Bidirectional multienzyme scaffold for the biosynthesis of cannabinoids

Abstract

This article deals with the biosynthesis of cannabinoids in recombinant hosts using a bidirectional multienzyme scaffold.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application Serial No. 62 / 836,265 filed on April 19, 2019 and U.S. Application Serial No. 62 / 771,839 filed on November 27, 2018. The disclosure of the prior application is considered part of the disclosure of the present application and is incorporated in its entirety into the present application. Technical Field

[0003] The present invention relates to methods and materials for biosynthesizing cannabinoids, and more particularly, to biosynthesizing cannabinoids using a bidirectional multi-enzyme scaffold. Background Art

[0004] The emerging therapeutic potential of cannabinoids makes it necessary to produce them on an industrial scale to meet future comprehensive needs. Traditional cannabinoid production efforts rely on large-scale cultivation of cannabis herb (Cannabis sativa L). However, agricultural cannabinoid production is problematic due to issues such as uncontrollable environmental factors and scale limitations. Summary of the invention

[0005] The present invention is based, at least in part, on the discovery that a bidirectional multi-enzyme scaffold can be engineered to allow high-throughput cannabinoid production in recombinant host cells. The multi-enzyme scaffold described herein allows for flux-optimized cannabinoid biosynthesis in genetically engineered host cells by controlling the localization, spatial orientation, and stoichiometry of enzymes that catalyze the biosynthesis of cannabinoids and cannabinoid precursors.

[0006] In one aspect, the present invention 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 comprises at least three different exogenous nucleic acids, wherein the first and second exogenous nucleic acids each encode a plurality of engineered enzymes selected from the group consisting of acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase, β-ketothiolase, trans-enoyl-CoA reductase, HMG-CoA synthetase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate delta isomerase, geranyl diphosphate synthase, olivetol synthase, olivetate cyclase, and CBGA synthase; wherein each engineered enzyme comprises 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 one another; 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 capable of binding to the first or second peptide motif of one of the heterologous interaction domains. The plurality of engineered enzymes may also include ATP citrate lyase and acetyl-CoA carboxylase. The host cell may also include exogenous nucleic acids encoding cannabidiol acid synthase (CBDAS) and cannabidiol acid synthase (CBCAS). The host cell may include exogenous CBDAS. The host cell may include exogenous CBCAS. The host cell may include exogenous CBDAS and exogenous CBCAS. The host cell may include exogenous hexanoyl-CoA synthetase. The host cell may include at least four different exogenous nucleic acids, wherein the first, second and fourth nucleic acids each encode a variety of engineered enzymes. The host cell may include at least five different exogenous nucleic acids, wherein the first, second, fourth and fifth nucleic acids each encode a variety of engineered enzymes. The host cell may include at least six different exogenous nucleic acids, wherein the first, second, fourth, fifth and sixth nucleic acids each encode a variety of engineered enzymes. Each exogenous nucleic acid may include a constitutive promoter operably connected to a sequence encoding an engineered enzyme or a polypeptide scaffold or an inducible promoter operably connected to a sequence encoding an engineered enzyme or a polypeptide scaffold. In some embodiments, the promoter is a GAL1-10 promoter. In some embodiments, the constitutive promoter used to express the polypeptide scaffold has a weaker constitutive activity level than the constitutive promoter used to express the engineered enzyme. In some embodiments, the constitutive promoter is used to express the engineered enzyme, and the inducible promoter is used to express the polypeptide scaffold. In some embodiments, the inducible promoter is used to express the engineered enzyme, and the constitutive promoter is used to express the polypeptide scaffold.

[0007] Any host cell can be a bacterium, yeast, algae or plant cell. Bacterial cells can be selected from the group consisting of Escherichia coli, Bacillus, Brevibacterium, Streptomyces and Pseudomonas cells. Yeast cells can be selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Yarrowialipolytica, Kluyveromyces marxianus, and Komagataella phaffii cells. Algae cells can be Dunaliella sp., Chlorella variabilis, Euglena mutabilis, or Chlamydomonas reinhardtii cells. The plant cell may be a Cannabis or a tobacco cell.

[0008] In some embodiments, each engineered enzyme has the following formula: enzyme-linker 1- Spacer-Joint 2- Motif 1- Connectors 3- Motif 2, wherein linkers 1, 2 and 3 may be the same or different, motif 1 and motif 2 may be the same or different, and motif 1 and motif 2 form a heterologous interaction domain. The scaffold polypeptide may have the following formula: N-terminus - [ligand 1 - linker - ligand 2 - spacer] n - (optionally labeled) C-terminus, wherein n is the number of heterologous interaction domains, and wherein ligand 1 and ligand 2 bind to motif 1 and motif 2 of the heterologous interaction domain, respectively. The scaffold polypeptide may be tagged with a MYC tag, a FLAG tag or an HA tag. The host cell may also include a nucleic acid encoding a second polypeptide scaffold comprising a plurality of peptide ligands, wherein each peptide ligand comprises an amino acid sequence capable of binding to a different motif of the heterologous interaction domain. The linker may have a flexible GS-rich sequence flanked by a rigid α-helical portion. The spacer may be a cTPR6 spacer.

[0009] The present invention also features a method of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabichromenic acid, and cannabicyclyl phenolic acid. The method may include culturing any host cell described herein under conditions such that the host cell produces the one or more cannabinoids. The host cell may be cultured in a medium supplemented with citrate, glucose, caproic acid, and / or other carbon sources and / or in a medium supplemented with malonyl-CoA. The method may also include extracting the one or more cannabinoids from the host cell.

[0010] Unless otherwise defined, the meaning of all technical and scientific terms used herein is the same as that generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to those described herein may be used in the implementation 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 herein by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. In addition, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0011] Other features and advantages of the present invention will become readily apparent from the following detailed description and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A Schematic diagram of a representative embodiment of a multi-enzyme cannabinoid scaffold in a cell. The multi-enzyme scaffold includes enzymes of the hexanoyl-CoA pathway, enzymes of the upper cannabinoid pathway, and enzymes of the mevalonate pathway. The schematic diagram also depicts a second scaffold according to an embodiment comprising enzymes of the malonyl-CoA pathway, and depicts a non-scaffolded cannabidiol acid synthase (CBDAS) and a non-scaffolded cannabidiol acid synthase (CBCAS). ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; and the scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products cannabidiol acid (CBGA), cannabidiol (CBG), cannabidiol acid (CBDA), cannabidiol (CBD), cannabidiol acid (CBCA), and cannabidiol (CBC) are highlighted by box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0013] Figure 1BSchematic diagram of a representative embodiment of a bidirectional multi-enzyme scaffold in a cell (e.g., a yeast cell). The multi-enzyme scaffold (referred to as the intranuclear SCF gene box) includes an enzyme of the hexanoyl-CoA pathway (referred to as the intranuclear HCA box), an enzyme of the upper cannabinoid pathway (referred to as the intranuclear CAN box), and an enzyme of the mevalonate pathway (referred to as the intranuclear GPP box). The schematic diagram also depicts a second scaffold according to an embodiment of an enzyme comprising a malonyl-CoA pathway, and depicts non-scaffolded CBDAS and non-scaffolded CBCAS. ID refers to an enzyme-linked interaction domain; cTPR6 refers to a spacer sequence; and a scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific to each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are emphasized with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0014] Figure 2A is a schematic diagram of a gene cassette according to one embodiment for engineering cannabinoidergic cells.

[0015] Figure 2B is a schematic diagram of the gene cassettes used in Examples 2-4 for biosynthesis of cannabinoids in yeast.

[0016] Figure 3 is an example of an enzyme-scaffold complex.

[0017] Figure 4 Schematic diagram of a representative embodiment of a multi-enzyme cannabinoid scaffold in a cell. The multi-enzyme scaffold includes enzymes of the hexanoyl-CoA pathway, enzymes of the upper cannabinoid pathway, and enzymes of the mevalonate pathway. The schematic diagram also depicts a second scaffold according to an embodiment comprising enzymes of the malonyl-CoA pathway, and depicts non-scaffolded CBDAS and non-scaffolded CBCAS. Pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase (E2) replace ATP citrate lyase in the two shown scaffolds. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0018] Figure 5Schematic diagram of a representative embodiment of a multi-enzyme cannabinoid scaffold in a cell. The multi-enzyme scaffold includes enzymes of the hexanoyl-CoA pathway, enzymes of the upper cannabinoid pathway, and enzymes of the MEP (2-C-methylerythritol 4-phosphate) pathway. The schematic diagram also depicts a second scaffold according to an embodiment comprising enzymes of the malonyl-CoA pathway, and depicts non-scaffolded CBDAS and non-scaffolded CBCAS. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; and the scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0019] Fig. 6A The invention comprises the amino acid sequence of each of the following enzymes: ATP citrate lyase (SEQ ID NO:83), acetyl-CoA acetyltransferase (atoB) (SEQ ID NO:84), 3-hydroxybutyryl-CoA dehydrogenase (SEQ ID NO:85), enoyl-CoA hydratase (SEQ ID NO:86), trans-enoyl-CoA reductase (SEQ ID NO:88), beta-ketothiolase (bktB) (SEQ ID NO:87), HMG-CoA synthase (SEQ ID NO:90), truncated HMG-CoA reductase (SEQ ID NO:91), mevalonate kinase (SEQ ID NO:92), phosphomevalonate kinase (SEQ ID NO:93), diphosphomevalonate decarboxylase (SEQ ID NO:94), isopentenyl-diphosphate delta isomerase (SEQ ID NO:95), mutant geranyl-diphosphate synthase (ERG20 WW )(SEQ ID NO:96), olivetol synthase (SEQ ID NO:98), olivetate cyclase (SEQ ID NO:99), CBGA synthase (SEQ ID NO:100), acetyl-CoA carboxylase (SEQ ID NO:100), acetyl-CoA carboxylase (SEQ ID NO:97), CBDA synthase (SEQ ID NO:101), CBCA synthase (SEQ ID NO:102), and hexanoyl-CoA synthetase (SEQ ID NO:89).

[0020] Figure 6BAn amino acid sequence of an engineered enzyme comprising the following formula: enzyme-enzyme linker-cTPR6 spacer-ID linker-ID motif #1-ID motif linker-ID motif #2, wherein the linkers (enzyme linker, ID linker, and ID motif linker) can be the same or different, and ID motif #1 and ID motif #2 can be the same or different. The amino acid sequences of the following engineered enzymes are provided: ATP citrate lyase (ID1) (SEQ ID NO: 103), acetyl-CoA acetyltransferase (atoB) (ID2) (SEQ ID NO: 104), 3-hydroxybutyryl-CoA dehydrogenase (ID3) (SEQ ID NO: 105), enoyl-CoA hydratase (ID4) (SEQ ID NO: 106), trans-enoyl-CoA reductase (ID5) (SEQ ID NO: 107), β-ketothiolase (bktB) (ID6) (SEQ ID NO: 108), HMG-CoA synthase (ID7) (SEQ ID NO: 109), truncated HMG-CoA reductase (ID8) (SEQ ID NO: 110), mevalonate kinase (ID9) (SEQ ID NO: 111), phosphomevalonate kinase (ID10) (SEQ ID NO: 112), diphosphomevalonate decarboxylase (ID11) (SEQ ID NO: 113). NO:113), isopentenyl diphosphate delta isomerase (ID12) (SEQ ID NO:114), mutant geranyl diphosphate synthase (ERG20 WW )(ID13)(SEQ ID NO:115), olivetol synthase (ID14)(SEQ ID NO:116), olivetate cyclase (ID15)(SEQ ID NO:117), CBGA synthase (ID16)(SEQ ID NO:118) and acetyl-CoA carboxylase (ID17)(SEQ ID NO:211).

[0021] Figure 6C An amino acid sequence comprising a polypeptide scaffold having the formula: N-terminus - [ligand #1 - ID motif #1 ligand - linker - ID motif #2 ligand - scaffolded ID-binding site spacer]n - (Myc)3-tagged C-terminus, wherein n is 16 and the ID motif ligands correspond to the motifs of ID 1-16 as shown in Table 2. See SEQ ID NO: 119.

[0022] Fig.6DAn amino acid sequence comprising a polypeptide scaffold having the formula: N-terminus - [ligand #1 - ID motif #1 ligand - linker - ID motif #2 ligand - scaffolded ID-binding site spacer]n - (FLAG)3-tagged C-terminus, wherein n is 2 and the ID motif ligands correspond to the motifs of IDs 1 and 17, as shown in Table 2. See SEQ ID NO: 120.

[0023] Figure 7 Schematic diagram of a representative embodiment of a scaffold with minimal requirements for cannabinoid acid synthesis. The scaffold contains enzymes of the upper cannabinoid pathway. In this embodiment, non-scaffolded hexanoyl-CoA synthetase (HCS), non-scaffolded CBDAS, and non-scaffolded CBCAS are also used. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0024] Figure 8 is a schematic diagram of a representative embodiment of a bidirectional scaffold, which contains HCS at the N-terminus of the scaffold, geranyl pyrophosphate synthase (GPPS) at the C-terminus of the scaffold, and an enzyme of the upper cannabinoid pathway between the HCS and the GPPS. In this embodiment, non-scaffolded CBDAS and non-scaffolded CBCAS can also be used. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; the scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0025] Fig. 9Schematic diagram of a representative embodiment of a one-way scaffold containing enzymes of the upper cannabinoid pathway, showing soluble enzymes from precursor pathways (hexanoyl-CoA pathway, mevalonate pathway, and malonyl-CoA pathway), and soluble CBDAS and CBCAS. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0026] Fig.10 Schematic diagram of a representative embodiment of a multi-enzyme cannabinoid scaffold in a cell. The multi-enzyme scaffold includes enzymes of the malonyl-CoA (CMA) pathway, enzymes of the upper cannabinoid pathway, and enzymes of the mevalonate pathway. The schematic diagram also depicts separate scaffolds according to an embodiment comprising enzymes of the hexanoyl-CoA pathway, and depicts non-scaffolded CBDAS and non-scaffolded CBCAS. ID refers to the enzyme-linked interaction domain; cTPR6 refers to the spacer sequence; and the scaffolded ligand refers to a tandem peptide ligand that forms a scaffold binding site specific for each enzyme-linked ID. The target products CBGA, CBG, CBDA, CBD, CBCA, and CBC are highlighted with box marks. CBG can be produced by decarboxylation of CBGA, CBD can be produced by decarboxylation of CBDA, and CBC can be produced by decarboxylation of CBCA. For each decarboxylation, the "Δ" symbol represents heat and the "hν" symbol represents light.

[0027] Fig.11 is a schematic diagram of one representative embodiment of a multiple enzymatic cannabinoid scaffold in the dual compartments of the cell, cytoplasm and mitochondria / plastids.

[0028] Fig. 12AThe invention comprises a nucleotide sequence encoding ATP citrate lyase (SEQ ID NO: 121), acetyl-CoA acetyltransferase (atoB) (SEQ ID NO: 122), 3-hydroxybutyryl-CoA dehydrogenase (SEQ ID NO: 123), enoyl-CoA hydratase (SEQ ID NO: 124), trans-enoyl-CoA reductase (SEQ ID NO: 125), beta-ketothiolase (bktB) (SEQ ID NO: 126), HMG-CoA synthase (SEQ ID NO: 127), truncated HMG-CoA reductase (SEQ ID NO: 128), mevalonate kinase (SEQ ID NO: 129), phosphomevalonate kinase (SEQ ID NO: 130), diphosphomevalonate decarboxylase (SEQ ID NO: 131), isopentenyl-diphosphate delta isomerase (SEQ ID NO: 132), geranyl-diphosphate synthase (ERG20 WW )(SEQ ID NO: 133), olivetol synthase (SEQ ID NO: 134), olivetate cyclase (SEQ ID NO: 135), CBGA synthase (SEQ ID NO: 136), acetyl-CoA carboxylase (SEQ ID NO: 137), CBDA synthase (SEQ ID NO: 138), CBCA synthase (SEQ ID NO: 139), and hexanoyl-CoA synthetase (SEQ ID NO: 140).

[0029] Fig. 12Bcomprising a nucleotide sequence encoding an engineered enzyme having the following formula: enzyme-enzyme linker-cTPR6 spacer-ID linker-ID motif #1-ID motif linker-ID motif #2, wherein the enzyme linker, ID linker and ID motif linker may be the same or different, and wherein ID motif #1 and ID motif #2 may be the same or different. Provided are nucleotide sequences encoding the following engineered enzymes: ATP citrate lyase (ID1) (SEQ ID NO: 141), acetyl-CoA acetyltransferase (atoB) (ID2) (SEQ ID NO: 142), 3-hydroxybutyryl-CoA dehydrogenase (ID3) (SEQ ID NO: 143), enoyl-CoA hydratase (ID4) (SEQ ID NO: 144), trans-enoyl-CoA reductase (ID5) (SEQ ID NO: 145), bktB (ID6) (SEQ ID NO: 146), HMG-CoA synthase (ID7) (SEQ ID NO: 147), truncated HMG-CoA reductase (ID8) (SEQ ID NO: 148), mevalonate kinase (ID9) (SEQ ID NO: 149), phosphomevalonate kinase (ID10) (SEQ ID NO: 150), diphosphomevalonate decarboxylase (ID11) (SEQ ID NO: 151), isopentenyl diphosphate delta isomerase (ID12) (SEQ ID NO: 152). NO:152), mutant geranyl-diphosphate synthase (ERG20 WW )(ID13)(SEQ ID NO:153), olivetol synthase (ID14)(SEQ ID NO:154), olivetate cyclase (ID15)(SEQ ID NO:155), CBGA synthase (ID16)(SEQ ID NO:156) and acetyl-CoA carboxylase (ID17)(SEQ ID NO:157).

