WO2024254488A1 - Superpositions améliorées pour la production de cannabinoïdes - Google Patents

Superpositions améliorées pour la production de cannabinoïdes Download PDF

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WO2024254488A1
WO2024254488A1 PCT/US2024/033062 US2024033062W WO2024254488A1 WO 2024254488 A1 WO2024254488 A1 WO 2024254488A1 US 2024033062 W US2024033062 W US 2024033062W WO 2024254488 A1 WO2024254488 A1 WO 2024254488A1
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oil
amino acid
acid sequence
cannabinoid
seq
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Joshua LENG
Si CHEN
Minkyung Kang
Felipe BARATHO BEATO
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Amyris Inc
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Amyris Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y121/00—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21)
    • C12Y121/03—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21) with oxygen as acceptor (1.21.3)
    • C12Y121/03008—Cannabidiolic acid synthase (1.21.3.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01102—Geranyl-pyrophosphate—olivetolic acid geranyltransferase (2.5.1.102)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01026—Olivetolic acid cyclase (4.4.1.26)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y602/00—Ligases forming carbon-sulfur bonds (6.2)
    • C12Y602/01—Acid-Thiol Ligases (6.2.1)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0492—Applications, solvents used

Definitions

  • Cannabinoids are chemical compounds such as cannabigerols (CBG), cannabi chromene (CBC), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabinodiol (CBDL), cannabicyclol (CBL), cannabielsoin (CBE), cannabitriol (CBT), and tetrahydrocannabinolic acid (THCa), as well as acid forms thereof, which are produced by the cannabis plant. Cannabinoids may be used to improve various aspects of human health.
  • a cannabinoid using oil overlays also referred to herein as overlays
  • a mixture of substantially equal amounts by weight percentage e.g., 40%:60% to 60%:40% of a branched, saturated C12-C20 alcohol (e.g., 2-hexyl- 1 -decanol, commercially available as JARCOLTM 1-16) and an oil (e.g., high-oleic sunflower oil (“HOSUN”)).
  • a branched, saturated C12-C20 alcohol e.g., 2-hexyl- 1 -decanol, commercially available as JARCOLTM 1-16
  • an oil e.g., high-oleic sunflower oil (“HOSUN”)
  • the oil overlay was found to increase the yield and productivity of cannabinoid production compared to other percent ratios of a branched, saturated C12-C20 alcohol and an oil, or oil alone, or a branched, saturated C12-C20 alcohol alone.
  • One aspect the invention provides for a method of producing a cannabinoid where the method includes the steps of i) culturing a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway in a culture medium and under conditions suitable for the population of host cells to produce the cannabinoid, thereby producing a fermentation composition, ii) contacting the fermentation composition with an oil overlay, wherein the oil overlay comprises a 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil; and iii) optionally, recovering one or more cannabinoids from the fermentation composition and/or the oil overlay.
  • the fermentation composition is in contact with the oil overlay while the population of host cells are undergoing fermentation.
  • the oil comprises a vegetable oil (e.g., avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, the like, and any combination thereof).
  • the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyl dodecanol, 2-hexyl-l -decanol, and 2-octyl decanol.
  • the host cell contains one or more heterologous nucleic acids that each, independently, encode (a) an acyl activating enzyme (AAE), and/or (b) a tetraketide synthase (TKS), and/or (c) a cannabigerolic acid synthase (CBGaS), and/or (d) a geranyl pyrophosphate (GPP) synthase.
  • the host cell contains heterologous nucleic acids that independently encode (a) an AAE, (b) a TKS, (c) a CBGaS, and (d) a GPP synthase.
  • the host cells comprise a heterologous nucleic acid that encodes olivetolic acid cyclase (OAC).
  • OAC olivetolic acid cyclase
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59.
  • the host cell contains a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • the host cell contains a heterologous nucleic acid encoding a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell contains heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell further contains one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl-CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the host cell is cultured with a precursor required to make the cannabinoid.
  • the precursor required to make the cannabinoid is hexanoate, hexanoic acid, or olivetolic acid.
  • the cannabinoid is cannabidiolic acid (CBDA), cannabidiol (CBD) or an acid form thereof, cannabigerolic acid (CBGA), cannabigerol (CBG) or an acid form thereof, tetrahydrocannabinol (THC) or an acid form thereof, or tetrahydrocannabinolic acid (THCa).
  • the host cell is a yeast cell or yeast strain. In the preferred embodiment, the yeast cell is 5. cerevisiae.
  • Another aspect the invention provides for a method of producing a cannabinoid where the method includes the steps of i) providing a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway in a culture medium and under conditions suitable for the population of host cells to produce the cannabinoid, thereby producing a fermentation composition, ii) contacting the fermentation composition with an oil overlay, wherein the oil overlay comprises a 40%: 60% to 60%:40% (e.g., 50%/50%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil; and iii) optionally, recovering one or more cannabinoids from the fermentation composition and/or the oil overlay.
  • the fermentation composition is in contact with the oil overlay while the population of host cells are undergoing fermentation.
  • the oil comprises a vegetable oil (e.g., avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, the like, and any combination thereof).
  • the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyldodecanol, 2-hexyl-l -decanol, and 2-octyl decanol.
  • the host cell contains one or more heterologous nucleic acids that each, independently, encode (a) an acyl activating enzyme (AAE), and/or (b) a tetraketide synthase (TKS), and/or (c) a cannabigerolic acid synthase (CBGaS), and/or (d) a geranyl pyrophosphate (GPP) synthase.
  • the host cell contains heterologous nucleic acids that independently encode (a) an AAE, (b) a TKS, (c) a CBGaS, and (d) a GPP synthase.
  • the host cells comprise a heterologous nucleic acid that encodes OAC.
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59.
  • the host cell contains a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • the host cell contains a heterologous nucleic acid encoding a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell contains heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell further contains one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl-CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the host cell is cultured with a precursor required to make the cannabinoid.
  • the precursor required to make the cannabinoid is hexanoate, hexanoic acid, or olivetolic acid.
  • the cannabinoid is CBDA, CBD or an acid form thereof, CBGA, CBG or an acid form thereof, THC or an acid form thereof, or THCa.
  • the host cell is a yeast cell or yeast strain. In the preferred embodiment, the yeast cell is 5. cerevisiae.
  • the invention provides for a method of producing a cannabinoid where the method includes the steps of i) providing a mixture comprising a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway, a culture medium, and an oil overlay, wherein the oil overlay comprises a 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil; and ii) recovering the cannabinoid from the mixture.
  • the oil comprises a vegetable oil (e.g., avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil jojoba oil, sunflower oil, the like, and any combination thereof).
  • the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyl dodecanol, 2-hexyl-l -decanol, and 2-octyl decanol.
  • the recovering of the one or more cannabinoids comprises separating the mixture into an aqueous liquid fraction, an oily liquid fraction, and a pellet by centrifugation.
  • the host cell contains one or more heterologous nucleic acids that each, independently, encode (a) an acyl activating enzyme (AAE), and/or (b) a tetraketide synthase (TKS), and/or (c) a cannabigerolic acid synthase (CBGaS), and/or (d) a geranyl pyrophosphate (GPP) synthase.
  • the host cell contains heterologous nucleic acids that independently encode (a) an AAE, (b) a TKS, (c) a CBGaS, and (d) a GPP synthase.
  • the host cells comprise a heterologous nucleic acid that encodes OAC.
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59.
  • the host cell contains a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • the host cell contains a heterologous nucleic acid encoding a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell contains heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell further contains one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl-CoA thiolase, an HMG-CoA synthase, an HMG- CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the host cell is cultured with a precursor required to make the cannabinoid.
  • the precursor required to make the cannabinoid is hexanoate, hexanoic acid, or olivetolic acid.
  • the cannabinoid is CBDA, CBD or an acid form thereof, CBGA, CBG or an acid form thereof, THC or an acid form thereof, or THCa.
