase (OMT) gene sequence that encodes an O- methyltransferase (OMT or I-OMT) amino acid sequence of Table 37, including OMT lp - 0MT 1 Ip (SEQ ID Nos. 327-337), or a sequence having about 90% sequence identity to any of the OMT amino acid sequences.

[0115] In some embodiments, the method comprises an indole ring hydroxylase (Ox - or Il OH), a p450 monooxygenase that can catalyze the 10-hydroxylation of ibogamine to produce noribogaine and 10-hydroxylation of coronaridine to produce 10-hydroxycoronaridine. In some embodiments, the method comprises a hydroxylase (Ox) gene of Table 1 1, including Ox ln - Ox_20n (SEQ ID Nos. 128-147), or a sequence having about 90% sequence identity to the Ox sequences. In some embodiments, the polynucleotide comprises a hydroxylase (Ox) gene sequence that encodes a hydroxylase amino acid sequence of Table 38, including Ox lp - Ox_20p (SEQ ID Nos. 338-357), or a sequence having about 90% sequence identity to any of the Ox amino acid sequences.

[0116] In some embodiments, the polynucleotide comprises an enol O-methyltransferase (enolMT) that catalyzes the methylation of the enol moiety, leading to the conversion of compounds such as geissoschizine to produce geissoschizine methyl ether. In some embodiments, the polynucleotide comprises an enol -O-methyltransferase (enolMT) gene comprising any of the sequences in Table 15, including enolMT ln, enolMT_2n, enolMT_3n, enolMT_4n, enolMT_5n, enolMT_6n, or enolMT_7n (SEQ ID Nos. 160-166), or a sequence having about 90% sequence identity to the enolMT sequences. In some embodiments, the polynucleotide comprises an enol-O-methyltransferase (enolMT) gene sequence that encodes an enol-O-methyltransferase (enolMT) amino acid sequence in Table 42, including including enolMT lp, enolMT_2p, enolMT_3p, enolMT_4p, enolMT_5p, enolMT_6p, or enolMT_7p (SEQ ID Nos. 370-376), or a sequence having about 90% sequence identity to any of the enolMT amino acid sequences.

[0117] In some embodiments, the polynucleotide comprises a cytochrome P450 reductase (CPR) gene comprising any of the sequences in Table 13, including CPR ln, CPR_2n, CPR_3n, or CPR_4n (SEQ ID Nos. 150-153), or a sequence having about 90% sequence identity to the CPR sequences. In some embodiments, the polynucleotide comprises a cytochrome P450 reductase (CPR) gene sequence that encodes a cytochrome P450 reductase (CPR) amino acid sequence in Table 40, including CPR lp, CPR_2p, CPR_3p, or CPR_4p (SEQ ID Nos. 360-363), or a sequence having about 90% sequence identity to any of the CPR amino acid sequences.

[0118] In some embodiments, the sequences comprise trxporter genes that are in the Nitrate / Peptide Family (NPF) transporters that uptake exogenous iridoid glucosides including loganic acid and loganin, increasing the intracellular concentrations of these iridoid glucosides. In some embodiments, the polynucleotide comprises a transporter (trxporter) gene comprising any of the sequences in Table 14, including trxporter ln, trxporter_2n, trxportcr_3n, trxporter_4n, trxporter_5n, or trxporter_6n (SEQ ID Nos. 154-159), or a sequence having about 90% sequence identity to the trxporter sequences. In some embodiments, the polynucleotide comprises a transporter (trxporter) gene sequence thatencodes a transporter (trxporter) amino acid sequence in Table 41, including trxporter lp, trxporter_2p, trxporter_3p, trxporter_4p, trxporter_5p, or trxporter_6p (SEQ ID Nos. 364- 369), or a sequence having about 90% sequence identity to any of the trxporter amino acid sequences.

[0119] In some embodiments, the sequences comprise a tabersonine synthase (TabS or TS), which generates tabersonine from dehydrosecodine. In some embodiments, the polynucleotide comprises a tabersonine synthase gene comprising any of the sequences in Table 47, including TS ln (SEQ ID Nos. 413), or a sequence having about 90% sequence identity to the TS sequences. In some embodiments, the polynucleotide comprises a coronaridine synthase (TS) gene sequence that encodes a tabersonine synthase amino acid sequence in Table 50, including TS lp (SEQ ID Nos. 425), or a sequence having about 90% sequence identity to any of the TS amino acid sequence(s).

[0120] In some embodiments, the polynucleotide comprises a cytochrome b5 (CYB5) gene, which together with CPR can relay electrons from NADPH to a cytochrome p450 enzyme using FAD and FMN as cofactors. Coexpression of CPR and CYB5 proteins can enhance enzymatic oxidations, such as increasing the activity of the P450 enzymes described herein. In some embodiments, the polynucleotide comprises a CYB5 gene comprising any of the sequences in Table 12, including CYB5_ln, or CYB_2n (SEQ ID Nos. 148-149), or a sequence having about 90% sequence identity' to the CYB5 sequences. In some embodiments, the polynucleotide comprises a cytochrome b5 (CYB5) gene sequence that encodes a cytochrome b5 (CYB5) amino acid sequence in Table 39, including CYB5_lp, or CYB5_2p (SEQ ID Nos. 358-359), or a sequence having about 90% sequence identity to any of the CYB5 amino acid sequences.

[0121] In some embodiments, the polynucleotide comprises a tryptophan importer (TAT2) gene comprising any of the sequences in Table 24, including TAT2_ln (SEQ ID No. 207) or a sequence having about 90% sequence identity' to the TAT2 sequences. In some embodiments, the polynucleotide comprises a try'ptophan importer (TAT2) gene sequence that encodes a tryptophan importer (TAT2) amino acid sequence.

[0122] In some embodiments, the polynucleotide comprises a S AMe importer (SAM3) gene comprising any of the sequences in Table 22, including SAM3_ln (SEQ ID No. 205) or a sequence having about 90% sequence identity' to the SAM3 sequences. In some embodiments, the polynucleotide comprises a SAMe importer (SAM3) gene sequence that encodes a SAMe importer (SAM3) amino acid sequence.

[0123] In some embodiments, the polynucleotide comprises a methionine importer (MUP1) gene comprising any of the sequences in Table 23, including MUPl ln (SEQ ID No. 206) or a sequence having about 90% sequence identity to the MUP1 sequences. In some embodiments, the polynucleotide comprises a methionine importer (MUP1) gene sequence that encodes a methionine importer (MUP1) amino acid sequence.

[0124] In some embodiments, the polynucleotide comprises a mRNA stabilizer (1ME4) gene, a P450 / CYP activity enhancer (ICE2 and / or INO2) gene, a SAMe pathway enhancer (ADK1 and / or SAM2) gene comprising any of the sequences identified in the Tables (e g., Tables 18-21, 25), including IME4_ln (SEQ ID No. 208), ICE2_ln (SEQ ID No. 201), INO2_ln (SEQ ID No. 202), ADKl ln (SEQ ID No. 204), and / or SAM2_ln (SEQ ID No. 203) or a sequence having about 90% sequence identity to these sequences. In some embodiments, the polynucleotide comprises a mRNA stabilizer (IME4) gene, a P450 / CYP activity enhancer (ICE2 and / or INO2) gene, a SAMe pathway enhancer (ADK1 and / or SAM2) gene that encodes the corresponding amino acid sequence, i.e., (IME4), (ICE2), (INO2), (ADK1), and / or (SAM2) amino acid sequence.

[0125] In some embodiments, N terminal peptide sequences for membrane localization are fused to the N terminus of P450s such as the GO, PAS, Ox, and CPR proteins. In some embodiments, membrane localization signals LclTag_24p-28p, encoded by LclTag_24n-28n (SEQ ID Nos. 193-197), are fused to any of the GO, PAS, Ox, and / or CPR sequences described herein. Such fusion polypeptides when expressed may lead to increased P450 / CYP and CPR activity due to enhanced membrane localization.

[0126] Hmx 1 is a heme oxygenase involved in the degradation of heme, and can be incorporated in accordance with some aspects and example embodiments of the disclosure. For example, in some embodiments, the HMX1 gene of the host is deleted to reduce heme degradation. Heme depletion is a major source of cellular stress and cells are particularly susceptible to this source of stress when several p450 monooxygenase enzymes are expressed in the cell. In other embodiments, enzymes to increase cellular heme are expressed, including Hem3, Hem2 and Hem 12. Iron (II) citrate and 5-aminolevulinic acid can be fed into fermentation media to contribute to heme production in a recombinant host cell.

[0127] The tryptophan importer (TAT2) is a high affinity tryptophan and tyrosine permease. Heterologous pathway enzymes that are expressed to produce compounds with an indole core such as tryptamine and serotonin use L-tryptophan as a directing molecule. Try ptophan production in cells is normally tightly regulated. Tryptophan accumulation in a recombinant host is increased by overexpressing a recombinant L-tryptophan transporter(TAT2). This allows for exogenous tryptophan to be fed to the cells and transported in the recombinant host. See also, WO / 2021 / 248087.

[0128] SAMe importer (SAM3) is a high-affinity S-adenosylmethionine permease. In some embodiments, a recombinant host is modified to increase the accumulation of the methyl donor, SAMe, which is used in the upstream pathway by enzymes such as the recombinant LAMT enzymes to methylate loganic acid. SAMe accumulation in the recombinant host cell can be increased by overexpressing this permease and feeding exogenous SAMe into the media. See also international patent application WO / 2021 / 248087.

[0129] The methionine importer (MUP1) is a high affinity methionine permease. SAMe is a robust methyl donor synthesized from methionine and ATP. In some embodiments, to increase the uptake of exogenous L-methionine fed into the SAMe pathway, recombinant Mupl is overexpressed. See also international patent application WO / 2021 / 248087.

[0130] In some embodiments, the nucleic acids described herewith encode a polypeptide or oligopeptide having an amino acid sequence that is naturally occurring. In other embodiments, the nucleic acids encode a polypeptide or oligopeptide having an amino acid sequence that is not naturally occurring. The encoded polypeptides or oligopeptides that are not naturally occurring can vary from a naturally occurring polypeptide or oligopeptide, or portion thereof, by a small amount (e.g., one conservative amino acid substitution or a histidine tag) or extensively (e.g., further comprising a fusion peptide, a substituted or added domain from another protein, a scaffold, etc.).

[0131] In some of the above embodiments, the nucleic acid provided herein comprises the sequence of any one of the nucleic acid sequences referred to in Table 1 A below.Table 1A: Summary of tabulated sequences

[0132] Nucleic acid sequences in accordance with the disclosure are synthesized and cloned using techniques known in the art. Gene expression can be controlled by inducible or constitutive promoter systems using the appropriate expression vectors. Genes are transformed into an organism using standard yeast or fungus transformation methods to generate modified host strains (i.e., the recombinant host organism). The modified strains express the genes for biosynthetic pathways that generate indole alkaloid products in the ibogaine biosynthetic pathway, which can be fed with compounds produced in an upstream biosynthetic pathway pathway (e.g., loganin, loganic acid, strictosidine, strictosidine aglycone, etc.). The indole alkaloid pathway genes herein can be integrated into the genome of the cell or maintained as an episomal plasmid. Recombinant host fermentation samples are: (i) prepared and extracted using a combination of fermentation, dissolution, and purification steps; and (ii) analyzed by HPLC for the presence of directing molecules, precursor molecules, intermediate molecules, and target molecules such as those illustrated in the Examples and otherwise disclosed herein.

[0133] The polynucleotides can be used in, or used to generate, modified strains of host cells, which produce target compounds including ibogaine alkaloids, precursors, intermediates, or metabolites thereof.

[0134] In some embodiments, the nucleic acid sequence (i.e., polynucleotide) encoding a gene or a complementary nucleic acid sequence to such a coding sequence can be codon optimized for production in a selected microorganism. A number of factors can be used in determining a codon-optimized sequence (see, e.g., US Patent No. 10,435,727). Factors can include, for example, (1) selecting a codon for each amino acid residue in the recombinant polypeptide based on the usage frequency of each codon in the heterologous host cell (e.g., Saccharomyces cerevisiae) genome; (2) removing sequences that provide for restriction sites for enzymes to prevent DNA cleavage; (3) modifying long repeats (e.g., consecutive sequences of 5 or more nucleotide) to prevent low-complexity regions; (4) adding a ribosome binding site to the N-terminus; (5) adding a stop codon; (6) changing nucleotides that encode amino acids susceptible to undesirable post-translational modifications (e.g., changing codons for a surface exposed LYS to an ARG codon to avoid ubiquitination); (7) removing or replacing a localization signal sequence.

[0135] In various embodiments, the nucleic acid sequences can further comprise additional sequence encoding amino acids that are not part of the included enzymes or regulatory proteins herein. In some of these embodiments, the additional sequences encode additional amino acids present when the nucleic acid is translated, encoding, for example, aco-folding peptide, as disclosed herein, or an additional protein domain, with or without a linker sequence, creating a fusion protein. Other examples are localization sequences, i.e., signals directing the localization of the folded protein to a specific subcellular compartment or membrane. Additional non-limiting examples are an affibody tag, a localization scaffold, a vacuolar localization tag, a secretion signal, and a histidine tag (e.g., 6xhis tag). Additional examples include cleavage sites, such as a TEV protease recognition sequence or 2A -selfcleaving peptides encoded between two or more genes for cleavage of individual proteins post-translation and enabling polycistronic gene expression.