[0030] Fig. 12C comprising a nucleotide sequence encoding a scaffold polypeptide (SEQ ID NO: 158) comprising a peptide ligand corresponding to IDs 1-16 shown in Table 2 and a three-repeat myc tag at the C-terminus.

[0031] Fig.12D The invention comprises a nucleic acid sequence encoding a scaffold polypeptide (SEQ ID NO: 159), the scaffold polypeptide comprising peptide ligands corresponding to ID1 and 17, and a three-repeat FLAG tag at the C-terminus.

[0032] Fig.13AThe amino acid sequence comprising the scaffold-bound engineered enzyme and a soluble hexanoyl-CoA synthetase (HCS) (SEQ ID NO: 209) (encoded by the HCA gene cassette). The scaffold-binding engineered enzymes are ATP citrate lyase (ACL) (ACL-enzyme linker-cTPR6 spacer-ID linker-ID1) (SEQ ID NO: 160); acetyl-CoA acetyltransferase (atoB) (atoB-enzyme linker-cTPR6 spacer-ID linker-ID2) (SEQ ID NO: 161); 3-hydroxybutyryl-CoA dehydrogenase (BHBD) (BHBD-enzyme linker-cTPR6 spacer-ID linker-ID3) (SEQ ID NO: 162); enoyl-CoA hydratase (ECH) (ECH-enzyme linker-cTPR6 spacer-ID linker-ID4) (SEQ ID NO: 163); trans-enoyl-CoA reductase (ECR) (ECR-enzyme linker-cTPR6 spacer-ID linker-ID5) (SEQ ID NO: 164); and β-ketothiolase (bktB) (bktB-enzyme linker-cTPR6 spacer-ID linker-ID6) (SEQ ID NO:165).

[0033] Fig. 13B The invention comprises an amino acid sequence of a scaffold-binding engineered enzyme encoded by a GPP gene cassette. The scaffold-binding engineered enzyme is HMG-CoA synthase (HMGS) (HMGS-enzyme linker-cTPR6 spacer-ID linker-ID7) (SEQ ID NO: 166); truncated HMG-CoA reductase (tHMGR) (tHMGR-enzyme linker-cTPR6 spacer-ID linker-ID8) (SEQ ID NO: 167); mevalonate kinase (ERG12) (ERG12-enzyme linker-cTPR6 spacer-ID linker-ID9) (SEQ ID NO: 168); phosphomevalonate kinase (ERG8) (ERG8-enzyme linker-cTPR6 spacer-ID linker-ID10) (SEQ ID NO: 169); diphosphomevalonate decarboxylase (MVD1) (MVD1-enzyme linker-cTPR6 spacer-ID linker-ID11) (SEQ ID NO: 161); ID NO: 170); isopentenyl diphosphate delta-isomerase (IDI1) (IDI1-enzyme linker-cTPR6 spacer-ID linker-ID12) (SEQ ID NO: 171); and geranyl-diphosphate synthase (ERG20WW) (ERG20WW-enzyme linker-cTPR6 spacer-ID linker-ID13) (SEQ ID NO: 172).

[0034] Fig. 13CThe amino acid sequences of the scaffold-bound engineered enzymes, soluble CBDA synthase (SEQ ID NO: 173) and soluble CBCA synthase (SEQ ID NO: 174) (encoded by the CAN gene cassette). The scaffold-bound engineered enzymes are olivetol synthase (OS) (OS-enzyme linker-cTPR6 spacer-ID linker-ID14) SEQ ID NO: 175); olivetate cyclase (OAC) (OAC-enzyme linker-cTPR6 spacer-ID linker-ID15) (SEQ ID NO: 176); CBGA synthase (CBGAS-enzyme linker-cTPR6 spacer-ID linker-ID16) (SEQ ID NO: 177); and, acetyl-CoA carboxylase (ACC) (ACC-enzyme linker-cTPR6 spacer-ID linker-ID17) (SEQ ID NO: 178).

[0035] Fig.13D The amino acid sequences comprising the cannabinoid metabolic scaffold (CBSCFLD)-(Myc)3 (SEQ ID NO: 179) and the malonyl-CoA metabolic scaffold (MCASCFLD)-(FLAG)3 (SEQ ID NO: 180).

[0036] Fig.14A Contains code Fig.13A The codon-optimized nucleotide sequences of the enzymes described herein (SEQ ID NOs: 181-187).

[0037] Fig. 14B Contains code Fig. 13B The codon-optimized nucleotide sequences of the enzymes described herein (SEQ ID NOs: 188-194).

[0038] Fig. 14C Contains code Fig. 13C The codon-optimized nucleotide sequences of the enzymes described herein (SEQ ID NOs: 195-200).

[0039] Fig.14D Contains code Fig.13D The codon-optimized nucleotide sequences of the scaffolds (SEQ ID NO: 201 and SEQ ID NO: 202).

[0040] Fig.15A A nucleotide sequence comprising the HCA gene cassette (SEQ ID NO: 203).

[0041] Fig. 15B A nucleotide sequence comprising a GPP gene cassette (SEQ ID NO: 204).

[0042] Fig. 15CA nucleotide sequence comprising the CAN gene cassette (SEQ ID NO: 205).

[0043] Fig.15D A nucleotide sequence comprising the SCF gene cassette (SEQ ID NO: 206).

[0044] Fig.15E A nucleotide sequence comprising the SOL gene cassette (SEQ ID NO: 207).

[0045] Fig.16 is a map of the pCCI-Brick plasmid construct.

[0046] Fig.17 is a map of the pESC-TRP ("vHCA") vector construct. In this figure, the vector contains the TRP gene, allowing selection in a medium lacking tryptophan. Similar vectors were also prepared in which the TRP gene was replaced with the LEU gene, which allows selection in a medium lacking leucine, the HIS3 gene, which allows selection in a medium lacking histidine, or the URA3 gene, which allows selection in a medium lacking uracil.

[0047] Fig.18 Graph showing proliferation curves of yCBSCF and yCBSOL cultures. Line graph depicting cell proliferation curves obtained by recording cell density measurements (OD 600nm ). The initial cell density of all cultures was normalized to OD 600nm = 0.3. For all measurements, n = 3 biological replicates of yCBSCF and yCBSOL cultures. Floating data points represent mean values ​​with 95% confidence intervals. Dashed lines represent 95% confidence intervals of regression curve fits.

[0048] Figure 19 shows a comparison of cannabinoid and precursor titers for scaffolded and soluble cannabinoid biosynthesis. Representative mass spectra of target analytes isolated from (A) yCBSOL and (B) yCBSCF cultures incubated for 48 hours in basal medium. Bar graphs depict (C) total (polymeric) cannabinoid (CBGA+CBDA+CBCA+CBG+CBD+CBC) titers, (D) cannabinoid precursor (OVA) titers and total parent and decarboxylated derivative (CBGA+CBG, CBDA+CBD, and CBCA+CBC) cannabinoid titers, and (E) isolated parent (COO(H)) cannabinoid (CBGA, CBDA, and CBCA) and decarboxylated derivative (ΔCOOH) cannabinoid (CBG, CBD, and CBC) titers for 48 hour yCBSOL (left) and yCBSCF (right) cultures grown in basal medium. For all measurements, n=3 biological replicates for yCBSCF and yCBSOL cultures. CB, cannabinoids; cannabigerolic acid, CBGA; cannabigerol, CBG; cannabidiolic acid, CBDA; cannabidiol, CBD; cannabicycline acid, CBCA; cannabicycline phenol, CBC; olive acid, OVA. Floating asterisks indicate that the inter-strain differences between yCBSCF vs. yCBSOL cultures are statistically significant (determined by Bonferroni multiple comparison post hoc test; α = 0.05). Bar graphs depict mean values ​​with 95% confidence intervals. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0049] Fig. 20 is a bar graph of the effects of citrate and hexanoate supplementation on scaffolding and soluble cannabinoid biosynthesis. Total cannabinoid (CBGA+CBDA+CBCA+CBG+CBD+CBC) titers of yCBSOL and yCBSCF cultures incubated for 48 hours in basal, hexanoate (300 mg / L) supplemented and buffered (pH 6.0) citrate (300 mg / L) supplemented media. Floating asterisks indicate that the inter-strain differences between yCBSCF and yCBSOL cultures are statistically significant (determined by Bonferroni multiple comparison post hoc test; α=0.05). The asterisked line indicates that the intra-strain differences between the total cannabinoid titers of the basal medium of yCBSCF cultures and the total cannabinoid titers of the medium supplemented with citrate are statistically significant (determined by Bonferroni multiple comparison post hoc test; α=0.05). The bar graph depicts the mean with 95% confidence intervals. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0050] Fig.21Scaffolding and concentration-response parameterization of soluble cannabinoid biosynthesis from citrate are shown. Fig.21 In A, a line graph is shown depicting eight-point concentration ([citrate])-response (total cannabinoid titer) curves fit by asymmetric sigmoidal (five-parameter) logistic regression, and in Fig.21 In B, a bar graph is shown depicting 48-hour yCB SCF and yCB SOL The concentration-response parameter estimate (CB 最大 , estimated maximum total cannabinoid titer and citrate EC 50 , estimated citrate concentration producing half-maximal total cannabinoid titer), the cultures were incubated for 48 h in medium supplemented with 0, 10, 30, 100, 300, 1000, 3000, or 10000 mg / L buffered (pH 6.0) citrate. For all measurements, yCB SCF and yCB SOL n = 3 biological replicates of culture. Floating asterisks indicate yCB SCF With yCB SOL The differences between strains of cultures were statistically significant (determined by Bonferroni multiple comparison post hoc test; α=0.05). Floating data points and bar graphs depict means with 95% confidence intervals. Dashed lines represent 95% confidence intervals of regression curve fits. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0052] This document provides methods and materials for producing cannabinoids in host cells or in vitro using a bidirectional multi-enzyme scaffold that can control the localization and stoichiometry of enzymes that catalyze the biosynthesis of cannabinoids and cannabinoid precursors. As described herein, a bidirectional multi-enzyme scaffold and one or more soluble cannabinoid synthases can be used to produce one or more cannabinoids, including cannabigerolic acid (CBGA), cannabidiol acid (CBDA), cannabicycleric acid (CBCA) and tetrahydrocannabinolic acid (tetrahydrocannabinolic acid), and the conjugate bases of these cannabinoids, cannabigerol acid, cannabicycleric acid, cannabicycleric acid and tetrahydrocannabinolic acid, respectively, and the decarboxylation products of these cannabinoids, cannabigerol (CBG), cannabidiol (CBD), cannacycleric acid (CBC) and tetrahydrocannabinol, respectively, and can also be the oxidation product of tetrahydrocannabinolic acid, cannabicycleric acid and its decarboxylation product, cannabinol. The bidirectional multi-enzyme scaffold described herein results in a significant increase in the production of cannabinoids in a recombinant host, including the production of total cannabinoids, CBGA, CBG, CBDA, CBD, CBCA, CBC, and olivetate precursors, compared to the production of cannabinoids in a recombinant host using the same enzyme not bound to the scaffold. As used herein, enzymes not bound to the scaffold are referred to as soluble or non-scaffolded. Although specific forms of cannabinoids or other compounds may be mentioned herein, it should be understood that any neutral or ionized form thereof, including any salt form thereof or its decarboxylated derivatives (e.g., produced in the presence of heat and light), are also included in the scope of this article unless otherwise stated. It is understood by those skilled in the art that the specific form will depend on factors such as pH and carboxylation state.

[0053] In general, the enzymes described herein can be co-localized on one or more scaffolds and used to produce cannabinoids or cannabinoid precursors that are engineered to include an interaction domain (ID) that can be separated from the N-terminus or C-terminus of the enzyme by an amino acid spacer sequence. The ID can contain two or more scaffold binding motifs. The engineered enzyme can also include one or more linkers between the enzyme, the spacer and / or the ID. The engineered enzyme can be bound to a scaffold that is a polypeptide comprising a unique ID binding domain, i.e., a tandem peptide ligand, such as Figure 1A and Figure 1BAs shown, the enzyme is co-localized in the support. In other words, each enzyme can be engineered to include a protein-protein interaction domain, which is specific to one or more ligands (binding sites) on the support, so that the enzyme can be located at discrete positions along the support by non-covalent interactions. In some cases, the engineered enzyme can be a chimeric enzyme. Amino acid linkers or spacers can be used to separate the scaffolding ligands. For example, see Horn and Sticht, Frontiers in Bioengineering and Biotechnology, 2015, Vol. 3, P. 191; Whitaker and Dueber, Methods in Enzymology, Chapter 19, "Metabolic Pathway Flux Enhancement by Synthetic Protein Scaffolding", Vol. 497, 2011, for describing ID, binding domain, linker and spacer. ID can also be referred to as an adaptor domain.

[0054] Typically, each interaction domain consists of two tandem scaffold binding motifs that continue / extend from the C-terminus of the engineered enzyme and are capable of binding to their corresponding scaffolded peptide ligands that are constructed in tandem along the scaffold. The dual binding of the enzyme to the scaffold ensures a fixed spatial orientation, increases the binding specificity of each ID scaffold interaction, and better tethers each enzyme to the scaffold, all of which can improve pathway flux by substrate channeling of each enzymatic step in the scaffolded biosynthetic pathway.

[0055] In some embodiments, there are more than two, for example three, four, five, six, seven, eight, nine or ten or more molecules of each enzyme positioned in the support. In addition, the ratio of any given enzyme and any other enzyme in the biosynthetic pathway is variable. For example, the ratio of a kind of engineered enzyme in the approach and the second engineered enzyme in the same approach is variable, for example from about 1:5 to about 5:1, for example from about 1:5 to about 2:5, about 2:5 to about 3:5, about 3:5 to about 5:5, about 5:5 to about 5:3, about 5:3 to about 5:2 or about 5:2 to about 5:1.

[0056] Peptide ligands are typically short peptide sequences with a length of 3-50 amino acid residues. For example, the length of the peptide ligand can be 3-10, 7-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45 or 40-50 amino acids. There is a database with more than 200 different motifs on the elm.eu.org website that can be used as described herein. See, for example, Dinkel et al., Nucleic Acids Res. 2014; 42(database content): D259–D266.

[0057] The ID can be a peptide sequence ranging in length from 3 to 200 amino acid residues. For example, the ID can be 3-10, 7-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 45-55, 50-60, 65-75, 70-80, 85-95, 90-100, 100-110, 105-115, 110-120, 115-125, 120-130, 125-135, 130-140, 10-15, 13-150, 135-145, 140-150, 145-155, 150-160, 165-175, 170-180, 175-185, 180-190, 185-195, or 190-200 amino acids in length. For example, the ID can be an SH2 domain, an SH3 domain, a PDZ domain, a GTPase binding domain (GBD), a leucine zipper domain, a PTB domain, a FHA domain, a WW domain, a 14-3-3 domain, a death domain, a caspase recruitment domain, a bromodomain, a chromatin organization modifier, a shadow staining domain, an F-box domain, a HECT domain, a ring finger domain, a sterile alpha motif domain, a glycine-tyrosine-phenylalanine domain, a SNAP domain, a VHS domain, an A NK repeats, armadillo repeats, WD40 repeats, MH2 domains, calmodulin homology domains, Dbl homology domains, gelsolin homology domains, PB1 domains, SOCS box, RGS domains, Toll / IL-1 receptor domains, tetratricopeptide repeats, TRAF domains, Bcl-2 homology domains, coiled-coil domains, bZIP domains, fibronectin receptor domains, FNDC domains, SAMD domains, WBP domains and / or SASH domains. See, e.g., U.S. Pat. No. 9,856,460 for a list of domains that can be used as IDs as described herein.

[0058] For example, the ID can be a "Src homology 2" (SH2) or "Src homology 3" (SH3) domain. The SH2 domain is a highly conserved structure of approximately 100 amino acid residues, including two α-helices and seven β-strands. The SH2 domain can have promiscuous or strict specificity for a motif of 3-5 amino acids flanking the phosphorylated tyrosine. See Horn and Sticht, 2015, supra. For example, an SH2 domain that can be used as an ID as described herein can be residues 5-122 of the mouse Ct10 regulon of the kinase adaptor (Crk) protein having GenBank accession number AAH31149.

