Compositions and methods for biosynthesis of terpenoids or cannabinoids in heterologous systems

Abstract

To provide improved methods, compositions and host cells for improved prenylation of aromatic substrates or production of downstream metabolites thereof in host cells.SOLUTION: Provided herein are methods and compositions for producing cannabinoids and other metabolites in a host cell.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 814,823, filed March 6, 2019, and U.S. Provisional Application No. 62 / 814,816, filed March 6, 2019, the entire contents of each of which are incorporated by reference for all purposes.

[0002] Cannabinoids and their derivatives possess several properties with therapeutic potential. Activation or blockage of CB-1 and / or CB-2 receptors by cannabinoids can modulate downstream signal transduction and metabolic pathways, which can subsequently affect synaptic transmission, including pain and other peripheral sensory signal transduction, immune responses, and inflammation. For this reason, interest has grown in the use of natural or synthetic cannabinoids for therapeutic purposes. However, low extraction yields and high separation costs make the use of naturally derived cannabinoids uneconomical. Similarly, fully synthetic methods for producing cannabinoids are hindered by the complexity of these compounds.

[0003] Heterologous systems for the production of cannabinoids known in the art rely on eukaryotic host organisms to produce and secrete cannabinoid synthases, which are then used in in vitro enzyme-catalyzed reactions to produce cannabinoid products. For example, U.S. Patent Nos. 9,587,212, 9,512,391, 9,394,512, 9,526,715, and 9,359,625 each describe methods, compositions, and bioreactors for producing cannabinoids in vitro using recombinant Pichia pastoris that secrete THCA synthase or CBDA synthase. Unfortunately, however, this system requires the use of a eukaryotic host and additional means for generating suitable substrates for the secreted enzymes.

[0004] Regarding in vivo cannabinoid production schemes, Carvalho A, et al., FEMS Yeast Res. 2017, teaches that prokaryotic production of the enzymes in the cannabinoid pathway is not feasible due to the enzymes' need for membrane binding, glycosylation, and disulfide bond formation. Specifically, Carvalho discloses that expression of CBGAS in E. coli is unlikely, and the use of prokaryotic hosts to express functional THCAS or CBDAS is precluded.

[0005] Furthermore, olivetolate, the substrate of aromatic prenyltransferase CBGAS required for CBGA production, is not produced endogenously, if at all, at useful levels in typical prokaryotic systems. Therefore, olivetolate must be exogenously supplied to the cell culture medium or by expression of an alternative biosynthetic pathway for heterologous production of olivetolate. However, biosynthetic production of olivetolate imposes a metabolic burden that can dramatically reduce microbial production. Similarly, olivetolate is not efficiently transported into cells from the surrounding medium, and therefore, exogenously supplied olivetolate represents a rate-limiting step in the production of downstream metabolites. Other substrates of aromatic prenyltransferase, such as divalinolic acid (DVA), face the same issues of endogenous production, the metabolic burden of heterologous production, and rate-limiting membrane transport. Thus, there is a long-felt but unmet need to develop cost-effective heterologous systems for in vivo cannabinoid production. Summary of the Invention

[0006] Described herein are improved methods, compositions, and host cells for improved aromatic substrate prenylation or production of downstream metabolites in (e.g., prokaryotic) host cells. The inventors have identified membrane transporters that are functional when expressed as heterologous transporters in host cells and can enhance the transport of extracellular aromatic prenyltransferase substrates, such as olivetolate, into (e.g., prokaryotic) host cells. For example, the inventors have identified major facilitator superfamily (MFS) aromatic acid antiporters that are functional and can enhance the transport of extracellular aromatic prenyltransferase substrates, such as olivetolate, into (e.g., prokaryotic) host cells. Independently, the inventors have identified outer membrane porin (OMP) superfamily transporters that are functional and can enhance the transport of extracellular aromatic prenyltransferase substrates, such as olivetolate, into (e.g., prokaryotic) host cells. Without wishing to be bound by theory, the inventors hypothesize that enhancing the transport of aromatic prenyltransferase substrates, such as olivetolate, into cells via, for example, antiporters or porins, increases the flux of the aromatic prenylation step, thereby improving production of downstream metabolic products. In some cases, increased flux reduces the (e.g., steady-state) intracellular concentration of deleterious intermediates, such as geranyl pyrophosphate (GPP), thereby improving production of downstream metabolic products.

[0007] Thus, the present invention provides a host cell comprising: a) an expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding a transporter; and b) an exogenous aromatic substrate for the transporter. In embodiments, the host cell is capable of enhanced import of the aromatic substrate for the transporter into the host cell compared to a control prokaryotic host cell lacking the expression cassette of a).

[0008] For example, in one aspect, the present invention provides a host cell comprising: a) an expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding a major facilitator superfamily (MFS) aromatic acid antiporter; and b) an exogenous aromatic substrate for the MFS aromatic acid antiporter. In embodiments, the host cell is capable of enhancing import of the aromatic substrate for the MFS aromatic acid antiporter into the host cell compared to a control prokaryotic host cell lacking the expression cassette of a). By way of another example, in one aspect, the present invention provides a host cell comprising: a) an expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding an OMP superfamily porin; and b) an exogenous aromatic substrate for the OMP superfamily porin. In embodiments, the host cell is capable of enhancing import of the aromatic substrate for the OMP superfamily porin into the host cell compared to a control prokaryotic host cell lacking the expression cassette of a).

[0009] In some embodiments, the aromatic substrate of the transporter is a substrate of a heterologous aromatic prenyltransferase expressed in the host cell. For example, the aromatic substrate of the transporter can be a prenyl acceptor of a heterologous aromatic prenyltransferase expressed in the host cell. In some embodiments, the aromatic substrate of the transporter is an aromatic acid. In some embodiments, the aromatic substrate of the transporter is olivetolate and / or divalinolic acid. In some embodiments, the aromatic substrate of the transporter is a decarboxylated derivative of an aromatic acid. In some embodiments, the substrate of the transporter is olivetol. In some embodiments, the substrate of the transporter is divalinol. In some embodiments, the substrate of the transporter is resveratrol, naringenin, or florisovalerophenone, or a combination thereof. In some embodiments, the substrate of the transporter is apigenin, daidzein, genistein, naringenin, olivetol, OA, or resveratrol, or a combination thereof.

[0010] In some embodiments, the host cell is a prokaryote. Optionally, the prokaryotic host cell is selected from the group consisting of prokaryotes of the genus Escherichia, Panteoa, Bacillus, Corynebacterium, or Lactococcus. In some embodiments, the cell is Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, or L. lactis. In some embodiments, the cell is E. coli. In some embodiments, the host cell is a prokaryotic host cell comprising: a) an expression cassette comprising a prokaryotic promoter operably linked to a heterologous nucleic acid encoding a transporter, such as a major facilitator superfamily (MFS) aromatic acid antiporter (e.g., pcaK) or an OMP superfamily porin, e.g., an OprD family porin (e.g., pp3656).

[0011] In some embodiments, the host cell is a eukaryotic organism. In some embodiments, the eukaryotic organism is a fungal cell, an insect cell, or a mammalian cell. In some embodiments, the eukaryotic organism is a fungal cell. In some embodiments, the eukaryotic organism is selected from the group consisting of eukaryotic organisms of the genera Saccharomyces, Schizosaccharomyces, Hansela, Kluyveromyces, Yarrowia, Spodoptera, Drosophila, Aedes, Trichoplusia, Estigmene, Bombyx, and Autographica. In some embodiments, the cell is Saccharomyces cerevisiae or Pichia pastoris. In some embodiments, the cell is Saccharomyces cerevisiae. In some embodiments, the host cell is a eukaryotic host cell comprising: a) an expression cassette comprising a eukaryotic promoter operably linked to a heterologous nucleic acid encoding a major facilitator superfamily (MFS) aromatic acid antiporter or an outer membrane porin (OMP).

[0012] In some embodiments, the MFS aromatic acid antiporter is pcaK or a functional fragment thereof. In some embodiments, the MFS aromatic acid antiporter is pcaK, a nucleotide sequence of SEQ ID NO: 6 (MNQAQTNVGKSLDVQSFINQQPLSRYQWRVVLLCFLIVFLDGLDTAAMGFIAPALSQEWGIDRASLGPVMSAALIGMVFGALGSGPLADRFGRKGVLVGAVLVFGGFSLASAYATNVDQLLVLRFLTGLGLGAGMPNATTLLSEYTPERLKSLLVTSMFCGFNLGMAGGGFISAKMIPAYGWHSLLVIGGVLPLLLALVLMIWLPESARFLVVRNRGTDKVRKTLSPIAPQVVAEAGSFSVPEQKAV AARNVFAVIFSGTYGLGTVLLWLTYFMGLVIVYLLTSWLPTLMRDSGASMEQAAFIGALFQFGGVLSAVGVGWAMDRFNPHKVIGIFYLLAGVFAYAVGQSLGNITLLATLVLVAGMCVNGAQSAMPSLAARFYPTQGRATGVSWMLGIGRFGAILGAWSGATLLGLGWSFEQVLTALLVPAALATVGVVVKGLVSHADAT). In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 100 consecutive amino acids of the sequence set forth in SEQ ID NO:6. In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to, or identical to, 150 consecutive amino acids of the sequence set forth in SEQ ID NO:6.

[0013] In some embodiments, the MFS aromatic acid antiporter is pcaK or a functional fragment thereof. In some embodiments, the MFS aromatic acid antiporter is pcaK, a nucleotide sequence of SEQ ID NO: 8 (MNQAQTNVGKSLDVQSFINQQPLSRYQWRVVLLCFLIVFLDGLDTAAMGFIAPALSQEWGIDRASLGPVMSAALIGMVFGALGSGPLADRFGRKGVLVGAVLVFGGFSLASAYATNVDQLLVLRFLTGLGLGAGMPNATTLLSEYTPERLKSLLVTSMFCGFNLGMAGGGFISAKMIPAYGWHSLLVIGGVLPLLLALVLMVWLPESARFLVVRNRGTDKVRKTLSPIAPQVVAEAGSFSVPEQKAV AARNVFAVIFSGTYGLGTVLLWLTYFMGLVIVYLLTSWLPTLMRDSGASMEQAAFIGALFQFGGVLSAVGVGWAMDRFNPHKVIGIFYLLAGVFAYAVGQSLGNITLLATLVLVAGMCVNGAQSAMPSLAARFYPTQGRATGVSWMLGIGRFGAILGAWSGATLLGLGWSFEQVLTALLVPAALATVGVVVKGLVSHADAT). In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 100 consecutive amino acids of the sequence set forth in SEQ ID NO:8. In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to, or identical to, 150 consecutive amino acids of the sequence set forth in SEQ ID NO:8.

[0014] In some embodiments, the OMP is an OprD family porin. In some embodiments, the OprD family porin is pp3656 or a functional fragment thereof. In some embodiments, the OprD family porin is selected from the group consisting of SEQ ID NO: 7 (MSIAFKKTLACSATLLVAPYASAAFVEDFKGSLELRNFYYNRDFRNDGATQSKRDEWAQGFILNLQSGFTEGPVGFGIDAMGLLGVKLDSSPDRTGSGLLAYDSDRQVEDEYGKFVATAKARMGKTELRIGGVNPLMPLLWSNNSRLLPQVFRGGSLTVNDIDKLTVTATRINAVKQRNSTDFESLTATGYAPVEADHYNYLAFDFKPAKDMTFSLHAAELEDLYKSYFAGIKV In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to 100 consecutive amino acids of the sequence set forth in SEQ ID NO:7. In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to, or identical to, 150 consecutive amino acids of the sequence set forth in SEQ ID NO:7.

[0015] In some embodiments, the OMP is an OprD family porin. In some embodiments, the OprD family porin is pp3656 or a functional fragment thereof. In some embodiments, the OprD family porin is selected from the group consisting of SEQ ID NO: 9 (MSIAFKKTLACSATLLVAPYASAAFVEDFKGSLELRNFYYNRDFRNDGATQSKRDEWAQGFTLNLQSGFTEGPVGFGIDAMGLLGVKLDSSPDRTGSGLLAYDSDRQVEDEYGKFVATAKARMGKTELRIGGVNPLMPLLWSNNSRLLPQIFRGGSLTVNDIDKLTVTATRVNAVKQRNSTDFESLTATGYAPVEADHYNYLAFDFKPAKDMTFSLHAAELEDLYKSYFAGIKV In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to 100 consecutive amino acids of the sequence set forth in SEQ ID NO:9. In some embodiments, the MFS aromatic acid antiporter is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to, or identical to, 150 consecutive amino acids of the sequence set forth in SEQ ID NO:9.

[0016] In some embodiments, the (e.g., prokaryotic) host cell further comprises an aromatic prenyltransferase or a functional fragment and / or variant thereof, wherein the aromatic prenyltransferase is functional and capable of prenylating an aromatic acid substrate of a transporter (e.g., an MFS aromatic acid antiporter or an OMP superfamily porin). In some embodiments, the aromatic acid substrate is olivetolate, and the aromatic prenyltransferase is functional and capable of prenylating olivetolate. In some cases, the aromatic prenyltransferase is functional and capable of prenylating olivetolate to produce cannabigerolic acid.

[0017] In some embodiments, the aromatic prenyltransferase is CBGAS or NphB, or a functional fragment thereof. In some embodiments, the aromatic prenyltransferase is CsPT4 (Lou et al. Nature February 28, 2019), or a functional fragment thereof and / or a variant thereof.

[0018] In some embodiments, the aromatic prenyltransferase is a functional fragment of CBGAS, such as SEQ ID NO: 3 (CBGAS, AJN57774.1)MGLSSVCTFSFQTNYHTLLNPHNNNPKTSLLCYRHPKTPIKYSYNNFPSKHCSTKSFHLQNKCSESLSIAKNSIRAATTNQTEPPESDNHSVATKILNFGKACWKLQRPYTIIAFTSCACGLFGKELLHNTNLISWSLMFKAFFFLVAILCIASFTTTINQIYDLHIDRINKPDLPLASGEISVNTAWIMSIIVALFGLIITIKMKGGPLYIFGYC In some embodiments, the CBGAS is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to a 50 contiguous amino acid stretch of the sequence set forth in SEQ ID NO:3. In some embodiments, the CBGAS is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to a stretch of 150 contiguous amino acids of the sequence set forth in SEQ ID NO:3.

[0019] In some cases, the host cell further comprises a (e.g., prokaryotic) promoter operably linked to a nucleic acid encoding an aromatic prenyltransferase, such as CBGA synthase (CBGAS). In some embodiments, the CBGAS is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to a 50 contiguous amino acid stretch of the sequence set forth in SEQ ID NO: 3. In some embodiments, the CBGAS is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to a 100 contiguous amino acid stretch of the sequence set forth in SEQ ID NO: 3. In some embodiments, the CBGAS is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to a 150 contiguous amino acid stretch of the sequence set forth in SEQ ID NO: 3.

[0020] In some cases, the aromatic prenyltransferase (e.g., CBGAS) comprises an N-terminal truncation that lacks a plastid or chloroplast retention signal. In some cases, the aromatic prenyltransferase (e.g., CBGAS) comprises an N-terminal truncation that lacks a plastid retention signal.

[0021] In some embodiments, the aromatic prenyltransferase is a functional fragment of NphB. In some embodiments, NphB is SEQ ID NO: 4 (NphB, AFD38743.1) MSEAADVERVYAAMEEAAGLLGVACARDKIYPLLSTFQDTLVEGGSVVVFSMASGRHSTELDFSISVPTSHGDPYATVVEKGLFPATGHPVDDLLADTQKHLPVSMFAIDGEVTGGFKKTYAFFPTDNMPGVAELSAIPSMPPAVAENAELFARYGLDKVQMTSMDYKKRQVN LYFSELSAQTLEAESVLALVRELGLHVPNELGLKFCKRSFSVYPTLNWETGKIDRLCFAVISNDPTLVPSSDEGDIEKFHNYATKAPYAYVGEKRTLVYGLTLSPKEEYYKLGAYYHITDVQRGLLKAFDSLED. In some embodiments, the aromatic prenyltransferase is a functional fragment of NphB. In some embodiments, the NphB is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 100 consecutive amino acids of the sequence set forth in SEQ ID NO:4. In some embodiments, the aromatic prenyltransferase is a functional fragment of NphB. In some embodiments, NphB is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to, or identical to, 150 contiguous amino acids of the sequence set forth in SEQ ID NO: 4. Optionally, NphB contains one or more or all of the following mutations: Y288A, Y288N, G286S, A232S, F213H, and / or Y288V. In some embodiments, NphB contains one of the following combinations of mutations: Y288N / G286S, Y288A / G286S, Y288A / G286S / A232S, Y288A / G286S / A232S / F213H, Y288V / G286S, Y288V / A232S, or Y288A / A232S.See Valliere et al. Nature Communications 2019 10:565.

[0022] In some cases, the host cell further comprises a (e.g., prokaryotic) promoter operably linked to a nucleic acid encoding an aromatic prenyltransferase, such as NphB. In some embodiments, NphB is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to 50 contiguous amino acids of the sequence set forth in SEQ ID NO: 4. In some embodiments, NphB is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to 100 contiguous amino acids of the sequence set forth in SEQ ID NO: 4. In some embodiments, NphB is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to or identical to 150 contiguous amino acids of the sequence set forth in SEQ ID NO: 4.

[0023] In some embodiments, the host cell comprises an expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding at least one (eg, prokaryotic) chaperone.

[0024] In some cases, the host cell comprises a cannabinoid synthase. In some cases, the host cell comprises an expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding the cannabinoid synthase. In some cases, the cannabinoid synthase is CBDAS. In some cases, the cannabinoid synthase is THCAS.

[0025] In some embodiments, the cannabinoid synthase is SEQ ID NO: 1 (Cannabidiolic acid synthase. A6P6V9.1. Signal peptide removed)NPRENFLKCFSQYIPNNATNLKLVYTQNNPLYMSVLNSTIHNLRFTSDTTPKPLVIVTPSHVSHIQGTILCSKKVGLQIRTRSGGHDSEGMSYISQVPFVIVDLRNMRSIKIDVHSQTAWVEAGATLGEVYYWVNEKNENLSLAAGYCPTVCAGGHFGGGGYGPLMRNYGLAADNIIDAHLVNVHGKVLDRKSMGEDLFWALRGGGAESFGIIVAWKIRLVAVPKSTMFSVKKIMEIHELVKLVNKWQNIAYKYDKDLLLMTHFITRNITD NQGKNKTAIHTYFSSVFLGGVDSLVDLMNKSFPELGIKKTDCRQLSWIDTIIFYSGVVNYDTDNFNKEILLDRSAGQNGAFKIKLDYVKKPIPESVFVQILEKLYEEDIGAGMYALYPYGGIMDEISESAIPFPHRAGILYELWYICSWEKQEDNEKHLNWIRNIYNFMTPYVSKNPRLAYLNYRDLDIGINDPKNPNNYTQARIWGEKYFGKNFDRLVKVKTLVDPNNFFRNEQSIPPLPRHRH.

[0026] In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 100 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 150 contiguous amino acids of the sequence set forth in SEQ ID NO: 1. In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to SEQ ID NO: 1.