[0136] In some embodiments, two or more recombinant polynucleotide sequences can be fused together (i.e., co-expressed) by including a nucleotide linker sequence between a polynucleotide sequence and an additional polynucleotide sequence or polynucleotide sequences, such as the sequences disclosed herein. The resulting chimeric genes encode for fusions (i .e., a polypeptide fused to another polypeptide or polypeptides), such as the recombinant amino acid sequences disclosed herein. In embodiments, the linker region(s) can be the same or different, and can comprise from 3 to 50 amino acids. In such embodiments the fused polynucleotide sequences encoding fused polypeptides, such as fusions between the sequences disclosed herein (GS and GO, redox 1 and redox2, PAS and DPAS, among other combinations), can lead to enhanced yields by increasing the local concentration of substrate and active enzyme due to the increased co-localization of the fused enzymes. These embodiments relating to fusions of one or more sequences in accordance with the disclosure also confer a benefit that can allow for a single promoter to control expression of multiple recombinant proteins, which can decrease the size of expression cassettes. The sequences illustrated herein include several exemplary fusion pairs in accordance with aspects and embodiments of the disclosure such as, for example, the 2A_Pair ln-7n genes encoding enzymes 2A_Pairlp-7p that include a polycistronic message / expression of the example sequences GO-2A-GS, Redoxl-2A-Redox2, PAS-2A-SAT, DPAS-2A-DPAS (e.g. 2X DPASes to express multiple variants of this reductase as desired), I10H-2A-IOMT, and further constructs relevant to the upstream biosynthetic pathway (e.g., STR-2 A-SGD, and CPR-2A-TDC), and which demonstrate efficient space saving gene expression in a recombinant host for MIA production. Other non-limiting examples of such combinations include linked CORS-DPAS, such as DPAS-2A-CORS, or CORS-2A-DPAS, can also be implemented in accordance with the disclosure (see, e.g., Tables 48-49 (SEQ ID Nos. 414- 424) and Tables 51-52 (SEQ ID Nos. 426-436).

[0137] In further embodiments, sequences disclosed herein can include gene and enzyme fusions whereby a flexible linker is introduced as described above to force localization between enzymes, which may benefit on-pathway precursor and intermediate flux and deter shunt product (i.e., undesired) formation. Certain enzyme functions can benefit from forced localization, for example, whenone or more intermediates are unstable. Such fusions are included as illustrative gene Fusion_Pairs_ln-4n (Table 49, SEQ ID Nos. 421-424) encoding enzymes Fusion_Pairs_lp-4p (Table 52, SEQ ID Nos 433-436), which include pairs of CORS-DPAS and DPAS-CORS fusions, linked genetically by codons for a flexible Gly-Ser amino acid sequence, forcing proximity but also allowing for enough distance for proper protein folding and preventing other potentially inhibitory effects from too rigid or too short a linker.

[0138] In some embodiments, the nucleic acid comprises additional nucleotide sequences that are not translated. Non-limiting examples include promoters, terminators, barcodes, Kozak sequences, targeting sequences, and enhancer elements. In some particular embodiments the polynucleotide sequences comprise a promoter that is functional in yeast, fungi, and bacteria. In embodiments, expression of a gene encoding an enzyme or regulatory protein is controlled by the promoter operably linked to the gene sequence. For a gene to be expressed, a promoter must be present within 1 ,000 nucleotides upstream of the gene. A gene is generally cloned under the control of a desired promoter. Hie promoter is placed upstream of the gene in the genome or on an episomal plasmid. The promoter regulates the amount of enzyme expressed in the cell and the timing of expression, or expression in response to external factors such as carbon source.

[0139] Any promoter can be utilized to drive the expression of the enzymes and regulatory proteins described herein. Listings of various promoters in organisms such as yeast are readily available (See, e.g., the registry of Standard Biological Parts for yeast at the website: parts igem.org / Y east). Several of the exemplary promoters listed in Table 1 below drive strong expression, constant gene expression, medium or weak gene expression, or provide inducible gene expression. Inducible or repressible gene expression is dependent on the presence or absence of a certain molecule (inducer / repressor). For example, the GALI, GAL7, and GAL10 promoters are activated by the presence of galactose and are repressed by the presence of glucose. The HO promoter is active and drives gene expression only in the presence of the alpha factor peptide. The HXT1 promoter is activated by the presence of glucose while the ADH2 promoter is repressed by the presence of glucose.Table 2A: Exemplary promoters

[0140] As discussed herein, various embodiments of the disclosure provide the nucleic acid sequence as an expression cassette, e.g., a yeast expression cassette. Any yeast expression cassette capable of expressing the enzyme in a yeast cell can be utilized. Additional regulatory elements can also be present in the expression cassette, including restriction enzyme cleavage sites, antibiotic resistance genes, integration sites, auxotrophic selection markers, origins of replication, and degrons.

[0141] The expression cassette can be present in a vector that, when transformed into a host cell, either integrates into chromosomal DNA or remains episomal in the host cell. Such vectors are well-known in the art. One non-limiting example of a yeast vector is a yeastepisomal plasmid (YEp) that contains the pBluescript II SK(+) phagemid backbone, an auxotrophic selectable marker, yeast and bacterial origins of replication and multiple cloning sites enabling gene cloning under a suitable promoter (see Table 2A). Other non-limiting vectors include pRS series plasmids.

[0142] Mutations introduced into the DNA can provide enzyme variations that can prevent or promote post translational modifications of the protein. Non-limiting examples of post-translational modifications include phosphorylation, acetylation, methylation, SUMOylation, ubiquitination, proteolytic cleavage, lipidation, prenylation such as famesylation or myristoylation, glycosylation, nitrosylation and biotinylation.

[0143] The nucleic acid sequences can be modified from a gene from any source, e.g., any microorganism, protist, virus, plant, or animal. In some embodiments, the gene encoding an enzyme or regulatory protein is derived from a bacterium. For example, the bacterium can be from phylum Abditibacteriota, including class Abdihbactena. including order Abditibacteriales\ phylum Abyssubacteria or Acidobacteria, including class Acidobacteriia, Blastocatellia, Holophagae, Thermoanaerobaculia, or Vicinamibacteria, including order Acidobacteriales, Bryobacterales, Blastocatellales, Acanthopleuribacterales, Holophagales, Thermotomaculales, Ihermoanaerobaculales, or Vicinamibacteraceae; phylum Actinobacteria, including class Acidimicrobiia, Actinobacteria, Actinomarinidae, Coriobacteriia, Nitriliruptoria, Rubrobacteria, or Thermoleophilia, including orders Acidimicrobiales, Acidothermales, Actinomycetales, Actinopolysporales, Bifidobacteriales, Nanopelagicales, Catenidisporales, Corunebacteriales, Cryptosporangiales, Frankiales, Geodermatophilales, Glycomycetales, Jiangellales, Micrococcales, Micromonosporales, Nakamurellales, Propionibacteriales, Pseudonocardiales, Sporichthyales, Streptomycetales, Streptosporangiales, Actinomarinales, Coriobacteriales, Eggerthellales, Egibacterales, Egicoccales, Euzebyales, Nitriliruptorales, Gaiellales, Rubrobacterales, Solirubrobacterales, or Thermoleophilales,' phylum Aquificae, including class Aquificae, including order Aquificales or Desulfurobacteriales; phylum Armatimonadetes, including class Armatimonadia, including order Armatimonadales, Capsulimonadales , Chthonomonadetes, Chthonomonadales, Fimbriimonadia, or Fimbriimonadales-, phylum Aureabacteria or Bacteroidetes, including class Armatimonadia, Bacteroidia, Chitinophagia, Cytophagia, Flavobacteria, Saprospiria or Sphingobacteriia, including order Bacteroidales, Marinilabiliales, Chitinophagales, Cytophagales, Flavobacteriales, Saprospirales, or Sphingopacteriales: phylum Balneolaeota, Caldiserica. Calditrichaeota, or Chlamydiae, including class Balneolia, Caldisericia, Calditrichae, or Chlamydia, including orderBalneolales, Caldisericales, Calditrichales, Anoxychlamydiales, Chlamydiales, or Parachlamydiales; phylum Chlorobi or Chloroflexi, including class Chlorobia. Anaerolineae, Ardenticatenia, Caldilineae, Thermofonsia, Chloroflexia. Dehalococcoidia. Ktedonobacteria, Tepidiformia, Thermoflexia, Thermomicrobia, or Sphaerobacteridae, including order Chlorobiales, Anaerolineales, Ardenticatenales, Caldilineales, Chloroflexales. Herpetosiphonales, Kallotenuales, Dehalococcoidales, Dehalogenimonas, Ktedonobacterales, Thermogemmatisporales, Tepidiformales, Thermoflexales, Thermomicrobiales, or Sphaerobacterales; phylum Chrysiogenetes, Cloacimonetes, Coprothermobacterota, Cryosericota, or Cyanobacteria, including class Chrysiogenetes, Coprothermobacteria, Gloeobacteria, or Oscillatoriophycideae, including order Chrysiogenales, Coprothermobacterales, Chroococcidiopsidales, Gloeoemargaritales, Nostocales, Pleurocapsales, Spirulinales, Synechococcales, Gloeobacterales, Chroococcales, or Oscillatoriales; phyla: Eferribacieres, Deinococcus-thermus, Dictyoglomi. Dormibacteraeota, Elusimicrobia, Eremiobacteraeota, Fermentibacteria, or Fibrobacteres, including class Deferribacteres, Demococci, Dictyoglomia, Elusimicrobia, Endomicrobia, Chitinispirillia, Chitinivibrionia, or Fibrobacteria, including order Deferribacterales, Deinococcales, Thermales, Dictyoglomales, Elusimicrobiales, Endomicrobiales, Chitinspirillales, Chitinvibrionales, Fibrobacterales, or Fibromonadales; phylum Firmicutes, Fusobacteria, Gemmatimonadetes, or Hydrogenedentes, including class Bacilli, Clostridia, Ery>sipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, Tissierellia, Fusobacteriia. Gemmatimonadetes, Longimicrobia, including order Bacillales, Ixictobacillales, Borkfalkiales, Clostridiales, Halanaerobiales, Natranaerobiales, Thermoanaerobacterales, Erysipelotrichales, Limnochordales, Acidaminococcales, Selenomonadales, Veillonellales, Thermolithobacterales, Tissierellales, E'usobacteriales, Gemmatimonadales, or longimicrobia; phylum Hydrogenedentes, Ignavibacteriae, Kapabacteria, Kiritimatiellaeota, Krumholzibacteriota, Kryptonia, lotescibacteria, LCP-89, Lentisphaerae, Margulisbacteria, Marinimicrobia, Melainabacteria, Nitrospinae, or Omnitrophica. including class Ignavibacteria, Kiritimatiellae, Krumholzibacteria, lontisphaeria, Oligosphaeria, or Nitrospinae, including order Ignavibacteriales, Kiritimatiellales, Krumholzibacteriales, Lentisphaerales, Victivallales, Oligosphaerales, or Nitrospinia; phylum Omnitrophica or Planctomycetes, including class Brocadiae, Phycisphaerae, Planctomycetia, or Phycisphaerales, including order Sedimentisphaerales, Tepidisphaerales, Gemmatales, Isosphaerales, Pirellulales, or Planctomycetales; phylum Proteobacteria including class Acidithiobacillia, Alphaproteobacteria, Betaproteobacteria,Izimbdaproteobactena, Muproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Hydrogenophilaha, Oligoflexia, or Zetaproteobacteria, including order Acidithiobacillales, Caulobacterales, Emcibacterales, Holosporales, lodidimonadales, Kiloniellales, Kopriimonadales, Kordiimonadales, Magnetococcales, Micropepsales, Mmwuiales, Parvularculales, Pelagibacterales, Rhizobiales, Rhodobacterales, Rhodospirillales, Rhodothalassiales, Rickettsiales, Sneathiellales, Sphingomonadales, Burkholderiales, Ferritrophicales, Ferrovales, Neisseriales, Nitrosomonadales, Procabacteriales, Rhodocyclales, Bradymonadales, Acidulodesulfobacterales, Desulfarculales, Desulfobacterales, Desulfovibrionales. Desulfurellales, Desulfuromonadales, Myxococcales, Syntrophobacterales, Campylobacterales, Nautiliales, Acidiferrobacterales, Aeromonadales, Alteromonadales, Arenicellales, Cardiobacteriales, Cellvibrionales, Chromatiales, Enterobacterales, Immundisolibacterales, Legionellales, Methylococcales, Nevskiales, Oceanospirillales, Orbales, Pasteurellales Pseudomonadales, Salinisphaerales, Thiotrichales, Vibrionales, Xanthomonadales, Hydrogenophilales, Bacteriovoracales, Bdellovibrionales, Oligoflexales, Silvanigrellales, or Mariprofundales; phylum Rhodothermaeota, Saganbacteria. Sericytochromatia, Spirochaetes, Synergistetes, Tectomicrobia, or Tenericutes, including class Rhodothermia, Spirochaetia, Synergistia, Izimaplasma, or Mollicutes, including order Rhodothermales, Brachyspirales, Brevinematales, Leptospirales, Spirochaetales, Synergistales, Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, or Mycoplasmatales; phylum Thermodesulfobacteria, Thermotogae, Verrucomicrobia, or Zixibacteria. including class Thermode sulfobacteria, Thermotogae, Methylacidiphilae, Opitutae, Spartobacteria, or Verrucomicrobiae, including order lhermodesulfobactenales. Kosmotogales, Mesoaciditogales, Petrotogales. Thermotogales, Methylacidiphilales, Opitutales, Puniceicoccales, Xiphinematobacter, Chthoniobacterales, Terrimicrobium, or Verrucomicrobiales.