[0059] SH3 domains are small modules of approximately 60 residues that can bind proline-rich ligands that bind to the domain surface at three shallow grooves formed by conserved aromatic residues and exhibit two different binding orientations. See Horn and Sticht, 2015, supra. In some embodiments, the proline-rich ligand can have a core PXXP motif flanked by positively charged residues. Class I PZP domains recognize ligands that conform to the consensus +XXPXXP (where + is Arg or Lys), while class II domains recognize the PXXPX+ motif and bind to the ligand in the opposite orientation. See Teyra et al., FEBS Lett., 2012 586(17):2631-7. Individual SH3 domains do not interact measurably with other SH3 domain family ligands in vivo, thereby minimizing crosstalk and increasing the number of domain / ligand pairs that can be used simultaneously. See Whitaker and Dueber, 2011, supra. For example, an SH3 domain useful as an ID as described herein may be residues 134-190 of the mouse Crk protein having GenBank Accession No. AAH31149, and its peptide ligand may be PPPALPPKRRR (SEQ ID NO: 1).

[0060] For example, the ID may be a PDZ (PSD-95 / Discs-large / ZO1) domain. PDZ domains are approximately 100 amino acid residues in length and target a specific motif at the C-terminus of a binding partner. Peptide ligands adopt β-strands and extend existing β-sheets within the PDZ domain upon binding. At least four different classes of ligands are known for PDZ domains that display different binding specificities. See Horn and Sticht, 2015, supra. For example, PDZ domains are divided into two main specificity classes based on different ligand characteristics: Class I PDZ domains recognize Motif, class II PDZ domain recognition motif, while class III PDZ domains recognize motif, where X is any residue, is a hydrophobic amino acid. See, Teyra et al., 2012, supra. PDZ and SH3 domains are found throughout eukaryotic and eubacterial genomes. For example, a PDZ domain that can be used as an ID as described herein can be residues 77-171 of mouse α-syntrophin with GenBank accession number EDL06069, and the peptide ligand can be GVKESLV (SEQ ID NO: 208).

[0061] For example, the ID can be a GBD domain from a protein, such as Wiskott-Aldrich syndrome-like protein (N-WASP). An isolated GBD domain does not adopt a single, discrete structure under physiological conditions, but rather exhibits multiple loose conformations in solution. The corresponding peptide ligands are derived from the autoinhibitory form of the GBD. See Horn and Sticht, 2015, supra. For example, a GBD domain that can be used as an ID as described herein can include residues 196-274 of the rat N-WASP protein having GenBank accession number BAA21534 and its peptide ligand, which can be LVGALMHVMQKRSRAIHSSDEGEDQAGDEDED (SEQ ID NO: 2), which can be used as a peptide ligand as described herein.

[0062] For example, the ID can have a leucine zipper or a synthetic coiled coil domain. The leucine zipper domain can include multiple interspersed leucine residues, separated by approximately seven amino acid residues. Havranek and Harbury ((2003), Nat. Struct. Biol. 10, 45-52) identified new homodimers or heterodimers by changing the residues between the leucine zipper pairs based on computational predictions. Reinke et al. ((2010). J. Am. Chem. Soc. 132, 6025–6031) identified three pairs of synthetic coiled coils that did not show measurable self-association. See Whitaker and Dueber, 2011, supra. An example of an ID that may be used as described herein may be ITIRAAFLEKENTALRTEIAELEKEVGRCENIVSKYETRYGPL (SEQ ID NO:3), and its peptide ligand used as described herein may be LEIRAAFLEKENTALRTRAAELRKRVGRCRNIVSKYETRYGPL (SEQ ID NO:4).

[0063] For example, the ID can be a cohesion dockerin polypeptide that can be localized to a specific cohesion dockerin polypeptide on a scaffold as described herein. Cohesion-dockerin pairs are particularly useful for ex vivo applications because binding is calcium dependent. See Whitaker and Dueber, 2011, supra.

[0064] The combination of IDs with high affinity and high specificity, i.e., minimal cross-reactivity, for its peptide ligand can be used as described herein to allow a variety of different enzymes to bind to the scaffolds provided herein. For example, at least three different enzymes can be positioned on the scaffold. In some embodiments, at least four different enzymes can be positioned on the scaffold. In some embodiments, at least five different enzymes can be positioned on the scaffold. In some embodiments, at least six different enzymes can be positioned on the scaffold. In some embodiments, at least seven different enzymes can be positioned on the scaffold. In some embodiments, at least eight different enzymes can be positioned on the scaffold. In some embodiments, at least nine different enzymes can be positioned on the scaffold. In some embodiments, at least ten different enzymes can be positioned on the scaffold. In some embodiments, at least eleven different enzymes can be positioned on the scaffold. In some embodiments, at least twelve different enzymes can be positioned on the scaffold. In some embodiments, at least fifteen different enzymes can be positioned on the scaffold. In some embodiments, at least seventeen different enzymes can be positioned on the scaffold. In some embodiments, at least eighteen different enzymes can be positioned on the scaffold. In some embodiments, at least twenty different enzymes can be positioned on the scaffold. In some embodiments, at least twenty-one different enzymes may be positioned on the scaffold.

[0065] Table 1 provides exemplary combinations of heterologous IDs, i.e., IDs that are different from one another, that can be used for seventeen different engineered enzymes, and Table 2 provides exemplary combinations of corresponding peptide ligands that can be used to localize seventeen different enzymes to one or more scaffolds. In the embodiments shown in Tables 1 and 2, each ID comprises two tandem peptide motifs, such as corresponding peptide ligands that interact with the tandem peptide motifs. It should be understood that any of the enzymes listed in Tables 1 and 2 can be used in combination with any listed combination of IDs and corresponding peptide ligands.

[0066] Table 1

[0067] Interaction domain motif sequences in engineered enzymes

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074] Table 2

[0075] Tandem peptide ligand sequences in scaffolds

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] The spacer or joint of ligase and ID, and the binding domain on the scaffold, can be a peptide sequence of 6 to 250 amino acid residues in length. The term "spacer" generally refers to a longer peptide sequence with a more rigid structure, while the term "joint" generally refers to a shorter peptide sequence with a more flexible structure. In an embodiment in which these two terms are used simultaneously, a joint generally refers to a sequence of about 3 to about 50 amino acids in length, while a spacer generally refers to a longer sequence (e.g., a length of about 36 to about 250 amino acids). For example, the length of the joint can be 6-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45 or 40-50 amino acids. The spacer length can be, for example, 36-40, 40-50, 45-55, 50-60, 55-65, 60-70, 65-75, 70-80, 75-85, 90-100, 95-105, 100-110, 105-115, 110-120, 115-125, 120-130, 125-135, 130-140, 135-145, 140-150 , 145-155, 150-160, 165-175, 170-180, 175-185, 180-190, 185-195, 190-200, 195-205, 200-210, 205-215, 210-220, 215-225, 220-230, 225-235, 230-240, 235-245, or 240-250 amino acids. See, e.g., Chen et al., Adv Drug Deliv Rev. 2013 65(10): 1357–1369. In either case, the linker / spacer can be a series of small and / or hydrophilic and / or other amino acid residues that can accommodate flexible and / or rigid structures. For example, the linker can be a series of glycine residues, a series of alanine residues, a series of serine residues, or a series of alternating glycine and serine (or threonine) residues, such as (GS)8 (SEQ ID NO: 60), (GS) 10 (SEQ ID NO:61) or (GS) 15(SEQ ID NO:62), or predominantly glycine residues, such as (GGGGS)3(SEQ ID NO:63) or (GGGGS)4(SEQ ID NO:64), or any other series of canonical or non-canonical amino acid residues or combinations thereof. In some embodiments, the linker may include glutamic acid, alanine, and lysine residues, such as (EAAAK)2(SEQ ID NO:65), (EAAAK)3(SEQ ID NO:66), or (EAAAK)4(SEQ ID NO:67). See Horn and Sticht, 2015, supra. In some embodiments, the linker may be a combination of glycine, alanine, proline, and methionine residues, such as AAAGGM(SEQ ID NO:68), AAAGGMPPAAAAGGM(SEQ ID NO:69), AAAGGM(SEQ ID NO:70), or PPAAAGGMM(SEQ ID NO:71). See, e.g., U.S. Pat. No. 9,856,460.

[0082] Depending on the amino acid composition, the linker or spacer can be structured or unstructured in nature. For example, in some embodiments, the spacer can have a sequence that adopts a more rigid α-helical conformation, while the linker can have a more flexible GS-rich peptide sequence. For example, in some embodiments, the linker can include a flexible GS-rich sequence flanked by one or more rigid α-helical portions, such as a GS-rich sequence flanked by a double, triple or quadruple α-helical portion. For example, in some embodiments, the linker or spacer can have the sequence GSAGSAAGSGEF (SEQ ID NO: 72), KLSGGGGSGGGGSGGGGS (SEQ ID NO: 73), GSAGSAAGSGEFGSAEAAAKEAAAKAGSAGSAAGSGEFGS (SEQ ID NO: 74), GSAGSAAGSGEFAEAAAKEAAAKAGSAGSAAGSGEF (SEQ ID NO: 75) or GSAGSAAGSGEFGSAEAAAKEAAAAKEAAAKEAAAKAGSAGSAAGSGEFGS (SEQ ID NO: 76).

[0083] In some embodiments, the ligands on the scaffold can be separated by a linker of 20-50 amino acid residues in length (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues in length). In some embodiments, the ID engineered at the C-terminus or N-terminus of each scaffolded enzyme can comprise a linker (e.g., a flexible linker) of 15 to 30 (e.g., 20) amino acid residues in length, flanked by a spacer of 15 to 50 (e.g., 36) amino acid residues. In some embodiments, the ID can be separated from the enzyme by a spacer sequence, such as a cTPR6 spacer sequence, which contains a six-repeat rigid α-helical portion and can have the following sequence: AEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNAEAWYNLGNAYYKQGDYQKAIEDYQKALELDPNNLQAEAWKNLGNAYYKQGDYQKAIEYYQKALELDPNNASAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNAKAWYRRGNAYYKQGDYQKAIEDYQKALELDPNNRSRSA (SEQ ID NO: 77).

[0084] In some embodiments, the engineered enzyme may have the following formula: enzyme-linker 1-spacer-linker 2-motif 1-linker 3-motif 2, wherein linkers 1, 2, and 3 may be the same or different, and motif 1 and motif 2 may be the same or different. In some embodiments, linker 1 may be referred to as an enzyme linker, i.e., it connects the enzyme to a spacer, such as a cTPR6 spacer, and may include a GC-rich flexible portion flanked by a rigid α-helical portion, such as KLSGGGGSGGGGSGGGGS (SEQ ID NO: 73). In some embodiments, linker 2 may be referred to as an ID linker, and may include, for example, a GS-rich flexible portion flanked by a rigid α-helical portion, such as GGGGSGGGGSGGGGAS (SEQ ID NO: 78). In some embodiments, linker 3 may be referred to as a motif linker, and may include a GS-rich flexible portion flanked by a rigid α-helical portion, such as GSAGSAAGSGEFGSAEAAAKEAAAKAGSAGSAAGSGEFGS (SEQ ID NO: 74). Table 1 provides non-limiting examples of Motif 1 and Motif 2 that are used together to form a heterologous ID. Figure 3 A schematic diagram comprising an exemplary engineered enzyme of the formula complexed with a scaffold. Figure 6B and Fig.13A -C contains the following amino acid sequences: ATP citrate lyase, atoB, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase, trans-enoyl-CoA reductase, β-ketothiolase (bktB), HMG-CoA synthase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate delta isomerase, geranyl diphosphate synthase (ERG20 WW ), olivetol synthase, olivetate cyclase, CBGA synthase and acetyl-CoA carboxylase, according to the formula. In some embodiments, linkers 1 and 2 can be (G4S)3, the spacer can be a cTPR6 sequence, and linker 3 can be (GS)8.

[0085] In some embodiments, the scaffold may have the formula: N-terminus-[ligand #1-linker-ligand #2-spacer]n-(optionally labeled) C-terminus, where n is the number of interaction domains. The linker may be referred to as a scaffolded ligand linker and may be used to connect and separate pairs of motif binding ligands that recruit / localize each enzyme to its scaffold binding site. Such a linker may include a GS-rich flexible portion flanked by a rigid α-helical portion and have a sequence such as GSAGSAAGSGEFAEAAAKEAAAKAGSAGSAAGSGEF (SEQ ID NO: 75). The spacer may be referred to as a scaffolded ID binding site spacer and may be used to connect and separate scaffold binding sites (comprising paired motif binding ligands) for each enzyme. Such a spacer may include a GS-rich flexible portion flanked by a rigid α-helical portion and have a sequence such as GSAGSAAGSGEFGSAEAAAKEAAAKEAAAKEAAAKAGSAGSAAGSGEFGS (SEQ ID NO: 76). The N-terminus may include a flexible GS-rich sequence to help stabilize and dissolve the scaffold. For example, the N-terminus can have the sequence GSAGSAAGSGEFGSAGSAAGSGEFGSAGSAAGSGEF (SEQ ID NO: 79). The C-terminus can include a flexible GS-rich sequence flanked by rigid α-helical portions to stabilize and dissolve the scaffold, and can optionally be tagged (e.g., with a MYC tag, FLAG tag, or other tags described below) to facilitate purification or detection of the scaffold. For example, a C-terminal sequence with a three-repeat MYC tag can have the sequence GSAGSAAGSGEFGSAEAAAKEAAAKEAAAKEAAAKAGSAGSAAGSGEFGSEQKLISEEDLEQKLISEEDLEQKLISEEDLGSAGSAGSAAGSGEFGSAGSAAGSGEFGSAGSAAGSGEFGSAGSAAGSGEF (SEQ ID NO: 80). For example, the C-terminal sequence with a three-repeat FLAG tag can have the sequence GSAGSAAGSGEFGSAEAAAKEAAAKEAAAKEAAAKAGSAGSAAGSGEFGSDYKDDDDKDYKDDDDKDYKDDDDKGSAGSAAGSGEFGSAGSAAGSGEFGSAGSAAGSGEF (SEQ ID NO: 81). Figure 6C and Fig.13D Each includes an example of a scaffold polypeptide having the formula, which includes a peptide ligand corresponding to IDS 1-16 shown in Table 2, and a three-repeat MYC tag at the C-terminus. For example, Fig.13D Examples of scaffold peptides containing a triple-repeat MYC tag (see Figure 2Bof the SCF gene cassette). Fig.6D and Fig.13D Each comprises an example of a scaffold polypeptide comprising peptide ligands corresponding to IDs 1 and 17 shown in Table 2 and a three-repeat FLAG tag at the C-terminus. Thus, the amino acid sequence of the scaffold may depend on the sequence of the peptide ligand that can bind to the selected ID motif of the enzyme.

[0086] In some embodiments, any of the enzymes can be engineered to contain an N-terminal or C-terminal linker motif that allows for covalent (iso-peptide) bonding to the scaffold. See, e.g., the SpyTag and SpyCatcher systems described in Zakeri et al., Proc. Natl. Acad. Sci., 2012 109(12) E690-E697.

[0087] In some embodiments related to the multi-enzyme scaffolds described herein, the first engineered enzyme of a biosynthetic pathway can produce a first product, which can be a substrate for a second engineered enzyme of the biosynthetic pathway, which can produce a second product, which can be a substrate for a third engineered enzyme of the biosynthetic pathway, and so on. In some cases, the second engineered enzyme can be fixed on a scaffold so that it is adjacent to or very close to the first engineered enzyme. The third engineered enzyme can be fixed on a scaffold so that it is adjacent to or very close to the second engineered enzyme. In this way, a high effective concentration of the first product can be obtained, and the second engineered enzyme can act efficiently on the first product, and the third engineered enzyme can act efficiently on the second product, and so on.

[0088] exist Figure 1A and 1B In one example of a multi-enzyme scaffold, an enzyme of the hexanoyl-CoA pathway at the N-terminus of the scaffold, an enzyme of the mevalonate pathway at the C-terminus of the scaffold, and an enzyme of the cannabinoid pathway in between. In any pathway, the enzymes may be from a single source, i.e., from one species or genus, or may be from multiple sources, i.e., different species or genera. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in public databases such as GenBank or EMBL (see below).

[0089] The fully assembled multi-enzyme scaffolds provided herein can adopt stoichiometric and spatial arrangements that can help maximize pathway flux and minimize the accumulation of pathway intermediates and byproducts. This scaffold can promote substrate access within and between cannabinoid and cannabinoid precursor pathways. Specifically, this scaffolded system can promote unidirectional flux through each major cannabinoid precursor pathway and converge near the midpoint of the scaffold. The hexanoyl-CoA / olive acid (OVA) pathway can start from the N-terminus of the scaffold, and the mevalonate or MEP pathway can start from the C-terminus of the scaffold. The enzyme that catalyzes the rate-limiting / key step in cannabinoid biosynthesis, CBGA synthase, can be positioned at the intersection of these precursor pathways near the midpoint of the scaffold.

