TECHNICAL FIELD

This document relates to methods and materials for biosynthesizing cannabinoids, and more particularly to using bidirectional multi-enzymatic scaffolds to biosynthesize cannabinoids.

BACKGROUND

The emerging therapeutic potential of cannabinoids warrants industrial-scale production to meet compounding future demands. Traditional cannabinoid production efforts rely on large-scale farming ofCannabis sativaL. However, agricultural cannabinoid production is problematic due to issues such as uncontrollable environmental factors and scaling limitations.

SUMMARY

This document is based, at least in part, on the discovery that a bidirectional, multi-enzymatic scaffold can be engineered to allow high-throughput cannabinoid production in recombinant host cells. By controlling the localization, spatial orientation, and stoichiometry of enzymes catalyzing the biosynthesis of cannabinoids and cannabinoid precursors, the multi-enzymatic scaffolds described herein allow flux-optimized cannabinoid biosynthesis in genetically-engineered host cells.

In one aspect, this document features a host cell capable of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabidiolic acid, and cannabichromenic acid. The host cell includes at least three different exogenous nucleic acids, wherein the first and the second exogenous nucleic acids each encode a plurality of engineered enzymes selected from the group consisting of acetyl-CoA acetyltransferase, a 3-hydroxybutyryl-CoA dehydrogenase, an enoyl-CoA hydratase, a beto-ketothiolase, a trans-enoyl-CoA reductase, an HMG-CoA synthetase, an HMG-CoA reductase, a mevalonate kinase, a phosphomevalonate kinase, a diphosphomevalonate decarboxylase, an isopentenyl-diphosphate delta isomerase, a geranyl-diphosphate synthase, an olivetol synthase, an olivetolic acid cyclase, and a CBGA synthase; wherein each of the engineered enzymes includes a heterologous interaction domain, wherein the heterologous interaction domain comprises a first and a second peptide motif, and wherein each heterologous interaction domain is different from each other; and wherein the third exogenous nucleic acid encodes a polypeptide scaffold comprising a plurality of peptide ligands, wherein each peptide ligand comprises an amino acid sequence that can bind to the first or the second peptide motif of one of the heterologous interaction domains. The plurality of engineered enzymes further can include an ATP citrate lyase and an acetyl-CoA carboxylase. The host cell further can include an exogenous nucleic acid encoding a cannabidiolic acid synthase (CBDAS) and a cannabichromenic acid synthase (CBCAS). The host cell can include an exogenous CBDAS. The host cell can include an exogneous CBCAS. The host cell can include an exogenous CBDAS and an exogenous CBCAS. The host cell can include an exogenous hexanoyl-CoA synthetase. The host cell can include at least four different exogenous nucleic acids, wherein the first, second, and fourth nucleic acids each encode a plurality of the engineered enzymes. The host cell can include at least five different exogenous nucleic acids, wherein the first, second, fourth, and fifth nucleic acid each encode a plurality of the engineered enzymes. The host cell can include at least six different exogenous nucleic acids, wherein the first, second, fourth, fifth, and sixth nucleic acids each encode a plurality of the engineered enzymes. Each exogenous nucleic acid can include a constitutive promoter operably linked to the sequence encoding the engineered enzyme or polypeptide scaffold or an inducible promoter operably linked to the sequence encoding the engineered enzyme or polypeptide scaffold. In some embodiments, the promoter is a GAL1-10 promoter. In some embodiments, a constitutive promoter used to express the polypeptide scaffold has weaker constitutive activity level than a constitutive promoter used to express the engineered enzymes. In some embodiments, a constitutive promoter is used to express the engineered enzymes and an inducible promoter is used to express the polypeptide scaffold. In some embodiments, an inducible promoter is used to express the engineered enzymes and a constitutive promoter is used to express the polypeptide scaffold.

Any of the host cells can be bacterial, yeast, algae, or plant cells. A bacterial cell can be selected from the group consisting ofEscherichia coli, Bacillus, Brevibacterium, Streptomyces, andPseudomonascells. A yeast cell can be selected from the group consisting ofPichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyvces marxianus, andKomagataella phafficells. An algae cell can beDunaliellasp.,Chlorella variabilis, Euglena mutabilis, orChlamydomonas reinhardtiicells. A plant cell can be aCannabisor tobacco cell.

In some embodiments, each of the engineered enzymes is of the formula: enzyme-linker1-spacer-linker2-motif1-linker3-motif2, where linkers 1, 2, and 3 can be the same or different, motif 1 and motif 2 can be the same or different, and where motif 1 and motif 2 form the heterologous interaction domain. A scaffold polypeptide can be of the formula: N-terminus-[Ligand 1-linker-Ligand 2-Spacer]n-(optionally-tagged)C-terminus, where n is the number of heterologous interaction domains, and where ligand 1 and ligand 2 bind motif 1 and motif 2, respectively, of the heterologous interaction domain. The scaffold polypeptide can be tagged with a MYC tag, FLAG tag, or HA tag. The host cell further can include a nucleic acid encoding a second polypeptide scaffold comprising a plurality of peptide ligands, wherein each peptide ligand comprises an amino acid sequence that can bind to a different motif of the heterologous interaction domain. The linker can have a flexible GS-rich sequence flanking a rigid α-helical moiety. The spacer can be the cTPR6 spacer.

This document also features a method of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabidiolic acid, and cannabichromenic acid. The method can include culturing any of the host cells described herein under conditions wherein the host cell produces the one or more cannabinoids. The host cells can be cultured in a culture medium supplemented with citrate, glucose, hexanoic acid, and/or other carbon source, and/or in a culture medium supplemented with malonyl-CoA. The method further can include extracting the one or more cannabinoids from the host cells.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

