The invention provides engineered biosynthetic pathways that can be used to produce cannabinoids from fatty acids, recombinant microorganisms incorporating such pathways, methods of biosynthetically producing cannabinoids from fatty acids, and cannabinoids so produced.

The present disclosure provides recombinant host cells comprising a pathway capable of producing a cannabinoid and/or cannabinoid precursor, wherein the pathway comprises an enzyme AAE from a source organism other than Cannabis sativa, such as Humulus lupulus. The disclosure also provides methods of using the host cells to produce rare cannabinoids and/or rare cannabinoid precursors.

Disclosed herein are microorganism and methods that can be used for the synthesis of cannabigerolic acid (CBGA) and cannabinoids. The methods disclosed can be used to produce CBGA, Δ.sup.9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), cannabichromenic acid (CBGA), Δ.sup.9-tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), Δ.sup.9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC). Enzymes useful for the synthesis of CBGA and cannabinoids, include but are not limited to acyl activating enzyme (AAE1), polyketide synthase (PKS), olivetolic acid cyclase (OAC), prenyltransferase (PT), THCA synthase (THCAS), CBDA synthase (CBDAS), CBC A synthase (CBCAS), HMG-Co reductase (HMG1), and/or famesyl pyrophosphate synthetase (ERG20). The microorganisms can also have one or more genes disrupted, such as gene that that controls beta oxidation of long chain fatty acids.

A nucleic acid molecule is disclosed for transiently transforming a plant to produce Δ9-tetrahydrocannabinolic acid synthase, cannabidiolic acid synthase, and/or cannabichromenic acid synthase. The nucleic acid molecule corresponds to a nucleotide sequence comprising at least one of a nucleotide sequence fragment encoding a polypeptide having at least 78% sequence identity to SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6 or comprising at least 15 contiguous nucleotides of the nucleotide sequence SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO:6. The nucleotide sequence further comprises a KDEL or HDEL retrieval tag for targeting the nucleotide sequence to the endoplasmic reticulum. Further aspects relate to a viral vector comprising such nucleic acid molecule, and to methods for producing THCAS, CBDAS, CBCAS, THCA, CBDA, CBCA, THC, CBD and/or CBC based on transient expression of the nucleic acid sequence in a host plant.

Provided herein are methods and compositions for producing cannabinoids and other metabolites in a host cell.

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

Provided is a Cannabis plant exhibiting altered tetrahydrocannabinolic acid (THCA) and/or cannabidiolic acid (CBDA) content. The plant includes a modified genomic locus involved in tetrahydrocannabinolic acid synthase (THCAS) and/or cannabidiolic acid synthase (CBDAS) gene expression, the genomic locus includes at least one targeted nucleotide modification within a regulatory region modulating the expression of at least one allele of the THCAS and/or CBDAS genes. Further provided are methods for producing the aforementioned Cannabis plant.

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).

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.

Enzymes involved in cannabinoid biosynthesis are recombinantly expressed in a host cell. The host cell may be a prokaryote (e.g. Escherichia coli) or a eukaryote (e.g. Yarrowia lipolytica). The enzymes include a heterologous cannabigerolic acid synthase as well as additional enzymes involved in the biosynthesis of cannabinoid precursors such as geranyl diphosphate, olivetol, olivetolic acid, divarin and/or divarinic acid. Methods are provided for producing C5-cannabinoids and/or C3-cannabinoids by fermentation of the recombinant host cell. Alternatively, cannabinoids can be produced by biotransformation of cannabinoid precursors in recombinant cells or by disrupted recombinant cells.