[0090] By this design, the two main precursors for cannabinoid biosynthesis, hexanoyl-CoA / olive acid and geranyl pyrophosphate, can be bidirectionally delivered to CBGA synthase at this junction. CBGA synthase can catalyze the biosynthesis of CBGA, the main cannabinoid from which all other cannabinoids are derived. According to the law of mass action, the passage of substrates within and between scaffolded pathways can accelerate the kinetics of the composite pathway.

[0091] like Figure 1A and 1B As shown, the N-terminal hexanoyl-CoA pathway can include ATP citrate lyase (ACL) (also known as ATP citrate synthase), acetyl-CoA acetyltransferase (atoB), two 3-hydroxy-acyl-CoA dehydrogenases (BHBD), two enoyl-CoA hydratases (ECH), a β-ketothiolase (bktB) and two trans-2-enoyl-CoA-reductases (ECR).

[0092] In such Figure 1A and 1BIn the hexanoyl-CoA pathway shown, citric acid from cell metabolism and / or supplemented in the growth medium can be used as a substrate for acetyl-CoA synthesis catalyzed by ACL. ACL is classified under EC2.3.3.8. Acetyl-CoA can be used as a substrate for acetoacetyl-CoA synthesis catalyzed by atoB. atoB is classified under EC2.3.1.9. Acetoacetyl-CoA can be used as a substrate for 3-hydroxybutyryl-CoA synthesis catalyzed by BHBD. BHBD is classified under EC1.1.1.157. 3-Hydroxybutyryl-CoA can be used as a substrate for trans-but-2-enoyl-CoA synthesis catalyzed by ECH. ECH is classified under EC4.2.1.17. Trans-but-2-enoyl-CoA can be used as a substrate for butyryl-CoA synthesis catalyzed by ECR. ECR is classified under EC1.3.8.1. Butyryl-CoA can serve as a substrate for the synthesis of 3-ketohexanoyl-CoA catalyzed by bktB. bktB is classified under EC2.3.1.9. The bktB that catalyzes the production of 3-ketohexanoyl-CoA from butyryl-CoA can be the same or different from the atoB that catalyzes the production of acetoacetyl-CoA from acetyl-CoA. 3-Ketohexanoyl-CoA is a substrate for the synthesis of 3-hydroxyhexanoyl-CoA catalyzed by BHBD. BHBD is classified under EC1.1.1.157. The BHBD that catalyzes the production of 3-hydroxyhexanoyl-CoA can be the same or different from the BHBD that catalyzes the production of 3-hydroxybutyryl-CoA. 3-Hydroxyhexanoyl-CoA can be a substrate for the synthesis of trans-hex-2-enoyl-CoA catalyzed by ECH. ECH is classified under 4.2.1.17. The ECH that catalyzes the production of trans-hex-2-enoyl-CoA can be the same or different from the ECH that catalyzes the production of trans-but-2-enoyl-CoA. Trans-hex-2-enoyl-CoA can be the substrate for the synthesis of hexanoyl-CoA catalyzed by ECR. ECR is classified under EC1.3.1.38 or EC1.3.1.44. The ECR that catalyzes the production of hexanoyl-CoA can be the same or different from the ECR that catalyzes the production of butyryl-CoA.

[0093] In some embodiments, a hexanoyl-CoA synthetase (HCS) enzyme can be included in a soluble form in place of or in addition to a scaffolded enzyme of a hexanoyl-CoA pathway, and in some embodiments, hexanoate can be added to the growth medium as a substrate for HCS-catalyzed hexanoyl-CoA production. The HCS can be included on a scaffold, located Figure 1A and 1B The N-terminus of the cannabinoid pathway is shown, and / or it can be non-scaffolded (soluble).

[0094] like Figure 1A and 1BAs shown, the C-terminal mevalonate pathway may include ACL, atoB, hydroxymethylglutaryl-CoA, HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (ERG12), phosphomevalonate kinase (ERG8), diphosphomevalonate decarboxylase (MVD1), isopentyl diphosphate isomerase (IDI1), and mutant GPP synthase (mGPPS). Figure 1A and 1B In the mevalonate pathway shown, citrate from cell metabolism and / or supplemented in the growth medium can be used as a substrate for ACL-catalyzed acetyl-CoA synthesis. ACL is classified under EC2.3.3. Acetyl-CoA can be used as a substrate for bktB-catalyzed acetoacetyl-CoA synthesis. bktB is classified under EC2.3.1.9. Acetoacetyl-CoA can be a substrate for HMG-CoA synthesis catalyzed by HMGS. HMG-CoA can be a substrate for mevalonate synthesis catalyzed by HMGR. HMGR is classified under EC1.1.1.88 or 1.1.1.34. Mevalonate can be a substrate for mevalonate 5-phosphate synthesis catalyzed by mevalonate kinase. Mevalonate kinase is classified under EC2.7.1.36. Mevalonate 5-phosphate can be a substrate for mevalonate pyrophosphate synthesis catalyzed by phosphomevalonate kinase. Phosphomevalonate kinase is classified under EC 2.7.4.2. Mevalonate pyrophosphate can be a substrate for the synthesis of isopentyl pyrophosphate catalyzed by diphosphomevalonate decarboxylase. Diphosphomevalonate decarboxylase is classified under EC 4.1.1.33. Isopentyl pyrophosphate can be a substrate for the synthesis of dimethylallyl pyrophosphate catalyzed by isopentyl diphosphate isomerase. Isopentyl diphosphate isomerase is classified under EC.5.3.3.2. Dimethylallyl pyrophosphate can be a substrate for the synthesis of geranyl pyrophosphate catalyzed by geranyl pyrophosphate synthase (GPPS). GPPS is classified under EC 2.5.1.1.

[0095] Since acetyl-CoA can be the initial substrate for the biosynthetic pathways of hexanoyl-CoA, mevalonate / geranyl pyrophosphate, and malonyl-CoA cannabinoid precursors, Figure 1A and 1B In some embodiments, the ACL enzyme is included only at the N-terminus and the C-terminus of the multi-enzyme scaffold. ACL can be directly coupled to the scaffolding pathway and the cell metabolism through the ACL-catalyzed generation of acetyl-CoA from citrate derived from the citric acid cycle. Citrate can also be supplemented to the culture medium (e.g., in a buffered citrate form). In some embodiments, the ACL enzyme is included only at the N-terminus of the scaffold. In some embodiments, the ACL enzyme is included only at the C-terminus of the scaffold. In some embodiments, the ACL enzyme is included in a soluble form.

[0096] In some embodiments, the 2-C-methylerythritol 4-phosphate (MEP) pathway, which can also produce geranyl pyrophosphate, can replace the scaffolded mevalonate pathway at the C-terminus of the scaffold, or can be included in soluble form in addition to the scaffolded mevalonate pathway. Figure 5 As shown, the C-terminus of the scaffold can include 1-deoxy-D-xylulose-5-phosphate (DOXP) synthase, DOXP reductoisomerase, MEP cytidine transferase, 4-diphosphocytidine-2-C-methylerythritol (CDPME) kinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase, 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) synthase, HMBPP reductase and GPPS. Pyruvate and glyceraldehyde-3-phosphate (G3P) can be used as substrates for DOXP synthesis catalyzed by DOXP synthase. DOXP is classified under EC2.2.1.7. DOXP can be a substrate for MEP synthesis catalyzed by DOXP reductoisomerase (DXR). DXR is classified under EC1.1.1.267. MEP can be a substrate for the synthesis of 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) catalyzed by 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (ISPD). ISPD is classified under EC2.7.7.60. CDP-ME can be a substrate for the synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-MEP) catalyzed by 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ISPE). ISPE is classified under EC2.7.1.148. CDP-MEP can be a substrate for the synthesis of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (cMEPP) catalyzed by 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (ISPF). ISPF is classified under EC4.6.1.12. cMEPP can be a substrate for the synthesis of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) catalyzed by HMB-PP synthase (ISPG). ISPG is classified under EC1.17.7.1. HMBPP can be a substrate for the synthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ISPH). ISPH is classified under EC1.17.1.2. IPP and DMAPP can be substrates for the synthesis of geranyl pyrophosphate catalyzed by GPPS. GPPS is classified under EC2.5.1.1.

[0097] In some embodiments, the mevalonate pathway can replace the scaffolded MEP pathway at the scaffolded C-terminus, or can be included in a soluble form in addition to the scaffolded MEP pathway.

[0098] In such Figure 1A and 1B In the illustrated embodiment, a second multi-enzyme scaffold can be co-expressed to increase the cytoplasmic titer of malonyl-CoA, another secondary substrate that can be used for cannabinoid biosynthesis. Such a scaffold can include ATP citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) in series. In some embodiments, ACL and ACC are paired in triplicate or direpeat along the scaffold. If ACL and ACC are paired in triplicate or direpeat, the three or two ACLs on the scaffold can be the same or different, and the three or two ACCs can be the same or different. In any of these embodiments, malonyl-CoA can be supplemented to the growth medium in place of or in addition to that provided by the scaffolded malonyl-CoA pathway.

[0099] In any embodiment using ACL enzyme, pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase (E2) can replace ACL. Figure 4 Shown in, pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase (E2) can be substituted in the upstream of scaffolding mevalonate, caproyl-CoA and malonyl-CoA approach.Using pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase can allow to use pyruvate rather than citric acid to produce acetyl-CoA as primary substrate.In such embodiment, pyruvate also can be supplemented in the growth medium.Pyruvate dehydrogenase and dihydrolipoyl transacetylase are the components of multienzyme pyruvate dehydrogenase complex, and it can catalyze and produce acetyl-CoA from pyruvate.E1 and E2 are present in bacterium and eukaryote.

[0100] like Figure 1A and Figure 1B As shown, the co-scaffolded upper cannabinoid pathway can include olivetol synthase (OS), olivetate cyclase (OAC) and aromatic isoprenyl transferase (APT), such as CBGA synthase (CBGAS). The upper cannabinoid pathway can begin to utilize hexanoyl-CoA and three malonyl-CoAs as substrates for the synthesis of 3,5,7-trioxadodecanoyl-CoA catalyzed by olivetol synthase. Olivetol synthase is classified under EC 2.3.1.206. 3,5,7-trioxadodecanoyl-CoA can be used as a substrate for the synthesis of olivetate catalyzed by OAC. OAC is classified under EC 4.4.1.26.

[0101] At the flux intersection point (near the midpoint of the scaffold) of the converging N-terminal hexanoyl-CoA / upper cannabinoid and C-terminal mevalonate / MEP pathways, APTs such as CBGAS can utilize olivetate from the hexanoyl-CoA / upper cannabinoid pathway and geranyl pyrophosphate from the mevalonate or MEP pathways as substrates for cannabinate synthesis. Suitable APTs are classified under EC 2.5.1.102.

[0102] In some embodiments, enzymes in the upper cannabinoid pathway can be scaffolded with hexanoyl-CoA synthetase (HCS) to biosynthesize cannabigerol esters. In some embodiments, soluble HCS can be used with scaffolded enzymes of the upper cannabinoid pathway to biosynthesize cannabigerol esters, such as Figure 7 Suitable enzymes for the upper cannabinoid pathway are described above.

[0103] In some embodiments, a minimal bidirectional stent may be used, e.g. Figure 8 The scaffold depicted in FIG. 1 , wherein HCS is at the N-terminus of the scaffold, GPPS is at the C-terminus of the scaffold, and the enzymes in the upper cannabinoid pathway are scaffolded between HCS and GPPS.

[0104] In some embodiments, for example Fig. 9 In the illustrated embodiment, the enzymes in the upper cannabinoid pathway can be scaffolded, while the enzymes in the caproyl-CoA pathway, the enzymes in the mevalonate pathway, and the enzymes in the malonyl-CoA pathway can be soluble. In some embodiments, the enzymes in the upper cannabinoid pathway can be scaffolded, while the enzymes in the caproyl-CoA pathway, the enzymes in the MEP pathway, and the enzymes in the malonyl-CoA pathway can be soluble. In such embodiments, HCS can replace the soluble form of the enzymes of the caproyl-CoA pathway. Suitable enzymes for each of these pathways are described above.

[0105] In some embodiments, the enzymes in the upper cannabinoid pathway may be scaffolded, while the enzymes in the hexanoyl-CoA synthase, mevalonate or MEP pathways, and the enzymes in the malonyl-CoA pathway may be soluble. Suitable enzymes for each of these pathways are described above.

[0106] In some embodiments, the HCS may be scaffolded at the N-terminus relative to the scaffolded enzymes in the upper cannabinoid pathway, while the enzymes in the mevalonate or MEP pathway and the enzymes in the malonyl-CoA pathway may be soluble. Suitable enzymes for each of these pathways are described above.

[0107] In some embodiments, the enzymes in the upper cannabinoid pathway can be scaffolded, while the enzymes in the hexanoyl-CoA pathway or hexanoyl-CoA synthase and the enzymes in the mevalonate or MEP pathways can be soluble. In some embodiments, the enzymes in the hexanoyl-CoA pathway or hexanoyl-CoA synthase can be scaffolded at the N-terminus relative to the enzymes in the upper cannabinoid pathway, and the enzymes in the mevalonate or MEP pathways can be soluble. In such embodiments, malonyl-CoA can be supplemented. Suitable enzymes for each of these pathways are described above.

[0108] In some embodiments, for example Fig.10In the embodiment shown, the bidirectional scaffold may include an enzyme of the malonyl-CoA (MCA) pathway at the N-terminus of the scaffold, an enzyme of the mevalonate pathway at the C-terminus of the scaffold, and an enzyme of the upper cannabinoid pathway therebetween. In some embodiments, the bidirectional scaffold may include an enzyme of the malonyl-CoA pathway at the N-terminus of the scaffold, an enzyme of the MEP pathway at the C-terminus of the scaffold, and an enzyme of the upper cannabinoid pathway therebetween. In such embodiments, the enzyme of the hexanoyl-CoA pathway may be on a separate scaffold or may be soluble. In some embodiments, HCS may replace the scaffolded enzyme or soluble enzyme of the hexanoyl-CoA pathway.

[0109] In some embodiments, each pathway is on a separate scaffold. For example, in one embodiment, enzymes for the upper cannabinoid pathway can be located on one scaffold, enzymes for the mevalonate or MEP pathway can be located on one scaffold, enzymes for the hexanoyl-CoA pathway can be located on one scaffold, and enzymes for the malonyl-CoA pathway can be located on another scaffold.

[0110] In any of the embodiments described herein, the biosynthesized cannabinoid acids may be isolated and / or may be used as substrates for the synthesis of other secondary and tertiary cannabinoids using downstream cannabinoid synthases. To produce a wider variety of cannabinoids, the downstream cannabinoid synthases are typically not scaffolded, as scaffolding favors the production of terminal cannabinoids. However, in some embodiments, one or more downstream cannabinoid synthases may be included on a scaffold as described herein.

[0111] For example, one or more of cannabidiolic acid synthase (CBDAS), cannabichromene acid synthase (CBCAS), tetrahydrocannabinolic acid synthase (THCAS) or other cannabinoid synthases can be used to produce other cannabinoids derived from cannabinoid salts / esters. For example, CBDAS; CBCAS; THCAS; CBDAS and CBCAS; CBDAS and THCAS; CBCAS and THCAS; or CBDAS, CBCAS and THCAS can be used to produce other cannabinoids derived from cannabinoid salts / esters, such as one or more of cannabidiolic acid, cannabichromene acid and delta-9 tetrahydrocannabinolic acid. CBDAS is classified under EC1.21.3.8 and can catalyze the synthesis of cannabidiolic acid from cannabichromene acid. CBCAS is classified under EC1.3.3- and can catalyze the synthesis of cannabichromene acid from cannabichromene acid. THCAS is classified under EC1.21.3.7 and can catalyze the synthesis of delta-9 tetrahydrocannabinolic acid from cannabichromene acid.

[0112] Host cells for cannabinoid production

[0113] Cannabinoids can be produced in host cells or in vitro using the multi-enzyme scaffolds described herein. Suitable host cells include any microorganism, eukaryotic or prokaryotic, such as bacteria (e.g., Escherichia coli, Bacillus, Brevibacterium, Streptomyces, or Pseudomonas), yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, or Komagataella phaffii) and other fungi (e.g., Neurospora crassa) and green algae (e.g., Dunaliella sp., Chlorella variabilis), Euglena variegated mutabilis or Chlamydomonas reinhardtii), as well as plant cells that can be maintained in culture (e.g., tobacco, cannabis or other photosynthetic plant cells), or in the case of plant cells (e.g., cells from tobacco or cannabis plants), that can be engineered in culture and grown as complete transgenic plants (e.g., cells from tobacco or cannabis plants). Such host cells or plants may or may not naturally produce cannabinoids.

[0114] Host cells can be modified to contain one or more exogenous nucleic acids encoding a support as described herein and one or more exogenous nucleic acids encoding an engineered enzyme. The term "nucleic acid" as used herein encompasses RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Nucleic acids can be double-stranded or single-stranded. In the case of a single strand, the nucleic acid can be a sense strand or an antisense strand. In addition, nucleic acids can be circular or linear.

