Application US20200165644

PRODUCTION OF CANNABINOIDS IN YEAST
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US20200165644

1. A genetically modified yeast comprising: (10) (5)

(a) one or more GPP producing genes and optionally, one or more GPP pathway genes;

(b) two or more olivetolic acid producing genes;

(d) one or more Hexanoyl-CoA producing genes, and

(e) at least 5% dry weight of fatty acids or fats.

2. The yeast of claim 1, wherein the one or more GPP producing gene comprises at least one of: (0) (8)

a) a mutated farnesyl diphosphate synthase;

b) a mutated S. cerevisiae ERG20 comprising a K197E substitution;

c) a double mutated S. cerevisiae ERG20 comprising F96W and N127W substitutions;

d) a mutated Y. lipolytica ERG20 comprising a K189E substitution;

e) a double mutated Y. lipolytica ERG20 comprising F88W and N119W substitutions;

f) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 1-4;

g) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 1-4; or

h) any combination of (a)-(g).

3. The yeast of claim 1, wherein the one or more GPP producing genes is selected from a GPP pathway gene. (1) (0)

4. The yeast of claim 3, wherein the GPP pathway gene is selected from: (0) (5)

a) a hydroxymethylglutaryl-CoA reductase (HMGR);

b) a truncated hydroxymethylglutaryl-CoA reductase (tHMGR);

c) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 5-6;

d) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 5-6; or

e) any combination of (a)-(d);

5. The yeast of claim 1, wherein the two or more olivetolic acid producing genes comprise: at least one of (a)-(d) and at least one of (e)-(h); (0) (10)

wherein (a)-(d) comprise:

a) an olivetol synthase (OLS);

b) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7;

c) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 7; or

d) any combination of (a)-(c);

and wherein (e)-(h) comprise:

e) an olivetolic acid cyclase (OAC);

f) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 8;

g) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

h) any combination of (e)-(g).

6. The yeast of claim 1, wherein the one or more cannabinoid precursor or cannabinoid producing genes comprise at least one of: (1) (12)

a) a soluble aromatic prenyltransferase;

b) a cannabigerolic acid synthase (CBGAS);

c) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 9-12;

d) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 9-12; or

e) any combination of (a)-(e);

alone or in combination with at least one of:

f) a tetrahydrocannabinolic acid synthase (THCAS);

g) a cannabidiolic acid synthase (CBDAS);

h) a cannabichromenic acid synthase (CBCAS);

i) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 13-15;

j) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOS: 13-15; or

k) any combination of (f)-(j).

7. The yeast of claim 6, wherein the soluble aromatic prenyltransferase is NphB from Streptomyces sp. strain CL190. (0)

8. The yeast of claim 1, wherein the one or more Hexanoyl-CoA producing genes comprise at least one of: (1) (11)

a) a hexanoyl-CoA synthase;

b) HexA and HexB;

c) StcJ and StcK;

d) a mutated FAS1 and a mutated FAS2;

e) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16 and a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17;

f) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 16 and a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17;

g) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18 and a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19;

h) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18 and a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 19;

i) a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 and a polynucleotide encoding a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21;

j) a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 20 and a polypeptide that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 21; or

k) any combination of (a)-(j).

9. The yeast of claim 8, wherein the mutated FAS1 and FAS2 genes comprise the genetic modifications selected from: (0) (3)

10. The yeast of claim 1, wherein the yeast comprises at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% dry weight of fatty acids or fats. (0)

11. The yeast of claim 1, wherein the yeast is genetically modified to produce at least 5%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% dry weight of fatty acids or fats. (0)

13. The yeast of claim 1, wherein the yeast is oleaginous. (0)

15. The yeast of claim 1, wherein the yeast is a Yarrowia lipolytica, a Lipomyces starkey, a Rhodosporidium toruloides, a Rhodotorula glutinis, a Trichosporon fermentans or a Cryptococcus curvatus. (0)

16. A method of producing at least one cannabinoid or cannabinoid precursor comprising contacting the yeast of claim 1 with a carbohydrate source under culture conditions and for a time sufficient to produce the at least one cannabinoid or cannabinoid precursor. (2) (0)

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Application US20200165644

PRODUCTION OF CANNABINOIDS IN YEAST

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