[0027] In some embodiments, the cannabinoid synthase is SEQ ID NO: 2 (tetrahydrocannabinolic acid synthase. AB057805.1. Secretion signal removed)NPRENFLKCFSKHIPNNVANPKLVYTQHDQLYMSILNSTIQNLRFISDTTPKPLVIVTPSNNSHIQATILCSKKVGLQIRTRSGGHDAEGMSYISQVPFVVVDLRNMHSIKIDVHSQTAWVEAGATLGEVYYWINEKNENLSFPGGYCPTVGVGGHFSGGGYGALMRNYGLAADNIIDAHLVNVDGKVLDRKSMGEDLFWAIRGGGGENFGIIAAWKIKLVAVPSKSTIFSVKKNMEIHGLVKLFNKWQNIAYKYDKDLVLMTHFITKN ITDNHGKNKTTVHGYFSSIFHGGVDSLVDLMNKSFPELGIKKTDCKEFSWIDTTIFYSGVVNFNTANFKKEILLDRSAGKKTAFSIKLDYVKKPIPETAMVKILEKLYEEDVGAGMYVLYPYGGIMEEISESAIPFPHRAGIMYELWYTASWEKQEDNEKHINWVRSVYNFTTPYVSQNPRLAYLNYRDLDLGKTNHASPNNYTQARIWGEKYFGKNFNRLVKVKTKVDPNNFFRNEQSIPPLPPHHH, or consisting of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 50 consecutive amino acids of the sequence set forth in

[0028] In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 100 contiguous amino acids of the sequence set forth in SEQ ID NO: 2. In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 150 contiguous amino acids of the sequence set forth in SEQ ID NO: 2. In some embodiments, the cannabinoid synthesis enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to SEQ ID NO: 2.

[0029] In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to 150 consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence that is at least 50% or 55% identical to 300 consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95% or 99% identical to 300 or all consecutive amino acids of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the cannabinoid synthase is a Cannabis sativa cannabinoid synthase.

[0030] In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to 150 consecutive amino acids of SEQ ID NO:3. In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence at least 50% or 55% identical to 300 consecutive amino acids of SEQ ID NO:3. In some embodiments, the cannabinoid synthase comprises or consists of an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to 300 or all consecutive amino acids of SEQ ID NO:3. In some embodiments, the host cell comprises a nucleic acid encoding a CBGA synthase and a nucleic acid encoding a cannabinoid synthase selected from the group consisting of THCA synthase and CBDA synthase, or a combination of one or more nucleic acids encoding two or all of them. Optionally, the host cell comprising the CBGA synthase expression cassette further comprises nucleic acids encoding THCA synthase and / or CBDA synthase, each synthase independently operably linked to a promoter in the same or different expression cassettes.

[0031] In some cases, a host cell comprising an expression cassette comprising a heterologous nucleic acid encoding a transporter (e.g., an MFS aromatic acid antiporter, e.g., pcaK, or an OMP superfamily porin, e.g., an OprD family porin, e.g., pp3656) further comprises a nucleic acid encoding an aromatic prenyltransferase, a THCA synthase, and / or a CBDA synthase, wherein the synthase and / or prenyltransferase are each independently operably linked to a promoter in the same or a different expression cassette. In some cases, a host cell comprising an expression cassette comprising a heterologous nucleic acid encoding a transporter (e.g., an MFS aromatic acid antiporter, e.g., pcaK, or an OMP superfamily porin, e.g., an OprD family porin, e.g., pp3656) further comprises a nucleic acid encoding an aromatic prenyltransferase independently operably linked to a promoter in the same or a different expression cassette. In some cases, a host cell containing an expression cassette comprising a heterologous nucleic acid encoding a transporter (e.g., an MFS aromatic acid antiporter, e.g., pcaK, or an OMP superfamily porin, e.g., an OprD family porin, e.g., pp3656) further comprises nucleic acids encoding an aromatic prenyltransferase and a CBDA synthase, each synthase and prenyltransferase independently operably linked to a promoter in the same or different expression cassettes.

[0032] In some embodiments, the cannabinoid synthase, or at least one encoded cannabinoid synthase, is a truncated cannabinoid synthase selected from the group consisting of a truncated THCA synthase and a truncated CBDA synthase, wherein the truncation is the deletion of all or a portion of a signal peptide, a plastid retention signal, and / or a chloroplast retention signal. In some embodiments, the cannabinoid synthase comprises a deletion of all or a portion of a transmembrane or membrane-associated region such that the cannabinoid synthase is not membrane-associated or is not membrane-associated when expressed in a eukaryotic system.

[0033] In some embodiments, the promoter operably linked to the nucleic acid encoding the transporter is a constitutive promoter. In some embodiments, the promoter operably linked to the nucleic acid encoding the transporter is an inducible promoter. Optionally, the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase is a constitutive promoter. In some embodiments, the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase is an inducible promoter. Optionally, the promoter operably linked to the nucleic acid encoding the transporter is a constitutive promoter and the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase is a constitutive promoter. Optionally, the promoter operably linked to the nucleic acid encoding the transporter is an inducible promoter and the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase is an inducible promoter. Optionally, the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase and the promoter operably linked to the nucleic acid encoding the transporter are the same promoter. Optionally, the promoter operably linked to the nucleic acid encoding the aromatic prenyl transferase and the promoter operably linked to the nucleic acid encoding the transporter are different promoters.

[0034] In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, each expression cassette comprises an inducible promoter operably linked to a cannabinoid synthesis enzyme. In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, at least one expression cassette comprises an inducible promoter operably linked to a cannabinoid synthesis enzyme. In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, at least one expression cassette comprises a constitutive promoter operably linked to a cannabinoid synthesis enzyme.

[0035] In some embodiments, the promoter operably linked to the nucleic acid encoding the cannabinoid synthesis enzyme is a constitutive promoter. In some embodiments, the promoter operably linked to the nucleic acid encoding the cannabinoid synthesis enzyme is an inducible promoter. In some embodiments, the host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, and each expression cassette comprises a constitutive promoter operably linked to a cannabinoid synthesis enzyme.

[0036] In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, each expression cassette comprises an inducible promoter operably linked to a cannabinoid synthesis enzyme. In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, at least one expression cassette comprises an inducible promoter operably linked to a cannabinoid synthesis enzyme. In some embodiments, a host cell comprises two or more expression cassettes comprising different cannabinoid synthesis enzymes, at least one expression cassette comprises a constitutive promoter operably linked to a cannabinoid synthesis enzyme.

[0037] In some embodiments, the host cell comprises, or further comprises, an expression cassette comprising a promoter operably linked to a nucleic acid encoding one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. Optionally, the host cell comprises, or further comprises, an expression cassette comprising a promoter operably linked to a nucleic acid encoding the bifunctional MEP pathway enzyme ispDF. Optionally, the expression cassette comprising the bifunctional ispDF enzyme further comprises one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. Optionally, the expression cassette comprising the bifunctional ispDF enzyme further comprises dxs and idi.

[0038] In some cases, the host cell comprises a higher level of expression of one or more MEP pathway genes compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDF enzyme. In some cases, the host cell comprises a higher level of expression of dxs and idi compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDF enzyme.

[0039] In some embodiments, the host cell comprises, or further comprises, an expression cassette comprising a promoter operably linked to a nucleic acid encoding an ispDE bifunctional MEP pathway enzyme. In some embodiments, the bifunctional MEP pathway enzyme comprises a flexible linker peptide between the ispD and ispE domains. In some embodiments, the flexible linker comprises the sequence SLGGGGSAAA. In some cases, the linker sequence has greater than 65% random coil formation as determined by the GOR algorithm, version IV (Methods in Enzymology 1996 RF Doolittle Ed., vol 266, 540-553).

[0040] In some embodiments, the ispDE bifunctional MEP pathway enzyme comprises or consists of an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identical to or identical to 50 consecutive amino acids of the sequence set forth in SEQ ID NO:10().

[0041] In some cases, the host cell comprises, or further comprises, an expression cassette comprising a promoter operably linked to a nucleic acid encoding the bifunctional MEP pathway enzyme ispDF. In some cases, the expression cassette comprising the bifunctional ispDE enzyme further comprises one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispF, ispG, ispH, and idi. In some cases, the expression cassette comprising the bifunctional ispDE enzyme further comprises dxs, ispF, and idi. In some cases, the expression cassette comprising the bifunctional ispDE enzyme further comprises the bifunctional ispDF enzyme (see PCT / CA2018 / 051074). In some cases, the expression cassette comprising the bifunctional ispDE enzyme further comprises one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispDF, ispG, ispH, and idi.

[0042] In some cases, the host cell comprises a higher level of expression of one or more MEP pathway genes compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDE enzyme. In some cases, the host cell comprises a higher level of expression of dxs and idi compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDE enzyme. In some cases, the host cell comprises a higher level of expression of one or more MEP pathway genes compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDE enzyme. In some cases, the host cell comprises a higher level of expression of dxs and idi compared to a control cell that does not comprise an expression cassette comprising the bifunctional ispDE enzyme.

[0043] In some embodiments, the host cell comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a GPP synthase.

[0044] In some embodiments, the host cells are in a culture medium that includes a substrate (e.g., olivetolate (OA)) of the transporter (e.g., an MFS aromatic acid antiporter or an OMP superfamily porin, e.g., an OprD family porin, e.g., pp3656). In some cases, the substrate (e.g., olivetolate (OA)) is exogenous to the host cells. For example, the substrate (e.g., OA) can be exogenously supplied to the culture medium in which the host cells are cultured.

[0045] In some embodiments, the host cell comprises a deletion in one, two, three, four, five, six, seven, eight or all of the genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps and atoDA.

[0046] In some embodiments, the host cell comprises a PDH bypass. See, e.g., Valliere et al. 2019. In some embodiments, the PDH bypass comprises heterologously expressed pyruvate oxidase and phosphate acetyltransferase.

[0047] In embodiments, one or more, or two or more, or all expression cassettes are integrated into the genome of the host cell. In further or alternative embodiments, one or more expression cassettes are not integrated into the genome of the host cell.

[0048] In a second aspect, the present invention provides methods for enhancing transport of an aromatic substrate of an MFS aromatic acid antiporter into a (e.g., prokaryotic) host cell. In some embodiments, the methods comprise culturing a host cell described herein in a culture medium containing the aromatic substrate under conditions suitable for expression of the transporter or a functional fragment thereof.

[0049] In another aspect, the invention provides methods for prenylating a substrate (e.g., olivetolate (OA) of a transporter (e.g., an MFS aromatic acid antiporter or an OMP superfamily porin, e.g., an OprD family porin, e.g., pp3656)). In some embodiments, the method includes culturing a host cell described herein in a culture medium containing an aromatic substrate of the transporter and an aromatic prenyltransferase, thereby prenylating the aromatic substrate of the transporter. In some embodiments, the substrate is olivetolate. In some embodiments, the aromatic prenyltransferase is functional and capable of transferring a geranyl moiety (e.g., from geranyl diphosphate) to the aromatic substrate. In some embodiments, the aromatic prenyltransferase is functional and capable of transferring a farnesyl moiety (e.g., from farnesyl diphosphate) to the aromatic substrate. In some embodiments, the aromatic prenyltransferase is functional and capable of transferring a neryl moiety (e.g., from neryl diphosphate) to the aromatic substrate. In some embodiments, the aromatic prenyltransferase is functional and capable of transferring a geranyl moiety (e.g., from geranyl diphosphate) and / or a neryl moiety (e.g., from neryl diphosphate) to an aromatic substrate. In some embodiments, the aromatic prenyltransferase is functional and capable of transferring a geranyl moiety (e.g., from geranyl diphosphate), a farnesyl moiety (e.g., from farnesyl diphosphate), and / or a neryl moiety (e.g., from neryl diphosphate) to an aromatic substrate.

[0050] In some embodiments, the aromatic prenyltransferase has geranyl diphosphate:olivetolate geranyltransferase activity. In some embodiments, the aromatic prenyltransferase is a CBGA synthase, an orthologue thereof, or a functional fragment thereof. In some embodiments, the aromatic prenyltransferase is a CBGA synthase having the sequence of SEQ ID NO: 3, or a functional fragment thereof. In some embodiments, the aromatic prenyltransferase is NphB, an orthologue thereof, or a functional fragment thereof. In some embodiments, the aromatic prenyltransferase is NphB having the sequence of SEQ ID NO: 4, or a functional fragment thereof. In some embodiments, the aromatic acid is olivetolate, the aromatic prenyltransferase is CBGA synthase or NphB, and the method comprises producing cannabigerolic acid.

[0051] In some embodiments, the method increases the production of a prenylated product of an aromatic prenyltransferase and an aromatic acid substrate compared to a control method performed under conditions in which the transporter is not expressed or is expressed in a lower amount or activity, hi some embodiments, the method increases the production of a prenylated olivetolate product compared to a control method performed under conditions in which the transporter is not expressed or is expressed in a lower amount or activity.

[0052] In some embodiments, the method comprises culturing a prokaryotic host cell described herein in a suitable culture medium under conditions suitable for inducing expression in one or more host cell expression cassettes, and then harvesting the cultured cells or spent medium, thereby obtaining a desired metabolic product. In some embodiments, the desired metabolic product is THCA, CBDA, CBCA, CBGA, CBN, CBC, THC, or CBD, or a mixture of one or more thereof. In some embodiments, the culture medium comprises exogenous olivetolate. In some embodiments, the culture medium comprises exogenous DVA. In some embodiments, the method comprises adding olivetolate to the culture medium and / or providing a culture medium comprising olivetolate, and culturing the host cell in the provided culture medium. In some embodiments, the method comprises adding DVA to the culture medium and / or providing a culture medium comprising DVA, and culturing the host cell in the provided culture medium.

[0053] In some embodiments, the method comprises harvesting and lysing the cultured cells, thereby producing a cell lysate. In some embodiments, the method comprises purifying the cannabinoid of interest from the cell lysate, thereby producing a purified cannabinoid of interest. In some embodiments, the method comprises purifying the cannabinoid of interest from spent culture medium, thereby producing a purified cannabinoid of interest.

[0054] In some embodiments, the metabolite of interest for purification is a cannabinoid and the method comprises formulating the cannabinoid into a pharmaceutical composition. In some embodiments, the metabolite of interest for purification is a cannabinoid and the method comprises forming a salt, prodrug, or solvate of the purified cannabinoid. In some embodiments, the metabolite of interest for purification is a cannabinoid and the method comprises forming a decarboxylated form of the purified cannabinoid. In some embodiments, the decarboxylated form is formed by heating the metabolite of interest for purification. In some embodiments, the method comprises heating the host cell, host cell lysate, or spent culture medium to decarboxylate the metabolite of interest.

[0055] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0056] [Figure 1] 1 shows a schematic diagram of a cannabinoid pathway for the production of one or more cannabinoids selected from the group consisting of CBGA, CBGVA, THCA, CBDA, CBCA, THCVA, CBCVA, CBDVA, CBN, THC, CBD, CBC, THCV, CBCV, and CBDV. [Figure 2] The pcaK (left) and pp3656 (right) expression plasmids are shown, where expression of the pcaK or pp3656 transgene is under the control of the arabinose promoter. [Figure 3] The B5 expression plasmid construct is shown. The B5 plasmid expresses the IspDF1 chimera, idi, and dxs for the non-mevalonate (MEP) pathway, GPP synthase for GPP production, and an optimized NphB variant aromatic prenyltransferase for CBGA production from OA and GPP. [Figure 4]SDS-PAGE analysis of E. coli expression cultures harboring the NphB expression plasmid and either the pcaK expression plasmid (B5-pcaK), the pp3656 expression plasmid (B5-3656), or the control expression plasmid (B5-pBAD) shows that the predicted sizes of pcaK, pp3656, and NphB are 47.1 kDa, 46.7 kDa, and 33.7 kDa, respectively. [Figure 5] 1 shows a comparison of olivetrate permeability in the presence or absence of aromatic transporters. [Figure 6] 1 shows a comparison of olivetrate cell permeability at different temperatures in the presence of the aromatic transporter pcaK. [Figure 7] Olivetrate cell permeability at different incubation times in the presence of aromatic transporter pcaK is shown. [Figure 8] Figure 1 shows enhanced olivetrate uptake into cells expressing pcaK or pp3656 compared to control cells in which no heterologous transporter was expressed. Enhanced olivetrate uptake into cells was detected over 24-48 hours after expression and induction of pBAD-pcaK and pBAD-3656 compared to BL21 controls in which no additional transporter was expressed. [Figure 9] We show increased production of CBGA in cells expressing NphB and either pcaK or pp3656 compared to control cells expressing an NphB mutant optimized for olivetrate prenylation (see Valliere et al. Nature Communications 2019 10:565) but not the heterologous transporter. [Figure 10] Expression constructs encoding non-mevalonate pathways for the production of IPP and DMAPP are shown. [Figure 11] An expression construct encoding the aromatic prenyltransferase, CBGAS enzyme, is shown. [Figure 12] An expression construct encoding the aromatic prenyltransferase NphB is shown. [Figure 13] An expression construct encoding the THCAS enzyme is shown. [Figure 14] Expression of novel IspDF in E. coli as demonstrated by SDS-PAGE analysis: Lanes 1 and 5 are total and purified IspDE1 extracts, respectively; lanes 2 and 6 are total and purified IspDF2 extracts, respectively; lanes 4 and 7 are total and purified IspDF3 extracts, respectively; and lanes 3 and 8 are protein ladders. [Figure 15] A protein sequence alignment of various IspDE fusion proteins is shown. [Figure 16] This shows an SDS / PAGE image of the soluble protein fraction of pSASDFI. Lane 1 is E. coli BL21(DE3), lane 2 is a protein ladder, and lanes 3 and 4 are SASDFI. The bands corresponding to the proteins are Dxs (band a, 68.2 kDa), IspD (band b, 25.7 kDa), IspF (band d, 16.9 kDa), and Idi (band c, 21.2 kDa). [Figure 17] (a)-(b) show the effect of the rate-limiting step on the MEP pathway flux. (a) Lycopene production, (b) Isoprene production. The IPTG concentration used for induction is indicated in the legend. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 18] (a)-(b) show the effect of novel IspDF fusions on MEP pathway flux. (a) Lycopene production; (b) Isoprene production. The IPTG concentrations used for induction are indicated in the legend. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 19] (a)–(d) show homology models for the fusion proteins generated by the SWISS-MODEL tool: (a) cjIspDF (Liu et al. Biosci Rep. 2018 Feb 28;38(1):BSR20171370), (b) IspDF1, (c) IspDF2, and (d) IspDF3. The IspD domain is colored pink, the IspF domain is colored blue, and the linker is colored green. N-terminal residues are colored black, and C-terminal residues are colored orange. [Figure 20] Figure 1 shows the effect of IspE overexpression on lycopene production. The IPTG concentrations used for induction were 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 21] (a)-(b) show the linker for IspDF1 and its effect on MEP pathway flux. (a) shows the strain overexpressing Dxs, the IspDF chimera, and Idi, and (b) shows the strain overexpressing Dxs, the IspDF chimera, IspE, and Idi. The IPTG concentrations used for induction were 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 22] (a)-(b) show the linker for the non-native fusion of IspD and IspF in E. coli and its effect on MEP pathway flux. (a) shows the strain overexpressing Dxs, the IspDF chimera, and Idi, and (b) shows the strain overexpressing Dxs, the IspDF chimera, IspE, and Idi. The IPTG concentrations used for induction were 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 23] The linker for the non-native fusion of E. coli IspD and IspF is shown for MEP pathway flux. The IPTG concentrations used for induction are 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 24] Figure 1 shows the effect of domain separation of IspDF1 on MEP pathway flux. The IPTG concentrations used for induction are 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 25]Figure 1 shows non-native fusions of IspE and their effect on MEP pathway flux. The IPTG concentrations used for induction are 0 μM, 25 μM, and 50 μM for each construct, from left to right. The first Y-axis is the terpene titer, and the second Y-axis is the normalized terpene titer. [Figure 26] Comparative plots showing lycopene production in the indicated ispDE-overexpressing strains compared to different control constructs are shown. Titer (left) and normalized titer (right) values ​​are shown. Blank spaces are represented by "-". DETAILED DESCRIPTION OF THE INVENTION

[0057] Described herein are host cell genetic engineering strategies for enhancing the transport of aromatic acids into prokaryotic host cells. The aromatic acid can then be provided intracellularly as a substrate for one or more downstream enzymatic steps to produce a desired target metabolic product. For example, the aromatic acid can be a substrate for a heterologous aromatic prenyltransferase. The aromatic prenyltransferase can prenylate the aromatic acid to produce a prenylated product. The prenyl donor can be an exogenous prenyl donor or a heterologous prenyl donor. In certain embodiments, the prenyl donor is geranyl diphosphate. In some embodiments, the prenyl donor is neryl pyrophosphate. In some embodiments, the prenyl donor is an organic pyrophosphate. In some embodiments, the prenyl donor is an organic pyrophosphate naturally occurring in Cannabis sativa. In some embodiments, the prenyl donor is an organic pyrophosphate naturally occurring in E. coli. In some embodiments, the prenyl donor is an organic pyrophosphate selected from the group consisting of isopentyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and isomers thereof, such as isomers of GPP neryl diphosphate.

