WO2024124165A2 - Procédés et compositions de purification de cannabinoïdes - Google Patents

Procédés et compositions de purification de cannabinoïdes Download PDF

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Publication number
WO2024124165A2
WO2024124165A2 PCT/US2023/083173 US2023083173W WO2024124165A2 WO 2024124165 A2 WO2024124165 A2 WO 2024124165A2 US 2023083173 W US2023083173 W US 2023083173W WO 2024124165 A2 WO2024124165 A2 WO 2024124165A2
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seq
amino acid
acid sequence
culture medium
synthase
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PCT/US2023/083173
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English (en)
Inventor
Cara TEO ONG
Brady GALEN
Jenna LLOYD-RANDOLFI
Jennifer DANGER NILL
William Draper
Victor HOLMES
Mark Wogulis
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Amyris, Inc.
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Publication of WO2024124165A2 publication Critical patent/WO2024124165A2/fr

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  • Processes developed for the production of minor cannabinoids typically fall into two categories: 1 ) the extraction and purification from the cannabis plant, and 2) the conversion of a cannabinoid precursor into various products, either chemically or biochemically.
  • Processes typically involved in the purification of plant-derived cannabinoids include extraction from the cannabis plant, decarboxylation to convert the acidic cannabinoids by heating the extract, evaporation to concentrate the product, crystallization, and a variety of chromatography steps to remove residual impurities.
  • the present disclosure features methods of isolating and purifying one or more cannabinoids from a fermentation composition.
  • the methods described herein include for the first time using an oil overlay in combination with a cannabichromenic acid (CBCa) synthase. It has been presently discovered that this combination results in near complete conversion of cannabigerolic acid (CBGa) to CBCa.
  • CBCa cannabichromenic acid
  • CBGa cannabigerolic acid
  • the present disclosure provides variant CBCa synthase polypeptides having one or more amino acid substitutions.
  • the variant CBCa synthases described herein show improved enzymatic activity in comparison to the wild-type enzyme.
  • the disclosure provides a method of making a cannabichromenic acid (CBCa).
  • the method includes (a) culturing a population of host cells capable of producing cannabigerolic acid (CBGa) in a culture medium comprising a fermentation broth and an overlay, under conditions suitable for the host cells to produce CBGa, and wherein the CBGa partitions into the overlay; (b) separating the overlay from the fermentation broth; (c) combining the separated overlay of step (b), with a CBCa synthase, thereby producing a bioconversion mixture; and (d) purifying the CBCa from the bioconversion mixture.
  • CBGa cannabigerolic acid
  • the overlay comprises a plant-based oil.
  • the plant-based oil is selected from soybean oil, sunflower oil, safflower oil, canola oil, grapeseed oil, or castor oil.
  • the plant-based oil is sunflower oil.
  • the overlay comprises a synthetic ester or a fatty alcohol.
  • the CBCa synthase is produced by culturing a population of host cells capable of producing a CBCa synthase in a culture medium and under conditions suitable for the host cells to produce the CBCa synthase, thereby producing the CBCa synthase.
  • the overlay and the fermentation broth are separated by centrifugation. In some embodiments, the overlay and the fermentation broth are separated by demulsification.
  • the demulsification includes contacting the fermentation broth with an oil.
  • the oil includes a mineral oil, a vegetable oil, a synthetic ester, or a fatty alcohol.
  • the oil includes a vegetable oil.
  • the vegetable oil is soybean oil, sunflower oil, safflower oil, canola oil, grapeseed oil, or castor oil.
  • the oil includes a synthetic ester, optionally wherein the synthetic ester is ESTEREXTM A51 .
  • the oil includes a fatty alcohol, optionally wherein the fatty alcohol is oleyl alcohol or JARCOLTM 1-16.
  • the oil has a concentration of between about 1% (w/v) and about 10% (w/v) (e.g., about 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5% (w/v), 6% (w/v), 7% (w/v), 8% (w/v), 9% (w/v), or 10% (w/v)). In some embodiments, the oil has a concentration of about 5% (w/v).
  • the overlay includes CBGa.
  • the CBGa has a concentration of between about 0.1% (w/v) or and 10% (w/v) (e.g., between 0.1% (w/v) and 8% (w/v), 0.1% (w/v) and 6% (w/v), 0.1% (w/v) and 4% (w/v), 0.1% (w/v) and 2% (w/v), 2% (w/v) and 10% (w/v), 4% (w/v) and 10% (w/v), 6% (w/v) and 10% (w/v), or 8% (w/v) and 10% (w/v)).
  • the CBGa has a concentration of between about 0.5% (w/v) and 5% (w/v) (e.g., between 0.5% (w/v) and 4% (w/v), 0.5% (w/v) and 3% (w/v), 0.5% (w/v) and 2% (w/v), 0.5% (w/v) and 1% (w/v), 1% (w/v) and 5% (w/v), 2% (w/v) and 5% (w/v), 3% (w/v) and 5% (w/v), or 4% (w/v) and 5% (w/v)).
  • 0.5% (w/v) and 5% (w/v) e.g., between 0.5% (w/v) and 4% (w/v), 0.5% (w/v) and 3% (w/v), 0.5% (w/v) and 2% (w/v), 0.5% (w/v), 1% (w/v) and 5% (w/v), or 4% (w/v)
  • the method further includes stirring the bioconversion mixture for between 12 hours and 144 hours before performing step (d) (e.g., between 12 hours 120 hours, 12 hours and 96 hours, 12 hours and 72 hours, 12 hours and 48 hours, 12 hours and 24 hours, 24 hours and 144 hours, 48 hours and 144 hours, 72 hours and 144 hours, 96 hours and 144 hours, or 120 hours and 144 hours).
  • the method includes stirring the bioconversion mixture for between 24 hours and 96 hours (e.g., between 24 hours and 84 hours, 24 hours and 72 hours, 24 hours and 60 hours, 24 hours and 48 hours, 48 hours and 96 hours, 60 hours and 96 hours, 72 hours and 96 hours, or 84 hours and 96 hours).
  • the method includes stirring the bioconversion mixture for about 48 hours.