  • the host cell is a yeast cell or yeast strain. In the preferred embodiment, the yeast cell is S. cerevisiae.
  • the invention provides for a mixture comprising: a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway; a culture medium; and an oil, wherein the oil comprises a 40%:60% to 60%:40% mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • the oil comprises a vegetable oil (e.g., avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, the like, and any combination thereof).
  • the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyldodecanol, 2-hexyl-l -decanol, and 2-octyl decanol.
  • cannabinoid refers to a chemical substance that binds or interacts with a cannabinoid receptor (for example, a human cannabinoid receptor) and includes, without limitation, chemical compounds such endocannabinoids, phytocannabinoids, and synthetic cannabinoids.
  • Synthetic compounds are chemicals made to mimic phytocannabinoids which are naturally found in the cannabis plant (e.g., Cannabis sativa), including, but not limited to, cannabigerols (CBG), cannabichromene (CBC), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabinodiol (CBDL), cannabicyclol (CBL), cannabielsoin (CBE), and cannabitriol (CBT).
  • CBD cannabigerols
  • CBC cannabichromene
  • CBD cannabidiol
  • THC tetrahydrocannabinol
  • CBN cannabinol
  • CBDL cannabinodiol
  • CBL cannabicyclol
  • CBE cannabielsoin
  • CBT cannabitriol
  • the term “capable of producing” refers to a host cell which is genetically modified to include the enzymes necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound.
  • a cell e.g., a yeast cell
  • “capable of producing” a cannabinoid is one that contains the enzymes necessary for production of the cannabinoid according to the cannabinoid biosynthetic pathway.
  • exogenous refers a substance or compound that originated outside an organism or cell. The exogenous substance or compound can retain its normal function or activity when introduced into an organism or host cell described herein.
  • the term “fermentation composition” refers to a composition which contains genetically modified host cells and products or metabolites produced by the genetically modified host cells.
  • An example of a fermentation composition is a whole cell broth, which may be the entire contents of a vessel, including cells, oil overlays or immiscible compounds, aqueous phase, and compounds produced from the genetically modified host cells.
  • the term “gene” refers to the segment of DNA involved in producing or encoding a polypeptide chain. It may include regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). Alternatively, the term “gene” can refer to the segment of DNA involved in producing or encoding a non-translated RNA, such as an rRNA, tRNA, gRNA, or micro RNA.
  • a first encoded enzyme uses a substrate to make a first product, which in turn is used as a substrate for a second encoded enzyme to make a second product.
  • the genetic pathway includes 3 or more members (e.g., 3, 4, 5, 6, 7, 8, 9, etc.), wherein the product of one encoded enzyme is the substrate for the next enzyme in the synthetic pathway.
  • the term “genetic switch” refers to one or more genetic elements that allow controlled expression of enzymes, e.g., enzymes that catalyze the reactions of cannabinoid biosynthesis pathways.
  • a genetic switch can include one or more promoters operably linked to one or more genes encoding a biosynthetic enzyme, or one or more promoters operably linked to a transcriptional regulator which regulates expression one or more biosynthetic enzymes.
  • genetically modified denotes a host cell that contains a heterologous nucleotide sequence.
  • the genetically modified host cells described herein typically do not exist in nature.
  • heterologous refers to what is not normally found in nature.
  • heterologous compound refers to the production of a compound by a cell that does not normally produce the compound, or to the production of a compound at a level not normally produced by the cell.
  • a cannabinoid can be a heterologous compound.
  • Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change.
  • a host cell includes cells into which a recombinant vector or a heterologous polynucleotide of the invention has been introduced, including by transformation, transfection, and the like.
  • medium refers to culture medium and/or fermentation medium.
  • modified refers to host cells or organisms that do not exist in nature, or express compounds, nucleic acids or proteins at levels that are not expressed by naturally occurring cells or organisms.
  • oil refers to a biologically compatible hydrophobic, lipophilic, carbon-containing substance including, but not limited to, geologically-derived crude oil, distillate fractions of geologically-derived crude oil, vegetable oil, algal oil, microbial lipids, or synthetic oils.
  • the oil is neither itself toxic to a biological molecule, a cell, a tissue, or a subject, nor does it degrade (if the oil degrades) at a rate that produces byproducts at toxic concentrations to a biological molecule, a cell, a tissue, or a subject.
  • Preferred examples of oils include, but are not limited to, avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, and sunflower oil (e.g., high-oleic sunflower oil).
  • HOSUN refers to high-oleic sunflower oil.
  • High-oleic sunflower oil is sunflower oil with a minimum of 70% oleic acid.
  • JARCOLTM 1-16 or “JARCOL” or “JARCOLTM” refers to 2-hexyl-l-decanol, which is a clear, high purity, colorless, fatty alcohol.
  • operably linked refers to a functional linkage between nucleic acid sequences such that the linked promoter and/or regulatory region functionally controls expression of the coding sequence.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid.
  • polynucleotide and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end.
  • a nucleic acid will generally contain phosphodiester bonds, although, in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones; and non-ribose backbones. Nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase.
  • Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus, the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. Nucleic acid sequences are presented in the 5’ to 3’ direction unless otherwise specified.
  • polypeptide As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • production generally refers to an amount of compound produced by a genetically modified host cell provided herein. In some embodiments, production is expressed as a yield of the compound by the host cell. In other embodiments, production is expressed as a productivity of the host cell in producing the compound.
  • productivity refers to production of a compound by a host cell, expressed as the amount of non-catabolic compound produced (by weight) per amount of fermentation broth in which the host cell is cultured (by volume) over time (per hour).
  • promoter refers to a synthetic or naturally derived nucleic acid that is capable of activating, increasing, or enhancing expression of a DNA coding sequence, or inactivating, decreasing, or inhibiting expression of a DNA coding sequence.
  • a promoter may contain one or more specific transcriptional regulatory sequences to further enhance or repress expression and/or to alter the spatial expression and/or temporal expression of the coding sequence.
  • a promoter may be positioned 5' (upstream) of the coding sequence under its control.
  • a promoter may also initiate transcription in the downstream (3’) direction, the upstream (5’) direction, or be designed to initiate transcription in both the downstream (3’) and upstream (5’) directions.
  • the distance between the promoter and a coding sequence to be expressed may be approximately the same as the distance between that promoter and the native nucleic acid sequence it controls. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
  • the term also includes a regulated promoter, which generally allows transcription of the nucleic acid sequence while in a permissive environment (e.g., microaerobic fermentation conditions, or the presence of maltose), but ceases transcription of the nucleic acid sequence while in a non-permissive environment (e.g., aerobic fermentation conditions, or in the absence of maltose). Promoters used herein can be constitutive, inducible, or repressible.
  • yield refers to production of a compound by a host cell, expressed as the amount of compound produced per amount of carbon source consumed by the host cell, by weight.
  • FIG. 1A is a plot of the feed rate (grams total reducing sugar/liter/hour, gTDS/L/h), oxygen uptake rate (OUR) (mmol/L/h), and oxygen tension (pO2) (%) for a fermentation using a control overlay (Hl 1845) of 100% HOSUN.
  • FIG. IB is a plot of the feed rate, OUR, and pO2 for a fermentation using an overlay (H12611) of 50%/50% JARCOLTM I-16/HOSUN by weight.
  • FIGS. 2A-2E are plots showing fermentation performance for a fermentation using a control overlay (Hl 1845) of 100% HOSUN or an overlay (H12611) of 50%/50% JARCOLTM I- 16/HOSUN by weight.
  • FIG. 2A is a plot of CBGa productivity over time.
  • FIG. 2B is a plot of CBGa titer over time.
  • FIG. 2C is a plot of packed cell volume (PCV) over time.
  • FIG. 2D is a plot of CBGa yield on total reducing sugar over time.
  • FIG. 2E is a plot of average OUR over time.