[0058] In some cases, some or all of the prenyl donors are produced or increased amounts of prenyl donors are provided by a heterologous expression cassette comprising a nucleic acid encoding a GPP synthase. In some cases, some or all of the prenyl donors are produced or increased amounts of prenyl donors are provided by a heterologous expression cassette comprising a nucleic acid encoding a non-mevalonate pathway component. In some cases, some or all of the prenyl donors are produced or increased amounts of prenyl donors are provided by a heterologous expression cassette comprising a nucleic acid encoding a bifunctional ispDF enzyme. In some cases, some or all of the prenyl donors are produced or increased amounts of prenyl donors are provided by a heterologous expression cassette comprising a nucleic acid encoding a bifunctional ispDE enzyme.

[0059] In embodiments in which the substrate of a heterologous transporter is a substrate for a heterologous aromatic prenyltransferase expressed in a host cell, the substrate is typically a prenyl acceptor. For example, the prenyl acceptor can be olivetolate or DVA. Accordingly, in some embodiments, methods and compositions for producing a prenylated olivetolate product are described herein. Additionally, or alternatively, methods and compositions for producing a prenylated divalinate product are described herein. In embodiments in which the prenyl donor is geranyl pyrophosphate and the prenyl acceptor is olivetolate, the prenylation product can be cannabigerolic acid (CBGA). In embodiments in which the prenyl donor is neryl pyrophosphate and the prenyl acceptor is olivetolate, the prenylation product can be cannabinerolate (CBNRA). In some embodiments, the prenyl acceptor is divalinate (DVA). Accordingly, in some embodiments, methods and compositions for producing a prenylated divalinate product are described herein. In embodiments where the prenyl donor is geranyl pyrophosphate and the prenyl acceptor is DVA, the prenylation product can be cannabigerovalic acid (CBGVA). In some embodiments, the prenyl donor is neryl pyrophosphate, the prenyl acceptor is olivetrate, the prenylation product is CBNRA, and the aromatic prenyltransferase is NphB or a functional fragment thereof.

[0060] A prenylated aromatic product (e.g., a prenylated aromatic acid), such as prenylated olivetolate, its downstream enzymatic product, or its decarboxylate can be isolated as a metabolite of interest from host cells, their lysates, or their spent culture medium. In some cases, the isolated metabolite of interest, its salt, its solvate, its derivative, and / or its decarboxylate can be used as an active drug ingredient in a pharmaceutical composition.

[0061] Thus, in embodiments where the prenylated aromatic product is prenylated olivetolate, olivetol, DVA, or divalinol, the methods and compositions described herein can be used to produce cannabinoids in host cells. For example, the host cell can co-express a heterologous cannabinoid synthase, such as CBDA synthase. Similarly, in some embodiments, the methods and compositions described herein can be used to produce cannabinoid precursors in host cells, which precursors can be isolated and used as reactants in one or more in vitro reactions to produce a desired product, such as a cannabinoid or a derivative thereof.

[0062] These in vitro reactions may include synthetic chemistry schemes to produce a desired product, such as a cannabinoid or a derivative thereof. These in vitro reactions may also, or alternatively, include one or more enzyme-catalyzed in vitro reactions. For example, a cannabinoid precursor may be contacted with a cannabinoid synthesis enzyme isolated from a host cell or contained in a host cell lysate. In yet another alternative, the cannabinoid precursor may be isolated and used as input to a second microbial synthesis step using a different prokaryotic or eukaryotic host in which the cannabinoid synthesis enzyme is heterologously expressed.

[0063] Also described herein are methods and compositions for co-expressing a heterologous transporter, a functional aromatic prenyltransferase capable of prenylating a substrate of the heterologous transporter, and one or more additional pathway components. As described herein, the one or more additional pathway components can include a cannabinoid synthase (e.g., THCAS and / or CBDAS) and one or more helper pathway components, thereby producing detectable amounts of cannabinoids in a (e.g., prokaryotic) host cell system. Another exemplary helper pathway component is a mevalonate-independent (MEP) pathway component, such as a bifunctional ispDF enzyme. Another exemplary helper pathway component is a mevalonate-independent (MEP) pathway component, such as a bifunctional ispDE enzyme. Another exemplary helper pathway component is a GPP synthase. Expression of one or more components of one or more helper pathways can be used to produce the cannabinoid of interest. Expression of nucleic acids encoding one or more of the heterologous transporter, aromatic prenyltransferase, cannabinoid synthase(s), one or more helper pathway component(s), and combinations thereof may be controlled by one or more heterologous promoters.

[0064] In some embodiments, the cannabinoid synthase is THCAS. In some embodiments, the cannabinoid synthase is CBDAS. In some embodiments, the prokaryotic host cell comprises an expression cassette comprising a promoter operably linked to THCAS, and an expression cassette comprising a promoter operably linked to CBDAS.

[0065] definition "THCAS" or "tetrahydrocannabinolic acid synthase" refers to the enzyme that catalyzes the conversion of cannabigerolic acid to tetrahydrocannabinolic acid.

[0066] "CBDAS" or "cannabidiolic acid synthase" refers to an enzyme that catalyzes the conversion of cannabigerolic acid to cannabidiolic acid.

[0067] "CBGAS" or "cannabigerolic acid synthase" refers to the enzyme that catalyzes the conversion of olivetrate and GPP to cannabigerolic acid.

[0068] The following abbreviations are used herein: "G3P" means glyceraldehyde 3-phosphate, "DOXP" means 1-deoxy-D-xylulose 5-phosphate, "MEP" means 2-C-methylerythritol 4-phosphate, "CDP-ME" means 4-diphosphocytidyl-2-C-methylerythritol, "CDP-MEP" means 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate, "MECPP" means 2-C-methyl-D-erythritol 2,4-cyclodiphosphate, "HMBPP" means (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, "IPP" means isopentenyl diphosphate, "DMAPP" means dimethylallyl diphosphate, and "GPP" means geranyl pyrophosphate.

[0069] "DXP pathway" and "MEP pathway" refer to the mevalonate-independent pathway, also known as the mevalonate-independent pathway. The genes in the MEP pathway are dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi.

[0070] "dxs" refers to DOXP synthase, "ispC" refers to DOXP reductase, "ispD" refers to 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, "ispE" refers to 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, "ispF" refers to 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, "ispG" refers to HMB-PP synthase, "ispH" refers to HMB-PP reductase, "idi" refers to isopentenyl / dimethylallyl diphosphate isomerase, and "ispA" refers to farnesyl diphosphate synthase, also known as "GPP synthase," which can convert DMAPP+IPP to GPP and GPP+IPP to farnesyl pyrophosphate.

[0071] The term "ispDF" refers to a bifunctional single-chain enzyme that has two distinct active sites and exhibits ispD activity (EC 2.7.7.60) and ispF activity (EC 4.6.1.12). Typically, an ispDF is a naturally occurring bifunctional enzyme or a derivative of a naturally occurring bifunctional enzyme that has one or more modifications, such as a deletion, insertion, or substitution of one or more amino acids.

[0072] "OA" refers to olivetrate, "CBGA" refers to cannabigerolic acid, "CBNRA" refers to cannabinerolic acid, "CBNA" refers to cannabinolic acid, "cannabinol" or "CBN" refers to 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol, "CBGVA" refers to cannabigerovaric acid, and "THCA" refers to Δ 9It refers to tetrahydrocannabinolic acid, including its isomers. "CBDV" refers to cannabidivarin, "CBC" refers to cannabichromene, "CBCA" refers to cannabichromenic acid, "CBCV" refers to cannabichromevarin, "CBG" refers to cannabigerol, "CBGV" refers to cannabigerovarin, "CBE" refers to cannabielsoin, "CBL" refers to cannabicyclol, "CBV" refers to cannabivarin, "CBT" refers to cannabiditriol, "THCV" refers to tetrahydrocannabivarin (THCV), "THC" refers to tetrahydrocannabinol, and "Δ 9 -THC" is Δ 9 -tetrahydrocannabinol, and "CBDA" refers to cannabidiolic acid.

[0073] As used herein, the terms "cannabidiol," "CBD," or "cannabidiol" refer to one or more of the following compounds, and are intended to be used interchangeably with the compound "ΔCBD," unless another specific stereoisomer or stereoisomers are specified: 2 -cannabidiol." These compounds include: (1) Δ 5 -Cannabidiol (2-(6-isopropenyl-3-methyl-5-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol), (2)Δ 4 -Cannabidiol (2-(6-isopropenyl-3-methyl-4-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol), (3)Δ 3 -Cannabidiol (2-(6-isopropenyl-3-methyl-3-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol), (4)Δ 3,7 -Cannabidiol (2-(6-isopropenyl-3-methylenecyclohex-1-yl)-5-pentyl-1,3-benzenediol), (5)Δ 2 -Cannabidiol (2-(6-isopropenyl-3-methyl-2-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol), (6)Δ 1-Cannabidiol (2-(6-isopropenyl-3-methyl-1-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol), and (7)Δ 6 -Cannabidiol (2-(6-isopropenyl-3-methyl-6-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol).

[0074] These compounds have one or more asymmetric centers and two or more stereoisomers as shown below: (1) Δ 5 -Cannabidiol has two asymmetric centers and four stereoisomers, (2)Δ 4 -Cannabidiol has three asymmetric centers and eight stereoisomers, (3)Δ 3 -Cannabidiol has two asymmetric centers and four stereoisomers, (4)Δ 3,7 -Cannabidiol has two asymmetric centers and four isomers, (5)Δ 2 -Cannabidiol has two asymmetric centers and four stereoisomers, (6)Δ 1 -Cannabidiol has two asymmetric centers and four stereoisomers, (7)Δ 6 -Cannabidiol has one asymmetric center and two stereoisomers. In a preferred embodiment, cannabidiol is specifically Δ 2 -cannabidiol. Unless specifically indicated, reference to "cannabidiol," "CBD," or "cannabidiol," or to any of the specific cannabidiol compounds (1)-(7) mentioned above, includes all possible stereoisomers of any compound included by reference. In one embodiment, "Δ 2 -cannabidiol" is a Δ 2 -Cannabidiol may be a mixture of stereoisomers.

[0075] "Isoprenoid" or "terpenoid" refers to any compound containing one or more five-carbon isoprene units, including linear and cyclic terpenoids. As used herein, the term "terpene" is interchangeable with terpenoid and isoprenoid. When terpenes are chemically modified, for example, by oxidation or rearrangement of the carbon chain, the resulting compounds are collectively referred to as terpenoids, also known as isoprenoids.

[0076] Terpenoids can be named according to the number of carbon atoms present, using the carbon-5 and carbon-10 groups as a standard. For example, hemiterpenoids (C5) have one isoprene unit (half a terpenoid), monoterpenoids (C10) have two isoprene units (one terpenoid), sesquiterpenoids (C15) have three isoprene units (1.5 terpenoids), and diterpenoids (C20) have four isoprene units (or two terpenoids). Typically, monoterpenoids are naturally produced from the C10 terpenoid precursor geranyl pyrophosphate (GPP). Similarly, "cyclic monoterpene" refers to a cyclic or aromatic (i.e., containing a ring structure) terpenoid. It is made from two isoprene building blocks, typically GPP. Linear monoterpenes include, but are not limited to, geraniol, linalool, ocimene, and myrcene. Cyclic monoterpenes (monocyclic, bicyclic and tricyclic) include, but are not limited to, limonene, pinene, carene, terpineol, terpinolene, phellandrene, thujene, tricyclene, borneol, sabinene and camphene.

[0077] "Terpenoid synthase" refers to an enzyme that can catalyze the conversion of one terpenoid or terpenoid precursor to another terpenoid or terpenoid precursor. For example, GPP synthase is an enzyme that catalyzes the formation of GPP, e.g., from the terpenoid precursors IPP and DMAPP. Similarly, FPP synthase is an enzyme that catalyzes the production of FPP, e.g., from GPP and IPP. Terpene synthase is an enzyme that catalyzes the conversion of a prenyl diphosphate (e.g., GPP) to an isoprenoid or isoprenoid precursor. The term includes both linear and cyclic terpene synthases.

[0078] "Cyclic terpenoid synthase" refers to an enzyme that can catalyze a reaction that modifies a terpenoid or terpenoid precursor to result in a ring structure. For example, a cyclic monoterpenoid synthase refers to an enzyme that can use a linear monoterpene as a substrate to produce a cyclic or aromatic (ring-containing) monoterpenoid compound. An example would be sabinene synthase, which can catalyze the formation of the cyclic monoterpene sabinene from the linear monoterpene precursor GPP. As used herein, the term "terpene synthase" is interchangeable with terpenoid synthase.

[0079] Prenyltransferases or isoprenyltransferases, also called prenyl or isoprenyl synthases, are enzymes that can catalyze the production of pyrophosphate precursors of terpenoid or isoprenoid compounds. An exemplary prenyltransferase or isoprenyltransferase is ispA, which can catalyze the formation of geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) in the presence of a suitable substrate.

[0080] Aromatic prenyltransferases are enzymes that can catalyze the transfer of a prenyl group to an aromatic substrate. An exemplary aromatic prenyltransferase is CBGAS. Another exemplary aromatic prenyltransferase is NphB. Yet another exemplary aromatic prenyltransferase is CsPT4.

[0081] "Cannabinoid synthase" refers to an enzyme that catalyzes one or more of the following activities: cyclization of CBGA to THCA, CBDA, or CBCA, cyclization of CBGVA to THCVA, CBCVA, CBDVA, prenylation of olivetolate to form CBGA, and combinations thereof. Exemplary cannabinoid synthases include, but are not limited to, those found naturally occurring in plants of the Cannabis genus, such as the THCA synthase, CBDA synthase, and CBCA synthase of Cannabis sativa.

[0082] Exemplary isoprenoid, terpenoid, cannabinoid, and MEP pathway polypeptides and nucleic acids include those described in the KEGG database, which contains the amino acid and nucleic acid sequences of numerous exemplary isoprenoid, terpenoid, cannabinoid, and MEP pathway polypeptides and nucleic acids (see, e.g., the World Wide Web at "genome.jp / kegg / pathway / map / map00100.html," and the sequences therein, each of which is hereby incorporated by reference in its entirety, particularly with respect to the amino acid and nucleic acid sequences of isoprenoid, terpenoid, cannabinoid, and MEP pathway polypeptides and nucleic acids).

[0083] As used herein, the term "heterologous" refers to any two components that are not found together in nature. For example, a nucleic acid encoding a gene heterologous to an operably linked promoter is a nucleic acid whose expression in its natural state (e.g., in an unmodified cell) is not controlled by the promoter operably linked to it in a particular genome. As provided herein, all genes operably linked to a non-naturally occurring promoter are considered "heterologous." Similarly, a gene that is "heterologous" to a host cell is a gene that is not found in unmodified cells of a particular organism, or that is found in a different genomic or non-genomic (e.g., plasmid) location, or that is operably linked to a different promoter in unmodified cells. In addition, a promoter that is "heterologous" to a host cell is a promoter that is not found in unmodified cells of a particular organism, or that is found in a different genomic or non-genomic (e.g., plasmid) location, or that is operably linked to a different nucleic acid in unmodified cells.

[0084] As used herein, "expression cassette" refers to a polynucleotide sequence comprising a promoter polynucleotide operably linked to at least one gene of interest, where the promoter is heterologous to the at least one operably linked gene, the promoter is heterologous to the host cell in which it resides, or the at least one operably linked gene is heterologous to the host cell, or a combination thereof. In embodiments describing expression cassettes containing promoters operably linked to nucleic acids encoding two or more proteins, it is understood that alternative embodiments are contemplated in which the two or more proteins are contained in different expression cassettes. Similarly, it is understood that separate expression cassettes may be combined. In typical embodiments, one or more or all expression cassettes comprise a promoter operably linked to a codon-optimized nucleic acid encoding one or more polypeptides. In exemplary embodiments, the nucleic acid encoding a heterologous transporter is codon-optimized.

[0085] "Salt" refers to the acid or basic salt of the compound used in the method of the present invention. Examples of pharmaceutically acceptable salts are mineral acid salts (such as hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acid salts (such as acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium salts (such as methyl iodide, ethyl iodide, etc.). It is understood that pharmaceutically acceptable salts are harmless. Further information about suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0086] As used herein, the term "solvate" refers to a compound formed by solvation (a combination of a solvent molecule with a solute molecule or ion), or an aggregate consisting of a solute ion or molecule, i.e., a compound of the present invention, and one or more solvent molecules. When water is the solvent, the corresponding solvate is a "hydrate." Examples of hydrates include, but are not limited to, hemihydrate, monohydrate, dihydrate, trihydrate, hexahydrate, and other water-containing species. Those skilled in the art will understand that pharmaceutically acceptable salts and / or prodrugs of a compound may also exist in a solvated form. Solvates are typically formed by hydration, either as part of the preparation of the compound or by natural absorption of water by anhydrous compounds of the present invention. In general, all physical forms are intended to be encompassed within the scope of the present invention.