[0115] As used herein, the term "exogenous" with respect to nucleic acids and specific host cells refers to any nucleic acid that is not derived from the specific host cell present in nature. Therefore, once introduced into a host cell, non-naturally produced nucleic acids are considered to be exogenous nucleic acids relative to the host cell. It is important to note that non-naturally produced nucleic acids can include nucleic acid sequences or fragments of nucleic acid sequences present in nature, provided that the nucleic acid does not exist in nature as a whole. For example, a nucleic acid molecule comprising a genomic DNA sequence in an expression vector is a non-naturally produced nucleic acid, and therefore, once introduced into a host cell, it is exogenous relative to the host cell because the nucleic acid molecule does not exist in nature as a whole (genomic DNA plus carrier DNA). Therefore, any vector, autonomously replicating plasmid or virus (such as retrovirus, adenovirus or herpes virus) that does not exist in nature as a whole is considered to be a non-naturally produced nucleic acid. Therefore, it is believed that genomic DNA fragments and cDNA produced by PCR or restriction endonuclease treatment are non-naturally produced nucleic acids because they exist in the form of separate molecules that do not exist in nature. It can also be inferred that any nucleic acid comprising a promoter sequence and a polypeptide coding sequence (eg, cDNA or genomic DNA) in an arrangement that does not occur in nature is a non-naturally occurring nucleic acid.

[0116] Naturally occurring nucleic acids can be exogenous to a particular cell. For example, an intact chromosome isolated from a cell of organism X is an exogenous nucleic acid to a cell of organism Y once the chromosome is introduced into the cell of Y.

[0117] It should be noted that an exogenous nucleic acid molecule encoding a polypeptide having an enzymatic activity that catalyzes the production of a compound not normally produced by the host cell can be administered to the host cell. Alternatively or additionally, an exogenous nucleic acid molecule encoding a polypeptide having an enzymatic activity that catalyzes the production of a compound normally produced by the host cell can be administered to the host cell. In this case, the recombinant host cell can produce more compounds, or can produce compounds more efficiently, compared to similar host cells without genetic modification.

[0118] The enzyme with a specific enzyme activity can be a naturally occurring or non-naturally occurring polypeptide. A naturally occurring polypeptide is any polypeptide having a naturally occurring amino acid sequence, including wild-type and polymorphic polypeptides. Such naturally occurring polypeptides can be obtained from any species, including but not limited to animals (e.g., mammals), plants, fungi, and bacterial species. A non-naturally occurring polypeptide is any polypeptide having an amino acid sequence that does not exist in nature. Therefore, a non-naturally occurring polypeptide can be a mutant form of a naturally occurring polypeptide, or an engineered polypeptide, such as an engineered enzyme comprising an ID as described herein. For example, a non-naturally occurring polypeptide having geranyl pyrophosphate synthase activity can be a mutant form of a naturally occurring polypeptide having geranyl pyrophosphate synthase activity. For example, a GPPS encoded by Erg20 can include a substitution of phenylalanine at position 96 by tryptophan and a substitution of asparagine at position 127 by tryptophan (referred to as Erg20 WW ). Erg20 WW It is advantageous to generate geranyl pyrophosphate instead of farnesyl pyrophosphate. See Jiang et al., Metab Eng. 2017, 41: 57-66. For example, a truncated HMGR (tHMGR) can be used, such as an N-terminal truncated HMGR comprising a catalytic domain but not comprising a transmembrane or regulatory domain of HMGR. For example, HMGR from Arabidopsis thaliana (A. thaliana) (GenBank accession number J04537) or HMGR from Saccharomyces cerevisiae (containing only residues 646-1025) can be truncated to remove the transmembrane and / or regulatory domains and used in the scaffold to eliminate the bottleneck in the mevalonate pathway. HMGR catalyzes the rate-limiting step in the mevalonate pathway (see, for example, Song et al., 2017, Scientific reports, doi: 10.1038 / s41598-017-15005-4). For example, a nucleic acid encoding atoB from Saccharomyces cerevisiae can be modified to include a synthetic 5'UTR (e.g., a synthetic 5'UTR sequence: 5'-cggcacccctacaaacagaaggaatataaa-3' (SEQ ID NO: 82)), and can be used in a scaffold because it can alter atoB expression to promote flux rebalancing, which favors the generation of acetoacetyl-CoA rather than the reverse reaction product butyryl-CoA (see Kim et al., 2018, Bioresour Technol, doi: 10.1016 / j.biortech.2017.10.014). A polypeptide can be mutated by, for example, sequence addition, deletion, substitution, or a combination thereof.

[0119] Any enzyme described herein that can be used to produce one or more cannabinoids may have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It should be understood that sequence identity can be determined based on the mature enzyme (e.g., removing any signal sequence).

[0120] For example, ACL can be compared to Homo sapiens ACL (see SEQ ID NO: 83, Fig. 6A ), or an amino acid sequence from Rattus norvegicus, Mus musculus, or Ciona intestinalis (e.g., GenBank Accession Nos. AAA74463, AAK56081, and BAB00624, respectively).

[0121] For example, acetyl-CoA acetyltransferase (atoB) can be expressed as E. coli atoB (see SEQ ID NO: 84, Fig. 6A ) or an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) from atoB of Cupriavidus necator, Clostridium acetobutylicum or Arabidopsis thaliana (e.g., GenBank Accession Nos. CAJ92573, AAK80816 and AAM67058, respectively). In some embodiments, a malonyl-CoA acyl carrier protein transacylase from Saccharomyces cerevisiae, Homo sapiens, Serratia plymuthica or Dickeya paradisiaca can replace atoB, e.g., GenBank Accession Nos. DAA10992, AAH30985, AGO55277 and ACS85236, respectively.

[0122] For example, 3-hydroxy-butyryl-CoA dehydrogenase (BHBD) can be expressed as Clostridium acetobutylicum BHBD (see SEQ ID NO: 85, Fig. 6A) or an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to a BHBD from Escherichia coli, Treponema denticola, or Arabidopsis thaliana (e.g., GenBank Accession Nos. AIZ91493, AAS11105, and AAN17431, respectively).

[0123] For example, the enoyl-CoA hydratase (ECH) can be combined with the C. acetobutylicum ECH (see SEQ ID NO: 86, Fig. 6A ) or an amino acid sequence from ECH of Acinetobacter oleivorans, Cupribotium necrotum, or Acinetobacter baumannii (e.g., GenBank Accession Nos. ADI91469, CAJ91294, and ACJ57023, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0124] For example, β-ketothiolase (bktB) can be conjugated to Cupriavidus necrotus bktB (see SEQ ID NO: 87, Fig. 6A ) or the amino acid sequence of bktB from Escherichia coli, Lactobacillus casei, or Clostridium acetobutylicum (e.g., GenBank Accession Nos. ALI39443, CAQ67083, and AAK80816, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0125] For example, trans-2-enoyl-CoA-reductase (ECR) can be conjugated to the ECR of Treponema denticola (see SEQ ID NO: 88, Fig. 6A ), or an amino acid sequence from ECRs of Coptis nectarini, Saccharomyces cerevisiae, or Klebsiella michiganensis (e.g., GenBank Accession Nos. AAP86010, DAA07148, and AIE72439, respectively), having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0126] For example, hexanoyl-CoA synthetase (HCS), which is an acyl-activating enzyme (AAE), can be expressed as C. sativa AAE1 (see SEQ ID NO: 89, Fig. 6A , GenBank Accession No. AFD33345) or Cannabis sativa L. AAE3 (GenBank Accession No. AFD33347) has at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) to the amino acid sequence of Cannabis sativa L. AAE1 and AAE3 can each utilize hexanoate / ester as a substrate. See Stout et al., Plant J., 71(3):353-365 (2012). In some embodiments, an AAE encoded by CsAAE1 can be used. For the coding sequence, see GenBank Accession No. JN717233. In some embodiments, an AAE encoded by CsAAE3 can be used. For the coding sequence, see GenBank Accession No. JN717233. In some embodiments, both CsAAE1 and CsAAE3 can be used.

[0127] For example, HMG-CoA synthase (HMGS) can be conjugated to Saccharomyces cerevisiae HMGS (see SEQ ID NO: 90, Fig. 6A ) or an amino acid sequence from Arabidopsis thaliana, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. AEE83052, CAQ67081, and AAA62411, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0128] For example, an N-terminally truncated or canonical HMG-CoA reductase (HMGR) can be conjugated to Saccharomyces cerevisiae HMGS (see SEQ ID NO: 91, Fig. 6A ) or an amino acid sequence having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to an HMGR from Arabidopsis thaliana, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. AEE35849, CAQ67082, and AAA52679, respectively).

[0129] For example, mevalonate kinase can be conjugated to Saccharomyces cerevisiae mevalonate kinase (see SEQ ID NO:92, Fig. 6A ) or the amino acid sequence of mevalonate kinase from Arabidopsis thaliana, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. AAD31719, CAQ66794, and AAF82407, respectively).

[0130] For example, phosphomevalonate kinase can be coupled to Saccharomyces cerevisiae phosphomevalonate kinase (see SEQ ID NO:93, Fig. 6A ), or the amino acid sequence of the mevalonate kinase from Scheffersomyces stipitis, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. EAZ63544, CAQ6630609, and AAH06089, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0131] For example, the mevalonate diphosphate decarboxylase can be combined with the Saccharomyces cerevisiae mevalonate diphosphate decarboxylase (see SEQ ID NO: 94, Fig. 6A ) or the amino acid sequence of mevalonate diphosphotyrosine decarboxylase from Arabidopsis thaliana, Lactobacillus casei or Homo sapiens (e.g., GenBank Accession Nos. AAC67348, CAQ66795 and AAC50440, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%).

[0132] For example, isopentyl diphosphate isomerase can be combined with Saccharomyces cerevisiae isopentyl diphosphate isomerase (see SEQ ID NO:95, Fig. 6A ) or the amino acid sequence of isopentyl diphosphate isomerase from Arabidopsis thaliana, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. AAC49920, CAQ66796, and AAP35407, respectively).

[0133] For example, a geranyl pyrophosphate synthase (GPPS) (also known as a geranyl-diphosphate synthase) can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of Saccharomyces cerevisiae GPS or GPPS from Acinetobacter baumannii, Lactobacillus casei, or Homo sapiens (e.g., GenBank Accession Nos. ACJ56139, CAQ66932, and AAH100, respectively). In some embodiments, a mutant GPPS can be used. For example, the GPPS encoded by Erg20 can include a substitution of phenylalanine at position 96 by tryptophan and a substitution of asparagine at position 127 by tryptophan (referred to as Erg20 WW ) (see SEQ ID NO:96, Fig. 6A ). Erg20 WWFavors the formation of geranyl pyrophosphate rather than farnesyl pyrophosphate. See Jiang et al., Metab Eng. 2017, 41: 57-66. In some cases, glutamate has an effect on Erg20 (Erg20 K179E ) can be used to produce GPPS that is beneficial for producing geranyl pyrophosphate. See WO2016010827A1.

[0134] For example, the DOXP synthase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, Clostridium acetobutylicum, Treponema denticola, or Arabidopsis thaliana DOXP synthase (e.g., GenBank Accession Nos. CDH63925, AAK80036, AAS12424, and ANM65835, respectively).

[0135] For example, the DOXP reductoisomerase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, a Clostridium acetobutylicum, a Treponema denticola, or an Arabidopsis thaliana DOXP reductoisomerase (e.g., GenBank Accession Nos. CDH63708, AAK79760, AAS12860, and AAM61343, respectively).

[0136] For example, the MEP cytidine transferase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, Clostridium acetobutylicum, Treponema denticola, or Arabidopsis thaliana MEP cytidine transferase (e.g., GenBank Accession Nos. CDH66380, AAK81121, AAS12810, and BAB21592, respectively).

[0137] For example, the CDPME kinase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of Escherichia coli, Clostridium acetobutylicum, Treponema denticola, or Arabidopsis thaliana CDPME kinase (e.g., GenBank Accession Nos. CDH64802, AAK80844, AAS11855, and AEC07908, respectively).

[0138] For example, the MECDP synthase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, Nicotiana tabacum, Treponema denticola, or Acinetobacter baumannii MECDP synthase (e.g., GenBank Accession Nos. CDH66379, AHM22925, AAS12811, and ACJ59227, respectively).

[0139] For example, the HMBPP synthase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, Acinetobacter baumannii, Treponema denticola, or Arabidopsis thaliana HMBPP synthase (e.g., GenBank Accession Nos. AAN81487, ACJ58210, AAS11783, and AED97354, respectively).

[0140] For example, the HMBPP reductase can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli, Acinetobacter baumannii, Treponema denticola, or Arabidopsis thaliana HMBPP reductase (e.g., GenBank Accession Nos. CDH63564, ACJ57384, AAS11585, and AEE86362, respectively).

[0141] For example, acetyl-CoA carboxylase (ACC) can be conjugated to Saccharomyces cerevisiae acetyl-CoA carboxylase (see SEQ ID NO: 97, Fig. 6A ) or an acetyl-CoA carboxylase from Homo sapiens, Treponema denticola, or Cupriavidus necrotizer (e.g., GenBank Accession Nos. AAP94122, AAS11086, and CAQ67359, respectively) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%).

[0142] For example, pyruvate dehydrogenase (E1) and dihydrolipoyl transacetylase (E2) can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequences of Saccharomyces cerevisiae, Escherichia coli, Clostridium acetobutylicum, or Cupriavidus necrotizingus E1 and E2 (e.g., GenBank Accession Nos. DAA07337, AMC97367, CAQ66617, and CAJ92510 for E1, and DAA10474, AUG14916, CAQ66619, and CAJ92511 for E2, respectively).

[0143] For example, olivetol synthase (OS) can be combined with OS from cannabis (shown in SEQ ID NO: 98 ( Fig. 6A )) or the amino acid sequence of OS from Cannabis sativa L. (having GenBank accession number BAG14339) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%). See, e.g., Taura et al., FEBS Letters 583 (2009) 2061-2066.

[0144] For example, olivetate cyclase (OAC) can be conjugated to OAC from cannabis as shown in SEQ ID NO:99 ( Fig. 6A ) or the amino acid sequence of OAC from cannabis sativa L. (having GenBank accession number AFN42527) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%). See, e.g., Gagne et al., Proc. Natl. Acad. Sci. USA, 2012 109(31)12811-12816.

[0145] For example, CBGAS can be combined with an aromatic isoprenyl transferase (APT) from cannabis (e.g., as shown in SEQ ID NO: 100 ( Fig. 6A ) has at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) to the amino acid sequence of CBGAS) shown in ). See, for example, U.S. Patent Publication No. 20120144523A1 and U.S. Patent No. 8,884,100B2. In some embodiments, a soluble APT (e.g., NphB) from Streptomyces can be used. See, for example, Carvalho et al., FEMS Yeast Research, 17, 2017, fox037.

[0146] For example, cannabidiolic acid synthase (CBDAS) can be combined with CBDAS from cannabis as shown in SEQ ID NO: 101 ( Fig. 6A ) or the amino acid sequence of CBDAS from cannabis (having GenBank accession number BAF65033) having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%). See, e.g., Taura et al., FEBS Lett. 581(16), 2929-2934(2007).

[0147] For example, cannabichromene acid synthase (CBCAS) can be combined with CBCAS from cannabis as shown in SEQ ID NO: 102 ( Fig. 6A ) or the amino acid sequence of the CBCAS of cannabis shown in SEQ ID NO:2 of WO 2015 / 196275 A1 has at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%). SEQ ID NO:2 of WO 2015 / 196275 A1 includes a signal peptide of 28 amino acids at the N-terminus. All or part of the signal peptide can be removed from the sequence. CBDAS from Indian cannabis (C.indica) or weed cannabis (C.ruderalis) can also be used. In some embodiments, the cannabis CBCAS encoding nucleic acid sequences shown in SEQ ID NO:8 and 9 of WO 2015 / 196275 A1, respectively, which are optimized for Escherichia coli or yeast, can be used.

[0148] For example, tetrahydrocannabinolic acid synthase (THCAS) can have at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%) to the amino acid sequence of THCAS from Cannabis sativa L. (having GenBank Accession No. BAC41356). See, e.g., Sirikantaramas et al., J. Biol. Chem. 279 (38), 39767-39774 (2004).

[0149] The percent identity (homology) between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST2 sequence (B12seq) program from a standalone version of BLASTZ containing BLASTP version 2.0.14. The standalone version of BLASTZ can be obtained from the website of Fish & Richardson (e.g., www.fr.com / blast / ) or the website of the National Center for Biotechnology Information of the U.S. government (www.ncbi.nlm.nih.gov). Instructions for explaining how to use the B12seq program can be found in the readme file that comes with BLASTZ. B12seq uses the BLASTP algorithm to compare two amino acid sequences. To compare two amino acid sequences, the options for B12seq are set as follows: -i is set to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are set to their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences have homology (identity), the specified output file will display those homologous regions in the form of aligned sequences. If the two compared sequences do not have homology (identity), the specified output file will not display aligned sequences. Similar operations can be followed for nucleic acid sequences, except that blastn is used.

