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  • C12P7/00—Preparation of oxygen-containing organic compounds
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  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
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  • C12Y404/00—Carbon-sulfur lyases (4.4)
  • C12Y404/01—Carbon-sulfur lyases (4.4.1)
  • C12Y404/01026—Olivetolic acid cyclase (4.4.1.26)
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  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae

Definitions

• processes preferably scalable, commercially relevant processes, of producing olivetol and olivetolic acid, or an analog thereof, or a salt of each thereof by fermentation employing a recombinant heterologous host microorganism.
• Olivetol and olivetolic acid are key gateway molecules for preparing cannabinoids. And yet, there is very little if any report of a scalable, commercially viable, production of olivetol and olivetolic acid by fermentation.
• divarin which is l,3-dihydroxy-5-propylbenzene and divarinic acid, which is 2,4- dihydroxy-6-propylbenzoic acid
• divarinic acid which is 2,4- dihydroxy-6-propylbenzoic acid
• Minor cannabinoids are naturally obtained in quantities smaller than major cannabinoids such as tetrahydrocannabinol (THC).
• THC tetrahydrocannabinol
• R is optionally substituted C 1 -C 8 alkyl, optionally substituted C 2 -C 6 alkenyl, or optionally substituted C 2 -C 8 alkynyl.
• other R groups such as optionally substituted cycloalkyl, preferably optionally substituted C 3 -C 8 cycloalkyl; optionally substituted heterocyclyl; optionally substituted aryl, preferably optionally substituted phenyl; and optionally substituted heteroaryl are contemplated as employed according to the present invention.
• the compound produced is of formula IA.
• the compound produced is of formula IB.
• Certain of these processes utilize a recombinant, heterologous, host cells or host microorganism.
• Certain of the host microorganisms comprise a recombinant olivetol synthase (OLS or OS), which is a tetraketide synthase (TKS).
• Certain of the host microorganisms comprise a recombinant Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS).
• Certain of the host microorganisms comprise a recombinant olivetolic acid cyclase (OAC). Certain of the heterologous microorganisms comprise a recombinant Cannabis sativa olivetolic acid cyclase (csOAC). Certain of the host microorganisms comprise an acyl activating enzyme (AAE). Certain of the heterologous microorganisms comprise a recombinant Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAEl.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csOLS. In another embodiment, the recombinant host microorganism comprises 4-20, or 6-16 copies of csOAC. In another embodiment, the recombinant host microorganism comprises 4-20, or 6-16 copies of csAAEl.
• glucose is fermented.
• galactose is fermented.
• the process further comprises a carboxylic acid of formula R-CO2H or a salt thereof.
• the microorganism is a yeast.
• the microorganism is Socchoromyces cerevisiae.
• the microorganism is a bacteria.
• the microorganism is Escherichia coli.
• the process further comprises contacting: an aqueous phase comprising glucose and RCO2H or a salt thereof and an organic phase immiscible with the aqueous phase.
• R is Ci-Cs alkyl. In another embodiment, R is C1-C4 alkyl. In another embodiment, R is C 6 -C 8 alkyl. In another embodiment, R is substituted Ci-Cs alkyl.
• R is C 2 -C 8 alkenyl. In another embodiment, R is substituted C 2 - C 8 alkenyl.
• R is C 2 -C 8 alkynyl. In another embodiment, R is substituted C 2 - Cg alkynyl.
• the compounds of formula IA and IB are provided in a combined amount of at least about 2 g/liter over about 4 to about 7 days. In another embodiment, the compounds of formula IA and IB are provided in a combined amount of at least about 3 g/liter over about 4 to about 7 days. In another embodiment, the compounds of formula IA and IB are provided in a combined amount of at least about4 g/liter over about 4 to about 7 days. In another embodiment, the compounds of formula IA and IB are provided in a combined amount of at least about 5 g/liter over about 4 to about 7 days. In another embodiment, the compounds of formula IA and IB are provided in a combined amount of at least about 10 g/liter over about 4 to about 7 days.
• This invention arises in part from the surprising discovery that recombinant host microorganisms produce commercially relevant amounts of olivetol and olivetolic acid by fermentation.
• processes of producing olivetol, olivetolic acid, or a salt thereof are commercially viable for producing olivetol, olivetolic acid or a salt thereof, which are key gateway compounds for preparing a variety of cannabinoids.
• Certain of these processes utilize a recombinant, heterologous, host microorganism.
• Certain of the host microorganisms include a recombinant Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS).
• heterologous microorganisms include a recombinant Cannabis sativa olivetolic acid cyclase (csOAC).
• Certain of the heterologous microorganisms include a recombinant Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAEl.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csOLS.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csOAC.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csAAEl.
• glucose is fermented.
• galactose is fermented.
• the fermentation further comprises hexanoic acid or a salt thereof. Certain of these processes provide olivetol and olivetolic acid in a combined amount of at least 3 g/liter.
• the microorganism is Saccharomyces cerevisiae.
• This invention arises in another part from the surprising discovery that recombinant host microorganisms produce divarin and divarinic acid by fermentation.
• processes of producing divarin and/or divarinic acid or a salt thereof are commercially viable for producing divarin and divarinic acid, which are key gateway compounds for preparing a variety of minor cannabinoids.
• Certain of these processes utilize a recombinant, heterologous, host microorganism.
• Certain of the host microorganisms include a recombinant Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS).
• heterologous microorganisms include a recombinant Cannabis sativa olivetolic acid cyclase (csOAC).
• Certain of the heterologous microorganisms include a recombinant Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAEl.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csOLS.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csOAC.
• the recombinant host microorganism comprises 4-20, or 6-16 copies of csAAEl.
• glucose is fermented.
• the fermentation further comprises butyric acid or a salt thereof.
• these processes provide divarin and/or divarinic acid or a salt thereof in a combined amount of at least about 0.25 - about 8 g/liter, about 1 - about 7 g/liter, about 0.25 - about 2 g/liter, about 0.25 - about 2 g/liter, about 0.5 - about 1 g/liter, or about 2 - about 4 g/liter.
• Certain of these processes provide divarin and/or divarinic acid or a salt thereof in a combined amount of at least about 2 - about 5, preferably about 3 - about 4 g/liter.
• the combined amount of divarin and/or divarinic acid is provided over 2-7 or 4-7 days, such as 2,3, 4, 5, 6, or 7 days.
• the microorganism is Saccharomyces cerevisiae.
• the fermentation is performed as a batch/fed batch fermentation with a fixed batch duration. In one embodiment, the fermentation is performed as a "semi- continuous" fermentation operating mode. In one embodiment, the fermentation is performed as a continuous fermentation operating mode.
• the continuous mode may be a fill-and-draw, or a true continuous operation.
• a mixture of compounds of formula IA and IB provided by fermentation is extracted from a fermentation media by alkaline extraction.
• the alkaline extraction is an aqueous alkaline extraction.
• the alkaline extraction is performed at a pH of about 12 - about 14.
• the alkaline extraction is performed at a pH of about 13.
• the alkaline extraction is performed under milder alkaline conditions.
• the alkaline extraction is performed at a pH of about 7 - about 12.
• the alkaline extraction is performed at a pH of about 7 - about 10.
• the extracted mixture of compounds of formula IA and IB are decarboxylated to provide a compound of formula IA.
• the decarboxylation is performed by heating. In some embodiments, the heating is performed at about 100°C - about 140°C, or preferably at about 110°C - about 130°C. In some embodiments, the heating is performed at about 120°C.
• Post decarboxylation, the compound of formula IA provided comprises by weight about 2% or less, or preferably about 1% or less of a compound of formula IB or a salt thereof.
• the extracted mixture of compounds of formula IA and IB are acidified before decarboxylation. In some embodiments, the decarboxylation is performed at a pH of about 5 - about 8. In some embodiments, the decarboxylation is performed at a pH of about 6.5.
• the compound of formula IA provided by decarboxylation is extracted into an organic solvent (e.g., a water immiscible organic solvent) to provide a solution of the compound of formula IA in the organic solvent.
• the organic solvent is a solvent capable of dissolving a compound of formula IA; formula IA comprises an aromatic ring and polar hydroxy groups.
• the organic solvent comprises an aromatic hydrocarbon solvent.
• the organic solvent comprises toluene.
• the organic solvent is toluene.
• the organic solvent comprises aliphatic or alicyclic hydrocarbon solvents.
• the compound of formula IA present as a solution in the organic solvent, is reacted with a terpene alcohol, a terpenal (i.e., a terpene aldehyde), and the likes.
• the solution of the compound of formula IA in the organic solvent is employed for reacting the compound of formula IA with a terpene alcohol.
• the solution of the compound of formula IA in the organic solvent is employed for reacting the compound of formula IA with a terpenal.
• the terpene alcohol is geraniol.
• the terpene alcohol is farnesol.
• the terpene alcohol is menthadienol (trans 2,8-menthadienol or PMD). In one embodiment, the terpene alcohol is or a diastereomer thereof, or an ester of each thereof. In one embodiment, the the hydroxy form (unesterified) is employed. In one embodiment, the terpene alcohol is:
• the terpenal is citral.
• the reaction with a terpenal further comprises a primary amine.
• the primary amine is tertiary butyl amine.
• the reaction of a compound of formula IA with a terpene alcohol, a terpenal, or the likes provides a cannabinoid.
• the cannabinoid is cannabigerol (CBG).
• the cannabinoid is cannabichromene (CBC).
• the cannabinoid is cannabidiol (CBD).
• the cannabinoid is tetrahydrocannabinol (THC).
• the cannabinoid is cannabinol (CBN).
• the cannabinoid is the varin analog (CBGV, CBCV, CBDV, THCV, CBNV) of CBG, CBC, CBD, THC, CBN.
• a varin analog is a compound where the n- pentyl chain of a cannabinoid, e.g., and without limitation, CBG, CBC, CBD, or THC is replaced by an n-propyl chain.
• the cannabinoids obtained are purified by a variety of purification methods.
• the purification method comprises chromatography.
• the purification method comprises distillation.
• the chromatography comprises a reverse phase chromatography.
• R is n-pentyl. In another embodiment, R is n-propyl. In another embodiment, R is n-heptyl.
• the initial engineering of Saccharomyces cerevisiae was done by introducing a gene fragment containing csOLS, csOAC, and csAAEl under the control of galactose regulatable elements called promoters.
• the csOLS and csOAC were physically linked to each other on the gene with a genetic element called T2A in all examples.
• CEN.PK is useful as a wild type Saccharomyces cerevisiae.
• Saccharomyces cerevisiae uptakes foreign DNA and stably utilize the foreign DNA is called recombination.
• the final Saccharomyces cerevisiae strains that took up the foreign DNA and utilized the DNA are called recombinants.
• Saccharomyces cerevisiae that did not undergo recombination are the wild type.
• the process of selecting recombinants in a preferred media is termed prototrophy rescue. To separate recombinants from wild type prototrophy rescue was utilized.
• Examples herein below provide a method to create recombinants that produce various levels of O/OA by varying how many of those gene fragments are uptaken by JK9-3d.
• recombinants that produce O/OA under the control of galactose are disclosed.
• Another example discloses, how the number of exogenous fragments taken up from Saccharomyces cerevisiae correlates with O/OA concentrations in the media.
• the number of genetic fragments recombinants contain can be determined by sequencing the DNA of the recombinant and by using the PCR method to quantify the number of amplicons generated. Amplicons are quantified by quantitative real time polymerase chain reaction (qPCR). qPCR and direct sequencing, and how those quantitative values of the genetic elements relate to the quantitative levels of O and OA are exemplified and provided herein.
• Figure 1 schematically illustrates recovery of olivetol and other compounds of formula IA in accordance with the present invention.
• FIG. 2 schematically illustrates the semisynthesis of cannabinoids (CBG) by prenylation of fermented olivetol.
• FIG. 3 schematically illustrates the semisynthesis of cannabinoids (CBC) by prenylation of fermented olivetol.
• Figure 4A graphically illustrates the time course of total product titer (olivetol and olivetolic acid) in g/L.
• Figure 4B graphically illustrates the time course of titer/time (g/L/day).
• Figure 5A graphically illustrates the time course of total product titer (divarin and divarinic acid) in g/L.
• Figure 5B graphically illustrates the time course of titer/time (g/L/day).
• an "expression vector” includes a single expression vector as well as a plurality of expression vectors, either the same ( e.g the same operon) or different; reference to "cell” includes a single cell as well as a plurality of cells; and the like.
• compositions consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method.
• Consisting of shall mean excluding more than trace elements of other ingredients. Embodiments defined by each of these transition terms are within the scope of this technology.
• Alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. Higher carbon atom containing alkyl groups are also contemplated in certain embodiments, as the context will indicate.
• This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2), -n- propyl- (CH3CH2CH2-), isopropyl ((CH3)CH), -n-butyl- (CH3CH2CH2CH2-), isobutyl ((CH 3 )2CHCH 2 -), sec-butyl ((CH3)(CH 3 CH 2 )CH), -t-butyl- ((CH 3 ) 3 C), -n- pentyl- (CH3CH2CH2CH2CH2- ), and neopentyl ((CH 3 ) 3 CCH 2 -).
• linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2), -n- propyl- (CH3CH2CH2-), isopropyl ((CH3)CH), -n-butyl- (CH3CH2CH2
• Alkynyl refers to straight or branched monovalent hydrocarbyl groups having from 2 to 10 carbon atoms and preferably 2 to 6 carbon atoms or preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of acetylenic (-CoC-) unsaturation. Higher carbon atom containing alkynyl groups are also contemplated in certain embodiments, as the context will indicate. Examples of such alkynyl groups include acetylenyl (-CoCH), and propargyl (-CH2CoCH).
• Substituted alkyl refers to an alkyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
• Heteroalkyl refers to an alkyl group one or more carbons is replaced with -0-, -S-, SO2, a phosphorous (P) containing moiety, or -NR Q - moieties where R Q is H or C1-C6 alkyl.
• Substituted heteroalkyl refers to a heteroalkyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkyl
• Substituted alkenyl refers to alkenyl groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio,
• Heteroalkenyl refers to an alkenyl group where one or more carbons is replaced with one or more -0-, -S-, SO 2 , P containing moiety, or -NR Q - moieties where R Q is H or C 1 -C 6 alkyl.
• Substituted heteroalkenyl refers to a heteroalkenyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloal
• Substituted alkynyl refers to alkynyl groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkyloxy
• Heteroalkynyl refers to an alkynyl group one or more carbons is replaced with -O- , -S- , SO2, P containing moiety, or -NR Q - moieties where R Q is H or C1-C6 alkyl.
• Substituted heteroalkynyl refers to a heteroalkynyl group having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycl
• Alkylene refers to divalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms, preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained- or branched. Higher carbon atom containing alkenyl groups are also contemplated in certain embodiments, as the context will indicate.
• alkenylene and alkynylene refer to an alkylene moiety containing respective 1 or 2 carbon-carbon double bonds or a carbon-carbon triple bond.
• Substituted alkylene refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxyl ester, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, and oxo wherein said substituents are defined herein.
• Alkynylene refers to straight or branched divalent hydrocarbyl groups having from 2 to 10 carbon atoms and preferably 2 to 6 carbon atoms or preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of acetylenic (-CoC-) unsaturation. Higher carbon atom containing alkynylene groups are also contemplated in certain embodiments, as the context will indicate. Examples of such alkynylene groups include -CoC- and -CH2CoC-.