[0087] Thus, if a therapeutically active agent, such as, but not limited to, a cannabinoid or terpenoid, made in a method or included in a composition of the present invention, possesses a functional group that is sufficiently acidic, sufficiently basic, or both sufficiently acidic and sufficiently basic, then that group or groups may react with any of a number of inorganic or organic bases and inorganic and organic acids to form a pharmaceutically acceptable salt. Exemplary pharmaceutically acceptable salts include salts prepared by reaction of a pharmacologically active compound with a mineral or organic acid or inorganic base, such as sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, etc. salts including butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, and mandelate salts. When a pharmacologically active compound possesses one or more basic functional groups, the desired pharmaceutically acceptable salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, or with an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid such as glucuronic acid or galacturonic acid, an alpha hydroxy acid such as citric acid or tartaric acid, an amino acid such as aspartic acid or glutamic acid, an aromatic acid such as benzoic acid or cinnamic acid, a sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, and the like.When a pharmacologically active compound possesses one or more acidic functional groups, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide, or an alkaline earth metal hydroxide. Illustrative examples of suitable salts include organic salts derived from amino acids, such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, and cyclic amines, such as piperidine, morpholine, and piperazine, and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0088] As used herein, "composition" is intended to encompass a product containing the specified ingredients in the specified amounts, and any product obtained by combining the specified ingredients in the specified amounts. "Pharmaceutically acceptable" means the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0089] "Pharmaceutically acceptable excipient" refers to a substance that aids in the administration of an active agent to a subject and its absorption by the subject. Pharmaceutical excipients useful in the present invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, and coloring agents. One skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0090] In some cases, compounds used in the methods or compositions of the present invention may contain protecting groups. The use of such protecting groups is done to prevent subsequent hydration or other reactions that may occur in vivo and may degrade the compound. Groups that may be protected include alcohols, amines, carbonyls, carboxylic acids, phosphates, and terminal alkynes. Protecting groups useful for protecting alcohols include, but are not limited to, acetyl, benzoyl, benzyl, β-methoxyethoxyethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuran, trityl, silyl ether, methyl ether, and ethoxyethyl ether. Protecting groups useful for protecting amines include carbobenzyloxy, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethyl chloroformate, and sulfonamide. Protecting groups useful for protecting carbonyls include acetals, ketals, acylals, and dithianes. Protecting groups useful for protecting carboxylic acids include methyl esters, benzyl esters, t-butyl esters, esters of 2,6-disubstituted phenols, silyl esters, orthoesters, and oxazolines. Protecting groups useful for protecting phosphate groups include 2-cyanoethyl and methyl. Protecting groups useful for protecting terminal alkynes include propargyl alcohol and silyl groups. Other protecting groups are known in the art.

[0091] As used herein, the term "prodrug" refers to a precursor compound that liberates a biologically active compound through some chemical or physiological process in vivo after administration (e.g., a prodrug is converted to a biologically active compound when physiological pH is reached or by enzymatic action). The prodrug itself may either lack or possess the desired biological activity. Thus, the term "prodrug" refers to a pharmaceutically acceptable precursor of a biologically active compound. In some cases, a prodrug has improved physical and / or delivery properties over the parent compound from which the prodrug is derived. Prodrugs often offer advantages of solubility, tissue compatibility, or slow release in mammalian organisms (H. Bundgard, Design of Prodrugs (Elsevier, Amsterdam, 1988), pp. 7-9, 21-24). A description of prodrugs is provided in T. Higuchi et al., "Pro-Drugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and E B Roche, ed., Bioreversible Carriers in Drug Design (American Pharmaceutical Association & Pergamon Press, 1987). Exemplary advantages of prodrugs may include, but are not limited to, their physical properties, such as enhanced drug stability during long-term storage.

[0092] The term "prodrug" is also meant to include any covalently bonded carrier that liberates the active compound in vivo upon administration of the prodrug to a subject. Prodrugs of the therapeutically active compounds described herein can be prepared by modifying one or more functional groups present in therapeutically active compounds, including cannabinoids, terpenoids, and other therapeutically active compounds used in the methods or included in the compositions of the present invention, in such a way that the modification is cleaved, either by routine manipulation or in vivo, to yield the parent therapeutically active compound. Prodrugs include compounds having a hydroxy, amino, or mercapto group covalently bonded to any group that is cleaved to form a free hydroxy, free amino, or free mercapto group, respectively, upon administration of the prodrug of the active compound to a subject. Examples of prodrugs include, but are not limited to, fumaric acid or benzoic acid derivatives of alcohols, or acetamide, formamide, or benzamide derivatives of therapeutically active agents bearing a reactive amine function.

[0093] For example, if the therapeutically active agent, or a pharmaceutically acceptable form of the therapeutically active agent, contains a carboxylic acid functional group, the prodrug may be 1-8 Alkyl, C 2-12Alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)aminomethyl having 4 to 10 carbon atoms, and esters formed by replacing a hydrogen atom of a carboxylic acid group with a group such as N,N-di(C-C)alkylcarbamoyl-(C-C)alkyl, N,N-di(C-C)alkylcarbamoyl-(C-C)alkyl, and piperidino-, pyrrolidino-, or morpholino(C-C)alkyl.

[0094] Similarly, when a disclosed compound or a pharmaceutically acceptable form of the compound contains an alcohol functional group, a prodrug can be formed by replacing the hydrogen atom of the alcohol group with a group such as (C-C)alkanoyloxymethyl, 1-((C-C))alkanoyloxy)ethyl, 1-methyl-1-((C-C)alkanoyloxy)ethyl(C-C)alkoxycarbonyloxymethyl, N(C-C)alkoxycarbonylaminomethyl, succinoyl, (C-C)alkanoyl, α-amino(C-C)alkanoyl, arylacyl, and α-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independently selected from naturally occurring L-amino acids, P(O)(OH), P(O)(O(C-C)alkyl), or glycosyl (a radical obtained by removal of the hydroxy group of the hemiacetal form of a carbohydrate).

[0095] When a disclosed compound or a pharmaceutically acceptable form of said compound incorporates an amine functional group, a prodrug can be prepared by replacing R and R′ each independently with (C1-C 10 R-carbonyl, RO-carbonyl, NRR'-carbonyl, Y, where R-carbonyl is a natural α-aminoacyl or a natural α-aminoacyl-natural α-aminoacyl. 1 C(OH)C(O)OY with H, (C1-C6) alkyl or benzyl 1 , Y 2 is (C1-C4) alkyl and Y 3 C(OY) is (C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C1-C4)alkyl, or mono-N or di-N,N(C1-C6)alkylaminoalkyl 2 )Y 3 , Y 4 is H or methyl, and Y 5 is mono-N or di-N,N(C1-C6) alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl; 4 )Y 5 These groups may be formed by replacing a hydrogen atom in an amine group with a group such as

[0096] The use of prodrug systems is described in T. Jarvinen et al., "Design and Pharmaceutical Applications of Prodrugs" in Drug Discovery Handbook (S.C.G.A., ed., Wiley-Interscience, Hoboken, NJ, 2005), ch. 17, pp. 733-796. Other alternatives for prodrug construction and use are known in the art. Where the methods or pharmaceutical compositions of the present invention use or include a prodrug of a cannabinoid, terpenoid, or other therapeutically active agent, the prodrug and active metabolites of the compound can be identified using routine techniques known in the art. For example, Bertolini et al., J.Med.Chem.,40,2011-2016(1997), Shan et al.,J.Pharm.Sci.,86(7),765-767, Bagshawe,Drug Dev.Res.,34,220-230(1995), Bodor,Advances in Drug Res., 13, 224-331 (1984), Bundgaard, Design of Prodrugs (Elsevier Press 1985), Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991), Dear et al. al., J. Chromatogr. B, 748, 281-293 (2000), Spraul et al., J. Pharmaceutical & Biomedical Analysis, 10, 601-605 (1992), and Prox et al., Xenobiol., 3, 103-112 (1992).

[0097] It will be understood that when used herein to disclose or claim polypeptides such as OMP superfamily porins, e.g., OrpD family porins, e.g., pp3656, MFS aromatic acid antiporters, aromatic prenyltransferases, cannabinoid synthases and / or non-mevalonate pathway components, orthologs of the recited polypeptides are alternatively contemplated.

[0098] cannabinoids Cannabinoids are a group of chemicals known to activate cannabinoid receptors in cells throughout the human body, including the skin. Phytocannabinoids are cannabinoids derived from the Cannabis sativa plant. They can be isolated from the plant or produced synthetically. Endocannabinoids are endogenous cannabinoids found in the human body. Classical phytocannabinoids are ABC tricyclic terpenoid compounds that possess a benzopyran moiety.

[0099] Cannabinoids exert their effects by interacting with cannabinoid receptors present on the surface of cells. To date, two types of cannabinoid receptors have been identified: CB1 and CB2 receptors. These two receptors share approximately 48% amino acid sequence identity, are distributed in different tissues, and have different signaling mechanisms. They also differ in their sensitivity to agonists and antagonists.

[0100] Thus, described herein are in vitro and in vivo methods for screening, identifying, making and using genes, promoters, helper pathway components and expression cassettes for the in vivo production of cannabinoids.

[0101] Typically, the methods and compositions described herein may be used for the production or enhanced production of one or more cannabinoids in a host cell, or the production of one or more cannabinoid precursors in a host cell. Optionally, the cannabinoid or precursor thereof may be purified, derivatized (e.g., to form a prodrug, solvate or salt, or to form the cannabinoid of interest from the precursor), and / or formulated into a pharmaceutical composition.

[0102] Cannabinoids that may be produced according to the methods and / or using the compositions of the present invention include, but are not limited to, phytocannabinoids. In some cases, cannabinoids include cannabinol, cannabidiol, Δ 9 -Tetrahydrocannabinol (Δ 9 Cannabinoids include, but are not limited to, cannabidivarin (CBDV), cannabichromene (CBC), cannabichromevarin (CBCV), cannabigerol (CBG), cannabigerovarin (CBGV), cannabielsoin (CBE), cannabicyclol (CBL), cannabivarin (CBV), and cannabiditriol (CBT). Still other cannabinoids include tetrahydrocannabivarin (THCV) and cannabigerol monomethyl ether (CBGM). Additional cannabinoids include cannabichromenic acid (CBCA), Δ 9 - tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), these further cannabinoids being characterized by the presence of a carboxylic acid group in their structure.

[0103] Additional cannabinoids include nabilone, rimonabant, JWH-018 (naphthalen-1-yl-(1-pentylindol-3-yl)methanone), JWH-073 naphthalen-1-yl-(1-butylindol-3-yl)methanone, CP-55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol), dimethylheptylpyran, and HU-331 (3-hydroxy-2-[(1R)-6-isopropenyl-3-methyloctan-2-yl]phenol). ethyl-cyclohex-2-en-1-yl]-5-pentyl-1,4-benzoquinone), SR144528 (5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-1H-pyrazole-3-carboxamide), WIN55,212-2 ((11R)-2-methyl-11-[(morpholin-4-yl)methyl]-3-(naphthalene-1-carbonyl)-9-oxa-1-azatricyclo[6.3.1.0] 4 , 12 ]dodeca-2,4(12),5,7-tetraene), JWH-133 ((6aR,10aR)-3-(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran), levonatradol, and AM-2201 (1-[(5-fluoropentyl)-1H-indol-3-yl]-(naphthalen-1-yl)methanone). Other cannabinoids include Δ 8 -Tetrahydrocannabinol (Δ 8 -THC), 11-hydroxy-Δ 9 -Tetrahydrocannabinol, Δ 11 -tetrahydrocannabinol, and 11-hydroxy-tetracannabinol.

[0104] In another alternative, these cannabinoid analogs or derivatives may be obtained by production of cannabinoid precursors and further derivatization, for example by synthetic means. Synthetic cannabinoids include, but are not limited to, those described in U.S. Patent No. 9,394,267 to Attala et al., U.S. Patent No. 9,376,367 to Herkenroth et al., U.S. Patent No. 9,284,303 to Gijsen et al., U.S. Patent No. 9,173,867 to Travis, U.S. Patent No. 9,133,128 to Fulp et al., U.S. Patent No. 8,778,950 to Jones et al., U.S. Patent No. 7,700,634 to Adam-Worrall et al., U.S. Patent No. 7,504,522 to Davidson et al., U.S. Patent No. 7,294,645 to Barth et al., U.S. Patent No. 7,109,216 to Kruse et al., U.S. Patent No. 6,825,209 to Thomas et al., and U.S. Patent No. 6,284,788 to Mittendorf et al.

[0105] In another alternative, the cannabinoid may be an endocannabinoid or a derivative or analog thereof. Endocannabinoids include, but are not limited to, anandamide, 2-arachidonoylglycerol, 2-arachidonylglyceryl ether, N-arachidonoyldopamine, and virodamine. Several analogs of endocannabinoids are also known, including 7,10,13,16-docosatetraenoylethanolamide, oleamide, stearoylethanolamide, and homo-γ-linolenoylethanolamine.

[0106] Cannabinoids produced by the methods and compositions of the present invention may be selective for the CB2 cannabinoid receptor, or may be non-selective across the two cannabinoid receptors and bind to either the CB1 or CB2 cannabinoid receptor. In some cases, cannabinoids produced by the methods and compositions of the present invention are selective for the CB2 cannabinoid receptor. In some cases, the cannabinoid, or one of the cannabinoids in the mixture of cannabinoids, is a CB2 antagonist (e.g., a selective or non-selective antagonist). In some cases, cannabinoids produced by the methods and compositions of the present invention are selective for the CB2 cannabinoid receptor. In some cases, the cannabinoid, or one of the cannabinoids in the mixture of cannabinoids, is a CB1 antagonist (e.g., a selective or non-selective antagonist).

[0107] Expression cassette The present specification describes an expression cassette suitable for expressing one or more genes of interest in host cells.The expression cassette described herein can be a component of a plasmid or can be integrated into the genome of a host cell.A single plasmid can contain one or more expression cassettes described herein.When used herein to describe two or more expression cassettes, it is understood that alternatively, at least two of the two or more expression cassettes can be combined to reduce the number of expression cassettes.Similarly, when multiple genes of interest are described as being operably linked to a single promoter, and thus as components of a single expression cassette, it is understood that the single expression cassette can be subdivided into two or more expression cassettes that contain overlapping or non-overlapping subsets of the single described expression cassette.

[0108] The expression cassettes described herein may contain any suitable promoter known in the art. In some cases, the promoter is a constitutive promoter. In other examples, the promoter is an inducible promoter. In a preferred embodiment for use in or with a prokaryotic host, the promoter may be a T5 promoter, a T7 promoter, a Trc promoter, a Lac promoter, a Tac promoter, a Trp promoter, a tip promoter, a λP promoter, or the like. L Promoter, λP R Promoter, λP R P L promoter, such as the arabinose promoter (araBAD). In some embodiments, the promoter is selected from the group consisting of promoters described in Lee et al., Applied and Environmental Microbiology, September 2007, p. 5711-15, which are hereby incorporated by reference in their entirety, particularly with respect to promoters, expression cassettes, including plasmids, for expression of nucleic acids of interest, genes of interest, host cells, and combinations thereof described herein. In some embodiments, the promoter is selected from the group consisting of E. coli promoters described in Zaslaver et al., Nat Methods. 2006 Aug;3(8):623-8, which are hereby incorporated by reference in their entirety, particularly with respect to promoters, expression cassettes, including plasmids, for expression of nucleic acids of interest, genes of interest, host cells, and combinations thereof described herein. Promoters useful for driving expression of one or more genes of interest in various host cells are numerous and familiar to those of skill in the art (see, e.g., WO2004 / 033646, US8,507,235, US8,715,962, and WO2011 / 017798, and the references cited therein, each of which is hereby incorporated by reference in its entirety, particularly with respect to promoters, expression cassettes, including plasmids, for expression of nucleic acids of interest, genes of interest, host cells, and combinations thereof described herein).

[0109] The methods and compositions described herein can be used to express a functional heterologous transporter, e.g., an MFS aromatic acid antiporter (e.g., pcaK), or an OMP superfamily porin, e.g., a porin of the OprD family (e.g., pp3656). The methods and compositions described herein can also be used to express a functional aromatic prenyltransferase. In some cases, the methods and compositions described herein can also be used to increase the production of prenyl donors, e.g., via the non-mevalonate pathway, e.g., by expression of a bifunctional ispDF enzyme and / or a bifunctional ispDE enzyme. The methods and compositions described herein can also be used to express a functional cannabinoid synthase, e.g., THCAS and / or CBDAS.

[0110] Typically, functional THCAS and / or CBDAS is provided by one or more helper pathway components and / or co-expression of one or more components of one or more helper pathways.

[0111] A heterologous transporter can be modified for expression in a host. For example, one or more transmembrane or signal peptide domains can be truncated or used in place of a transmembrane or signal peptide domain compatible with expression in a host cell. Additionally or alternatively, one or more glycosylation sites can be deleted (e.g., by mutation of the primary amino acid sequence). Similarly, one or more or all of the cysteines found in the intramolecular disulfide bonds of the native protein in the native host can be mutated, for example, to serine. Similarly, one or more or all of the cysteines found in the intermolecular disulfide bonds of the native protein in the native host can be mutated, for example, to serine.

[0112] The methods and compositions described herein can be used for expression of GPP synthase in a suitable (e.g., prokaryotic) host cell in combination with expression of a heterologous transporter, and optionally, an aromatic prenyltransferase. For example, the host cell can contain an expression cassette having a promoter operably linked to a heterologous nucleic acid encoding GPP synthase.

[0113] The methods and compositions described herein can be used for the expression of one or more genes of the MEP pathway in a suitable (e.g., prokaryotic) host cell, in combination with the expression of a heterologous transporter, and optionally, an aromatic prenyltransferase. In some embodiments, MEP pathway flux is increased by overexpression of one or more endogenous components of the host cell by amplifying gene copy number and / or operably linking the endogenous gene (or a copy thereof) to a strong constitutive or inducible heterologous promoter. Thus, in one embodiment, an expression cassette is provided that includes a promoter operably linked to a nucleic acid encoding one or more genes of the MEP pathway. In E. coli, the endogenous MEP pathway genes are dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi.

[0114] In some cases, the promoter of the expression cassette is operably linked to nucleic acids encoding two or more genes of the MEP pathway. In some cases, the promoter of the expression cassette is operably linked to nucleic acids encoding three or more genes of the MEP pathway. In some cases, the promoter of the expression cassette is operably linked to nucleic acids encoding four, five, six, or all endogenous genes of the MEP pathway, or one, two, three, four, five, six, or all orthologues thereof. In some cases, the MEP pathway genes provided in the expression cassette are prokaryotic genes. In some cases, the MEP pathway genes provided in the expression cassette are E. coli genes. In some cases, one or more of the MEP pathway genes provided in the expression cassette are genes heterologous to wild-type E. coli. In some cases, one or more genes of the MEP pathway are provided in a first expression cassette and one or more genes of the MEP pathway are provided in a second expression cassette. In a preferred embodiment, an expression cassette is provided comprising a promoter operably linked to dxs.