[0150] After alignment, the number of matches is determined by counting the number of positions where the same amino acid residue exists in the two sequences. The percent identity (homology) is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence and then multiplying the resulting value by 100. It should be noted that the percent identity (homology) values ​​are rounded to the first decimal place. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It should also be noted that the length value is always an integer.

[0151] It is understood that a polypeptide having a particular amino acid sequence can be encoded by many nucleic acids. The degeneracy of the genetic code is well known in the art; that is, for many amino acids, there is more than one nucleotide triplet as a codon for that amino acid. For example, codons in the coding sequence of a given enzyme can be modified using an appropriate codon bias table to obtain optimal expression in a particular species (e.g., bacteria or fungi). For example, Fig. 12A The nucleotide sequence shown is encoding ATP citrate lyase, atoB, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase, β-ketothiolase (bktB), trans-enoyl-CoA reductase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyl diphosphate delta isomerase, geranyl-diphosphate synthase (ERG20 WW ), olivetol synthase, olivetate cyclase, CBGA synthase, CBDA synthase, CBCA synthase, acetyl-CoA carboxylase, and hexanoyl-CoA synthetase nucleic acid sequences. The nucleic acid sequences of ATP citrate lyase, atoB, 3-hydroxybutyryl-CoA dehydrogenase, enoyl-CoA hydratase, trans-enoyl-CoA reductase, bktB, olivetol synthase, olivetate cyclase, CBGA synthase, CBDA synthase, and CBCA synthase were codon-optimized for expression in yeast. Figures 14A-14C Contains code Figures 13A-13C Codon-optimized (for expression in yeast) nucleic acid sequences of engineered enzymes.

[0152] In addition to sequence similarity, it is understood that enzymes and scaffolds having structural and / or functional similarities to the enzymes and scaffolds described herein are also included within the scope herein.

[0153] Provided herein are recombinant host cells that can be used to produce one or more cannabinoids as described herein. For example, individual host cells can contain exogenous nucleic acids to express the scaffold polypeptide and each enzyme fixed on the scaffold. Importantly, it is noted that such host cells can contain exogenous nucleic acid molecules of any number and / or combination. For example, a specific host cell may contain exogenous nucleic acids encoding a scaffold, and enzymes encoding malonyl-CoA pathways, enzymes of hexanoyl-CoA pathways, or other exogenous nucleic acids encoding HCS and mevalonate or MEP pathways. A single exogenous nucleic acid can encode one enzyme or more than one enzyme (e.g., 1 to 10 (or more) enzymes, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or one or more copies of 2 to 3 enzymes). Therefore, the number of different exogenous nucleic acids required to produce the engineered enzymes positioned on the scaffold will depend on the design and / or specific embodiment of the scaffold. Figure 2A and Figure 2BNon-limiting schematic diagrams of suitable gene cassettes for expression of scaffolds and enzymes are each provided. Fig. 12C Provided are nucleic acid sequences encoding scaffold polypeptides comprising peptide ligands corresponding to IDs 1-16 shown in Table 2 and a three-repeat MYC tag. See also Fig.14D , which is the encoding Fig.13D A codon-optimized nucleic acid sequence of a scaffold polypeptide. Fig.12D A nucleic acid sequence encoding a scaffold polypeptide is provided, the scaffold polypeptide comprising a peptide ligand corresponding to IDs 1 and 17 and a three-repeat FLAG tag. See also Fig.14D .

[0154] In some embodiments, a nucleic acid sequence encoding a self-cleaving peptide can be used to separate multiple nucleic acids encoding polypeptides (e.g., Figure 2A or Figure 2B The nucleic acid of the gene cassette in ( ) is connected together. During the translation of the transcript, the growing polypeptide can be cut at the 2A peptide, and the translation continues to the next polypeptide. When designing a vector for expressing a polypeptide in the form of a multicistronic unit, the nucleic acid encoding the polypeptide and the self-cleaving peptide (e.g., 2A peptide) can be designed so that they are in the translation frame with each other. Examples of 2A peptides that can be used as described herein include, but are not limited to, the 2A peptide of foot-and-mouth disease virus (FMDV), the 2A peptide of equine rhinitis A virus (ERAVO), the 2A peptide of the bright vein flat moth virus (TaV), or the 2A peptide of porcine teschovirus-1 (PTV-1) or porcine teschovirus-2 (PTV-2). The 2A peptides from PTV-1 and PTV-2 are referred to as P2A peptides. See, for example, SEQ ID NO: 212, a codon-optimized nucleotide sequence (for Saccharomyces cerevisiae) encoding the P2A peptide.

[0155] In addition, the cells described herein may contain a single copy or multiple copies (e.g., about 5, 10, 20, 35, 50, 75, 100, or 150 copies) of a particular exogenous nucleic acid molecule. Likewise, the cells described herein may contain more than one particular exogenous nucleic acid molecule and / or its copies. For example, a particular cell may contain about 50 copies of an exogenous nucleic acid molecule X and about 75 copies of an exogenous nucleic acid molecule Y.

[0156] Any method can be used to introduce exogenous nucleic acid molecules into host cells. In fact, many methods for introducing nucleic acids into host cells such as bacteria and yeast are well known to those skilled in the art. For example, heat shock, lipofection, electroporation, nuclear transfection, conjugation, protoplast fusion and gene gun delivery are common methods for introducing nucleic acids into bacteria and yeast cells. See, for example, Ito et al., J. Bacterol. 153: 163-168 (1983); Durrens et al., Curr. Genet. 18: 7-12 (1990); and Becker and Guarente, Methods in Enzymology 194: 182-187 (1991).

[0157] The exogenous nucleic acid molecule contained in the specific host cell can be maintained in the host cell in any form. For example, the exogenous nucleic acid molecule can be integrated into the genome of the microorganism or remain in a free state. In other words, the microorganism can be a stable or transient transformant. Equally, the microorganism as described herein can contain a single copy or multiple copies (for example, about 5, 10, 20, 35, 50, 75, 100 or 150 copies) of the specific exogenous nucleic acid molecule as described herein.

[0158] Suitable nucleic acid constructs for expressing engineered enzymes and scaffolds include, for example, CRISPR plasmids, baculovirus vectors, phage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g., viral vectors based on vaccinia virus, polio virus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, etc.), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and other vectors. Typically, such constructs include regulatory elements that promote the expression of nucleic acid sequences encoding polypeptides. Typically, regulatory elements are DNA sequences that regulate the expression of other DNA sequences at the transcriptional level. Therefore, regulatory elements include, but are not limited to, promoters, enhancers, etc. Any type of promoter can be used to express amino acid sequences from exogenous nucleic acid molecules. Examples of promoters include, but are not limited to, constitutive promoters, tissue-specific promoters, inducible or repressible promoters that respond or do not respond to specific stimuli (e.g., light, oxygen, chemical concentrations, sound, etc.).

[0159] In some embodiments, endogenous yeast promoters with different constitutive activity levels can be used to express engineered enzymes and / or scaffolds. In order to maintain an excess of enzyme relative to the scaffold molecule, the scaffold can be expressed under the control of the weakest promoter. For example, one or more of the following yeast promoters can be used: a promoter from the gene encoding the transcription elongation factor EF-1α (pTEF1), a promoter from the gene encoding phosphoglycerate kinase (PGK1), a promoter from the gene encoding triosephosphate isomerase (pTPI1), a promoter from the gene encoding the hexose transporter (pHXT7), HXT7, a promoter from the gene encoding pyruvate kinase 1 (pPYK1), a promoter from the gene encoding alcohol dehydrogenase 1 (pADH1), or a promoter from the gene encoding triphosphate dehydrogenase (pTDH3). For example, in Figure 2A In the embodiment shown, the pTPI1 promoter can be used to express enzymes of the upper hexanoyl-CoA (HCA) pathway, enzymes of the lower HCA pathway, enzymes of the upper mevalonate (MVA) pathway, enzymes of the lower MVA pathway, and enzymes of the lower cannabinoid (CB) pathway, while the pTEF1 promoter can be used to express enzymes of the upper CB pathway, atoB enzymes, and enzymes of the malonyl-CoA pathway, and the pADH1 promoter can be used to express the scaffold. Among these promoters, the pADH1 promoter has the weakest activity ( Figure 2A The pTEF1 promoter has the strongest activity ( Figure 2A +++ in the pTPI1 promoter), the activity of the pTPI1 promoter is between the other two ( Figure 2A ++ in ). In some embodiments, the Gal 1-10 promoter (e.g., from Saccharomyces cerevisiae) can be used. See, e.g. Fig.17 .

[0160] The nucleic acid construct can also include a selective marker, such as a selective marker for antibiotics such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance or kanamycin resistance. In some embodiments, a nutritional marker gene providing a prototrophic type of essential nutrients such as tryptophan (TRP1), uracil (URA3), histidine (HIS3), leucine (LEU2), lysine (LYS2) or methionine can be included on the nucleic acid construct. See, e.g. Fig.17. As shown in Example 3, four different auxotrophic markers are used to sequentially select transformed cells containing the desired combination of nucleic acids encoding enzymes and scaffolds. For example, yeast cells transformed with a vector containing a TRP gene and a nucleic acid encoding an enzyme of the hexanoyl-CoA pathway are grown in a culture medium lacking tryptophan. Transformed cells grown in a culture medium lacking tryptophan are selected and further transformed with a vector containing the LEU gene and a nucleic acid encoding a mevalonate pathway enzyme. The resulting transformed cells grow on a culture medium lacking tryptophan and leucine, and cells grown in a culture medium lacking tryptophan and leucine are transformed with a vector containing the HIS gene and a nucleic acid encoding an upper cannabinoid pathway enzyme. The resulting transformed cells grow on a culture medium lacking tryptophan, leucine, and histidine, while cells grown in a culture medium lacking tryptophan, leucine, and histidine are transformed with a vector containing the URA3 gene and a nucleic acid encoding a scaffold. The resulting transformed cells grow on a culture medium lacking tryptophan, leucine, histidine, and uracil. Cells grown in medium lacking tryptophan, leucine, histidine, and uracil contain the desired combination of enzymes and scaffolds, e.g. Figure 1B shown.

[0161] In some embodiments, the encoded enzymes (e.g., one or more enzymes from the cannabinoid biosynthetic pathway, the mevalonate pathway, the MEP pathway, the hexanoyl-CoA pathway, or the hexanoyl-CoA synthetase) and / or scaffolds can include targeting sequences that can be used to direct the enzymes or scaffolds to one of several different intracellular compartments, including, for example, the endoplasmic reticulum (ER), mitochondria, plastids (e.g., chloroplasts), vacuoles, Golgi apparatus, or protein storage vesicles (PSVs). For example, mitochondrial or plastid targeting sequences can be used to promote mitochondrial or plastid compartmentalization of cannabinoid / cannabinoid precursor biosynthesis, such that the encoded enzymes and scaffolds are expressed in the mitochondria or plastids of the host cell.

[0162] In some embodiments, cannabinoid / cannabinoid precursor biosynthesis can be performed by co-expressing one or more engineered enzymes and scaffolds in both the cytosolic compartment and the plastids or mitochondria of the host cell. See e.g. Fig.11 It should be understood that although Fig.11Depicted are scaffolds comprising enzymes of the hexanoyl-CoA pathway, enzymes of the upper cannabinoid pathway, and enzymes of the mevalonate pathway, and dual compartment engineering can be performed with any of the scaffolds and enzymes described herein. For example, dual compartment engineering can be performed in two compartments by co-expressing the scaffold and enzymes of the hexanoyl-CoA pathway, enzymes of the upper cannabinoid pathway, and enzymes of the MEP pathway in both the cytosolic compartment and the mitochondrial plastid of the host cell. Dual compartment engineering can also be performed by engineering separate haploid yeast strains for cytosolic and mitochondrial / plastid cannabinoid biosynthesis, and then hybridizing the two haploid strains to produce a diploid lineage that is heterozygous for cytosolic and mitochondrial / plastid cannabinoid biosynthesis.

[0163] In some embodiments, the engineered enzyme and / or scaffold further comprises a tag (e.g., c-myc, FLAG, polyhistidine (e.g., hexa-histidine), hemagglutinin (HA), glutathione-S-transferase (GST), or maltose binding protein (MBP)) that can be used to purify the recombinant protein, or a detectable marker (e.g., luciferase, green fluorescent protein (GFP), or chloramphenicol acetyltransferase (CAT)). For example, in Figure 6C and 6D In the illustrated embodiment, the scaffold may include a myc tag (eg, (Myc)3 tag) or a FLAG tag (FLAG)3 tag at the C-terminus.

[0164] In some embodiments, host cells can be engineered to increase the availability of acetyl-CoA for the biosynthesis of cannabinoids and cannabinoid precursors. For example, the mitochondrial enzyme isocitrate dehydrogenase-1 (IDH1) can be placed under transient microRNA-mediated induction inhibition. Since mitochondrial IDH1 is primarily responsible for the consumption of the cellular citric acid pool, microRNA-mediated IDH1 inhibition can increase the availability and cytoplasmic shuttling of citric acid to produce acetyl-CoA by ATP citrate lyase. The resulting increase in acetyl-CoA bioavailability can further improve the titer of downstream hexanoyl-CoA and geranyl pyrophosphate by improving the initial substrate availability of hexanoyl-CoA and mevalonate pathways. The combined metabolic engineering of acetyl-CoA can alleviate the problems associated with siphoning acetyl-CoA from the endogenous metabolism of host cells.

[0165] In some embodiments, one or more conventional and / or modern gene editing techniques can be used to produce a recombinant host. For example, clustered, regularly spaced, short palindromic repeats (CRISPR) technology can be used to modify the expression of endogenous nucleic acids. The CRISPR / Cas system includes components of the prokaryotic adaptive immune system, which is functionally similar to eukaryotic RNA interference, using RNA base pairing to guide DNA or RNA cutting. The function of the Cas9 protein is an endonuclease, and the CRISPR RNA (crRNA) and trans-activating RNA (tracerRNA) sequences are complexed with the Cas9 enzyme to target the target DNA sequence (Makarova et al., Nat Rev Microbiol 9 (6): 467-477, 2011). Modification of a single targeting RNA may be sufficient to change the nucleotide target of the Cas protein. In some cases, crRNA and tracrRNA can be engineered into a cr / tracrRNA hybrid (also referred to as "guide RNA" or "gRNA") to guide Cas9 cutting activity (Jinek et al., Science, 337 (6096): 816-821, 2012). The CRISPR / Cas system can be used in various prokaryotic and eukaryotic organisms (see, e.g., Jiang et al., Nat Biotechnol, 31(3):233-239, 2013; Dicarlo et al., Nucleic Acids Res, doi:10.1093 / nar / gkt135, 2013; Cong et al., Science, 339(6121):819-823, 2013; Mali et al., Science, 339(6121):823-826, 2013; Cho et al., Nat Biotechnol, 31(3):230-232, 2013; and Hwang et al., Nat Biotechnol, 31(3):227-229, 2013).

[0166] Another gene editing technology can include sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of, for example, folded endonucleases. Original and customized TALE nucleases ("TALENs") are fused to direct DNA double-strand breaks at specific target sites. See, for example, Christian et al., Genetics 186: 757-761 (2010) and U.S. Patent Publication No. 20110145940.

[0167] Other suitable gene insertion techniques include the use of retroviral vectors and gene gun particle gene delivery systems (commonly known as "gene guns").

[0168] Methods for identifying and / or selecting host cells containing exogenous nucleic acids or modified endogenous nucleic acids are well known to those skilled in the art. Such methods include, but are not limited to, introducing and expressing negative selection markers such as antibiotic resistance genes, PCR and nucleic acid hybridization techniques such as Northern and Southern analysis. In some cases, immunohistochemistry and biochemical techniques can be used to determine whether a microorganism contains a specific nucleic acid by detecting the expression of an enzymatic polypeptide encoded by a specific nucleic acid molecule. For example, antibodies specific to the encoded enzyme can be used to determine whether a specific cell contains the encoded enzyme. In addition, biochemical techniques can be used to determine whether a cell contains a specific nucleic acid molecule encoding an enzymatic polypeptide by detecting the organic products produced by the expression of the enzymatic polypeptide.

[0169] Also provided herein are isolated nucleic acid molecules. The term "isolated" used herein with respect to nucleic acids refers to naturally occurring nucleic acids that are not adjacent to the two sequences (one at the 5' end and one at the 3' end) that are adjacent to them in the naturally occurring genome of the organism from which they are derived. For example, an isolated nucleic acid can be, but is not limited to, a recombinant DNA molecule of any length, provided that one of the nucleic acid sequences that are usually closely flanked by the recombinant DNA molecule in the naturally occurring genome is removed or does not exist. Therefore, isolated nucleic acids include, but are not limited to: recombinant DNA that exists as an isolated molecule independently of other sequences (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment), and recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryotic or eukaryotic organism. In addition, an isolated nucleic acid may include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

[0170] The term "isolated" used herein for nucleic acids also includes any non-naturally produced nucleic acids, because such non-naturally produced nucleic acid sequences do not exist in nature, and there is no immediately adjacent sequence in the naturally produced genome. For example, non-naturally produced nucleic acids such as engineered nucleic acids are considered to be isolated nucleic acids. Engineered nucleic acids can be manufactured using general molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally produced nucleic acids can be independent of other sequences, or incorporated into vectors, self-replicating plasmids, viruses (e.g., retroviruses, adenoviruses or herpes viruses) or genomic DNA of prokaryotes or eukaryotes. In addition, non-naturally produced nucleic acids can include nucleic acid molecules that are part of hybridization or fusion nucleic acid sequences.