• Substituted alkynylene refers to alkynylene groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkyloxy
• Heteroalkylene refers to an alkylene group wherein one or more carbons is replaced with -0-, -S-, SO 2 , a P containing moiety, or -NR Q - moieties where R Q is H or C 1 -C 6 alkyl.
• Substituted heteroalkylene refers to heteroalkynylene groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the substituents disclosed for substituted alkylene.
• Heteroalkenylene refers to an alkenylene group wherein one or more carbons is replaced with -0-, -S-, SO 2 , a P containing moiety, or -NR Q - moieties where R Q is H or C 1 -C 6 alkyl.
• Substituted heteroalkenylene refers to heteroalkynylene groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the substituents disclosed for substituted alkenylene.
• Heteroalkynylene refers to an alkynylene group wherein one or more carbons is replaced with -0-, -S-, SO 2 , a P containing moiety, or -NR Q - moieties where R Q is H or C 1 -C 6 alkyl.
• Substituted heteroalkynylene refers to heteroalkynylene groups having from 1 to 3 substituents, and preferably 1 to 2 substituents, selected from the substituents disclosed for substituted alkynylene.
• Alkoxy refers to the group -O-alkyl wherein alkyl is defined herein. Alkoxy includes, by way of example, methoxy-, ethoxy, n-propoxy, isopropoxy, n-butoxy, t- butoxy, -sec-butoxy, and- n-pentoxy. "Substituted alkoxy” refers to the group -0-(substituted alkyl) wherein substituted alkyl is defined herein.
• Acyl refers to the groups H-C(O), -alkyl-C-(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl- C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclic-C(O), and substituted- heterocyclic-C-(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substitute
• Acylamino refers to the groups -NR 40 C(O)alkyl, -NR 40 C(O)substituted alkyl, - NR 40 C(O)cycloalkyl, -NR 40 C(O)substituted cycloalkyl, -NR 40 C(O)cycloalkenyl, - NR 40 C(O)substituted cycloalkenyl, -NR 40 C(O)alkenyl, -NR 40 C(O)substituted alkenyl, - NR 40 C(O)alkynyl, -NR 40 C(O)substituted alkynyl, -NR 40 C(O)aryl, -NR 40 C(O)substituted aryl, - NR 40 C(O)heteroaryl, -NR 40 C(O)substituted heteroaryl, -NR 40 C(O)heterocyclic, and
• Acyloxy refers to the groups alkyl-C-(0)0, substituted-alkyl-C-(0)0-, alkenyl-C(0)0-, substituted alkenyl-C(0)0-, alkynyl-C(0)0-, substituted alkynyl-C(0)0-, aryl-C(0)0, substituted- aryl-C-(0)0-, cycloalkyl-C(0)0-, substituted cycloalkyl-C(0)0-, cycloalkenyl-C(0)0-, substituted cycloalkenyl-C(0)0-, heteroaryl-C(0)0-, substituted heteroaryl-C(0)0, -heterocyclic-C-(0)0, and substituted-heterocyclic-C-(0)0- wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkenyl, substituted substituted substituted substituted substituted al
• amino refers to the group -IMH2.
• substituted amino refers to the group -NR 41 R 42 where R 41 and R 40 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, -SCh-alkyl, -SCh-substituted alkyl, -SCh-alkenyl, -SCh-substituted alkenyl, -SCh-cycloalkyl, -SCh-substituted cylcoalkyl, -SCh-cycloalkenyl, -S0 2 -substituted c
• R 41 is hydrogen and R 42 is alkyl
• the substituted amino group is sometimes referred to herein as alkylamino.
• R 41 and R 42 are alkyl
• the substituted amino group is sometimes referred to herein as dialkylamino.
• a monosubstituted amino it is meant that either R 41 or R 42 is hydrogen but not both.
• a disubstituted amino it is meant that neither R 41 nor R 42 are hydrogen.
• Aminocarbonyl refers to the group -C(O)NR 50 R 51 where R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl
• Aminothiocarbonyl refers to the group -C(S)NR 50 R 51 where R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted substituted
• Aminocarbonylamino refers to the group -NR 40 C(O)NR 50 R 51 where R 40 is hydrogen or alkyl and R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic, and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkenyl, substituted cyclo
• Aminothiocarbonylamino refers to the group -NR 40 C(S)NR 50 R 51 where R 40 is hydrogen or alkyl and R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cyclo
• Aminocarbonyloxy refers to the group -O-C(O)NR 50 R 51 where R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R50 and R51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted
• Aminosulfonyl refers to the group -SO2NR 50 R 51 where R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl
• Aminosulfonyloxy refers to the group -O-SO2NR 50 R 51 where R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted
• Aminosulfonylamino refers to the group -NR 40 SO2NR 50 R 51 where R 40 is hydrogen or alkyl and R 50 and R 51 are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R 50 and R 51 are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cyclo
• Aryl or “Ar” refers to an aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-l,4-benzoxazin- 3(4H)-one-7-yl, and the like) provided that the point of attachment is at an aromatic carbon atom.
• Certain, preferred aryl groups include phenyl and naphthyl.
• Substituted aryl refers to aryl groups which are substituted with 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloal
• Arylene refers to a divalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring or multiple condensed rings.
• Substituted arylene refers to an arylene having from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents as defined for aryl groups.
• Heteroarylene refers to a divalent aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring.
• Substituted heteroarylene refers to heteroarylene groups that are substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl. Unless otherwise noted, the context will clearly indicate, whether an aryl or heteroaryl moiety is monovalent or divalent.
• Aryloxy refers to the group-O-aryl-, where aryl is as defined herein, that includes, by way of example, phenoxy and naphthoxy.
• Substituted aryloxy refers to the group -0-(substituted aryl) where substituted aryl is as defined herein.
• Arylthio refers to the group-S-aryl-, where aryl is as defined herein.
• Substituted arylthio refers to the group -S-(substituted aryl), where substituted aryl is as defined herein.
• Carboxyl or”carboxy refers to -COOH or salts thereof.
• Carboxyl ester or “carboxy ester” refers to the group -C(0)(0)-alkyl, -C(0)(0)- substituted alkyl, -C(0)0-alkenyl, -C(0)(0)-substituted alkenyl, -C(0)(0)-alkynyl, - C(0)(0)- substituted alkynyl, -C(0)(0)-aryl, -C(0)(0)-substituted-aryl, -C(0)(0)-cycloalkyl, -C(0)(0)- substituted cycloalkyl, -C(0)(0)-cycloalkenyl, -C(0)(0)-substituted cycloalkenyl, -C(0)(0)- heteroaryl, -C(0)(0)-substituted heteroaryl, -C(0)(0)-heterocyclic, and -C(0)(0)- substituted heterocyclic wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl
• (Carboxyl ester)amino refers to the group-NR 40 C(O)(O)-alkyl, -NR 40 C(O)(O)- substituted alkyl, -NR 40 C(O)O-alkenyl, -NR 40 C(O)(O)-substituted alkenyl, -NR 40 C(O)(O)- alkynyl, - NR 40 C(O)(O)-substituted alkynyl, -NR 40 C(O)(O)-aryl, -NR 40 C(O)(O)- substituted-aryl, - NR 40 C(O)(O)-cycloalkyl, -NR 40 C(O)(O)-substituted cycloalkyl, -NR 40 C(O)(O)-cycloalkenyl, - NR 40 C(O)(O)-substituted cycloalkenyl, -NR 40 C
• (Carboxyl ester)oxy refers to the group -0-C(0)0-alkyl, -0-C(0)0-substituted alkyl, -O- C(0)0-alkenyl, -0-C(0)0-substituted alkenyl, -0-C(0)0-alkynyl, -0-C(0)(0)- substituted alkynyl, -0-C(0)0-aryl, -0-C(0)0-substituted-aryl, -0-C(0)0-cycloalkyl, - 0-C(0)0-substituted cycloalkyl, -0-C(0)0-cycloalkenyl, -0-C(0)0-substituted cycloalkenyl, -0-C(0)0-heteroaryl, -0C(0)-0- substituted heteroaryl, -0C(0)-0- heterocyclic-, and -0C(0)-0-substituted heterocyclic wherein alkyl, substituted alkyl, alkenyl, substituted al
• Cyano refers to the group -CN.
• Cycloalkyl refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems, and further includes cycloalkenyl.
• the fused ring can be an aryl ring provided that the non aryl part is joined to the rest of the molecule.
• suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclooctyl.
• Substituted cycloalkyl and “substituted cycloalkenyl” refers to a cycloalkyl or cycloalkenyl group having from 1 to 5 or preferably 1 to 3 substituents selected from the group consisting of oxo, thioxo, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl,
• Cycloalkyloxy refers to-O-cycloalkyl-.
• Substituted cycloalkyloxy refers to -0-(substituted cycloalkyl).
• Cycloalkylthio refers to -S-cycloalkyk
• Substituted cycloalkylthio refers to -S-(substituted cycloalkyl).
• Cycloalkenyloxy refers to-O-cycloalkenyl-.
• Substituted cycloalkenyloxy refers to -0-(substituted cycloalkenyl).
• Cycloalkenylthio refers to-S-cycloalkenyl-.
• Substituted cycloalkenylthio refers to -S-(substituted cycloalkenyl).
• Halo or halogen refers to fluoro, chloro, bromo and iodo.
• Heteroaryl refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen and sulfur within the ring.
• Such heteroaryl groups can have a single ring (e.g., pyridinyl orfuryl) or multiple condensed rings (e.g., indolizinyl or benzothienyl) wherein the condensed rings may or may not be aromatic and/or contain a heteroatom provided that the point of attachment is through an atom of the aromatic heteroaryl group.
• the nitrogen and/or the sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N—O), sulfinyl, or sulfonyl moieties.
• N—O N-oxide
• sulfinyl N-oxide
• sulfonyl moieties N-oxide
• Certain non-limiting examples include pyridinyl, pyrrolyl, indolyl, thiophenyl, oxazolyl, thizolyl, and- furanyl.
• Substituted heteroaryl refers to heteroaryl groups that are substituted with from 1 to 5, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.
• Heteroaryloxy refers to -O-heteroaryl.
• Substituted heteroaryloxy refers to the group -0-(substituted heteroaryl).
• Heteroarylthio refers to the group-S-heteroaryl-.
• Substituted heteroarylthio refers to the group -S-(substituted heteroaryl).
• Heterocycle or “heterocyclic” or “heterocycloalkyl” or “heterocyclyl” refers to a saturated or partially saturated, but not aromatic, group having from 1 to 10 ring carbon atoms and from 1 to 4 ring heteroatoms selected from the group consisting of nitrogen, sulfur, or oxygen. Heterocycle encompasses single ring or multiple condensed rings, including fused bridged and spiro ring systems. In fused ring systems, one or more the rings can be cycloalkyl, aryl, or heteroaryl provided that the point of attachment is through a nonaromatic ring. In one embodiment, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the-N-oxide, sulfinyl-, or sulfonyl moieties.
• Substituted heterocyclic or “substituted heterocycloalkyl” or “substituted heterocyclyl” refers to heterocyclyl groups that are substituted with from 1 to 5 or preferably 1 to 3 of the same substituents as defined for substituted cycloalkyl.
• Heterocyclyloxy refers to the group -O-heterocycyl.
• Substituted heterocyclyloxy refers to the group -0-(substituted heterocycyl).
• Heterocyclylthio refers to the group -S-heterocycyl.
• Substituted heterocyclylthio refers to the group -S-(substituted heterocycyl).
• heterocycle and heteroaryls include, but are not limited to, azetidine, pyrrole, furan, thiophene, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydrois
• Niro refers to the group -NO2.
• Phenylene refers to a divalent aryl ring, where the ring contains 6 carbon atoms.
• Substituted phenylene refers to phenylenes which are substituted with 1 to 4, preferably 1 to 3, or more preferably 1 to 2 substituents selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cyclo
• Spirocycloalkyl and “spiro ring systems” refers to divalent cyclic groups from 3 to 10 carbon atoms having a cycloalkyl or heterocycloalkyl ring with a spiro union (the union formed by a single atom which is the only common member of the rings).
• Sulfonyl refers to the divalent group -S(0) 2 -.
• Substituted sulfonyl refers to the group -SCh-alkyl-, -SO2- substituted-alkyl, -50 2 - alkenyl, -S0 2 -substituted alkenyl, -S0 2 -cycloalkyl, -S0 2 -substituted cylcoalkyl, -S0 2 -cycloalkenyl, -S0 2 -substituted cylcoalkenyl, -S0 2 -aryl, -S0 2 -substituted aryl, -S0 2 -heteroaryl, -S0 2 -substituted heteroaryl, -S0 2 -heterocyclic, -S0 2 -substituted-heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted
• Substituted sulfonyloxy refers to the group -OSCh-alkyl, -OSCh-substituted- alkyl, - 0S0 2 -alkenyl, -OSCh-substituted alkenyl, -OSCh-cycloalkyl, -0S0 2 -substituted cylcoalkyl, -OSO 2 - cycloalkenyl, -OSCh-substituted cylcoalkenyl,-OS0 2 -aryl, -0S0 2 -substituted aryl, -OSO 2 - heteroaryl, -OSCh-substituted heteroaryl, -OS0 2 -heterocyclic, -0S0 2 -substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alky
• Thioacyl refers to the groups H-C(S)-, alkyl-C(S)-, substituted alkyl-C(S), -alkenyl-C-(S), substituted-alkenyl-C-(S)-, alkynyl-C(S)-, substituted alkynyl-C(S)-, cycloalkyl-C(S)-, substituted cycloalkyl-C(S)-, cycloalkenyl-C(S)-, substituted cycloalkenyl-C(S)-, aryl-C(S)-, substituted aryl- C(S)-, heteroaryl-C(S)-, substituted heteroaryl-C(S)-, heterocyclic-C(S), and substituted- heterocyclic-C-(S)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substitute
• Thiol refers to the group -SH.
• Alkylthio refers to the group-S-alkyl- wherein alkyl is as defined herein.
• Substituted alkylthio refers to the group -S-(substituted alkyl) wherein substituted alkyl is as defined herein.
• Optionally substituted refers to a group selected from that group and a substituted form of that group.
• Substituents are such as those defined hereinabove.
• substituents can be selected from monovalent and divalent groups, such as, C 1 -C 10 or C 1 -C 6 alkyl, substituted C 1 -C 10 or C 1 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 6 -C 10 aryl, C 3 -C 8 cycloalkyl, C 2 -C 10 heterocyclyl, C 1 -C 10 heteroaryl, substituted C 2 -C 6 alkenyl, substituted C 2 -C 6 alkynyl, substituted C 6 -C 10 aryl, substituted C 3 -C 8 cycloalkyl, substituted C 2 -C 10 heterocyclyl, substituted C 1 -C 10 heteroaryl, halo,
• impermissible substitution patterns e.g., methyl substituted with 5 fluoro groups.
• impermissible substitution patterns are well known to the skilled artisan.
• a “salt” is derived from a variety of organic and inorganic counter ions well known in the art and include, when the compound has an acidic functionality, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium; and when the molecule has a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Salts include acid addition salts formed with inorganic acids or organic acids.
• Inorganic acids suitable for forming acid addition salts include, by way of example and not limitation, hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.
• hydrohalide acids e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, etc.
