Metabolic engineering of E. coli for cannabinoid product biosynthesis

Abstract

Provided herein are methods and compositions for producing terpenoids in host cells. In some cases, the terpenoids are cannabinoids. [Selected Figure] Figure 1

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 554,494, the entire content of which is incorporated herein by reference and is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, the entire content of which is incorporated herein by reference. The ASCII copy was created on October 17, 2018, named NMD - 003_PCT_SL.txt, and is 109,691 bytes in size.

Background Art

[0003] The glandular trichomes of the plant Cannabis sativa accumulate various terpenophenol compounds (cannabinoids). Natural products derived from these plants can directly interact with cannabinoid receptors (CB1 and CB2) found throughout the animal and human body. The CB - 1 receptor is mainly found in the nervous system, and the CB - 2 receptor is mainly found in the immune system or immune - derived cells.

[0004] Cannabinoids and their derivatives have several properties that hold potential for treatment. When a cannabinoid activates or blocks the CB - 1 and / or CB - 2 receptors, downstream signal transduction and metabolic pathways are regulated, which then affects synaptic transmission such as pain and other sensory signal transmission at the periphery, immune response, and inflammation. Therefore, there is interest in the use of natural or synthetic cannabinoids for therapeutic purposes. However, the use of naturally - derived cannabinoids has been uneconomical due to low extraction yields and high separation costs. Similarly, a fully synthetic method for cannabinoid production has not yet been realized due to the complexity of these compounds.

[0005] Heterologous systems for the production of cannabinoids known in the art rely on eukaryotic host organisms to produce and secrete cannabinoid synthase, and use the system in an in vitro enzymatic reaction to produce cannabinoid products. For example, U.S. Patent Nos. 9,587,212; 9,512,391; 9,394,512; 9,526,715; 9,359,625 describe methods and compositions for creating cannabinoids using recombinant Pichia pasloris that secretes THCA synthase or CBDA synthase, and describe bioreactors therein. However, unfortunately, this system requires additional means for producing substrates suitable for the secreted enzyme. Therefore, there has long been a desire to develop a cost-effective heterologous system for producing cannabinoids in vivo, and it has not yet been realized. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0006] This specification describes methods, compositions, and host cells for the production of cannabinoids and terpenoids.

[0007] In some embodiments, the present invention provides an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme. In some embodiments, the expression cassette increases the flux through the MEP pathway in the host cell in which the expression cassette is present. The increase in flux through the MEP pathway can increase the production of isoprenoid precursors suitable for increasing the downstream production of geranyl phosphate (GPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), terpenoids, isoprene, lycopene, cannabinoids (e.g., CBGA), monoterpenes, sesquiterpenes, diterpenes, and / or carotenoids. Therefore, in some embodiments, the expression cassette optionally includes components of the lycopene synthesis pathway (e.g., crtE, crt1, and / or crtB), isoprene synthase, GPP synthase (e.g., ispA, or plant-derived GPP synthase), monoterpene synthase, and / or cannabinoid synthase.

[0008] In some embodiments, the bifunctional ispDF enzyme is the following ispDF enzymes: H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 CFF8240_0409 differs from this one in at least one amino acid. In some cases, the bifunctional ispDF enzyme in question is the following ispDF enzymes: H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 Any one of CFF8240_0409 is identical to 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 90%, or less than 95%. In some embodiments, the bifunctional ispDF enzyme is identical to CJ-ispDF using the default BLAST2.7.0 protein:protein alignment setting to 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, or less than 25%.

[0009] In some embodiments, the promoter functionally linked to the nucleic acid encoding the difunctional ispDF enzyme is an inductive promoter. In some embodiments, the promoter functionally linked to the nucleic acid encoding the difunctional ispDF enzyme is a constitutive promoter. In some embodiments, the difunctional ispDF enzyme includes an amino acid sequence in which at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are identical, or an amino acid sequence in which 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 are identical. In some embodiments, the bifunctional ispDF enzyme contains an amino acid sequence that is identical or identical to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0010] In some embodiments, the nucleic acid encoding the bifunctional ispDF enzyme is codon-optimized. In some embodiments, the expression cassette further comprises nucleic acids encoding one or more, two or more, or all of the enzymes selected from the group consisting of dxs, idi, and ispE. In some embodiments, the expression cassette further comprises nucleic acids encoding dxs and idi, and optionally, GPP synthase (e.g., ispA, or plant-derived GPP synthase), monoterpene synthase, and / or cannabinoid synthase. In some embodiments, the expression cassette further comprises nucleic acids encoding dxs, idi, and ispE, and optionally, GPP synthase (e.g., ispA, or plant-derived GPP synthase), monoterpene synthase, and / or cannabinoid synthase. In some embodiments, the cannabinoid synthase is CBGA synthase, preferably Cannabis CBGA synthase.

[0011] In some embodiments, the cannabinoid synthase is a cleaved cannabinoid synthase selected from the group consisting of THCA synthase, CBDA synthase, and CBCA synthase, and the cleavage is the deletion of all or part of the signal peptide.

[0012] In one embodiment, the present invention provides a plasmid comprising at least one, two, three, or more expression cassettes or fragments thereof, according to any of the expression cassette embodiments, embodiments, examples, or examples described herein. In another embodiment, the present invention provides a plurality of plasmids comprising at least two, three, four, or more expression cassettes or fragments thereof, according to any one of the expression cassette embodiments, embodiments, examples, or examples described herein.

[0013] In some embodiments, the plasmid(s) include an expression cassette containing a nucleic acid encoding isoprene synthase (ispS). In some embodiments, the plasmid(s) include an expression cassette containing a nucleic acid encoding GPP synthase. In some embodiments, the GPP synthase is a eukaryotic GPP synthase. In some embodiments, the GPP synthase is a plant-derived GPP synthase. In some embodiments, the GPP synthase is codon-optimized, for example, expression in the host cell.

[0014] In some embodiments, the plasmid(s) may include nucleic acids encoding one or more components of the lycopene synthesis pathway (e.g., crtE, crtI, and / or crtB), diterpene synthase, sesquiterpene synthase, or monoterpene synthase. In some embodiments, the plasmid(s) may include nucleic acids encoding calen synthase, myrcene synthase, or limonene synthase. In some embodiments, the plasmid(s) may include nucleic acids encoding cannabinoid synthase.

[0015] In some cases, the cannabinoid synthase is selected from the group consisting of CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase. In some embodiments, the cannabinoid synthase is selected from the group consisting of Cannabis CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase. In some embodiments, the cannabinoid synthase is, for example, Cannabis CBGA synthase. In some embodiments, the cannabinoid synthase is a cleaved cannabinoid synthase selected from the group consisting of THCA synthase, CBDA synthase, and CBCA synthase, and the cleavage is the deletion of all or part of the signal peptide.

[0016] In some embodiments, the present invention provides a host cell comprising one or more of the expression cassettes described herein and / or one or more of the plasmids described herein. In some embodiments, one or more, or all, of the expression cassettes of the host cell are incorporated into the genome of the host cell at one or more loci.

[0017] In some embodiments, the present invention provides a host cell comprising: a) an expression cassette comprising a promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme; and b) an expression cassette comprising a promoter functionally linked to a nucleic acid encoding a terpenoid synthase. In some embodiments: i.) one or both of the cannabinoid synthase, the ispDF, or the promoter are heterogeneous to the host cell; ii.) the nucleic acid encoding the bifunctional ispDF enzyme is heterogeneous to the functionally linked promoter; and / or iii.) the nucleic acid encoding the cannabinoid synthase is heterogeneous to the functionally linked promoter.

[0018] In some embodiments, the terpenoid synthase is isoprene synthase. In some embodiments, the terpenoid synthase is a component of the lycopene synthase pathway (e.g., crtI, crtE, or crtB). In some embodiments, a host cell containing nucleic acids encoding components of the lycopene synthesis pathway contains one or more nucleic acids encoding crtI, crtE, and crtB, where crtI, crtE, and crtB are located on the same or different expression cassettes.