[0144] In other embodiments, the gene encoding the enzyme or regulatory protein is modified from an archaeon. For example, the archaeon can be from: phylum Euryarchaeota, including class Archaeoglobi, Hadesarchaea, Halobacteria, Methanobacteria, Methanococci, Methanofastidiosa, Methanomicrobia, Methanopyri, Nanohaloarchaea, Theionarchaea, Thermococci, or Thermoplasmata, including order Archaeoglobales, Hadesarchaeales, Halobacteriales, Methanobacteriales, Methanococcales, Methanocellales, Methanomicrobiales, Methanophagales, Methanosarcinales, Methanopyrales, Thermococcales, Methanomassiliicoccales, Thermoplasmatales, or Nanoarchaeales; DP ANNsuperphylum, including subphyla Aenigmarcheota, Altiarchaeota, Diapherotrites, Micrarchaeota, Nanoarchaeota, Pacearchaeota, Parvarchaeota, or Woesearchaeota; I ACK superphylum, including subphylum Korarchaeota, Crenarchaeota, Aigarchaeota, Geoarchaeota, Thaumarchaeota, or Bathyarchaeota: Asgard superphylum including subphylum Odinarchaeota, Thorarchaeota, Loldarchaeota, Helarchaeota, or Heimdallarchaeota.

[0145] In additional embodiments, the gene encoding the enzyme or regulatory protein is modified from a fungus. For example, the fungus can be from: phyla Chytridiomycota, Basidiomycota, Ascomycota, Blastocladiomycota, Ascomycota, Microsporidia, Basidiomycota, Glomeromycota, Symbiomycota, and Neocallimastigomycota; phylum Ascomycota, including classes and orders Pezizomycotina, Arthoniomycetes, Coniocybomycetes, Dothideomycetes, Eurotiomycetes. Geoglossomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, Orbiliomycetes, Pezizomycetes. Sordariomycetes, Xylonomycetes, Ixihmiales, Itchiclahmadion, Triblidiales. Saccharomycotina, Saccharomycetes, Taphrinomycotina, Archaeorhizomyces, Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes, Taphrinomycetes; phylum Basidiomycota including subphyla or classes Pucciniomycotina, Ustilaginomycotina, Wallemiomycetes, and Entorrhizomycetes; subphylum Agaricomycotina including classes Tremellomycetes, Dacrymycetes, and Agaricomycetes; phylum Symbiomycota, including class Entorrhizomycota; subphylum Ustilaginomycotina including classes Ushlagmomycetes and Exobasidiomycetes; phylum Glomeromycota including classes Archaeosporomycetes, Glome romycetes, and Paraglomeromycetes; subphylum Pucciniomycotina including orders and classes: Pucciniomycotina, Cystobasidiomycetes, Agaricostilbomycetes, Microbotryomycetes, Atractiellomycetes. Classiculomycetes, Mixiomycetes, and Cryptomycocolacomycetes; subphylum incertae sedis Mucoromyceta including orders Calcarisporiellomycota and Mucoromycota; phylum Mortierellomyceta including class Mortierellomycota; subphylum incertae sedis Entomophthoromycotina including order Entomophthorales; phylum Zoopagomyceta including classes Basidiobolomycota, Entomophthoromy’cota, Kickxellomycota, and Zoopagomycotina; subphylum incertae sedis Mucoromycotina including orders Mucorales, Endogonales, and Mortierellales; phylum Neocallimastigomycota including class Neocallimastigomycetes; phylum Blastocladiomycota including classes Physodermatomycetes and Blastocladiomycetes; phylum Rozellomyceta including classes Rozellomycota and Microsporidia; phylum Aphelidiomyceta including class Aphelidiomycota; phylum Chytridiomyceta including classes Chytridiomycetes andMonoblepharidomycetes; phylum Oomycota including classes or orders I-eptomitales, Myzocytiopsidales, Olpidiopsidales, Peronosporales, Pythiales, Rhipidiales, Salilagenidiales, Saprolegniales, Sclerosporales, Anisolpidiales, Ixigenismatales, Rozellopsidales, and Haptoglossales.[0146[ In additional embodiments, the gene for the enzyme or regulatory protein is derived from the organism below. This includes but is not limited to: Acanthurus tractus, Aplysina aerophoba, Stevia rebaudiana, Bos Taurus, Bufo bufo. Bufotes viridis, Chrysochloris asiatica. Fukomys damarensis, Streptomyces reticuliscabie, Homo sapiens, Ratlus norvegicus, Rhinella marina, Rhinella spinulosa, Schistosoma mansoni, Xenopus laevis, Xenopus tropicalis, Acacia koa, Arabidopsis thaliana, Psilocybe cubensis, Brassica oleracea, Citrus sinensis, Hordeum vulgare, Juglans cinereal, Juglans regia, Lophophora williamsii. Nymphaea colorata, Oryza sativa, Ipomoea violaceae, Rivea corymbosa, Argyreia nervosa, Gentiana rigescens, Nyssa sinensis, Camptotheca acuminata, Merremia tuberose, Mitragyna speciosa, Tabemanthe iboga, Tabemaemontana elegans, Voacanga africana, Tabemaemontana undulata, Ricinus communis, Solarium lycopersicum. Sorghum bicolor, Theobroma cacao, and Triticum aestivum, Catharanthus roseus, Actinidia polygama, Corydalis cheilanthifolia, Lonicera japonica, Olea europaea, Antirrhinum majus. Salix suchowensis, Sesamum indicum, Salvia splendens, Nepeta racemosa. Nothapodytes nimmonian, Vinca minor, Rhazya stricta, Corydalis yanhusuo. Linum usitatissimum, Rauvolfia serpentina, Ophiorrhiza pumila, Kalopanax septemlobus. Amsonia hubrichtii, Phaedon cochleariae, Aphis glycines, Pogonomyrmex barbatus, Thermobifida fusca YX, Nocardiopsis kunsanensis, Actinorugispora endophytica, Swertia mussotii, Aedes aegypti. Arabidopsis thaliana, Melianthus tuberosus, Phratora vitellinae, Macadamia integrifolia, Mentha canadensis, Coptis chinensis, Durio zibethinus, Hibiscus syriacus, Populus alba, Capsicum pubescens, Uncaria rhynchophylla, Coffea canephora and Coffea eugenioides.Enzymes and Amino Acid Sequences

[0147] The disclosure provides for non-naturally occurring amino acid sequences (i e.. enzymes or regulatory proteins) comprising a sequence encoded by any of the nucleic acids described above. In some embodiments, the amino acid sequence is 85%, 90%, 95%, 98%, or 100% identical to any one of the sequences in the amino acid sequence Tables disclosed herein (e.g., listed in Table 1A; Tables 28-46). In these embodiments, the enzyme or regulatory protein can be isolated in vitro and used in vitro to provide enzyme activity.Alternatively, the enzyme can be expressed in a recombinant organism as described herein. In some embodiments, the recombinant microorganism for the recombinant production of an amino acid sequence is a bacterium, for example an E. colt. In other embodiments, the recombinant microorganism is a yeast or fungal cell, e.g., a species ofSaccharomyces (for example 5. cerevisiae\ Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, Aspergillus or Yarrowia.

[0148] In some embodiments relating to production and purification of the recombinant amino acid sequences, the gene encoding the enzyme and / or regulators' protein is cloned into an expression vector such as the pET expression vectors from Novagen, transformed into a protease deficient strain of E. colt such as BL21 and expressed by induction with IPTG. The protein of interest may be tagged with an affinity tag to facilitate purification, e.g. hexahistidine, GST, calmodulin, TAP, AP, CAT, HA, FLAG, MBP, etc. Coexpression of a bacterial chaperone such as dnaK, GroES / GroEL or SecY may help facilitate protein folding. See Green and Sambrook (2012).

[0149] In some aspects relating to sequences comprising amino acids (e.g., enzymes) and nucleotides (e.g., polynucleotides / genes), the disclosure provides sequence variants of the sequences disclosed herein. With regard to amino acid sequences, variants (e.g., substitution, deletion, addition) can be considered as "similar" to the original polypeptide or to have a certain "percent identity" to the original polypeptide, and include those polypeptides wherein one or more amino acid residues of a polypeptide are removed and replaced with alternative residues. In embodiments, the substitutions in amino acid sequences are conservative in nature, however, the disclosure embraces substitutions that are also non-conservative.

[0150] For amino acid sequences, sequence identity and / or similarity can be determined by using standard techniques known in the art such as, for example, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, NucL Acid Res. 12:387-395, using the default settings, or by inspection. Various alignment parameters can be set according to known methods (e.g., "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.). Additional usefulalgorithms include PILEUP, which can align multiple sequences from a group of related sequences using progressive, painvise alignments; BLAST, including gapped BLAST, WU- BLAST-2 (see, e.g., Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; Altschul et al., 1996, Methods in Enzymology 266:460-480; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787).

[0151] Generally, the amino acid homology , similarity, or identity' between sequences are at least 80%, including at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and from 99% to almost 100% identity. Similarly, the "percent (%) nucleic acid sequence identity " with respect to the nucleic acid sequences described herein refers to the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotides disclosed herein, in the gene or coding sequence of the related polypeptide. Specific methods can include the default parameters of algorithms such as, for example, BLASTN (WU-BLAST-2).Recombinant host cells

[0152] In an aspect the disclosure provides recombinant cells that comprise the polynucleotides and polypeptides described herein. In embodiments, the host cells can comprise any type of cell that is adaptable to genetic manipulation and / or expression of foreign genes and proteins. In some embodiments, host cells can include any species of filamentous fungus, including but not limited to any species of Aspergillus, which may be optionally genetically altered to accumulate and / or produce target, precursor, or intermediate indole alkaloid molecules. In embodiments, host cells may also be any species of bacteria, including but not limited to Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or lactobacillus . In some embodiments, the host cell is a yeast cell capable of being genetically engineered and can be utilized in these embodiments. Nonlimiting examples of such yeast cells include species of Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffer somyces, Blakeslea, Rhodotorula, or Yarrowia.

[0153] These cells can achieve gene expression controlled by inducible promoter systems, natural or induced mutagenesis, recombination, and / or shuffling of genes, pathways, and whole cells performed sequentially or in cycles; overexpression and / or deletion of single or multiple genes and reducing or eliminating parasitic side pathways that reduce target compounds, intermediate or precursor concentrations.

[0154] The host cells of the recombinant organism may also be engineered to produce any or all precursor molecules necessary for the biosynthesis of the target, precursor, or intermediate indole alkaloid compounds and can comprise any of the disclosed polynucleotide sequences, vectors or expression cassettes that are capable of expressing the recombinant enzyme encoded therein.

[0155] Construction of modified host cells such as Saccharomyces cerevisiae strains expressing the enzymes and regulatory' proteins provided herein is carried out via expression of a gene which encodes for the enzyme. The gene encoding the enzyme can be cloned into vectors with the proper regulatoiy elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the gene encoding the enzyme may be inserted into the recombinant host genome. Integration may be achieved by a single or double cross-over insertion event of a plasmid, or by nuclease-based genome editing methods, as are known in the art e.g. CRISPR, TALEN and ZFR. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing. See, e.g., Green and Sambrook (2012).

[0156] As described herein, the recombinant cell may be any species of yeast, including but not limited to any species of Saccharomyces. Candida, Schizosaccharomyces, Yarrowia, etc., which have been genetically altered to produce monoterpene indole alkaloid (MIA) molecules, including precursors and intermediates. Additionally, genetically engineered host cells may be any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules such as GPP, geraniol, tryptamine, serotonin, 4-hydroxytryptamine, 5-methoxytryptamine, 4- methoxytryptamine, and strictosidine for use as precurors for the downstream MIA compound production in accordance with the present disclosure. Some of the species of yeast include but are not limited to: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica.