• sulfuric acid nitric acid
• phosphoric acid phosphoric acid
• Organic acids suitable for forming acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4- hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2- ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2- naphthalenes
• Salts also include salts formed when an acidic proton present in the parent compound is either replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion) or by an ammonium ion (e.g., an ammonium ion derived from an organic base, such as, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, dimethylamine, diethylamine, triethylamine, and ammonia).
• a metal ion e.g., an alkali metal ion, an alkaline earth metal ion, or an aluminum ion
• an ammonium ion e.g., an ammonium ion derived from an organic base, such as, ethanolamine, diethanolamine, triethanolamine, morpholine, piperidine, dimethylamine, diethylamine, triethylamine, and ammonia.
• Amino acids in a protein coding sequence are identified herein by the following abbreviations and symbols. Specific amino acids are identified by a three-letter abbreviation, as follows: Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine, Gin is glutamine, Glu is glutamic acid, Gly is glycine, His is histidine, Leu is leucine, lie is isoleucine, Lys is lysine, Met is methionine, Phe is phenylalanine, Pro is proline, Ser is serine, Thr is threonine, Trp is tryptophan, Tyr is tyrosine, and Val is valine, or by a one-letter abbreviation, as follows: A is alanine, R is arginine, N is asparagine, D is aspartic acid, C is cysteine, Q is glutamine, E is glutamic acid, G is
• a dash (-) in a consensus sequence indicates that there is no amino acid at the specified position.
• a plus (+) in a consensus sequence indicates any amino acid may be present at the specified position.
• a plus in a consensus sequence herein indicates a position at which the amino acid is generally non-conserved; a homologous enzyme sequence, when aligned with the consensus sequence, can have any amino acid at the indicated "+" position.
• Specific amino acids in a protein coding sequence are identified by their respective one-letter abbreviation followed by the amino acid position in the protein coding sequence where 1 corresponds to the amino acid (typically methionine) at the N-terminus of the protein. For example, G204 in C.
• sativa wild type OLS refers to the glycine at position 204 from the OLS N-terminal methionine (/.e., Ml). Amino acid substitutions (i.e., point mutations) are indicated by identifying the mutated (i.e., progeny) amino acid after the one-letter code and number in the parental protein coding sequence; for example, G204A in C. sativa OLS refers to substitution of glycine by alanine at position 204 in the OLS protein coding sequence. The mutation may also be identified in parentheticals, for example OLS (G204A).
• OLS G204A/Q205N indicates that mutations G204A and Q205N are both present in the OLS protein coding sequence.
• the number of mutations introduced into some examples has been annotated by a dash followed by the number of mutations, preceding the parenthetical identification of the mutation (e.g., B1Q2B6-1 (G204A)).
• B1Q2B6-1 (G204A) B1Q2B6 (G204A)).
• the term “express”, when used in connection with a nucleic acid encoding an enzyme or an enzyme itself in a cell, means that the enzyme, which may be an endogenous or exogenous (heterologous) enzyme, is produced in the cell.
• the term “overexpress”, in these contexts, means that the enzyme is produced at a higher level, i.e., enzyme levels are increased, as compared to the wild type, in the case of an endogenous enzyme.
• overexpression of an enzyme can be achieved by increasing the strength or changing the type of the promoter used to drive expression of a coding sequence, increasing the strength of the ribosome binding site or Kozak sequence, increasing the stability of the mRNA transcript, altering the codon usage, increasing the stability of the enzyme, and the like.
• expression vector refers to a nucleic acid and/or a composition comprising a nucleic acid that can be introduced into a host cell, e.g., by transduction, transformation, or infection, such that the cell then produces (“expresses") nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell, that are contained in or encoded by the nucleic acid so introduced.
• an "expression vector” contains nucleic acids (ordinarily DNA) to be expressed by the host cell.
• the expression vector can be contained in materials to aid in achieving entry of the nucleic acid into the host cell, such as the materials associated with a virus, liposome, protein coating, or the like.
• Expression vectors suitable for use in various aspects and embodiments include those into which a nucleic acid sequence can be, or has been, inserted, along with any preferred or required operational elements.
• an expression vector can be transferred into a host cell and, typically, replicated therein (although, one can also employ, in some embodiments, non- replicable vectors that provide for "transient" expression).
• an expression vector that integrates into chromosomal, mitochondrial, or plastid DNA is employed.
• an expression vector that replicates extrachromosomally is employed.
• Typical expression vectors include plasmids, and expression vectors typically contain the operational elements required for transcription of a nucleic acid in the vector. Such plasmids, as well as other expression vectors, are described herein or are well known to those of ordinary skill in the art.
• ferment refers herein to describe culturing host cells and microbes under conditions to produce useful chemicals, including but not limited to conditions under which microbial growth, be it aerobic or anaerobic, occurs.
• heterologous refers to a material that is non-native to a cell.
• a nucleic acid is heterologous to a cell, and so is a "heterologous nucleic acid" with respect to that cell, if at least one of the following is true: (a) the nucleic acid is not naturally found in that cell (that is, it is an "exogenous” nucleic acid); (b) the nucleic acid is naturally found in a given host cell (that is, “endogenous to”), but the nucleic acid or the RNA or protein resulting from transcription and translation of this nucleic acid is produced or present in the host cell in an unnatural ( e.g ., greater or lesser than naturally present) amount; (c) the nucleic acid comprises a nucleotide sequence that encodes a protein endogenous to a host cell but differs in sequence from the endogenous nucleotide sequence that encodes that same protein (having the same or substantially the
• a protein is heterologous to a host cell if it is produced by translation of RNA or the corresponding RNA is produced by transcription of a heterologous nucleic acid; a protein is also heterologous to a host cell if it is a mutated version of an endogenous protein, and the mutation was introduced by genetic engineering.
• host cell and "host microorganism” are used interchangeably herein to refer to a living cell that can perform one or more steps of the cannabinoid pathway, e.g. and without limitation, converting malonyl-CoA and hexanoyl-CoA (or another acyl-CoA) to olivetol and olivetolic acid.
• a host cell can be (or is) transformed via insertion of an expression vector.
• a host microorganism or cell as described herein may be a prokaryotic cell (e.g., a microorganism of the kingdom Eubacteria) or a eukaryotic cell.
• a prokaryotic cell lacks a membrane-bound nucleus, while a eukaryotic cell has a membrane- bound nucleus.
• a host cell is part of a multi-cellular organism.
• PKSs Polyketide synthases
• OLS organic radical synthase
• OS olivetol synthase
• TKS tetraketide synthase
• olivetolic synthase as described herein or elsewhere typically refers to any enzyme capable of converting three molecules of malonyl-CoA and one molecule of hexanoyl-CoA or another acyl-CoA to olivetol or an olivetol analog.
• a wild type example of an OLS is the native C. sotivo OLS enzyme (UniProt ID: B1Q2B6; SEQ ID NO: 1).
• Olivetolic acid cyclase (“OAC”, EC: 4.4.1.26) is a polyketide cyclase derived from C. sativa which functions in concert with an OLS enzyme or a tetraketide synthase ("TKS”) to form OLA. See, e.g.:
• Sequence ID 2A OAC MAVKHLIVLKFKDE!TEAQKEEFFKTYVNLVN!!PAMKDVYWGKDVTQKKEEGYTHIVEVTFESVETIQDYI!HP
• cannabinoid pathway refers gene rally to a biosynthetic pathway that facilitates the synthesis and production of olivetol, olivetolic acid, and olivetolic acid-derived compounds.
• This biosynthetic pathway utilizes a variety of enzymes, catalysts, and intermediate compounds.
• cannabigerolic acid synthase EC: 2.5.1.102
• cannabigerolic acid synthase is used to convert OA to cannabigerolic acid, which is a key intermediate acted upon by a variety of enzymes during THC synthesis.
• Cannabidiolic acid synthase (EC: 1.21.3.7) is used to convert cannabigerolic acid into cannabidiolic acid.
• Tetrahydrocannabinolic acid synthase (EC: 1.21.3.8) is used to convert cannabigerolic acid into D 9 -tetrahydrocannabinolic acid.
• a cannabichromenic acid synthase is used to convert cannabigerolic acid into cannabichromenic acid (CAS# 20408-52-0).
• cannabidiol results from cannabidiolic acid decarboxylation
• D 9 -tetrahydrocannabinol results from D 9 - tetrahydrocannabinolic acid decarboxylation
• subsequent isomerization of D 9 - tetrahydrocannabinol results in A 6 -tetrahydrocannabinol.
• recombinant refers to the alteration of genetic material by human intervention. Typically, recombinant refers to the manipulation of DNA or RNA in a cell or virus or expression vector by molecular biology (recombinant DNA technology) methods, including cloning and recombination. Recombinant can also refer to manipulation of DNA or RNA in a cell or virus by random or directed mutagenesis.
• a "recombinant" cell or nucleic acid can typically be described with reference to how it differs from a naturally occurring counterpart (the "wild type").
• the terms “transduce”, “transform”, “transfect”, and variations thereof as used herein refers to the introduction of one or more nucleic acids into a cell.
• the nucleic acid must be stably maintained or replicated by the cell for a sufficient period of time to enable the function(s) or product(s) it encodes to be expressed for the cell to be referred to as “transduced”, “transformed”, or “transfected”.
• stable maintenance or replication of a nucleic acid may take place either by incorporation of the sequence of nucleic acids into the cellular chromosomal DNA, e.g., the genome, as occurs by chromosomal integration, or by replication extrachromosomally, as occurs with a freely- replicating plasmid.
• a virus can be stably maintained or replicated when it is "infective”: when it transduces a host microorganism, replicates, and (without the benefit of any complementary virus or vector) spreads progeny expression vectors, e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.
• progeny expression vectors e.g., viruses, of the same type as the original transducing expression vector to other microorganisms, wherein the progeny expression vectors possess the same ability to reproduce.
• a process comprising: contacting an aqueous phase comprising glucose and hexanoic acid or a salt thereof and an organic phase immiscible with the aqueous phase with a heterologous microorganism comprising a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS), Cannabis sativa olivetolic acid cyclase (csOAC), and a Cannabis sativa acyl activating enzyme (csAAE) to provide olivetol and olivetolic acid or a salt thereof, wherein the olivetol and olivetolic acid is provided in a combined amount of at least about 3 g/liter over about 4 to about 7 days.
• a heterologous microorganism comprising a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS), Cannabis sativa olivetolic acid cyclase (csOAC),
• a process comprising: contacting an aqueous phase comprising glucose and butyric acid or a salt thereof and an organic phase immiscible with the aqueous phase with a heterologous microorganism comprising a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS), Cannabis sativa olivetolic acid cyclase (csOAC), and a Cannabis sativa acyl activating enzyme (csAAE) to provide divarin and/or divarinic acid or a salt thereof.
• a heterologous microorganism comprising a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS), Cannabis sativa olivetolic acid cyclase (csOAC), and a Cannabis sativa acyl activating enzyme (csAAE) to provide divarin and/or divarinic acid or a salt
• microorganisms such as those utilize herein, utilized herein are useful in accordance with certain embodiments of this process.
• the divarin and/or divarinic acid is provided in a combined amount of at least about 0.25 - about 2 g/liter, or about 0.5 - about lg/liter over about 4 to about 7 days.
• the fermenting is performed in the absence of galactose.
• the aqueous phase comprises galactose.
• the fermenting is performed in the absence of galactose.
• the aqueous phase comprises galactose.
• the organic phase immiscible with aqueous phase comprises an alkane, an alcohol with carbon number greater than 4, an ester (such as isopropyl myristate), a triglyceride (including commercially available vegetable oils such as sunflower oil, soybean oil, or olive oil), a diester (such as dialkyl malonate), a ketone, or a glyme.
• an ester such as isopropyl myristate
• a triglyceride including commercially available vegetable oils such as sunflower oil, soybean oil, or olive oil
• a diester such as dialkyl malonate
• ketone such as dialkyl malonate
• a glyme glyme
• Other organic solvents immiscible with water or the aqueous phase employed can be utilized.
• the organic phase comprises isopropyl myristate. Suitable solvents include without limitation, other esters, aromatic solvents, and the likes.
• the organic phase comprises an aromatic solvent.
• In-situ liquid-liquid extraction is a strategy that can be employed in accordance with the present invention for physical separation of product from microorganisms via partitioning into the water immiscible organic liquid phase from an aqueous culture phase.
• the organic liquid phase or organic phase is present as either an overlay if its density is less than that of the aqueous phase, or an underlay if its density is greater than that of the aqueous phase.
• Certain properties of the overlay or underlay are considered for production of olivetolic acid/olivetol and other resorcinols such as of formulas IA and IB: (1) non-toxic or low toxicity for growth of the host strain, and/or (2) a favorable partition coefficient of the product in the organic phase vs.
• the aqueous phase and/or (3) preferably a lower partition coefficient for fed hexanoic acid (for olivetolic acid/olivetol) or other fatty acid such as RCO2H (for other resorcinols) in the organic phase vs. the aqueous phase.
• hexanoic acid for olivetolic acid/olivetol
• RCO2H for other resorcinols
• Additional properties of the organic phase enhance its suitability for downstream conversion, e.g. and without limitation, to cannabigerol and other cannabinoid compounds, including suitability as a solvent or co-solvent during downstream prenylation or other reactions, and boiling point if downstream separation by distillation is employed.
• dialkyl ester chain length from diethyl to diisopropyl to dibutyl in a dialkyl adipate series reduced toxicity. Growth was observed with diisopropyl adipate and no apparent toxicity observed in dibutyl adipate. Dibutyl sebacate was also completely non-inhibitory to growth and accordingly, non-toxic.
• the minimum non-toxic internal alkyl chain length of diethyl diesters is sebacate. In certain embodiments, shorter internal alkyl chain length down to adipate is possible with diisopropyl diesters.
• growth-suitable monoester overlays for resorcinol or cannabinoid production include hexyl hexanoate or any higher chain length alkyl hexanoate ester, C 3 chain-length or higher (e.g., and without limitation C 6 -Cs or higher) alkyl octanoate esters, and methyl (Ci) or higher (e.g., and without limitation C 6 -Cs or higher) alkyl decanoates, laurates, or myristates.
• esters and diesters are employed as the organic phase in accordance with the present invention.
• Fatty alcohols are mostly solids above C 10 saturated chain length. Decanol, a liquid, was toxic to growth under test conditions. However, oleyl alcohol supported robust growth. In certain embodiments, longer chain length (C 12 or higher) unsaturated fatty alcohols can be suitable overlays supporting S. cerevisiae or another fermenting organism's growth. In various embodiments, fatty alcohols, preferably C 12 or higher alcohols, are employed as the organic phase in accordance with the present invention.
• alkanes and paraffins support robust growth. Lack of toxicity was observed for dodecane, tetradecane, hexadecane, light and heavy paraffin oils, and isopar M.
• C 12 and higher paraffins are suitable overlays supporting S. cerevisiae or another fermenting organism's growth.
• fatty alcohols preferably C 12 or higher alcohols, are employed as the organic phase in accordance with the present invention.
• triacylglycerols were tested, including tricaprylin, coconut oil, and canola oil (vegetable oils having different average chain length compositions of fatty acid chains, with coconut oil being predominantly C 12 -C 14 saturated fatty acids, and canola being predominantly Ci6-Ci8and a mixture of saturated and unsaturated fatty acids).
• Tricaprylin a synthetic oil containing three Cs fatty acid chains, was fairly toxic, however allowed some growth of strains.
• coconut and canola oil were non-toxic to growth.