  • a cannabinoid may be produced from a fermentation composition produced by culturing host cells genetically modified to express one or more enzyme of a cannabinoid biosynthetic pathways in a culture medium.
  • the fermentation composition may be contacted with an oil overlay comprising a 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • an oil overlay comprising a 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • the disclosure provides methods for producing a cannabinoid from a fermentation composition.
  • the method uses a mixture of a branched, saturated C12-C20 alcohol and an oil.
  • the mixture is a 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of the branched, saturated C12-C20 alcohol and the oil.
  • the fermentation occurs in the presence of a 40%: 60% to 60%:40% (e.g., 40%:60% to 50%:50%, 45%:55% to 55%:45%, 50%:50% to 60%:40%, 40%/60%, 42%/58%, 44%/56%, 46%/54%, 48%/52%, 50%/50%, 52%/48%, 54%/46%, 56%/44%, 58%/42%, or 60%/40%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • the mixture is present in the fermentation composition at the beginning of the fermentation reaction and is present until the fermentation reaches completion.
  • the fermentation is allowed to reach completion prior to the addition of the mixture.
  • the mixture is added into the fermentation composition after the fermentation is completed, and the mixture is mixed with the fermentation composition for 1 min to 600 min (e.g., 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min,
  • the population of host cells, the fermentation composition, and the 40%:60% to 60%:40% (e.g., 50%/50%) mixture by weight of the branched, saturated C12-C20 alcohol and the oil are separated into an aqueous liquid fraction, an oily liquid fraction, and a pellet by way of centrifugation.
  • the oil fraction is recovered from the liquid fraction following centrifugation.
  • the oil fraction undergoes centrifugation more than one time (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times) to ensure separation from the aqueous liquid fraction.
  • the fermentation composition may be mixed for a time and at a temperature sufficient to allow for demulsification of the fermentation composition before the cannabinoid undergoes decarboxylation, and the cannabinoid may be recovered.
  • at least one demulsification step e.g., 2 steps, 3 steps, 4 steps, 5 steps, 6 steps, 7 steps, 8 steps, 9 steps, or 10 steps is performed between centrifugation steps.
  • Demulsification may, in some embodiments, be conducted using a demulsification aid, such as an enzymatic composition including a serine protease (e.g., TERGAZYME®).
  • a demulsification aid such as an enzymatic composition including a serine protease (e.g., TERGAZYME®).
  • the enzymatic composition includes between 0.003% and 20% serine protease by weight (e.g., between 0.003% and 15%, between 0.005% and 10%, between 0.007% and 7%, and between 0.01% and 5% serine protease by weight).
  • the enzymatic composition includes between 0.01% and 10% serine protease by weight (e.g., between 0.01% and 10%, between 0.02% and 9%, between 0.03% and 8%, between 0.04% and 7%, between 0.05% and 6%, between 0.06% and 5%, between 0.07% and 4%, between 0.08% and 3%, between 0.08% and 2%, between 0.09% and 1%, and between 0.1% and 1% serine protease by weight).
  • serine protease by weight e.g., between 0.01% and 10%, between 0.02% and 9%, between 0.03% and 8%, between 0.04% and 7%, between 0.05% and 6%, between 0.06% and 5%, between 0.07% and 4%, between 0.08% and 3%, between 0.08% and 2%, between 0.09% and 1%, and between 0.1% and 1% serine protease by weight).
  • the enzymatic composition includes between 0.01% and 5% by serine protease by weight (e.g., between 0.01% and 5%, between 0.05% and 4%, between 0.1% and 3%, between 0.5% and 2% serine protease by weight).
  • the serine protease is a subtilisin.
  • the subtilisin is from Bacillus licheniformis .
  • the subtilisin is subtilisin Carlsberg.
  • the serine protease is deactivated by exposure to 300 ppm hypochlorite at a temperature of 85 °F for less than one minute; 3.5 ppm hypochlorite at a temperature of 100 °F for 2 min; a pH below 4 for 30 min at a temperature of 140 °F; or by heating to a temperature of 175 °F for 10 min.
  • the enzymatic composition includes an alkylaryl sulfonate salt. In some embodiments, the alkylaryl sulfonate includes a linear alkylaryl sulfonate salt. In some embodiments, the enzymatic composition includes a phosphate salt. In some embodiments, the enzymatic composition includes a carbonate salt. In some embodiments, the salt is a sodium salt. [0045] In some embodiments, the enzymatic composition has a pH of between 8.5 and 11 (e g., between pH 8.7 and pH 10.5, between pH 9.0 and pH 10, and between pH 9.2 and pH 9.7) in a 1% (w/v) solution. In some embodiments, the enzymatic composition has a pH of about 9.5 in a 1% (w/v) solution.
  • the oil fraction contains the cannabinoid, and the oil fraction undergoes further purification using distillation (e.g., using an evaporator).
  • distillation is performed to evaporate the oil solvent to recover crystalized cannabinoid.
  • distillation is performed more than one time (e.g., 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times) to improve purity.
  • the recovered cannabinoid has a purity between 50% and 100% (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
  • the cannabinoid is recovered with one or more impurities.
  • the impurities are present in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% w/w.
  • the impurity is present in an amount of about 0.1% w/w.
  • the impurity is present in an amount of about 0.3% w/w.
  • the impurity is present in an amount of about 0.6% w/w.
  • the one or more impurities include one or more of cannabidivarinic acid, cannabidivarin, cannabidiolic acid, SCBGa, cannabigerol, tetrahydrocannabivarinic acid, cannabinol, cannabinolic acid, delta-9-tetrahydrocannabinol, delta-8-tetrahydrocannabinol, cannabicyclol, cannabichromene, tetrahydrocannabinolic acid, cannabichromenic acid, and cannabidiol.
  • the molar yield of the cannabinoid is between 60% and 100% (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%).
  • a host cell described herein includes one or more nucleic acids encoding one or more enzymes of a heterologous genetic pathway that produces a cannabinoid or a precursor of a cannabinoid.
  • the cannabinoid biosynthetic pathway may begin with hexanoic acid as the substrate for an acyl activating enzyme (AAE) to produce hexanoyl-CoA, which is used as the substrate of a tetraketide synthase (TKS) to produce tetraketide-CoA, which is used by an OAC to produce olivetolic acid, which is then used to produce a cannabigerolic acid by a geranyl pyrophosphate (GPP) synthase and a cannabigerolic acid synthase (CBGaS).
  • GEP geranyl pyrophosphate
  • CBGaS cannabigerolic acid synthase
  • the cannabinoid precursor that is produced is a substrate in the cannabinoid pathway (e.g., hexanoate or olivetolic acid).
  • the precursor is a substrate for an AAE, a TKS, an OAC, a CBGaS, or a GPP synthase.
  • the precursor, substrate, or intermediate in the cannabinoid pathway is hexanoate, olivetol, or olivetolic acid.
  • the precursor is hexanoate.
  • the host cell does not contain the precursor, substrate or intermediate in an amount sufficient to produce the cannabinoid or a precursor of the cannabinoid.
  • the host cell does not contain hexanoate at a level or in an amount sufficient to produce the cannabinoid in an amount over 10 mg/L.
  • the heterologous genetic pathway encodes at least one enzyme selected from the group consisting of an AAE, a TKS, an OAC, a CBGaS, or a GPP synthase.
  • the genetically modified host cell includes an AAE, TKS, OAC, CBGaS, and a GPP synthase.
  • the cannabinoid pathway is described in Keasling et al. (U.S. Patent No. 10,563,211), the disclosure of which is incorporated herein by reference.
  • Some embodiments concern a host cell that includes a heterologous AAE such that the host cell is capable of producing a cannabinoid.
  • the AAE may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have AAE activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid.
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-24).
  • the AAE has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1-24 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-24).
  • the AAE has the amino acid sequence of any one of SEQ ID NO: 1-24.