[0157] In various embodiments, the recombinant cell can comprise combinations of endogenously upregulated and / or codon optimized genes encoding for proteins which can improve: protein production, correct protein folding, protein secretion, proper protein intracellular localization, enzyme activity, correct post-translational modifications for protein features, mRNA stability7, cell tolerance to stress, cellular metabolic activity', availability of cofactors for enzyme activity, glycolysis, fatty' acid metabolism, feedstock conversion, amino acid biosynthesis, mevalonate pathway flux, coenzyme A (CoA) flux, acetyl-CoA production,tolerance to oxidative stress (e.g. H2O2) from increased protein production, tolerance to oxidative stress from monoterpene indole alkaloid (MIA) and related precursor enzymatic pathway steps, titer of alkaloids and alkaloid pathway precursors, intermediates, and compounds.[0158J As discussed herein, the recombinant cells and modified strains express the genes for biosynthetic pathways that generate monoterpene indole alkaloid pathway products, such as coronaridine, voacangine, noribogaine, ibogamine, ibogaine, etc., and / or all intermediate monoterpene indole alkaloids compounds and precursors described herein. The monoterpene indole alkaloid pathway genes herein can be integrated into the genome of the cell or maintained as an episomal plasmid. Recombinant host fermentation samples are: (i) prepared and extracted using a combination of fermentation, dissolution, and purification steps; and (ii) analyzed by HPLC for the presence of target molecules, precursor molecules, and / or intermediate molecules. In some embodiments, the product MIA pathway products are generated from an upstream pathway feed that comprises compounds such as geraniol, nepetalactol, loganic acid, loganin, secologanin, tryptamine, serotonin, 5 -methoxytryptamine, 4-methoxytryptamine, 4-hydroxytryptamme, strictosidine, any of which can also be detected.[0159J In some related embodiments, the genes and proteins that may be expressed by the recombinant cell (with reference to exemplary Genbank protein accession numbers) can include one or more of:• rRNA processing proteins for ribosome biogenesis, such as BFR2 (QHB07760. 1)• 14-3-3 proteins which regulate vesicle and protein transport, such as BMH2 (CAA59275.1)• Transcription factors which upregulate the unfolded protein response, such asMAC I (QHB08305.1)• Karyopherin overexpression to help the transport and translocation of hydrophobic and / or chimeric proteins, such as PSEl (QHBH037.1)• Ribosomal proteins for maintaining translational productivity through cell stress, such as RPPO (QHB 10483.1),• Oxidative stress protection, such as CCSl (QHB10774.1), SODl, (CAA89634.1), and / or SSA4,(KZVH87l. l)• ER stress sensors which can upregulate protein folding chaperones and heat shock responses, such as IRE1 (QHB09072.1)• Proteins which can increase cellular glycosylation status, such as PSA1,(CAA98617.1)• Peroxide consuming proteins including catalase to protect from oxidative damage, such as CTT1, (CAA97090.1)• Protein folding chaperones and chaperon ins, such as SSE 1 ,(KZV 07411.1), JEM1,(QHBO9554.1), KAR2,(CAA89325.1), LHSl,(CAA81910.1), SCJ 1 ,(CAA41529. 1 ), SIL 1 ,(QHB 11576.1 ), GroEL (QEP09777.1 ), GeoES (QEP09776.1), and / or SSS1,(CAA989O6.1)• Post-translational support, such as UBI4, (CAA97489.1)• Isomerases for proper protein folding, such as CPR5 (KZV 12543. 1 ) and / or CPR2 (KZV10787.1)• Heat shock response ER membrane proteins, including calnexin, such CNE1 , (QHB06590.1)• Endoplasmic reticulum redox balance for proper protein folding including disulfide bond formation, such as ER01, (QHB 10609.1), PDI1, (KZV 12810.1), and / or EUG1,(KZV12759.1),• Protein translocation and protein folding enhancers, such as SBH 1, (KZV 11852. 1), and / or SEC61 (CAA44215.1)• Enhancers of protein trafficking, vesicle formation, tethering, and fusion between organelles and membranes, SEC31(CAA98772.1), SLY1,(CAA38221.1), BOS1,(CAA97636.1), BET1,(CAA86247.1), SEC22,(QHB 10420.1), SED5,(CAA97549.1), COG7,(QHB08640.1), COY1,(QHB09825.1), IMH1, (QHB 10458.1), SEC1,(QHBO7623.1), SEC4,(QHB08330.1), SNC1, (KZV 13437.1), SNC2,(CAA89974.1), SSO1,(CAA97949.1), and / or SS02,(KZV09033.1)• Secretory pathway processing enzymes, including glycosyltransferase, KEX2,(CAA96143.1), MNN1,(AAA53676.1),MNN2,(QHB06786.1), MNN9,(KZV07467.1), MNN10 (QHB07704.1),MNN 11 ,(QHB09454. 1), and / or OCH 1 ,(CAA96740.1 )• Cell wall related proteins for stress tolerance and cell wall stability , such as CCW12 (KZV09357.1 ), CWP2,(CAA81937. 1 ), YPS 1 , (KZV09366. 1), MKC7,(KZV 12382. 1 ), and / or SED1 (BAI99734.1)• Flavin cofactor synthesis and recycling proteins for FAD+ / FADH use by enzymes expressed in a modified host, such as FADI (CAA98604.1) or FMN1 (NM_001180544.1)• Nicotinamide cofactor synthesis and recycling proteins for NAD(P)+ / NAD(P)H use by enzymes expressed in a modified host, such as UTR1 (CAA89577. 1), POS5 (CAA97900.1),• Upregulation of the pentose phosphate pathway (PPP) such as increased expression of pentose phosphate genes encoding for enzymes in the PPP, such as GND1 (KZV 10926.1) and / or ZWF1 (CAA96146.1)• Enzymes to increase acetyl-CoA pool including alcohol and aldehyde dehydrogenases and acetyl-CoA synthetases such as ALD6 (QEP09776.1), ADH2 (QEP09776.1), and / or ACS (AAL23099.1)• Enzymes to upregulate heme biosynthesis, such as delta-aminolevulinic acid dehydratase (Hem2) (CAA96742.1), porphobilinogen deaminase (Hem3) (CAA98783.1), uroporphyrinogen decarboxylase (Hem l2) (QHB07515.1), oxygendependent coproporphyrinogen III oxidase (Hem 13) (QHB07512.1), and / or ferrochelatase (Heml5) (CAA99385.1).• Transporters to increase metal ion import, such as FET3 (QHB 10789.1), FTR1(QHBO8222.1), and / or FET4(QHB11048.1)• Metal ion oxidoreductases to convert metals into usable forms, such as the ferric and copper reductases, FREI (QHB10365.1) and / or FRE3(KZV08274.1)

[0160] In various embodiments, the recombinant cell comprising endogenous genes encoding proteins for an improved effects) for the expression of a target compound (monoterpene indole alkaloid (MIA), intermediate, precursor, or metabolite thereof) may be adapted or manipulated (i.e., genetically modified) in a way that modifies endogenous geneexpression. Non-limiting examples include swapping an endogenous promoter for the gene of interest with a stronger promoter (constitutive or inducible, such as a glycolytic TEF1 promoter or a galactose inducible GALI promoter); codon optimized gene sequence encoding proteins (e.g., genetic integration, episomal plasmids, or artificial chromosomes of such genes in an expression cassette (e.g. promoter, coding region, terminator)) any of which can be accomplished using methods known in the art.

[0161] The recombinant cell can contain combinations of modifications to host genes where genes or combinations thereof have downregulaied or no (i.e. knocked out) expression, and / or silenced message, which can improve: protein production, correct protein folding, protein secretion, proper protein intracellular localization, enzyme activity, correct post- translational modifications for protein features, mRNA stability, cell tolerance to stress, cellular metabolic activity, availability' of cofactors for enzyme activity, glycolysis, fatty acid metabolism, feedstock conversion, amino acid biosynthesis, mevalonate pathway flux, coenzyme A (CoA) flux, acetyl-CoA production, tolerance to oxidative stress (e.g. H2O2) from increased protein production, tolerance to oxidative stress from monoterpene indole alkaloid (MIA) and related precursor enzymatic pathway steps, titer of alkaloids and alkaloid pathway precursors, intermediates, and compounds.

[0162] Such genes (with examples of Genbank nucleotide accession numbers) include combinations thereof:• T ranscription factors such as ROX 1 (N M OO 1184162.1)• Heme depletors, including oxygensases, such as HMX1 (NM 001 182092)• Proteases, such as PEP4 (NM_001 183968.1), PRC1 (NM_001 182806.1), PRB1 (NM_001178875.1)• Ubiquitin ligases, such as BUL1 (NM_001182782. 1) and / or BUL2 (NM_001 182473.1)• O-acetyltransferases, such as ATF1 (NM 001183797) and / or ATF2 (NM_001181306.1) e Dehydrogenases and reductases, such as ARI 1 (NM 001181022), ADH6(NM_001 182831.3), OYE2 (NM_001 179310), and / or OYE3 (NM_001183985.1)• Genes encoding for proteins involved in amino acid metabolism, such as BNA2 (NM_001181736.3), ARO 10 (KU050081.1), PDC6 (NM_001181216.3)• Genes that promote cell death in response to toxicity of high MIA precursor concentrations, such as YCA1 (AY692832.1).Biosynthetic Methods

[0163] In some aspects, the disclosure provide a biosynthetic method for producing one or more target compounds (e.g., an iboga compound, an indole alkaloid, or an intermediate, precursor, or metabolite thereof), comprising: (i) generating a recombinant host cell; (ii) growing the recombinant host cell under conditions effective to produce the target compound(s), or precursors) thereof; and (iii) isolating the target compound(s) from the recombinant host cell. In some embodiments of the method, the method comprises growing the recombinant host cell under conditions effective to express the genes encoding the enzymes and / or proteins, and fermenting the recombinant host cell to produce the target compound(s). Endogenous pathways of the recombinant host can be modified by the systems and methods herein to produce high purity target compounds.

[0164] In various embodiments of the biosynthetic methods to produce the desired monoterpene indole alkaloids and related intermediates and precursors, the nucleic acid encoding the enzyme(s) and / or regulatory protein(s) are introduced into a host cell using standard cell (e.g., yeast) transformation techniques (Green and Sambrook, 2012). Cells are subjected to fermentation under conditions that activate the promoter controlling the synthesis of the enzyme and / or regulatory protein. The broth may be subsequently subjected to HPLC analysis to determine the presence or yield of the desired monoterpene indole alkaloid and / or related intermediate and / or precursor products.

[0165] In various embodiments, the host cells are provided with various feedstocks to drive production of the desired monoterpene indole alkaloids and the related precursors or pathway intermediates, (e.g., feedstocks comprising glucose, fructose, sucrose, galactose, raffinose, maltose, ethanol, xylose, fatty- acids, glycerol, acetate, molasses, malt syrup, com steep liquor, dairy, flour, protein powder, olive mill waste, fish waste, etc., as is known in the art.

[0166] In some embodiments, the biosynthesis of MI As, intermediates, and analogs, is carried out by cell-free lysate, crude or purified, or via purified enzymes from a recombinant host, or a combination of modified hosts, which has been modified to express MIA biosynthetic pathway enzymes. As described above, the modified strains express the genes for biosynthetic pathways that generate downstream biosynthetic pathway s to generate, e.g.,coronaridine, voacangine, noribogaine, ibogamine, ibogaine, stemmadenine, coronaridine, and / or other intermediate monoterpene indole alkaloids compounds and precursors.

[0167] Recombinant host cells expressing the MIA pathway to generate precursors, intermediates, and end products of the MIA pathway can be grown, fermented, and produce products on various carbon and nitrogen sources as described herein. In some embodiments, additions such as vitamin mixes and trace minerals are added to a fermentation media, which can include choline chloride, niacin, pyridoxine hydrochloride, riboflavin, calcium pantothenate, para-aminobenzoic acid (PABA), thiamine HC1, biotin, cyanocobalamin, and / or folic acid, and mineral mixes, which can include calcium chloride dihydrate, ferrous sulfate heptahydrate, manganese (II) sulfate monohydrate, copper sulfate pentahydrate, zinc sulfate heptahydrate, magnesium chloride, and solutes, such as glycerol.

[0168] In some applications, MIA precursors and products generated from a recombinant host are captured using in situ product removal (ISPR). In some embodiments, 1-30% volume to volume of fermentation broth to ISPR reagent is used. Such ISPR reagents can include, but are not limited to, isopropyl myristate, vegetable oils such as com oil or olive oil, oleic acid, MTBE, 1 -decanol, oleyl alcohol, bis(2-ethylhexyl)phthalate, alkanes such as octane and dodecane, styrene, silicone oil, non-polar macroporous resin such as C8 or C18 resin, ionexchange resin, trioctylamine, activated carbon, ethyl oleate, lauryl acetate, famesane, TWEEN-80, TX-100, squalene, glycerin, and / or propylene glycol. For example, ISPR and analysis of MIA products or precursors can be carried out by an ISPR process using dodecane. In this case, a 20% volume to volume dodecane layer is added to a recombinant host fermentation. After 72h of fermentation, the dodecane is removed, diluted 1 : 10 with ethanol, and filtered for volatile compound detection via HPLC. The broth extraction and subsequent HPLC detection can be carried out as described herein and / or as inWO / 2021 / 248087 (PCT / US21 / 36031). The use of these reagents also contributes to increased MIA yield by reducing the amount of pathway intermediate lost to evaporation.

[0169] Isolation and detection of MIA products and precursors from recombinant cell, lysate, or co-culture fermentation broth can be performed by solvent extraction, filtration, and analytical methods including high performance liquid chromatography (HPLC), liquid chromatography mass spectrometiy (LC-MS), high resolution mass spectrometiy (HUMS), gas chromatography (GC), gas chromatography mass spectrometry (GC-MS), and / or nuclear magnetic resonance (NMR) methods, as known to those skilled in the art. For example, recombinant host strains expressing the MIA biosynthesis pathway can be mixed with a solvent such as acetonitrile, ethanol, isopropanol, methanol, ethyl acetate, acetone, water, andmixtures thereof, which can include additives in amounts of 0.01-20% volume to volume formic acid (FA), ammonium carbonate, or trifluoroacetic acid (TFA). Such solvent mixtures can be applied to whole broth, cell pellets, or fermentation supernatant in combination with centrifugation, vortexing, bead-beating, homogenization, filtration, such as tangential flow filtration (TFF), and / or shaking and incubation, at varying temperatures from 4 -100 degrees Celsius from 1-120 minutes. Such extractions can then be filtered for running on analytical equipment, such as dead-end filtration through a 0. 1-0.22 micron filter of polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), nylon, cellulose, or polyethersulfone (PES) to remove unwanted debris from sensitive analytical procedures.Generating Downstream Ibogaine Alkaloids

[0170] As discussed throughout the disclosure, methods are provided for the biosynthetic production of molecules that are part of the downstream alkaloid biosynthetic pathway related to ibogaine, its precursors, and analogs (e.g., including isotopic analogs (e.g., deuterium analogs), substituted analogs (e g., oxa- or thia- tryptamines; halogenated compounds), and stereoisomers).

[0171] In some embodiments, the methods for generating a downstream alkaloid (ibogaine) compound comprises one or a combination of polynucleotides, enzymes, proteins, or recombinant cells as described herein that are maintained under conditions that allow for the expression and / or activity of the polynucleotide(s) and / or enzymes or proteins and which are sufficient for the synthesis and production for the downstream alkaloid compound.

[0172] In some embodiments of the methods, the disclosure provides for an increase in the production yield and / or titer of one or more of the target compound(s) that are generated. In further embodiments, the increase in the production yield and / or titer of the target compound(s) is relative to the amount or titer of the target compound(s) that is present in a naturally-occurring source. In further embodiments, the increase in the production yield and / or titer of the target compound(s) is relative to the amount or titer of the target compound(s) that can be generated in alternative chemical synthetic methods. In further embodiments, the increase in the production yield and / or titer of the target compound(s) is relative to the amount or titer of the target compound(s) that can be produced from alternative recombinant host cells.

[0173] In some embodiments, the method comprises a geissoschizine synthase (GS) involved in NADPH-dependent iminium reduction on 4,21-dehydrogeissoschizine to produce19E-geissoschizine. In some embodiments, the method comprises a geissoschizine synthase (GS) gene of Table 1, including GS ln - GS_13n (SEQ ID Nos. 1-13), or a geissoschizine synthase (GS) amino acid sequence of Table 28, including GS lp - GS_13p (SEQ ID Nos. 211-223), or a sequence having about 90% sequence identity to any of the GS sequences.