• IPM isopropyl myristate
• DBE diesters - dibasic esters
• DBE diethyl sebacate
• di-tert-butyl malonate were explored to investigate if lower percentage mixtures of these compounds in non-toxic IPM or isopar M would mitigate their toxicity toward growth of a microorganism such as S. cerevisioe, as they may also advantageously alter partitioning properties of olivetolic acid, olivetol, and other analogues into the overlay and could offer advantages with alternative downstream separations processes.
• a DBE which may be toxic by itself as an underlay, was much less toxic at concentrations of between 1 and 2.5% (v/v) in IPM and especially isopar M.
• Di-tert- butyl malonate also exhibited much lower toxicity at 1-10% (v/v), and particularly 1-2.5% (v/v), in IPM and isopar M.
• mixtures of longer chain monoesters or paraffins with moderately to very toxic diesters are useful according to the present invention.
• the aqueous phase further comprises histidine.
• the pH of the aqueous phase is at a pH of about 4 to about 8.
• the olivetol and olivetolic acid is provided in a combined amount of at least about 4 g/liter over about 4 to about 7 days. In another embodiment, the olivetol and olivetolic acid is provided in a combined amount of at least about 4.5 g/liter over about 4 to about 7 days. In another embodiment, the olivetol and olivetolic acid is provided in a combined amount of at least about 5 g/liter over about 4 to about 7 days. In another embodiment, the olivetol and olivetolic acid is provided in a combined amount of at least about 7 g/liter over about 4 to about 7 days. In another embodiment, the olivetol and olivetolic acid is provided in a combined amount of at least about 9 g/liter over about 4 to about 7 days.
• the combined amount of olivetol and olivetolic acid provided herein is provided over 4 days. In another embodiment, the combined amount of olivetol and olivetolic acid provided herein, is provided over 5 days. In another embodiment, the combined amount of olivetol and olivetolic acid provided herein, is provided over 6 days. In another embodiment, the combined amount of olivetol and olivetolic acid provided herein, is provided over 7 days.
• the fermentation is performed in a semi-continuous mode ("fill- and-draw"). In another embodiment, the fermentation is performed in a continuous mode. In one embodiment, the overall combined productivity of olivetol and olivetolic acid is greater than 0.3 g per L of total volume (including aqueous and immiscible liquid phases) per day of operation. In another embodiment, the fermentation is performed in a total volume of 15 liters or a larger volume such as 1,000 liters, 10,000 liters, 20,000 or 50,000 liters, or an even larger volume.
• the combined yield of O and OA obtained in such large scale fermentation performed according to the present invention over 2-7 or 4-7 days, such as over 2, 3, 4, 5, 6, or 7 days is unexpectedly high.
• the combined amount of O/OA obtained, even in large scale fermentations, e.g., and without limitation in 10,000 liters fermentations, is about 7 - about 10 g/liter. In some embodiment, the combined amount of O/OA obtained, even in large scale fermentations, e.g., and without limitation in 20,000 liters fermentations, is about 7 - about 10 g/liter.
• the functional OLS has a Sequence ID 1. In another embodiment, the functional OLS has an at least 95% sequence identity with Sequence ID 1. In another embodiment, the functional olivetolic acid cyclase has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID 1.
• the functional OAC has a Sequence ID 2A. In another embodiment, the functional OAC has an at least 95% sequence identity with Sequence ID 2A. In another embodiment, the functional olivetolic acid cyclase has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 2A. In another embodiment, the functional olivetolic acid cyclase is of SEQ ID NO: 2B. In another embodiment, the functional olivetolic acid cyclase has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 2B.
• the functional AAE has a Sequence ID 3A. In another embodiment, the functional AAE has an at least 95% sequence identity with Sequence ID 3A.
• Sequence ID 3A Cannabis sativa acyl activating enzyme (CsAAEl)
• the functional AAE polypeptide comprises an amino acid sequence SEQ ID NO: 3B. In another embodiment, the functional AAE polypeptide comprises an amino acid sequence that has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 3B. In another embodiment, the functional AAE polypeptide comprises an amino acid sequence SEQ ID NO: 3C. In another embodiment, the functional AAE polypeptide comprises an amino acid sequence that has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 3C. In another embodiment, the functional AAE polypeptide comprises an amino acid sequence that is SEQ ID NO: 3D. In another embodiment, the functional AAE polypeptide has at least 50%, at least 75%, or at least 95% sequence identity to SEQ ID NO: 3D.
• SEQ ID NO 3B Cannabis sativa acyl activating enzyme (CsAAE3) MEKSGYGRDGIYRSLRPPLHLPNNNNLSMVSFLFRNSSSYPQKPALIDS ETNQILSFSHFKSTVIKVSHGFLNLGIKKNDWLIYAPNSIHFPVCFLGIIA SGAIATTSNPLYTVSELSKQVKDSNPKLIITVPQLLEKVKGFNLPTILIGP DSEQESSSDKVMTFNDLVNLGGSSGSEFPIVDDFKQSDTAALLYSSGTT GMSKGWLTHKNFIASSLMVTMEQDLVGEMDNVFLCFLPMFHVFGLAI ITYAQLQRGNTVISARFDLEKMLKDVEKYVTHLWWPPVILALSKNSM VKFNLSSIKYIGSGAAPLGKDLMEECSKWPYGIVAQGYGMTETCGIVS MEDIRGGKRNSGSAGMLASGVEAQIVSVDTLKPLPPNQLGEIWVKGPN
• SEQ ID NO 3C Truncated Cannabis sativa acyl activating enzyme MEKSGYGRDGIYRSLRPPLHLPNNNNLSMVSFLFRNSSSYPQKPALIDS ETNQILSFSHFKSTVIKVSHGFLNLGIKKNDWLIYAPNSIHFPVCFLGIIA SGAIATTSNPLYTVSELSKQVKDSNPKLIITVPQLLEKVKGFNLPTILIGP DSEQESSSDKVMTFNDLVNLGGSSGSEFPIVDDFKQSDTAALLYSSGTT
• SEQ ID NO 3D Escherichia coli hexanoyl-CoA synthetase amino acid sequence MHPTGPHLGPDVLFRESNMKVTLTFNEQRRAAYRQQGLWGDASLADYWQQTARAMPDKIAVVDNHGA SYTYSALDHAASCLANWMLAKGIESGDRIAFQLPGWCEFTVIYLACLKIGAVSVPLLPSWREAELVWVLNKC QAKMFFAPTLFKQTRPVDLILPLQNQLPQLQQIVGVDKLAPATSSLSLSQIIADNTSLTTAITTHGDELAAVLFT SGTEGLPKGVMLTHNNILASERAYCARLNLTWQDVFMMPAPLGHATGFLHGVTAPFLIGARSVLLDIFTPDA CLALLEQQRCTCMLGATPFVYDLLNVLEKQPADLSALRFFLCGGTTIPKKVARECQQRGIKLLSVYGSTESSPH AVVNLDDPLSRFMHTDGYAAAGVEIK
• sequence identity is at least 50%. In another embodiment, the sequence identity is at least 75%. In another embodiment, the sequence identity is at least 95%.
• sequence identity is at least 99% with a protein sequence utilized herein. In another embodiment, the sequence identity is at least 99% with a nucleic acid sequence utilized herein.
• the heterologous microorganism is antibiotic-marker free.
• the heterologous microorganism is an FAA2 (peroxisomal medium chain fatty acyl-CoA synthetase) knock out or has a lowered FAA2 activity.
• the heterologous microorganism is a PXA1 (part of the heterodimeric peroxisomal fatty acid and/or acyl-CoA ABC transport complex with PXA2) knockout or has a lowered PXA2 activity.
• the heterologous microorganism is a PEX11 (peroxisomal protein required for medium-chain fatty acid oxidation) knockout or has a lowered PEX11 activity.
• the heterologous microorganism is an ANTI (peroxisomal adenine nucleotide transporter, which exchanges AMP generated in peroxisomes by acyl-CoA synthetases for ATP, that is consumed in that reaction, from the cytosol) knockout or has a lowered ANTI activity.
• ANTI peroxisomal adenine nucleotide transporter, which exchanges AMP generated in peroxisomes by acyl-CoA synthetases for ATP, that is consumed in that reaction, from the cytosol
• the microorganism is Saccharomyces cerevisiae.
• Saccharomyces cerevisiae comprises galactose regulatable promoters for the heterologous genes (csOLS, csOAC, csAAE, and the likes).
• the Saccharomyces cerevisiae does not include galactose regulatable promoters for the heterologous genes.
• the Saccharomyces cerevisiae is haploid.
• the Saccharomyces cerevisiae is diploid.
• Saccharomyces cerevisiae The initial engineering of Saccharomyces cerevisiae was done by introducing a gene fragment containing csOLS, csOAC, and csAAEl under the control of galactose regulatable elements called promoters.
• the csOLS and csOAC were physically linked to each other on the gene with a genetic element called T2A in all examples.
• T2A a genetic element that allows growth on nutrient preferred media was utilized.
• the process by which Saccharomyces cerevisiae uptake foreign DNA and stably utilize the foreign DNA is called recombination.
• Saccharomyces cerevisiae strains that took up the foreign DNA and utilized the DNA are called recombinants. Saccharomyces cerevisiae that did not undergo recombination is called wild type.
• the process of selecting in preferred media is defined as prototrophy rescue. In order to separate recombinants from wild type we utilized prototrophy rescue. In some cases, recombinants we added foreign genes that contained genetic elements that controlled the resistance to antibiotics. Antibiotics such as G418 or hygromycin will normally kill Saccharomyces cerevisiae.
• foreign genes were introduced into O/OA producing lines in order to rescue survival of G418 or hygromycin for the purpose of removing genes native to the Saccharomyces cerevisiae and allowing them to survive in antibiotics while decreasing the need for galactose utilization.
• a recombinant host cell modified by genetic engineering as disclosed herein.
• a recombinant polyketide synthase such as an OLS enzyme
• an aromatic prenyltransferase is introduced.
• the modification increases the production of malonyl-CoA, hexanoyl-CoA or a R-CoA.
• the host cell is engineered via recombinant DNA technology to express heterologous nucleic acids that encode a cannabinoid pathway enzyme such as an OLS enzyme, which is either a mutated version of a naturally occurring enzyme, or a non-naturally occurring enzyme as provided herein.
• the invention includes methods of generating a polynucleotide that expresses one or more of the SEQ IDs related to a mutant or modified OLS provided or utilized herein.
• the proteins of the invention are expressed using any of a number of systems, such as in whole plants, as well as plant cell and/or yeast suspension cultures.
• the polynucleotide that encodes the OLS is placed under the control of a promoter that is functional in the desired host cell.
• a promoter that is functional in the desired host cell.
• An extremely wide variety of promoters may be available and can be used in the expression vectors of the invention, depending on the particular application. Ordinarily, the promoter selected depends on the cell in which the promoter is to be active.
• Other expression control sequences such as ribosome binding sites, transcription termination sites and the like are also optionally included.
• Nucleic acid constructs provided and utilized herein include expression vectors that comprise nucleic acids encoding one or more polyketide synthase enzymes.
• the nucleic acids encoding the enzymes are operably linked to promoters and optionally other control sequences such that the subject enzymes are expressed in a host cell containing the expression vector when cultured under suitable conditions.
• the promoters and control sequences employed depend on the host cell selected for the production of olivetol, olivetolic acid (OLA or OA), OLA- derived compound, or another cannabinoid or cannabinoid derivative.
• OLA or OA olivetolic acid
• OLA- derived compound OLA- derived compound
• another cannabinoid or cannabinoid derivative cannabinoid derivative.
• Nucleic acids encoding the polyketide synthase enzymes can be prepared by any suitable method known to those of ordinary skill in the art, including, for example, direct chemical synthesis and cloning. Further, nucleic acid sequences for use in the invention can be obtained from commercial vendors that provide de novo synthesis of the nucleic acids.
• a nucleic acid encoding the desired enzyme can be incorporated into an expression vector by known methods that include, for example, the use of restriction enzymes to cleave specific sites in an expression vector, e.g., plasmid, thereby producing an expression vector of the invention.
• restriction enzymes produce single stranded ends that may be annealed to a nucleic acid sequence having, or synthesized to have, a terminus with a sequence complementary to the ends of the cleaved expression vector. The ends are then covalently linked using an appropriate enzyme, e.g., DNA ligase.
• DNA linkers may be used to facilitate linking of nucleic acids sequences into an expression vector.
• a set of individual nucleic acid sequences can also be combined by utilizing polymerase chain reaction (PCR)-based methods known to those of skill in the art.
• PCR polymerase chain reaction
• each of the desired nucleic acid sequences can be initially generated in a separate PCR. Thereafter, specific primers are designed such that the ends of the PCR products contain complementary sequences.
• specific primers are designed such that the ends of the PCR products contain complementary sequences.
• a typical expression vector contains the desired nucleic acid sequence preceded and optionally followed by one or more control sequences or regulatory regions, including a promoter and, when the gene product is a protein, ribosome binding site, e.g., a nucleotide sequence that is generally 3-9 nucleotides in length and generally located 3-11 nucleotides upstream of the initiation codon that precede the coding sequence, which is followed by a transcription terminator in the case of E. coli or other prokaryotic hosts. See Shine et a!., Nature 254:34 (1975) and Steitz, in Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger), vol. 1, p. 349 (1979) Plenum Publishing, N.Y.
• a typical expression vector contains the desired nucleic acid coding sequence preceded by one or more regulatory regions, along with a Kozak sequence to initiate translation and followed by a terminator. See Kozak, Nature 308:241-246 (1984).
• Regulatory regions or control sequences include, for example, those regions that contain a promoter and an operator.
• a promoter is operably linked to the desired nucleic acid coding sequence, thereby initiating transcription of the nucleic acid sequence via an RNA polymerase.
• An operator is a sequence of nucleic acids adjacent to the promoter, which contains a protein-binding domain where a transcription factor can bind. Transcription factors activate or repress transcription initiation from a promoter. In this way, control of transcription is accomplished, based upon the particular regulatory regions used and the presence or absence of the corresponding transcription factor.
• Non-limiting examples for prokaryotic expression include lactose promoters (Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator) and tryptophan promoters (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator).
• lactose promoters Lad repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator
• tryptophan promoters when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator.
• Non-limiting examples of promoters to use for eukaryotic expression include pTDH3, pTEFl, pTEF2, pRNR2, pRPL18B, pREVl, pGALl, pGALlO, pGAPDH, pCUPl, pMET3, pPGKl, pPYKl, pHXT7, pPDCl, pFBAl, pTDH2, pPGIl, pPDCl, pTPIl, pEN02, pADHl, and pADH2.
• a variety of expression vectors and components thereof are useful.
• expression vectors include, without limitation: plasmids, such as pESC, pTEF, p414CYCl, p414GALS, pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRIOO, pCR4, pBAD24, pUC19, pRS series; and bacteriophages, such as M13 phage and l phage.
• plasmids such as pESC, pTEF, p414CYCl, p414GALS, pSClOl, pBR322, pBBRlMCS-3, pUR, pEX, pMRIOO, pCR4, pBAD24, pUC19, pRS series
• bacteriophages such as M13 phage and l phage.
• Such expression vectors may only be suitable for particular host cells or for expression of particular polyketide synthases.
• the expression vector can be introduced into the host cell, which is then monitored for viability and expression of the sequences contained in the vector.
• relevant texts and literature describe expression vectors and their suitability to any particular host cell.
• strains are built where expression cassettes are directly integrated into the host genome.