  • the bioconversion mixture is at a temperature of between 4 °C and 50 °C.
  • the bioconversion mixture is at a temperature of between 20 °C and 40 °C.
  • the bioconversion mixture is at a temperature of about 35 °C.
  • the second mixture further includes one or more amphiphilic moieties.
  • the one or more amphiphilic moieties includes a cyclodextrin, plant derived silica, cellulose, or a combination thereof.
  • the cyclodextrin includes randomly methylated cyclodextrin, 2, 6-Di-O-methyl-p-cyclodextrin, or a combination thereof.
  • the demulsification includes one or more of: (i) contacting the culture medium with an enzymatic composition comprising a serine protease, (ii)contacting the culture medium with a surfactant; and (iii) contacting the culture medium with NaOH to adjust the culture medium to a pH of between pH 7 and pH 9.
  • the demulsification includes contacting the culture medium with the enzymatic composition including the serine protease.
  • the purifying comprises contacting the culture medium with the surfactant.
  • the culture medium is contacted with the enzymatic composition or surfactant after the mixture is adjusted to a pH of between about pH 7 and pH 9.
  • the culture medium is contacted with the enzymatic composition or surfactant after the culture medium is adjusted to a pH of pH 8.
  • the final concentration of the enzymatic composition is between about 0.5% (w/v) to about 3% (w/v) (e.g., between about 0.5% (w/v) and 2% (w/v), 0.5% (w/v) and 1 % (w/v), 1 % (w/v) and 3% (w/v), or 2% (w/v) and 3% (w/v)) after contacting the culture medium including a cannabinoid with the enzymatic composition.
  • the culture medium is contacted with the enzymatic composition at a final concentration of about 1 % (w/v). In some embodiments, the culture medium is mixed with the enzymatic composition for between 0.5 hours and 2 hours (e.g., between 0.5 hours and 1 .5 hours, 0.5 hours and 1 hour, 1 hour and 2 hours, or 1 .5 hours and 2 hours).
  • the demulsification includes centrifugation of the culture medium. In some embodiments, the centrifugation includes liquid-liquid centrifugation. In some embodiments, the centrifugation results in a crude oil light phase and an aqueous heavy phase. In some embodiments, the demulsification further includes a decarboxylation step comprising evaporating the culture medium. In some embodiments, the decarboxylation includes evaporating the crude oil light phase. In some embodiments, evaporating includes one or more passes. In some embodiments, evaporating includes a first pass and a second pass.
  • the first pass is performed at a temperature of between about 100 °C and about 500 °C (e.g., between about 100 °C and 400 °C, 100 °C and 300 °C, 100 °C and 200 °C, 200 °C and 500 °C, 300 °C and 500 °C, or 400 °C and 500 °C). In some embodiments, the first pass is performed at a temperature of about 180 °C.
  • the first pass is performed at a pressure of between about 0.5 torr and 760 torr (e.g., between 0.5 torr and 700 torr, 0.5 torr and 500 torr, 0.5 torr and 200 torr, 0.5 torr and 50 torr, 50 torr and 760 torr, 200 torr and 760 torr, 400 torr and 760 torr, or 600 torr and 760 torr). In some embodiments, the first pass is performed at a pressure of about 1 torr.
  • the second pass is performed at a temperature of between 150 °C and 300 °C (e.g., between 150 °C and 250 °C, 150 °C and 200 °C, 200 °C and 300 °C, or 250 °C and 300 °C). In some embodiments, the second pass is performed at a temperature of about 240 C.
  • the second pass is performed at a pressure of between about 0.5 torr and 760 torr (e.g., between 0.5 torr and 700 torr, 0.5 torr and 500 torr, 0.5 torr and 200 torr, 0.5 torr and 50 torr, 50 torr and 760 torr, 200 torr and 760 torr, 400 torr and 760 torr, or 600 torr and 760 torr). In some embodiments, the second pass is performed at a pressure of about 1 torr.
  • purifying includes one or more of a liquid-liquid extraction, chromatography, or saponification.
  • the host cells include a nucleic acid sequence encoding a cannabichromenic acid (CBCa) synthase.
  • CBCa synthase includes one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase includes an amino acid substitution at a residue selected from Q75, F82, T130, S140, V169, N240, V294, A299, K305, T335, R340, H354, L435, Y461 , K535, S540, and T545 of SEQ ID NO: 1.
  • the one or more amino acid substitutions include an amino acid substitution at residue Q75 of SEQ ID NO: 1 .
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 substitutes Q75 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 is a Q75L substitution.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 substitutes Q75 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 is a Q75E substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue S140 of SEQ ID NO: 1 .
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 substitutes S140 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 is a S140A substitution.
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 is a S140T substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue V169 of SEQ ID NO: 1 .
  • the amino acid substitution at residue V169 of SEQ ID NO: 1 substitutes V169 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue V169 of SEQ ID NO: 1 is a V169E substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue N240 of SEQ ID NO: 1 .
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 substitutes N240 with an amino acid including a polar, uncharged side chain at physiological pH.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 is a N240Q substitution.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 substitutes N240 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 is a N240M substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue V294 of SEQ ID NO: 1 .
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including a polar, uncharged side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294S substitution.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294E substitution.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including a cationic side chain at physiological pH. In some embodiments, the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294R substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue A299 of SEQ ID NO: 1 .
  • the amino acid substitution at residue A299 of SEQ ID NO: 1 substitutes A299 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue A299 of SEQ ID NO: 1 is an A299V substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue K305 of SEQ ID NO: 1 .
  • the amino acid substitution at residue K305 of SEQ ID NO: 1 substitutes K305 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue K305 of SEQ ID NO: 1 is a K305C substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue D328 of SEQ ID NO: 1 .
  • the amino acid substitution at residue D328 of SEQ ID NO: 1 is a D328P substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue T335 of SEQ ID NO: 1 .
  • the amino acid substitution at residue T335 of SEQ ID NO: 1 substitutes T335 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue T335 of SEQ ID NO: 1 is a T335L substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue R340 of SEQ ID NO: 1 .
