Described herein are variant, novel cannabinoid synthases, nucleic acids encoding same, and various uses thereof.

Described are methods for producing cannabinoid prodrugs as well as methods for formulating such prodrugs in a pharmaceutically acceptable form and their use as therapeutic agents for treating diseases.

The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase Δ9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (αA, αB and αC) where a disulfide bond is not formed between alpha helix αA and alpha helix αC, wherein the non-natural cannabinoid synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into a cannabinoid. The invention further relates to a non-natural Δ.sup.9-tetrahydrocannabinolic acid synthase (THCAS), a non-natural cannabidiolic acid synthase (CBDAS), and a non-natural cannabichromenic acid synthase (CBCAS) comprising at least one amino acid variation as compared to a wild type THCAS, CBDAS, or CBCAS, respectively, comprising three alpha helices (αA, αB and αC) and wherein a disulfide bond is not formed between alpha helix αA and alpha helix αC. The invention also relates to a nucleic acid, expression construct, and engineered cell for making the non-natural THCAS, CBDAS, and/or CBCAS. Also provided are compositions comprising the non-natural THCAS, CBDAS, and/or CBCAS; isolated non-natural THCAS, CBDAS, and/or CBCAS enzymes; methods of making the isolated enzymes; cell extracts comprising cannabinoids; and methods of making cannabinoids.

The present invention provides methods for producing cannabinoid prodrugs. Also described are pharmaceuticals acceptable compositions of the prodrugs and a system for the large-scale production of the prodrugs.

The present invention provides methods for producing cannabinoids. More specifically, the invention is directed to the bio-enzymatic synthesis of THC-v, CBV and CBN by contacting a compound according to Formula I with a cannabinoid synthase enzyme. Also described is a system for producing these pharmaceutically important cannabinoids and the use of such cannabinoids as therapeutic agents.

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.

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro.

The present disclosure relates to the production of cannabinoids in yeast. In as aspect there is provided a genetically modified yeast comprising: one or more GPP producing genes and optionally, one or more GPP pathway genes; two or more olivetolic acid producing genes; one or more cannabinoid precursor or cannabinoid producing genes; one or more Hexanoyl-CoA producing genes, and at least 5% dry weight of fatty acids or fats.

An expression system and method for producing a cannabinoid in algae are provided. The method includes expressing in an algae cell an enzyme for converting hexanoic acid to hexanoyl-CoA, enzymes for converting hexanoyl-CoA to olivetolic acid (OA), an enzyme for converting olivetolic acid (OA) to cannabigerolic acid (CbGA) and an enzyme for converting cannabigerolic acid (CbGA) to a cannabinoid.

The methods and systems disclose enzymes that function to import malonic acid or malonates into a cell. The enzymes increase the output of precursor molecules by enriching certain pathways in the cell. The precursor molecules can be converted to cannabinoids. The enzymes are a family of proteins which have a majority of common alignments.