[0174] In some embodiments, the method comprises a geissoschizine oxidase (GO) that catalyzes the oxidation of, for example, 19E-geissoschizine to form an unstable intermediate that eventually converts to the stable akuammicine. In some embodiments, the method comprises a geissoschizine oxidase (GO) gene of Table 2, including GO ln - GO_1 In (SEQ ID Nos. 14-24), or a geissoschizine oxidase (GO) amino acid sequence of Table 29, including GO lp - GO_1 Ip (SEQ ID Nos. 224-234), or a sequence having about 90% sequence identity7to any of the GO sequences.

[0175] In some embodiments, the method comprises a Redox 1 (CAD) and / or a Redox2 (AKR) that together perform an iminium reduction of two unstable intermediates to form stemmadenine, for example, from preakuammicine. In some embodiments, the method comprises a Redoxl (CAD) dehydrogenase gene of Table 3, including Redoxl ln - Redoxl lOn (SEQ ID Nos. 25-34), or a Redoxl (CAD) amino acid sequence of Table 30, including Redoxl lp - RedoxlOp (SEQ ID Nos. 235-244), or a sequence having about 90% sequence identity to any of the Redoxl sequences.

[0176] In some embodiments, the method comprises a Redox2 (AKR) dehydrogenase gene of Table 4, including Redox2_ln - Redox2_9n (SEQ ID Nos. 35-43), or a Redox2 (AKR) amino acid sequence of Table 31, including Redox2_lp - Redox2_9p (SEQ ID Nos. 245-253), or a sequence having about 90% sequence identity to any of the Redox2 sequences.

[0177] In some embodiments, the method comprises a stemmadenine O-acetyltransferase (SAT) that O-acetylates compounds such as stemmadenine to stemmadenine acetate. In some embodiments, the method comprises a stemmadenine O-acetyltransferase (SAT) gene of Table 5, including SAT ln - SAT_9n (SEQ ID Nos. 44-52), or a stemmadenine O- acetyltransferase (SAT) amino acid sequence of Table 32, including SAT lp - SAT_9p (SEQ ID Nos. 254-262), or a sequence having about 90% sequence identity' to any of the SAT sequences.

[0178] In some embodiments, the method comprises a precondylocarpine acetate synthase (PAS) that catalyzes the oxidation of stemmadenine acetate for example to precondylocarpine acetate. In some embodiments, the method comprises a precondylocarpine acetate synthase (PAS) gene of Table 6, including PAS ln - PAS_14n (SEQ ID Nos. 53-66), or a precondylocarpine acetate synthase (PAS) amino acid sequence ofTable 33, including PAS lp - PAS_14p (SEQ ID Nos. 263-276), or a sequence having about 90% sequence identity to any of the PAS sequences.

[0179] In some embodiments, the method comprises a dihydroprecondylocarpine acetate synthase (DPAS) that together with NADPH catalyzes, for example, the reduction of precondylocarpine acetate to dihydroprecondylocarpine acetate. In some embodiments the DPAS over-reduces the precondylocarpine acetate to form secodine or dehydrosecodine. In some embodiments, with excess NADPH, DPAS catalyzes the formation of vincadifformine. In some embodiments, the method comprises a dihydroprecondylocarpine acetate synthase (DPAS) gene of Table 7, including DPASJn - DPAS_23n (SEQ ID Nos. 67-89), or a dihydroprecondylocarpine acetate synthase (DPAS) amino acid sequence of Table 34, including DPAS lp - DPAS_23p (SEQ ID Nos. 277-299), or a sequence having about 90% sequence identity to any of the DPAS sequences.

[0180] In some embodiments, the method comprises a coronaridine synthase (CorS) that generates the unique scaffold, (-)-coronaridine from dehydrosecodine. In some embodiments, the method comprises a coronaridine synthase (CorS) gene of Table 8, including CorS ln - CorS_18n (SEQ ID Nos. 90-107), or a coronaridine synthase (CorS) amino acid sequence of Table 35, including CorS lp - CorS_18p (SEQ ID Nos. 300-317), or a sequence having about 90% sequence identity to any of the CorS sequences.

[0181] In some embodiments, the method comprises a polyneuridine aldehyde esterase (PNAE) that de-esterifies (-)-voacangine and which can be decarboxylated (with heat) to (-)- ibogaine. In some embodiments, the method comprises a polyneuridine Aldehyde Esterase (PNAE) gene of Table 9, including PNAE ln - PNAE_9n (SEQ ID Nos. 108-116), or a polyneuridine Aldehyde Esterase (PNAE) amino acid sequence of Table 36, including PNAE lp - PNAE_9p (SEQ ID Nos. 318-326), or a sequence having about 90% sequence identity to any of the PNAE sequences.

[0182] In some embodiments, the method comprises an O-methyltransferase (OMT) that catalyzes, for example, the 10-O-methylation of noribogaine to produce ibogaine and 10-0- methylation of 10-hydroxycoronaridine to voacangine. In some embodiments, the method comprises an O-methyltransferase (OMT) gene of Table 10, including OMT ln - 0MT 1 In (SEQ ID Nos. 1 17-127), or an O-methyltransferase (OMT) amino acid sequence of Table 37, including OMT lp - 0MT 1 Ip (SEQ ID Nos. 327-337), or a sequence having about 90% sequence identity to any of the OMT sequences.

[0183] In some embodiments, the method comprises an indole ring hydroxy lase (Ox) (Il OH) which is a p450 monooxygenase that can catalyze the 10-hydroxylation of ibogamineto produce noribogaine and 10-hydroxylation of coronaridne to produce 10- hydroxycoronaridine. In some embodiments, the method comprises a hydroxylase (Ox) gene of Table 11, including Ox ln - Ox_20n (SEQ ID Nos. 128-147), or a hydroxylase (Ox) amino acid sequence of Table 38, including Ox lp - Ox_20p (SEQ ID Nos. 338-357), or a sequence having about 90% sequence identity to any of the Ox sequences.

[0184] In some embodiments, the method comprises an enol-O-methyltransferase (enolMT) that catalyzes the methylation of, for example, geissoschizine into geissoschizine methyl ether. In some embodiments, the method comprises an enolMT gene of Table 15, including enolMT ln - enolMT_7n (SEQ ID Nos. 160-166), or an enolMT amino acid sequence of Table 42, including enolMT lp - enolMT_7p (SEQ ID Nos. 370-376), or a sequence having about 90% sequence identity to any of the enolMT sequences.

[0185] In some embodiments, the method comprises a cytochrome P450 reductase (CPR) gene of Table 13, including any one or more of sequences CPR ln - CPR_4n (SEQ ID Nos. 150-153), or a cytochrome P450 reductase (CPR) amino acid sequence of Table 40, including any one or more of sequences CPR lp - CPR_4p (SEQ ID Nos. 360-363), or a sequence having at least 90% sequence identity to any of the CPR sequences.

[0186] In some embodiments, the method comprises a cytochrome b5 (CYB5) gene, which together with CPR can participate in electron transfer reactions. Coexpression of CPR and CYB5 proteins can enhance enzymatic oxidations, such as increasing the activity of the P450 enzymes described herein. In some embodiments, the method comprises a CYB5 gene of Table 12, including CYB5_ln, or CYB_2n (SEQ ID Nos. 148-149), or a cytochrome b5 (CYB5) amino acid sequence of Table 39, including CYB5_lp, or CYB5_2p (SEQ ID Nos. 358-359), or a sequence having at least 90% sequence identity to any of the CYB5 sequences.

[0187] In some embodiments, the method comprises a transporter (trxporter) gene of Table 14, including any one or more of sequences trxporter ln - trxporter_6n (SEQ ID Nos. 154-159), or a transporter (trxporter) amino acid sequence of Table 41, including any one or more of sequences trxporter lp - trxporter_6p (SEQ ID Nos. 364-369), or a sequence having at least 90% sequence identity to any of the trxporter sequences.

[0188] In some embodiments, the method comprises a tryptophan importer (TAT2) gene of Table 24, including TAT2_ln (SEQ ID No. 207) or a tryptophan importer (TAT2) amino acid sequence, or a sequence having at least 90% sequence identity to any of the TAT2 sequences.

[0189] In some embodiments, the method comprises a SAMe importer (SAM3) gene of Table 22, including SAM3_ln (SEQ ID No. 205) or a SAMe importer (SAM3) amino acidsequence, or a sequence having at least 90% sequence identity to any of the SAM3 sequences.

[0190] In some embodiments, the method comprises a methionine importer (MUP1) gene of Table 23, including MUPl ln (SEQ ID No. 206) or a methionine importer (MUP1) amino acid sequence, or a sequence having at least 90% sequence identity to any of the MUP1 sequences.

[0191] In some embodiments, the method can include production of one or more labile intermediate compounds that can form spontaneously and which can feed into a separate portion of the pathway for production of indole alkaloids including ibogaine.

[0192] For purposes of illustration and clarity, FIG. 1 depicts the tryptamine and alkaloid skeletons. The portion of the molecule from the tryptophan indole is highlighted. The portion of the molecule from the monoterpenoid is highlighted. In embodiments, engineering of the native yeast tryptophan pathway to enable high flux toward tryptophan and tryptamine production is used in the systems herein, and is described in, e.g., WO 2021 / 248087 (PCT / US2021 / 036031).

[0193] The terpene moiety of strictosodine, secologanin, can be fermentation-derived by the enzyme-engineered host as described herein or fed into the media. Secologanin exogenously provided to a fermentation media can be crude extract material from plants which contain MIA precursors. For example, extracts from plants from the Symphoricarpos genus, such as snowberry' (Symphoricarpos albus), and other plants such as Silvervine (Actinidia polygama), Lonicera japonica and Honeysuckle (Lonicera caerulea) can be a source of exogenous MIA precursors such as secologanin. In other embodiments, material and extracts thereof from plants of the Gentianales order, which can include Mitragyna speciosa (kratom), Uncaria tomentosa, Uncaria rhynchophylla, Voacanga africana, Voacanga thouarsii, Tabemanthe ihoga, Tabemanthe mannii, Picralima nitida, Tabernaemontana elegans, and / or Catharanthus roseus. In some embodiments the secologanin is provided by the plant and / or berry extract for the host cell to uptake as a starting substrate to feed into the biosynthetic pathway for MIA production. Likewise, other iridoid precursors for MIA production can be sourced and extracted, whether to high purity or remaining as a crude extract, from the plants listed herein. For example, snowberry (Symphoricarpos albus) extract and Honeysuckle (Lonicera caerulea) extract contain loganic acid and loganin. In some embodiments the loganic acid and / or loganin is provided by plant and / or berry extract for the host to uptake as starting substrate to feed into the biosynthetic pathway for indole alkaloid production.

[0194] In embodiments wherein plant material is incorporated as a fermentation media supplement for recombinant hosts to produce MIAs, the plant material comprising MIA precursors is processed by methods known by those skilled in the art. For example, in some embodiments, the plant material, such as Honey suckle, is crushed up into powder using a mortar and pestle, a homogenizer, vortexing, or a blender, and can be extracted using a variety of organic solvents ranging in polarity, including but not limited to methanol, ethanol, butanol, ethyl acetate, acetonitrile, methyl tertiary-butyl ether (MTBE), and / or acetone. To lyse plant cells for efficient extraction of indole alkaloid pathway compounds, the powder is dissolved in water (e.g., ddFhO) and subjected to either bead beating for 3-6 min. repeated one to five times, homogenization, or freeze-thaw cycles (e.g., alternating boiling water bath and frozen ethanol bath or liquid nitrogen) for three to six times. Organic solvent listed above is then added to the lysate and incubated with the lysate for 1-24 hrs at 20-50 °C. The organic layer is then collected, dried and resuspended in acetonitrile for analytical detection and quantification. The leftover aqueous layer is also extracted with acetonitrile and subjected to analy sis by HPLC, LC-MS, HRMS, and LC-MS / MS. The dried plant extract containing MIA precursors can be used as a fermentation media supplement for MIA production by recombinant host cells.

[0195] For selective detection and characterization of MIA compounds and precursors including ketones and aldehydes produced by recombinant hosts or present in plant extracts, derivatization techniques known to those skilled in the art can be used. Reagents (e.g., modifying or derivatizing agents) that can be used for detection and characterization of MIA compounds can include, but not be limited to, 2 -amino benzamidoxine (ABAO), para- methoxy-2 -amino benzamidoxime (PMA), 4-bromo-N-methylbenzylamine, 9- fluoromethylchloroformate (FMOC), 3-methyl-2-benzothiazolinone hydrazone (MBTH), 1- (5-fluoro-2,4-dinitrophenyl)-4-methylpiperazine (PPZ), 2,4-dinitrophenylhydrazine (DNPH) and 4-bromo-N-methylbenzylamine (4-BNMA). For example, in some embodiments, fermentation broth or plant material extracts are derivatized using 2-amino benzamidoxime (ABAO). In such embodiments, fermentation broth or plant material extracts (e g., in acetonitrile) are subjected to reacting with 0.1-lmM ABAO at pH 4-7. The reaction can be performed at 30-37°C and incubated for at least 15 min or allowed to incubate for hours (i.e., overnight). In embodiments comprising an ABAO derivatization of a MIA precursor and / or product, the derivatized product formation is monitored with fluorescence excitation at 360 nm and emission at 528 nm. Alternatively, both the ABAO reagent and derivatized adductscan be monitored with a UV absorbance at 405 nm. All derivatized products can be further analyzed with LC-UV-MS.

[0196] In some embodiments, sugars and nitrogen from plant and berry' extracts also provide the nutrients necessary for the growth and fermentation of recombinant host cells in addition to providing iridoid precursors for MIA production, minimizing or eliminating any other additional media additives for the recombinant host.