[0115] In some cases, an expression cassette is provided that encodes one or more genes in the MEP pathway and further comprises a promoter operably linked to a nucleic acid encoding a GPP synthase, a cannabinoid synthase, or an isoprene synthase, or a functional fragment thereof. In some cases, an expression cassette is provided that encodes one or more genes in the MEP pathway and further comprises a promoter operably linked to a nucleic acid encoding a THCA synthase, or a functional fragment thereof. In some cases, an expression cassette is provided that encodes one or more genes in the MEP pathway and further comprises a promoter operably linked to a nucleic acid encoding a CBGA synthase, or a functional fragment thereof. In some cases, an expression cassette is provided that encodes one or more genes in the MEP pathway and further comprises a promoter operably linked to a nucleic acid encoding a CBDA synthase, or a functional fragment thereof. In some cases, an expression cassette is provided that encodes one or more genes in the MEP pathway and further comprises a promoter operably linked to a nucleic acid encoding an NphB, or a functional fragment thereof.

[0116] In some embodiments, an expression cassette is provided that contains a promoter operably linked to a nucleic acid encoding a bifunctional ispDF enzyme. The ispDF gene can be used in addition to, or as an alternative to, overexpression of native ispD and / or ispF in a host cell. Optionally, the nucleic acid encodes an ispDF protein having the following amino acid sequence (SEQ ID NO:5): MIALQRSLSMHVTAIIAAAAGEGRRLGAPLPKQLLDIGGRSILERSVMAFARHERIDDVIVVLPPALAAAPPDWIAASGRVPAVHVVSGGERRQDSVANAFDRVPAQSDVVLVHDAARPFVTAELISRAIDGAMQHGAAIVAVPVRDTVKRVDPDGEHPVITGTIPRDTIYLAQTPQAFRRDVLGAAVALGRSGVSATDE AMLAEQAGHRVHVVEGDPANVKITTSADLDQARQRLRSAVAARIGTGYDLHRLIEGRPLIIGGVAVPCDKGALGHSDADVACHAVIDALLGAAGAGNVGQHYPDTDPRWKGASSIGLLRDALRLVQERGFTVENVDVCVVLERPKIAPFIPEIRARIAGALGIDPERVSVKGKTNEGVDAVGRGEAIAAHAVALLSES.

[0117] In other embodiments, the ispDF nucleic acid encodes an ispDF protein identical to or having at least 32%, 40%, 45%, 50%, 52%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 99% identity to SEQ ID NO:5.

[0118] In some cases, the bifunctional ispDF is selected from the group consisting of H. pylori HP1020, H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis Sheeba strain Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168 Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 It has a primary amino acid sequence that is 75% or less identical to at least 300 consecutive amino acids of CFF8240_0409. In some cases, the bifunctional ispDF is not H. pylori HP1020, H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis Sheeba strain Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168 Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, or C. fetus subsp. fetus 82-40 CFF8240_0409.

[0119] Exemplary ispDF bifunctional enzymes are described herein. Further examples of bifunctional ispDF enzymes include, but are not limited to, those shown in the table below:

[0120] [Table 1-1] [Table 1-2] [Table 1-3]

[0121] Exemplary ispDF enzymes further include those having at least 80% identity (or 85%, or 90%, or 95%, or 99%, or 100% identity) to an ispDF enzyme sequence set forth herein (e.g., IspDF1, IspDF2, or IspDF3). Further exemplary ispDF enzymes include those having an ispF domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to an ispF domain sequence set forth in the table above. Further exemplary ispDF enzymes include those having an ispD domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to an ispD domain sequence set forth in the table above.

[0122] The bifunctional ispDF may be encoded by a nucleic acid in a plasmid. Alternatively, the bifunctional ispDF may be encoded by a nucleic acid integrated into the genome of a heterologous host cell. Optionally, a heterologous promoter is operably linked to the nucleic acid encoding the bifunctional ispDF. Additionally, or alternatively, the host cell may be heterologous to the nucleic acid encoding the bifunctional ispDF. The bifunctional ispDF enzyme and methods for its use, for example, in cannabinoid production in a host cell (e.g., a prokaryotic host cell), are described, for example, in PCT / CA2018 / 051074, the entire contents of which are incorporated by reference for all purposes.

[0123] The nucleic acid encoding the bifunctional ispDF can be in an MEP pathway expression cassette, such as any one of the expression cassettes described above containing a nucleic acid encoding a MEP pathway gene. Optionally, the nucleic acid encoding the bifunctional ispDF can be in an expression cassette containing a nucleic acid encoding a cannabinoid synthase. Optionally, the nucleic acid encoding the bifunctional ispDF can be in an expression cassette containing a nucleic acid encoding a GPP synthase. Optionally, the nucleic acid encoding the bifunctional ispDF can be in an expression cassette containing a nucleic acid encoding an isoprene synthase.

[0124] In some embodiments, an expression cassette is provided that contains a promoter operably linked to a nucleic acid encoding a bifunctional ispDE enzyme. The ispDE gene can be used in addition to, or as an alternative to, overexpression of native ispD and / or ispF and / or heterologous ispDF in a host cell. In some cases, the nucleic acid encodes an ispDF protein having a native ispD amino acid sequence, or a functional fragment thereof, fused via a linker to a native ispE amino acid sequence, or a functional fragment thereof.

[0125] Exemplary ispDE bifunctional enzymes are described herein. Further examples of bifunctional ispDE enzymes include, but are not limited to, those shown in the table below (linker sequences are in bold and underlined):

[0126] [Table 2]

[0127] Exemplary ispDE enzymes further include those having at least 80% identity (or 85%, or 90%, or 95%, or 99%, or 100% identity) to the ispDE enzyme sequences set forth herein (e.g., SEQ ID NO: 10). Further exemplary ispDE enzymes include those having an ispE domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to the ispE domain sequences set forth in the tables above. Further exemplary ispDE enzymes include those having an ispD domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to the ispD domain sequences (e.g., excluding linker sequences) set forth in the tables above. Further exemplary ispDE enzymes include those having an ispD domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to the ispD domain sequence shown in the table above, including the linker sequence.

[0128] The bifunctional ispDE can be encoded by a nucleic acid in a plasmid. Alternatively, the bifunctional ispDE can be encoded by a nucleic acid integrated into the genome of a heterologous host cell. In some cases, a heterologous promoter is operably linked to the nucleic acid encoding the bifunctional ispDE. Additionally or alternatively, the host cell can be heterologous to the nucleic acid encoding the bifunctional ispDE.

[0129] In some embodiments, ispEF bifunctional enzymes, or nucleic acids encoding such ispEF bifunctional enzymes, are provided. Exemplary ispEF bifunctional enzymes include, but are not limited to, those shown in the table below, and ispEF bifunctional enzymes having 80% identity (or 85%, or 90%, or 95%, or 99%, or 100% identity) to the ispEF enzyme sequences set forth in the table below.

[0130] [Table 3]

[0131] Further exemplary ispEF enzymes include those having an ispF domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to the ispF domain sequence shown in the table above. Further exemplary ispEF enzymes include those having an ispE domain that is at least 80% identical (or 85%, or 90%, or 95%, or 99%, or 100% identical) to the ispE domain sequence shown in the table above.

[0132] The bifunctional ispEF can be encoded by a nucleic acid in a plasmid. Alternatively, the bifunctional ispEF can be encoded by a nucleic acid integrated into the genome of a heterologous host cell. In some cases, a heterologous promoter is operably linked to the nucleic acid encoding the bifunctional ispDF. Additionally or alternatively, the host cell can be heterologous to the nucleic acid encoding the bifunctional ispEF.

[0133] In some cases, the nucleic acid encodes an ispDE protein having an ispD amino acid sequence that is at least 32%, 40%, 45%, 50%, 52%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% identical to, or identical to, a functional fragment of a native E. coli ispD amino acid sequence. In some cases, the nucleic acid encodes, or further encodes, an ispDE protein having an ispE amino acid sequence that is at least 32%, 40%, 45%, 50%, 52%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% identical to, or identical to, a functional fragment of a native E. coli ispE amino acid sequence.

[0134] In some cases, the nucleic acid encoding the ispDE protein encodes a flexible peptide linker between the ispE domain and the ispD domain. In some cases, the flexible linker is 6-15 amino acids in length. In some cases, the flexible linker is 7-12 amino acids in length. In some cases, the flexible linker comprises at least 65% or at least 70% random coil formation as predicted by the GOR algorithm, version IV.

[0135] The bifunctional ispDE can be encoded by a nucleic acid in a plasmid. Alternatively, the bifunctional ispDE can be encoded by a nucleic acid integrated into the genome of a heterologous host cell. In some cases, a heterologous promoter is operably linked to the nucleic acid encoding the bifunctional ispDE. Additionally or alternatively, the host cell can be heterologous to the nucleic acid encoding the bifunctional ispDE.

[0136] The ispDE bifunctional enzymes described herein may be useful for producing isoprene. The ispDE bifunctional enzymes described herein may be useful for producing one or more terpenoids, such as hemiterpenoids, monoterpenoids, sesquiterpenoids, diterpenoids, indole diterpenes, triterpenoids, cyclic terpenoids, and linear terpenoids. Exemplary terpenoid products include, but are not limited to, lycopene, geraniol, linalool, ocimene and myrcene, taxol, limonene, pinene, carene, terpineol, terpinolene, phellandrene, thujene, tricyclene, borneol, sabinene, or camphene. The ispDE bifunctional enzymes described herein may be useful for producing taxol and / or taxol derivatives. The ispDE bifunctional enzymes described herein may be useful for producing steroids, N-glycans, carotenoids, ubiquinone, zeatin, and / or polyprenols.

[0137] In some embodiments, the bifunctional MEP pathway enzyme comprises a flexible linker peptide between the ispD domain, or a functional fragment thereof, and the ispE domain, or a functional fragment thereof. In some embodiments, the flexible linker comprises the sequence SLGGGGSAAA. Optionally, the linker sequence has greater than 65% random coil formation as determined by the GOR algorithm, version IV (Methods in Enzymology 1996 RF Doolittle Ed., vol. 266, 540-553). Optionally, the nucleic acid encoding the ispDE protein encodes a flexible peptide linker between the ispE domain and the ispD domain. Optionally, the flexible linker is 6-15 amino acids in length. Optionally, the flexible linker is 7-12 amino acids in length. Optionally, the flexible linker comprises at least 65% or at least 70% random coil formation as predicted by the GOR algorithm, version IV.

[0138] In one aspect, one or more of the bifunctional ispDE enzymes described herein can be encoded by a nucleic acid in an expression cassette, e.g., in a host cell. In some embodiments, one or more bifunctional ispDE enzymes are heterologously expressed in a host cell. In some cases, one or more bifunctional ispDE enzymes are co-expressed with one or more components of the MEP pathway in the same or a different expression cassette. MEP pathway components include, for example, dxs, ispC, ispF, ispG, ispH, and idi. In some embodiments, an expression cassette comprising a promoter operably linked to a nucleic acid encoding a bifunctional ispDE enzyme further comprises one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispF, ispG, ispH, and idi. In one embodiment, an expression cassette comprising a promoter operably linked to a bifunctional ispDE enzyme further comprises dxs, ispF, and idi. In one embodiment, the expression cassette comprising a promoter operably linked to a nucleic acid encoding a bifunctional ispDE pathway enzyme further comprises a bifunctional ispDF enzyme as described in International Application No. PCT / CA2018 / 051074, the disclosure of which is expressly incorporated herein by reference.

[0139] In some cases, one or more bifunctional ispDE enzymes are co-expressed with one or more aromatic prenyltransferases in the same or different expression cassettes. In some cases, one or more bifunctional ispDE enzymes are co-expressed with one or more cannabinoid synthases in the same or different expression cassettes. In some embodiments, the invention provides expression cassettes or expression cassette systems for heterologous expression in a host cell of a cannabinoid synthase (e.g., CBDAS or THCAS, preferably CBDAS) and a bifunctional ispDE enzyme.

[0140] In some embodiments, the invention provides expression cassettes or expression cassette systems for heterologous expression in host cells of one or more bifunctional ispDE enzymes and one or more terpenoid synthases, including, but not limited to, isoprene synthase or lycopene synthase. In some embodiments, the expression cassettes or expression cassette systems include nucleic acids encoding one or more components of a lycopene synthesis pathway (e.g., crtE, crtI, and / or crtB), a diterpene synthase, a sesquiterpene synthase, or a monoterpene synthase. In some embodiments, the expression cassettes or expression cassette systems include nucleic acids encoding carene synthase, myrcene synthase, or limonene synthase. In some embodiments, the expression cassettes or expression cassette systems optionally include components of a lycopene synthesis pathway (e.g., crtE, crtI, and / or crtB), an isoprene synthase, a GPP synthase (e.g., ispA or plant-derived GPP synthase), a monoterpene synthase, and / or a cannabinoid synthase.

[0141] In some cases, one or more bifunctional ispDE enzymes are co-expressed with one or more aromatic prenyltransferases and one or more cannabinoid synthases (e.g., CBDAS and / or THCAS) in the same or different expression cassettes. In some embodiments, the cannabinoid synthase is selected from the group consisting of Cannabis CBGA synthase.

[0142] The nucleic acid encoding the bifunctional ispDE can be in an MEP pathway expression cassette, such as any one of the expression cassettes described above containing a nucleic acid encoding a MEP pathway gene. Optionally, the nucleic acid encoding the bifunctional ispDE can be in an expression cassette containing a nucleic acid encoding a cannabinoid synthase. Optionally, the nucleic acid encoding the bifunctional ispDE can be in an expression cassette containing a nucleic acid encoding a GPP synthase. Optionally, the nucleic acid encoding the bifunctional ispDE can be in an expression cassette containing a nucleic acid encoding an isoprene synthase.

[0143] The methods and compositions described herein can be used to produce GPP from precursors produced in the MEP pathway in a suitable (e.g., prokaryotic) host cell, where GPP is the prenyl donor substrate for an aromatic prenyltransferase and an aromatic acid is the prenyl acceptor for the aromatic prenyltransferase. Thus, in some embodiments, an expression cassette is provided comprising a promoter operably linked to a nucleic acid encoding a GPP synthase. The GPP synthase can be in an expression cassette that also contains a nucleic acid encoding a MEP pathway gene. Additionally or alternatively, the GPP synthase can be in an expression cassette that also contains a nucleic acid encoding a cannabinoid synthase. In some cases, the promoter of the expression cassette operably linked to the nucleic acid encoding the GPP synthase is also operably linked to a cannabinoid synthase. Additionally or alternatively, the GPP synthase can be in an expression cassette that also contains a nucleic acid encoding an isoprene synthase.

[0144] host cell Any of the above expression cassettes, and combinations thereof, can be introduced into a suitable host cell and used to produce a desired metabolite, such as a cannabinoid or a prenylated aromatic acid. Suitable host cells include, but are not limited to, prokaryotes, such as those of the genera Escherichia, Panteoa, Corynebacterium, Bacillus, or Lactococcus. Preferred prokaryotic host cells include, but are not limited to, Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, and Lactococcus lactis. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the expression cassettes described herein comprise a promoter (e.g., a heterologous promoter) operably linked to a nucleic acid encoding one or more genes of interest (e.g., an MFS aromatic acid antiporter (e.g., pcaK), an OMP superfamily porin, an OprD family porin (e.g., pp3656), an aromatic prenyltransferase, an MEP pathway gene, a cannabinoid synthase gene, ispA, ispS, ispDF, or a GPP synthase), wherein the nucleic acid encoding the one or more genes of interest is codon-optimized for a host cell comprising the expression cassette.

[0145] In some cases, the host cell comprises one or more products of the MEP pathway, such as DMAPP and / or IPP. For example, a host cell containing a MEP pathway expression cassette described herein may comprise increased amounts of a MEP pathway product, such as DMAPP and / or IPP, compared to a host cell that does not contain the MEP pathway expression cassette.

[0146] In some cases, the host cell may contain one or more downstream products of the MEP pathway. For example, a host cell containing a GPP synthase expression cassette may contain an increased amount of GPP compared to a host cell lacking the GPP synthase expression cassette. In another example, a host cell containing an isoprene synthase expression cassette may contain an increased amount of isoprene compared to a host cell lacking the isoprene synthase expression cassette.

[0147] In yet another example, a host cell comprising a cannabinoid synthesis enzyme expression cassette may contain increased amounts of cannabinoids compared to a host cell lacking an expression cassette containing a heterologous nucleic acid encoding a heterologous transporter or a functional fragment thereof. In some cases, the cannabinoid is CBGA. In some cases, the cannabinoid is CBCA. In some cases, the cannabinoid is CBDA. In some cases, the cannabinoid is THCA. In some cases, the cannabinoid is CBNA or CBN. In some cases, the cannabinoid is CBD. In some cases, the cannabinoid is THC. In some cases, the cannabinoid is CBC. In some cases, the cannabinoid is THCV. In some cases, the cannabinoid is CBDV. In some cases, the cannabinoid is CBCV.

[0148] Similarly, when host cells are cultured under conditions suitable for inducing expression from the expression cassette, the host cells may contain an increased amount of the product of one or more enzymes encoded by the expression cassette within the host cells compared to non-induced conditions. For example, when induced, the host cells may contain an increased intracellular amount of an aromatic acid substrate of a heterologous transporter, or an increased intracellular accumulation rate of an aromatic acid substrate, compared to the same host cells cultured in the absence of an inducer. As another example, when induced, the host cells may contain an increased amount or increased production rate of a product of an aromatic prenyltransferase compared to the same host cells cultured in the absence of an inducer (e.g., in the absence of IPTG, arabinose, etc.). As another example, when induced, the host cells may exhibit increased DMAPP and / or IPP compared to the same host cells cultured in the absence of an inducer (e.g., in the absence of IPTG, arabinose, etc.). As another example, when induced, the host cells may exhibit increased GPP compared to the same host cells cultured in the absence of an inducer (e.g., in the absence of IPTG, arabinose, etc.). As another example, a host cell when induced may exhibit increased isoprene compared to the same host cell cultured in the absence of an inducer (e.g., in the absence of IPTG, arabinose, etc.) As another example, a host cell when induced may exhibit increased cannabinoids compared to the same host cell cultured in the absence of an inducer (e.g., in the absence of IPTG, arabinose, etc.).

[0149] In some embodiments, the host cells comprise olivetolate (OA). OA can be introduced into the host cells by culturing the host cells in a medium containing OA. In some embodiments, the host cells comprise divaleric acid (DVA). DVA can be introduced into the host cells by culturing the host cells in a medium containing DVA. In typical embodiments, OA and / or DVA are substrates for a heterologous transporter.

[0150] In some embodiments, host cells are genetically modified to delete or reduce the expression of one or more genes encoding endogenous enzymes that reduce flux through the MEP pathway. In some embodiments, host cells are genetically modified to delete or reduce the amount or activity of endogenous enzymes that reduce flux through the MEP pathway. For example, pyruvate and glyceraldehyde-3 phosphate (G3P) are substrates for the first enzyme in the MEP pathway, dxs. Endogenous pathways that consume pyruvate and G3P can be modified to increase the amount of pyruvate and G3P, thus increasing flux through the MEP pathway. In some cases, one or more endogenous host cell genes or gene products selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA are modified to increase pyruvate or G3P levels.

[0151] Culture method The present invention further provides a process for culturing a host cell of the present invention in a suitable medium under inducing conditions to result in the production of a metabolite of interest. The metabolite of interest may be a cannabinoid, a terpenoid, or a precursor thereof. The method may include concentrating the metabolite in spent medium and / or within the host cell.