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 substitutes R340 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a R340M substitution.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a R340G substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue H354 of SEQ ID NO: 1 .
  • the amino acid substitution at residue H354 of SEQ ID NO: 1 substitutes H354 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a H354V substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue L435 of SEQ ID NO: 1 .
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 substitutes L435 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 is a L435A substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue Y461 of SEQ ID NO: 1 .
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 substitutes Y461 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 is a Y4611 substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue K535 of SEQ ID NO: 1 .
  • the amino acid substitution at residue K535 of SEQ ID NO: 1 substitutes K535 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 is a K535M substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue S540 of SEQ ID NO: 1 .
  • the amino acid substitution at residue S540 of SEQ ID NO: 1 substitutes S540 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue S540 of SEQ ID NO: 1 is a S540D substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue T545 of SEQ ID NO: 1 .
  • the amino acid substitution at residue T545 of SEQ ID NO: 1 substitutes T545 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue T545 of SEQ ID NO: 1 is a T545E substitution.
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A Y461 I, K535, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, N240M, V294S, A299V, T335L, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, A299V, K305C, T335L, R340M, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V294S, A299V, K305C, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, S140A, V169E, N240M, K305C, T335L, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, V169E, V294S, A299V, R340M, H354V, L435A, K535M, or S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, V294S, A299V, T335L, H354V, L435A, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, V294S, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, T335L, R340M, H354V, L435A, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V294S, K305C, T335L, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including T130L, S140A, V169E, V294S, A299V, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, N240M, V294S, A299V, T335L, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, A299V, K305C, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, V294S, A299V, K305C, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, V169E, V294S, A299V, K305C, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, K305C, T335L, R340M, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, A299V, T335L, R340M, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, A299V, T335L, R340M, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, V169E, N240M, V294S, A299V, K305C, T335L, L435A, Y461 1, and K535M of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is from about 85% to about 99.7% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., about 85%, 96%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7%).
  • the CBCa synthase has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is from about 90% to about 99.7% (e.g., about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is from about 95% to about 99.7% (e.g., about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of (i) the one or more amino acid substitutions or deletions and, optionally, (ii) one or more additional, conservative amino acid substitutions. In some embodiments, the CBCa synthase has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of the one or more amino acid substitutions or deletions. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 2-67.
  • the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 29-67.
  • the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 29-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 29-67. In some embodiments, the CBCa synthase has the amino acid sequence of any one of SEQ
  • the host cell includes 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.
  • AAE acyl activating enzyme
  • TKS tetraketide synthase
  • CBGaS cannabigerolic acid synthase
  • GPP geranyl pyrophosphate
  • the host cell includes heterologous nucleic acids that independently encode (a) an AAE, (b) a TKS, (c) a CBGaS, and (d) a GPP synthase.
  • the host cell includes 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: 68-91 .
  • 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: 68-91 .
  • the AAE has the amino acid sequence of any one of SEQ ID NO: 68-91 .
  • the host cell includes 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: 68-80.
  • 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: 68-80. In some embodiments, the AAE has the amino acid sequence of any one of SEQ ID NO: 68-80. In some embodiments, the host cell includes 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: 68-72. In some embodiments, 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: 68-72. In some embodiments, the AAE has the amino acid sequence of any one of SEQ ID NO: 68-72.
  • the host cell includes 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: 92-126. In some embodiments, 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: 92-126. In some embodiments, the TKS has the amino acid sequence of any one of SEQ ID NO: 92-126. In some embodiments, the host cell includes 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: 92-95.
  • 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: 92-95. In some embodiments, the TKS has the amino acid sequence of any one of SEQ ID NO: 92-95. In some embodiments, the host cell includes 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: 92. In some embodiments, the TKS has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92, optionally wherein the TKS has the amino acid sequence of SEQ ID NO: 92.
  • the host cell includes 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: 127-131 .
  • 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: 127-131 .
  • the CBGaS has the amino acid sequence of any one of SEQ ID NO: 127-131 .
  • the host cell includes 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: 132-137. In some embodiments, 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: 132- 137. In some embodiments, the GPP has the amino acid sequence of any one of SEQ ID NO: 132- 137. In some embodiments, the host cell includes 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 SEQ ID NO: 132. In some embodiments, the GPP synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132. In some embodiments, the GPP has the amino acid sequence of SEQ ID NO: 132.
  • the host cell includes heterologous nucleic acids that independently encode an AAE having the amino acid sequence of any one of SEQ ID NO: 68-91 , a TKS having the amino acid sequence of any one of SEQ ID NO: 92-126, a CBGaS having the amino acid sequences of any one of SEQ ID NO: 127-131 , and a GPP synthase having the amino acid sequence of any one of SEQ ID NO: 132-137.
  • the host cell further includes 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.
  • the host cell includes heterologous nucleic acids that independently encode 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 further includes a heterologous nucleic acid that encodes an olivetolic acid cyclase (OAC).
  • OAC olivetolic acid cyclase
  • the OAC has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 138.
  • the OAC has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138.
  • the OAC has the amino acid sequence of SEQ ID NO: 138.
  • the host cell further includes one or more heterologous nucleic acids that each, independently, encode an acetyl-CoA synthase, and/or an aldehyde dehydrogenase, and/or a pyruvate decarboxylase.
  • the acetyl-CoA synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 139.
  • the acetyl-CoA synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 139.
  • the acetyl-CoA synthase has the amino acid sequence of SEQ ID NO: 139.
  • the acetyl-CoA synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the acetyl-CoA synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the acetyl-CoA synthase has the amino acid sequence of SEQ ID NO: 140. In some embodiments, the aldehyde dehydrogenase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 141 .
  • the aldehyde dehydrogenase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141 . In some embodiments, the aldehyde dehydrogenase synthase has the amino acid sequence of SEQ ID NO: 141 . In some embodiments, the pyruvate decarboxylase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the pyruvate decarboxylase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the pyruvate decarboxylase has the amino acid sequence of SEQ ID NO: 142.
  • expression of the one or more heterologous nucleic acids is regulated by an exogenous agent.
  • the exogenous agent includes a regulator of gene expression.