[0019] In some embodiments, the terpenoid synthase is a cannabinoid synthase. In some embodiments, the cannabinoid synthase is selected from the group consisting of CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase. In some embodiments, the cannabinoid synthase is selected from the group consisting of Cannabis CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase. In some embodiments, the cannabinoid synthase is Cannabis sativa cannabinoid synthase. In some embodiments, the host cell contains a nucleic acid encoding CBGA synthase and a nucleic acid encoding another cannabinoid synthase selected from the group consisting of THCA synthase, CBDA synthase, and CBCA synthase, or a combination of one or more nucleic acids encoding two or all of them.

[0020] In some embodiments, the host cell contains an expression cassette comprising a promoter functionally linked to a nucleic acid encoding a cannabinoid synthase, where the cannabinoid synthase is CBGA synthase. In some cases, the host cell containing the CBGA synthase expression cassette further comprises nucleic acids encoding THCA synthase, CBDA synthase, and / or CBCA synthase, each cannabinoid synthase being functionally linked independently to a promoter in the same or different expression cassettes.

[0021] In some embodiments, the cannabinoid synthase, or at least one encoded cannabinoid synthase, is a cleaved cannabinoid synthase selected from the group consisting of THCA synthase, CBDA synthase, and CBCA synthase, wherein the cleavage is the deletion of all or part of the signal peptide. In some embodiments, the cannabinoid synthase includes the deletion of all or part of a transmembrane or membrane-related region, and the cannabinoid synthase is not membrane-related.

[0022] In some embodiments, the host cell includes an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding GPP synthase. In some embodiments, the expression cassette in a) or b) further comprises a nucleic acid encoding GPP synthase. In some embodiments, the host cell does not include a heterologous nucleic acid encoding ispC, ispE, ispG, ispH, or a combination thereof, or all of them. In some embodiments, the host cell does not include a heterologous nucleic acid encoding ispC, ispG, ispH, or a combination thereof, or all of them.

[0023] In some embodiments, the host cell is a prokaryote, such as a prokaryote of the genus Escherichia, Panteoa, Bacillus, Corynebacterium, or Lactococcus. In some embodiments, the cell is Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, or L. lactis.

[0024] In some embodiments, the expression cassettes a) and / or b) are incorporated into the genome of the host cell. In some embodiments, the expression cassette a) is incorporated into the genome of the host cell, but the expression cassette b) is not incorporated, or is incorporated into a different locus of the genome of the host cell. In some embodiments, the expression cassette b) is incorporated into the genome of the host cell, but the expression cassette a) is not incorporated, or is incorporated into a different locus of the genome of the host cell. In some embodiments, the expression cassette a) and the expression cassette b) are incorporated into the genome of the host cell at the same or different loci.

[0025] In some embodiments, the expression cassettes a) and / or b) are located on a plasmid within the host cell. In some embodiments, the expression cassette a) and the expression cassette b) are located on a plasmid within the host cell. In some embodiments, the host cell contains a plasmid containing the expression cassettes a) and / or b). In some embodiments, the host cell contains a plasmid containing the expression cassette a) and / or b). In some embodiments, the expression cassette a) and the expression cassette b) are located on the same plasmid. In some embodiments, the expression cassette a) is located on a different plasmid than the expression cassette b).

[0026] In some embodiments, the expression cassette, which includes a promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme, also includes the same promoter functionally linked to a nucleic acid encoding a cannabinoid synthase. In some cases, the cannabinoid synthase is CBGA synthase. In some embodiments, the host cell includes a nucleic acid encoding a cannabinoid synthase (e.g., CBGA synthase) functionally linked to a constitutive promoter. In some embodiments, the host cell includes a nucleic acid encoding a cannabinoid synthase (e.g., CBGA synthase) functionally linked to an inductive promoter.

[0027] In some embodiments, the promoter functionally linked to the nucleic acid encoding the difunctional ispDF enzyme is a constitutive promoter. In some embodiments, the promoter functionally linked to the nucleic acid encoding the difunctional ispDF enzyme is an inductive promoter. In some embodiments, if the host cell contains two or more expression cassettes containing different cannabinoid synthases, each expression cassette contains a constitutive promoter functionally linked to a cannabinoid synthase, each expression cassette contains an inductive promoter functionally linked to a cannabinoid synthase, or one or more expression cassettes contain a constitutive promoter functionally linked to a cannabinoid synthase, and one or more expression cassettes contain an inductive promoter functionally linked to a cannabinoid synthase.

[0028] In some embodiments, if the host cell contains two or more expression cassettes containing different cannabinoid synthases, each expression cassette contains an inductive promoter functionally linked to the cannabinoid synthase. In some embodiments, if the host cell contains two or more expression cassettes containing different cannabinoid synthases, at least one expression cassette contains an inductive promoter functionally linked to the cannabinoid synthase. In some embodiments, if the host cell contains two or more expression cassettes containing different cannabinoid synthases, at least one expression cassette contains a constitutive promoter functionally linked to the cannabinoid synthase.

[0029] In some embodiments, the difunctional ispDF enzyme contains an amino acid sequence that is identical to, or identical to, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or to 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the difunctional ispDF enzyme contains an amino acid sequence that is identical to, or identical to, SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of, SEQ ID NO: 3.

[0030] In some embodiments, the bifunctional ispDF enzyme is the following ispDF enzymes: H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 CFF8240_0409 differs from this one in at least one amino acid. In some cases, the bifunctional ispDF enzyme in question is the following ispDF enzymes: H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 One of the CFF8240_0409 values ​​is identical to a value less than 50%, 80%, 90%, or 95%.

[0031] In some embodiments, the host cell includes, or further includes, an expression cassette comprising a promoter functionally linked to a nucleic acid encoding one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. In some cases, the expression cassette containing the difunctional ispDF enzyme further includes a nucleic acid encoding one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. In some cases, the expression cassette containing the difunctional ispDF enzyme further includes nucleic acids encoding dxs and idi. In some cases, the expression cassette containing the difunctional ispDF enzyme further includes a nucleic acid encoding ispE. In some cases, the expression cassette containing the difunctional ispDF enzyme further includes dxs, idi, and ispE. In some cases, the expression cassette containing the difunctional ispDF enzyme does not contain nucleic acid sequences encoding one or more of ispC, ispE, ispF, ispG, or ispH.

[0032] In some cases, the host cells exhibit high levels of expression of one or more MEP pathway genes compared to control cells that do not contain the expression cassette containing the difunctional ispDF enzyme. In some cases, the host cells exhibit high levels of expression of dxs and idi compared to control cells that do not contain the expression cassette containing the difunctional ispDF enzyme. In some cases, the host cells show increased flux through the MEP pathway compared to control cells that do not contain one or more expression cassettes and / or at least one of one or more plasmids described herein.

[0033] In some embodiments, the host cell includes an expression cassette comprising a promoter functionally linked to a nucleic acid encoding GPP synthase. In some cases, the expression cassette in a) further comprises a nucleic acid encoding GPP synthase. In some cases, the expression cassette in b) further comprises a nucleic acid encoding GPP synthase. In some cases, the expression cassette in a) and the expression cassette in b) are different expression cassettes. In some cases, the expression cassette in a) and the expression cassette in b) are the same expression cassette.

[0034] In some embodiments, the host cells further contain olivetolic acid (OA). In some cases, the olivetolic acid is exogenous to the host cells. For example, the OA can be exogenously applied to the culture medium in which the host cells are cultured.

[0035] In some embodiments, the host cell includes an expression cassette comprising a promoter functionally linked to a nucleic acid encoding one or more glycosylation pathway genes: a) the glycosylation pathway gene is heterogeneous to the host cell; b) the promoter is heterogeneous to the host cell; c) the promoter is heterogeneous to one or more of the glycosylation pathway genes; or d) the expression cassette is heterogeneous to the host cell. In some embodiments, the host cell includes deletions in one, two, three, four, five, six, seven, eight, or all of the genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA.