[0197] In some embodiments, these precursors are converted to MIAs in a cell-free system, such as via crude or purified lysate, microsomes, or purified enzymes from a recombinant host or combinations of recombinant hosts which contain the biosynthetic enzymes for MIA production.

[0198] In some further downstream biosynthetic pathway embodiments, FIG. 3 shows example enzyme conversion of strictosidine aglycone to ibogaine, norvoacangine to ibogaine, or de-esterifying coronaridine to ibogaine. Such a pathway can be carried out in a recombinant host expressing combinations of sequences described here in, including genes encoding enzymes for GS, GO, PAS, DPAS, REDOX 1, REDOX2, CPR, CYB5, SAT, Ox, OMT, CorS, and / or PNAE, in addition to the genes described herein to generate MIA pathway precursors and intermediates.

[0199] FIG. 1C depicts substituted ibogaine molecules with a modified or substituted indole ring. Sources of modified indole rings can stem from substituted tryptophan or tryptamine -like molecules can be found in WO / 2021 / 248087. R substitutions on the indole ring positions include single or multiple independent substitutions of H, OR2, NR2 R2, N+R2, SR2, S+R2, P+R2, P(O)R2, P(O)R2, S(O)R2, SO2R2, SO3R2, CO2R2, CN, oxo, a halide, a haloalkyl and substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkylamino, dialkylamino and alkylthio, wherein R2 independently comprises H, alkyl, alkenyl, alkynyl, oxoalkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkylamino, a halide, a haloalkyl, dialkylamino and alkylthio. Additional variants of the indole ring nitrogen can also be incorporated including, for example, the substitutions of the indole ring nitrogen with an oxygen atom (i.e. benzofurans), a sulfur atom, a carbon atom (optionally substituted), or a substituted ring nitrogen (such as with R2 as defined above (e.g., methylated) or a halogenated) as indicated by Z in FIG. 1 (where Z can be O, S, C optionally substituted (e.g., with R2), or N optionally substituted (e.g., with R2)). Such substitutions of the nitrogen of the indole ring can lead to substituted strictosidines, as well as derivates to downstream indole alkaloids such as, for example, coronaridines, voacangines, ibogamines, and ibogaines. A nonlimiting example of these substituted MIAs where the indole nitrogen is replaced, forinstance with an oxygen (e.g. benzofuran derivatives) can lead to ‘oxa’ versions of MIAs, including oxa-strictosodine, oxa-coronaridine, oxa-voacangine, oxa-ibogamine, oxanoribogaine, oxa-ibogaine (see FIG. ID) and many more MIAs, where the benzofuran moiety can originate from a benzofuran precursor added to a fermentation or cell free reaction. Another nonlimiting example can stem from a 1- (or N-) methylated substituted ibogaine molecule (see FIG. ID) where the N-methylated indole moiety' can originate from a 1 -methylated tryptophan or tryptamine precursor added to a fermentation or cell free reaction. FIG. IE depicts a structural genus for a variety of downstream MIA compounds, reflecting preferred stereoisomer arrangements, where R and Z are as described above.

[0200] FIG. 3 depicts example embodiments comprising modified cell hosts expressing gene combinations for generating MIA pathway products, in accordance with the disclosure, and embodiments that utilize the compounds generated by the disclosed methods to feed into various downstream MIA biosynthetic pathways.

[0201] To illustrate some additional embodiments, FIG. 4A depicts an example of bioconversion by a modified cell host of an MIA precursor into a MI A target compound. Exogenous pure precursor or crude extract of precursors, e.g., containing iridoids, loganic acid, loganin, secologanin, strictosidine, among others, can be fed to recombinant cells expressing the MIA biosynthesis pathway. FIG. 4B shows an example of bioconversion using multiple organisms via a modified cell host expressing a portion of the MIA pathway, for example producing up to strictosidine, while another modified host cell expressing a downstream MIA pathway uses the products from the first host cell to produce MIAs such as ibogaine. Precursors made by modified cell host 1 can be excreted or derived from crude or purified lysate and fed to cells expressing downstream pathway enzymes.

[0202] In embodiments, the de novo biosynthesis pathway of L-tryptophan and SAM / SAMe are utilized as precursor molecules and cofactors in the systems and methods herein. The precursor molecules lead to target molecules of the tryptamine pathways, when incorporated on-pathway. In some embodiments of the systems (e.g., organisms) and methods herein, glycolysis leads to chorismate (e.g., comprising chorismate synthase) via the shikimate pathway; glutamate biosynthesis pathway leads to L-glutamine via L-glutamate; and the L-serine biosynthesis pathway leads to L-serine via 3-phospho-L-serine (i.e., dephosphorylation). Chorismate, glutamine, and L-serine can be combined to form L- tryptophan as a precursor molecule, and which can be incorporated into the biosynthetic pathway that can yield tryptamine. In some embodiments of the systems (e.g., organisms) and methods herein, L-methionine is a direct precursor that can produce SAMe, when combinedwith ATP in the presence of Sam2 and Adkl enzymes. Methods described or referenced herein to increase flux through the tryptophan and SAMe biosynthesis pathways, such as overexpressing genes involved in the shikimate pathway, chorismate pathway, and methionine pathway, can lead to increased MIA product yield.

[0203] Describing this further, in some embodiments, tryptophan accumulation in a recombinant host cell can be increased by one or more of: (a) overexpressing feedbackresistant versions of tryptophan-syTithesizing enzymes; (b) knocking out and / or inhibiting expression of off-pathway tryptophan-consuming genes and enzymes; and / or (c) overexpressing a recombinant L-tryptophan transporter for exogenous tryptophan import (see also U.S. PGPub 2021 / 0147888 and International publication WO 2021 / 248087). In some embodiments the biosynthesis can comprise (over)expression of enzymes (e.g., TRP1, TRP2, TRP3, TRP4 and / or TRP5) which can increase intracellular tryptophan and maintain or direct tryptophan flux through the pathway. In some embodiments the biosynthesis can comprise (over)expression of one or more genes useful for tryptophan biosynthesis, such as ARO1, ARO4, AROL, SERI, SER3, SER33, and / or SER2, and including feedback resistance mutant versions thereof, which increase shikimate pathway flux and / or supply serine.

[0204] The following Examples illustrate several aspects and embodiments in accordance with the disclosure and in no way serve to limit the scope or spirit of the claims.EXAMPLESExample 1 - Method of Cell Growth

[0205] Modified host cells that yield MIA precursors and compounds, such as the downstream alkaloid-producing strains described herein, express engineered genes and enzymes. More specifically, the downstream alkaloid-producing strains herein are grown in a minimal, complete culture media containing yeast nitrogen base, amino acids, vitamins, ammonium sulfate, and a carbon source of glucose and galactose. The recombinant host cells are grown in 24-well plates or shake flasks in a volume range of 2 mL to 100 mL of media starting from an inoculation density of ODbOOnm = 1 . Exogenous compounds (e.g., upstream precursor compounds, such as strictosidine, strictosidine aglycone, or coronaridine) up to 1% can be added to media to supplement the precursor pool for downstream alkaloid-production. In addition or alternatively, early (upstream) pathway precursors can be imported into host cells. Exogenous L-tryptophan can be taken up by strains expressing the TAT2 L-tryptophanimporter protein and exogenous L-methionine can be taken up by strains expressing the MUP1 L-methionine permease protein. The strains herein can be harvested during a fermentation period ranging from 12 hours onward from the start of pathway enzyme induction.

[0206] Alternatively, strains such as Saccharomyces cerevisiae that are modified to produce downstream alkaloids can be grown and fermented in media containing in the juice of Snowberry {Symphoricarpos albus) or Honeysuckle (Ivnicera caeruled) to provide the secologanin precursor. Platform strains are grown in berry juice supplemented with compounds essential for growth (20 mg / L adenine, 20 mg / L L-histidine, 30 mg / L lysine, 20 mg / L L-leucine, and 20 mg / L tryptophan, pH 5.8). Carbon source, such as sucrose, glucose, galactose, xylose, lactose, and / or glycerol at 10-60 g / L is added to the media. Alternatively, secologanin can be provided as a feed-in to the media at 2mM final concentration.

[0207] Some embodiments can include, during the fermentation, addition of substituted substrate molecules (e.g ., substituted tryptophan or tryptamine) that allow for the synthesis and production of substituted downstream MIAs. For example, oxa-tryptamine (i.e. a benzofuran) can be added to a fermentation at a concentration of 0.1-0.5 g / L, and maintained at such concentrations for 12-48 hrs by bolus addition of a concentrated oxa-tryptamine stock solution or fed-in at a rate to maintain the concentration of the substrate in the fermentation, to generate ‘oxa’ analogs of downstream MIAs such as, for example, an oxa-ibogaine. Similarly, 1-methyl-tryptamine (i.e., N-methylated at the indole ring N) is added to a fermentation at a concentration of 0. 1-0.5 g / L, and maintained at such concentrations for 12- 48 hrs by bolus addition of a concentrated 1-methyl-tryptamine stock solution or fed-in at a rate to maintain the concentration of the substrate in the fermentation to generate N-methyl analogs of downstream MIAs such as, for example, 1-N-methyl-ibogaine (see FIG. ID). In addition the non-limiting examples mentioned above, the methods can incorporate deuterated molecules (e.g., as feedstocks, solvents, precursors, etc. comprising deuterium), in order to generate and synthesize deuterated forms of the alkaloid compounds generated by the methods described herein. Similarly halogenated molecules can be incorporated in a similar fashion in order to generate and synthesize halogenated forms of the alkaloid compounds generated in accordance with the aspects and embodiments of the disclosure.

[0208] After fermentation, the broth can be extracted as described herein, for analysis of MIA pathway products.Example 2 - Detection of Isolated Product

[0209] To identify fermentation-derived monoterpene indole alkaloids (MIAs), their precursors, and all other products of a recombinant host expressing an engineered biosynthetic pathway for alkaloids, an Agilent 1100 series liquid chromatography (LC) system and Agilent 6125 coupled to an Agilent 1260 LC stack, equipped with a reverse phase Cl 8 column (Agilent Eclipse Plus Cl 8, Santa Clara, CA, USA) is used. For high accuracy small molecule analysis and high resolution mass spectrometry (HRMS), an Agilent 6230 LC system coupled with Time-of-Flight (TOF) is used with a reverse phase C18 column (Agilent Eclipse Plus Cl 8, Santa Clara, CA, USA). A gradient is composed of mobile phase A (ultraviolet (UV) grade H2O + 0. 1% formic acid) and mobile phase B (UV grade acetonitrile + 0.1% formic acid). Column temperature is set at 30 °C. Compound absorbance is measured at 210nm, 235nm, 270nm, and 305nm using a diode array detector (DAD) and spectral analysis from 200nm to 400nm wavelengths. A mass range of m / z 10-2000 is enabled for mass spectrometiy analysis of small molecules. A 0. 1 mg / mL analytical standard is made from certified reference material for each compound (Cayman Chemical Company, USA). Each sample is prepared by diluting fermentation biomass from a recombinant host expressing the engineered biosynthesis pathway 1 :3 or 1 :20 in 100% acetonitrile and filtered in 0.2 tun nanofilter vials. The retention time and UV-visible absorption spectrum (i.e., spectral fingerprint) of the samples are compared to the analytical standard retention time and UV-visible spectra (i.e. spectral fingerprint) when identifying the monoterpene indole alkaloids, their precursors, and all other related compounds.

[0210] In addition or alternatively, mass fragmentation of analytes can be performed for additional compound identification. LC-MS and LC-MS / MS, as depicted in FIG. 5, FIG. 6, FIG.7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12, FIG. 13 and FIG. 14, was carried out using an Agilent Eclipse C 18, 4.6 X 100 mm, 2.5 pm particles column, at 40°C, a flow rate of Iml / min, with mixtures of solvents A (H2O with 0. 1% Formic acid) and solvents B (Acetonitrile with 0.1% Formic acid), with UV-vis detection and with a Thermo LTQ Orbitrap. FIG. 7 depicts LC-MS / MS fragmentation pattern data identifying derived ibogaine produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with an PNAE gene (PNAE_3n) encoding an PNAE enzyme (PNAE_3p), as further described below. FIG. 8 depicts LC-MS data identifying geissoschizine methyl ether produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with GS and enolMT genes (GS_4n and enolMT ln) encoding GS and enolMT enzyme (GS_4p andenolMT lp). FIG. 9 depicts LC-MS and LC-MS / MS data identifying akuammicine produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with GS and GO genes (GS_4n and GO ln) encoding GS and GO enzyme (GS_4p and GO lp). ). FIG. 10 depicts LC-MS and data identifying tabersonine, which shares the same precursors as coronaridine, produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with PAS, DPAS, and TS genes (PAS ln, DPAS ln, and TS ln) encoding PAS, DPAS, and TS enzymes (PAS lp, DPAS lp and TA lp). FIG. 11 depicts LC-MS / MS EIC and fragmentation pattern data identifying ibogamine produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with an PNAE gene (PNAE_3n) encoding an PNAE enzyme (PNAE_3p). FIG. 12 and FIG. 13 depict LC-MS data identifying norvoacangine (hydroxycoronaridine) produced by recombinant Saccharomyces cerevisiae cells or lysate derived from such cells (e.g. cell-free) expressing the MIA pathway with an Ox (I10H) gene (Ox i In and Ox_20n) encoding an Ox (I10H) enzyme (Ox i Ip and Ox_20p). FIG. 14 depicts LC-MS data identifying norvoacangine (EIC 355 m / z) and voacangine (EIC 369 m / z), produced by a co-culture of recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with an Ox (Il OH) gene (Ox i In) encoding an Ox (Il OH) enzyme (0x 1 Ip) and recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with an 0MT gene (0MT_8n) encoding an 0MT enzyme (0MT_8p).