  • the exogenous agent decreases production of the cannabinoid.
  • the exogenous agent is maltose.
  • the exogenous agent increases production of the cannabinoid.
  • the exogenous agent is galactose.
  • the exogenous agent is galactose and expression of one or more heterologous nucleic acids encoding the AAE, TKS, and CBGaS enzymes is under the control of a GAL promoter.
  • expression of one or more heterologous nucleic acids encoding the AAE, TKS, and CBGaS enzymes is under the control of a galactose-responsive promoter, a maltose-responsive promoter, or a combination of both.
  • the method further includes culturing the host cell with a precursor required to make the cannabinoid.
  • the precursor required to make the cannabinoid is hexanoate.
  • the cannabinoid is cannabichromene (CBC), cannabinol (CBN), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), cannabichromevarin (CBCV), 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 cannabinoid is CBC.
  • the CBC has a purity of at least about 50% (w/v) following purifying. In some embodiments, the CBC has a purity of between about 50% (w/w) and 100% (w/w) (e.g., between 50% (w/w) and 80 % (w/w), 50% (w/w) and 60% (w/w), 60% (w/w) and 100% (w/w), or 80% (w/w) and 100% (w/w)) following purifying. In some embodiments, the CBC has a purity of about 70% (w/w).
  • the host cell is a yeast cell or yeast strain.
  • the yeast cell is S. cerevisiae.
  • the disclosure provides a composition including CBC produced by any one of the methods described herein.
  • the CBC has a purity of at least about 50% (w/w).
  • the CBC has a purity of between about 50% (w/w) to about 100% (w/w) (e.g., between 50% (w/w) and 80 % (w/w), 50% (w/w) and 60% (w/w), 60% (w/w) and 100% (w/w), or 80% (w/w) and 100% (w/w)).
  • the CBC has a purity of between about 70% (w/w) to about 100% (w/w) (e.g., between 70% (w/w) and 90% (w/w), 70% (w/w) and 80% (w/w), 80% (w/w) and 100% (w/w), or 90% (w/w) and 100% (w/w)). In some embodiments, the CBC has a purity of from about 99.5% (w/w) to about 100% (w/w) (e.g., about 99.5% (w/w), 99.6% (w/w), 99.7% (w/w), 99.8% (w/w), or 99.9% (w/w)).
  • the disclosure provides a variant CBCa synthase polypeptide including one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase includes an amino acid substitution at a residue selected from Q75, F82, T130, S140, V169, N240, V294, A299, K305, T335, R340, H354, L435, Y461 , K535, S540, of SEQ ID NO: 1 .
  • the one or more amino acid substitutions include an amino acid substitution at residue Q75 of SEQ ID NO: 1 .
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 substitutes Q75 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 is a Q75L substitution.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 substitutes Q75 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue Q75 of SEQ ID NO: 1 is a Q75E substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue S140 of SEQ ID NO: 1 .
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 substitutes S140 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 is a S140A substitution.
  • the amino acid substitution at residue S140 of SEQ ID NO: 1 is a S140T substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue V169 of SEQ ID NO: 1 .
  • the amino acid substitution at residue V169 of SEQ ID NO: 1 substitutes V169 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue V169 of SEQ ID NO: 1 is a V169E substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue N240 of SEQ ID NO: 1 .
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 substitutes N240 with an amino acid including a polar, uncharged side chain at physiological pH.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 is a N240Q substitution.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 substitutes N240 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue N240 of SEQ ID NO: 1 is a N240M substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue V294 of SEQ ID NO: 1 .
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including a polar, uncharged side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294S substitution.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294E substitution.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including a cationic side chain at physiological pH. In some embodiments, the amino acid substitution at residue V294 of SEQ ID NO: 1 is a V294R substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue A299 of SEQ ID NO: 1 .
  • the amino acid substitution at residue A299 of SEQ ID NO: 1 substitutes A299 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue A299 of SEQ ID NO: 1 is an A299V substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue K305 of SEQ ID NO: 1 .
  • the amino acid substitution at residue K305 of SEQ ID NO: 1 substitutes K305 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue K305 of SEQ ID NO: 1 is a K305C substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue D328 of SEQ ID NO: 1 .
  • the amino acid substitution at residue D328 of SEQ ID NO: 1 is a D328P substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue T335 of SEQ ID NO: 1 .
  • the amino acid substitution at residue T335 of SEQ ID NO: 1 substitutes T335 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue T335 of SEQ ID NO: 1 is a T335L substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue R340 of SEQ ID NO: 1 .
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 substitutes R340 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a R340M substitution.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a R340G substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue H354 of SEQ ID NO: 1 .
  • the amino acid substitution at residue H354 of SEQ ID NO: 1 substitutes H354 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 is a H354V substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue L435 of SEQ ID NO: 1 .
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 substitutes L435 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 is a L435A substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue Y461 of SEQ ID NO: 1 .
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 substitutes Y461 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 is a Y4611 substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue K535 of SEQ ID NO: 1 .
  • the amino acid substitution at residue K535 of SEQ ID NO: 1 substitutes K535 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue Y461 of SEQ ID NO: 1 is a K535M substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue S540 of SEQ ID NO: 1 .
  • the amino acid substitution at residue S540 of SEQ ID NO: 1 substitutes S540 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue S540 of SEQ ID NO: 1 is a S540D substitution.
  • the one or more amino acid substitutions include an amino acid substitution at residue T545 of SEQ ID NO: 1 .
  • the amino acid substitution at residue T545 of SEQ ID NO: 1 substitutes T545 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue T545 of SEQ ID NO: 1 is a T545E substitution.