• the expression vectors are introduced or transferred, e.g., by transduction, transfection, or transformation, into the host cell.
• Such methods for introducing expression vectors into host cells are well known to those of ordinary skill in the art.
• one method for transforming P. kudriavzevii with an expression vector involves a calcium chloride treatment wherein the expression vector is introduced via a calcium precipitate.
• a variety of methods are available. For example, potentially transformed host cells in a culture are separated, using a suitable dilution, into individual cells and thereafter individually grown and tested for expression of a desired gene product of a gene contained in the introduced nucleic acid.
• an often-used practice involves the selection of cells based upon antibiotic resistance that has been conferred by antibiotic resistance-conferring genes in the expression vector, such as the beta lactamase (amp), aminoglycoside phosphotransferase (neo), and hygromycin phosphotransferase (hyg, hph, hpt) genes.
• a host cell of the disclosure is transformed with at least one expression vector.
• the vector will typically contain a polyketide synthase gene.
• the host cell is cultured in a suitable medium containing a carbon source, such as a sugar ( e.g ., glucose).
• a carbon source such as a sugar (e.g ., glucose).
• a sugar e.g ., glucose
• these OLS(s) and other enzymes provided and utilized herein convert three molecules of malonyl-CoA and one molecule of hexanoyl-CoA or R-CoA, wherein R is defined as herein, to olivetol or a compound of formula (I).
• a host cell of the invention is to include more than one heterologous gene
• the multiple genes can be expressed from one or more vectors.
• a single expression vector can comprise one, two, or more genes encoding one, two, or more mutant OLS enzyme(s), other enzymes of the cannabinoid pathway, e.g., improved malonyl-CoA production, hexanoyl-CoA, or R-CoA production, etc.
• the heterologous genes can be contained in a vector replicated episomally or in a vector integrated into the host cell genome, and where more than one vector is employed, then all vectors may replicate episomally (extrachromasomally), or all vectors may integrate, or some may integrate and some may replicate episomally.
• a "gene" is generally composed of a single promoter and a single coding sequence, in certain host cells, two or more coding sequences are controlled by one promoter in an operon. In some embodiments, a two or three operon system is used.
• the coding sequences employed have been modified, relative to some reference sequence, to reflect the codon preference of a selected host cell. Codon usage tables for numerous organisms are readily available and can be used to guide sequence design. The use of prevalent codons of a given host organism generally improves translation of the target sequence in the host cell.
• the subject nucleic acid sequences will be modified for yeast codon preference (see, for example, Bennetzen et al., J. Biol. Chem. 257: 3026-3031 (1982)).
• the nucleotide sequences will be modified for P.
• nucleotide sequences are modified to include codons optimized forS. cerevisiae codon preference.
• Nucleic acids can be prepared by a variety of routine recombinant techniques. Briefly, the subject nucleic acids can be prepared from genomic DNA fragments, cDNAs, and RNAs, all of which can be extracted directly from a cell or recombinantly produced by various amplification processes including but not limited to PCR and rt-PCR. Subject nucleic acids can also be prepared by a direct chemical synthesis.
• the nucleic acid transcription levels in a host microorganism can be increased (or decreased) using numerous techniques.
• the copy number of the nucleic acid can be increased through use of higher copy number expression vectors comprising the nucleic acid sequence, or through integration of multiple copies of the desired nucleic acid into the host microorganism's genome.
• Non-limiting examples of integrating a desired nucleic acid sequence onto the host chromosome include recA-mediated recombination, lambda phage recombinase- mediated recombination and transposon insertion.
• Nucleic acid transcript levels can be increased by changing the order of the coding regions on a polycistronic mRNA or breaking up a polycistronic operon into multiple poly- or mono-cistronic operons each with its own promoter. RNA levels can be increased (or decreased) by increasing (or decreasing) the strength of the promoter to which the protein-coding region is operably linked.
• the translation level of a desired polypeptide sequence in a host microorganism can also be increased in a number of ways. Non-limiting examples include increasing the mRNA stability, modifying the ribosome binding site (or Kozak) sequence, modifying the distance or sequence between the ribosome binding site (or Kozak sequence) and the start codon of the nucleic acid sequence coding for the desired polypeptide, modifying the intercistronic region located 5' to the start codon of the nucleic acid sequence coding for the desired polypeptide, stabilizing the 3'-end of the mRNA transcript, modifying the codon usage of the polypeptide, altering expression of low-use/rare codon tRNAs used in the biosynthesis of the polypeptide. Determination of preferred codons and low-use/rare codon tRNAs can be based on a sequence analysis of genes derived from the host microorganism.
• the polypeptide half-life, or stability can be increased through mutation of the nucleic acid sequence coding for the desired polypeptide, resulting in modification of the desired polypeptide sequence relative to the control polypeptide sequence.
• the modified polypeptide is an enzyme
• the activity of the enzyme in a host is altered due to increased solubility in the host cell, improved function at the desired pH, removal of a domain inhibiting enzyme activity, improved kinetic parameters (lower Km or higher k cat values) for the desired substrate, removal of allosteric regulation by an intracellular metabolite, and the like.
• Altered/modified enzymes can also be isolated through random mutagenesis of an enzyme, such that the altered/modified enzyme can be expressed from an episomal vector or from a recombinant gene integrated into the genome of a host microorganism.
• the recombinant host cell is a eukaryote.
• the eukaryote is a yeast strain selected from the non-limiting list of example genera: Candida, Cryptococcus, Hansenula, Issatchenkia, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, or Yarrowia.
• the host cell is Saccharomyces cerevisiae. In other embodiments, the host cell is Pichia kudriavzevii. In other embodiments of the invention, the eukaryotic host cell is a fungus or algae. In yet other embodiments, the recombinant host cell is a prokaryote selected from the non-limited example genera: Bacillus, Clostridium, Corynebacterium, Escherichia, Pseudomonas, Rhodobacter, and Streptomyces. In various embodiments, the host cell is P. kudriavzevii.
• the host cell is part of a multicellular organism.
• the multicellular organism is a plant.
• the plant is a cannabis plant.
• the plant is a tobacco plant.
• a host cell provided or utilized herein is further engineered to include a genetic modification useful for converting pyruvate to malonyl-CoA, wherein the genetic modification produces and/or provides a pyruvate decarboxylase, an acetaldehyde dehydrogenase, an acetyl-CoA synthetase, an acetyl- CoA carboxylase, and a carbonic anhydrase.
• an engineered host cell provided or utilized herein is a Saccharomyces cerevisiae host cell.
• an engineered host cell comprises heterologous enzymes that are overexpressed to increase malonyl-CoA production, thereby facilitating production of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative.
• the engineered host cell comprises heterologous enzymes selected from the group consisting of an acetyl Co-A carboxylase, such as P. kudriavzevii acetyl-CoA carboxylase, S. cerevisiae aldehyde dehydrogenase, Yarrowia lipolytica acetyl-CoA synthetase, and S. cerevisiae pyruvate decarboxylase.
• the host cell is a Saccharomyces cerevisiae host cell.
• a yeast host cell expressing an OLS is used to produce olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative.
• an oleaginous yeast host cell expressing an OLS is used to produce olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative.
• a mutated OLS comprising a mutated active site, vectors for expressing the mutant, and host cells that express the mutant.
• the host cell further produces olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative.
• Introduction of mutations in the region comprising D198 to G209 of OLA increases the turnover rate (i.e., k cat values) of the mutated OLS.
• One or more point mutations at amino acid positions D198 to G209 can be introduced alone or in any desired combination.
• the recombinant host cell can be, without limitation, a P. kudriavzevii or yeast, including but not limited to S. cerevisiae or other yeast, host cell.
• recombinant host cells preferably host cells suitable for producing olivetol (including OLA and/or OLA-derived compounds) and other cannabinoids and cannabinoid derivatives in accordance with the methods provided herein, the host cells comprising one or more heterologous OLS enzymes, preferably OLS enzymes having an increased k cat value as compared to wild type or homologous OLS enzymes, wherein the recombinant host cells provide increase olivetol titer, yield, and/or productivity relative to a host cell not comprising a heterologous OLS enzyme.
• recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased malonyl-CoA biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased hexanoyl-CoA synthetase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased pyruvate dehydrogenase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased acetaldehyde dehydrogenase biosynthesis.
• recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased acetyl-CoA synthetase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased acetyl-CoA carboxylase biosynthesis. In some aspects, provided herein are recombinant host cells suitable for producing olivetol in accordance with the methods of the invention comprising increased carbonic anhydrase biosynthesis.
• the invention provides host cells, vectors, enzymes, and methods relating thereto.
• Malonyl-CoA is produced in host cells through the activity of an acetyl-CoA carboxylase (EC 6.4.1.2) catalyzing the formation of malonyl-CoA from acetyl-CoA and carbon dioxide.
• the invention provides recombinant host cells for producing olivetol that express a heterologous acetyl-CoA carboxylase (ACC).
• ACC heterologous acetyl-CoA carboxylase
• the host cell is a S.
• cerevisiae cell comprising a heterologous S. cerevisiae acetyl-CoA carboxylase ACC1 or an enzyme homologous thereto.
• the host cell modified for heterologous expression of an ACC such as S. cerevisiae ACC1 is further modified to eliminate ACC1 post-translational regulation by genetic modification of S. cerevisiae SN FI protein kinase or an enzyme homologous thereto.
• the disclosure also provides a recombinant host cell suitable for producing olivetol in accordance with the invention that is an E. coli cell that comprises a heterologous nucleic acid coding for expression of E. coli acetyl-CoA carboxylase complex proteins AccA, AccB, AccC and AccD or one or more enzymes homologous thereto.
• the recombinant host cell comprises a heterologous nucleic acid encoding a mutant OLS enzyme or another mutant cannabinoid pathway enzyme, that results in increased production of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative relative to host cells not comprising the mutant OLS enzyme and/or an OLS enzyme.
• an OLS enzyme other than, or in addition to, OLS derived from C. sativa can be used for biological synthesis of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative in a recombinant host.
• the recombinant host is P. kudriavzevii.
• the recombinant host is S. cerevisiae.
• the recombinant host is E. coli.
• the recombinant host is a yeast other than P. kudriavzevii.
• the host is modified to express a mutated OLS enzyme and/or an OLS enzyme provided or utilized herein. In various embodiments, the host is further modified to express one or more heterologous enzymes that are overexpressed to increase malonyl-CoA production. In various embodiments, the host is further modified to express or overexpress a functional hexanoyl-CoA synthetase.
• recombinant host cells suitable for biological production of cannabinoids and derivatives, such as without limitation olivetol, OLA, OLA- derived compound, or another cannabinoid or cannabinoid derivative. Any suitable host cell is useful in practice of the methods provided herein.
• the host cell is a recombinant host microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), either to produce olivetol, or to increase yield, titer, and/or productivity of olivetol relative to a "wild type", "control cell", “parental cell”, or “reference cell”.
• a "control cell” can be used for comparative purposes, and is typically a wild type or recombinant parental cell that does not contain one or more of the modification(s) made to the host cell of interest.
• the invention provides a recombinant host cell that has been modified to produce one or more enzymes that facilitate malonyl-CoA production. In some embodiments, the invention provides a recombinant host cell that has been modified to produce one or more enzymes of the cannabinoid pathway. In some embodiments, the invention provides a recombinant host cell that has been modified to produce an OLS, such as, without limitation, an engineered or modified OLS, for example, olivetol synthase, having improved k cat values. In some embodiments, the invention provides a recombinant host cell that has been modified to produce an OLS, such as, without limitation, an engineered or modified OLS having improved solubility in the host.
• the invention provides a recombinant host cell that has been modified to produce an OLS, such as, without limitation, an engineered or modified OLS or having improved stability in the host.
• OLS an OLS
• various embodiments of the invention provide recombinant host cells capable of producing increased amounts of olivetol, OLA, OLA-derived compound, or another cannabinoid or cannabinoid derivative (i.e., product) per unit time.
• various embodiments of the invention provide recombinant host cells capable of achieving higher titers of product over shorter fermentation run times.
• the recombinant host cells provided or utilized herein produce titer levels that exceed production titer levels of control cells.
• the recombinant host cells provided or utilized herein produce titer levels that are suitable for commercial production, for example approximately 1-20 g/L, such as 2-10 g/L or 3-8 g/L, or greater.
• the recombinant host cells described herein promote high titer levels of product(s) in at least two ways.
• the recombinant host cells produce mutated OLS enzymes having improved synthetase kinetics (i.e., an increase in k cat ), which allows for faster product production, thereby increasing the rate and ease at which a desired titer level can be achieved.
• the materials and methods provided or utilized herein provide and facilitate in situ extraction of the product into an organic phase, as described below. By adding the organic phase directly to the broth during the fermentation, the product can be quickly and continuously separated from the fermentation process, thereby decreasing undesirable effects of the product on the fermentation process, such as toxicity and product inhibition feedback on the pathway enzymes, thereby further increasing the titer levels of the product(s).
• various genetic modifications provided or utilized herein are useful for increasing the provision of malonyl-CoA, which is a substrate for OLS.
• yeast cells suitable for the production of cannabinoids and derivatives such as, without limitation, olivetol, at levels sufficient for subsequent purification and use as described herein.
• Yeast host cells are excellent host cells for construction of recombinant metabolic pathways comprising heterologous enzymes catalyzing production of small molecule products.
• molecular biology techniques and nucleic acids encoding genetic elements necessary for construction of yeast expression vectors including, but not limited to, promoters, origins of replication, antibiotic resistance markers, auxotrophic markers, terminators, and the like.
• techniques for integration of nucleic acids into the yeast chromosome are well established. Yeast also offers a number of advantages as an industrial fermentation host.
• Yeast can tolerate high concentrations of organic acids and maintain cell viability at low pH and can grow under both aerobic and anaerobic culture conditions, and there are established fermentation broths and fermentation protocols.
• the ability of a strain to propagate and/or produce desired product under low pH provides a number of advantages. First, this characteristic provides tolerance to the environment created by the production of malonic acid. Second, from a process standpoint, the ability to maintain a low pH environment limits the number of organisms that are able to contaminate and spoil a batch.
• the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is a eukaryote.
• the eukaryote is a yeast selected from the non-limiting list of genera; Saccharomyces, Candida, Cryptococcus, Hansenula, Issatchenki, Kluyveromyces, Komagataella, Lipomyces, Pichia, Rhodosporidium, Rhodotorula, or Yarrowia species.
• the yeast is of a species selected from the group consisting of Candida albicans, Candida ethanolica, Candida krusei, Candida methanosorbosa, Candida sonorensis, Candida tropicalis, Cryptococcus curvotus, Hansenula polymorpha, Issotchenkio orientalis, Kluyveromyces loctis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Komagataella pastoris, Lipomyces starkeyi, Pichia angusta, Pichia deserticola, Pichia galeiformis, Pichia kodamae, Pichia kudriavzevii, Pichia membranaefaciens, Pichia methanolica, Pichia pastoris, Pichia salictaria, Pichia stipitis, Pichia thermotolerans, Pichia trehalophila, Rhodosporidium toruloides, Rhodot
• recombinant host cells include without limitation, eukaryotic, prokaryotic, and archaea cells.
• eukaryotic cells include, but are not limited to: Aspergillus niger, Aspergillus oryzae, Crypthecodinium cohnii,
• non-pathogenic strains include but are not limited to: Pichia pastoris and Saccharomyces cerevisiae.