[0211] FIG. 15 depicts LC-MS and LC-MS / MS data identifying coronaridine produced by recombinant Saccharomyces cerevisiae cells expressing the MIA pathway with a CORS- DPAS fusion gene (Fusion_Pair_2n) encoding CORS-DPAS fusion enzyme (Fusion_Pair_2p).Example 3 - Expression of coronaridine 10-hydroxylase in a modified host organism to produce 10-hydroxycoronaridine

[0212] P450 (Ox) enzymes which act as a coronaridine 10-hydroxylase (110H) can convert coronaridine to 10-hydroxycoronaridine and / or convert ibogamine to noribogaine. P450 hydroxylases are membrane bound enzymes that require the expression of accessory' proteins such as cytochrome B5 (CYB5) and cytochrome P450 reductase (CPR) for electron transport. In some embodiments, cytochrome P450 hydroxylases also require the presence of heme. Knocking out the host HMX1 gene involved in the degradation of heme can increase the presence of cellular heme required for proper hydroxylase function. Construction ofSaccharomyces cerevisiae production of 1 O-hydroxycoronaridine can be carried out via knocking out HMX1 gene and the expression of optimized Ox, CYB5 and CPR genes.

[0213] Production of norvoacangine via a recombinant host as disclosed herein can be carried out by producing strains expressing a coronaridine 10-hydroxylase (Ox, also called Il OH) in the presence of endogenously derived coronaridine, such as from a modified strain generating coronaridine, or conversion of exogenous coronaridine into norvoacangine. Such strains can be grown as in Example 1, with norvoacangine isolated and detected as in Example 2. Further, modified strains expressing II OH and CPR (i.e. the P450 & CPR pairing for efficient electron transport described above), are grown in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 and fermented for 48-96 hrs at 30°C. For applications where the promoter of the relevant genes (e.g. Ox) are inducible, galactose, glycerol, or lactose (0.01-2% v / v) is added to the culture media as an inducer 24-48hrs of growth. After the initial 24-48 hrs of growth, 0.01-0.5 g / L coronaridine is added as exogenous substrate for conversion of coronaridine into norvoacangine. The formation of norvoacangine is monitored using liquid chromatography with mass spectrometry' (LC-MS). Detection of products is carried out as described in Example 2. In FIG. 12, hydroxylase activity leading to norvoacangine was detected as above only in recombinant hosts expressing active hydroxylase (Ox or II OH) and not in a negative control strain. Norvoacangine production was also validated by comparing the fermentation derived norvoacangine to an analytical standard. The Ox (Il OH) genes depicted are the Ox 1 In and Ox_20n genes encoding the Ox 1 Ip and Ox_20p enzymes.Example 4 - Expression of recombinant polyneuridine Aldehyde Esterase (PNAE) in a modified host organism to produce de-ester-(-)-voacangine and (-) ibogaine.

[0214] Recombinant Saccharomyces cerevisiae strains with activity to 1) convert voacangine to de-ester voacangine to ibogaine, 2) convert coronaridine to de-ester coronaridine to ibogamine, and / or 3) convert norvoacangine to de-ester norvoacangine to noribogaine, are generated via expression of the PNAE gene (PNAE_ln-9n) which encodes the MIA esterase enzyme (PNAE_lp-9p). The optimized PNAE genes are synthesized using DNA synthesis techniques known in the art. The optimized genes are cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression froman episomal plasmid, the optimized PNAE gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.

[0215] Ibogaine produced from recombinant hosts expressing the PNAEs disclosed herein, can be detected and isolated as shown in FIG. 6 with further LC-MS / MS data in FIG. 7. Here, recombinant Saccharomyces cerevisiae cells are grown in 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4, for 24 hrs in an incubator shaking at 300 RPM. Cells are induced with minimal media containing 20 g / L galactose. Voacangine is fed to this modified yeast cell culture at a concentration of 0. Img / mL and the culture is incubated for an additional 72h at 30°C under the same conditions. Products are extracted and isolated as described in Example 2. In FIG. 6 and FIG. 7, esterase activity is detected only in recombinant hosts expressing PNAE and not in negative control strains. The PNAEs depicted are PNAE_2n, 3n genes encoding PNAE_2p, 3p enzymes. The de-ester-voacangine intermediate is unstable and is spontaneously converted to ibogaine as seen in FIG. 6 and FIG. 7.

[0216] Similarly, ibogamine can be generated using coronaridine as a substrate for the PNAE activity, where coronaridine substitutes for voacangine in the above experimental conditions. Ibogamine is extracted and isolated as described in Example 2. In FIG. 11, esterase activity is detected only in recombinant hosts expressing PNAE and not in negative control strains. The PNAE depicted is the PNAE 3n gene encoding the PNAE_3p enzyme.Example 5 - Construction of Saccharomyces cerevisiae platform strains with accumulation of MIA pathway intermediates and products.

[0217] Construction of the Saccharomyces cerevisiae platform strain with accumulated intermediates in the ibogaine biosynthetic pathways is carried out via knocking out transporter genes that can be implicated in the export of aforementioned intermediates. MIA upstream biosynthetic pathway intermediates and products such as nepetalactol, loganic acid, loganin, secologanin, strictosidine, and strictosidine aglycone, as well as downstream ibogaine biosynthetic pathway compounds such as, stemmadinine, stemmadenine acetate, coronaridine, norvoacangine, voacangine, ibogaine, and / or other MIA products intermediates, can exit cells via transporters, permeases, and exporters. The loss of MIA pathway intermediates to the extracellular space decreases the availability of these pathway intermediates inside the cells to act as substrates for the MIA biosynthetic enzymes andlowers the flux of the pathway towards ibogaine MI As. To accumulate these key intermediates in the cells, the endogenous Saccharomyces cerevisiae genes, including but not limited to ERC1, TPO1, FLR1, SNQ2, STE6, YOR1, AUS1, MDL1, MDL2, PTR2, NPF, PDR5, PDR10, PDR12 and PDR15, are deleted as single or combinatorial knockouts. Knockout of these transporter genes is performed by placement of an early stop codon in the coding region of the corresponding gene sequences using techniques known in the art. Such insertions can proceed via homologous recombination using the host machinery and make use of a synthesized DNA framing (oligonucleotide) with 40-800 nucleotides on either or both sides of a STOP codon that are complementary to the gene locus to be knocked out. Further verification of the modification in said strain can be carried out by genome sequencing and analyzed by the techniques disclosed in U.S. Patent 10,671,632. Once the Saccharomyces cerevisiae platform strains with single and / or multiple transporter knockouts are completed, combinatorial episomal expression of MIA pathway enzymes, such as those encoded by, for example, Ox and OMT genes can consume the intracellular trapped MIA precursors to generate additional amounts of downstream MIA products, including ibogaine.EXAMPLE 6 - Cell-free production of MIA compounds using a recombinant MIA pathway-expressing Saccharomyces cerevisiae strain

[0218] For enzymes such as P450s that are tethered to a membrane, membrane fractions must be prepared and included in the reaction mixture. Ibogamine 10 hydroxylase (Ox or I10H) is one such enzyme. To provide this activity in a cell free system, the following procedure is followed. Cells expressing the Ox enzymes (Ox_lp-19p) encoded by the Ox genes (Ox_ln-19n) are inoculated in 25-50mL of appropriately selective media and cultured at 30°C at 250rpm for 40-56 hours, where the media contains 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4. Inducer such as 0.2- 10% v / v galactose is added to the culture media and induction proceeds under the same conditions as growth for a further 18-30 hrs. Cell growth via optical density (OD) is measured and cells are harvested by centrifugation 1 lOOrcf for 10 min. Cell pellets are normalized by OD. Cell pellets are washed once with 25mL cold water. Pellets areresuspended in water and aliquoted at 30 OD. Cells are pelleted once again at 1 min at 8000 ref and then the supernatant removed. Remaining cells can be frozen and stored at -80°C for MIA producing reactions as needed, or used immediately. Cells are then lysed by adding 250 uL lysis buffer per pellet. Lysis buffer consists of 0. IM sorbitol, 50mM potassium acetate, 20mM HEPES, and ImM DTT. If pellets were previously frozen, pellets are first thawed on ice. Resuspended pellets are added to a tube with glass beads and cells are disrupted in a bead beater 3X 3 min at 2400rpm with 1 min on ice in between. Fractionation buffer is added to the lysed pellet, and the resulting lysate is aliquoted to a new tube and spun for 5 min at 0.8 ref at 4°C. Fractionation buffer consists of 150mM potassium acetate, 5mM magnesium chloride, and 250mM sorbitol. Supernatant is then taken to a new tube and spun 18,000rcf for 20 min at 4°C. The pellet is then collected, washed and respun. The resulting membrane fraction with MIA enzyme activity is then resuspended in buffer, along with addition of substrate. Substrate is added at 0.01-1 mg per ml. Substrates can include coronaridine, voacangine, norvoacangine, ibogamine, and / or 10-hydroxy-ibogamine. Buffer is generally 10-lOOmM Tris pH 7-8, with 0.1-2 mM NADPH. Reactions proceed 2-24 hrs at 30°C. Samples are extracted, isolated, and analyzed via methods described herein (See Example 2).

[0219] In certain cases, cell-free systems, such as crude or clarified lysate, or purified enzyme components are desired to carry out reactions. Such reactions can be usefid to avoid cell toxicity, to facilitate enzyme-substrate reactions, and / or to quickly screen enzymes and conditions for activity on substrates. Recombinant cells expressing components of the MIA pathway, or the complete MIA pathway, can be used to generate cell-free systems for generating complex MIAs and their analogs. For example, lysate generated from recombinant strains expressing hydroxyl ase(s) (Ox or II OH) can be used to convert coronaridine into norvoacangine ( 10-hydroxy coronaridine).

[0220] To provide this activity in a cell-free system, the following procedure is followed. Cells expressing the MIA enzymes (e g. the hydroxylase enzymes Ox_lp-20p encoded by the Ox gene Ox_ln-20n) are inoculated in 2-50mL of minimal media and cultured at 30°C at 250rpm for 18-72 hours, where the media contains 20-60 g / L glucose, 5- 25 g / L ammonium sulfate, 1-5 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 5-100 mM KH2PO4 and 1-20 mM K2HPO4. If the genes of interest are under inducible promoters), an inducer such as 0.02-10% v / v galactose, glycerol, or lactose is added to the culture media after 18-24 hours of growth and induction proceeds under the same conditions as growth for a further 6-48 hrs. Cell growthvia optical density (OD) is measured (OD600) and cells are harvested by centrifugation l,100rcf for 10 min. Cell pellets are normalized by OD. Cell pellets are washed once with 5- 25mL cold water. Pellets are resuspended in water and aliquoted at 5-50 OD. Cells are pelleted once again at 1 min at 5,000 - 10,000 ref and then the supernatant removed.Remaining cells can be frozen and stored at -80°C for MIA producing reactions as needed, or used immediately. Cells are then lysed by adding 0.2-1 ml sterile deionized water or a lysis buffer per 0.05-0.2 ml pellet. Lysis buffer consists of either 0. IM sorbitol, 50mM potassium acetate, 20mM HEPES, and ImM DTT, or a lysis buffer of 20- 100m M Glycine at pH 8-9.5 with ImM DTT, or a lysis buffer consisting of 10-150 mM carbonate at pH 8-9.5 with ImM DTT. The resuspended cells are lysed by bead beating 2-4 times for 2-4 minutes each, with 1-2 minutes on ice between each cycle. The resulting crude lysate can either be used directly for cell-free assays, or further centrifuged to prepare clarified cell-free lysate by centrifugation at 10,000-100,000 ref for 3-7 minutes, after w'hich the clarified lysate is transferred to a new tube and kept on ice. For reactions using such lysate, additional lysis buffer can be added. Cofactors such as FAD (final concentration 0. 1-0.5 mM) and NADPH (final concentration 0.5-2 mM) are added as needed. Metal ions such as zinc and manganese can also be supplemented at concentrations ranging from 10 pM to 10 mM. The substrate, such as coronaridine, is added to the reaction mixture at a final concentration of 0.05-0.5 mg / mL. The reaction is incubated at 25-35°C with shaking at 250-400 rpm for 2-8 hours. Samples are then extracted with 2-3 volumes of 70-90% acetonitrile in water, mixed, and filtered for analysis. FIG. 13 shows a cell-free reaction where exogenous coronaridine is converted into norvocangine, where norvoacangine is detected as described in Example 2. In FIG. 13, hydroxylase activity leading to norvoacangine was detected as above only in cell- free lysate prepared from recombinant hosts expressing active hydroxylase and not in a negative control strain. Norvoacangine production was also validated by comparing the fermentation derived norvoacangine to an analytical standard. The Ox (Il OH) genes depicted are the Ox 1 In and Ox_20n genes encoding the Ox l ip and Ox_20p enzymes.EXAMPLE 7 - Expression of recombinant noribogaine methyltransf erase (OMT) in a modified host organism to produce voacangine

[0221] To catalyze the methyltransferase reaction converting norvoacangine to voacangine (FIG. 5), recombinant cells expressing an OMT (OMT_lp-l Ip) encoded by an OMT gene (OMT_ln-l In) containing a methyltransferase enzyme are grown, induced andlysed as above. The resulting lysate is spun for 10 min at 18,000 ref at 4 °C and the supernatant is used as the source of methyltransferase activity'. S-adenosylmethionine (SAMe) is added as a methyl donor cofactor at 200uM. Norvoacangine substrate is dissolved in Methanol to a concentration of Img / mL and added to reaction mixture at 5% by volume. Assay buffer contained 50mM Tris pH8.0, 10% glycerol, 3mM MgCh an lOOug / mL BSA, ImM DTT and protease inhibitors. Reaction proceeded overnight at 30°C with shaking. The assay samples are extracted with organic solvents such as acetonitrile and the product formation analyzed by LC-UV-MS. In FIG. 5, methyltransferase activity leading to voacangine production was detected as above only in recombinant hosts expressing OMT and not in negative control strains. The OMT depicted is the OMT 8n gene encoding the OMT 8p enzyme.Example 8 - Expression of a SAM-dependent enol methyltransferase (enoIMT) in a modified host organism to produce geissoschizine methyl ether