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A Y461 I, K535, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, N240M, V294S, A299V, T335L, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, A299V, K305C, T335L, R340M, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V294S, A299V, K305C, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, S140A, V169E, N240M, K305C, T335L, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, V169E, V294S, A299V, R340M, H354V, L435A, K535M, or S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, V294S, A299V, T335L, H354V, L435A, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, V294S, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, T335L, R340M, H354V, L435A, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V294S, K305C, T335L, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y4611, and K535M of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including T130L, S140A, V169E, V294S, A299V, R340M, H354V, L435A, Y4611, K535M, and S540D of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, N240M, V294S, A299V, T335L, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, A299V, K305C, R340M, H354V, L435A, Y461 1, and K535M of SEQ ID NO: 1 . In some embodiments, the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, T130L, S140A, V169E, V294S, A299V, K305C, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, V169E, V294S, A299V, K305C, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, K305C, T335L, R340M, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, A299V, T335L, R340M, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, A299V, T335L, R340M, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, F82I, T130L, S140A, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the polypeptide has amino acid substitutions including Q75E, V169E, N240M, V294S, A299V, K305C, T335L, L435A, Y461 1, and K535M of SEQ ID NO: 1 .
  • the CBCa synthase comprises an amino acid substitution at a residue selected from Q75, F82, T130, S140, V169, N240, V294, A299, K305, T335, R340, H354, L435, Y461 , K535, S540, and T545 of SEQ ID NO: 1
  • the CBCa synthase has an amino acid sequence that is from about 85% to about 99.7% identical to the amino acid sequence of SEQ ID NO: 1 (e.g., about 85%, 96%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7%).
  • the CBCa synthase has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is from about 90% to about 99.7% (e.g., about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase has an amino acid sequence that is from about 95% to about 99.7% (e.g., about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1.
  • the CBCa synthase has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of (i) the one or more amino acid substitutions or deletions and, optionally, (ii) one or more additional, conservative amino acid substitutions. In some embodiments, the CBCa synthase has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 only by way of the one or more amino acid substitutions or deletions.
  • the CBCa synthase includes an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
  • the CBCa synthase has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 2-67. In some embodiments, the CBCa synthase has the amino acid sequence of any one of SEQ ID NO: 2-67.
  • the CBCa synthase has an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NO: 29-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO: 29-67. In some embodiments, the CBCa synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 29-67.
  • the CBCa synthase has the amino acid sequence selected from SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 , SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61
  • the disclosure provides a variant CBCa synthase polypeptide, wherein the polypeptide is capable producing a yield of CBCa concentration of at least 50 mg/L.
  • the disclosure provides a nucleic acid encoding any one of the variant polypeptides described herein.
  • the disclosure provides a host cell including any one of the variant polypeptides or nucleic acids described herein.
  • FIG. 1 is a graph showing a schematic of the Production process for generating cannabichromene (CBC) by combining cannabigerolic acid (CBGa) in high oleic sunflower oil (HOSUN) overlay with aqueous CBCa synthase, chemically or mechanically demulsifying and decarboxylating CBCa, distilling the separated overlay to remove HOSUN, and purifying the resulting material.
  • CBDa cannabigerolic acid
  • HOSUN high oleic sunflower oil
  • FIG. 2 is a graph showing the bioconversion time course titers of cannabichromenic acid (CBCa), CBGa, and cannabigerol (CBG).
  • CBCa cannabichromenic acid
  • CBGa cannabigerol
  • FIG. 3 is a graph showing the reaction rate as a function of CBGa concentration in the reaction and in the overlay for a given CBGa reaction, as the overlay % was decreased as the CBGa concentration in the overlay increased.
  • FIG. 4 is a graph showing the changeover base case bioconversion for BiosilicaTM (silica derived from sugar cane bagasse) (purple square), microcrystalline cellulose, randomly methylated cyclodextrin, and 2,6-Di-O-methyl-beta-cyclodextrin.
  • FIG. 5 is a graph showing the reaction rates of the recycle experiments described in Example 1 without cyclodextrin and with cyclodextrin.
  • FIG. 6 is a graph showing the concentration of CBCa produced by CBGa synthase enzymes having a point mutation in comparison to the wild-type CBCa synthase.
  • FIG. 7 is a graph showing the concentration of CBCa produced by CBGa synthase enzymes having a combination of point mutations in comparison to the wild-type CBCa synthase.
  • bioconversion mixture refers to a composition including a substrate, for example, cannabigerolic acid (CBGa), and an enzyme capable of transforming the substrate, for example, cannabichromenic acid (CBCa) synthase, to generate a product, for example, cannabichromenic acid (CBCa).
  • CBDa cannabigerolic acid
  • CBCa cannabichromenic acid synthase
  • 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).
  • Cannabinoids includes but not limited to cannabigerols (CBG), cannabichromens (CBC), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN), cannabinodiol (CBDL), cannabicyclol (CBL), cannabielsoin (CBE), cannabitriol (CBT), cannabinol (CBN), cannabichromene (CBC), cannabidioloic acid (CBDA), cannabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA), cannabinolic acid (CBNA), cannabidivarin (CBDV), tetrahydrocannabivarin (THCV), cannabigerovarin (CBGV), cannabichromevarin (CBCV), and others.
  • CBD cannabigerols
  • CBD cannabidiol
  • the term “capable of producing” refers to a host cell that is genetically modified to express the enzyme(s) necessary for the production of a given compound in accordance with a biochemical pathway that produces the compound.
  • a host cell e.g., a yeast cell
  • a cannabinoid is one that expresses the enzymes necessary for production of the cannabinoid according to the cannabinoid biosynthetic pathway.
  • endogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • a particular organism e.g., a human
  • a particular location within an organism e.g., an organ, a tissue, or a cell, such as a human cell.
  • exogenous describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).
  • Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted there from.
  • the term "express” refers to any one or more of the following events: (1 ) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Expression of a gene of interest in a cell, tissue sample, or subject can manifest, for example, as: an increase in the quantity or concentration of mRNA encoding a corresponding protein (as assessed, e.g., using RNA detection procedures described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques), an increase in the quantity or concentration of a corresponding protein (as assessed, e.g., using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assays (ELISA), among others), and/or an increase in the activity of a corresponding protein (e.g., in the case of an enzyme, as assessed using an enzymatic activity assay described herein or known in the art).
  • RNA detection procedures described herein or known in the art such as quantitative polymerase chain reaction (qPCR) and RNA seq techniques
  • qPCR quantitative polymerase chain reaction
  • RNA seq techniques an increase in the quantity or concentration of a corresponding protein (
  • expression cassette or “expression construct” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively.
