Method for producing olivetolic acid in an amoebozoa host species
The present invention relates to a method for the recombinant production of olivetolic acid (OA) in a host species selected from amoebozoa, based on a hybrid-gene or enzyme of polyketide synthase 37 (PKS37) in which the C-terminal type III PKS domain from an amoeba is replaced by an olivetol synthase (OLS) from a plant, and is expressed together with an olivetolic acid cyclase from a multi-gene expression vector. Further provided is a recombinant amoebozoa host species, and an improved method for producing Δ 9 -tetrahydrocannabinol (THC) or other cannabinoids.
1 . A hybrid protein comprising a polyketide synthase 37 (PKS37) having β-ketoacyl synthase activity and lacking a functional C-terminal type III PKS domain, and the hybrid protein comprising a functional plant olivetol synthase (OLS) or an active fragment thereof with olivetol synthase activity at C-terminus of the hybrid protein.
2 . The hybrid protein according to claim 1 , wherein said hybrid protein comprises amino acids 1 to 2618 of PKS37 from Dictyostelium discoideum as encoded by a polynucleotide amplifiable from genome of Dictyostelium discoideum AX2 using primer pair SEQ ID NOs: 31 and 32, and amino acids 2 to 385 of OLS from Cannabis sativa as encoded by a polynucleotide amplifiable from genome of Cannabis sativa using primer pair SEQ ID NOs: 36 and 38.
3 . The hybrid protein according to claim 1 , wherein said pks37 is from Dictyostelium discoideum.
4 . A polynucleotide sequence that encodes the hybrid protein of claim 1 .
5 . The nucleotide sequence according to claim 4 , wherein said hybrid protein comprises amino acids 1 to 2618 of PKS37 from Dictyostelium discoideum as encoded by a polynucleotide amplifiable from genome of Dictyostelium discoideum AX2 using primer pair SEQ ID NOs: 31 and 32, and amino acids 2 to 385 of OLS from Cannabis sativa as encoded by a polynucleotide amplifiable from genome of Cannabis sativa using primer pair SEQ ID NOs: 36 and 38.
6 . A multi-gene expression vector for the recombinant production of olivetolic acid (OA) in a host species selected from amoebozoa, comprising and expressing genes that encode the hybrid protein according to claim 1 , a plant olivetolic acid cyclase (OAC), and Tobacco etch virus (TEV) peptidase, wherein said genes are transcribed as a single polycistronic mRNA spaced apart by viral 2A sequences.
7 . A method for the recombinant production of olivetolic acid (OA) in a species selected from amoebozoa, comprising the steps of a) culturing a recombinant amoebozoa species comprising and expressing the multi-gene expression vector according to claim 6 in a suitable culture medium without a need of hexanoic acid supplementation, and b) isolating said OA from said culture of step a).
8 . A method for producing Δ 9 -tetrahydrocannabinol or another cannabinoid, comprising the steps of performing the method according to claim 7 , and chemically and/or enzymatically converting the olivetolic acid (OA) as produced into Δ 9 -tetrahydrocannabinol or another cannabinoid.
9 . The multi-gene expression vector according to claim 6 , wherein said plant olivetol synthase (OLS) and/or plant olivetolic acid cyclase (OAC) is from C. sativa.
10 . The multi-gene expression vector according to claim 6 , wherein said genes are codon optimized for expression in said amoebozoa host species, wherein said species is Dictyostelium discoideum.
11 . A recombinant amoebozoa host species, comprising and expressing the multi-gene expression vector according to claim 6 .