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-13 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-13).
  • the AAE has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1-13 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-13).
  • the AAE has the amino acid sequence of any one of SEQ ID NO: 1-13.
  • the host cell contains a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-5 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1 -5).
  • the AAE has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 1-5 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 1-5).
  • the AAE has the amino acid sequence of any one of SEQ ID NO: 1-5.
  • Some embodiments concern a host cell that includes a heterologous TKS such that the host cell is capable of producing a cannabinoid.
  • a TKS uses the hexanoyl-CoA precursor to generate tetraketide-CoA.
  • the TKS may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have TKS activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid precursor olivetolic acid.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 25-59).
  • the TKS has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 25-59 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 25-59).
  • the TKS has the amino acid sequence of any one of SEQ ID NO: 25-59.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-28 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 25-28).
  • a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-28 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 25-28).
  • the TKS has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 25-28 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 25-28). In some embodiments, the TKS has the amino acid sequence of any one of SEQ ID NO: 25-28.
  • the host cell contains a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 25 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25).
  • the TKS has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 25 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25).
  • the TKS has the amino acid sequence of SEQ ID NO: 25.
  • Some embodiments concern a host cell that includes a heterologous CBGaS such that the host cell is capable of producing a cannabinoid.
  • a CBGaS uses the olivetolic acid precursor and GPP precursor to generate cannabigerolic acid.
  • the CBGaS may be from Cannabis sativa or may be an enzyme from another plant or fungal source which has been shown to have CBGaS activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid cannabigerolic acid.
  • the host cell contains a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 60-64).
  • a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 60-64).
  • the CBGaS has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 60-64 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 60-64). In some embodiments, the CBGaS has the amino acid sequence of any one of SEQ ID NO: 60-64.
  • Some embodiments concern a host cell that includes a heterologous GPP synthase such that the host cell is capable of producing a cannabinoid.
  • a GPP synthase uses the product of the isoprenoid biosynthesis pathway precursor to generate cannabigerolic acid together with a prenyltransferase enzyme.
  • the GPP synthase may be from Cannabis sativa or may be an enzyme from another plant or bacterial source which has been shown to have GPP synthase activity in the cannabinoid biosynthetic pathway, resulting in the production of the cannabinoid cannabigerolic acid.
  • the host cell contains a heterologous nucleic acid that encodes a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 65-70).
  • a heterologous nucleic acid that encodes a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 65-70).
  • the GPP synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 65-70 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 65-70). In some embodiments, the GPP synthase has the amino acid sequence of any one of SEQ ID NO: 65-70.
  • the host cell contains a heterologous nucleic acid that encodes a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 65 (e g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65).
  • the GPP synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 65 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65).
  • the GPP synthase has the amino acid sequence of SEQ ID NO: 65.
  • the host cell may further express other heterologous enzymes in addition to the AAE, TKS, CBGaS, and/or GPP synthase.
  • the host cell may include an olivetolic acid cyclase (OAC) as part of the cannabinoid biosynthetic pathway.
  • OAC olivetolic acid cyclase
  • the OAC may have an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 71.
  • the OAC has an amino acid sequence of SEQ ID NO: 71.
  • the host cell may include a heterologous nucleic acid that encodes at least one enzyme from the mevalonate biosynthetic pathway.
  • Enzymes which make up the mevalonate biosynthetic pathway may include, but are not limited to, an acetyl-CoA thiolase, a HMG-CoA synthase, a HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the host cell includes a heterologous nucleic acid that encodes the acetyl-CoA thiolase, the HMG-CoA synthase, the HMG-CoA reductase, the mevalonate kinase, the phosphomevalonate kinase, the mevalonate pyrophosphate decarboxylase, and the IPP:DMAPP isomerase of the mevalonate biosynthesis pathway.
  • the host cell may express heterologous enzymes of the central carbon metabolism. Enzymes of the central carbon metabolism may include an acetyl-CoA synthase, an aldehyde dehydrogenase, and a pyruvate decarboxylase. In some embodiments, the host cell includes heterologous nucleic acids that independently encode an acetyl-CoA synthase, and/or an aldehyde dehydrogenase, and/or a pyruvate decarboxylase.
  • the acetyl-CoA synthase and the aldehyde dehydrogenase from Saccharomyces cerevisiae, and the pyruvate decarboxylase from Zymomonas mobilis has an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%>, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 72. In some embodiments, the acetyl-CoA synthase has the amino acid sequence of SEQ ID NO: 72.
  • the host cell expresses a heterologous acetyl-CoA synthase having an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94 >, 95%, 96%, 97%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 73.
  • the acetyl-CoA synthase has the amino acid sequence of SEQ ID NO: 73.
  • the aldehyde dehydrogenase has an amino acid sequence that is at least 90%> (e.g., at least 91%>, 92%, 93%, 94%o, 95%, 96%, 97%, 98%o, or 99%) identical to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the aldehyde dehydrogenase has the amino acid sequence of SEQ ID NO: 74.
  • the pyruvate dehydrogenase has an amino acid sequence that is at least 90%o (e.g., at least 91 %>, 92%, 93%>, 94%o, 95%o, 96%>, 97%, 98%>, or 99%o) identical to the amino acid sequence of SEQ ID NO: 75.
  • the pyruvate decarboxylase has an amino acid sequence of SEQ ID NO: 75.
  • polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding the protein components of the heterologous genetic pathway described herein.
  • Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called “codon optimization” or “controlling for species codon bias.”
  • Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.
  • Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively.
  • a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity.
  • the disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide.
  • the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.
  • homologs of enzymes useful for the compositions and methods provided herein are encompassed by the disclosure.
  • two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • a “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
  • R group side chain
  • a conservative amino acid substitution will not substantially change the functional properties of a protein.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (e.g., Pearson W. R., 1994, Methods in Mol Biol 25: 365-89).
  • Sequence homology for polypeptides is typically measured using sequence analysis software.
  • a typical algorithm used for comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences.
  • any of the genes encoding the foregoing enzymes may be optimized by genetic/protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in a host cell, for example, a yeast.
  • genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed in the host cell.
  • a variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorphs, Candida spp., Trichosporon spp., Yamadazyma spp., including Y.
  • Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp.
  • Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia coli, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., and Salmonella spp.
  • Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes.
  • analogous genes and/or analogous enzymes can be identified by functional analysis and will have functional similarities.
  • Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes.
  • techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of an ADA gene/enzyme or by degenerate PCR using degenerate primers designed to amplify a conserved region among ADA genes.
  • Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence.
  • analogous genes and/or analogous enzymes or proteins techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, JGI Phyzome vl2.1, BLAST, NCBI RefSeq, UniProt KB, or MetaCYC Protein annotations in the UniProt Knowledgebase may also be used to identify enzymes which have a similar function in addition to the National Center for Biotechnology Information RefSeq database.
  • the candidate gene or enzyme may be identified within the above-mentioned databases in accordance with the teachings herein.
  • host cells comprising at least one enzyme of the cannabinoid biosynthetic pathway.
  • the cannabinoid biosynthetic pathway contains a genetic regulatory element, such as a nucleic acid sequence, that is regulated by an exogenous agent.
  • the exogenous agent acts to regulate expression of the heterologous genetic pathway.
  • the exogenous agent can be a regulator of gene expression.
  • the exogenous agent can be used as a carbon source by the host cell.
  • the same exogenous agent can both regulate production of a cannabinoid and provide a carbon source for growth of the host cell.
  • the exogenous agent is galactose.
  • the exogenous agent is maltose.
  • the genetic regulatory element is a nucleic acid sequence, such as a promoter.
  • the genetic regulatory element is a galactose-responsive promoter.
  • galactose positively regulates expression of the cannabinoid biosynthetic pathway, thereby increasing production of the cannabinoid.
  • the galactose-responsive promoter is a GALI promoter.