• certain strains, including Saccharomyces cerevisiae have been designated by the Food and Drug Administration as Generally Regarded As Safe (or GRAS) and so can be conveniently employed in various embodiments of the methods of the invention.
• Illustrative and non-limiting examples of recombinant prokaryotic host cells include, Bacillus subtilis, Brevibacterium ammoniagenes, Clostridium beigerinckii, Corynebacterium glutamicum, Escherichia coli, Enterobacter sakazakii, Lactobacillus acidophilus, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter capsulatus, Rhodobacter sphaeroides, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella flexneri, Staphylococcus aureus, Streptomyces ambofaciens, Streptomyces aureofaciens, Streptomyces aureus, Streptomyces fungicidicus, Streptomyces griseochromogenes,
• Certain of these cells including Bacillus subtilis, Corynebacterium glutamicum, and Lactobacillus acidophilus, have been designated by the Food and Drug Administration as Generally Regarded As Safe (or GRAS) and so are employed in various embodiments of the methods of the invention. While desirable from public safety and regulatory standpoints, GRAS status does not impact the ability of a host strain to be used in the practice of this invention; hence, non-GRAS and even pathogenic organisms are included in the list of illustrative host strains suitable for use in the practice of this invention.
• Escherichia coli and Corynebacterium glutamicum are suitable prokaryotic host cells for metabolic pathway construction. Wild type E. coli can catabolize both pentose and hexose sugars as carbon sources.
• E. coli host cells suitable for the production of malonate as described herein.
• the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is an E. coli cell.
• the recombinant host cell comprising a heterologous nucleic acid provided or utilized herein is a C. glutamicum cell.
• the fermentation is performed at a pH of about 5-6, preferably at about 5.5. In one embodiment, the fermentation is performed at a temperature of about 30°C. In one embodiment, the organic solvent immiscible with aqueous phase employed in the fermentation is loaded at about 26% of total fermentation tank volume. In one embodiment, the organic solvent immiscible with aqueous phase employed in the fermentation is loaded at about 40% of initial fermentation tank volume. In one embodiment, Isopropyl myristate is the aqueous phase immiscible organic solvent. In one embodiment, the aqueous phase immiscible organic solvent is added about 12 to about 36 hours post inoculation.
• the fermentation is performed wherein the compound of formula RCO2H or a salt thereof is present in an amount of 0.1-0.3 moles/500 g of glucose in feed. In one embodiment, the fermentation is performed wherein the compound of formula RCO2H or a salt thereof is present in an amount of 0.14-0.25 moles/500 g of glucose in feed. In one embodiment, the fermentation is performed wherein the compound of formula RCO2H or a salt thereof is present in an amount of 0.14-0.21 moles/500 g of glucose in feed. In one embodiment, the fermentation is performed wherein the sodium hexanoate/glucose in feed ratio was in the range of about 20 to about 28 g sodium hexanoate/ 500 g glucose. In one embodiment, the fermentation is performed wherein the sodium hexanoate/glucose in feed ratio was in the range of about 23 to about 28 g sodium hexanoate/ 500 g glucose.
• the oxygen transmission rate is about 60 - about 80 mmoles/L/hr. In one embodiment, an oxygen uptake rate (OUR) of about 100 - about 110 mmoles/L/hr is achieved.
• the pulse parameter was about 1.7 g glucose/L initial tank volume/pulse with a feed rate of about 10 g/L of initial tank volume/hr. In one embodiment, the batch glucose concentration employed in the fermentation was about 10 - about 20 g/L.
• a cannabinoid in some aspects, provided herein are methods of producing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative.
• the methods may involve culturing a genetically modified host cell of the present disclosure in a suitable medium and recovering the produced cannabinoid, the cannabinoid precursor, the cannabinoid precursor derivative, or the cannabinoid derivative.
• the methods may also involve cell-free production of cannabinoids, cannabinoid precursors, cannabinoid precursor derivatives, or cannabinoid derivatives using one or more polypeptides disclosed herein expressed or overexpressed by a genetically modified host cell of the disclosure.
• methods of producing a cannabinoid or a cannabinoid derivative may involve culturing a genetically modified host cell of the present disclosure in a suitable medium and recovering the produced cannabinoid or cannabinoid derivative.
• the methods may also involve cell-free production of cannabinoids or cannabinoid derivatives using one or more polypeptides disclosed herein expressed or overexpressed by a genetically modified host cell of the disclosure.
• Cannabinoids, cannabinoid derivatives, cannabinoid precursors, or cannabinoid precursor derivatives that can be produced according to the present disclosure may include, but are not limited to, cannabichromene (CBC) type (e.g., cannabichromenic acid), cannabigerol (CBG) type (e.g., cannabigerolic acid), cannabidiol (CBD) type (e.g., cannabidiolic acid), D 9 -trans- tetrahydrocannabinol (D 9 -THC) type (e.g., A 9 -tetrahydrocannabinolic acid), A 8 -trans- tetrahydrocannabinol (D 8 -THC) type, cannabicyclol (CBL) type, cannabielsoin (CBE) type, cannabinol (CBN) type, cannabinodiol (CBND) type, cannabit
• Cannabinoids or cannabinoid derivatives that can be produced with the methods or genetically modified host cells of the present disclosure may also include, but are not limited to, cannabigerolic acid (CBGA), cannabigerolic acid monomethylether, (CBGAM), cannabigerol (CBG), cannabigerol monomethylether (CBGM), cannabigerovarinic acid (CBGVA), cannabigerovarin (CBGV), cannabichromenic acid (CBCA), cannabichromene (CBC), cannabichromevarinic acid (CBCV), cannabichromevarin (CBCV), cannabidiolic acid (CBDA), cannabidiol (CBD), cannabidiol monomethylether (CBDM), cannabidiol-C4 (CBD-C4), cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabidiorcol (CBD-Ci), D
• Additional cannabinoid derivatives that can be produced with the methods or genetically modified host cells of the present disclosure may also include, but are not limited to, 2-geranyl-5-pentyl-resorcylic acid, 2-geranyl-5-(4-pentynyl)-resorcylic acid, 2-geranyl-5- (trans- 2-pentenyl)-resorcylic acid, 2-geranyl-5-(4-methylhexyl)-resorcylic acid, 2-geranyl-5- (5-hexynyl) resorcylic acid, 2-geranyl-5-(trans-2-hexenyl)-resorcylic acid, 2-geranyl-5-(5- hexenyl)-resorcylic acid, 2-geranyl-5-heptyl-resorcylic acid, 2-geranyl-5-(6-heptynoic)- resorcylic acid, 2-geranyl-5- oc
• LSC3-2 was iteratively constructed by transforming chemically competent JK9-3d (LSC3- 1) with pAG304GalllOOSOACCSAAEl, pAG305GalllOOSOACCSAAEl and pAG306GalllOOSOACCSAAEl and controlling their genomic copy number at specific genomic loci.
• pAG304GalllOOSOACCSAAEl and pAG306GalllOOSOACCSAAEl were constructed by first amplifying the yeast shuttle vectors designated as pAG304 and pAG306 with the primers pAG304_fwd and pAG304_rev to amplify the Saccharomyces cerevisiae prototrophy genetic elements in addition, E.
• the dual promoter system pGall and pGallO was amplified from genomic DNA of JK9- 3d with primers Gall_10_fwd and Gall_10_rev.
• the csAAEl and OS-T2A-OAC fragments were amplified from sequences that were stored in pUC19 subcloning vectors. Amplified DNA fragments were mixed at equimolar concentrations with their respective shuttle vector sequences (pAG304 or pAG306) and preassembled by Gibson Assembly using the NEBuilder HiFi DNA Assembly Mix (NEB E5520S).
• the final sequences pAG304GalllOOSOACCSAAEl and pAG306GalllOOSOACCSAAEl were generated.
• the template GalllOCBGA was amplified with Gall_10_fwd and Gall_10_rev to generate the yeast shuttle vector containing leucine prototrophy, dual expression promoter pGall and pGallO, and the OS-T2A-OAC fragment.
• the csAAEl fragment was amplified from a pUC19 subcloning containing the csAAEl gene fragment using primers CB_CSAAEl_fwd and CB_CSAAEl_rev.
• the amplified sequences were mixed at equimolar concentrations and assembled by Gibson Assembly using the NEBuilder HiFi DNA Assembly Mix.
• LSC3-16 was generated by transforming 2 microgram (ug) of Aflll (NEB R0520S) linearized pAG305GalllOOSOACCSAAEl into chemically competent JK9-3d mating type alpha cells that are auxotrophic to leucine, histidine, tryptophan, and uracil. Selection for pAG305GalllOOSOACCSAAEl integration was done with leucine prototrophy rescue on yeast nitrogen base agar plates with dropout amino acid mixes deficient in leucine supplemented with 100 mg/L glucose. Genetic copy number of pAG305GalllOOSOACCSAAEl integrated at chromosome III was initially quantitated by qPCR through isolation of sister clones from the transformation. The highest copy integrant was taken and designated as LSC3-16.
• Example IB LSC3-4 strain
• LSC3-4 was generated from LSC3-2 by integrating a cassette containing PkHIS4 (HIS4 from Pichia kudriavzevii) preceded by the TEF1 promoter from Saccharomyces cerevisiae (pScTEFl) into the GAL80 locus using PCR fragments amplified from Pichia kudriavzevii genomic DNA using primers Y011334 and Y011335, and from S. cerevisiae genomic DNA using primers YO11307 and YO11308.
• PkHIS4 HIS4 from Pichia kudriavzevii
• pScTEFl Saccharomyces cerevisiae
• the two PCR fragments were transformed into chemically competent LSC3-2, and colonies were selected on defined media containing Complete Supplement Mixture (CSM; Formedium, Hunstanton, UK) without histidine (CSM-His). Integration of the cassette at the GAL80 locus was confirmed by colony PCR.
• CSM Complete Supplement Mixture
• Hunstanton, UK Hunstanton, UK
• Example 1C LSC3-13 strain
• LSC3-13 was generated from LSC3-4 by integrating a cassette (HygR) containing the hph gene encoding hygromycin-B 4-O-kinase from Escherichia coli (with a GGT codon inserted immediately after the start codon) flanked by the 379 bp TEF1 promoter and 240 bp TEF1 terminator from Ashbya gossypii, into the MIG1 locus.
• a PCR fragment containing the HygR cassette was amplified from an in-house plasmid (pLYG-001) using primers Y011337 and Y011338. The PCR fragment was transformed into chemically competent LSC3-4, and colonies were selected on YPD medium containing 300 mg/mL hygromycin B. Integration of the cassette at the MIG1 locus was confirmed by colony PCR.
• LSC3-18 was generated from LSC3-4 by integrating the HygR cassette into the GAL1 locus.
• a PCR fragment containing the HygR cassette was amplified from an in-house plasmid using primers Y011436 and Y011437. The PCR fragment was transformed into chemically competent LSC3-4, and colonies were selected on YPD medium containing 300 mg/mL hygromycin. Integration of the cassette at the GAL1 locus was confirmed by colony PCR.
• Example IE LSC3-13 gall A strain (LSC3-64. LSC3-65)
• LSC3-64 and LSC3-65 were generated by transforming two PCR fragments into chemically competent LSC3-13 that together compose a split KanMX marker (with a TEF1 promoter and terminator from Ashbya gossypii flanking a kanR gene encoding an aminoglycoside phosphotransferase conferring G418 resistance) flanked by lox66 and lox71 recombination sites, replacing the GAL1 gene region.
• This cassette is hereafter referred to as a loxKanMX cassette.
• the first fragment was amplified from an internal plasmid containing the assembled KanMX cassette flanked by lox sites (pLOA-058) using primers Y011791 and Y0316, binding internally in the KanMX cassette.
• the second PCR fragment was generated by PCR from the same internal plasmid template using primers Y0949, binding internally in the KanMX cassette, and Y011792. Colonies were selected on YPD agar plates containing 200 mg/mL G418, and two isolates (LSC3-64 and LSC3-65) containing the full length integration at the desired locus were confirmed by colony PCR.
• LSC3-133 and LSC3-134 An antibiotic marker-free version of LSC3-13 (LSC3-133 and LSC3-134) was generated by first integrating a cassette containing the HIS4 gene from S. cerevisiae CEN.PK2-1C MATa, together with its native upstream promoter and downstream terminator regions, and flanked by lox66 and lox71 recombination sites, into the GAL80 locus.
• This "loxHIS4" cassette was amplified in two fragments from an in-house constructed vector (pLOA-027) using primers Y011438 and Y011431, and Y011432 and Y011439. The PCR fragments were transformed into chemically competent LSC3-2, and colonies were selected on CSM-His agar plates.
• the integrated functional HIS4 marker was looped out by transforming an in-house vector (pLYG-005) expressing Cre recombinase and harboring the CEN/ARS origin of replication. Transformants were selected on YPD plates containing 200 mg/mL G418 and up to 50 colonies were restruck on both G418 and YPD plates to screen for colonies that were spontaneously cured of pLYG-005 (grow on YPD but not on YPD plus G418).
• loxPkHIS4 cassette consisting of the PkHIS4 marker with promoter and terminator as previously described, flanked with lox66 and lox71 recombination sites (hereafter referred to as a loxPkHIS4 cassette), was amplified from an in-house vector (pLOA-093) in two PCR fragments and integrated into the MIG1 locus of LSC3-103. The first fragment was amplified using primers YO12096 and YO12098, and the second fragment was amplified using primers YO12018 and YO12097. Both fragments were transformed into the chemically competent loopout strain and colonies were selected on CSM- His agar plates. Integration of the cassette into the MIG1 locus was confirmed by colony PCR.
• LSC3-133A and LSC3-134A An alternative marker-free version of LSC3-13 (LSC3-133A and LSC3-134A) was generated by integrating a cassette containing the HygR cassette previously described, flanked by the lox66 and lox71 recombination sites (hereafter referred to as a loxHygR cassette).
• Two PCR fragments were amplified from an in-house vector containing the loxHygR cassette (pLOA- 094) using primers YO12096 and Y0343, and Y0189 and YO12097. The two PCR fragments were transformed into chemically competent LSC3-4 and colonies selected on YPD medium containing 300 mg/mL hygromycin B.
• HygR cassette was looped out by transforming the resulting strain above with pLYG-005, expressing Cre recombinase and harboring the CEN/ARS origin of replication. Transformants were selected on YPD plates containing 200 mg/mL G418 and up to 50 colonies were restruck on both YPD plus G418 and YPD plates to screen for colonies that were spontaneously cured of pLYG-005. Cured isolates were confirmed for loss of HygR by colony PCR and checking for lack of growth on YPD plus 300 mg/mL hygromycin-B plates.
• LSC3-89 and LSC3-90 were generated by transforming two PCR fragments into chemically competent LSC3-13 that together comprise a split loxKanMX cassette as described above into the GPD1 locus.
• the first fragment was amplified as described above using primers YO11970 and Y0316, and the second PCR fragment was amplified as described above using primers Y0949 and Y011971.
• LSC3-91 and LSC3-92 were generated in an identical way except into chemically competent LSC3-18. Colonies were selected on YPD medium containing 200 mg/mL G418 and integration in the GPD1 locus was confirmed by colony PCR.
• genes were individually disrupted and tested in the LSC3-2 background. These included FAA2 (peroxisomal medium chain fatty acyl-CoA synthetase), PXA1 (part of the heterodimeric peroxisomal fatty acid and/or acyl-CoA ABC transport complex with PXA2),
• PEX11 peroxisomal protein required for medium-chain fatty acid oxidation
• ANTI peroxisomal adenine nucleotide transporter, which exchanges AMP generated in peroxisomes by acyl-CoA synthetases for ATP, that is consumed in that reaction, from the cytosol.
