The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of olivetolic acid and phytocannabinoids. A method of producing olivetolic acid (OVLa) and/or a phytocannabinoid in a heterologous host cell having OVLa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and culturing the transformed host cell to produce OVLa and/or phytocannabinoids therefrom. The variant OAC protein (SEQ ID NO:92) has at least 85% sequence identity with the wild type OAC protein (SEQ ID NO:91). Exemplary variants having improved OVLa or phytocannabinoid production capacity are described.
The present disclosure relates generally to methods and cells for the production of substituted phytocannabinoids or substituted phytocannabinoid precursors in host cells that produce the phytocannabinoid or the phytocannabinoid precursor. Methods are described which comprise transforming host cells with a sequence encoding an enzyme for derivatizing the phytocannabinoid or precursor with a substituent, such as O-methyl, glycosyl, or halogen. The transformed cells are cultured to produce substituted phytocannabinoids or substituted phytocannabinoid precursors.
C07C 65/05 - Compounds having carboxyl groups bound to carbon atoms of six-membered aromatic rings and containing any of the groups OH, O-metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
3.
CANNABIDIOLIC ACID SYNTHASE VARIANTS WITH IMPROVED ACTIVITY FOR USE IN PRODUCTION OF PHYTOCANNABINOIDS
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of cannabidiolic acid (CBDa), cannabigerolic acid (CBCa), and other phytocannabinoids. A method of producing CBDa, CBCa, and/or other phytocannabinoids in a heterologous host cell having CBDa-producing, CBCa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant CBDa synthase protein having a serine insertion between residues P224 and K225 and one or more other amino acid mutation relative to wild type CBDa synthase, and culturing the transformed host cell to produce CBDa, CBCa, and/or other phytocannabinoids therefrom. The variant CBDa synthase protein has at least 85% sequence identity with the wild type CBDa synthase protein sequence OXC52 according to SEQ ID NO:140, with serine insertion (SEQ ID NO:141). Exemplary variants having good phytocannabinoid production capacity are described.
The present disclosure relates generally to methods and cell lines for the production of phytocannabinoids, phytocannabinoid precursors or intermediates, or phytocannabinoid analogue. Methods for transformation of host cells, such as yeast cells, are described. Cells may be transformed, for example, with a polynucleotide encoding a polyketide synthase (PKS) enzyme, a polynucleotide encoding an olivetolic acid cyclase (OAC) enzyme, and/or a polynucleotide encoding a prenyltransferase (PT) enzyme; and optionally a polynucleotide encoding an acyl-CoA synthase (Alk) enzyme; a polynucleotide encoding a fatty acyl CoA activating (CsAAE) enzyme; and/or a polynucleotide encoding a THCa synthase (OXC) enzyme.
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of olivetolic acid and phytocannabinoids. A method of producing olivetolic acid (OVLa) and/or a phytocannabinoid in a heterologous host cell having OVLa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and culturing the transformed host cell to produce OVLa and/or phytocannabinoids therefrom. The variant OAC protein (SEQ ID NO:92) has at least 85% sequence identity with the wild type OAC protein (SEQ ID NO:91). Exemplary variants having improved OVLa or phytocannabinoid production capacity are described.
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of olivetolic acid and phytocannabinoids. A method of producing olivetolic acid (OVLa) and/or a phytocannabinoid in a heterologous host cell having OVLa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and culturing the transformed host cell to produce OVLa and/or phytocannabinoids therefrom. The variant OAC protein (SEQ ID NO:92) has at least 85% sequence identity with the wild type OAC protein (SEQ ID NO:91). Exemplary variants having improved OVLa or phytocannabinoid production capacity are described.
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of cannabidiolic acid (CBDa) and other phytocannabinoids. A method of producing CBDa and/or a phytocannabinoid in a heterologous host cell having CBDa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant CBDa synthase protein having a serine insertion between residues P224 and K225 and one or more other amino acid mutation relative to wild type CBDa synthase, and culturing the transformed host cell to produce CBDa and/or phytocannabinoids therefrom. The variant CBDa synthase protein has at least 85% sequence identity with the wild type CBDa synthase protein sequence OXC52 according to SEQ ID NO:140, with serine insertion (SEQ ID NO:141). Exemplary variants having improved CBDa or phytocannabinoid production capacity are described.
The present disclosure relates generally to methods and cells for the production of substituted phytocannabinoids or substituted phytocannabinoid precursors in host cells that produce the phytocannabinoid or the phytocannabinoid precursor. Methods are described which comprise transforming host cells with a sequence encoding an enzyme for derivatizing the phytocannabinoid or precursor with a substituent, such as O-methyl, glycosyl, or halogen. The transformed cells are cultured to produce substituted phytocannabinoids or substituted phytocannabinoid precursors.