[0171] It is apparent to one skilled in the art that a nucleic acid present among hundreds to millions of other nucleic acid molecules in, for example, a cDNA or genomic library or a gel slice containing restriction enzyme digests of genomic DNA is not considered to be an isolated nucleic acid.

[0172] In some embodiments, the production of one or more cannabinoids in vitro can be performed using the scaffolds and immobilized enzymes described herein, using lysates from recombinant host cells (e.g., buffered cell lysates) as a source of scaffolds and enzymes, using multiple lysates from different host cells as a source of scaffolds and enzymes, or using a cell-free reaction buffer such as a synthetic reaction buffer. For example, after immunoprecipitation of a C-terminally Myc / Flag-spiked enzyme-bound scaffold, the scaffold-enzyme complex can be maintained in a reaction buffer supplemented with citric acid and / or supplemented with glucose (or supplemented with other carbon sources), which allows for in vitro scaffolded cannabinoid biosynthesis.

[0173] Production of cannabinoids using recombinant hosts

[0174] Typically, one or more cannabinoids can be produced by providing a recombinant host such as a recombinant microorganism and culturing the microorganism with a culture medium. Generally speaking, the culture medium and / or culture conditions can allow the microorganism to grow to a sufficient density and efficiently produce cannabinoids. For example, aerobic batch fermentation can be performed on the microorganism. In some embodiments, one or more precursors (e.g., citrate, glucose, caproic acid and / or other carbon sources and / or malonyl-CoA) are supplemented in the culture medium. In some embodiments, about 30 mg / L to about 10,000 mg / L (e.g., about 100 mg / L to about 5,000 mg / L, about 200 mg / L to about 4,000 mg / L, about 300 mg / L to about 3,000 mg / L, or about 350 mg / L to about 1,000 mg / L) of buffered citrate, pH 6.0 can be added to the culture medium.

[0175] For large-scale production processes, any method can be used, such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2nd Edition, Editors: AL Demain and JEDavies, ASM Publishing Company; and Principles of Fermentation Technology, PF Stanbury and A. Whitaker, Pergamon). In short, a large container (e.g., a 100-gallon, 200-gallon, 500-gallon or larger volume container) containing a suitable culture medium is inoculated with a specific microorganism. After inoculation, the microorganism is incubated to produce biomass. Once the desired biomass or cell confluence is reached, part or all of the broth containing the microorganism can be transferred to a second container. The second container can be of any size. For example, the second container can be larger, smaller or of the same size as the first container. Typically, the second container is larger than the first container so that other culture media can be added to the broth from the first container. In addition, the culture media in the second container can be the same or different from the culture media used in the first container. The system can be expanded to include an array consisting of any number of individual containers.

[0176] After transfer, the microorganisms can be incubated to produce one or more cannabinoids. Once produced, the cannabinoids can be isolated by any method. For example, the biomass can be removed from the broth using conventional separation techniques, and the cannabinoids can be obtained from the biomass using conventional separation procedures (e.g., extraction, such as non-polar extraction with hexane followed by ethyl acetate), high performance liquid chromatography (e.g., HPLC diode array detector (HPLC-DAD)), gas chromatography-flame ionization detection (GC-FID), or ion exchange procedures).

[0177] The host cells described herein can produce one or more cannabinoids at a concentration of at least about 10 mg / L (e.g., at least about 15 mg / L, 25 mg / L, 50 mg / L, 75 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L or more). For example, in some embodiments, total cannabinoids (the total amount of CBG, CBGA, CBD, CBDA, CBC, and CBCA) can be produced at a concentration of at least about 10 mg / L, 15 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L or 100 mg / L or more. For example, in some embodiments, the total cannabinoids (the total amount of CBG, CBGA, CBD, CBDA, CBC, and CBCA) can be produced at a concentration of about 10 mg / L to about 500 mg / L (e.g., 20 mg / L to 450 mg / L, 40 mg / L to 380 mg / L, 60 mg / L to 280 mg / L, 60 mg / L to 250 mg / L, 60 mg / L to 150 mg / L, 80 mg / L to 400 mg / L, 80 mg / L to 300 mg / L, 80 mg / L to 250 mg / L, 80 mg / L to 200 mg / L, 80 mg / L to 175 mg / L, 90 mg / L to 400 mg / L, 90 mg / L to 300 mg / L, 90 mg / L to 250 mg / L, or 90 mg / L to 150 mg / L). In some embodiments, one or more individual cannabinoids (e.g., one or more of CBG, CBGA, CBD, CBDA, CBC, and CBCA) may be produced at a concentration of at least about 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, 100 mg / L or more.For example, in some embodiments, one or more individual cannabinoids may be produced at a concentration of about 1 mg / L to about 100 mg / L (e.g., 2 to 90 mg / L, 2 to 80 mg / L, 2 to 70 mg / L, 2 to 60 mg / L, 2 to 50 mg / L, 2 to 40 mg / L, 2 to 30 mg / L, 2 to 20 mg / L, 2 to 15 mg / L, 3 to 90 mg / L, 3 to 80 mg / L, 3 to 70 mg / L, 3 to 60 mg / L, 3 to 50 mg / L, 3 to 40 mg / L, 3 to 30 mg / L, 3 to 20 mg / L, 3 to 15 mg / L, 4 to 90 mg / L, 4 to 80 mg / L, 4 to 70 mg / L, 4 to 60 mg / L, 4 to 50 mg / L, 4 to 40 mg / L, 4 to 30 mg / L, 4 to 20 mg / L, or 4 to 15 mg / L).

[0178] The present invention is further illustrated by the following examples, which do not limit the scope of the present invention described in the claims. Example

[0179] Example 1 - General Methods

[0180] Enzymatic constructs

[0181] Each enzyme construct is designed to include an interaction domain (ID) comprising two tandem N-terminal or C-terminal ligand binding motifs separated from a given enzyme and from each other by an amino acid sequence containing a flexible, GS-rich linker flanked by a rigid alpha-helical spacer sequence. The motifs comprising each enzyme ID are capable of specific binding to a tandem peptide ligand that forms an ID binding site at discrete positions along the synthetic intracellular polypeptide scaffold. The expression of each enzyme is controlled by a constitutive or inducible promoter. The nucleic acids encoding the enzymes can be codon-optimized, for example, for expression in yeast.

[0182] Scaffolded constructs

[0183] ID binding sites comprising tandem peptide ligands specific for the tandem scaffold binding motifs, comprising the ID of each enzyme, are inserted at discrete locations along the intracellular polypeptide scaffold.

[0184] The tandem ligands comprising each scaffolded ID binding site are separated from each other by a sequence of 36 amino acid residues comprising a flexible, GS-rich linker flanking a rigid α-helical spacer sequence, while the scaffolded ID binding sites themselves are separated from each other by a sequence of 50 amino acid residues (or any other number of amino acid residues) comprising a flexible, GS-rich linker flanking a rigid α-helical spacer sequence. Specifically, the scaffold binding site for each enzyme in the hexanoyl-CoA pathway (in catalytic order) is located proximal to the ATP citrate lyase and acetyl-CoA acetyltransferase at the N-terminus of the primary scaffold. The scaffold binding site for each enzyme in the upper cannabinoid pathway is located proximal to (immediately downstream of) the binding site for the hexanoyl-CoA pathway enzyme. The scaffold binding site for each enzyme in the mevalonate (or MEP) pathway (in catalytic order) is located proximal to the ATP citrate lyase and acetyl-CoA acetyltransferase at the C-terminus of the primary scaffold. The enzyme catalyzing the rate-limiting / critical step in cannabinoid biosynthesis (CBGA synthase, the final enzymatic step in the upper cannabinoid pathway) is located at the intersection of converging cannabinoid precursor pathways near the midpoint of the scaffold.

[0185] Assessment of cannabinoidergic potential by transient transfection

[0186] Competent yeast and / or green algae cells were transiently transfected with plasmids encoding various arrangements of scaffolds and enzymes. To establish baseline cannabinoid capacity, cells were first transiently transfected with enzymes required for cannabinoid biosynthesis (but not scaffolds), and then the biosynthesized cannabinoids were extracted, isolated and quantified as described below (see "Cannabinoid Extraction, Isolation and Analytical Characterization"). To measure the improvement in cannabinoid capacity conferred by multi-enzymatic scaffolding, a subset of the above cells were co-transfected with plasmids encoding one or more multi-enzymatic scaffolds described herein, and the biosynthesized cannabinoids were extracted, isolated and quantified. The presence of plasmid DNA was confirmed by PCR, functional gene expression was confirmed by qRT-PCR, protein / polypeptide production was confirmed by Western blotting, and the scaffold of each enzyme was confirmed by co-immunoprecipitation of the C-terminal myc / flag-spiked scaffold, followed by Western blot analysis of each co-immunoprecipitated enzyme.

[0187] Engineering of stable cannabinoid-ergic cell lines

[0188] The construct can be integrated into the genome of a host cell (e.g., yeast, green algae, or other suitable host) by stable transfection. Gene integration is confirmed by PCR, functional gene expression is confirmed by qRT-PCR, and protein / polypeptide production is confirmed by Western blotting. Gene expression / protein synthesis is confirmed by comparing qRT-PCR and Western blotting results between samples with and without genetic engineering. In order to evaluate the improvement in cannabinoid capacity brought about by the multi-enzyme scaffold to the stably engineered cannabinoid cell line, cannabinoid biosynthesis is compared between cells stimulated for enzyme but no scaffold expression and cells stimulated for enzyme and scaffold expression.

[0189] Validation of multi-enzyme scaffolding

[0190] To verify the success of the multi-enzyme scaffold in transient transfection and stable engineered cells, a myc tag (or other immunoprecipitation tag) was inserted at the N-terminus or C-terminus of the polypeptide scaffold. The scaffolded enzymes were selectively co-immunoprecipitated by affinity chromatography using anti-myc affinity beads. Western blotting was performed to detect and quantify each co-immunoprecipitated enzyme.

[0191] Aerobic batch fermentation

[0192] Stably engineered cannabinoid-producing yeast, green algae or other host cells are grown in a bioreactor (or any other vessel) by aerobic batch fermentation (or any other culture technique).

[0193] Extraction, separation and analytical characterization of cannabinoids

[0194] After fully initiating cannabinoid biosynthesis, the engineered yeast / green algae cells are precipitated by centrifugation and washed with TBS. The supernatant (liquid culture medium) is poured out and collected. After washing with TBS, the precipitated cells are resuspended in ethanol adjusted with NaOH and lysed by repeated freeze-thaw and ultrasonic treatment. The biosynthesized cannabinoid fermentation product is then harvested from the lysate and supernatant by triple non-polar extraction using hexane and then ethyl acetate. The resulting organic fractions are combined and rotary evaporated. The biosynthesized cannabinoids are then quantitatively and qualitatively measured using high performance liquid chromatography with a diode array detector (HPLC-DAD) or gas chromatography-flame ionization detector (GC-FID).

[0195] In the following examples, each 48-hour culture was lysed / homogenized by ultrasonic wave. The ultrasonically treated samples were then subjected to triple liquid-liquid extraction with ethyl acetate (one volume equivalent of ethyl acetate was extracted each time). After separation, the ethyl acetate fractions collected from each sample were combined, and the combined samples were centrifuged and filtered. Ethyl acetate was then removed from each sample in a vacuum oven, and the residual sample was resuspended in 10 mL of methanol for analytical characterization. The analytical characterization of all samples was performed by a licensed independent third-party analytical testing agency (Precision Plant Molecules, Denver, Colorado). HPLC-DAD was used to quantitatively and qualitatively measure each parent and derivative cannabinoid and cannabinoid precursor OVA.

[0196] Example 2- Synthetic gene cassette assembly / synthesis, plasmid preparation and polycistronic vector construction

[0197] Five synthetic gene cassettes (named HCA, GPP, CAN, SCF, and SOL) were constructed for the biosynthesis of cannabinoids in heterologous cells or in cell-free reaction buffers. Figure 2B These cassettes collectively encode all scaffold-binding engineered enzymes and the polypeptide scaffolds to which the engineered enzymes can bind.

[0198] The HCA gene cassette encodes scaffold-bound engineered enzymes for scaffolded hexanoyl-CoA biosynthesis, namely ACL, atoB, BHBD, ECH, ECR, and bktB, and encodes a soluble HCS for the production of additional hexanoyl-CoA from hexanoate-supplemented culture medium or cell-free reaction buffer. Fig.13A The GPP gene cassette encodes scaffold-binding engineered enzymes for scaffolded geranyl pyrophosphate (GPP) biosynthesis, namely HMGS, tHMGR, ERG12, ERG8, MVD1, IDI1, and ERG20 WW See also Fig. 13B The CAN gene cassette encodes scaffold-binding engineered enzymes for the biosynthesis of scaffolded OAC, malonyl-CoA, and CBGA, i.e., OS and OAC, ACC, and CBGAS, respectively, and all enzymes for the biosynthesis of soluble (non-scaffolded) CBDA and CBCA, i.e., CBDAS and CBCAS, respectively. See Fig. 13C The SCF gene cassette encodes a polypeptide scaffold for bidirectional scaffolding of cannabinoid biosynthesis and scaffolding of malonyl-CoA biosynthesis, i.e., a cannabinoid metabolic scaffold (CBSCF) and a malonyl-CoA metabolic scaffold (MCASCF), respectively, and additional copies of ACL and atoB to enhance acetyl-CoA biosynthesis from supplemented and / or endogenous citrate and acetoacetyl-CoA biosynthesis from acetyl-CoA, respectively. See Fig.13DThe SOL gene cassette lacks the polypeptide scaffold for bidirectional scaffolded cannabinoid biosynthesis and scaffolded malonyl-CoA biosynthesis (i.e., it is for soluble cannabinoid biosynthesis), but, like the SCF gene cassette, encodes additional copies of ACL and atoB to enhance acetyl-CoA biosynthesis from supplemented and / or endogenous citrate and acetoacetyl-CoA biosynthesis from acetyl-CoA. The amino acid sequences of the ACL and atoB engineered enzymes are shown in Fig.13A .

[0199] Gene cassettes were assembled / synthesized using self-cleaving 2A peptides (P2A) to link multiple codon-optimized (for S. cerevisiae) gene sequences assigned to each cassette. To improve P2A cleavage, a GSG linker (comprising a single serine residue flanked by a single glycine residue) was inserted at the interface between each constituent gene sequence and the P2A linker sequence fused thereto (having the format: gene cassette sequence 1–SG–P2A linker–gene cassette sequence 2–GSG–P2A linker–gene cassette sequence 3–GSG–P2A linker-), etc. Codon-optimized nucleic acid sequences encoding engineered enzymes and scaffolds are described in Figures 14A-14D After assembly, each synthetic gene cassette was inserted into the pCCI-Brick plasmid, resulting in plasmids named pHCA, pGPP, pCAN, pSCF, and pSOL as described in Table 3. The complete gene cassettes inserted into the plasmids are shown in Figures 15A-15E Each of these plasmids was then used to amplify each synthetic gene cassette by standard plasmid preparation. Plasmid DNA encoding each complete synthetic gene cassette was cloned into the SpeI / XhoI cloning site of a polycistronic yeast auxotrophic selection vector, generating vectors named vHCA, vGPP, vCAN, vSCF, and vSOL, as described in Table 3, to allow iterative antibiotic / auxotrophic selection of only those cells that were transformants of one or more of these polycistronic vectors.

[0200] Table 3

[0201]

[0202]

[0203]

[0204] The genes assigned to each synthetic gene cassette and the plasmids and vectors into which each synthetic gene cassette is inserted are listed in Table 3 , and the amino acid sequence encoded by each synthetic gene cassette is provided in Figures 13A-13D The codon-optimized nucleotide sequence fragments comprising each synthetic gene cassette are described in detail in Figures 14A-14D The complete nucleotide sequence of each fully assembled synthetic gene cassette (the complete insert sequence of each plasmid and expression vector) is provided at Figures 15A-15EThe general map of the pCCI-Brick plasmid is shown in Fig.16 In, and a general map of a polycistronic yeast auxotrophic selection vector is shown in Fig.17 middle.

[0205] Example 3 - Engineering of cannabinoid cells

[0206] To engineer a new heterologous pathway for the biosynthesis of cannabinoids from citrate, and to evaluate the impact of bidirectional multi-enzyme scaffolding thereon, competent Saccharomyces cerevisiae cells were sequentially / iteratively transformed with, and selected in an auxotrophic manner for, the expression of vHCA, vGPP, vCAN, and, either vSCF (for scaffolded cannabinoid biosynthesis) or vSOL (for non-scaffolded / soluble cannabinoid biosynthesis) constructs.