  • the galactoseresponsive promoter is a GAL10 promoter.
  • the galactose-responsive promoter is a GAL2, GAL3, or GAL7 promoter.
  • heterologous genetic pathway contains the galactose-responsive regulatory elements described in Westfall et al.
  • the host cell lacks the GALI gene and is unable to metabolize galactose, but galactose can still induce galactose-regulated genes.
  • the galactose regulation system used to control expression of one or more enzymes of the cannabinoid biosynthetic pathway is re-configured such that it is no longer induced by the presence of galactose. Instead, the gene of interest will be expressed unless repressors, which may be maltose in some strains, are present in the medium.
  • the genetic regulatory element is a maltose-responsive promoter.
  • maltose negatively regulates expression of the cannabinoid biosynthetic pathway, thereby decreasing production of the cannabinoid.
  • the maltoseresponsive promoter is selected from the group consisting of pMALl, pMAL2, pMALl 1, pMAL12, pMAL31 and pMAL32.
  • the maltose genetic regulatory element can be designed to both activate expression of some genes and repress expression of others, depending on whether maltose is present or absent in the medium. Maltose regulation of gene expression and maltoseresponsive promoters are described in U.S.
  • Patent Publication 2016/0177341 which is hereby incorporated by reference. Genetic regulation of maltose metabolism is described in Novak et al., “Maltose Transport and Metabolism in S. cerevisiae,” Food Technol. Biotechnol. 42 (3) 213-218 (2004).
  • the heterologous genetic pathway is regulated by a combination of the maltose and galactose regulons.
  • the recombinant host cell does not contain, or expresses a very low level of (for example, an undetectable amount), a precursor (e.g., hexanoate) required to make the cannabinoid.
  • a precursor e.g., hexanoate
  • the precursor is a substrate of an enzyme in the cannabinoid biosynthetic pathway.
  • yeast strains useful in the present methods include yeasts that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc.) and belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea,
  • the strain is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (previously called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorphs (now known as Pichia angusta).
  • the host microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii , Candida krusei, Candida pseudotropicalis, or Candida utilis.
  • the strain is Saccharomyces cerevisiae.
  • the host is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CEN.PK, CEN.PK2, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME- 2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1.
  • the strain of Saccharomyces cerevisiae is CEN.PK.
  • the strain is a microbe that is suitable for industrial fermentation.
  • the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.
  • mixtures of the host cells described herein, a culture medium, and an oil overlay comprising a 40%:60% to 60%:40% (e.g., 40%:60% to 50%:50%, 45%:55% to 55%:45%, 50%:50% to 60%:40%, 40%/60%, 42%/58%, 44%/56%, 46%/54%, 48%/52%, 50%/50%, 52%/48%, 54%/46%, 56%/44%, 58%/42%, or 60%/40%) mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • oils that may be used in the oil overlay may include, but are not limited to, geologically-derived crude oil, distillate fractions of geologically-derived crude oil, vegetable oil, algal oil, microbial lipids, or synthetic oils, the like, and any combination thereof.
  • Preferred oils are vegetable oils, which may include, but are not limited to, avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, the like, and any combination thereof.
  • the sunflower oil may be HOSUN.
  • Examples of branched, saturated C12-C20 alcohols that may be used in the oil overlay may include, but are not limited to, 2-butyl octanol, octyldodecanol, 2 -hexyl- 1 -decanol, 2-octyl decanol, the like, and any combination thereof.
  • the culture medium contains an exogenous agent that decreases production of a cannabinoid.
  • the exogenous agent that decreases production of the cannabinoid is maltose.
  • the exogenous agent that decreases production of a cannabinoid is maltose.
  • the culture medium contains an exogenous agent described herein. In some embodiments, the culture medium contains an exogenous agent that increases production of the cannabinoid. In some embodiments, the exogenous agent that increases production of the cannabinoid is galactose. In some embodiments, the culture medium contains a precursor or substrate required to make the cannabinoid. In some embodiments, the precursor required to make the cannabinoid is hexanoate. In some embodiments, the precursor required to make the cannabinoid is olivetolic acid.
  • the culture medium contains an exogenous agent that increases production of the cannabinoid and a precursor or substrate required to make the cannabinoid.
  • the exogenous agent that increases production of the cannabinoid is galactose, and the precursor or substrate required to make the cannabinoid is hexanoate.
  • the culture medium contains a precursor required to make the cannabinoid.
  • the precursor is hexanoate.
  • the methods include transforming a host cell with the heterologous nucleic acid constructs described herein which encode the proteins expressed by a heterologous genetic pathway described herein.
  • Methods for transforming host cells are described in “Laboratory Methods in Enzymology: DNA,” Edited by Jon Lorsch, Volume 529, (2013); and US Patent No. 9,200,270 to Hsieh, Chung-Ming, et al., and references cited therein.
  • methods are provided for producing a cannabinoid are described herein.
  • the method decreases expression of the cannabinoid.
  • the method includes culturing a host cell comprising at least one enzyme of the cannabinoid biosynthetic pathway described herein in a medium comprising an exogenous agent, wherein the exogenous agent decreases the expression of the cannabinoid.
  • the exogenous agent is maltose.
  • the method results in less than 0.001 mg/L of cannabinoid or a precursor thereof.
  • the method is for decreasing expression of a cannabinoid or precursor thereof.
  • the method includes culturing a host cell comprising an AAE, and/or a TKS, and/or a CBGaS, and/or a GPP synthase described herein in a medium comprising an exogenous agent, wherein the exogenous agent decreases the expression of the cannabinoid.
  • the exogenous agent is maltose.
  • the method results in the production of less than 0.001 mg/L of a cannabinoid or a precursor thereof.
  • the method increases the expression of a cannabinoid.
  • the method includes culturing a host cell comprising an AAE, and/or a TKS, and/or a CBGaS, and/or a GPP synthase described herein in a medium comprising the exogenous agent, wherein the exogenous agent increases expression of the cannabinoid.
  • the exogenous agent is galactose.
  • the method further includes culturing the host cell with the precursor or substrate required to make the cannabinoid. [0095] In some embodiments, the method increases the expression of a cannabinoid or precursor thereof.
  • the method includes culturing a host cell comprising a heterologous cannabinoid pathway described herein in a medium comprising an exogenous agent, wherein the exogenous agent increases the expression of the cannabinoid or a precursor thereof.
  • the exogenous agent is galactose.
  • the method further includes culturing the host cell with a precursor or substrate required to make the cannabinoid or precursor thereof.
  • the precursor required to make the cannabinoid or precursor thereof is hexanoate.
  • the combination of the exogenous agent and the precursor or substrate required to make the cannabinoid or precursor thereof produces a higher yield of cannabinoid than the exogenous agent alone.
  • the cannabinoid or a precursor thereof is CBDA, CBD, CBGA, or CBG.
  • the methods of producing cannabinoids provided herein may be performed in a suitable culture medium in a suitable container, including, but not limited to, a cell culture plate, a flask, or a fermenter. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermenter may be used including a stirred tank fermenter, an airlift fermenter, a bubble fermenter, or any combination thereof.
  • strains can be grown in a fermenter as described in detail by Kosaric, et al, in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co. KDaA, Weinheim, Germany.
  • the culture medium is any culture medium in which a genetically modified microorganism capable of producing a heterologous product can subsist, i.e., maintain growth and viability.
  • the culture medium is an aqueous medium comprising assimilable carbon, nitrogen, and phosphate sources. Such a medium can also include appropriate salts, minerals, metals, and other nutrients.
  • the carbon source and each of the essential cell nutrients are added incrementally or continuously to the fermentation medium, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass.
  • Suitable conditions and suitable medium for culturing microorganisms are well known in the art.
  • the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic to select for microorganisms comprising the genetic modifications).
  • an inducer e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter
  • a repressor e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter
  • a selection agent e.g., an antibiotic
  • the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof.
  • suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof.
  • suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.
  • suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof.
  • suitable non-fermentable carbon sources include acetate and glycerol.
  • the concentration of a carbon source, such as glucose or sucrose, in the culture medium should promote cell growth, but not be so high as to repress growth of the microorganism used.
  • a carbon source such as glucose or sucrose
  • concentration of a carbon source, such as glucose or sucrose, in the culture medium is greater than about 1 g/L, preferably greater than about 2 g/L, and more preferably greater than about 5 g/L.
  • the concentration of a carbon source, such as glucose or sucrose, in the culture medium is typically less than about 100 g/L, preferably less than about 50 g/L, and more preferably less than about 20 g/L. It should be noted that references to culture component concentrations can refer to both initial and/or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during culture.
  • Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources.
  • Such nitrogen sources include anhydrous ammonia, ammonium salts and substances of animal, vegetable and/or microbial origin.
  • Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids.
  • the concentration of the nitrogen sources, in the culture medium is greater than about 0.1 g/L, preferably greater than about 0.25 g/L, and more preferably greater than about 1.0 g/L.
  • the addition of a nitrogen source to the culture medium is not advantageous for the growth of the microorganisms.
  • the concentration of the nitrogen sources, in the culture medium is less than about 20 g/L, preferably less than about 10 g/L and more preferably less than about 5 g/L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culture.
  • the effective culture medium can contain other compounds such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in carbon, nitrogen, or mineral sources in the effective medium or can be added specifically to the medium.
  • the culture medium can also contain a suitable phosphate source.
  • phosphate sources include both inorganic and organic phosphate sources.
  • Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate, and mixtures thereof.
  • the concentration of phosphate in the culture medium is greater than about 1.0 g/L, preferably greater than about 2.0 g/L, and more preferably greater than about 5.0 g/L. Beyond certain concentrations, however, the addition of phosphate to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of phosphate in the culture medium is typically less than about 20 g/L, preferably less than about 15 g/L, and more preferably less than about 10 g/L-
  • a suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
  • a source of magnesium preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used.
  • the concentration of magnesium in the culture medium is greater than about 0.5 g/L, preferably greater than about 1.0 g/L, and more preferably greater than about 2.0 g/L.
  • the addition of magnesium to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of magnesium in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 3 g/L. Further, in some instances, it may be desirable to allow the culture medium to become depleted of a magnesium source during culture.
  • the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate.
  • a biologically acceptable chelating agent such as the dihydrate of trisodium citrate.
  • the concentration of a chelating agent in the culture medium is greater than about 0.2 g/L, preferably greater than about 0.5 g/L, and more preferably greater than about 1 g/L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of a chelating agent in the culture medium is typically less than about 10 g/L, preferably less than about 5 g/L, and more preferably less than about 2 g/L.
  • the culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium.
  • Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and mixtures thereof.
  • Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide, and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.
  • the culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride.
  • a biologically acceptable calcium source including, but not limited to, calcium chloride.
  • the concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg/L to about 2000 mg/L, preferably within the range of from about 20 mg/L to about 1000 mg/L, and more preferably in the range of from about 50 mg/L to about 500 mg/L.
  • the culture medium can also include sodium chloride.
  • the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g/L to about 5 g/L, preferably within the range of from about 1 g/L to about 4 g/L, and more preferably in the range of from about 2 g/L to about 4 g/L.
  • the culture medium can also include trace metals. Such trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium.
  • the amount of such a trace metals solution added to the culture medium is greater than about 1 mL/L, preferably greater than about 5 mL/L, and more preferably greater than about 10 mL/L. Beyond certain concentrations, however, the addition of a trace metals to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the amount of such a trace metals solution added to the culture medium is typically less than about 100 mL/L, preferably less than about 50 mL/L, and more preferably less than about 30 mL/L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.
  • the culture medium can include other vitamins, such as pantothenate, biotin, calcium, pantothenate, inositol, pyridoxine-HCl, and thiamine-HCl.
  • vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of the microorganisms.
  • the culture medium may be supplemented with hexanoic acid or hexanoate as a precursor for the cannabinoid biosynthetic pathway.
  • the hexanoic acid may have a concentration of less than 3 mM hexanoic acid (e.g., from 1 nM to 2.9 mM hexanoic acid, from 10 nM to 2.9 mM hexanoic acid, from 100 nM to 2.9 mM hexanoic acid, or from 1 pM to 2.9 mM hexanoic acid).
  • the fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous and semi- continuous.
  • the fermentation is carried out in fed-batch mode.
  • some of the components of the medium are depleted during culture, including pantothenate during the production stage of the fermentation.
  • the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and/or production is supported for a period of time before additions are required.
  • the preferred ranges of these components are maintained throughout the culture by making additions as levels are depleted by culture.
  • Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations.
  • additions can be made at timed intervals corresponding to known levels at particular times throughout the culture.
  • rate of consumption of nutrient increases during culture as the cell density of the medium increases.
  • addition is performed using aseptic addition methods, as are known in the art.
  • anti-foaming agent may be added during the culture.
  • the temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and/or production of compounds of interest.
  • the culture medium prior to inoculation of the culture medium with an inoculum, can be brought to and maintained at a temperature in the range of from about 20 °C to about 45 °C, preferably to a temperature in the range of from about 25 °C to about 40 °C and more preferably in the range of from about 28 °C to about 32 °C.
  • the pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium.
  • the pH is maintained from about 3.0 to about 8.0, more preferably from about 3.5 to about 7.0, and most preferably from about 4.0 to about 6.5.
  • the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture.
  • Glucose or sucrose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium.
  • the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g/L and can be determined readily by trial.
  • the glucose when glucose is used as a carbon source the glucose is preferably fed to the fermenter and maintained below detection limits.
  • the glucose concentration in the culture medium is maintained in the range of from about 1 g/L to about 100 g/L, more preferably in the range of from about 2 g/L to about 50 g/L, and yet more preferably in the range of from about 5 g/L to about 20 g/L.
  • the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium.
  • the use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium (e.g., the nitrogen and phosphate sources) can be maintained simultaneously.
  • the trace metals concentrations can be maintained in the culture medium by addition of aliquots of the trace metals solution.
  • Embodiment 2 A method of producing a cannabinoid, the method comprising: i) providing a fermentation composition that has been produced by culturing a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway in a culture medium and under conditions suitable for the host cells to produce the cannabinoid; ii) contacting the fermentation composition with an oil overlay, wherein the oil overlay comprises a 40%:60% to 60%:40% mixture by weight of a branched, saturated C12-C20 alcohol and an oil; and iii) optionally, recovering the cannabinoid from the fermentation composition, the oil overlay, or both.
  • Embodiment 3 The method of Embodiment 1 or 2, wherein the fermentation composition is in contact with the oil overlay while the population of host cells are undergoing fermentation.
  • Embodiment 4 The method of any one of Embodiments 1-3, wherein the oil comprises a vegetable oil.
  • Embodiment 5. The method of Embodiment 4, wherein the vegetable oil is selected from the group consisting of avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, and any combination thereof.
  • Embodiment 6 The method of any one of Embodiments 1-5, wherein the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyldodecanol, 2-hexyl-l- decanol, and 2-octyl decanol.
  • Embodiment 8 The method of Embodiment 7, wherein the host cells comprise heterologous nucleic acids that independently encode (a) the AAE, (b) the TKS, (c) the CBGaS, and (d) the GPP synthase.
  • Embodiment 9 The method of Embodiment 7 or 8, wherein the host cells comprise a heterologous nucleic acid that encodes an OAC.
  • Embodiment 10 The method of any one of Embodiments 1-9, wherein the host cells comprise a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • Embodiment 11 The method of any one of Embodiments 1-10, wherein the host cells comprise a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59.