C07C 65/05 - Compounds having carboxyl groups bound to carbon atoms of six-membered aromatic rings and containing any of the groups OH, O-metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
C12N 15/52 - Genes encoding for enzymes or proenzymes
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
C12N 9/00 - Enzymes, e.g. ligases (6.)ProenzymesCompositions thereofProcesses for preparing, activating, inhibiting, separating, or purifying enzymes
C12P 17/06 - Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
C12P 19/56 - Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
C12P 19/60 - Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
C12P 7/40 - Preparation of oxygen-containing organic compounds containing a carboxyl group
The present disclosure relates generally to methods and cell lines for the production of phytocannabinoids, phytocannabinoid precursors or intermediates, or phytocannabinoid analogue. Methods for transformation of host cells, such as yeast cells, are described. Cells may be transformed, for example, with a polynucleotide encoding a polyketide synthase (PKS) enzyme, a polynucleotide encoding an olivetolic acid cyclase (OAC) enzyme, and/or a polynucleotide encoding a prenyltransferase (PT) enzyme; and optionally a polynucleotide encoding an acyl-CoA synthase (Alk) enzyme; a polynucleotide encoding a fatty acyl CoA activating (CsAAE) enzyme; and/or a polynucleotide encoding a THCa synthase (OXC) enzyme.
C12P 17/06 - Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
C12P 17/18 - Preparation of heterocyclic carbon compounds with only O, N, S, Se, or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
C12P 7/22 - Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
10.
Method and cell line for production of phytocannabinoids and phytocannabinoid analogues in yeast
Streptomyces sp CL190. The yeast cell may be modified to mitigate depletion of geranyl pyrophosphate for increasing available geranyl pyrophosphate for prenylation.
Dictyostelium discoideum polyketide synthase (“DiPKS”). Wild type DiPKS produces methyl-olivetol only. DiPKS may be modified to produce olivetol only or a mixture of both olivetol and methyl-olivetol. The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol.
Dictyostelium discoideum polyketide synthase (“DiPKS”). Wild type DiPKS produces methyl-olivetol only. DiPKS may be modified to produce olivetol only or a mixture of both olivetol and methyl-olivetol. The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol.
A method and cell line for producing polyketides in yeast. The method applies, and the cell line includes, a yeast cell transformed with a polyketide synthase coding sequence. The polyketide synthase enzyme catalyzes synthesis of olivetol or methyl-olivetol, and may include Dictyostelium discoideum polyketide synthase ("DiPKS"). Wild type DiPKS produces methyl-olivetol only. DiPKS may be modified to produce olivetol only or a mixture of both olivetol and methyl-olivetol. The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol.
A method and cell line for producing phytocannabinoids and phytocannabinoid analogues in yeast. The method applies, and the cell line includes, a yeast cell transformed with a polyketide synthase CDS and a cytosolic prenyltransferase CDS. The polyketide synthase enzyme catalyzes synthesis of olivetol or methyl-olivetol, and may include Cannabis sativa olivetolic acid synthase or Dictyostelium discoideum polyketide synthase ("DiPKS"). The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol. The prenyltransferase enzyme catalyzes synthesis of cannabigerol or a cannabigerol analogue, and may include an αββα cytosolic prenyltransferase enzyme from Streptomyces sp CL190. The yeast cell may be modified to mitigate depletion of geranyl pyrophosphate for increasing available geranyl pyrophosphate for prenylation.
C07C 65/05 - Compounds having carboxyl groups bound to carbon atoms of six-membered aromatic rings and containing any of the groups OH, O-metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
C12N 1/19 - YeastsCulture media therefor modified by introduction of foreign genetic material
C12N 15/52 - Genes encoding for enzymes or proenzymes
A method and cell line for producing phytocannabinoids and phytocannabinoid analogues in yeast. The method applies, and the cell line includes, a yeast cell transformed with a polyketide synthase CDS and a cytosolic prenyltransferase CDS. The polyketide synthase enzyme catalyzes synthesis of olivetol or methyl-olivetol, and may include Cannabis sativa olivetolic acid synthase or Dictyostelium discoideum polyketide synthase ("DiPKS"). The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol. The prenyltransferase enzyme catalyzes synthesis of cannabigerol or a cannabigerol analogue, and may include an aßßa cytosolic prenyltransferase enzyme from Streptomyces sp CL190. The yeast cell may be modified to mitigate depletion of geranyl pyrophosphate for increasing available geranyl pyrophosphate for prenylation.