  • expression of transgenes one of skill will recognize that the inserted polynucleotide sequence need not be identical but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence.
  • an expression cassette is a polynucleotide construct that includes a polynucleotide sequence encoding a polypeptide for use in the invention operably linked to a promoter, e.g., its native promoter, where the expression cassette is introduced into a heterologous microorganism.
  • an expression cassette includes a polynucleotide sequence encoding a polypeptide of the invention where the polynucleotide that is targeted to a position in the genome of a microorganism such that expression of the polynucleotide sequence is driven by a promoter that is present in the microorganism.
  • 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.
  • the term “fermentation broth” refers to a composition which contains host cells and products, or metabolites produced by the host cells.
  • An example of a fermentation broth is a whole cell broth, which may be the entire contents of a vessel, including cells, aqueous phase, and compounds produced from the host cells.
  • a “genetic pathway” or “biosynthetic pathway” as used herein refers to a set of at least two different coding sequences, where the coding sequences encode enzymes that catalyze different parts of a synthetic pathway to form a desired product (e.g., a cannabinoid).
  • 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.
  • host cell refers to a microorganism, such as yeast, and includes an individual cell or cell culture including a heterologous vector or heterologous polynucleotide as described herein.
  • 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.
  • 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 as used in the present disclosure will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, including, 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 ribonucleotides, 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.
  • precursor cannabinoid refers to a small molecule which by way of an enzymatic reaction is transformed to generate the cannabinoid of interest.
  • the small molecule may be considered to be a cannabinoid itself.
  • Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e. , 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithm or by manual alignment and visual inspection as described above.
  • the identity exists over a region that is at least about 50 nucleotides (or 20 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 50, 100, or 200 or more amino acids) in length.
  • Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • amino acid sequences one of skill will recognize that individual substitutions in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • amino acid groups defined in this manner can include: a "charged/polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R) and His (Histidine or H); an "aromatic or cyclic group” including Pro (Proline or P), Phe (Phenylalanine or F), Tyr (Tyrosine or Y) and Trp (Tryptophan or W); and an "aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Vai (Valine or V), Leu (Leucine or L), lie (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T) and Cys (Cysteine or C).
  • a "charged/polar group” including Glu (Glutamic acid
  • subgroups can also be identified.
  • the group of charged/polar amino acids can be sub-divided into sub-groups including: the "positively-charged subgroup” including Lys, Arg and His; the "negatively-charged sub-group” comprising Glu and Asp; and the "polar sub-group” comprising Asn and Gin.
  • the aromatic or cyclic group can be sub-divided into sub-groups including: the "nitrogen ring sub-group” comprising Pro, His and Trp; and the "phenyl sub-group” comprising Phe and Tyr.
  • the aliphatic group can be sub-divided into sub-groups including: the "large aliphatic non-polar sub-group” comprising Vai, Leu, and lie; the "aliphatic slightly-polar sub-group” comprising Met, Ser, Thr and Cys; and the "small-residue sub-group” comprising Gly and Ala.
  • conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained.
  • the following six groups each contain amino acids that further provide illustrative conservative substitutions for one another.
  • the terms “conservative mutation,” “conservative substitution,” and “conservative amino acid substitution” refer to a substitution of one or more amino acids for one or more different amino acids that exhibit similar physicochemical properties, such as polarity, electrostatic charge, and steric volume. These properties are summarized for each of the twenty naturally occurring amino acids in Table 1 , below. Table 1. Representative physicochemical properties of naturally occurring amino acids
  • 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.
  • 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.
  • heterologous refers to what is not normally found in nature.
  • heterologous nucleic acid refers to a nucleic acid not normally found in a given cell in nature.
  • a heterologous nucleic acid can be: (a) foreign to its host cell, i.e., exogenous to the host cell such that a host cell does not naturally contain the nucleic acid; (b) naturally found in the host cell, i.e., endogenous or native to the host cell, but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus.
  • a “heterologous” polypeptide refers to a polypeptide that is encoded by a “heterologous nucleic acid.”
  • a “heterologous” polypeptide may be naturally produced by a host cell but is encoded by a heterologous nucleic acid that has been introduced into the host cell by genetic engineering.
  • a “heterologous” polypeptide can include embodiments in which an endogenous polypeptide is produced by an expression construct and is overexpressed in the host cell compared to native levels of the polypeptide produced by the host cell.
  • the term “introducing” in the context of a nucleic acid or protein in a host cell refers to any process that results in the presence of a heterologous nucleic acid or polypeptide inside the host cell.
  • the term encompasses introducing a nucleic acid molecule (e.g., a plasmid or a linear nucleic acid) that encodes the nucleic acid of interest (e.g., an RNA molecule) or polypeptide of interest and results in the transcription of the RNA molecules and translation of the polypeptides.
  • the term also encompasses integrating the nucleic acid encoding the RNA molecules or polypeptides into the genome of a progenitor cell.
  • nucleic acid is then passed through subsequent generations to the host cell, so that, for example, a nucleic acid encoding an RNA-guided endonuclease is “pre-integrated” into the host cell genome.
  • introducing refers to translocation of a nucleic acid or polypeptide from outside the host cell to inside the host cell.
  • Various methods of introducing nucleic acids, polypeptides and other biomolecules into host cells are contemplated, including but not limited to, electroporation, contact with nanowires or nanotubes, spheroplasting, PEG 1000-mediated transformation, biolistics, lithium acetate transformation, lithium chloride transformation, and the like.
  • transformation refers to a genetic alteration of a host cell resulting from the introduction of exogenous genetic material, e.g., nucleic acids, into the host cell.
  • mutation refers to a change in the nucleotide sequence of a gene. Mutations in a gene may occur naturally as a result of, for example, errors in DNA replication, DNA repair, irradiation, and exposure to carcinogens or mutations may be induced as a result of administration of a transgene expressing a mutant gene. Mutations may result from a single nucleotide substitution or deletion.
  • 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.
  • operably linked refers to a functional linkage between nucleic acid sequences such that the sequences encode a desired function.