• LSC3-48 and LSC3-49 were generated by integrating a loxHIS4 cassette as 2 PCR fragments in the 3' portion (starting at nucleotide position 412) of the FAA2 locus.
• the immediate 5' portion of the gene containing the first 411 nucleotides of FAA2 and its upstream region were preserved due to overlap with the BUD25 locus transcribed from the complement strand.
• the two PCR fragments were amplified from pLOA-027 using primers Y011478 and Y011431, and Y011432 and Y011479, transformed into chemically competent LSC3-2, and colonies were selected CSM-His agar plates.
• LSC3-63 PXA1 knockout was generated by integrating a loxHIS4 cassette as 2 PCR fragments in the PXA1 locus.
• the two PCR fragments were amplified from pLOA-027 using primers Y011795 and Y011431, and Y011432 and Y011796, transformed into chemically competent LSC3-2, and colonies were selected on CSM- His agar plates.
• the native non-functional HIS4 locus was first knocked out in LSC3-2 by integrating a HygR cassette, generating strain LSC3-52.
• a PCR fragment was amplified from pLYG-001 using primers YO11709 and YO11710, transformed into chemically competent LSC3-2, and colonies were selected on YPD plus 300 mg/mL hygromycin B, with integration at the HIS4 locus confirmed by colony PCR.
• This strain exhibited enhanced efficiency of desired integrations using the loxHIS4 cassette, due to reduced homology with the native HIS4 locus.
• LSC3-74 and LSC3-75 (PEX11 knockouts) were subsequently generated by integrating a loxHIS4 cassette as 2 PCR fragments into the PEX11 locus of LSC3-52.
• the two PCR fragments were amplified from pLOA-027 using primers Y011498 and Y011431, and Y011432 and Y011499, transformed into chemically competent LSC3-52, and colonies were selected on CSM-His agar plates.
• LSC3-76 and LSC3-77 (ANTI knockouts) were generated by integrating a loxHIS4 cassette as 2 PCR fragments into the ANTI locus of LSC3-52.
• the two PCR fragments were amplified from pLOA-027 using primers YO11680 and Y011431, and Y011432 and Y011681, transformed into chemically competent LSC3-52, and colonies were selected on CSM-His agar plates. Integrations of cassettes into the desired loci were all confirmed by colony PCR for all strains.
• LSC3-47 (harboring a knockout of PRB1, encoding vacuolar proteinase B) was generated by integrating a loxHIS4 cassette in the PRB1 locus using 2 PCR fragments. The two PCR fragments were amplified from pLOA-027 using primers Y011488 and Y011431, and Y011432 and Y011489, transformed into chemically competent LSC3-2, and colonies were selected on CSM-His agar plates.
• LSC3-87 and LSC3-88 (harboring knockouts of PEP4, encoding vacuolar aspartyl protease/proteinase A) were generated by integrating a loxHIS4 cassette at the PEP4 locus in LSC3-52 using two PCR fragments.
• the two PCR fragments were amplified from pLOA-027 using primers Y011687 and Y011431, and Y011432 and Y011688, transformed into chemically competent LSC3-52, and colonies were selected on CSM-His agar plates. Integrations of cassettes into both desired loci were confirmed by colony PCR.
• Additional knockouts can subsequently be combined from any combination of integrated lox66/lox71 flanked cassettes by transforming into strains where the previous marker was looped out by transforming pLYG-005, isolating colonies spontaneously cured for pLYG-005 with confirmed loopout by colony PCR and phenotypic checks, and integrating the next lox site flanked marker into a new locus.
• a strain can harbor knockouts in modifications that allow production from glucose (GAL80 knockout in combination with either MIG1 or GAL1 knockouts), knockouts in genes involved in hexanoic acid or hexanoyl-CoA degradation (e.g. FAA2 and ANTI knockouts), and/or knockouts in one or multiple proteases involved in degradation of expressed heterologous pathway proteins (e.g. PRB1 and PEP4 knockouts).
• Example 1G Small-scale strain screening examples
• Main cultures were inoculated using between 1 to 3 mL of preculture in 250 mL baffled shake flasks containing 30 mL of YP + 0.02-0.04% (w/v) hexanoic acid + 2% (w/v) galactose or 2% (w/v) glucose + 5 mL of isopropyl myristate (IPM).
• IPM isopropyl myristate
• the percentage of galactose or glucose was altered, the percent hexanoic acid added was modified, or the overlay was intentionally not added or was replaced with alternative overlay candidates, such as diethyl sebacate, di-tert-butyl malonate, or methyl soyate.
• Sampling time was between 24 to 50 hours as indicated.
• the shake flask experiments were scaled down to 96 well deepwell plate format. Precultures from colonies of each strain were grown in 300 pL YP + 2% (w/v) glucose. Main cultures containing 300 pL YP + 2% (w/v) galactose (or glucose or combinations of galactose and glucose) + 0.04% (w/v) hexanoic acid + 20% (v/v) IPM (60 pL) or alternative overlay candidates, were grown at 30°C with 950 rpm shaking and 80% humidity in an Infors Multitron plate shaker, and the IPM or diethyl sebacate overlay was sampled at different elapsed times between 18 and 48 hours post-inoculation, following acidification of the media with 10 pL of 5 M phosphoric acid. Overlay from the cultures was diluted 2:1 with methanol prior to HPLC analysis.
• YNB medium was initially optimized and consisted of 100 mL/L of a 10X YNB stock solution (containing 68 g/L yeast nitrogen base without amino acids from Sigma-Aldrich, product number Y0626), optionally 1 mL/L of 10% BactoTM casamino acids (BD Biosciences), 300 mL/L of 1 M MES buffer (pH 6.5), optionally 3.6 mL/L of a trace element solution (containing 130 g/L citric acid monohydrate, 0.574 g/L copper (II) sulfate pentahydrate, 8.07 g/L iron (III) chloride hexahydrate, 0.5 g/L boric acid, 0.333 g/L manganese (II) chloride, 0.2 g/L sodium molybdate, and 4.67 g/L zinc sulfate heptahydrate), and optionally 1 mL/L of a vitamin
• Delft CSM medium consisted of (per liter solution) 7.5 g ammonium sulfate, 14.4 g potassium phosphate monobasic, 0.5 g magnesium sulfate heptahydrate (with these first three components prepared as an 0.9X solution and adjusted to pH 6.5 with sodium hydroxide prior to autoclaving), 3.6 mL of a trace metal solution (consisting of 130 g/L citric acid monohydrate, 0.574 g/L copper (II) sulfate pentahydrate, 8.07 g/L iron (III) chloride hexahydrate, 0.5 g/L boric acid, 0.333 g/L manganese (II) chloride, 0.2 g/L sodium molybdate, and 4.67 g/L zinc sulfate heptahydrate), 1.0 mL of a vitamin solution (0.008 g/L biotin, 1.6 g/L calcium pantothenate, 0.008 g/L folic acid
• Titers for both screening methods are presented as mg/L values on the basis of the entire volume of broth and overlay (total volume of 35 mL for shake flasks and 0.36 mL for 96 well deep well plates).
• “olivetol equivalents” are depicted, which is the titer of olivetol, plus the titer of olivetolic acid multiplied by the molecular weight of olivetol divided by the molecular weight of olivetolic acid.
• “olivetolic acid equivalents” are depicted, which is the titer of olivetolic acid, plus the titer of olivetol multiplied by molecular weight of olivetolic acid divided by the molecular weight of olivetol.
• the sampling time at 18 hours is indicative of productivity/rate of product formation due to hexanoic acid not yet being depleted.
• the sampling time at 48 hours represents a total conversion of hexanoic acid after hexanoic acid is fully utilized.
• LSC3-48, LSC3-74, LSC3-76, and LSC3-77 both improved 18 and 48 hour titers were observed, indicating more efficient incorporation of hexanoic acid into olivetolic acid and olivetol.
• LSC3-48 and LSC3-76/77 had the highest overall conversions of hexanoic acid to olivetolic acid and olivetol, therefore these FAA2 and ANTI were selected for further combinatorial knockouts and introduction into galactose independent strains.
• LSC3-47 had a higher 48 hour titer of olivetolic acid, indicating a potential role in proteolysis of CsOAC.
• LSC3- 64 and LSC3-65 combine the features of LSC3-13 and LSC3-18, with greatly enhanced productivities up to at least 24 hours compared to LSC3-13 in YP + 2% glucose that are more similar to productivities from LSC3-18, as well as reducing the galactose-dependent inhibition of production of LSC3-13 after 48 hours.
• CACG G AT AT CACCG G GTTT AAG AT GT ATTTTTTTTTTCCAC AG AAATT AAAGTT CCAG CGTTT ACCAAAGT
• AACATT ACCCCT G AAGGTTT AG AAAACAT CGGT AG AGCT GTT AT GTGCGTTGCCAAG AAGG AGGGT CT A
• CACG G AT AT CACCG G GTTT AAG AT GT ATTTTTTTTTTCCAC AG AAATT AAAGTT CCAG CGTTT ACCAAAGT
• CAGTT AG ATT C ACCCGTTTT ATTT G CTCCTTACAAG CCTG AT ATG ATG CAAATCTC AG AG AAG CTAAAAA
• AAG CTTT AG CG CT ACCG AG AGTCG AC ATT GTT AG AAAAT GT ATT GC ACATT CC ACT AC AATT GT AGT CAA
• CACG G AT AT CACCG G GTTT AAG AT GT ATTTTTTTTTTCCAC AG AAATT AAAGTT CCAG CGTTT ACCAAAGT
• This example demonstrates producing a surprisingly high and commercially relevant yield of a combined amount of olivetol and olivetolic acid of about 4.5 g/liter over 4-5 days.
• Genotype of parent strain o pGallO-CsAAEl-tCycl-pGall-OST2AOAC-tCycl::leu2-3, pGa 110-CsAAE 1-tCycl-pGa I l-OST2AOAC-tCycl : : u ra 3-52, pGallO-CsAAEl-tCycl-pGall-OST2AOAC-tCycl::trpl, pGallO-HMGK2R-tADHl-pGall-IDIl-tCycl-KanMX::YORWA22
• a shake flask containing 50 mL YPD with 20 g/L glucose was inoculated with freshly streaked LSC3-4. Strain grew at 30 C for 24 hours to an OD600 of 8. 40 mL of this culture was used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose Batch media:
• Pulse feeding was used for both growth phase and production phase during the run. Fermentation batch was inoculated with 40 mL of inoculum. Feeding was triggered at the end of batch phase when batch glucose was completely exhausted and p02 was increased by 20% (or more). Feed media (growth or production media) was delivered in pulses. Each pulse delivered 2 g glucose /starting batch volume with maximum feed rate not exceeding 20 g glucose/L/hr. pH was maintained at 6 throughout the run. Temperature of fermenter was maintained at 30 C. Airflow rate was maintained at 1.25 L/min. Agitation was 800 rpm. Summary of Metrices:
• a shake flask containing 50 mL YPD with 20 g/L glucose was inoculated with freshly streaked LSC3-13. Strain grew at 30 C for 24 hours to an OD600 of 8. 40 mL of this culture was used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose Batch media:
• Pulse feeding was used for both growth phase and production phase during the run. Fermentation batch was inoculated with 40 mL of inoculum. Feeding was triggered at the end of batch phase when batch glucose was completely exhausted and p02 was increased by 20% (or more). Feed media (growth or production media) was delivered in pulses. Each pulse delivered 2 g glucose /starting batch volume with maximum feed rate not exceeding 20 g glucose/L/hr. pH was maintained at 6 throughout the run. Temperature of fermenter was maintained at 30 C. Airflow rate was maintained at 1.25 L/min. Agitation was 800 rpm. Summary of Metrices:
• This example demonstrates a high yielding fermentation of O and OA.
• a shake flask containing 50 mL YPD with 20 g/L glucose was inoculated with freshly streaked LSC3-13. Strain grew at 30 C for 24 hours to an OD600 of 4. 40 mL of this culture was used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose
• Fermentation batch was inoculated with 40 mL of inoculum. Feeding was triggered at the end of batch phase when batch glucose was completely exhausted and p02 was increased by 20% (or more). Feed media (growth or production media) was delivered in pulses. Each pulse delivered 2 g glucose /starting batch volume with maximum feed rate not exceeding 40 g glucose/L/hr. pH was maintained at 6 throughout the run. Temperature of fermenter was maintained at 30C. Air flow rate was maintained at 1.25 L/min. Agitation was 800 rpm. This fermentation works at pH range of 5-6. It is contemplated that in some embodiments, the fermentation is carried out at about pH 5.0.
• a pulse rate of about 1.7 g/L/pulse, with maximum feed rate of about 10 g/L/hr is employed. It is contemplated that in some embodiments, a pulse rate of about 1.7 g/L/pulse is employed. It is contemplated that in some embodiments a maximum feed rate of about 10 g/L/hr is employed.
• Genotype of parent strain leu2 A ::ScLEU2 ⁇ pScLEU2/tScCYCl>CsAEEKpScGAL10/pScGALl>CsTkS-T2A-CsOAC ⁇ tScCY
• Seed Media 10 g/L yeast extract ⁇ 20 g/L peptones ⁇ 20 g/L glucose
• Fermentation batch was inoculated with 17 mL of inoculum. Feeding was triggered at the end of batch phase when batch glucose was completely exhausted and p02 was increased by 10% (or more). Feed media (production media) was delivered in pulses. Each pulse delivered 1.7 g glucose /starting batch volume with maximum feed rate not exceeding 10 g glucose/L/hr. pH was maintained at 5.5 throughout the run. Temperature of fermenter was maintained at 30C. Air flow rate was maintained at 1.25 L/min. Agitation was 800 rpm. Median oxygen uptake rate (OUR) was 60-80 mmoles/L/hr. A maximum OUR of 100-110 moles/L/hr was achieved during the process.
• OUR Median oxygen uptake rate
• Optimum time to add IPM We found that the optimum time to add IPM is between 12 and 36 hours post inoculation.
• Pulse metric parameters We found that the optimum pulse parameters for our process was 1.7 g glucose/L initial tank volume/pulse with a maximum feed rate of 10 g/L of initial tank volume/hr. Effect of overlay: Isopropyl myristate is used as an overlay in our process. The optimum IPM loading for our process at pH 5.5 is 26% of total tank volume or 40% of initial tank volume.
• Seed train condition We found that the optimum seed train condition was to inoculate an initial flask containing YPD (10 g/L yeast extract, 20 g/L peptones and 20 g/L glucose) with 1 mL seed vial. All subsequent seed tanks would be inoculated with 2% inoculum and will run as batch tanks with pH control (pH set at 5.5). The production tank will be inoculated with 3.5% inoculum from the last seed train stage.
• YPD g/L yeast extract, 20 g/L peptones and 20 g/L glucose
• Precultures of LSC3-2 were grown in 50 mL tubes containing 10 mL of YP glucose overnight. These were used to inoculate 50 mL tubes containing 10 mL of YP + 2% (w/v) galactose + 2 mM (176 mg/L) butyric acid (BA) + 20% (v/v) isopropyl myristate (IPM) and were grown for 48 hours with 180 rpm shaking at 30 C. D/DA present in IPM layer only, is tabulated below based on standard curves run for DA and D, and titers are based on the whole volume of broth plus overlay (12 mL total).