[0036] In a second aspect, the present invention provides a host cell comprising an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme, wherein the bifunctional ispDF enzyme comprises amino acids that are identical to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or amino acids that are identical to 25, 50, 75, 100, 125, 150, 175, 200, or 225 consecutive amino acids of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the bifunctional ispDF enzyme contains an amino acid sequence that is identical or identical to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0037] In some embodiments, the host cell further comprises an expression cassette containing a promoter functionally linked to a nucleic acid encoding one or more MEP pathway enzymes selected from the group consisting of dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi, wherein: a) the promoter is heterogeneous to one or more MEP pathway enzymes; or b) the promoter or one or more MEP pathway enzymes are heterogeneous to the host cell. In some embodiments, the host cell further comprises an expression cassette containing a promoter functionally linked to nucleic acids encoding dxs and idi. In some embodiments, the host cell further comprises an expression cassette containing a promoter functionally linked to nucleic acids encoding dxs, idi, and ispE.

[0038] In some embodiments, the host cell further comprises an expression cassette containing a promoter functionally linked to a nucleic acid encoding the ispS enzyme. In some embodiments, the host cell further comprises an expression cassette containing a promoter functionally linked to a nucleic acid encoding the GPP synthase enzyme. In some embodiments, the host cell contains deletions in one, two, three, four, five, six, seven, eight, or all of the genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA. In some embodiments, the host cell further comprises a cannabinoid synthase.

[0039] In some embodiments, the host cell is a prokaryote, such as a prokaryote of the genus Escherichia, Panteoa, Bacillus, Corynebacterium, or Lactococcus. In some embodiments, the cell is Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, or L. lactis.

[0040] In another embodiment, the present invention provides a method for obtaining a target metabolite (e.g., a terpenoid or cannabinoid), the method comprising culturing host cells in a suitable culture medium under conditions suitable for inducing expression in one or more host cell expression cassettes, according to any one of the embodiments, embodiments, examples, or examples described herein, and then collecting the cultured cells or used medium to obtain the target metabolite. In some embodiments, the method comprises culturing host cells according to any one of the embodiments, embodiments, examples, or examples described herein, and the metabolite is a cannabinoid. In some embodiments, the cannabinoid is THCA, CBDA, CBCA, CBN, THC, CBD, or CBC, or a mixture of one or more thereof.

[0041] In some embodiments, the method comprises culturing host cells according to any one of the embodiments, examples, cases, or examples described herein, wherein the metabolite is a terpenoid or isoprene. In some embodiments, the method comprises harvesting and lysing the cultured cells to produce a cell lysate. In some embodiments, the method comprises purifying the target metabolite from the cell lysate to produce a purified target metabolite. In some embodiments, the method comprises purifying the target metabolite from the used culture medium to produce a purified target metabolite.

[0042] In some embodiments, the purified target metabolite is a cannabinoid, and the method includes formulating the cannabinoid into a pharmaceutical composition. In some embodiments, the purified target metabolite is a cannabinoid, and the method includes forming a salt, prodrug, or solvate of the purified cannabinoid.

[0043] References to literature All publications, patents, and patent applications referenced herein are incorporated as part of this specification, in the same manner as individually and specifically indicating the use of the content of each publication, patent, and patent application. [Brief explanation of the drawing]

[0044] [Figure 1] This figure shows a schematic of cannabinoid synthesis in Cannabis Sativa. [Figure 2]This diagram illustrates the mevalonate-independent (MEP) pathway for the biosynthesis of isoprenoid precursors in E. coli. The substrates and products shown at the top are as follows: G3P (glyceraldehyde 3-phosphate), DOXP (1-deoxy-D-xylulose 5-phosphate), MEP (2-C-methylerythritol 4-phosphate), CDP-ME (4-diphosphocytidyl-2-C-methylerythritol), CDP-MEP (4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate), MECPP (2-C-methyl-D-erythritol 2,4-cyclodiphosphate), HMBPP ((E)-4-hydroxy-3-methyl-buto-2-enylpyrophosphate), IPP (isopentenyl diphosphate), DMAPP (dimethylallyl diphosphate), GPP (geranyl pyrophosphate). The corresponding enzymes listed below are as follows: dxs (DOXP synthase), ispC (DOXP reductase), ispD (2-C-methyl-D-erythritol 4-phosphate cytidyltransferase), ispE (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), ispF (2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase), ispG (HMB-PP synthase), ispH (HMB-PP reductase), and idi (isopentenyl / dimethylallyl diphosphate isomerase). [Figure 3] Figure 3A-B shows two different expression cassettes for overexpression of the MEP pathway in host cells. [Figure 4] This shows an SDS-PAGE gel illustrating heterologous expression of dxs, ispD, idi, and ispF in host cells. [Figure 5] This shows an expression cassette for heterologous expression of cannabigerol acid synthase (CBGAS) in host cells. [Figure 6]This SDS-PAGE gel shows the expression and purification of polyhistidine (6x-His)-tagged aromatic prenyltransferase ("6x-His," disclosed as SEQ ID NO: 43) in E. coli host cells. Clarified cell lysates were loaded without purification. a: Uninducible cell lysates. b-f: Cell lysates induced with 1 mM IPTG. c-f: Flow through the nickel-NTA column in the fractions. [Figure 7] This is a chromatogram showing the in vitro production of cannabigerol acid (CBGA). Line a is the chromatogram of a 0.0625 μg / mL CBGA standard; line b is the reaction mixture containing CBGAS-induced cell lysate, olivetolic acid (OA), and GPP; and line c is the same reaction mixture without GPP. [Figure 8] This chromatogram shows the in vivo production of cannabigerol acid (CBGA) in E. coli. Line a is the chromatogram of a 0.5 μg / mL CBGA standard; line b is the used culture medium in which E. coli expressing CBGAS was grown, with OA and GPP added; and line c is the same reaction mixture without OA. [Figure 9] This figure illustrates an expression cassette for the production of Δ(9)-tetrahydrocannabinolate synthase (THCAS) in E. coli. [Figure 10] This figure illustrates host cells containing a THCAS expression cassette and a chaperone co-expression cassette. [Figure 11] This figure illustrates host cells containing a THCAS expression cassette and an expression cassette for a heterologous glycosylation system. [Figure 12] This figure illustrates a low-copy-number CBGAS expression cassette. [Figure 13] This figure illustrates host cells containing mevalonate-independent pathway expression cassettes, as well as GPP synthase (GPPS) and CBGAS expression cassettes. [Figure 14] The table below shows the optimized inducer concentrations of the expression constructs shown for E. coli. [Figure 15]The OD measurements of E. coli under different inducer concentrations are shown. Strains B-D contain the GPPS CBGAS expression cassette pBAD33_GPPS_CBGAS. As illustrated in Figure 13, strains F-J contain both the pBAD33_GPPS_CBGAS expression cassette and the MEP pathway expression cassette pTRC_RDE. [Figure 16] The results of SDS-PAGE analysis of cell lysates after expression of pBAD33_GPPS_CGGAS, or co-expression of pBAD33_GPPS_CBGAS and pTRC_RDE, are shown. [Figure 17] The following shows the different protein concentrations in cell lysates from inducible expression cultures having the indicated inducible substance concentrations. Strains A-D contain plasmid pBAD33_GPPS_CBGAS. Strains E-J contain plasmid pBAD33_GPPS_CBGAS and pTRCRDE. [Figure 18] The results of SDS-PAGE analysis of cell lysates after ispDF expression in a strain containing the pTRC_RDE* expression construct are shown. [Figure 19] The protein sequence alignments of the bifunctional ispDF enzymes identified from the metagenomics screening assay (ispDF1) (SEQ ID NO: 1) using native ispD, ispF (ispD-ispF) (SEQ ID NO: 42), and C. jejuni ispDF (CJ-ispDF) (SEQ ID NO: 41) are illustrated. [Figure 20] Expression constructs of pTRC_RDEE and pTRC_RDE*E are shown. pTRC_RDE*E contains the bifunctional ispDF enzyme instead of the ispD and ispF enzymes. [Figure 21] This document outlines the constructs and strains tested for increased flux through the MEP pathway. [Figure 22] The constructs and data for isoprene production are shown. Construct SA04 is identical to construct SA03 except that the nucleic acid encoding ispDF is codon-optimized. [Figure 23]This shows lycopene production in four different strains of E. coli: SA01: pAC-LYC; SA02: pAC-LYC and pTRC-RDE; SA03: pAC-LYC and pTRC-RDEE; SA04: pAC-LYC and pTRC-RDE*; and SA05: pAC-LYC and pTRC-RDE*E. [Figure 24] Let's take lycopene production as an example. [Figure 25] This document shows the structures and data for the production of monoterpene carenes. [Figure 26] The peptide sequences of the bifunctional enzymes ispDF1, ispDF2, and ispDF3 are shown. [Modes for carrying out the invention]