[0152] The microorganisms produced can be cultivated continuously, for example as described in WO 05 / 021772, or discontinuously in a batch process (batch culture) or in a fed-batch or repeated fed-batch process, for the purpose of producing the desired organic chemical compound. Overviews of the general nature of known cultivation methods can be found in the textbooks by Chmiel (BioprozeBtechnik.1:Einfiihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or by Storhas (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0153] The culture or fermentation medium used must meet the requirements of each strain in a suitable manner. Culture media for various microorganisms are described in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington, DC, USA, 1981). The terms culture medium and fermentation medium are interchangeable.

[0154] As carbon sources, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing liquids derived from sugar beet or sugar cane processing, starch, starch hydrolysates and cellulose; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol, methanol and ethanol; and organic acids such as acetic acid or lactic acid can be used.

[0155] As nitrogen sources, organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, and urea; or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

[0156] As phosphorus source it is possible to use phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts.

[0157] The culture medium may further contain salts required for growth, for example in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium and iron, such as magnesium sulfate or ferrous sulfate. Finally, in addition to the above substances, essential growth factors such as amino acids, for example homoserine, and vitamins such as thiamine, biotin or pantothenic acid may be employed.

[0158] The starting materials may be added to the culture in a single batch or may be fed in any suitable manner during the culture.

[0159] The pH of the culture can be controlled by using basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia; or acidic compounds, such as phosphoric acid or sulfuric acid, in a suitable manner. The pH is usually adjusted to 6.0 to 8.5, preferably 6.5 to 8. To control foaming, antifoaming agents, such as fatty acid polyglycol esters, can be used. To maintain plasmid stability, suitable selection substances, such as antibiotics, can be added to the medium. Cultivation is preferably carried out under aerobic conditions. To maintain these conditions, oxygen or an oxygen-containing gas mixture, such as air, is introduced into the culture. It is also possible to use a liquid enriched in hydrogen peroxide. If appropriate, cultivation is carried out under elevated pressure, for example, at a pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually 20 to 45°C, preferably 25 to 40°C, and particularly preferably 30 to 37°C. In a batch or fed-batch process, cultivation is preferably continued until a sufficient amount of the desired organic chemical compound is formed to be recovered. This goal is typically achieved within 10-160 hours (e.g., within 10-72 hours, within 10-48 hours, within 10-24 hours, or within 10-16 hours). Longer incubation times are possible in continuous processes. Microbial activity results in the concentration (accumulation) of organic chemical compounds in the fermentation medium and / or cells of the microorganisms.

[0160] Examples of suitable culture media can be found in, inter alia, US 5,770,409, US 5,990,350, US 5,275,940, WO2007 / 012078, US 5,827,698, WO2009 / 043803, US 5,756,345, and US 7,138,266.

[0161] Analysis of the metabolites of interest to determine their concentration one or more times during cultivation can occur by separating the metabolites by means of chromatography, preferably reverse phase chromatography.

[0162] Detection can be photometric (absorbance, fluorescence).

[0163] The performance of a culture method using a host cell containing one or more expression cassettes of the present invention with respect to one or more parameters selected from the group consisting of concentration (target metabolite formed per unit volume), yield (target metabolite formed per unit carbon source consumed), formation (target metabolite formed per unit volume and unit time), and specific formation (target metabolite per unit cell dry matter or unit dry biomass and unit time, or compound formed per unit cell protein and unit time), or other process parameters, and combinations thereof, can be improved by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative to a culture method using a host cell not containing an expression cassette of the present invention. This is believed to be highly valuable for large-scale industrial processes.

[0164] A product containing the desired metabolite may then be produced or recovered in liquid or solid form.

[0165] Spent medium refers to a culture medium in which host cells have been cultured for a certain period of time at a certain temperature. The culture medium or medium(s) employed during the culture contain all substances or components that ensure the production of the desired target metabolic product and typically growth and viability. Thus, upon completion of the culture, the resulting spent medium contains a) the microbial biomass (cell mass) produced by the growth of the microbial cells, b) the desired target metabolic product formed during the culture, c) organic by-products that may be formed during the culture, and d) constituents of the employed culture medium or of the starting material that were not consumed during the culture, such as vitamins such as biotin or salts such as magnesium sulfate.

[0166] Organic by-products include substances produced, and possibly secreted, by the microorganism employed in the culture in addition to the specific desired compound. Spent medium can be removed from the culture vessel or fermentation tank, harvested if appropriate, and used to provide a product containing the desired metabolic product in liquid or solid form. In the simplest example, the spent medium containing the desired metabolic product removed from the fermentation tank itself constitutes the recovered product.

[0167] In some cases, recovery of the metabolic products of interest (e.g., terpenoids, cannabinoids, or precursors thereof) can be achieved by, but not limited to, a) partial (greater than 0% to less than 80%) to all (100%) or substantially all (greater than 80%, 90%, 95%, 96%, 97%, 98%, or 99%) removal of water; b) partial (greater than 0% to less than 80%) to all (100%) or substantially all (greater than 80%, 90%, 95%, 96%, 97%, 98%, or 99%) removal of biomass, optionally with inactivation of the latter prior to removal; c) organic by-products formed during cultivation. a) partially (greater than 0% to less than 80%), completely (100%), or substantially completely (greater than 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.7%) removing constituents of the employed fermentation medium or of the starting material that were not consumed during cultivation; and b) partially (greater than 0%), completely (100%), or substantially completely (greater than 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.3%, or 99.7%) removing constituents of the employed fermentation medium or of the starting material that were not consumed during cultivation from the spent medium, thereby achieving concentration or purification of the desired target metabolic product. In some cases, the target metabolic product is produced intracellularly and recovered by a method involving lysis of cultured host cells of the invention. In some cases, the method for recovering a metabolic product of interest comprises providing a lysate of cultured host cells of the invention and isolating the metabolic product of interest from the lysate, thereby isolating a composition having a desired content of the metabolic product of interest. Lysing the cultured host cells can be performed, for example, after isolating the host cells from spent culture medium.

[0168] Partial (more than 0% and less than 80%), complete (100%), or substantially complete (more than 80% and less than 100%) removal of water (means a)) is also referred to as drying.

[0169] In one variation of the process, all or substantially all of the water, biomass, organic by-products, and unconsumed constituents of the employed fermentation medium are removed to yield a pure (greater than 80%, greater than 90% by weight) or highly pure (greater than 95%, greater than 97%, or greater than 99% by weight) product form of the desired target metabolic product. Many technical instructions for means a) are available in the art.

[0170] Depending on the requirements, the biomass can be totally or partially removed from the spent medium by separation methods such as, for example, centrifugation, filtration, decantation or a combination thereof, or can be left all in it. Where appropriate, the biomass, or the spent medium containing the biomass, is inactivated during a suitable process step, for example, by thermal treatment (heating) or by the addition of alkali or acid.

[0171] In some procedures, all or substantially all of the biomass may be removed, such that no biomass (0%), or at most 30%, at most 20%, at most 10%, at most 5%, at most 1%, or at most 0.1% remains in the prepared product. In further procedures, none or only a small percentage of the biomass is removed, such that all (100%), or more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, or more than 99.9% of the biomass remains in the prepared product. Thus, in some processes according to the invention, more than 0% to less than 100% of the biomass is removed. Finally, the fermentation broth obtained after fermentation can be adjusted to an acidic pH with inorganic acids, such as hydrochloric acid, sulfuric acid, or phosphoric acid, or organic acids, such as propionic acid, before or after complete or partial removal of the biomass to improve the handling properties of the final product (see, for example, GB 1,439,728 or EP 1331220). It is also possible to acidify the fermentation broth containing all the biomass. Finally, stabilization of the broth can also be performed by adding sodium bisulfite (NaHCO3, GB 1,439,728) or another salt, such as an ammonium, alkali metal, or alkaline earth metal salt of sulfite.

[0172] During biomass removal, any organic or inorganic solids present in the spent medium may be partially or completely removed. Dissolved organic by-products in the spent medium and dissolved constituents of the unconsumed fermentation medium (starting materials) may remain in the product at least partially (greater than 0%), sometimes as much as at least 25%, sometimes as much as at least 50%, and sometimes as much as at least 75%. Where appropriate, they may also remain in the product entirely (100%), or substantially entirely, meaning more than 95%, or more than 98%, or more than 99%.

[0173] Subsequently, water can be removed from the spent medium or the spent medium can be thickened or concentrated by known methods, for example by using a rotary evaporator, thin film evaporator, falling film evaporator, reverse osmosis or nanofiltration. This concentrated spent medium can then be post-processed by methods such as freeze-drying, spray-drying, spray-granulation or other processes such as in a circulating fluidized bed as described in PCT / EP2004 / 006655 to form a fluid product, in particular a fine powder or preferably coarse granules.

[0174] References The following publications are incorporated herein by this reference. These publications are referred to herein by the numbers indicated below. The inclusion of any publication in this publication list should not be construed as an admission that any publication mentioned herein is prior art. ·JAMA.2006;295(7):761-775 ·Comput Struct Biotechnol J,2012,3,1-11 ·Biotechnol.Bioeng.2004 88,909-915. ·Science 2002,298(5599),1790-3. ·Sonal R.Ayakar(2019),Biocatalysis and bioprocess engineering for terpenoid production,PhD thesis,University of British Columbia,Canada [Example]

[0175] Example 1: Aromatic prenyltransferase substrate transporter expression in E. coli Cloning: Two different transporters, PcaK and PP3656, were amplified by PCR from Pseudomonas putida KT2440 and cloned into a plasmid under the pTrc promoter. The plasmid was then transformed into BL21 DE3 for expression and used to transport aromatic compounds into BL21 DE3.

[0176] Preparation of seed culture Single colonies were picked from agar plates previously streaked with glycerol stocks (BL21 DE3 cells and BL21 DE3 cells containing plasmids pTrc-PcaK or pTrc-PP3656) and grown at 37°C (overnight, BL21 DE3 containing plasmids) [typically 16 hours] in LB medium (5 ml) containing 100 μg / ml carbenicillin.

[0177] Inoculation, induction and expression The overnight seed culture was inoculated into 5 ml of fresh LB medium at an OD of 0.1 and grown at 37°C until the OD reached 0.6 (typically, this takes 2.5–3 hours). For the BL21 DE3 strain containing the plasmid, the cell culture was induced with 100 μM IPTG. Both cell lines were then fed with 0.1 mM olivetrate and grown at 30°C and / or 22°C for 6, 24, and 48 hours.

[0178] culture: After this time (typically 14-16 hours), cells were harvested by centrifugation of the overnight culture at 3500 rpm for 20 minutes. The cell pellet was either lysed or stored at -80°C overnight. The supernatant was stored at -20°C for HPLC analysis (Supernatant 1).

[0179] Cell lysis: Cells were lysed by resuspending the entire pellet from a 5 mL culture in 300 μl of lysis buffer (lysis buffer composition: 50 mM Tris pH 8, 10% glycerol, 0.1% Triton X 100, 100 μg / mL lysozyme, 1 mM PMSF, DNase 3U, 2 mM MgCl2) and subsequently sonicating the cell pellet using a probe sonicator. The cell pellet resuspended in lysis buffer was kept on ice throughout the lysis, and sonication was performed for 10 cycles (15-second pulses followed by a 30-second pause on ice). After lysis, the crude cell lysate supernatant was collected by centrifugation at 14,000 rpm for 20 minutes at 4°C. The supernatant was used for HPLC analysis or stored at -80°C (supernatant 2).

[0180] HPLC analysis: Supernatant 1 was filtered through a 0.1 μm filter and 300 μL of the filtrate was used for HPLC analysis. Supernatant 2 was centrifuged at 14,000 rpm for 10 minutes and 300 μL of the upper clear supernatant was used for HPLC analysis. HPLC analysis was performed on a Perkin Elmer HPLC equipped with a Flexar PDA plus multi-wavelength detector and Chromera software. The HPLC analysis conditions were as follows: HPLC column: LUNA OMEGA 3μm Polar C18 column (150×4.6mm) Mobile phase: 75% ACN, 25% water, 0.1% formic acid ·Flow rate: 1ml / min Detection wavelength: 230 and 270 nm Oven temperature: 25℃ Injection volume: 10 μL ·Analysis time: 18 minutes

[0181] The results are shown in Figures 5 to 7.

[0182] Example 2: Aromatic prenyltransferase substrate transporter expression and cannabinoid production in E. coli Preparation of seed culture The following experimental host cells were tested: (1) E. coli transformed with a plasmid encoding the arabinose-inducible transporter pcaK or pp3656, and (2) E. coli transformed with a plasmid encoding the arabinose-inducible transporter pcaK or pp3656 and the B5 plasmid encoding the ispDF1 enzyme, GPPS, and an optimized mutant NphB (see Valliere et al.).

[0183] The seed culture of (1) from the glycerol stock is inoculated into 5 mL of LB containing 34 μg / mL chloramphenicol and incubated overnight at 30° C. The seed culture of (2) from the glycerol stock is inoculated into 5 mL of LB containing 34 μg / mL chloramphenicol and 50 μg / mL kanamycin and incubated overnight at 30° C.

[0184] Inoculation, induction and expression: The induction culture (1) from the seed culture was inoculated into TB medium containing 0.1 mM OA in a total culture volume of 5 mL and grown at 30°C until the OD reached 0.8. The culture was induced by adding arabinose and magnesium to a final concentration of 5 mM arabinose and 5 mM MgCl. During induction, the culture was incubated at 30°C. Samples of the induction culture were taken at 24 and 48 hours after the start of induction.

[0185] The induction culture (2) from the seed culture was inoculated into TB medium containing 0.5 mM OA and 5 mM MgCl in a total culture volume of 5 mL and grown at 30°C until an OD600 of 0.8 was reached. The culture was induced by adding arabinose to a final concentration of 5 mM and IPTG to a final concentration of 100 μM. The culture was incubated at 30°C during induction. Samples of the induction culture were taken at 24 and 48 hours after the start of induction.

[0186] Extraction of OA or CBGA: The culture was first centrifuged at 3000 rpm for 10 minutes to separate the pellet and culture medium supernatant fractions. The pellet was also washed twice with PBS. The cell pellet was lysed in B-PER complete reagent according to the manufacturer's protocol. Briefly, the pellet was resuspended in B-PER, incubated at 25°C for 20 minutes, and the insoluble material was spun down by centrifugation at 14000 rpm for 20 minutes. The soluble material was stored as cell lysate. A sample of the cell lysate was analyzed by SDS-PAGE analysis. See Figure 4.

[0187] To extract OA from the cell lysate, ethyl acetate was added to the soluble lysate fraction in a 1:1 volume ratio and mixed vigorously. The organic and aqueous fractions were separated by centrifugation at 14,000 rpm for 20 minutes. The organic phase was evaporated using a speed vacuum and resuspended in the HPLC mobile phase (75% ACN, 25% water, 0.1% formic acid) for analysis. The analytical results are shown in Figure 8.

[0188] To extract CBGA from the cell lysate, ethyl acetate was added to the culture medium supernatant in a 1:1 volume ratio and mixed vigorously. The organic and aqueous fractions were separated by centrifugation at 14,000 rpm for 20 minutes. The organic phase was evaporated using a speed vacuum and resuspended in HPLC mobile phase (75% ACN, 25% water, 0.1% formic acid) for analysis. The analytical results are shown in Figure 9.

[0189] Conclusion: Host cells expressing a heterologous aromatic prenyltransferase and a transporter capable of transporting the aromatic prenyltransferase substrate (e.g., olivetolate) into the cell exhibit increased production of one or more products of the aromatic prenyltransferase when cultured in a medium containing exogenously applied aromatic prenyltransferase substrate (e.g., olivetolate). See Figures 1-4 and 8-9.

[0190] Example 3: ispDE Expression and Analysis introduction It has been reported that very low flux through the MEP pathway in E. coli due to disruption of pathway genes is lethal in E. coli. 63,64 The pathway downstream of the Dxs catalytic step can be complemented by heterologous expression of the rate-limiting enzymes of the MVA pathway. 65 Dxs deficiency cannot be complemented by the MVA pathway due to its role in the biosynthesis of vitamins B6 and B1. 30 In contrast, IPP and DMAPP bind to t-RNA 66 and quinone 67 It is essential for the prenylation of

[0191] As described herein, MEP operates at a higher theoretical yield and is thermodynamically more favorable than the MVA route. 23 Experimentally observed MEP pathway yields are far from the theoretical maximum. The MEP pathway can be used to generate the most robust heterologous backbone for isoprenoid biosynthesis upon optimization.

[0192] Improving precursor supply for the MEP pathway Glycolysis and pyruvate are metabolic products from the glycolytic pathway involved in central carbon metabolism. Efforts to improve flux through glycolysis have been limited to attempts to increase the rate of sugar uptake. 68-70 Because glucose transporters were made more active, various steps in the glycolytic pathway lost their metabolic control. 71 The thermodynamics of the conversion of fructose-1,6-diphosphate to DHAP and GAP pushes the equilibrium toward the substrate. 72 Isomerization of DHAP and GAP favors DHAP. Some successful efforts have been directed toward the pentose phosphate pathway and the ED pathway for isopentenol production. 73 The partitioning of GAP and pyruvate plays a role in directing flux to the MEP pathway, and redirecting flux from GAP to pyruvate leads to improved downstream lycopene production. 74 The same study also reported that feeding GAP and pyruvate did not substantially change flux.

[0193] MEP Route Optimization Improvements in genome sequencing, genome mining, proteomics, metabolomics and bioinformatics tools have provided the field of metabolic engineering to find wider applications.

[0194] A well-studied strategy is optimization using metabolic engineering tools. Heterologous overexpression of the constricted portion of the homologous MEP pathway has been shown to significantly enhance the synthesis of terminal isoprenoid products. Overexpression of four genes—dxs, ispD, ispF, and idi—has been shown to improve taxol yield in E. coli. 24 In contrast, overexpression of dxs, ispD, ispF, and ispH increased lycopene yield by 15-fold in Bacillus subtilis. 75 .

[0195] MEP flux can be upregulated by expression of more active heterologous MEP pathway enzymes. This requires replacement of a single enzyme or the entire pathway framework. Expression of Dxs from Arabidopsis thaliana in transgenic Lavandula latifolia resulted in a five-fold higher total terpenoid yield. 76 .

[0196] Genes involved in the MEP pathway are controlled by constitutive promoters. In Corynebacterium glutamicum, the strong promoter P tuf Chromosomal exchange of the dxs promoter with β-lactamase resulted in a 60% increase in Dxs activity and a doubling of lycopene production. 47 .

[0197] Flux limitations are due to one or more of these factors: low activity, low stability, low expression level, low solubility, feedback regulation, or toxicity. Strategies to modify these enzymes at the genetic level by mutation have been attempted. In E. coli, directed co-evolution of Dxs, Dxr, and Idi resulted in a 60% improvement.77 .

[0198] Dxs, IspG, IspH, and IDI suffer from low solubility and inactive inclusion bodies upon overexpression. Improving their solubility would lead to enhanced activity. Lowering the incubation temperature, co-expression with chaperone proteins, and protein mutagenesis improve the solubility of otherwise insoluble proteins. Another strategy, supplementing the growth medium with betaine and sorbitol, increased the solubility of Dxs by 60%, which also led to an overall improvement in MEP pathway flux. 78 .