• the shake flask/tube experiments were scaled down to 96 well deepwell plate format. Precultures from colonies of each strain were grown in 300 pL YP + 2% (w/v) glucose. Main cultures containing 300 pL YP + 2% (w/v) galactose + 0.02-0.08% (w/v) butyric acid + 20% (v/v) IPM (60 pL) or 20% (v/v) diethyl sebacate (60 pL) for strain LSC3-2, or the same but with 2% (w/v) glucose instead of galactose for strains LSC3-4, LSC3-13, and LSC3-18, were grown for 48 hours and the IPM or diethyl sebacate overlay was sampled at 24 and 48 hours following acidification of the media with 10 pL of 5 M phosphoric acid. Three replicates of each strain/media condition were tested and averages and standard deviations for detected divarinic acid, divarin, and total divarin equivalents are reported in the table
• Example 3A Divarinic acid/divarin production in LSC3-2 and derived strains in defined media with varying pH
• a defined medium Delft CSM medium
• a defined medium consisted of (per liter solution) 7.5 g ammonium sulfate, 14.4 g potassium phosphate monobasic, 0.5 g magnesium sulfate heptahydrate (with these first three components prepared as an 0.9X solution and adjusted to pH 6.5 with sodium hydroxide prior to autoclaving), 3.6 mL of a trace metal solution (consisting of 130 g/L citric acid monohydrate, 0.574 g/L copper (II) sulfate pentahydrate, 8.07 g/L iron (III) chloride hexahydrate, 0.5 g/L boric acid, 0.333 g/L manganese (II) chloride, 0.2 g/L sodium molybdate,
• LSC3-2, LSC3-4, LSC3-13, and LSC3-18 were then tested in 96 well deepwell plates as described in the last experiment in Example 2, with precultures containing 2% (w/v) glucose as carbon source, and production cultures containing 2% (w/v) galactose for LSC3-2, 2% (w/v) glucose for LSC3-4, LSC3-13, and LSC3-18, 20% (v/v) IPM overlay, plus 0.04% (w/v) butyric acid as substrate. After 48 hours, production cultures were acidified with 10 pL of 5 M phosphoric acid, and the IPM layer was sampled.
• Divarinic acid and divarin were quantified by HPLC and detected whole broth + overlay titers, averaged across 3 biological replicates. Up to 59.6 mg/L divarinic acid plus 19.0 mg/L divarin were produced by strain LSC3-13 in full-strength Delft CSM medium plus 2% (w/v) glucose. In reduced salt strength buffered media, addition of MES pH 5.0 (leading to a final medium pH of 5.69) appeared optimal for most strains, with less of a pH dependence in production observed for LSC3-13.
• This example provides a process of producing divarin and/or divarinic acid .
• Genotype of parent strain o pGallO-CsAAEl-tCycl-pGall-OST2AOAC-tCycl::leu2-3, pGa 110-CsAAE 1-tCycl-pGa I l-OST2AOAC-tCycl : : u ra 3-52, pGallO-CsAAEl-tCycl-pGall-OST2AOAC-tCycl::trpl, pGallO-HMGK2R-tADHl-pGall-IDIl-tCycl-KanMX::YORWA22
• a shake flask containing 50 mL YPD with 20 g/L glucose is inoculated with freshly streaked LSC3- 4.
• Strain grows at 30 C for 24 hours to an OD600 of 8. 40 mL of this culture is used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose Batch media:
• Pulse feeding is used for both growth phase and production phase during the run. Fermentation batch is inoculated with 40 mL of inoculum. Feeding is triggered at the end of batch phase when batch glucose is completely exhausted and p02 is increased by 20% (or more). Feed media (growth or production media) is delivered in pulses. Each pulse delivered 2 g glucose /starting batch volume with maximum feed rate not exceeding 20 g glucose/L/hr. pH is maintained at 6 throughout the run. Temperature of fermenter is maintained at 30C. Air flow rate is maintained at 1.25 L/min. Agitation is 800 rpm.
• a shake flask containing 50 mL YPD with 20 g/L glucose is inoculated with freshly streaked LSC3- 13. Strain grew at 30 C for 24 hours to an OD600 of 8. 40 mL of this culture is used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose Batch media:
• Pulse feeding is used for both growth phase and production phase during the run. Fermentation batch is inoculated with 40 mL of inoculum. Feeding is triggered at the end of batch phase when batch glucose is completely exhausted and p02 is increased by 20% (or more). Feed media (growth or production media) is delivered in pulses. Each pulse delivered 2 g glucose /starting batch volume with maximum feed rate not exceeding 20 g glucose/L/hr. pH is maintained at 6 throughout the run. Temperature of fermenter is maintained at 30 C. Air flow rate is maintained at 1.25 L/min. Agitation is 800 rpm.
• Example 3B In a fermentation experiment run substantially as Example 3B, and employing strain LSC3-134A or LSC3-134 as disclosed here, surprisingly, a titer of 2 g/L of divarin equivalent was obtained.
• Example 4A Growth of 5. cerevisiae strains in overlay/underlay candidates ln-situ liquid-liquid extraction (biphasic fermentation) is a strategy that can be employed in accordance with the present invention for physical separation of product from cells via partitioning into a second liquid phase from an aqueous culture phase.
• the second or organic liquid phase is present as either an overlay if its density is less than that of the aqueous phase, or underlay if its density is greater than that of the aqueous phase.
• Certain properties of the overlay or underlay are considered for production of olivetolic acid/olivetol and other resorcinols such as formulas IA and IB: (1) non-toxic or low toxicity for growth of the host strain, (2) a favorable partition coefficient of the product in the organic phase vs. the aqueous phase, and (3) preferably a lower partition coefficient for fed hexanoic acid (for olivetolic acid/olivetol) or other fatty acid such as RCO2H (for other resorcinols) in the organic phase vs. the aqueous phase.
• Additional properties of the organic phase enhance its suitability for downstream conversion, e.g. and without limitation, to cannabigerol and other cannabinoid compounds, including suitability as a solvent or co-solvent during downstream prenylation or other reactions, and boiling point if downstream separation by distillation is employed.
• biological triplicate cultures were sampled and optical density at 600 nm (OD600) was measured as a proxy for biomass growth on a SpectraMax plate reader, with dilution in water to allow measurements to be within the linear range of instrumental readings. Averaged OD600s across at least three biological replicates of each strain/overlay condition at each sampling time. Multiple sets of measurements were performed on large groups of overlay and underlay candidates in different batches.
• Double colons indicate replacement of the indicated locus to the left of the colons with the integration cassette to the right of the colons.
• PkHIS4 is the HIS4 gene from Pichia kudriavzevii under control of a TEF1 promoter from S. cerevisiae.
• HygR is a hygromycin resistance cassette. Defective genes that generate auxotrophies are indicated in parentheses and strains that have none listed are fully prototrophic.
• dialkyl ester chain length from diethyl to diisopropyl to dibutyl in a dialkyl adipate series reduced toxicity. Some growth was observed with diisopropyl adipate and no apparent toxicity in dibutyl adipate. Dibutyl sebacate was also completely non-inhibitory to growth and accordingly, non-toxic.
• the minimum non-toxic internal alkyl chain length of diethyl diesters is sebacate. In certain embodiments, shorter internal alkyl chain length down to adipate is possible with diisopropyl diesters.
• octyl acetate was toxic and for the hexanoate series, growth was only observed starting with hexyl hexanoate, which was moderately non-toxic. Isopropyl octanoate was moderately inhibitory but allowed for some growth. For the decanoate series, methyl decanoate was moderately inhibitory to growth but still allowed for growth. Texanol, a monoester alcohol (2,2,4-trimethyl-l,3-pentanediol monoisobutyrate), was inhibitory to growth under the conditions tested.
• growth-suitable monoester overlays for resorcinol or cannabinoid production include hexyl hexanoate or any higher chain length alkyl hexanoate ester, C 3 chain-length or higher alkyl octanoate esters, and methyl (Ci) or higher alkyl decanoates, laurates, or myristates.
• esters and diesters are employed as the organic phase in accordance with the present invention.
• Fatty alcohols are mostly solids above C 10 saturated chain length.
• Decanol is a liquid however it was toxic to growth.
• oleyl alcohol supports robust growth.
• longer chain length (C 12 or higher) unsaturated fatty alcohols can be suitable overlays supporting S. cerevisiae or another fermenting organism's growth.
• fatty alcohols preferably C 12 or higher alcohols, are employed as the organic phase in accordance with the present invention.
• alkanes and paraffins support robust growth. Lack of toxicity was observed for dodecane, tetradecane, hexadecane, light and heavy paraffin oils, and isopar M.
• all C12 and higher paraffins are suitable overlays supporting S. cerevisiae or another fermenting organism's growth.
• fatty alcohols preferably C12 or higher alcohols, are employed as the organic phase in accordance with the present invention.
• triacylglycerols were tested, including tricaprylin, coconut oil, and canola oil (vegetable oils having different average chain length compositions of fatty acid chains, with coconut oil being predominantly C12-C14 saturated fatty acids, and canola being predominantly Ci 6 -Ci 8 and a mixture of saturated and unsaturated fatty acids).
• Tricaprylin a synthetic oil containing three Cs fatty acid chains, was fairly toxic, however allowed some growth of all strains in YP + 2% glucose.
• coconut and canola oil were non-toxic to growth.
• Di-tert-butyl malonate also exhibited much lower toxicity at 1-10% (v/v), and particularly 1- 2.5% (v/v), in IPM and isopar M.
• mixtures of longer chain monoesters or paraffins with moderately to very toxic diesters are useful according to the present invention.
• Precultures of LSC3-2 and LSC3-18 were inoculated from YPD streak plates and grown in 30 mL of YP + 2% (w/v) glucose in baffled 250 mL shake flasks for approximately 18 hours overnight at 30°C with 200 rpm shaking.
• Baffled 250 mL shake flasks containing 30 mL of YP + 2% (w/v) galactose, 5 mL of IPM or diethyl sebacate, and 0.04% (w/v) hexanoic acid were inoculated with 1 mL of preculture and grown for at 30°C with 200 rpm shaking.
• samples of cell culture broth plus overlay were sampled into microcentrifuge tubes and stored at -20°C at least overnight. Sample tubes were thawed and aqueous sample and overlay sample were pipetted into plates for HPLC analysis. Overlay samples were diluted 1:1 v/v with methanol in the HPLC plate prior to analysis.
• Total olivetol equivalents are defined as the concentration of olivetol in mg/L, plus the concentration of olivetolic acid in mg/L multiplied by the ratio of the molecular weight of olivetol to olivetolic acid.
• Lower total olivetol equivalents were observed with diethyl sebacate overlayer as compared to IPM. With IPM overlay, OA partitioned between the IPM and aqueous phases, with a substantial amount of OA remaining in the aqueous phase in these culturing conditions. By contrast, OA entirely partitioned into diethyl sebacate with none present in the aqueous phase. The reduction in total production levels with a diethyl sebacate overlay may occur due to a reduction in O ⁇ eoo due to moderately growth inhibitory properties of diethyl sebacate.
• LSC3-2 precultures were inoculated from YPD streak plates and grown in 300 m ⁇ YP + 2% (w/v) glucose in round-bottom square well 96 well deepwell plates for approximately 18 hours overnight at 30°C with 950 rpm shaking in an Infors plate shaker.
• 96 well deepwell plate wells containing 300 m ⁇ of YP + 2% (w/v) galactose, 60 m ⁇ of IPM, diethyl sebacate, di-tert-butyl malonate, or methyl soyate, and 0.04% (w/v) hexanoic acid were inoculated with 10 m ⁇ of preculture and grown for 30°C with 950 rpm shaking.
• LSC3-13 precultures were inoculated from YPD streak plates and grown in 300 m ⁇ Delft medium + 0.79 g/L complete supplement mixture (CSM) (ForMedium, Norfolk, UK) + 2% (w/v) glucose in round-bottom square well 96 well deepwell plates for approximately 18 hours overnight at 30°C with 950 rpm shaking in an Infors plate shaker.
• CSM complete supplement mixture
• Delft medium contains (per liter solution) 7.5 g ammonium sulfate, 14.4 g potassium phosphate monobasic (added from a 1 M stock solution adjusted to pH 6.5 with sodium hydroxide), 0.5 g magnesium sulfate heptahydrate, 3.6 mL of a trace metal solution (consisting of 130 g/L citric acid monohydrate, 0.574 g/L copper (II) sulfate pentahydrate, 8.07 g/L iron (III) chloride hexahydrate, 0.5 g/L boric acid, 0.333 g/L manganese (II) chloride, 0.2 g/L sodium molybdate, and 4.67 g/L zinc sulfate heptahydrate), and 1.0 mL of a vitamin solution (0.008 g/L biotin, 1.6 g/L calcium pantothenate, 0.008 g/L folic acid, 8 g/L myo-inosi
• the aqueous layer and overlay were sampled on a Bravo automated liquid handling platform (Agilent) by first removing 200 m ⁇ of aqueous sample into a 96 well filter plate, adding 180 m ⁇ of IPM to each well, mixing on a shaking platform for several minutes, centrifuging the plate at 3000 rpm for 5 minutes to separate phases, and pipetting 100 m ⁇ of overlay from each well into an HPLC plate.
• Overlay samples were diluted 1:1 v/v with methanol in the HPLC plate, sealed and analyzed by HPLC.
• Aqueous samples were centrifuged in the 96 well filter plate at 3000 rpm for 5 minutes into an HPLC plate, sealed, and analyzed by HPLC.
• Example 4C Divarinic acid and divarin production with organic solvent overlays
• LSC3-13 precultures were inoculated from YPD streak plates and grown in 300 m ⁇ .
• Delft medium + 0.79 g/L complete supplement mixture (CSM) (ForMedium, Norfolk, UK) + 2% (w/v) glucose in round-bottom square well 96 well deepwell plates for approximately 18 hours overnight at 30°C with 950 rpm shaking in an Infors plate shaker.
• 96 well deepwell plate wells containing 300 m ⁇ . of Delft medium + CSM + 2% (w/v) glucose, 60 m ⁇ .
• Genotype of parent strain leu2 A ::ScLEU2 ⁇ pScLEU2/tScCYCl>CsAEEKpScGAL10/pScGALl>CsTKS-T2A-CsOAC ⁇ tScCY
• a shake flask containing 50 mL YPD with 20 g/L glucose was inoculated with a seed vial containing LSC3-134. Strain grew at 30 C for 24 hours to an OD600 of 3-4. 15 mL of this culture was used to inoculate the fermentation tank.
• Seed Media YP with 20 g/L glucose
• Fermentation batch was inoculated with 15 mL of inoculum. Feeding was triggered at the end of batch phase when batch glucose was completely exhausted and p02 was increased by 10% (or more). Feed media (growth or production media) was delivered in pulses. Each pulse delivered 1.7 g glucose /starting batch volume with maximum feed rate not exceeding 10 g glucose/L/hr. pH was maintained at 5.5 throughout the run. Temperature of fermenter was maintained at 30 C. Air flow rate was maintained at 1.25 L/min. Agitation was 800 rpm.

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• 2021
  • 2021-05-03 WO PCT/US2021/030452 patent/WO2021225952A1/en unknown
  • 2021-05-03 CA CA3177968A patent/CA3177968A1/en active Pending
  • 2021-05-03 EP EP21799490.4A patent/EP4146809A1/de active Pending

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