[0045] This specification describes metabolic engineering strategies for modifying the mevalonate-independent (MEP) pathway to increase terpenoid production. The MEP pathway, or terpenoid pathway, produces geranyl pyrophosphate (GPP), a product usable in various downstream processes for the production of commercially valuable terpenoids and other compounds.

[0046] Furthermore, this specification also describes the bifunctional enzyme ispDF, which can catalyze both reactions induced by native E. coli ispD and ispF. This bifunctional enzyme can be used in a variety of in vitro or in vivo isoprene, terpenoid, or cannabinoid production systems. In some embodiments, the metabolic engineering strategies described herein can be used to increase the production of isoprene, GPP, or downstream terpenoids in heterologous host cells, with or without ispDF.

[0047] GPP is a substrate for cannabigerol acid synthase (CBGAS), an aromatic prenyltransferase enzyme, which is the first enzyme in the cannabinoid pathway (Figure 1). CBGAS uses the substrate GPP and olivetolic acid (OA) to produce cannabigerol acid (CBGA). In some cases, CBGA is a substrate for further in vitro or in vivo enzyme-catalyzed reactions, such as those catalyzed by THCA synthase (THCAS), which then Δ 9 - Tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA) can be used as a substrate for the production of cannabidiolic acid (CBDA) via a reaction catalyzed by CBDA synthase (CBDAS). Further pathways for cannabinoid production are described in, but are not limited to, Thakur et al., Life Sciences, 78(2005)454-466. The literature of Thakur et al., as an integral part of this specification, is incorporated herein by reference in its entirety, including enzymes, products, enzyme substrates, pathways, and some thereof, as well as the synthetic schemes.

[0048] In some embodiments, the products of downstream enzyme-catalyzed reactions involving substrates such as CBGA, THCA, CBDA, CBCA, THCVA, CBCVA, CBDVA, and combinations thereof, can be decarboxylated in vitro or in vivo using chemical, enzymatic, or thermal means to produce various cannabinoids, for example, as shown in Figure 1.

[0049] definition In this specification, the following abbreviations are used: "G3P" means glyceraldehyde 3-phosphate; "DOXP" means 1-deoxy-D-xylulose 5-phosphate; "MEP" means 2-C-methylerythritol 4-phosphate; "CDP-ME" means 4-diphosphocytidyl-2-C-methylerythritol; "CDP-MEP" means 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate; "MECPP" means 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; "HMBPP" means (E)-4-hydroxy-3-methyl-buto-2-enylpyrophosphate; "IPP" means isopentenyl diphosphate; "DMAPP" means dimethylallyl diphosphate; "GPP" means geranyl pyrophosphate.

[0050] The "DXP pathway" and "MEP pathway" refer to the mevalonate-independent pathway, also known as the mevalonate-independent pathway. The genes of the MEP pathway are dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi. With respect to the DXP or MEP pathway genes, gene products, or the nucleic acids that encode them, such genes can be, for example, native genes in host cells where heterologous nucleic acids are present, codon-optimized versions thereof, genes derived from different organisms (e.g., codon-optimized) or orthologues thereof.

[0051] "dxs" refers to DOXP synthase; "ispC" refers to DOXP reductase; "ispD" refers to 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; "ispE" refers to 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; "ispF" refers to 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate synthase; "ispG" refers to HMB-PP synthase; "ispH" refers to HMB-PP reductase; "idi" refers to isopentenyl / dimethylallyl pyrophosphate isomerase; "ispA" refers to farnesyl pyrophosphate synthase, also known as "GPP synthase," which can convert DMAPP+IPP to GPP and GPP+IPP to farnesyl pyrophosphate.

[0052] The term "ispDF" refers to a bifunctional single-chain enzyme that has two distinct active sites and exhibits both ispD activity (EC 2.7.7.60) and ispF activity (EC 4.6.1.12). Generally, ispDF is a naturally occurring bifunctional enzyme or a derivative of a naturally occurring bifunctional enzyme that has one or more modifications, such as the deletion, insertion, or substitution of one or more amino acids. In some cases, the gene is of plant origin or is a Cannabis gene. In some cases, the gene is an E. coli gene or its orthologue.

[0053] "OA" refers to olivetolic acid; "CBGA" refers to cannabigerolic acid; "CBNA" refers to cannabinerolic acid; "Cannabinol" or "CBN" refers to 6,6,9-trimethyl-3-pentylbenzo[c]chromen-1-ol; "CBGVA" refers to cannabigerivalic acid; "THCA" refers to tetrahydrocannabinolic acid, Δ 9Isomers included: "CBDV" refers to cannabidivarin; "CBC" refers to cannabichromene; "CBCA" refers to cannabichromic acid; "CBCV" refers to cannabiclomevalin; "CBG" refers to cannabigerol; "CBGB" refers to cannabigerovarin; "CBE" refers to cannabiersolin; "CBL" refers to cannabicyclol; "CBV" refers to cannabivarin; "CBT" refers to cannabitriol; "THCV" refers to tetrahydrocannabivarin (THCV); "THC" refers to tetrahydrocannabinol, and "Δ 9 -THC is Δ 9 -This refers to tetrahydrocannabinol; "CBDA" refers to cannabidiolic acid.

[0054] As used herein, "increase in flux through the MEP pathway" refers to an increase in the production of IPP and / or DMAPP. Typically, the production of IPP and / or DMAPP is determined indirectly by detecting the products formed by the action of a reporter enzyme that utilizes IPP and / or DMAPP as reactants. For example, by using isoprene synthase (ispS) as the reporter, an increase in flux through the MEP pathway can be detected as an increase in isoprene production. Alternatively, an increase in flux through the MEP pathway can be detected as an increase in GPP production by using GPP synthase as the reporter. In some cases, a reporter enzyme is used to detect GPP production. For example, by using a GPP synthase enzyme and a lycopene synthase reporter enzyme, an increase in lycopene production can be detected, which in turn can detect an increase in flux through the MEP pathway, thereby detecting an increase in GPP production. As another example, an increase in GPP production can be detected by using a GPP synthase enzyme and a monoterpene (e.g., limonene, carene, myrcene) synthase reporter enzyme to detect an increase in monoterpenes (e.g., limonene, carene, myrcene), thereby detecting an increase in flux through the MEP pathway. As yet another example, an increase in GPP production can be detected by using a GPP synthase enzyme and a cannabinoid (e.g., CBGA) synthase reporter enzyme to detect an increase in cannabinoid (e.g., CBGA) production, thereby detecting an increase in flux through the MEP pathway. Generally, this is an increase of at least 10% compared to a control strain lacking one or more heterologous expression cassettes in the test strain. In some cases, the increase is at least twofold compared to a control strain lacking one or more heterologous expression cassettes in the test strain.

