The present disclosure provides genetically modified host cells that produce a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative. The present disclosure provides methods of synthesizing a cannabinoid, a cannabinoid derivative, a cannabinoid precursor, or a cannabinoid precursor derivative.

Described herein are prenyltransferases including non-natural variants thereof having at least one amino acid substitution as compared to its corresponding natural or unmodified prenyltransferases and that are capable of at least two-fold greater rate of formation of cannabinoids such as cannabigerolic acid, cannabigerovarinic acid, cannabigerorcinic acid, and cannabigerol, as compared to a wild type control. Prenyltransferase variants also accept different hydrophobic substrates (e.g., “donor” molecules), compared to wild type controls, to create different minor and novel cannabinoids. Prenyltransferase variants also demonstrated regioselectivity to desired cannabinoid isomers such as CBGA (3-GOLA), 3-GDVA, 3-GOSA, and CBG (2-GOL). The prenyltransferase variants can be used to form prenylated aromatic compounds, and can be expressed in an engineered microbe having a pathway to such compounds, which include 3-GOLA, 3-GDVA, 3-GOSA, and CBG. 3-GOLA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

The present disclosure relates to recombinant prenyltransferase enzymes with increased thermostability and activity and the use of these enzymes in compositions and methods for biosynthesis involving prenylation reactions, including compositions and methods for the preparation of cannabinoids.

The present disclosure relates to recombinant prenyltransferase enzymes with increased thermostability and activity and the use of these enzymes in compositions and methods for biosynthesis involving prenylation reactions, including compositions and methods for the preparation of cannabinoids.

Described herein are non-natural variants of prenyltronsfcrases having at least one amino acid substitution as compared to its corresponding natural or unmodified prenyltransferascs. The variants are capable of an increased rate of formation of prenylated aromatic compounds, such as cannabinoids, as compared to a wild type control The prcnyltransferase variants can be expressed in an engineered microbe having a pathway to such cannabinoids, and optionally can include one or more other pathway transgencs to promote formation of substrate(s) for the prcnyltransferases. Therapeutically useful cannabinoids can be purified from engineered cells and cell cultures.

The present invention relates generally to production methods, enzymes and recombinant yeast strains for the biosynthesis of clinically important cannabinoid compounds.

Disclosed is yeast cells having peroxisomally localized GPP synthase and a peroxisomally localized enzyme that converts GPP into a monoterpenoids, cannabinoids, monoterpene indole alkaloids and prenylated aromatic compounds; or a precursor therefore, which yeast cells are capable of producing improved amounts of monoterpenoids, cannabinoids, monoterpene indole alkaloids and prenylated aromatic compounds, compared with the same yeast cells where the GPP synthase and the enzyme that converts GPP are located in the cytoplasm. Further disclosed is the use of the yeast cell for producing monoterpenoids, cannabinoids, monoterpene indole alkaloids and prenylated aromatic compounds.

Described herein are prenyltransferases including non-natural variants thereof having at least one amino acid substitution as compared to its corresponding natural or unmodified prenyltransferases and that are capable of at least two-fold greater rate of formation of cannabinoids such as cannabigerolic acid, cannabigerovarinic acid, cannabigerorcinic acid, and cannabigerol, as compared to a wild type control. Prenyltransferase variants also demonstrated regioselectivity to desired cannabinoid isomers such as CDBA (3-GOLA), 3-GDVA, 3-GOSA, and CBG (2-GOL). The prenyltransferase variants can be used to form prenylated aromatic compounds, and can be expressed in an engineered microbe having a pathway to such compounds, which include 3-GOLA, 3-GDVA, 3-GOSA, and CBG. 3-GOLA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

In an aspect, the disclosure provides methods for making neurotransmitters in a host organism. The neurotransmitters can be cannabinoids and derivatives of cannabinoids. The host cells can be microalgae, fungi or other host cells. In a related aspect, the disclosure provides host cells engineered to have biochemical pathways for making neurotransmitters such as cannabinoids.

The disclosure relates to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro comprising the use of disclosed prenyltransferase variants.