[0199] The occurrence of fused IspDF enzymes is common in α- and ε-proteobacterial genomes, but not in β- and γ-proteobacterial genomes. 79 Campylobacter jejuni 79 , Mesorhizobium loti 80 , and Agrobacterium tumefaciens 81 IspDF has been isolated from and studied in detail.

[0200] The bifunctional gene was first isolated from Campylobacter jejuni 79 The product (cjIspDF, a 42 kDa polypeptide) was synthesized by two reactions catalyzed by IspD and IspF, respectively, at 3.9 μmol mg -1 .min -1 and 0.8 μmol.mg -1 .min -1 cjIspDF showed higher similarity to E. coli IspF (approximately 48%) than to ispD (approximately 25%). 13 C-labeled MEP was employed to react in vitro with purified His-tagged proteins from recombinant E. coli to generate CDP-ME, which reacts with Zn as a cofactor. +2 Addition of ions resulted in the highest rate (18.5 μmol mg -1 .min -1) and the Km values ​​of CTP and MEP at pH 5 were 3 μM and 20 μM, respectively. The presence of ATP significantly reduced the Ca +2 The reaction kinetics did not change until IspE was added when CDP-MEP was used as a cofactor, resulting in the formation of MEcPP at the highest activity, with a Km value of 19 μM for CDP-MEP. In cjIspDF, the estimated shortest distance between the two catalytic centers of the IspD and IspF subunits is approximately 38 Å. cjIspDF has been reported to exist as a trimer, hexamer, and dodecamer when analyzed by size exclusion chromatography. 79 , whereas the crystal structure is a hexamer 62 It also shows that each domain contains two distinct domains connected by a linker sequence. The hexameric assembly contains two trimers of IspD domain dimers and two trimers of IspF domain trimers. In this hexameric complex, one IspF domain of the corresponding dimeric IspD domain associates to form a trimer. This means that individual domains of the same bifunctional polypeptide do not associate.

[0201] Another well-studied bifunctional IspDF from Mesorhizobium loti (mlIspDF) was expressed in E. coli and found to also exhibit the catalytic activities of both IspD and IspF. 80 The IspD subunit shared 46% similarity with E. coli IspD, whereas the IspF subunit shared 44% similarity with E. coli IspF. Size-exclusion chromatography of protein samples revealed the presence of monomeric units and dimeric complexes of mlIspDF. No higher molecular complexes were observed.

[0202] Three combinations of monomeric E. coli enzymes were analyzed by sedimentation velocity: (a) IspD and IspE; (b) IspE and IspF; and (c) IspD, IspE, and IspF. These studies revealed the assembly of three IspD dimers, three IspE dimers, and two IspF trimers. 62 The same study also revealed that domains IspD and IspF from IspDF associate with IspE to form a large complex. 62 This suggests that cjIspDF and atIspDF 81 It has been reported in both IspDF and IspD (IspDF from Agrobacterium tumefaciens). A trimer of IspD dimers and IspE dimers forms a complex with a dimer of IspF trimer to form an assembly of 18 catalytic centers. atIspDF was also detected to associate at a higher molecular weight ratio. In the case of cjIspDF, the distance between the two catalytic centers of the same multimer is 35 Å for the IspD subunit and 30 Å for the IspF subunit, which is shorter than the distance between the two catalytic centers of cjIspDF.

[0203] On the other hand, similar studies were performed on IspDF and IspE isolated from Agrobacterium tumefaciens (atIspDF and atIspE, respectively). 80Based on sedimentation velocity experiments, these enzymes were not found to associate. Further validation was performed by adding an inactive form of atIspE with an A152A point mutation in vitro. The inactive IspE did not alter the reaction pathway of MEP to MEcPP conversion by the atIspDF and atIspE cascade. If the enzymes associated to facilitate substrate channeling, the mutant IspE should have interacted with the complex and reduced the overall rate of the reaction. Another example of a fusion in which the active site does not channel the substrate is the GlmU enzyme from E. coli, involved in peptidoglycan biosynthesis, which is a bifunctional enzyme catalyzing successive steps in the pathway; however, intermediates are released from the first active site, accumulate in the environment, and are acted upon by the second functional group. 82 .

[0204] Natural occurrence of fused enzymes catalyzing non-sequential steps in biosynthetic pathways is rare. 21 Gram-positive bacteria such as Enterococcus faecalis and Enterococcus faecium are involved in the MVA pathway and encode a bifunctional enzyme, MvaE, that possesses both 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities, separated by a single step catalyzed by HMG-CoA synthase. 83,84 However, no associated complexes have been reported. A second example is involved in the carotenoid biosynthesis pathway. The carRA gene, which encodes a fusion for phytoene synthase and lycopene cyclase, was identified in the fungi Phycomyces blakesleeanus and Mucor circinelloides. 85,86Phytoene synthase is a prenyltransferase that catalyzes the synthesis of phytoene (GGPP) by the condensation of two GPP molecules. Phytoene is then converted to lycopene by a dehydrogenase encoded by CarB. β-Carotene is then synthesized by cyclization catalyzed by lycopene cyclase. While reports acknowledge the existence of these fusion anomalies, they do not justify the cause or demonstrate any utility of these fusions.

[0205] The occurrence of enzyme fusions at the gene level is common. The fatty acid and polyketide synthesis pathways involve bifunctional enzymes, all of which catalyze successive steps in the pathway. The reason for the existence of fusions such as IspDF, MvaE, and CraAR remains unclear. However, some researchers have argued for their relevance at the metabolic control level.

[0206] There is a gap between the theoretical maximum and experimentally achievable yields of the MEP pathway. Many efforts have been made in the fields of genome, protein, and metabolic engineering to bridge this gap. Strategies involving replacing the constriction step with a homologous enzyme with greater activity and / or stability do not appear to be widely adopted. Bifunctional enzymes reported to be involved in the pathway are promising targets. There have been no reports on the effect of these bifunctional IspDFs on MEP flux in vivo. Efforts have been directed toward studying purified proteins for their in vitro activity.

[0207] In this study, we performed a metagenomic screen to identify fusions of enzymes in the MEP pathway with the aim of enhancing substrate channeling. All fusions discovered were with IspD and IspF. These enzymes have been reported to catalyze non-sequential steps in the MEP pathway. We performed an in-depth study of the linker properties and their influence on MEP pathway flux. The linker sequence connecting the two domains in a bifunctional enzyme may alter enzyme activity. 87,88The flexibility and rigidity of the linker play a role in maintaining the independence of domain motion. We non-naturally fused IspE to each of IspD and IspF to mimic natural fusions. Such robust, high-yielding MEP pathway backbone strains can thus be utilized to produce isoprenoids and mine new compounds.

[0208] Synthetic fusion proteins with more than one catalytic activity are designed to either broaden the catalytic range of a protein or improve its catalytic efficiency. Expressing a single fusion protein also significantly reduces production costs, leading to greater industrial applicability. 89 Chemical catalysis has widely embraced the strategy of multifunctional catalysts adapted to catalyze more than one type of reaction and has gained popularity in industry. 90,91 .

[0209] There are two main approaches to generating non-natural fusions. 92 The first is at the genetic level, by replacing the transcription stop codon of the first gene and the transcription start codon of the second gene with nucleotide sequences that will form a peptide bond during translation. The second is by introducing a tag into the protein that triggers an association reaction that forms a peptide bond at the post-translational stage.

[0210] The conversion of 2-amino-1,2,3-butanetriol to L-erythrulose was catalyzed by the novel enzyme ω-aminotransferase, using serine as the amine donor. This reaction produced hydroxypyruvate as a by-product, which was transferred back to the substrate and regenerated the system as the amine donor by the action of the transketolase enzyme for the conversion of glycoaldehyde to L-erythrulose. Fusion of the aminotransferase and transketolase created an efficient closed-loop system. 93Another study combined four heterologously expressed enzymes to create a multienzyme reaction cascade in E. coli for the conversion of ethylbenzene to optically pure (R)-1-phenylethanamine, eliminating the need for additional cofactors. 94 .

[0211] There have been no reports of non-natural MEP pathway enzyme fusions. The absence or presence of fusions that serve to colocalize active sites and thus channel substrates for efficient conversion is a much-debated topic in the art. Moreover, fusions of IspD and IspF, which catalyze non-sequential steps in the pathway, have appeared, but fusions of IspE have never been reported.

[0212] As part of a long-term soil productivity (LTSP) study, soil samples were collected at the Skulow Lake site (SBS-3 WL), located at coordinates 52°20′N, 121°55′W. 95 High molecular weight genomic DNA was extracted and purified to generate large insert fosmid libraries. 96-98The NR fosmid library was generated from a Bt soil cross-section at a naturally disturbed reference site using the CopyControl™ Fosmid Library Production Kit (Epicentre) according to the manufacturer's protocol. Twenty 384-well plates from the library were subjected to Sanger end sequencing using pCC1-forward (5'-GGATGTGCTGCAAGGCGATTAAGTTGG) and pCC1-reverse (5'-CTCGTATGTTGTGTGGAATTGTGAGC) primers at the Michael Smith Genome Science Center (GSC), UBC, generating approximately 7680 pairs of end sequences. Based on phylogenetic genetic markers located at the fosmid ends and functional screening, approximately 530 fosmids were selected in silico and full-length sequenced on the Illumina HiSeq platform at GSC. Sequence analysis, including open reading frame (ORF) prediction and annotation, was performed using MetaPathways pipeline v2.5 with a set of reference databases (KEGG 2011-06-18, COG 2013-12-27, RefSeq 2014-01-18, and MetaCyc 2011-07-03). 95 Online HMMER Tool Version 2.17.3 99 A protein family search was performed using [translate] to confirm the functional annotations generated by the MetaPathways tool. The resulting MetaPathways output for fosmid ends and full-sequenced fosmids was searched to find the enzyme code (EC) of the bifunctional ispDF-encoding gene. A search of cognate nucleotide sequences was performed against the NCBI database using the online BLASTN search tool, and the resulting text file was uploaded to Megan 6.10.0 for taxonomic assignment using the LCA algorithm. 95Based on this analysis, the fosmid sequences of NR0032_N05, NR0032_O07, and NR0037_N05 were assigned to Acidobacteria and annotated to ispDF as ispDF1, ispDF2, and ispDF3, respectively.

[0213] All strains, plasmids, and genes used in this study are listed in Table 2.1, which contains the gene frameworks for the native monomeric and native fusion enzymes of the MEP pathway. The genes dxs, ispD, ispE, ispF, and idi were amplified by polymerase chain reaction from the E. coli strain K12 genome. The bifunctional genes ispDF1, ispDF2, and ispDF3, as well as ispS, were codon-optimized and synthesized by Genewiz Inc. pTrc-trGPPS(CO)-LS was a gift from Jay Keasling (Addgene plasmid 50603). 100 From this, the vector backbone was amplified to construct the plasmid variants. E. coli DH5α was used as the cloning host, and E. coli BL21(DE3) was used as the expression host.

[0214] [Table 4-1] [Table 4-2]

[0215] We constructed fusions with different linkers. The linkers used and their sequences are listed in Table 2.2. Linkers were added by PCR and generated by Gibson assembly.

[0216] [Table 5]

[0217] The CJ and XL linker sequences were generated by aligning the sequences of each fusion enzyme with E. coli IspD and IspF. Homology models for natural and unnatural chimeric fusions were constructed using the SWISS-MODEL server. The unnatural fusions are listed in Table 2.3. The IspD and IspF domains of IspDF1 were also expressed separately. This was achieved by adding a stop codon (TAA) to the end of the gene sequence for domain ispD, disrupting the gene sequence for the linker, and adding a RBS and start codon (ATG) in-frame with the gene sequence for IspF. This allowed for transcriptional separation of the two domains. The gene sequence encoding the IspD domain is designated ispD1, and the corresponding protein is designated IspD1. The gene sequence encoding the IspF domain is designated ispF1, and the corresponding protein is designated IspF1.

[0218] [Table 6]

[0219] The non-native fusion was cloned with other genes involved in the MEP pathway to assess its effect on pathway flux. These constructs and strains are listed in Table 2.4.

[0220] [Table 7]

[0221] Isoprene and lycopene starter cultures were grown overnight at 30°C in LB medium (Sigma-Aldrich) containing the appropriate constituent(s). The isoprene starter culture was then diluted to 15 mL with medium and analyzed at OD 600 The OD was adjusted to 0.2, induced with arabinose and / or IPTG, and grown in 25 mL sealed glass tubes for 24 hours at 30°C. Lycopene starter cultures were diluted to 5 mL with medium to reach an OD of 0.2. 600 The pH was adjusted to 0.2, induced with IPTG, and grown in a culture tube for 24 hours at 30°C in the dark.

[0222] Isoprene analysis was performed using a PerkinElmer Clarus 680 gas chromatograph and a PerkinElmer Clarus SQ 8T mass spectrometer (GC-MS). Because isoprene is a volatile monoterpene, sealed cultures were heated to 70°C for 1 min and vortexed for 5 s, followed by a 200 μL headspace sample taken using a gastight syringe. A standard curve for isoprene was generated in a similar manner for quantification. An HP-5MS capillary column (25 m long, 0.2 mm inner diameter, 0.33 μm film thickness; Agilent Technologies) was used with helium (1 mL / min) as the carrier gas. The oven temperature program was 35°C for 3 min, ramped to 200°C at 25°C / min, and held for 1 min. The injector was maintained at 60°C, and a 20:1 split ratio was maintained. Mass spectrometry acquisition was performed in SIR mode for ions at m / z 68 and m / z 67.

[0223] Lycopene is an intracellular product. Lycopene was extracted from the pellet by centrifuging 2 mL of cell culture at 8000 rpm for 5 min and extracting it with 1 mL of acetone. Extraction was performed at 55°C with intermittent vortexing for 20 min under low light conditions. The acetone suspension was centrifuged and filtered prior to analysis. Samples were analyzed on a PerkinElmer Flexar system equipped with a Zorbax C-18 column (4.6 × 250 mm, Agilent Technologies) maintained at 30°C. Samples were run at a flow rate of 1 mL / min with a mobile phase consisting of 66% (v / v) methanol, 30% (v / v) tetrahydrofuran, and 4% (v / v) water. Lycopene detection was performed by tracking absorbance at 474 nm using a photodiode detector.

[0224] result Soil metagenomic sequences were screened for orthologs of MEP pathway enzymes with higher activity and stability. This led to the discovery of novel fusions of two enzymes in the pathway—IspD and IspF. They were isolated from fosmids NR0032_N05, NR0032_O07, and NR0037_N05, and the corresponding genes were annotated as ispDF1, ispDF2, and ispDF3, respectively. The translated polypeptides were annotated as IspDF1 (41.6 kDa), IspDF2 (42.1 kDa), and IspDF3 (40.2 kDa), respectively. These genes were tagged for affinity-based isolation and expressed in E. coli BL21(DE3) using 0.5 mM IPTG as the inducer. Although the desired bands were observed on SDS-PAGE gels, the expression levels of IspDF were low. Denaturation and analysis of the insoluble cell debris showed that all three fusions formed inclusion bodies.

[0225] The sequences of IspDF1, IspDF2, and IspDF3 were aligned with IspD, IspF, and cjIspDF from E. coli (Table 2.5). The enzymes found were more similar to the native monofunctional enzyme from E. coli. Greater differences were observed when aligned against cjIspDF. 79 Although most residue functions were conserved among all five (IspDFs), clusters of differences existed. The amino acid region from 220 to 250 residues was highly variable and involved in connecting both domains. Other clusters of differences were found in the IspD domain of the fusion. All three IspDFs discovered have novel sequences and have never been reported before.

[0226] [Table 8]

[0227] Each domain of the fusion enzyme was aligned to E. coli IspD and E. coli IspF (Table 2.6). The IspF domain of the fusion shared higher sequence similarity with E. coli IspF than the similarity between the IspD domain and E. coli IspD. This result is consistent with the reported similarity of cjIspDF to the native E. coli enzyme. 62 The IspF domain of cjIspDF shares 48% sequence similarity with E. coli IspD, whereas the IspD domain shares 25% similarity with E. coli IspD.

[0228] [Table 9]

[0229] When the domains of the fusions were aligned with the cjIspDF domain, similar trends were observed (Table 2.7).

[0230] [Table 10]

[0231] The enzymatic steps catalyzed by Dxs, IspD, IspF, and Idi are rate-limiting steps of the MEP pathway in E. coli 24 The same framework was reconstituted (pSASDFI) and protein expression was analyzed. Soluble protein samples were run on an SDS / PAGE gel and stained with Coomassie dye.

[0232] SASDFI was tested for activity toward isoprene and lycopene production by coexpressing the scaffold with the downstream pathway (pSAIspS and pAC-LYC, respectively). To provide an explanation for the influence of IspD and IspF on improving MEP pathway flux, a clone expressing Dxs and Idi (pSASI) was constructed.

[0233] SALyc and SAIso produced very low yields of the corresponding terpenoids (Figures 17(a)-(b)). These strains reflect the natural expression levels of the MEP pathway. Induction had no substantial effect on terpenoid production. IPTG induction of SAIso adversely affected cell growth, thus resulting in a higher normalized yield. Higher IPTG induction levels prevented lycopene production and adversely affected growth. Overexpression of Dxs and Idi (strains SALyc-SI and SAIso-SI) resulted in 22-fold and 12-fold more terpenoids, respectively. Further expression of IspD and IspF (strains SALyc-SDFI and SAIso-SDFI) further enhanced terpenoid production by 47-fold and 15-fold, respectively. Uninduced cultures of SALyc-SI and SALyc-SDFI still produced lycopene at higher yields than SALyc.

[0234] All three fusions exhibited different effects on isoprene and lycopene production (Figure 18(a)-(b)). SALyc-SDF1I and SAIso-SDF1I performed best. Lycopene and isoprene production was improved by 20% and 75%, respectively, in strain IspDF1. Titers were reduced in the IspDF2 and IspDF3 variants. The OD of the strains 600 were in a similar range. The IspDF1 mutants showed higher normalized titers, indicating improved catalytic throughput. SALyc-SDF1I was tested at IPTG induction concentrations of 75 μM and 100 μM, but titers decreased, with the maximum titer being obtained at an IPTG concentration of 50 μM.

[0235] To assess the impact of the sole contribution from IspDF, strains SAIso-DF1, SAIso-DF2, and SAIso-DF3 were tested for isoprene production, and strains SALyc-DF1, SALyc-DF2, and SALyc-DF3 were tested for lycopene production. All six strains produced their respective terpenoids at levels equivalent to those of SAIso and SALyc (data not shown). Induction had no effect on terpenoid titers.

[0236] Homology models for the fusions were generated by SWISS-MODEL using cjIspDF as a template (Figure 19(a)-(d)). All four fusions have conserved subunit structures. IspDF1 and IspDF3 align well with cjIspDF, while IspDF2 has a longer linker. The active sites of the subunits are located at the opposite ends. The putative linker sequences are EAIARGTGERAVGERAA for IspDF2 and ERLIGARNTAGAM for IspDF3. IspDF1 improved terpenoid titer and was therefore used for further testing.