[0055] As used herein, the terms “cannabidiol,” “CBD,” or “cannabidiol” refer to one or more of the following compounds unless a specific other stereoisomer is identified, and the compound “Δ 2- It contains cannabidiol. These compounds are: (1) Δ 5 - Cannabidiol (2-(6-Isopropenyl-3-methyl-5-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol); (2) Δ 4 - Cannabidiol (2-(6-Isopropenyl-3-methyl-4-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol); (3) Δ 3 - Cannabidiol (2-(6-Isopropenyl-3-methyl-3-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol); (4) Δ 3,7 - Cannabidiol (2-(6-Isopropenyl-3-methylenecyclohex-1-yl)-5-pentyl-1,3-benzenediol); (5) Δ 2 - Cannabidiol (2-(6-Isopropenyl-3-methyl-2-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol); (6) Δ 1 - Cannabidiol (2-(6-Isopropenyl-3-methyl-1-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol); and, (7) Δ 6 - Cannabidiol (2-(6-Isopropenyl-3-methyl-6-cyclohexen-1-yl)-5-pentyl-1,3-benzenediol). [[ID=]16]

[0056] As described below, these compounds have one or more chiral centers and two or more stereoisomers: (1)(1) Δ 5 - Cannabidiol has two chiral centers and four stereoisomers; (2) Δ 4 - Cannabidiol has three chiral centers and eight stereoisomers; (3) Δ 3 - Cannabidiol has two chiral centers and four stereoisomers; (4) Δ 3,7 - Cannabidiol has two chiral centers and four stereoisomers; (5) Δ 2 - Cannabidiol has two chiral centers and four stereoisomers; (6) Δ 1-Cannabidiol has two chiral centers and four stereoisomers; and (7)Δ 6 -Cannabidiol has one chiral center and two stereoisomers. In a preferred embodiment, cannabidiol is specifically Δ 2 -Cannabidiol. Unless otherwise specified, the above references to "cannabidiol," "CBD," or "cannabidiol," or any of the specific cannabidiol compounds (1) to (7), include all possible stereoisomers of all compounds incorporated by reference. In some embodiments, "Δ 2 -Cannabidiol is produced partially or entirely in heterogeneous systems, Δ 2 -It can be a mixture of stereoisomers of cannabidiol.

[0057] The terms "isoprenoid" or "terpenoid" refer to any compound containing one or more five-carbon isoprene components, including linear and cyclic terpenoids. The term "terpene" as used herein is interchangeable with "terpenoid" and "isoprenoid." Compounds obtained when terpenes are chemically modified by oxidation or rearrangement of carbon chains are generally referred to as terpenoids, also known as isoprenoids.

[0058] Terpenoids can be named using groups of 5 and 10 carbon atoms as references, depending on the number of carbon atoms present. For example, hemiterpenoids (C5) have one isoprene unit (half a terpenoid); monoterpenoids (C10) have two isoprene units (one terpenoid); sesquiterpenoids (C15) have three isoprene units (1.5 terpenoids); and diterpenoids (C20) have four isoprene units (or two terpenoids). Monoterpenoids are typically produced spontaneously from the C10 terpenoid precursor geranyl pyrophosphate (GPP). Similarly, "cyclic monoterpenes" refer to cyclic or aromatic terpenoids (i.e., those containing a ring structure). They are made from two isoprene components, usually GPP. Linear monoterpenes include, but are not limited to, geraniol, linalool, ocimene, and myrcene. Cyclic monoterpenes (monocyclic, bicyclic, and tricyclic) include, but are not limited to, limonene, pinene, carene, terpineol, terpinolene, phellandrene, thujene, tricyclene, borneol, sabinene, and camphene.

[0059] A "terpenoid synthase" refers to an enzyme that can catalyze the conversion of one terpenoid or terpenoid precursor to another terpenoid or terpenoid precursor. For example, GPP synthase is an enzyme that catalyzes the formation of GPP from terpenoid precursors IPP and DMAPP. Similarly, FPP synthase is an enzyme that catalyzes the production of FPP from GPP and IPP. A terpene synthase is an enzyme that catalyzes the conversion of prenyl diphosphate (such as GPP) to an isoprenoid or an isoprenoid precursor. This term includes both linear and cyclic terpene synthases.

[0060] A "cyclic terpenoid synthase" refers to an enzyme that can catalyze a reaction modifying a terpenoid or terpenoid precursor to provide a cyclic structure. For example, a cyclic monoterpenoid synthase is an enzyme that can produce cyclic or aromatic (ring-containing) monoterpenoid compounds using a linear monoterpene as a substrate. One example is sabinene synthase, which can catalyze the formation of cyclic monoterpene sabinene from the linear monoterpene precursor GPP. The term "terpene synthase" as used herein is interchangeable with "terpenoid synthase."

[0061] Prenyltransferase, or isoprenyltransferase enzyme, also known as prenyl or isoprenyl synthase, is an enzyme that can catalyze the production of pyrophosphate precursors of terpenoid or isoprenoid compounds. An exemplary prenyltransferase, or isoprenyltransferase enzyme, ispA, which can catalyze the formation of geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) in the presence of a suitable substrate.

[0062] A "cannabinoid synthase" refers to an enzyme that catalyzes one or more of the following activities: cyclization of CBGA to THCA, CBDA, or CBCA; cyclization of CBGVA to THCVA, CBCVA, or CBDVA; prenylation of olivetolic acid to form CBGA; and combinations thereof. Examples of cannabinoid synthases include, but are not limited to, those commonly found in plants of the genus Cannabis, such as the THCA synthase, CBDA synthase, and CBCA synthase from Cannabis sativa.

[0063] Examples of isoprenoids, terpenoids, cannabinoids, and polypeptides and nucleic acids of the MEP pathway are listed in the KEGG database. This KEGG database stores amino acids and nucleic acid sequences of numerous examples of isoprenoids, terpenoids, cannabinoids, and polypeptides and nucleic acids of the MEP pathway (see, for example, the global web "genome.jp / kegg / pathway / map / map00100.html", which is incorporated in its entirety as part of this specification, and the sequences therein, particularly for amino acids and nucleic acid sequences of isoprenoids, terpenoids, cannabinoids, and polypeptides and nucleic acids of the MEP pathway). Polypeptides and nucleic acids that encode signal peptides described herein can be understood as further describing cleavage forms with or without the signal peptide.

[0064] As used herein, the term "heterogeneous" refers to any two components that do not exist together in nature. For example, a nucleic acid encoding a gene heterogeneous to a functionally linked promoter is a nucleic acid that, in a particular genome, has unregulated expression in its natural state (e.g., in a non-genetically modified cell) by a functionally linked promoter. As stated herein, all genes functionally linked to promoters that do not exist in nature are considered "heterogeneous." Similarly, a gene that is "heterogeneous" to a host cell is a gene not found in the non-genetically modified cells of a particular organism, or a gene found in a different genome, or at a non-genomic (e.g., plasmid) location, or a gene functionally linked to a different promoter within a non-genetically modified cell. In addition, a promoter that is "heterogeneous" to a host cell is a promoter not found in the non-genetically modified cells of a particular organism, or a promoter found in a different genome, or at a non-genomic (e.g., plasmid) location, or a promoter functionally linked to a different nucleic acid within a non-genetically modified cell.

[0065] As used herein, "expression cassette" means a polynucleotide sequence containing a promoter polynucleotide functionally linked to at least one target gene, wherein the promoter is heterogeneous to at least one functionally linked gene, the promoter is heterogeneous to the host cell in which it resides, or at least one functionally linked gene, or a combination thereof, is heterogeneous to the host cell.