  • Embodiment 12 The method of any one of Embodiments 1-11, wherein the host cells comprise a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • Embodiment 13 The method of any one of Embodiments 1-12, wherein the host cells comprise a heterologous nucleic acid encoding a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70.
  • Embodiment 14 The method of any one of Embodiments 1-13, wherein the host cells comprise heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • Embodiment 15 Embodiment 15.
  • the host cell further comprises one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • Embodiment 16 The method of any one of Embodiments 1-1 , wherein the culture medium is supplemented with a precursor for the cannabinoid biosynthetic pathway.
  • Embodiment 17 The method of Embodiment 16, wherein the precursor is hexanoate, hexanoic acid, or olivetolic acid.
  • Embodiment 18 The method of any one of Embodiments 1-17, wherein the cannabinoid is CBDA, CBD or an acid form thereof, CBGA, CBG or an acid form thereof, THC or an acid form thereof, or THC a.
  • Embodiment 19 The method of any one of Embodiments 1-18, wherein the host cell is a yeast cell or yeast strain.
  • Embodiment 20 The method of Embodiment 19, wherein the yeast cell is . cerevisiae.
  • Embodiment 21 A method of producing a cannabinoid, the method comprising: i) providing a mixture comprising a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway, a culture medium, and an oil, wherein the oil comprises a 40%: 60% to 60%: 40% mixture by weight of a branched, saturated C12-C20 alcohol and an oil; and ii) recovering the cannabinoid from the mixture.
  • Embodiment 22 The method of Embodiment 21, wherein the recovering of the one or more cannabinoids comprises separating the mixture into an aqueous liquid fraction, an oily liquid fraction, and a pellet by centrifugation.
  • Embodiment 23 The method of Embodiment 21 or 22, wherein the fermentation composition is in contact with the oil overlay while the population of host cells are undergoing fermentation.
  • Embodiment 24 The method of any one of Embodiments 21-23, wherein the oil comprises a vegetable oil.
  • Embodiment 25 The method of Embodiment 24, wherein the vegetable oil is selected from the group consisting of avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, and any combination thereof.
  • Embodiment 26 The method of any one of Embodiments 21-25, wherein the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyldodecanol, 2-hexyl-l- decanol, and 2-octyl decanol.
  • Embodiment 27 The method of any one of Embodiments 21-26, wherein the host cells comprise one or more heterologous nucleic acids that each, independently, encode (a) an AAE, and/or (b) a TKS, and/or (c) a CBGaS, and/or (d) a GPP synthase.
  • Embodiment 28 The method of Embodiment 27, wherein the host cells comprise heterologous nucleic acids that independently encode (a) the AAE, (b) the TKS, (c) the CBGaS, and (d) the GPP synthase.
  • Embodiment 29 The method of Embodiment 27 or 28, wherein the host cells comprise a heterologous nucleic acid that encodes an OAC.
  • Embodiment 30 The method of any one of Embodiments 21-29, wherein the host cells comprise a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • Embodiment 32 The method of any one of Embodiments 21-31, wherein the host cells comprise a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • Embodiment 34 The method of any one of Embodiments 21-33, wherein the host cells comprise heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • Embodiment 35 Embodiment 35.
  • the host cell further comprises one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • Embodiment 36 The method of any one of Embodiments 21-35, wherein the culture medium is supplemented with a precursor for the cannabinoid biosynthetic pathway.
  • Embodiment 37 The method of Embodiment 36, wherein the precursor is hexanoate, hexanoic acid, or olivetolic acid.
  • Embodiment 38 The method of any one of Embodiments 21-37, wherein the cannabinoid is CBDA, CBD or an acid form thereof, CBGA, CBG or an acid form thereof, THC or an acid form thereof, or THC a.
  • Embodiment 39 The method of any one of Embodiments 21-38, wherein the host cell is a yeast cell or yeast strain.
  • Embodiment 40 The method of Embodiment 39, wherein the yeast cell is . cerevisiae.
  • Embodiment 41 A mixture comprising: a population of host cells that are genetically modified to express one or more enzymes of a cannabinoid biosynthetic pathway; a culture medium; and an oil, wherein the oil comprises a 40%:60% to 60%:40% mixture by weight of a branched, saturated C12-C20 alcohol and an oil.
  • Embodiment 42 The mixture of Embodiment 41, wherein the oil comprises a vegetable oil.
  • Embodiment 43 The mixture of Embodiment 42, wherein the vegetable oil is selected from the group consisting of avocado oil, canola oil, grapeseed oil, hemp oil, soybean oil, jojoba oil, sunflower oil, and any combination thereof.
  • Embodiment 44 The mixture of any one of Embodiments 41-43, wherein the branched, saturated C12-C20 alcohol comprises one or more of: 2-butyl octanol, octyldodecanol, 2-hexyl-l- decanol, and 2-octyl decanol.
  • Embodiment 45 The mixture of any one of Embodiments 41-44, wherein the host cells comprise one or more heterologous nucleic acids that each, independently, encode (a) an acyl AAE, and/or (b) a TKS, and/or (c) a CBGaS, and/or (d) a GPP synthase.
  • Embodiment 46 The mixture of Embodiment 45, wherein the host cells comprise heterologous nucleic acids that independently encode (a) the AAE, (b) the TKS, (c) the CBGaS, and (d) the GPP synthase.
  • Embodiment 47 The mixture of Embodiment 45 or 46, wherein the host cells comprise a heterologous nucleic acid that encodes an OAC.
  • Embodiment 48 The mixture of any one of Embodiments 41-47, wherein the host cells comprise a heterologous nucleic acid that encodes an AAE having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 1-24.
  • Embodiment 49 The mixture of any one of Embodiments 41-48, wherein the host cells comprise a heterologous nucleic acid that encodes a TKS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 25-59.
  • Embodiment 50 The mixture of any one of Embodiments 41-49, wherein the host cells comprise a heterologous nucleic acid that encodes a CBGaS having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 60-64.
  • Embodiment 51 The mixture of any one of Embodiments 41-50, wherein the host cells comprise a heterologous nucleic acid encoding a GPP synthase having an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 65-70.
  • Embodiment 52 The mixture of any one of Embodiments 41-51, wherein the host cells comprise heterologous nucleic acids that independently encode a) an AAE having the amino acid sequence of any one of SEQ ID NO: 1-24, b) a TKS having the amino acid sequence of any one of SEQ ID NO: 25-59, c) a CBGaS having the amino acid sequences of any one of SEQ ID NO: 60-64, and d) a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 65-70.
  • Embodiment 53 Embodiment 53.
  • the host cell further comprises one or more heterologous nucleic acids that each, independently, encode an enzyme of the mevalonate biosynthetic pathway, wherein the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • the enzyme is selected from an acetyl- CoA thiolase, an HMG-CoA synthase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a mevalonate pyrophosphate decarboxylase, and an IPP:DMAPP isomerase.
  • Embodiment 54 The mixture of any one of Embodiments 41-53, wherein the culture medium is supplemented with a precursor for the cannabinoid biosynthetic pathway.
  • Embodiment 55 The mixture of Embodiment 54, wherein the precursor is hexanoate, hexanoic acid, or olivetolic acid.
  • Embodiment 56 The mixture of any one of Embodiments 41-55, wherein the cannabinoid is CBDA, CBD or an acid form thereof, CBGA, CBG or an acid form thereof, THC or an acid form thereof, or THC a.
  • Embodiment 57 The mixture of any one of Embodiments 41-56, wherein the host cell is a yeast cell or yeast strain.
  • Embodiment 58 The mixture of Embodiment 57, wherein the yeast cell is S. cerevisiae.
  • Example 1 Cannabinoid fermentation with 50/50 JARCOLTM I-16/HOSUN mixture overlays
  • JARCOLTM 1-12 (2-butyl octanol, available from Aurorium)
  • JARCOLTM 1-13 octyldodecanol, available from Aurorium
  • JARCOLTM 1-16 (2-hexyl-l -decanol, available from Aurorium)
  • JARCOLTM 1-20 (2-octyl decanol, available from Aurorium) were blended with HOSUN and added to yeast fermentation to identify an overlay mixture that improves cell health and cannabinoid production.
