Genetically modified yeast for the production of cannabigerolic acid, cannabichromenic acid and related cannabinoids
The present invention relates generally to production methods, enzymes and recombinant yeast strains for the biosynthesis of clinically important cannabinoid compounds.
1 . A modified recombinant yeast host cell comprising a first exogenous polynucleotide that encodes a prenyltransferase and a second exogenous polynucleotide that encodes a cannabichromenic acid (CBCA) synthase, a cannabidiolic acid (CBDA) synthase, or a tetrahydrocannabinolic acid (THCA) synthase, wherein the prenyltransferase comprises a geranyl-pyrophosphate-olivetolic acid geranyltransferase (GOT), and wherein the GOT is expressed as a fusion protein with a partner that confers enhanced expression of the active GOT.
2 . The modified recombinant yeast host cell of claim 1 , wherein the prenyltransferase amino acid sequence comprises SEQ ID NO:2.
3 . The modified recombinant yeast host cell of claim 1 , wherein the second exogenous polynucleotide encodes a CBCA synthase and the CBCA synthase comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOS:3-9.
4 . The modified recombinant yeast host cell of claim 1 , wherein the prenyltransferase amino acid sequence comprises SEQ ID NO:2; and the second exogenous polynucleotide encodes a CBDA synthase or THCA synthase.
5 . The modified recombinant yeast host cell of claim 1 , wherein the yeast cell is genetically modified to overexpress mevalonate pathway enzymes.
6 . The modified recombinant yeast host cell of claim 5 , wherein one or more mevalonate pathway enzymes is an endogenous enzyme.
7 . The modified recombinant yeast host cell of claim 5 , wherein one or more mevalonate pathway enzymes is an exogenous enzyme not natively expressed in the yeast host cell.
8 . The modified recombinant yeast host cell of claim 1 , wherein the yeast host cell is modified to overexpress erg10, erg13, thmgr, erg12, erg8, mvd1, idi1, and erg20 F96WN127W, and the mvaE, mvaS genes from Enterococcus faecalis.
9 . A method of producing a cannabinoid, the method comprising:
providing olivetolic acid or divarinic acid, or a fluorinated or chlorinated analog thereof to a modified recombinant yeast host cell according to claim 1 to produce the corresponding acidic cannabinoid CBCA, CBDA, or THCA, or fluorinated or chlorinated analog thereof, said acidic cannabinoid generated by prenylation of olivetolic acid or divarinic acid, or analog thereof;
decarboxylating the acidic cannabinoid enzymatically or chemically to produce the corresponding compound CBC, CBD, or THC, or analog thereof.
10 . The method of claim 9 , wherein the recombinant yeast host cell expresses a polynucleotide that encodes a CBCA synthase.
11 . The method of claim 10 , wherein CBCA produced is of greater than 96% enantiomeric purity.
12 . The method of claim 9 , wherein the decarboxylation step is performed on an extract of the yeast host cell prepared using an extraction reagent comprising an organic solvent or organic solvent/water mixture.
13 . The method of claim 9 , wherein the decarboxylation step comprises incubating the extract at a temperature of 20° to 100° C. in the presence of a metal catalyst.
14 . The method of claim 13 , wherein the metal catalyst is zinc, molybdenum, nickel, copper, platinum, palladium, or iron.
15 . The method of claim 13 , wherein the metal catalyst is provided as a metal-loaded zeolite catalyst.
16 . The method of claim 9 , wherein the decarboxylation step is performed with a decarboxylase enzyme.
17 . The method of claim 16 , wherein the decarboxylase is an Aspergillus nidulans orsB decarboxylase, a PatG enzyme from Aspergillus clavatus , or a 3,4-dihydroxybenzoic acid decarboxylase from Enterobacter cloacae.
18 . The method of claim 9 , wherein, the olivetolic acid or divarinic acid, or analog thereof, is produced by the host cell, which is genetically modified to express a polynucleotide that encodes an acyl-CoA synthase selected from the group consisting of Roseburia hominis butanoylCoA transferase, revS, CsAAE3, and CsAAE1; to express a polynucleotide that encodes olivetolic acid synthase; and to express a polynucleotide that encodes olivetolic acid cyclase.
19 . The method of claim 18 , wherein the acyl-CoA synthase is Roseburia hominis butanoylCoA transferase.
20 . The method of claim 18 , wherein the acyl-CoA synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:20; the olivetolic acid synthase comprises an amino acid sequence having at least 95% amino acid sequence identity to SEQ ID NO:13; and the olivetolic acid cyclase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.
21 . A modified recombinant yeast host cell comprising an exogenous polynucleotide that encodes a prenyltransferase, wherein the amino acid sequence of the prenyltransferase is SEQ ID NO:1; or comprising an exogenous polynucleotide that encodes a prenyltransferase polypeptide, wherein the prenyltransferase polypeptide comprises the amino acid sequence of SEQ ID NO:2.
22 . The modified recombinant yeast host cell of claim 21 , further comprising a second exogenous polynucleotide that encodes a cannabichromenic acid (CBCA) synthase, a cannabidiolic acid (CBDA) synthase, or a tetrahydrocannabinolic acid (THCA) synthase.
23 . The modified recombinant yeast host cell of claim 1 , wherein the partner that confers enhanced expression of the active GOT is selected from the group consisting of a superoxide dismutase, a yeast maltose binding protein (MBP), a human or yeast ubiquitin (Ub), a catalase, Saccharomyces cerevisiae catalase T (CTT1), or Saccharomyces cerevisiae peroxisomal catalase A (CTA1), transferrin, and human serum albumin (HSA).
24 . The modified recombinant yeast host cell of claim 23 , wherein the superoxide dismutase is human superoxide dismutase (hSOD).
25 . The modified recombinant yeast host cell of claim 23 , wherein the catalase is human catalase (hCAT).
26 . The modified recombinant yeast host cell of claim 1 , wherein the partner that confers enhanced expression of the active GOT is selected from the group consisting of human galectin, Escherichia coli maltose binding protein (MBP), yeast prepro alpha factor, and GB 1.