[0066] "Salt" refers to an acid salt or base salt of a compound used in the method of the present invention. Examples of pharmaceutically acceptable salts include salts of mineral acids (such as hydrochloric acid, hydrobromic acid, and phosphoric acid), salts of organic acids (such as acetic acid, propionic acid, glutamic acid, and citrate), and salts of quaternary ammonium compounds (such as methyl iodide and ethyl iodide). Pharmaceutically acceptable salts are understood to be non-toxic. Further information regarding suitable pharmaceutically acceptable salts can be obtained from Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference as part of this specification.

[0067] As used herein, the term “solvate” means a compound formed by solvation (a combination of a solvent molecule and a solute molecule or ion), or an aggregate having one or more solvent molecules consisting of solute ions or molecules, i.e., the compounds of the present invention. When water is the solvent, the corresponding solvate is a “hydrate.” Examples of hydrates include, but are not limited to, hemihydrates, monohydrates, dihydrates, trihydrates, hexahydrates, and other water-containing forms. Those skilled in the art will understand that pharmaceutically acceptable salts and / or prodrugs of compounds may also exist in the form of solvates. Such solvates are generally formed via hydration, which is part of the preparation of the compound, or by the spontaneous absorption of water by the anhydrous compounds of the present invention. Generally, all physical forms are intended to be within the scope of the present invention.

[0068] Therefore, if a therapeutic activator produced by the method of the present invention, or a therapeutic activator included in the composition of the present invention, such as cannabinoids or terpenoids, etc., is sufficiently acidic, sufficiently basic, or has both sufficiently acidic and sufficiently basic functional groups, these groups(s) can spontaneously react with a number of inorganic or organic bases, and either inorganic or organic acids, to form pharmaceutically acceptable salts. Exemplary pharmaceutically acceptable salts include pharmacologically active compounds and sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monophosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, isobutyrates, caproates, heptanoates, propionates, oxalates, malons, succinates, suberates, sebacinates, fumarates, maleates, butin-1,4-dioate, hexin-1,6-dioate These include salts prepared by reaction with mineral acids such as benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, β-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, and mandelates, or with organic or inorganic acids. If the pharmacologically active compound has one or more basic functional groups, a pharmaceutically acceptable salt of the desired compound can be prepared by treatment of a free base with any suitable method available in the art, for example, with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, or phosphoric acid, or with an organic acid such as pyranosidylic acid, citric acid, or alpha-hydroxy acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, glucuronic acid, or galacturonic acid, or alpha-hydroxy acid such as tartaric acid, aspartic acid, or amino acid such as glutamic acid, benzoic acid, or aromatic acid such as cinnamic acid, or sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid.If a pharmacologically active compound has one or more acidic functional groups, a pharmaceutically acceptable salt of the desired compound can be prepared by any suitable method available in the art, for example, by treatment with a free acid using an inorganic or organic base such as an amine (primary, secondary, or tertiary), alkali metal hydroxide, or alkaline earth metal hydroxide. Examples of suitable salts include amino acids such as glycine and arginine, ammonia, primary, secondary, and tertiary amines, as well as cyclic amines such as piperidine, morpholine, and piperazine, and organic salts derived from inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0069] As used herein, “composition” is intended to include products containing a specific amount of a specific component, as well as any product obtained from a specific combination of a specific component in a specific amount. “Pharmacovigilant” means that the carrier, diluent, or excipient must be compatible with the other components of the formulation and not harmful to its recipient.

[0070] A "pharmaceutically acceptable excipient" refers to a substance that assists in the administration of an activator to a target and its absorption by the target. Useful pharmaceutical excipients in this invention include, but are not limited to, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, and colorants. Those skilled in the art will recognize that other pharmaceutical excipients may also be useful in this invention.

[0071] In some cases, protecting groups can be included in the compounds used in the methods according to the present invention or in the compositions of the present invention. Such protecting groups prevent subsequent hydrolysis or other reactions that occur in vivo and can decompose the compounds. Protectable groups include alcohols, amines, carbonyls, carboxylic acids, phosphates, and terminal alkynes. Useful protecting groups for alcohols include, but are not limited to, acetyl, benzoyl, benzyl, β-methoxyethoxyethyl ether, dimethoxytrityl, methoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, methylthiomethyl ether, pivaloyl, tetrahydropyranyl, tetrahydrofuran, trityl, silyl ether, methyl ether, and ethoxyethyl ether. Useful protecting groups for amines include, but are not limited to, carbobenzyloxy, p-methoxybenzylcarbonyl, t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, acetyl, benzoyl, benzyl, carbamate, p-methoxybenzyl, 3,4-dimethoxybenzyl, p-methoxyphenyl, tosyl, trichloroethyl chloroformate, and sulfonamide. Useful protecting groups for carbonyls include acetals, ketals, acylals, and dithianes. Useful protecting groups for carboxylic acids include methyl esters, benzyl esters, t-butyl esters, esters of 2,6-disubstituted phenols, silyl esters, ortho esters, and oxazolines. Useful protecting groups for phosphate groups include 2-cyanoethyl and methyl. Useful protecting groups for terminal alkynes include propargyl alcohol and silyl groups. Other protecting groups are known in the art.

[0072] As used herein, the term "prodrug" refers to a precursor compound that, after administration, releases a biologically active compound in vivo via certain chemical or physiological processes (e.g., a prodrug that is converted to a biologically active compound by reaching a physiological pH or by enzymatic action). A prodrug itself may or may not possess the desired biological activity. Therefore, the term "prodrug" refers to a precursor of a pharmaceutically acceptable biologically active compound. In certain cases, a prodrug may have improved physical and / or delivery properties than its parent compound. Such prodrugs often offer advantages in mammalian organisms, such as solubility, histocompatibility, or delayed release (H. Bundgard, Design of Prodrugs (Elsevier, Amsterdam, 1988), pp. 7-9, 21-24). The concept of prodrugs is discussed in T. Higuchi et al., “Pro-Drugs as Novel Delivery Systems,” ACS Symposium Series, Vol. 14 and in EB Roche, ed., Bioreversible Carriers in Drug Design (American Pharmaceutical Association & Pergamon Press, 1987). Exemplary advantages of prodrugs include, but are not limited to, physical properties such as improved drug stability for long-term storage.

[0073] The term "prodrug" also includes any covalently bonded carrier that, upon administration to a target, releases an active compound in vivo. Prodrugs of therapeutic active compounds described herein may be prepared by modifying one or more functional groups present in the therapeutic active compound, including cannabinoids, terpenoids, and other therapeutic active compounds used in the methods of the present invention or included in the compositions of the present invention, by cleavage, either by conventional means or in vivo, to obtain a therapeutically active parent compound. Prodrugs include compounds in which a hydroxyl, amino, or mercapto group is covalently bonded to any group, and upon administration of the prodrug of the active compound to a target, it undergoes cleavage to form a free hydroxyl, free amino, or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, alcohols, or formate or benzoate derivatives of acetamide, formamide or benzamide derivatives of therapeutic activators having amine functional groups available for reaction.

[0074] For example, if a therapeutic agent, or a pharmaceutically acceptable form of a therapeutic agent, contains a carboxylic acid functional group, the prodrug may have a hydrogen atom of the carboxylic acid group, C 1-8 Alkyl, C 2-12Alkanoyloxymethyl, 1-(alkanoyloxy)ethyl with 4-9 carbon atoms, 1-methyl-1-(alkanoyloxy)ethyl with 5-10 carbon atoms, alkoxycarbonyloxymethyl with 3-6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl with 4-7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl with 5-8 carbon atoms, N-(alkoxycarbonyl)aminomethyl with 3-9 carbon atoms, 4-10 carbon atoms It may include esters formed by exchanging groups such as 1-(N-(alkoxycarbonyl)amino)ethyl, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N(C1-C2)alkylamino(C2-C3)alkyl((3-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl, and piperidino-, pyrrolidino-, or morpholino(C2-C3)alkyl.