  • a coding sequence for a gene of interest is in operable linkage with its promoter and/or regulatory sequences when the linked promoter and/or regulatory region functionally controls expression of the coding sequence. It also refers to the linkage between coding sequences such that they may be controlled by the same linked promoter and/or regulatory region; such linkage between coding sequences may also be referred to as being linked in frame or in the same coding frame.
  • “Operably linked” also refers to a linkage of functional but non-coding sequences, such as an autonomous propagation sequence or origin of replication. Such sequences are in operable linkage when they are able to perform their normal function, e.g., enabling the replication, propagation, and/or segregation of a vector bearing the sequence in a host cell.
  • the term “about” is used herein to mean a value that is ⁇ 10% of the recited value.
  • the present disclosure features methods of isolating and purifying one or more cannabinoids from a fermentation composition.
  • the fermentation composition may include host cells capable of producing one or more cannabinoids, such as for example, cannabichromene (CBC).
  • CBC cannabichromene
  • the methods described herein include using an oil overlay in combination with a cannabichromenic acid (CBCa) synthase to carry out near complete conversion of cannabigerolic acid (CBGa) to CBCa.
  • CBCa cannabichromenic acid
  • CBGa cannabigerolic acid
  • the present disclosure additionally features variant CBCa synthase polypeptides having one or more amino acid substitutions.
  • the variant CBCa synthase may have increased enzymatic activity in comparison to the wild-type enzyme.
  • the disclosure provides a method of making a cannabichromenic acid (CBCa).
  • the method may include (a) culturing a population of host cells capable of producing cannabigerolic acid (CBGa) in a culture medium comprising a fermentation broth and an overlay, under conditions suitable for the host cells to produce CBGa, and wherein the CBGa partitions into the overlay; (b) separating the overlay from the fermentation broth; (c) combining the separated overlay of step (b), with a CBCa synthase, thereby producing a bioconversion mixture; and (d) purifying the CBCa from the bioconversion mixture.
  • CBGa cannabigerolic acid
  • the demulsification may include contacting the second mixture containing the cannabinoid with an oil.
  • the oil may be a mineral oil, a vegetable oil, a synthetic ester, or a fatty alcohol.
  • the oil may be a vegetable oil.
  • the vegetable oil is soybean oil, sunflower oil, safflower oil, canola oil, grapeseed oil, or castor oil.
  • the oil is a synthetic ester, optionally wherein the synthetic ester is ESTEREXTM A51 .
  • the oil includes a fatty alcohol, optionally wherein the fatty alcohol is oleyl alcohol or JARCOLTM 1-16.
  • the oil may have a concentration of between about 1% (w/v) and about 10% (w/v) (e.g., about 1% (w/v), 2% (w/v), 3% (w/v), 4% (w/v), 5% (w/v), 6% (w/v), 7% (w/v), 8% (w/v), 9% (w/v), or 10% (w/v)).
  • the oil may have a concentration of is about 5% (w/v).
  • Th overlay further includes CBGa.
  • the CBGa has a concentration of between about 0.1% (w/v) and 10% (w/v) (e.g., between about 0.1 % (w/v) and 8% (w/v), 0.1% (w/v) and 6% (w/v), 0.1% (w/v) and 4% (w/v), 0.1% (w/v) and 2% (w/v), 0.1% (w/v) and 1% (w/v), 1% (w/v) and 10% (w/v), 2% (w/v) and 10% (w/v), 4% (w/v) and 10% (w/v), 6% (w/v) and 10% (w/v), or 8% (w/v) and 10% (w/v)).
  • the CBGa may have a concentration of between about 0.5% (w/v) or and 5% (w/v) (e.g., between about 0.5% (w/v) and 4% (w/v), 0.5% (w/v) and 3% (w/v), 0.5% (w/v) and 2% (w/v), 0.5% (w/v) and 1% (w/v), 1% (w/v) and 5% (w/v), 2% (w/v) and 5% (w/v), 3% (w/v) and 5% (w/v), or 4% (w/v) and 5% (w/v)).
  • the method may include stirring the bioconversion mixture.
  • the bioconversion mixture may be stirred may include stirring the second mixture for between 12 hours and 144 hours (e.g., between 12 hours 120 hours, 12 hours and 96 hours, 12 hours and 72 hours, 12 hours and 48 hours, 12 hours and 24 hours, 24 hours and 144 hours, 48 hours and 144 hours, 72 hours and 144 hours, 96 hours and 144 hours, or 120 hours and 144 hours) prior to purifying the CBCa from the bioconversion mixture.
  • 12 hours and 144 hours e.g., between 12 hours 120 hours, 12 hours and 96 hours, 12 hours and 72 hours, 12 hours and 48 hours, 12 hours and 24 hours, 24 hours and 144 hours, 48 hours and 144 hours, 72 hours and 144 hours, 96 hours and 144 hours, or 120 hours and 144 hours
  • the bioconversion mixture may be stirred for between 24 hours and 96 hours (e.g., between 24 hours and 84 hours, 24 hours and 72 hours, 24 hours and 60 hours, 24 hours and 48 hours, 48 hours and 96 hours, 60 hours and 96 hours, 72 hours and 96 hours, or 84 hours and 96 hours). In some embodiments, the bioconversion mixture may be stirred for about 48 hours.
  • the bioconversion mixture may be stirred at a rate of between about 50 rotations per minute (rpm) and 300 rpm (e.g., between about 50 rpm and 250 rpm, 50 rpm and 200 rpm, 50 rpm and 150 rpm, 50 rpm and 100 rpm, 100 rpm and 250 rpm, 150 rpm and 250 rpm, or 200 rpm and 250 rpm).
  • rpm rotations per minute
  • 300 rpm e.g., between about 50 rpm and 250 rpm, 50 rpm and 200 rpm, 50 rpm and 150 rpm, 50 rpm and 100 rpm, 100 rpm and 250 rpm, 150 rpm and 250 rpm, or 200 rpm and 250 rpm.
  • the bioconversion mixture may be stirred at a rate of about 150 rpm.
