This document relates to using bidirectional, multi-enzymatic scaffolds to biosynthesize cannabinoids in recombinant hosts.

The present invention relates to a process for the production of a compound of Formula (I), wherein the process comprises the step of reacting a plant extract having prenyltransferase activity with a compound of Formula (II) and geranyl pyrophosphate (GPP) having Formula (III) to produce the compound of Formula (I), wherein the plant extract is not a plant extract obtainable from a plant that naturally produces cannabinoids/phytocannabinoids. Accordingly, the present invention relates to a process for the production of a compound of Formula (I), wherein the process comprises the step of reacting a plant extract having prenyltransferase activity with a compound of Formula (II) and geranyl pyrophosphate (GPP) having Formula (III) to produce the compound of Formula (I), wherein the plant extract is not Cannabis sativa plant extract. Further, the present invention relates to the process for the production of a compound of Formula (IV), wherein the process comprises producing a compound of Formula (I) by the process as described herein and converting the compound of Formula (I) to the compound of Formula (IV) in the presence of the tetrahydrocannabinolic acid synthase (THCAS; EC 1.21.3.7). The present invention also relates to a process for the production of a compound of Formula (V) wherein the process comprises producing a compound of Formula (I) by the process as described herein and converting the compound of Formula (I) to a compound of Formula (V) in the presence of the THCAS (EC 1.21.3.7), cannabidiolic acid synthase (CBDAS; EC 1.21.3.8) and/or cannabichromenic acid synthase (CBCAS; EC 1.3.3.-). Further, the present invention relates to a process for the production of a compound of Formula (VI) wherein the process comprises producing a compound of Formula (I) by the process as described herein and converting the compound of Formula (I) to a compound of Formula (VI) in the presence of the CBDAS (EC 1.21.3.8).

The present disclosure provides methods and kits for characterizing Cannabis plants. Methods and kits of the present disclosure include detection/amplification of one or more enzymes involved in the production of cannabinoids, such as, for example, tetrahydrocannabinolic acid (THCA) synthase and/or cannabidiolic acid (CBDA) synthase.

Methods and systems for the alteration of cannabinoid expression and composition are described. Modular systems for processing cannabis and hemp are described. Described components of the modular system include improved recovery of high-value cannabis components and methods for utilizing residual biomass components after the cannabis plant has been fully processed.

The present disclosure provides engineered variants of a cannabidiolic acid synthase (CBDAS) polypeptide comprising an amino acid sequence of SEQ ID NO:3 with one or more amino acid substitutions, nucleic acids comprising nucleotide sequences encoding said engineered variants, methods of making modified host cells comprising said nucleic acids, modified host cells expressing said engineered variants, methods of producing cannabinoids or cannabinoid derivatives, and methods of screening engineered variants of the cannabidiolic acid synthase (CBDAS) polypeptide.

This document relates to using bidirectional, multi-enzymatic scaffolds to biosynthesize cannabinoids in recombinant hosts.

The invention relates to a non-natural cannabinoid synthase comprising at least one amino acid variation as compared to a wild type cannabinoid synthase 9-tetrahydrocannabinolic acid synthase (THCAS), comprising three alpha helices (A, B and C) where a disulfide bond is not formed between alpha helix A and alpha helix C, 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 .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 (A, B and C) and wherein a disulfide bond is not formed between alpha helix A and alpha helix C. 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 compositions and methods of the disclosure can be used to produce a cannabinoid in a host cell, such as a yeast cell. For example, the disclosure features host cells (e.g., yeast cells) modified to express one or more enzymes of a cannabinoid biosynthetic pathway, such as an acyl activating enzyme (AAE), a tetraketide synthase (TKS), a cannabigerolic acid synthase (CBGaS), and/or an olivetolic acid cyclase (OAC).