  • FIGS. 1A-1B are plots of the feed rate (grams total reducing sugar/liter/hour, gTDS/L/h), oxygen uptake rate (OUR) (mmol/L/h), and oxygen tension (pO2) (%) for the fermentation using a control overlay (Hl 1845) of 100% HOSUN (FIG.
  • FIGS. 2A-2E are plots showing fermentation performance for a fermentation using a control overlay (Hl 1845) of 100% HOSUN and an overlay (H12611) of 50%/50% JARCOLTM I-16/HOSUN by weight.
  • FIG. 2A is a plot of CBFa productivity over time.
  • FIG. 2B is a plot of CBFa titer over time.
  • FIG. 2C is a plot of packed cell volume (PCV) productivity over time.
  • FIG. 2D is a plot of CBFa yield on total reducing sugar over time.
  • FIG. 2E is a plot of average OUR over time.
  • the 50%/50% JARCOLTM I- 16/HOSUN overlay significantly raised the PCV from 6% to 12% (FIG.
  • CBGa cannabigerolic acid
  • the CBGa yield and productivity increased with the 50%/50% JARCOLTM I- 16/HOSUN fermentation compared to the fermentations with 100% HOSUN and 10%/90% JARCOLTM I-16/HOSUN. Further, the CBGa productivity with the 50%/50% JARCOLTM I- 16/HOSUN fermentation was similar compared to 100% JARCOLTM 1-16 fermentation. However, increasing the JARCOLTM 1-16 concentration further was less beneficial from a performance perspective because greater foaming was observed.
  • SEQ ID NO: 1 AAE candidate isolated from Pseudonocardia sp. N23
  • SEQ ID NO: 3 AAE candidate isolated from Streptomyces sp.ADI96-02
  • SEQ ID NO: 5 AAE candidate isolated from Saccharomyces cerevisiae
  • SEQ ID NO: 7 AAE candidate isolated from Bacillus subtilis (strain 168)
  • SEQ ID NO: 8 AAE candidate isolated from Saccharomyces cerevisiae
  • SEQ ID NO: 11 AAE candidate isolated from Alcaligenes xylosoxydans (Achromobacter xylosoxidans)
  • SEQ ID NO: 13 AAE candidate isolated from Arabidopsis thaliana (Mouse-ear cress)
  • SEQ ID NO: 18 AAE candidate isolated from Arabidopsis thaliana (Mouse-ear cress) [0237] Amino acid sequence
  • SEQ ID NO: 20 AAE candidate isolated from Nocardioides simplex (Arthrobacter simplex)
  • SEQ ID NO: 22 AAE candidate isolated from Pseudomonas putida (Arthrobacter siderocapsulatus)
  • SEQ ID NO: 23 AAE candidate isolated from Drosophila melanogaster (Fruit fly) [0252] Amino acid sequence
  • SEQ ID NO: 30 TKS candidate isolated from Vitis pseudoreticulata
  • SEQ ID NO: 48 TKS candidate isolated from Physcomitrella patens subsp. patens
  • SEQ ID NO: 50 TKS candidate isolated from Marchcmtia polymorpha subsp. ruderalis
  • SEQ ID NO: 54 TKS candidate isolated from Rhododendron dauricum
  • SEQ ID NO: 56 TKS candidate isolated from Garcinia mangostana
  • SEQ ID NO: 71 Olivetolic Acid Cyclase (OAC) from Cannabis sativa
  • VCEVS S ATTEDVEYAIEC ADRAFHDTEWATQDPRERGRLLSKLADELESQIDLVS SIEAL DNGKTLALARGDVTIAINCLRDAAAYADKVNGRTINTGDGYMNFTTLEPIGVCGQIIPW

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Abstract

La présente divulgation concerne des compositions et des procédés pour produire un ou plusieurs cannabinoïdes dans une cellule hôte qui est génétiquement modifiée pour exprimer les enzymes d'une voie de biosynthèse des cannabinoïdes. En utilisant les compositions et les procédés de la divulgation, une cellule hôte produisant des cannabinoïdes peut être mise en contact avec un mélange de 40%:60% à 60%:40% (par exemple, 50%/50%) en poids d'un alcool en C12-C20 ramifié, saturé et d'une huile de façon à améliorer la production du cannabinoïde à partir de la cellule hôte.
PCT/US2024/033062 2023-06-09 2024-06-07 Superpositions améliorées pour la production de cannabinoïdes Pending WO2024254488A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9200270B2 (en) 2008-03-03 2015-12-01 Abbvie Inc. Methods for transforming yeast
US20160177341A1 (en) 2013-08-07 2016-06-23 Amyris, Inc. Methods for stabilizing production of acetyl-coenzyme a derived compounds
US10563211B2 (en) 2017-04-27 2020-02-18 The Regents Of The University Of California Recombinant microorganisms and methods for producing cannabinoids and cannabinoid derivatives
WO2022040475A1 (fr) * 2020-08-19 2022-02-24 Amyris, Inc. Production microbienne de cannabinoïdes
WO2022256697A1 (fr) * 2021-06-04 2022-12-08 Amyris, Inc. Procédés de purification de cannabinoïdes
WO2023288188A1 (fr) * 2021-07-13 2023-01-19 Amyris, Inc. Production à haut rendement d'acide cannabigérolique et d'acide cannabidiolique

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9200270B2 (en) 2008-03-03 2015-12-01 Abbvie Inc. Methods for transforming yeast
US20160177341A1 (en) 2013-08-07 2016-06-23 Amyris, Inc. Methods for stabilizing production of acetyl-coenzyme a derived compounds
US10563211B2 (en) 2017-04-27 2020-02-18 The Regents Of The University Of California Recombinant microorganisms and methods for producing cannabinoids and cannabinoid derivatives
WO2022040475A1 (fr) * 2020-08-19 2022-02-24 Amyris, Inc. Production microbienne de cannabinoïdes
WO2022256697A1 (fr) * 2021-06-04 2022-12-08 Amyris, Inc. Procédés de purification de cannabinoïdes
WO2023288188A1 (fr) * 2021-07-13 2023-01-19 Amyris, Inc. Production à haut rendement d'acide cannabigérolique et d'acide cannabidiolique

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BAILEY ET AL.: "Biochemical Engineering Fundamentals", 1986, MCGRAW HILL
DALPHIN ET AL., NUCL ACIDS RES., vol. 24, 1996, pages 216 - 8
KIRITANI, K., BRANCHED-CHAIN AMINO ACIDS METHODS ENZYMOLOGY, 1970
KOSARIC ET AL.: "Ullmann's Encyclopedia of Industrial Chemistry", vol. 12, WILEY-VCH VERLAG GMBH & CO., pages: 398 - 473
LABORATORY METHODS IN ENZYMOLOGY: DNA, vol. 529, 2013
MURRAY ET AL., NUCL ACIDS RES., vol. 17, 1989, pages 477 - 508
NOVAK ET AL.: "Maltose Transport and Metabolism in S. cerevisiae", FOOD TECHNOL. BIOTECHNOL., vol. 42, no. 3, 2004, pages 213 - 218, XP002436253
PEARSON W. R., METHODS IN MOL BIOL, vol. 25, 1994, pages 365 - 89
SAINZ MARTINEZ AITOR ET AL: "Extraction techniques for bioactive compounds of cannabis", NATURAL PRODUCT REPORTS, vol. 40, no. 3, 1 January 2023 (2023-01-01), GB, pages 676 - 717, XP093218431, ISSN: 0265-0568, DOI: 10.1039/D2NP00059H *
WESTFALL, PNAS, vol. 109, 2012, pages 111 - 118

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