  • the second mixture may include one or more amphiphilic moieties in addition to the cannabinoid.
  • the one or more amphiphilic moieties may include a cyclodextrin, plant derived silica, cellulose, or a combination thereof.
  • the cyclodextrin may include randomly methylated cyclodextrin, 2, 6-Di-O-methyl-p-cyclodextrin, or a combination thereof.
  • the final concentration of the enzymatic composition may be from about 0.5% (w/v) to about 3% (w/v) (e.g., about 0.5% (w/v) and 2.5% (w/v), 0.5% (w/v) and 2% (w/v), 0.5% (w/v) and 1 .5% (w/v), 0.5% (w/v) and 1 % (w/v), 1 % (w/v) and 3% (w/v), 1 .5% (w/v) and 3% (w/v), 2% (w/v) and 3% (w/v), or 2.5% (w/v) and 3% (w/v)) after contacting the culture medium including a cannabinoid with the enzymatic composition.
  • the enzymatic composition may have a final concentration of about 1 % (w/v).
  • the demulsification may include centrifugation of the culture medium including a cannabinoid.
  • the centrifugation may include liquid-liquid centrifugation, which may result in a crude oil light phase and an aqueous heavy phase.
  • the variant polypeptide may include an amino acid substitution at residue V294 of SEQ ID NO: 1 .
  • This substitution may include an amino acid including a polar, uncharged side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including an anionic side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 substitutes V294 with an amino acid including a cationic side chain at physiological pH.
  • the amino acid substitution at residue V294 of SEQ ID NO: 1 may be a V294S substitution, a V294E substitution, or V294R substitution.
  • the variant polypeptide may have an amino acid substitution at residue H354 of SEQ ID NO: 1 .
  • the amino acid substitution at residue H354 of SEQ ID NO: 1 includes an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue R340 of SEQ ID NO: 1 may be a H354V substitution.
  • the variant CBCa synthase may include an amino acid substitution at residue L435 of SEQ ID NO: 1 .
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 may substitute L435 with an amino acid including a hydrophobic, uncharged side chain at physiological pH.
  • the amino acid substitution at residue L435 of SEQ ID NO: 1 may be a L435A substitution.
  • the variant polypeptide may have amino acid substitutions including Q75E, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, V169E, N240M, V294S, A299V, K305C, T335L, R340M, L435A, Y4611, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, T335L, R340M, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including T130L, S140A, V169E, V294S, A299V, R340M, H354V, L435A, Y461 I, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, N240M, V294S, A299V, T335L, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, A299V, K305C, R340M, H354V, L435A, Y461 1, and K535M of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, R340M, H354V, L435A, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, T130L, S140A, V169E, N240M, V294S, K305C, T335L, R340M, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including F82I, T130L, S140A, V169E, N240M, V294S, A299V, K305C, T335L, R340M, H354V, L435A, Y461 1, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including F82I, T130L, S140A, V169E, N240M, A299V, T335L, R340M, H354V, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the variant polypeptide may have amino acid substitutions including Q75E, F82I, S140A, V169E, N240M, V294S, A299V, K305C, T335L, L435A, Y461 1, K535M, and S540D of SEQ ID NO: 1 .
  • the CBCa synthase may have an amino acid sequence that is from about 90% to about 99.7% (e.g., about 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1 .
  • the CBCa synthase may have an amino acid sequence that is from about 95% to about 99.7% (e.g., about 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, or 99.7%) identical to the amino acid sequence of SEQ ID NO: 1 .
  • 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: 68-91 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 68-91 ). In some embodiments, the AAE has the amino acid sequence of any one of SEQ ID NO: 68-91 .
  • 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: 68-80 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 68-80). In some embodiments, the AAE has the amino acid sequence of any one of SEQ ID NO: 68-80.
  • 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: 92-95 (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: 92-95).
  • 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: 92-95 (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: 92-95).
  • 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: 127-131 (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: 127-131 ).
  • 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: 127-131 (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: 127-131 ).
  • 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: 127-131 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 127-131 ). In some embodiments, the CBGaS has the amino acid sequence of any one of SEQ ID NO: 127-131 .
  • 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 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: 132-137 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 132-137). In some embodiments, the GPP synthase has the amino acid sequence of any one of SEQ ID NO: 132-137.
  • 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: 132 (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: 132).
  • the GPP synthase has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132 (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 132).
  • the GPP synthase has the amino acid sequence of SEQ ID NO: 132.
  • 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: 138.
  • the OAC has an amino acid sequence of SEQ ID NO: 138.
  • 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, 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 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: 140.
  • the acetyl-CoA synthase has the amino acid sequence of SEQ ID NO: 140.
  • the aldehyde dehydrogenase 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: 141 . In some embodiments, the aldehyde dehydrogenase has the amino acid sequence of SEQ ID NO: 141 . In some embodiments, the pyruvate dehydrogenase 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: 142. In some embodiments, the pyruvate decarboxylase has an amino acid sequence of SEQ ID NO: 142.
  • any one of the polypeptide sequences disclosed herein may be encoded by DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure.
  • 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.
  • 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.
  • the genetic regulatory element is a nucleic acid sequence, such as a promoter.
  • 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, Holtermannia
  • 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. Typically, 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 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.
  • 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.
  • Example 1 Large scale bioconversion of cannabigerolic acid to cannabichromenic acid
  • WGEKYFGKNFNRLVKVKTKADPNNFFRNEQSIPPLPPHHH* SEQ ID NO: 51 - T130L/S140A/V169E/V294S/A299V/R340M/H354V/L435A/Y4611/K535M/S540D

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de production et d'isolement d'un cannabinoïde à partir d'une composition de fermentation comprenant des cellules hôtes capables de produire un cannabinoïde tel que par combinaison d'un mélange comprenant un acide cannabigérolique avec une synthase d'acide cannabichroménique (CBCa), éventuellement dans un mélange d'huile. L'invention concerne également des synthases CBCa ayant une activité améliorée par rapport à l'enzyme de type sauvage.
PCT/US2023/083173 2022-12-09 2023-12-08 Procédés et compositions de purification de cannabinoïdes WO2024124165A2 (fr)

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