[0075] Similarly, if the disclosed compound, or a pharmaceutically acceptable form of said compound, contains an alcohol functional group, the hydrogen atoms of said alcohol group are (C1-C6)alkanoyloxymethyl, 1-((C1-C6))alkanoyloxy)ethyl, 1-methyl-1-((C1-C6)alkanoyloxy)ethyl, (C1-C6)alkoxycarbonyloxymethyl, N(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl α-amino(C1-C4)alkanoyl, arylacyl, and α-aminoacyl, or α-aminoacyl-α-aminoacyl, in which each α-aminoacyl group is independently selected from naturally occurring L-amino acids, P(O)(OH)2, P(O)(O(C1-C6)alkyl)2, or glycosyl (a radical produced when the hydroxyl group of a carbohydrate's hemiacetal form is removed), can be used to form prodrugs.

[0076] If the disclosed compound, or a pharmaceutically acceptable form of said compound, contains an amine functional group, then the hydrogen atoms of said amine group are R-carbonyl, RO-carbonyl, NRR'-carbonyl, where R and R' are independently (C1-C 10 )alkyl, (C3-C7)cycloalkyl, benzyl, or R-carbonyl is a natural α-aminoacyl, or natural α-aminoacyl-natural α-aminoacyl, C(OH)C(O)OY 1 And in the formula, Y 1 is H, (C1-C6) alkyl, or benzyl, C(OY 2 )Y 3 And in the formula, Y 2 is (C1-C4) alkyl, and Y 3 This includes (C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C1-C4)alkyl, or mono-N, or di-N, N(C1-C6)alkylaminoalkyl, C(Y 4 )Y 5 And in the formula, Y 4 is H, or methyl, and Y 5 The group can be replaced with a mono-N, di-N, N(C1-C6)alkylamino, morpholino, piperidine-1-yl, or pyrrolidine-1-yl group to form a prodrug.

[0077] The use of prodrug systems is described in T. Jarvinen et al., “Design and Pharmaceutical Applications of Prodrugs” in Drug Discovery Handbook (SC Gad, ed., Wiley-Interscience, Hoboken, NJ, 2005), ch. 17, pp. 733-796. Other alternative means for constructing and using prodrugs are known in the art. If the method or pharmaceutical composition of the present invention uses or contains a prodrug of a cannabinoid, terpenoid, or other therapeutic agent, the prodrug of the compound and its active metabolite can be identified using common techniques known in the art. For example, Bertolini et al., J. Med. Chem., 40, 2011-2016 (1997);Shan et al., J. Pharm. Sci., 86 (7), 765-767;Bagshawe, Drug Dev. Res., 34, 220-230 (1995);Bodor, Advances in Drug Res., 13, 224-331 (1984);Bundgaard, Design of Prodrugs (Elsevier Press 1985);Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991);Dear et al., J. Chromatogr. B, 748, 281-293 (2000);Spraul et al., J. Pharmaceutical & See Biomedical Analysis, 10, 601-605 (1992); and Prox et al., Xenobiol., 3, 103-112 (1992).

[0078] Cannabinoids Cannabinoids are a group of chemicals known to activate cannabinoid receptors in cells throughout the human body, including in the skin. Phytocannabinoids are cannabinoids derived from the cannabis plant. They can be isolated from the plant or produced synthetically. Endogenous cannabinoids are cannabinoids found in the human body. Standard phytocannabinoids are ABC tricyclic terpenoid compounds supporting a benzopyran moiety.

[0079] Cannabinoids exert their effects by interacting with cannabinoid receptors present on the surface of cells. To date, two types of cannabinoid receptors, CB1 receptors and CB2 receptors, have been identified. These two receptors share approximately 48% amino acid sequence identity, are distributed in different tissues, and possess different signaling mechanisms. They also exhibit different sensitivities to agonists and antagonists.

[0080] Therefore, this specification describes in vitro and in vivo methods for screening and identifying genes, promoters, and expression cassettes for the in vivo production of cannabinoids.

[0081] Generally, the methods and compositions described herein can be used for the production or augmentation of one or more terpenoids, such as cannabinoids, in host cells, or for the production of one or more terpenoids or cannabinoid precursors in host cells. In some cases, terpenoids, or cannabinoids, or their precursors can be purified, derivatized (e.g., to form prodrugs, solvates, or salts, or to form the target terpenoid or cannabinoid from the precursor), and / or formulated into pharmaceutical compositions.

[0082] Cannabinoids that can be produced using the method and / or the composition of the present invention include, but are not limited to, phytocannabinoids. In some cases, such cannabinoids include cannabinol, cannabidiol, and Δ 9 -Tetrahydrocannabinol (Δ 9 Cannabinoids include, but are not limited to, THC, the synthetic cannabinoid HU-210(6aR,10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2-methyloctane-2-yl)-6H,6aH,7H,10H,10aH-benzo[c]isochromen-1-ol), cannabidivarin (CBDV), cannabichromen (CBC), cannabiclomevalin (CBCV), cannabigerol (CBG), cannabigerovalin (CBGV), cannabiersoin (CBE), cannabicyclol (CBL), cannabivarin (CBV), and cannabitriol (CBT). Further cannabinoids include tetrahydrocannivivarin (THCV) and cannabigerol monomethyl ether (CBGM). 9 - These include tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA); these further cannabinoids are characterized by the presence of a carboxylic acid group in their structure.

[0083] Furthermore, other cannabinoids include nabilone, limonabant, JWH-018 (naphthalene-1-yl-(1-pentylindole-3-yl)methanone), and JWH-073. Naphthalene-1-yl-(1-butylindole-3-yl)methanone, CP-55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctane-2-yl)phenol), dimethylheptylpyran, HU-331 (3-hydroxy-2-[(1R)-6-isopropenyl-3-methyl-cyclohex-2-en-1-yl]-5-pentyl-1,4-benzoquinone), SR144528 (5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl]-1H-pyrazole-3-carboxamide), WIN 55,212-2((11R)-2-methyl-11-[(morpholine-4-yl)methyl]-3-(naphthalene-1-carbonyl)-9-oxa-1-azatricyclo[6.3.1.0 4 , 12 ]Dodeca-2,4(12),5,7-tetraene), JWH-133((6aR,10aR)-3-(1,l-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran), levonatradol, and AM-2201(1-[(5-fluoropentyl)-1H-indole-3-yl]-(naphthalene-1-yl)methanone). Other cannabinoids include Δ 8 -Tetrahydrocannabinol (Δ 8 -THC), 11-hydroxy-Δ 9 - Tetrahydrocannabinol, Δ 11 -Tetrahydrocannabinol and 11-hydroxytetracannabinol are examples.

[0084] Alternatively, analogues or derivatives of these cannabinoids can be obtained by the production of cannabinoid precursors and further derivatization, for example, by synthetic means. As synthetic cannabinoids, there are U.S. Patent No. 9,394,267 by Attala et al.; U.S. Patent No. 9,376,367 by Herkenroth et al.; U.S. Patent No. 9,284,303 by Gijsen et al.; U.S. Patent No. 9,173,867 by Travis; U.S. Patent No. 9,133,128 by Fulp et al.; U.S. Patent No. 8,778,950 by Jones et al.; U.S. Patent No. 7,700,634 by Adam-Worrall et al.; U.S. Patent No. 7,504,522 by Davidson et al.; U.S. Patent No. 7,294,645 by Barth et al.; U.S. Patent No. 7,109,216 by Kruse et al.; U.S. Patent No. 6,825,209 by Thomas et al.; and Mittendorf et al. This includes, but is not limited to, those described in U.S. Patent No. 6,284,788 of al.

[0085] Alternatively, the cannabinoid in question may be an endocannabinoid, or a derivative or analog thereof. Endocannabinoids include, but are not limited to, anandamide, 2-arachidonylglycerol, 2-arachidonylglyceryl ether, N-arachidonyldopamine, and virodamine. Numerous analogs of endocannabinoids are known, such as 7,10,13,16-docosatetraenoylethanolamide, oleamide, stearoylethanolamide, and homo-γ-linolenoylethanolamine.