[0237] IspE has since been reported to affect flux by associating with IspD and IspF. 62 Thus, the associated complex facilitates efficient transfer and conversion of metabolites from MEP to MecPP. We investigated this phenomenon for lycopene production by testing recombinant E. coli strains expressing five enzymes: Dxs, IspD, IspF (or IspDF), IspE, and Idi. Both SALyc-SDFEI and SALyc-SDF1EI had lower lycopene titers than SALyc-SDFI and SALyc-SDF1I, respectively (Figure 20). The percent flux loss upon IspE overexpression was more pronounced in the IspDF1 clone than in the monofunctional native enzyme clone. This effect was the sum of a lower rate of lycopene production and a slower cell growth rate. OD in the IspE clone was significantly lower than in the IspDF1 clone. 600 was significantly lower (20-60). SALyc-SDFEI cultures had higher growth variability, reflected by the wider error bars.

[0238] To evaluate the role of the linker in enhancing the flux of SALyc-SDF1I, we replaced the putative linker sequence with three different linkers. The first was the linker identified from cjIspDF. The second was a glycine and serine linker called "FL," which confers flexibility to the domain. The third was "RL," which forms an α-helix and restricts free movement, imparting conformational rigidity. The effect of the linkers was tested in strains with and without IspE overexpression. The non-natural linkers did not improve the overall lycopene titer (Figures 21(a)-(b)), but they affected cell viability and increased the OD of the culture. 600 The normalized titer was SALyc-SD RL F1I is the highest, followed by SALyc-SD CJ The clones with flexible linkers exhibited the lowest lycopene titers in both sets.

[0239] The linker in the above section had a positive effect on the normalized titer, meaning that the linker improved flux at the expense of cell growth. Therefore, the same linker was employed together with the native linker of IspDF1 to link IspD and IspF in E. coli. For the strain in Figure 22(b), a lower normalized titer corresponds to a higher OD 600 This suggests a channeling of global carbon flux to cell growth metabolism. In contrast, for the strain shown in Figure 22(a), the fusion had no substantial effect on cell growth and adversely affected lycopene production.

[0240] Because the strains showed mixed responses to the CJ, FL, and RL linkers, fusions of IspD and IspF with the putative linker IspDF1 were constructed. These fusions reduced MEP flux and further reduced lycopene production (Figure 23). This effect was observed in the SALyc-SD XL FI.SALyc-SD XL This was particularly evident in FEI.

[0241] The negative effect of the XL linker on pathway flux suggested the need to study the IspDF1 domains in isolation (Figure 24). Isolation of the domains as individual enzymes had a more pronounced effect on SALyc-SD1F1EI.

[0242] Non-native fusions of IspE and their effect on MEP pathway flux To assess the natural origin of derivatives of enzymes catalyzing non-sequential steps in the MEP pathway, we constructed non-native fusions of IspE. The fusions were constructed using flexible linkers. The linking strategy was kept similar to that of native IspDF. An IspDE fusion was constructed by linking the C-terminus of IspD to the N-terminus of IspE. Additionally, an IspEF fusion was constructed by linking the C-terminus of IspE to the N-terminus of IspF. Figure 25 shows that the IspDE fusion exhibited a 20% improvement in lycopene production compared to SALyc-SDFI and a 2.3-fold improvement compared to SALyc-SDFEI. In contrast, the IspEF fusion significantly reduced lycopene production.

[0243] Figure 26 summarizes the results obtained so far. It is a comparative graph of the different constructs according to the highest titer and normalized titer values. Blank spaces are represented by "-".

[0244] Consideration The lycopene production framework is under the control of the endogenous promoter, and the MEP pathway framework is under the control of the trc promoter, which is reported to be leaky. 105-107 For these reasons, the lycopene cultures produced more lycopene than the base strain SALyc in the absence of induction. The higher normalized titers for both lycopene and isoprene fermentation indicate the abundance of C5 precursor metabolites—IPP and DMAPP—that shuttle through the respective downstream terpene synthesis pathways.

[0245] To study the role of the fusion and linker, it was necessary to construct a scaffold overexpressing Dxs, IspD, IspF, and Idi, which have been reported to increase taxol yield. 24 This strain containing the plasmid pSASDFI served as a basis for comparison in this study. Several reports have highlighted only the overexpression of Dxs and Idi for improving MEP pathway flux. 108,109 The results of this study (Figure 17) showed that further overexpression of IspD and IspF improved the titer for lycopene by 80%, but only 35% for isoprene. The microaerobic environment during isoprene cultivation may be a contributing factor to the difference in titers, as it is a highly oxygen-limited environment. The lycopene titers obtained in SALyc-SDFI are comparable to those reported in the literature. 110,111 Overall, the pSADFI backbone increased lycopene production by 47-fold and isoprene titer by 15-fold compared to the pSALyc and pSAIso strains, indicating that this strategy is effective in removing the constricted portion of the MEP pathway.

[0246] Dxs is the gatekeeper gene of the MEP pathway, and Idi catalyzes the final step, maintaining the balance of IPP and DMAPP concentrations required for downstream terpenoid biosynthesis. Therefore, overexpression of only IspD, IspF, and IspDF in the framework did not affect terpenoid titer. Terpenoid production by SAIso-DF1, SAIso-DF2, SAIso-DF3, SALyc-DF1, SALyc-DF2, and SALyc-DF3 was not significantly different from that of strains without MEP pathway overexpression (data not shown). Therefore, we decided to include the dxs and idi genes in future experiments to study the effects of intermediate steps.

[0247] The improvement in pathway flux upon expression of IspDF1 in the pSASDF1I operon may be the result of the role of the linker, which confers physical characteristics to the catalytic domain (e.g., flexibility or catalytic site proximity / substrate channeling) and / or confers greater stability and / or activity to IspDF1 than the native monofunctional enzyme. The IspF domain of IspDF1 shares the highest similarity with E. coli IspF compared to IspDF2 and IspDF3. The magnitude of the effect of IspDF1 overexpression in the lycopene strain differed from that in the isoprene strain. Because IspE catalyzes the step between IspD and IspF, further investigations were conducted to evaluate the role of IspE in the catalytic cascade. The step catalyzed by IspE has not been reported to be in the constricted portion of the pathway, and its overexpression imposed metabolic stress and reduced lycopene titer. SALyc-SDF1EI was stress-dependent despite expressing only four recombinant proteins compared to five in SALyc-SDFEI. This result highlights the presence of factor(s) other than metabolic stress.

[0248] The first factor investigated was the role of the linker. Flexible linkers were chosen to confer flexibility to the domains, as well as rigid linkers that form long helices that restrict domain movement. The linker from cjIspDF was also employed. For the non-native IspDF1 fusion, C flux was more diverted away from growth and towards the MEP pathway, resulting in higher normalized lycopene titers but less total lycopene production. SALyc-SD RL F1I was the best-performing strain, with a normalized titer 22% higher than SALyc-SDF1I and 33% higher than the base strain SALyc-SDFI, suggesting that the conformational rigidity of the fusion had a positive effect on catalytic activity. RL Homology modeling of F1 was inconclusive because the template was unable to accurately replicate the linker fold. On the other hand, when IspE was overexpressed (SALyc-SD RLF1EI strain), the production yield was reduced by 30% and the normalized titer was reduced by 80%. RL F1EI OD 600 SALyc-SD RL 50% higher than F1I. SALyc-SD RL Since F1EI expresses four heterologous enzymes, the effective dose of each enzyme is higher than that of SALyc-SD, which expresses three heterologous enzymes. RL Therefore, the MEP pathway flux was lower and the total C flux was diverted to biomass production.

[0249] To further deduce this effect, we constructed and compared non-native fusions of IspD and IspF in E. coli. In these examples (Figure 22), colocalization of the activities negatively affected lycopene production and normalized titers. However, in these examples, the overall OD of the Isp-overexpressing strains was significantly higher. 600 The activity of IspD in the mutants was 10-50% lower than that of their corresponding mutants without IspE overexpression, suggesting the involvement of IspE beyond its effects on cell health and growth. Furthermore, the putative IspDF1 linker behaved similarly to other non-native fusions when used to link E. coli IspD and IspF.

[0250] Chimeric enzymes with RL-type linkers have so far exhibited the highest flux through the MEP pathway at the expense of cell growth. This prompted a reevaluation of the effects of linkers and domain colocalization. The prevailing theory that enzyme fusion organization enhances the rate of reaction cascades is through lowering substrate diffusion limitations and substrate channeling. However, recent evidence indicates that the dynamics of fusions on metabolic cascades are more complex than previously assumed. 112 It is not simply the proximity of the enzymes that enhances the initial reaction rate; rather, colocalization increases the local concentration of the enzymes, thus increasing the opportunity for the diffusing substrate to interact with the active site cavity. 113 .

[0251] IspD1 and IspF1 retained their individual activities. The SALyc-SD1F1I strain produced 25% less lycopene, but the OD 600 The OD of SALyc-SD1F1EI was 30% lower. 600 , exhibiting an 82% increase in lycopene production. Both of these observations indicated a 3-fold improvement in normalized lycopene titer for SALyc-SD1F1EI over SALyc-SDF1EI. Although overall lycopene titer remained lower than SALyc-SDF1I due to the lower availability of enzyme copies due to the longer operon, the improvement in normalized titer reinforced the observation of higher stability and activity of IspDF1.

[0252] It is noteworthy that, in contrast to the many findings about IspDF, there is no literature on IspE fusions. The role of fusions of enzymes catalyzing non-sequential steps in the pathway, and the role of intermediate enzymes, is not only much debated but also rather unpredictable. The present inventors sought to elucidate this by constructing non-natural fusions of IspE. The performance of IspDE fusions was several-fold better than that of IspEF fusions. In fact, the IspDE fusions exhibited a 2.3-fold improvement in lycopene production and a 20% improvement in normalized titer compared to SALyc-SDFEI. SALyc-SD FL EFI OD 600 In contrast, the IspEF fusion reduced lycopene production by at least 65% and normalized titer by 85% compared to SALyc-SDFEI. FL The EFI strain was the best performing strain for lycopene production, and the MEP pathway flux was higher than that of SALyc-SD. RL This is due to the fact that the activity of the individual domains of IspDF1 is higher compared to the native IspD and IspF of E. coli. * * *

[0253] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," and "containing" should be read broadly and without limitation. In addition, the terms and phrases employed herein are used as terms of description rather than limitation, and the use of such terms and phrases is not intended to exclude any equivalents or any portions thereof hereafter shown and described, recognizing that various modifications are possible within the scope of the invention as claimed.

[0254] Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the invention disclosed herein may be available to those skilled in the art, and that such modifications and variations are deemed to be within the scope of the invention disclosed herein. The inventions are described broadly and generically herein. Each of the narrower species and subgroups falling within the scope of the generic disclosure also form part of these inventions. This includes the generic description of each invention with a postulate or negative limitation removing any subject matter from the generic concept, whether or not the removed material is specifically present therein.

[0255] Additionally, where features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also described by any individual member or subgroup of members of the Markush group. It is also to be understood that the above description is intended to be illustrative, and not limiting. Many embodiments will become apparent to those skilled in the art upon reading the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents entitled to such claims. The disclosures of all articles and references, including patent publications, are incorporated herein by reference.

Claims

1. A host cell comprising: a. An expression cassette comprising a promoter operably linked to a heterologous nucleic acid encoding a heterologous transporter or a functional fragment thereof. wherein the transporter is selected from the group consisting of major facilitator superfamily (MFS) aromatic acid antiporters and OprD family porins; b. An aromatic substrate selected from olivetolate, divalinolate (DVA), or a metabolite, derivative, or decarboxylate thereof. wherein the host cell is capable of enhancing import of the aromatic substrate into the host cell compared to a control host cell lacking the expression cassette of a).

2. 2. The host cell of claim 1, wherein the cell is a prokaryote, preferably the prokaryote is selected from the group consisting of prokaryotes of the genus Escherichia, Panteoa, Bacillus, Corynebacterium or Lactococcus.

3. 2. The host cell of claim 1, wherein the cell is Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, or L. lactis.

4. The host cell of claim 1 , wherein the cell is Escherichia coli (E. coli).

5. The host cell according to any one of claims 1 to 4, wherein the transporter is the MFS aromatic acid antiporter pcaK or a functional fragment thereof, or the transporter is the OprD family porin pp3656 or a functional fragment thereof.

6. 6. The host cell of any one of claims 1 to 5, wherein the transporter is at least 50% or 55% identical to, or identical to, 100 consecutive amino acids of the sequence shown in SEQ ID NO: 6, 7, 8 or 9.

7. 7. The host cell of any one of claims 1 to 6, wherein the host cell further comprises a heterologous aromatic prenyltransferase or a functional fragment thereof, wherein the aromatic prenyltransferase is functional and capable of prenylating the aromatic acid substrate.

8. 8. The host cell of claim 7, wherein the heterologous aromatic prenyltransferase is CBGAS or NphB, or a functional fragment thereof.

9. 9. The host cell of claim 8, wherein the heterologous aromatic prenyltransferase is a functional fragment of CBGAS.

10. 10. The host cell of claim 9, wherein the functional fragment of CBGAS is at least 50% or 55% identical to, or identical to, 100 consecutive amino acids of the sequence set forth in SEQ ID NO:

3.

11. 11. The host cell of any one of claims 1-10, wherein the host cell comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispDF, ispG, ispH, and idi, or variants thereof (e.g., variants that are at least 90%, 95%, or 99% identical to the respective native prokaryotic sequences).

12. The host cell of any one of claims 1 to 11, wherein the host cell comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding ispDF.

13. The host cell of any one of claims 1 to 12, wherein the host cell comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding an ispDE.

14. The host cell of any one of claims 1 to 13, wherein the host cell comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a GPP synthase.

15. 15. The host cell of any one of claims 1 to 14, wherein the host cell is in a culture medium comprising olivetolate, DVA, olivetol or divalinol, preferably wherein the host cell is in a culture medium comprising olivetolate and / or DVA.

16. 16. The host cell of any one of claims 1 to 15, wherein the host cell further comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a cannabinoid synthesis enzyme.

17. 17. The host cell of claim 16, wherein the cannabinoid synthase is CBDA synthase, CBCA synthase or THCA synthase, preferably the cannabinoid synthase is CBDA synthase.

18. 1. A method for enhancing the transport of olivetolate into a prokaryotic host cell, comprising: Culturing the host cell of any one of claims 1 to 17 in a culture medium containing an exogenous aromatic substrate of said transporter under conditions suitable for expression of said transporter. A method comprising:

19. 1. A method for prenylating olivetrate and / or DVA, comprising: Culturing the host cell of any one of claims 7 to 17 in a culture medium containing exogenous olivetolate and / or DVA under conditions suitable for expression of said transporter and said aromatic prenyltransferase, thereby prenylating said olivetolate and / or DVA. A method comprising:

20. 20. The method of claim 19, wherein the aromatic prenyltransferase is geranyldiphosphate:olivetrate geranyltransferase, and the method comprises producing cannabigerolic acid.

21. 21. The method of any one of claims 19 to 20, wherein the method increases the amount of prenylated olivetolate or DVA product produced compared to a control method carried out under conditions in which the transporter is not expressed or is expressed in a lower amount or activity.

22. 22. The method of any one of claims 19 to 21, wherein the method comprises harvesting and lysing the cultured cells, thereby producing a cell lysate.

23. 23. The method of claim 22, wherein the method comprises purifying the prenylated olivetolate or DVA product or a metabolite thereof from the cell lysate.

24. 22. The method of any one of claims 19 to 21, wherein the method comprises collecting spent culture medium produced by culturing the host cells.

25. 25. The method of claim 24, wherein the method comprises purifying the prenylated olivetolate or DVA product or a metabolite thereof from the spent culture medium.

26. 26. The method of claim 23 or claim 25, wherein the method comprises purifying CBGA or its decarboxylation products from the cell lysate or spent culture medium.

27. 26. The method of claim 21 or claim 25, wherein the method comprises purifying CBDA or a decarboxylation product thereof from the cell lysate or spent culture medium.

28. An expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDE enzyme or a functional fragment thereof.

29. An expression cassette comprising a heterologous promoter operably linked to a nucleic acid encoding a bifunctional ispDE, ispDF or ispEF enzyme or a functional fragment thereof, preferably wherein the nucleic acid encodes a bifunctional ispDE enzyme or a functional fragment thereof.

30. 29. The expression cassette of claim 28, wherein the bifunctional ispDE enzyme comprises a sequence that is at least 80% identical to the sequence set forth in SEQ ID NO:

10.

31. 31. The expression cassette of claims 28, 29, or 30, wherein the expression cassette comprises a promoter operably linked to a nucleic acid encoding at least one additional MEP pathway enzyme.

32. the at least one additional MEP pathway enzyme a. dxs, ispF and idi, or b. dxs, ispDF and idi 31. The expression cassette of claim 30, comprising:

33. A host cell comprising the expression cassette of any one of claims 28 to 32.

34. 34. The host cell of claim 33, wherein the host cell further comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a terpenoid synthase.

35. 35. The host cell of claim 33 or claim 34, wherein the host cell further comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a cannabinoid synthesis enzyme.

36. The host cell of any one of claims 33 to 35, wherein the host cell further comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding an aromatic prenyltransferase.

37. The host cell of any one of claims 33 to 36, wherein the host cell further comprises an expression cassette comprising a promoter operably linked to a nucleic acid encoding a GPP synthase.

38. The host cell a nucleic acid encoding ispDE; a nucleic acid encoding the GPP synthase; a nucleic acid encoding the aromatic prenyltransferase; and A nucleic acid encoding a cannabinoid synthase selected from the group consisting of CBDA synthase or a functional fragment thereof, CBCA synthase or a functional fragment thereof, and THCA synthase or a functional fragment thereof.

38. A host cell according to any one of claims 33 to 37, comprising:

39. The host cell of any one of claims 33 to 38, wherein the host cell further comprises olivetolate, olivetol, divalinolic acid, or divalinol.

40. 40. The host cell of claim 39, comprising olivetolate or divalinolic acid.

41. 41. The host cell of claim 40, comprising olivetrate.

42. 42. The host cell of any one of claims 33 to 41, wherein the host cell further comprises a heterologous expression cassette comprising a promoter operably linked to at least one prokaryotic chaperone.

43. The host cell a. a heterologous nucleic acid encoding ispDF, and optionally a heterologous nucleic acid encoding ispE; b. a heterologous nucleic acid encoding ispDE and, optionally, a heterologous nucleic acid encoding ispF, or c. A heterologous nucleic acid encoding ispEF, and optionally a heterologous nucleic acid encoding ispD. The host cell according to any one of claims 33 to 42, comprising:

44. 44. The host cell of any one of claims 33 to 43, wherein at least one, at least two, at least three, at least four or all of the heterologous expression cassettes are integrated into the genome of the host cell.

45. 44. The host cell of any one of claims 33 to 43, wherein at least one of the expression cassettes is not integrated into the genome of the host cell.

46. 46. ​​A method for producing a terpenoid, the method comprising culturing a host cell of any one of claims 33 to 45 under conditions suitable for expression of the ispDE bifunctional enzyme.

47. 47. The method of claim 46, wherein the method comprises culturing the host cell in a culture medium containing an exogenously supplied substrate for the heterologously expressed aromatic prenyltransferase.

48. 48. The method of claim 47, wherein the exogenously supplied substrate comprises olivetolate or divalinolic acid, preferably olivetolate.

Images