[0222] Construction of Saccharomyces cerevisiae strains is carried out via simultaneous expression of optimized GS (GS_ln-13n) genes encoding GS (GS_lp-13p) enzymes with optimized enoIMT (enolMT_ln-7n) genes encoding enoIMT (enolMT_lp-7p) enzymes to convert geissoschizine into geissoschizine methyl ether as a stable readout (see FIG. 8). Recombinant hosts capable of producing MI As (e g., able to produce precursors such as strictosidine and strictosidine aglycone) are grown in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 and fermented for 48-96 hrs at 30°C. After the initial 24-48 hrs of growth, 0. 1-0.5 g / L secologanin and 0. 1-0.5 g / L L-tryptophan are added to support MIA precursor flux to yield geissoschizine and geissoschizine methyl ether. The formation of geissoschizine methyl ether and related on-pathway intermediates in the late stage MIA pathway are monitored using liquid chromatography with mass spectrometiy (LC-MS). Detection of products are carried out as described in Example 2. In FIG. 8, enol O-methyltransferase activity leading to geissoschizine methyl ether production was detected as above only in recombinant hosts expressing enoIMT and not in negative control strains. The enoIMT depicted is the enoIMT In gene encoding the enoIMT Ip enzyme.Example 9 - Expression of a geissoschizine oxidase (GO) in a modified host organism to produce akuammicine

[0223] Construction of Saccharomyces cerevisiae strains is carried out via simultaneous expression of optimized GS (GS_ln-13n) genes encoding GS (GS_lp-13p) enzymes with optimized GO (GO_ln-l In) genes encoding GO (GO_lp-l Ip) enzymes to convert geissoschizine into akuammicine (see FIG. 9). Recombinant hosts capable of producing MIAs (e.g., able to produce precursors such as strictosidine and strictosidine aglycone) are grown in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 and fermented for 48-96 hrs at 30°C. After the initial 24-48 hrs of growth, 0.1-0.5 g / L secologanin and 0.1-0.5 g / L L -tryptophan are added to support MIA precursor flux to yield geissoschizine and akuammicine. The formation of akuammicine and related on-pathway intermediates in the late stage MIA pathway are monitored using liquid chromatography with mass spectrometry (LC-MS). Detection of products are carried out as described in Example 2, including LC-MS / MS fragmentation leading to known akuammicine fragment of 291 m / z. In FIG. 9, hydroxylation activity' leading to akuammicine production was detected as above only in recombinant hosts expressing GO and not in negative control strains. The GO depicted is the GO In gene encoding the GO Ip enzyme.Example 10 - Expression of a PAS, DPAS, and TabS (TS) in a modified host organism to produce tabersonine

[0224] Construction of Saccharomyces cerevisiae strains is carried out via simultaneous expression of optimized PAS (PAS_ln-4n) genes encoding PAS (PAS_lp-4p) enzymes with optimized DPAS (DPAS_ln-2n) genes encoding DPAS (DPAS_lp-2p) enzymes to generate precursors (e.g. precondylocarpine acetate, dehydrosecodine) for downstream cyclase enzymes such as tabersonine synthase, coronaridine synthase, catharanthine synthase, among other cyclases, leading to various downstream alkaloids. Expressing the optimized TabS (TS ln) gene encoding the TabS (TS lp) enzyme, tabersonine can be both a desired product and a readout for active enzyme and precursor presence from PAS and DPAS activity in a strain expressing a MIA biosynthesis pathway as described herein. Recombinant hosts capable of producing MIAs (e.g., able to produce precursors such as stemmadenine acetate) are grown in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 and fermented for 48-96 hrs at 30°C.After the initial 24-48 hrs of growth, 0. 1-0.5 g / L secologanin and 0. 1 -0.5 g / L L-tryptophan are added to support MIA precursor flux to yield PAS, DPAS, and TabS (TS) products, where tabersonine is a stable readout. Detection of tabersonine is carried out as described inExample 2. In FIG. 10, PAS, DPAS, and TabS (TS) activity leading to tabersonine production was detected in only recombinant hosts expressing PAS, DPAS, and TS and not in negative control strains. The PAS depicted is the PAS_3n gene encoding the PAS_3p enzyme. The DPAS depicted is the DPAS ln gene encoding the DPAS lp enzyme. The TabS depicted is the TS ln gene encoding the TS lp enzyme.Example 11 - Co-culture of recombinant host organisms to produce norvoacangine and voacangine

[0225] Combining recombinant hosts expressing different genes for different enzyme activities can be beneficial to 1) expand the product libraiy from common precursors and intermediates, 2) speed up enzyme or activity screening, and 3) allow for process separation such as a changing of conditions at different points within production cycle. As an example of this, MIA production of voacangine is shown in FIG. 14 where a co-culture of a recombinant strain expressing an Ox (Ox i In, Ox i Ip) and another recombinant strain expressing an OMT (0MT_8n, OMT_8p), convert coronaridine to norvoacangine and voacangine all within the same fermentation. Such strains can be grown as in Example 1, with norvoacangine and voacangine isolated and detected as in Example 2. In this example, strains are grown separately, and combined 8-48hrs post inoculation, whereby the co-culture reaction continues under standard conditions for another 6-72 hours. To expand further, a modified strain expressing Ox (Il OH) and CPR and a modified strain expressing OMT, are grown separately in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 for 8-48 hrs at 30°C shaking (200-300 rpm). For applications where the promoter of the relevant genes are inducible, galactose, glycerol, or lactose (0.01-2% v / v) is added to the culture media as an inducer after 8-48hrs of growth. After the initial 8-48 hrs of growth, for voacangine, 0.01-0.5 g / L coronaridine is added as exogenous substrate for conversion of coronaridine into norvoacangine and voacangine.

[0226] Combining broths of the various recombinant strains can occur before, during, or after addition of an exogenous precursor, where the respective broths are combined at avolume-to-volume ratio ranging from 1: 1 to 1 : 100. In this case, broths containing recombinant cells with Ox and OMT activity, respectively, are combined 1 : 1 during the addition of coronaridine, and the co-culture fermentation continues as before for an additional 6-72hrs. The formation of norvoacangine and voacangine is monitored using liquid chromatography with mass spectrometiy (LC-MS). Detection of products is carried out as described in Example 2. In FIG. 14, hydroxylase activity leading to norvoacangine was detected in samples where only the Ox strain is present or when the Ox strain is combined with an OMT strain. Similarly, the combined hydroxylase (Ox) and methyltransferase (OMT) activity leading to voacangine was only observed in the co-culture sample, and not in the sample where only the Ox strain is present.Example 12 - Expression of a CORS-DPAS fusion in a modified host organism to produce coronaridine

[0227] Construction of recombinant Saccharomyces cerevisiae strains for coronaridine production is carried out via expression of optimized CORS-DPAS and / or DPAS-CORS (Fusion_Pair_ln-4n) genes encoding the CORS-DPAS and / or DPAS-CORS (Fusion_Pair_lp-4p) enzymes. Recombinant hosts capable of producing MIAs (e.g., able to produce precursors such as precondylocarpine acetate) are grown in minimal media containing 40 g / L glucose, 15 g / L ammonium sulfate, 2 g / L yeast nitrogen base (YNB), 1 g / L complete minimal dropout media (CSM) supplement, buffered with 60 mM KH2PO4 and 10 mM K2HPO4 and fermented for 8-72 hrs at 30°C. For applications where the promoters of the relevant genes are inducible, galactose, glycerol, or lactose (0.01-2% v / v) is added to the culture media as an inducer after 8-48hrs of growth. After the initial 8-72 hrs of growth, 0. 1-5 g / L secologanin and 0.1-5 g / L L-tryptophan are added to bolster MIA precursor flax to yield coronaridine (E1C 339 m / z). Detection of coronaridine is carried out as described in Example 2. In FIG. ISA, fermentation-derived coronaridine is only detected by LC-MS (msl) at EIC 339 m / z in the replicates of a recombinant host expressing active CORS-DPAS fusion, and not in the negative control replicates, as compared to a coronaridine analytical standard. The coronaridine producing strains in this example express a CORS-DPAS fusion gene (Fusion_Pair_2n) encoding CORS-DPAS fusion enzyme (Fusion_Pair_2p). LC-MS / MS (ms2) in FIG 15B shows fragmentation consistent with coronaridine, including 1) 307 m / z from the loss of methylamine (CH3NH2) from the molecular ion via C-N cleavage and hydrogen rearrangement, 2) 279 m / z from alpha-cleavage of the methyl acetate group,resulting in the loss of CH3C00-, 3) 122 m / z from cleavage of the C-N bond between the indole and isoquinuclidine rings, followed by the loss of the ethyl side chain (protonated 3- methyleneindoline), among other masses, including a small 144 m / z fragment corresponding to heterolytic cleavage of the C-N bond in the isoquinuclidine ring, forming a stabilized cation.

[0228] Sequences described herein are enumerated in the tables below. The “SEQ REF’' column identifies the name of the sequence, as well as the corresponding SEQ ID NO in parentheses.SEQUENCESTable 1 - geissoschizine synthase (GS ) nucleotide sequencesCD 00CD CDONDGJChCD00CD00 o0000 ND00 CDg00 Ch00 CD0000 0000 CD<D O<D<D KDCD CD<D<D Ch$<D<D 00CD CDO OOO MO CDOO OiO CDOO 00O <DOMCDOiCD00<DK) OroK) CDSEAf InAf 2nLInLK) 2nL 3n L 4nL 5nL 6nL 7nL 8nL 9nK) OiCDK) 00K) <DCD OCDCD roW CDCDW OiW CDSRRRRRCDRRRCD 00CD CDOMWU10500cn ocncn rocn CDcncn OiCD CDcncn 00cn <DCD OCDO) MCD CDCDCD OiCD CDO)

Claims

CLAIMS1. A polynucleotide comprising a sequence as disclosed in any of Tables 1 -27 or 47-49, or a sequence having greater or equal to about 90% sequence identity to any one of said sequences.

2. The polynucleotide according to claim 1 , having greater or equal to about 95% sequence identity to any of of said sequences.

3. An expression vector comprising one or more polynucleotide sequences according to claim 1 and an operatively-linked promoter sequence that allows for expression of the one or more polynucleotide sequences in a cell.

4. A recombinant cell comprising a polynucleotide according to claim 1 , or the expression vector according to claim 3.

5. The recombinant cell according to claim 4, wherein the cell comprises a bacterial cell, a fungal cell, or a yeast cell.

6. The recombinant cell according to claim 5, wherein the cell is a species of yeast that includes Saccharomyces, Candida, Pichia, Schizosaccharomyces, Schejjersomyces, Blakeslea, Rhodotorula, or Yarrowia.

7. The recombinant cell according to claim 5, wherein the cell is a species of a filamentous fungus that includes Aspergillus or Penicillium.

8. The recombinant cell according to claim 3, wherein the cell is a species of bacteria that includes Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or Lactobacillus.

9. The recombinant cell according to any one of claims 4-8, wherein at least one copy of the polynucleotide sequence or polynucleotide sequences is located on at least one plasmid.

10. The recombinant cell according to any one of claims 4-8, wherein at least one copy of the polynucleotide sequence or polynucleotide sequences is stably integrated into the genome of the cell.

11. The recombinant cell according to any one of claims 9 or 10, wherein a plurality of copies of the polynucleotide sequence or polynucleotide sequences are stably integrated a plasmid or into the genome of the cell.

12. The recombinant cell according to any one of claims 4-11, wherein the polynucleotide sequence or polynucleotide sequences are the same sequence.

13. The recombinant cell according to any one of claims 4-11, wherein the polynucleotide sequence or polynucleotide sequences are different sequences.

14. The recombinant cell according to any one of claims 4-11 , or 13, wherein the polynucleotide sequence or polynucleotide sequences encode the same class of enzyme or amino acid sequence.

15. The recombinant cell according to any one of claims 4-11 , or 13, wherein the polynucleotide sequence or polynucleotide sequences encode a different class of enzyme or amino acid sequence.

16. A method for the biosynthesis of a downstream monoterpene indole alkaloid compound, or a precursor or metabolite thereof, the method comprising culturing a recombinant cell of any of claims 4-15 under conditions that allow for the biosynthesis of the compound.

17. The method according to claim 16, wherein the method further comprises contacting the recombinant cell with a feed comprising one or more exogenous substrate compounds,wherein the one or more exogenous substrate compounds comprise strictosidine, a strictosidine analog, coronaridine, stemmadenine, or a stemmadenine analog, or the feed comprises a plant extract that includes the one or more of the exogenous substrate compounds.

18. The method according to any one of claims 16-17, wherein the one or more exogenous substrate compounds are produced by a recombinant cell according to any one of claims 4-15.

19. The method according to any one of claims 16-18, wherein the method generates one or more of cathenamine, 4,21-dehydrogeissoschizine, 19E-geissoschizine, geissoschizine methyl ether, isositsirikine, tetrahydroalstonine, ajmalicine, 19-epi-ajmalicine, dehydropreakuammicine, preakuammicine, akuammicine, deshydroxymethylstemmadenine, stemmadenine, stemmadenine acetate, precondylocarpine acetate, dihydroprecondylocarpine acetate, secodine, dehydrosecodine, vincadifformine, tabersonine, catharanthine, coronaridine, 10- hydroxycoronaridine (norvoacangine), voacangine, de-ester-voacangine, ibogamine, noribogaine, and ibogaine.

20. An amino acid sequence comprising a sequence as disclosed in any of Tables 28-46, 50, 51, or 52 or a sequence having about 90% sequence identity to any one of said sequences.

21. An amino acid encoded by one or more of the polynucleotides of claims 1 or 2.