This invention is an improved method of robust and scalable production of precursors of active cannabinoids, including geranyl pyrophosphate (GPP) and/or cannabigerolic acid (CBGA), in a methylotrophic yeast host cell. The improved methods incorporate a polypeptide encoding an Erg20 variant (F98W/N128W) into a methylotrophic yeast host cell, for example Pichia pastoris (Komagataella phaffii), that biases the natural production of FPP and GPP towards GPP, a precursor to the intermediate CBGA, crucial to the synthesis of active cannabinoids.

The present invention relates generally to production methods, enzymes and recombinant yeast strains for the biosynthesis of clinically important prenylated polyketides of the cannabinoid family. Using readily available starting materials, heterologous enzymes are used to direct cannabinoid biosynthesis in yeast.

A genetically engineered microorganism for the production of a cannabinoid 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 using a genetically engineered microorganism.

The present disclosure relates to the production of cannabinoids in either recombinant microorganism or in cell-free systems using a combination of enzymes, including but not limited to a PKS enzyme, a npgA enzyme, a cs-OLAS-1, a pp-DVAS-1, a cs-HEX-1 and/or Butiryl synthase.

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

The invention relates to engineered microorganisms (e.g., E. coli) and associated improvements for increasing the production cannabinoids (e.g. CBGA) or precursors or derivatives thereof.

Described herein are olivetolic acid cyclases (OAC) including non-natural variants capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate. In some examples, the non-natural OAC is capable of forming a 2,4-dihydroxy-6-alkylbenzoic acid from a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate at a greater rate. In some examples, the non-natural OAC has a higher affinity for a 3,5,7-trioxoacyl-CoA or a 3,5,7-trioxocarboxylate substrate, as compared to the wild type OAC. The non-natural OAC can be used with olivetol synthase (OLS) to form the 2,4-dihydroxy-6-alkylbenzoic acid from malonyl-CoA and acyl-CoA through to a 3,5,7-trioxoacyl-CoAintermediate. The non-natural OAC (and OLS) can be expressed in an engineered cell having a pathway to form cannabinoids, which include CBGA, its analogs and derivatives. CBGA can be used for the preparation of cannabigerol (CBG), which can be used in therapeutic compositions.

The present disclosure provides recombinant host cells comprising a pathway capable of producing a cannabinoid and a nucleic acid derived from a Cannabis trichome mRNA that that does not encode an enzyme in the pathway but enhances the host cells' ability to produce the cannabinoid. The disclosure also provides methods of using host cells to produce cannabinoids.

A recombinant host cell capable of biosynthesizing cannabichromenic acid and a construction method thereof, and a method for biosynthesizing cannabichromenic acid through the recombinant host cell. Saccharomyces cerevisiae is taken as a host. First, cannabigerolic acid synthase and cannabichromenic acid synthase are over-expressed in the host; then, a metabolic pathway of a precursor compound, olivetolic acid, synthesizing cannabichromenic acid from saccharides is constructed in the host, a metabolic pathway for hexanoic acid to olivetolic acid is further constructed in the host, an endogenous mevalonate pathway of the host and a metabolic pathway of acetyl-CoA are optimized, cannabichromenic acid synthase is rationally designed, highly active cannabichromenic acid synthase is screened out, and finally, a cannabichromene pathway is located to peroxisomes and lipid droplets by using the cell compartmentalization principle to obtain recombinant Saccharomyces cerevisiae capable of biosynthesizing cannabichromenic acid.

Provided herein are processes, such as commercially viable processes, of producing alkyl resorcinols, such as olivetol and olivetolic acid, and analogs of each thereof. Certain of these processes utilize a recombinant, heterologous host microorganism. Certain of the heterologous microorganisms include a Cannabis sativa olivetol synthase (which is a tetraketide synthase, csOLS). Certain of the heterologous microorganisms include a Cannabis sativa olivetolic acid cyclase (csOAC). Certain of the heterologous microorganisms include a Cannabis sativa acyl activating enzyme (csAAE), such as, without limitation, csAAE1. In certain of these processes, glucose is fermented. In certain of these processes, the fermentation further comprises a carboxylic acid, RCO.sub.2H where R is defined as herein, or a salt thereof. Certain of these processes provide olivetol and olivetolic acid in a combined amount of at least 3 g/liter.