Recombinant microorganisms and methods for producing cannabinoids and cannabinoid derivatives

1. A genetically modified yeast cell for producing a cannabinoid or a cannabinoid derivative, the genetically modified yeast cell comprising one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO:110 or SEQ ID NO:100.

2. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell further comprises one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a tetraketide synthase (TKS) polypeptide comprising the amino acid sequence of SEQ ID NO:11 and one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding an olivetolic acid cyclase (OAC) polypeptide comprising the amino acid sequence of SEQ ID NO:10.

3. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell further comprises one or more of the following:

a) one or more heterologous nucleic acids encoding a polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative, wherein the polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative is an acyl-activating enzyme (AAE) polypeptide comprising the amino acid sequence of SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO:149; a fatty acyl-CoA ligase polypeptide comprising the amino acid sequence of SEQ ID NO:145 or SEQ ID NO:147; or a fatty acyl-CoA synthetase (FAA) polypeptide comprising the amino acid sequence of SEQ ID NO:169, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, or SEQ ID NO:200; or

b) one or more heterologous nucleic acids encoding a polypeptide that generates geranyl pyrophosphate, wherein the polypeptide that generates geranyl pyrophosphate is a geranyl pyrophosphate synthetase (GPPS) polypeptide comprising the amino acid sequence of SEQ ID NO:60.

4. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell further comprises one or more of the following:

a) one or more heterologous nucleic acids encoding a HMG-CoA synthase (HMGS) polypeptide comprising the amino acid sequence of SEQ ID NO:115;

b) one or more heterologous nucleic acids encoding a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) polypeptide comprising the amino acid sequence SEQ ID NO:208;

c) one or more heterologous nucleic acids encoding a mevalonate kinase (MK) polypeptide comprising the amino acid sequence of SEQ ID NO:64;

d) one or more heterologous nucleic acids encoding a phosphomevalonate kinase (PMK) polypeptide comprising the amino acid sequence of SEQ ID NO:205;

e) one or more heterologous nucleic acids encoding a mevalonate pyrophosphate decarboxylase (MVD) polypeptide comprising the amino acid sequence of SEQ ID NO:66; or

f) one or more heterologous nucleic acids encoding a isopentenyl diphosphate isomerase (IDI) polypeptide comprising the amino acid sequence of SEQ ID NO:58.

5. The genetically modified yeast cell of claim 4 , wherein the genetically modified yeast cell further comprises one or more heterologous nucleic acids encoding a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA and wherein the polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA is an acetoacetyl-CoA thiolase polypeptide comprising the amino acid sequence of SEQ ID NO:25.

6. The genetically modified yeast cell of claim 1 , wherein the yeast cell is Saccharomyces cerevisiae.

7. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell further comprises one or more heterologous nucleic acids encoding a cannabinoid synthase polypeptide, wherein the cannabinoid synthase polypeptide is a tetrahydrocannabinolic acid (THCA) synthase polypeptide comprising the amino acid sequence of SEQ ID NO:155 or a cannabidiolic acid (CBDA) synthase polypeptide comprising the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:151.

8. The genetically modified yeast cell of claim 1 , wherein the genetically modified yeast cell further comprises one or more of the following:

a) one or more heterologous nucleic acids encoding a polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative, wherein the polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative is an acyl-activating enzyme (AAE) polypeptide comprising the amino acid sequence of SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO:149; a fatty acyl-CoA ligase polypeptide comprising the amino acid sequence of SEQ ID NO:145 or SEQ ID NO:147; or a fatty acyl-CoA synthetase (FAA) polypeptide comprising the amino acid sequence of SEQ ID NO:169, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, or SEQ ID NO:200;

b) one or more heterologous nucleic acids encoding a polypeptide that generates geranyl pyrophosphate, wherein the polypeptide that generates geranyl pyrophosphate is a geranyl pyrophosphate synthetase (GPPS) polypeptide comprising the amino acid sequence of SEQ ID NO:60;

c) one or more heterologous nucleic acids encoding a HMG-CoA synthase (HMGS) polypeptide comprising the amino acid sequence of SEQ ID NO:115;

d) one or more heterologous nucleic acids encoding a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) polypeptide comprising the amino acid sequence of SEQ ID NO:208;

e) one or more heterologous nucleic acids encoding a mevalonate kinase (MK) polypeptide comprising the amino acid sequence of SEQ ID NO:64;

f) one or more heterologous nucleic acids encoding a phosphomevalonate kinase (PMK) polypeptide comprising the amino acid sequence of SEQ ID NO:205;

g) one or more heterologous nucleic acids encoding a mevalonate pyrophosphate decarboxylase (MVD) polypeptide comprising the amino acid sequence of SEQ ID NO:66;

h) one or more heterologous nucleic acids encoding a isopentenyl diphosphate isomerase (IDI) polypeptide comprising the amino acid sequence of SEQ ID NO:58;

i) one or more heterologous nucleic acids encoding a polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA, wherein the polypeptide that condenses two molecules of acetyl-CoA to generate acetoacetyl-CoA is an acetoacetyl-CoA thiolase polypeptide comprising the amino acid sequence of SEQ ID NO:25; or

j) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a tetraketide synthase (TKS) polypeptide comprising the amino acid sequence of SEQ ID NO:11 and one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding an olivetolic acid cyclase (OAC) polypeptide comprising the amino acid sequence of SEQ ID NO:10.

9. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising:

a) culturing a genetically modified yeast cell comprising:

i) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO:110 or SEQ ID NO:100; and

ii) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a tetraketide synthase (TKS) polypeptide comprising the amino acid sequence of SEQ ID NO:11; and/or

iii) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding an olivetolic acid cyclase (OAC) polypeptide comprising the amino acid sequence of SEQ ID NO:10;

 in a suitable medium containing a carboxylic acid; and

b) recovering the produced cannabinoid or cannabinoid derivative.

10. The method of claim 9 , wherein the carboxylic acid is hexanoic acid or a hexanoic acid derivative, butyric acid or a butyric acid derivative, or valeric acid or a valeric acid derivative.

11. The method of claim 9 , wherein the cannabinoid is cannabigerolic acid, cannabigerolic acid monomethylether (CBGAM), cannabigerol, Δ 9 -trans-tetrahydrocannabinol, Δ 9 -tetrahydrocannabinolic acid, Δ 9 -tetrahydrocannabinolic acid A, Δ 9 -tetrahydrocannabinolic acid B, Δ 9 -tetrahydrocannabinolic acid C 4 , Δ 9 -tetrahydrocannabinol C 4 , Δ 9 -tetrahydrocannabivarinic acid C 4 , 4 9 -tetrahydrocannabivarin, Δ 9 -tetrahydrocannabiorcolic acid, Δ 9 -tetrahydrocannabiorcol, Δ 7 -cis-iso-tetrahydrocannabivarin, Δ 9 -tetrahydrocannabinol, Δ 8 -tetrahydrocannabinolic acid, Δ 8 -trans-tetrahydrocannabinol, Δ 8 -tetrahydrocannabinol, cannabidiolic acid, cannabidiol, cannabidiol monomethylether (CBDM), cannabidiol-C 4 (CBD-C 4 ), cannabidiorcol (CBD-C 1 ), cannabitriol, cannabichromenic acid, cannabichromene, cannabinolic acid, cannabinol, cannabinol methylether, cannabinol-C4, cannabinol-C2, cannabidivarinic acid, cannabidivarin, tetrahydrocannabivarinic acid, tetrahydrocannabivarin, cannabichromevarinic acid, cannabichromevarin, cannabigerovarinic acid, cannabigerovarin, cannabicyclolic acid, cannabicyclol, cannabicyclovarin, cannabielsoic acid A, cannabielsoic acid B, cannabielsoinic acid, cannabielsoin, cannabicitranic acid, or cannabicitran.

12. The method of claim 10 , wherein the carboxylic acid is hexanoic acid and wherein the cell culture produces the cannabinoid or cannabinoid derivative at a concentration of about 15 mg/L to about 500 mg/L.

13. A method of producing a cannabinoid or cannabinoid derivative, the method comprising:

a) culturing a genetically modified yeast cell comprising:

i) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO:110 or SEQ ID NO:100;

ii) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding a tetraketide synthase (TKS) polypeptide comprising the amino acid sequence of SEQ ID NO:11;

iii) one or more heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell and encoding an olivetolic acid cyclase (OAC) polypeptide comprising the amino acid sequence of SEQ ID NO:10;

iv) one or more heterologous nucleic acids encoding a THCA synthase polypeptide comprising the amino acid sequence of SEQ ID NO:155 or a CBDA synthase polypeptide comprising the amino acid sequence of SEQ ID NO:88 or SEQ ID NO:151;

v) one or more heterologous nucleic acids encoding one or more polypeptides that generate geranyl pyrophosphate, wherein the polypeptide that generates geranyl pyrophosphate is a geranyl pyrophosphate synthetase (GPPS) polypeptide comprising the amino acid sequence of SEQ ID NO:60;

vi) one or more heterologous nucleic acids encoding one or more polypeptides that generate an acyl-CoA compound or an acyl-CoA compound derivative wherein the polypeptide that generates an acyl-CoA compound or an acyl-CoA compound derivative is an acyl-activating enzyme (AAE) polypeptide comprising the amino acid sequence of SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO:149; a fatty acyl-CoA ligase polypeptide comprising the amino acid sequence of SEQ ID NO:145 or SEQ ID NO:147; or a fatty acyl-CoA synthetase (FAA) polypeptide comprising the amino acid sequence of SEQ ID NO:169, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, or SEQ ID NO:200; and

vii) one or more heterologous nucleic acids encoding one or more polypeptides that generate malonyl-CoA, wherein the polypeptide that generates malonyl-CoA is an acetyl-CoA carboxylase-1 (ACC1) polypeptide comprising the amino acid sequence of SEQ ID NO:9, SEQ ID NO:97, or SEQ ID NO:207;

 in a suitable medium; and

b) recovering the produced cannabinoid or cannabinoid derivative.

14. The method of claim 13 , wherein at least one of the heterologous nucleic acids integrated into a chromosome of the genetically modified yeast cell is operably linked to an inducible promoter.

15. A method of producing a cannabinoid or a cannabinoid derivative, the method comprising use of a genetically modified yeast cell comprising a heterologous nucleic acid integrated into a chromosome of the genetically modified yeast cell and encoding a geranyl pyrophosphate:olivetolic acid geranyltransferase polypeptide comprising the amino acid sequence of SEQ ID NO:110 or SEQ ID NO:100.