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.

The disclosure relates to a non-natural flavin-dependent oxidase comprising at least one amino acid variation as compared to a wild type flavin-dependent oxidase, wherein the non-natural flavin-dependent oxidase does not comprise a disulfide bond, and wherein the non-natural flavin-dependent oxidase is capable of oxidative cyclization of a prenylated aromatic compound into a cannabinoid. The disclosure also relates to a nucleic acid, an expression construct, and an engineered cell for making the non-natural flavin-dependent oxidase. Also provided are compositions comprising the non-natural flavin-dependent oxidase; isolated non-natural flavin-dependent oxidase and methods of making the same; cell extracts comprising the non-natural flavin-dependent oxidase; and methods of making cannabinoids.

Provided herein are processes, including semi-synthetic, and synthetic processes for preparing cannabinoids, and cannabinoid compositions provided thereby.

A genetically engineered microorganism for the production of a cannabinoid biosynthetic pathway product is described. The genetically engineered microorganism comprises at least one nucleic acid molecule encoding at least one cannabinoid biosynthetic pathway enzyme. The disclosure also relates to methods for producing a cannabinoid biosynthetic pathway product using a genetically engineered microorganism.

Aspects of the disclosure relate to biosynthesis of cannabinoids and cannabinoid precursors in recombinant cells and in vitro. In particular, the disclosure relates to variants of olivetolic acid cyclase.

Provided is an engineered olivetolic acid cyclase (OAC) enzyme derived from a microorganism or non-Cannabis plant, wherein the enzyme catalyzes cyclization of a polyketide (I) into a resorcyclic acid derivative (II), where R.sub.1=CH.sub.3, CH.sub.2CH.sub.3, (CH.sub.2).sub.2CH.sub.3, (CH.sub.2).sub.3CH.sub.3, (CH.sub.2).sub.4CH.sub.3, (CH.sub.2).sub.5CH.sub.3, or (CH.sub.2).sub.6CH.sub.3

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Also provided is nucleic acids encoding the above OAC enzyme, expression cassettes comprising those nucleic acids, and recombinant microorganisms comprising those expression cassettes that express the OAC enzyme encoded therein.

Additionally provided is a method of converting a polyketide (I) into a resorcyclic acid derivative (II) comprising contacting the polyketide with the above-identified OAC enzyme in a manner and for a time sufficient to convert the polyketide (I) into the resorcyclic acid derivative (II).

The present disclosure relates generally to methods, isolated polypeptides and polynucleotides, expression vectors, and host cells for the production of cannabidiolic acid (CBDa), cannabigerolic acid (CBCa), and other phytocannabinoids. A method of producing CBDa, CBCa, and/or other phytocannabinoids in a heterologous host cell having CBDa-producing, CBCa-producing or phytocannabinoid-producing capacity comprises transforming the host cell with a nucleotide encoding a variant CBDa synthase protein having a serine insertion between residues P224 and K225 and one or more other amino acid mutation relative to wild type CBDa synthase, and culturing the transformed host cell to produce CBDa, CBCa, and/or other phytocannabinoids therefrom. The variant CBDa synthase protein has at least 85% sequence identity with the wild type CBDa synthase protein sequence OXC52 according to SEQ ID NO:140, with serine insertion (SEQ ID NO:141). Exemplary variants having good phytocannabinoid production capacity are described.

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.

The present invention provides methods and compositions for producing cannabinoids in photosynthetic microorganisms, e.g., cyanobacteria.

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.