C07C 65/05 - Compounds having carboxyl groups bound to carbon atoms of six-membered aromatic rings and containing any of the groups OH, O-metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
C12N 1/19 - YeastsCulture media therefor modified by introduction of foreign genetic material
C12N 9/00 - Enzymes, e.g. ligases (6.)ProenzymesCompositions thereofProcesses for preparing, activating, inhibiting, separating, or purifying enzymes
The present disclosure relates generally to methods and cell lines for the production of phytocannabinoids, phytocannabinoid precursors or intermediates, or phytocannabinoid analogue. Methods for transformation of host cells, such as yeast cells, are described. Cells may be transformed, for example, with a polynucleotide encoding a polyketide synthase (PKS) enzyme, a polynucleotide encoding an olivetolic acid cyclase (OAC) enzyme, and/or a polynucleotide encoding a prenyltransferase (PT) enzyme; and optionally a polynucleotide encoding an acyl-CoA synthase (Alk) enzyme; a polynucleotide encoding a fatty acyl CoA activating (CsAAE) enzyme; and/or a polynucleotide encoding a THCa synthase (OXC) enzyme.
C12N 15/63 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression
C12P 7/22 - Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
C12P 17/06 - Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
C12P 17/18 - Preparation of heterocyclic carbon compounds with only O, N, S, Se, or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
17.
OLIVETOLIC ACID CYCLASE VARIANTS WITH IMPROVED ACTIVITY FOR USE IN PRODUCTION OF PHYTOCANNABINOIDS
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of olivetolic acid and phytocannabinoids. A method of producing olivetolic acid (OVLa) and/or a phytocannabinoid in a heterologous host cell having OVLa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant olivetolic acid cyclase (OAC) protein having at least 6 amino acid mutations relative to the wild type OAC protein, and culturing the transformed host cell to produce OVLa and/or phytocannabinoids therefrom. The variant OAC protein (SEQ ID NO:92) has at least 85% sequence identity with the wild type OAC protein (SEQ ID NO:91). Exemplary variants having improved OVLa or phytocannabinoid production capacity are described.
A method and cell line for producing polyketides in yeast. The method applies, and the cell line includes, a yeast cell transformed with a polyketide synthase coding sequence. The polyketide synthase enzyme catalyzes synthesis of olivetol or methyl-olivetol, and may include Dictyostelium discoideum polyketide synthase ("DiPKS"). Wild type DiPKS produces methyl-olivetol only. DiPKS may be modified to produce olivetol only or a mixture of both olivetol and methyl-olivetol. The yeast cell may be modified to include a phosphopantethienyl transferase for increased activity of DiPKS. The yeast cell may be modified to mitigate mitochondrial acetaldehyde catabolism for increasing malonyl-CoA available for synthesizing olivetol or methyl-olivetol.
The present disclosure relates generally to methods and cells for the production of substituted phytocannabinoids or substituted phytocannabinoid precursors in host cells that produce the phytocannabinoid or the phytocannabinoid precursor. Methods are described which comprise transforming host cells with a sequence encoding an enzyme for derivatizing the phytocannabinoid or precursor with a substituent, such as O-methyl, glycosyl, or halogen. The transformed cells are cultured to produce substituted phytocannabinoids or substituted phytocannabinoid precursors.
C07C 65/05 - Compounds having carboxyl groups bound to carbon atoms of six-membered aromatic rings and containing any of the groups OH, O-metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring o-Hydroxy carboxylic acids
C12N 9/00 - Enzymes, e.g. ligases (6.)ProenzymesCompositions thereofProcesses for preparing, activating, inhibiting, separating, or purifying enzymes
C12P 17/06 - Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
C12P 19/56 - Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
C12P 19/60 - Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
20.
CANNABIDIOLIC ACID SYNTHASE VARIANTS WITH IMPROVED ACTIVITY FOR USE IN PRODUCTION OF PHYTOCANNABINOIDS
The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of cannabidiolic acid (CBDa) and other phytocannabinoids. A method of producing CBDa and/or a phytocannabinoid in a heterologous host cell having CBDa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant CBDa synthase protein having a serine insertion between residues P224 and K225 and one or more other amino acid mutation relative to wild type CBDa synthase, and culturing the transformed host cell to produce CBDa and/or phytocannabinoids therefrom. The variant CBDa synthase protein has at least 85% sequence identity with the wild type CBDa synthase protein sequence OXC52 according to SEQ ID NO:140, with serine insertion (SEQ ID NO:141). Exemplary variants having improved CBDa or phytocannabinoid production capacity are described.
patents