[0207] All vector transformations and auxotrophic selection procedures were performed as follows. An aliquot of an overnight S. cerevisiae culture was inoculated into 100 mL of YPD medium (10 g / L yeast nitrogen base, 20 g / L peptone, and 20 g / L D-(+)-glucose) to an OD of 600nm = 0.3 (stationary phase), and grown in an orbital shaker at 30°C and 225 RPM to OD 600nm =1.6. Then by centrifuging 3000xg for 3 minutes and then aspirating culture medium to harvest cells. The harvested cell pellet is subsequently washed 2 times with 50mL cooling nuclease-free water, and washed once with 50mL cooling electroporation buffer (1M sorbitol / 1mMCaCl2). The washed cells are conditioned by incubating in 20Ml 0.1M LiAc / 10mM DTT in an orbital oscillator at 30°C and 225RPM for 30 minutes, harvested, washed once with 50mL electroporation buffer, harvested, and resuspended in 100 μL electroporation buffer. By electroporation at 2.5kV and 25μF, a certain amount of vector containing 3μg target DNA insert (using the vector-insert ratio calculation of each vector) is used to transform the resuspended cells. Then 8 mL of YPD medium containing 1 M sorbitol was added to the electroporated cell suspension, and the resulting suspension was incubated in an orbital shaker at 30° C. and 225 RPM for 1 hour. To isolate the desired transformants by auxotrophic selection, cells were harvested, resuspended in the appropriate yeast nitrogen base (YNB) withdrawal (selection) medium, transferred to baffled culture bottles, and incubated overnight in an orbital shaker at 30° C. and 225 RPM as described later for each iterative transformation step. The transformation and selection protocols were used sequentially for each assigned vector.

[0208] Using the above method, the initial culture cells of electrocompetent Saccharomyces cerevisiae are first transformed with vHCA, which encodes the scaffold-binding engineered enzyme required for the biosynthesis of HCA from citric acid. Cells transformed with vHCA (named yHCA) are selected by resuspending and incubating in tryptophan-deficient YNB medium. The selected yHCA cells (i.e., cells grown in tryptophan-deficient YNB medium) are then transformed with vGPP, which encodes the scaffold-binding engineered enzyme required for the biosynthesis of GPP from citric acid. Cells co-transformed with vHCA and vGPP (called yHCAGPP) are selected by resuspending and incubating in tryptophan- and leucine-deficient YNB medium. The selected yHCAGPP cells (i.e., cells grown in tryptophan and leucine-deficient YNB medium) were then transformed with vCAN, which encodes the scaffold-bound engineered enzymes required for the biosynthesis of malonyl-CoA from citrate, the biosynthesis of olivetol from HCA and malonyl-CoA, the biosynthesis of OVA (oleic acid) from olivetol, and the biosynthesis of CBGA from OVA and GPP, as well as the soluble enzymes required for the biosynthesis of CBDA and CBCA from CBGA. Cells co-transformed with vHCA, vGPP, and vCAN (designated yCB 亲本 ) were selected by resuspending and incubating in YNB medium deficient in tryptophan, leucine, and histidine.

[0209] Then yCB containing cells grown in YNB medium deficient in tryptophan, leucine, and histidine 亲本 The culture was split into two separate cultures. The first split yCB 亲代 Cultures were transformed with vSCF, which encodes CBSCF (cannabinoid metabolism scaffold) and MCASCF (malonyl-CoA metabolism scaffold) as well as additional copies of ACL and atoB. Cells co-transformed with vHCA, vGPP, vCAN, and vSCF (designated yCB) were selected by resuspension and incubation in YNB medium deficient in tryptophan, leucine, histidine, and uracil. SCF ). The second separated yCB 亲本 Cultures were transformed with vSOL, which encodes additional copies of ACL and atoB but lacks CBSCF and MCASCF. Cells co-transformed with vHCA, vGPP, vCAN, and vSOL (designated yCB) were also selected by resuspending and incubating in YNB medium deficient in tryptophan, leucine, histidine, and uracil. SOL ).

[0210] To quantify the improvement in cannabinoid capacity conferred by multi-enzyme scaffolding, triplicate yCBs were cultured in 100 mL YPD medium at 30 °C and 400 RPM in an incubator shaker for 48 h. SOLand yCB SCF Cannabinoid titers were compared between cultures. SOL and yCB SCF The proliferation rate of each culture was initially diluted to OD 600nm = 0.3, and then record OD at 12-hour intervals 600nm Measurement results. Fig.18 Proliferation curves are depicted in Figure 2. Additional sum-of-squares F tests indicated that the proliferation curves of yCBSCF and yCBSOL cultures were not significantly different in any parameter over the 48-hour incubation period, indicating that the scaffold did not affect cell proliferation.

[0211] Total cannabinoid titers, parent (carboxylated) cannabinoids (CBGA, CBDA, and CBCA) titers, derivative (decarboxylated) cannabinoids (CBG, CBD, and CBC) titers, and pro-cannabinoid (OVA) titers were measured. As shown in Figure 19, mixed ANOVA tested strains (F 1,4 =943.8; p<0.0001) and analyte (cannabinoid and procannabinoid) titers (F 10,40 =216.4; p<0.0001), and a significant strain x analyte interaction (F 10,40 =131.4; p<0.0001). Relative to yCBSOL cultures, yCBSCF cultures showed increased titers of total cannabinoids (p<0.0001), OVA precursor (p<0.0001), CBG(A) (p<0.0001), CBD(A) (p<0.0001), CBC(A) (p<0.0001), CBGA (p<0.0001), CBDA (p<0.0001), CBCA (p<0.0001), CBG (p<0.0001), CBD (p<0.01), and CBC (p<0.001).

[0212] Example 4 - Effects of Citrate and Hexanoate Supplementation on Scaffolding and Soluble Cannabinoid Biosynthesis

[0213] To evaluate the effect of supplementation of the medium with citrate and hexanoate precursors, triplicate yCBs were grown in 100 mL YPD medium containing 300 mg / L buffered citrate (pH 6.0) or hexanoate at 30°C and 400 RPM on an orbital shaker for 48 h. SOL and yCB SCF Cannabinoid titers were compared in the cultures. All cultures were initially diluted to OD 600nm = 0.3. The cannabinoid titers of cultures grown in YPD medium, YPD medium supplemented with citrate, and YPD medium supplemented with caproic acid were evaluated and analyzed by ANOVA. Fig. 20As shown, mixed ANOVA tested the differences between strains (F 1,4 =457.5; p<0.0001) and medium supplements (F 2,8 =312.5; p<0.0001), and a significant strain x media supplement interaction (F 2,8 =289.6; p<0.0001). yCBSCF, but not yCBSOL, cultures showed increased total cannabinoid titers when cultured in medium supplemented with 300 mg / L citrate compared to basal medium cultures (p<0.0001). When cultured in medium supplemented with 300 mg / L hexanoate, the total cannabinoid titers of both yCBSCF and yCBSOL cultures were not different relative to basal medium. For all measurements, n=3 biological replicates for yCBSCF and yCBSOL cultures. In addition, relative to yCBSOL cultures, yCBSCF cultures showed increased total cannabinoid titers when cultured in basal medium (p<0.0001, data also reported in Figure 19) and medium supplemented with 300 mg / L citrate (p<0.0001) and hexanoate (p<0.0001).

[0214] To characterize the concentration-response relationship of citrate-supplemented media, triplicate yCBs grown in 100 mL YPD media containing 0, 10, 30, 100, 300, 1000, 3000, and 10,000 mg / L buffered citrate (pH 6.0) at 30°C and 400 RPM orbital shaker for 48 h were compared. SOL and yCB SCF Cannabinoid titers between cultures. All cultures were initially diluted to OD 600nm = 0.3. After quantification, an asymmetric sigmoidal (five-parameter) logistic regression was calculated to fit the concentration-response curves, from which yCB SOL With yCB SCF The maximum cannabinoid titer in the culture (CB 最大 ) estimates and citrate EC for cannabinoid biosynthesis 50 Concentration-response curve, CB 最大 The estimated values ​​and citrate EC50 estimates are as follows Fig.21 Mixed ANOVA detected strains (F 1,8 =69.9; p<0.0001) and parameters (F 1,8 = 66.7; p < 0.0001), and a significant strain x parameter interaction (F 1,8=5.3; p<0.05) for concentration-response parameter estimates (CBmax and citrate EC50). yCBSCF cultures showed significantly increased CBmax (p<0.0001) and citrate EC50 (p<0.001) estimates compared to yCBSOL cultures.

[0215] Other Implementations

[0216] It should be understood that although the invention has been described in conjunction with the specific description, the above description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages and improvements are also within the scope of the following claims.

Claims

1. A host cell capable of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabichromenic acid and cannabicycline acid, the host cell comprising (a) a first exogenous nucleic acid encoding a first polypeptide having CBGA synthase activity and comprising a first heterologous interaction domain, (b) a second exogenous nucleic acid encoding a second polypeptide having oliverate cyclase activity and comprising a second heterologous interaction domain, (c) a third exogenous nucleic acid encoding a third polypeptide having olivetol synthase activity and comprising a third heterologous interaction domain, (d) a fourth exogenous nucleic acid encoding a fourth polypeptide having trans-2-enoyl-CoA reductase activity and comprising a fourth heterologous interaction domain, (e) a fifth exogenous nucleic acid encoding a fifth polypeptide having enoyl-CoA hydratase activity and comprising a fifth heterologous interaction domain, (f) a sixth exogenous nucleic acid encoding a sixth polypeptide having 3-hydroxybutyryl-CoA dehydrogenase activity and comprising a sixth heterologous interaction domain, (g) a seventh exogenous nucleic acid encoding a seventh polypeptide having β-ketothiolase activity and comprising a seventh heterologous interaction domain, (h) an eighth exogenous nucleic acid encoding an eighth polypeptide having acetyl-CoA acetyltransferase activity and comprising an eighth heterologous interaction domain, (i) a ninth exogenous nucleic acid encoding a ninth polypeptide having ATP citrate lyase activity and comprising a ninth heterologous interaction domain, (j) a tenth exogenous nucleic acid encoding a tenth polypeptide having geranyl diphosphate synthase activity and comprising a tenth heterologous interaction domain, (k) an eleventh exogenous nucleic acid encoding an eleventh polypeptide having isopentenyl diphosphate isomerase activity and comprising an eleventh heterologous interaction domain, (l) a twelfth exogenous nucleic acid encoding a twelfth polypeptide having mevalonate diphosphate decarboxylase activity and comprising a twelfth heterologous interaction domain, (m) a thirteenth exogenous nucleic acid encoding a thirteenth polypeptide having phosphomevalonate kinase activity and comprising a thirteenth heterologous interaction domain, (n) a fourteenth exogenous nucleic acid encoding a fourteenth polypeptide having mevalonate kinase activity and comprising a fourteenth heterologous interaction domain, (o) a fifteenth exogenous nucleic acid encoding a fifteenth polypeptide having HMG-CoA reductase activity and comprising a fifteenth heterologous interaction domain, (p) a sixteenth exogenous nucleic acid encoding a sixteenth polypeptide having HMG-CoA synthase activity and comprising a sixteenth heterologous interaction domain, (q) a seventeenth exogenous nucleic acid encoding a polypeptide scaffold comprising a peptide ligand for each of the first to sixteenth heterologous interaction domains, wherein each of the first to sixteenth heterologous interaction domains are different, wherein the peptide ligand for each of the first to sixteenth heterologous interaction domains is different, wherein, in the order extending toward the first direction from the peptide ligand for the first heterologous interaction domain, the polypeptide scaffold comprises (1) the peptide ligand for the second heterologous interaction domain, (2) the peptide ligand for the third heterologous interaction domain, (3) the peptide ligand for the fourth heterologous interaction domain, (4) the peptide ligand for the fifth heterologous interaction domain, (5) the peptide ligand for the sixth heterologous interaction domain, (6) the peptide ligand for the seventh heterologous interaction domain, (7) the peptide ligand for the fourth heterologous interaction domain, (8) the peptide ligand for the fifth heterologous interaction domain, (9) the peptide ligand for the sixth heterologous interaction domain, (10) the peptide ligand for the eighth heterologous interaction domain, and (11) the peptide ligand for the ninth heterologous interaction domain, wherein, in the order of extending from the peptide ligand for the first heterologous interaction domain to the other direction, the polypeptide scaffold comprises (1) the peptide ligand for the tenth heterologous interaction domain, (2) the peptide ligand for the eleventh heterologous interaction domain, (3) the peptide ligand for the twelfth heterologous interaction domain, (4) the peptide ligand for the thirteenth heterologous interaction domain, (5) the peptide ligand for the fourteenth heterologous interaction domain, (6) the peptide ligand for the fifteenth heterologous interaction domain, (7) the peptide ligand for the sixteenth heterologous interaction domain, (8) the peptide ligand for the eighth heterologous interaction domain, and (9) the peptide ligand for the ninth heterologous interaction domain, and wherein the host cell is not a plant cell.

2. The host cell of claim 1, wherein the host cell further comprises (r) an eighteenth exogenous nucleic acid encoding an acetyl-CoA carboxylase and comprising an eighteenth heterologous interaction domain, and (s) a nineteenth exogenous nucleic acid encoding a polypeptide scaffold comprising a peptide ligand for each of the ninth and eighteenth heterologous interaction domains.

3. The host cell of claim 1, wherein the host cell further comprises exogenous nucleic acids encoding cannabidiolic acid synthase and cannabichromenic acid synthase.

4. The host cell of claim 1, wherein the host cell further comprises exogenous cannabidiol acid synthase.

5. The host cell of claim 1, wherein the host cell further comprises exogenous cannabichromenic acid synthase.

6. The host cell of any one of claims 1-5, wherein the host cell is a bacterial or yeast host cell.

7. The host cell of claim 6, wherein the bacterial cell is selected from the group consisting of: Escherichia coli ( Escherichia coli ), Bacillus ( Bacillus ), Brevibacterium ( Brevibacterium ), Streptomyces( Streptomyces ) and Pseudomonas ( Pseudomonas )cell 。 8. The host cell of claim 6, wherein the yeast host cell is selected from the group consisting of Pichia pastoris ( Pichia pastoris ), Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrowia lipolytica ), Kluyveromyces marxianus ( Kluyveromyces marxianus ), and Phaffia calvaria ( Komagataella phaffii )cell.

9. The host cell of any one of claims 1-5, wherein the host cell is an algae.

10. The host cell of claim 9, wherein the algae is Dunaliella salina ( Dunaliella sp.), Chlorella ( Chlorella variabilis ), Euglena mutans ( Euglena mutabilis ), or Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii )cell.

11. The host cell of claim 1 , wherein each of the first to sixteenth exogenous nucleic acids comprises a coding sequence having the following formula: enzyme-linker 1-spacer-linker 2-motif 1-linker 3-motif 2, wherein linkers 1, 2, and 3 are the same or different, motif 1 and motif 2 are the same or different, and wherein motif 1 and motif 2 form the heterologous interaction domain.

12. The host cell of claim 11, wherein the polypeptide scaffold has the following formula: N-terminus - [ligand 1 - linker - ligand 2 - spacer] n - C-terminus, wherein n is the number of heterologous interaction domains, and wherein ligand 1 and ligand 2 bind to motif 1 and motif 2 of the heterologous interaction domain, respectively.

13. The host cell of claim 12, wherein the C-terminus is tagged.

14. The host cell of claim 12, wherein the polypeptide scaffold is tagged with a MYC tag, a FLAG tag or a HA tag.

15. The host cell of claim 11, wherein the linker is a flexible GS-rich sequence flanked by rigid α-helical portions.

16. The host cell of claim 11, wherein the spacer is a cTPR6 spacer.

17. The host cell of claim 1, wherein each of the exogenous nucleic acids comprises a constitutive promoter operably linked to a sequence encoding each of the first to sixteenth polypeptides or the polypeptide scaffold.

18. The host cell of claim 17, wherein the constitutive activity level of the constitutive promoter used to express the polypeptide scaffold is weaker than that of the constitutive promoter used to express each of the first to sixteenth polypeptides.

19. The host cell of claim 1, wherein each of the exogenous nucleic acids comprises an inducible promoter operably linked to a sequence encoding each of the first to sixteenth polypeptides or the polypeptide scaffold.

20. A method of producing one or more cannabinoids selected from the group consisting of cannabigerolic acid, cannabichromenic acid and cannabichromenic acid, the method comprising culturing the host cell of claim 1 under conditions wherein the host cell produces the one or more cannabinoids.

21. The method of claim 20, wherein the host is cultured in a medium supplemented with buffered citrate, glucose, hexanoic acid and / or other carbon sources.

22. The method of claim 20 or claim 21, wherein the host is cultured in a medium supplemented with malonyl-CoA.

23. The method of claim 20, wherein the host is cultured in a medium supplemented with buffered citrate.

24. The method of claim 20, further comprising extracting the one or more cannabinoids from the host cell.

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