[0086] The cannabinoids and compositions produced by the method of the present invention can be selective to the CB2 cannabinoid receptor or non-selective to both cannabinoid receptors, binding to either the CB1 or CB2 cannabinoid receptor. In some cases, the cannabinoids and compositions produced by the method of the present invention are selective to the CB2 cannabinoid receptor. In some cases, one cannabinoid in the cannabinoids or mixture of cannabinoids is a CB2 antagonist (e.g., a selective or non-selective antagonist). In some cases, the cannabinoids and compositions produced by the method of the present invention are selective to the CB2 cannabinoid receptor. In some cases, one cannabinoid in the cannabinoids or mixture of cannabinoids is a CB1 antagonist (e.g., a selective or non-selective antagonist).

[0087] Expression Cassette This specification describes expression cassettes suitable for expressing one or more target genes in host cells. The expression cassettes described herein can be components of plasmids or can be incorporated into the host cell genome. A single plasmid may contain one or more expression cassettes described herein. For use herein, two or more expression cassettes are described, and it is understood that, alternatively, at least two of two or more expression cassettes can be combined to reduce the number of expression cassettes. Similarly, where multiple target genes are described as functionally linked to a single promoter, and therefore described as components of a single expression cassette, it is understood that the single expression cassette can be subdivided into two or more expression cassettes, including duplicate or non-duplicate subsets of the single expression cassette described.

[0088] The expression cassettes described herein may include suitable promoters known in the art. In some cases, the promoter is a constitutive promoter. In other cases, the promoter is an inducible promoter. In preferred embodiments, the promoter is a T5 promoter, a T7 promoter, a Trc promoter, a Lac promoter, a Tac promoter, a Trp promoter, a tip promoter, or a λP promoter. L promoter, λP R promoter, λP R P L Examples include promoters, such as the arabinose promoter (araBAD). In some embodiments, the promoter is selected from the group consisting of promoters and expression cassettes, particularly those described therein, which include plasmids for the expression of the nucleic acid of interest, target gene, host cell, and combination thereof, and which are incorporated in their entirety as part of this specification. There are many promoters useful for driving the expression of one or more target genes in various host cells, and are well known to those skilled in the art (see, for example, WO2004 / 033646;US8,507,235;US8,715,962; and WO2011 / 017798 and the references cited therein, particularly with respect to promoters and expression cassettes, particularly those described therein, which include plasmids for the expression of the nucleic acid of interest, target gene, host cell, and combination thereof, and which are incorporated in their entirety as part of this specification).

[0089] The methods and compositions described herein can be used for the expression of one or more genes of the MEP pathway in suitable host cells and / or for the production of one or more products of the MEP pathway. In some embodiments, the flux of the MEP pathway is increased by overexpressing one or more endogenous components of the host cell by amplifying the gene copy number and / or functionally linking the endogenous gene (or a copy thereof) to a strong constitutive or inducible xenopromoter. Thus, in some embodiments, an expression cassette is provided that includes a promoter functionally linked to a nucleic acid encoding one or more genes of the MEP pathway. In E. coli, the endogenous MEP pathway genes are dxs, ispC, ispD, ispE, ispF, ispG, ispH, and idi.

[0090] In some cases, the promoter of the expression cassette is functionally linked to nucleic acids encoding two or more genes of the MEP pathway. In some cases, the promoter of the expression cassette is functionally linked to nucleic acids encoding three or more genes of the MEP pathway. In some cases, the promoter of the expression cassette is functionally linked to nucleic acids encoding four, five, six, or all eight genes of the MEP pathway. In some cases, the MEP pathway genes provided by the expression cassette are E. coli genes. In other cases, one or more MEP pathway genes provided by the expression cassette are heterogeneous genes from wild-type E. coli. In some cases, one or more MEP pathway genes are provided in a first expression cassette, and one or more MEP pathway genes are provided in a second expression cassette. In a preferred embodiment, an expression cassette is provided that includes promoters functionally linked to dxs and idi.

[0091] In some cases, the present invention provides an expression cassette comprising a promoter functionally ligated to a nucleic acid encoding one or more genes of the MEP pathway and further encoding GPP synthase, cannabinoid synthase, or isoprene synthase. In some cases, the present invention provides an expression cassette comprising a promoter functionally ligated to a nucleic acid encoding one or more genes of the MEP pathway and further encoding THCA synthase. In some cases, the present invention provides an expression cassette comprising a promoter functionally ligated to a nucleic acid encoding one or more genes of the MEP pathway and further encoding CBGA synthase. In some cases, the present invention provides an expression cassette comprising a promoter functionally ligated to a nucleic acid encoding one or more genes of the MEP pathway and further encoding CBCA synthase. In some cases, the present invention provides an expression cassette comprising a promoter functionally ligated to a nucleic acid encoding one or more genes of the MEP pathway and further encoding CBDA synthase.

[0092] In some embodiments, an expression cassette is provided that includes a promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme. This ispDF gene can be used in addition to, or instead of, the overexpression of native ispD and / or ispF in host cells. In some cases, the nucleic acid encodes an ispDF protein having the following amino acid sequence (SEQ ID NO: 1): MIALQRSLSMHVTAIIAAAGEGRRLGAPLPKQLLDIGGRSILERSVMAFARHERIDDVIVVLPPAL AAAPPDWIAASGRVPAVHVVSGGERRQDSVANAFDRVPAQSDVVLVHDAARPFVTAELISRAI DGAMQHGAAIVAVPVRDTVKRVDPDGEHPVITGTIPRDTIYLAQTPQAFRRDVLGAAVALGRSG VSATDEAMLAEQAGHRVHVVEGDPANVKITTSADLDQARQRLRSAVAARIGTGYDLHRLIEGR PLIIGGVAVPCDKGALGHSDADVACHAVIDALLGAAGAGNVGQHYPDTDPRWKGASSIGLLRD ALRLVQERGFTVENVDVCVVLERPKIAPFIPEIRARIAGALGIDPERVSVKGKTNEGVDAVGRGE AIAAHAVALLSES.

[0093] In other embodiments, the ispDF nucleic acid encodes an ispDF protein having at least 32%, 40%, 45%, 50%, 52%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% identity with respect to SEQ ID NO: 1. In yet another embodiment, the ispDF nucleic acid encodes an ispDF protein having at least 32%, 40%, 45%, 50%, 52%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, or 99% identity with respect to at least 300 consecutive amino acids of SEQ ID NO: 1.

[0094] In some cases, the bifunctional ispDF is found in H. pylori HP1020, H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 It has a primary amino acid sequence that has 75% or less identity with at least 300 consecutive amino acids of CFF8240_0409. In some cases, the bifunctional ispDF is found in H. pylori HP1020, H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st.Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 It is not CFF8240_0409.

[0095] The bifunctional ispDF can be encoded by a nucleic acid within a plasmid, or by a nucleic acid integrated into the genome of a heterologous host cell. In some cases, a heterologous promoter functionally ligates to the nucleic acid encoding the bifunctional ispDF. Furthermore, or alternatively, the host cell can be heterologous to the nucleic acid encoding the bifunctional ispDF.

[0096] The nucleic acid encoding the bifunctional ispDF can be included in any of the above-mentioned expression cassettes containing nucleic acids encoding MEP pathway genes, or in some cases, in an expression cassette containing nucleic acids encoding cannabinoid synthase. In some cases, the nucleic acid encoding the bifunctional ispDF can be included in an expression cassette containing nucleic acids encoding GPP synthase. In some cases, the nucleic acid encoding the bifunctional ispDF can be included in an expression cassette containing nucleic acids encoding isoprene synthase.

[0097] The methods and compositions described herein can be used for the production of precursor-derived GPP produced in the MEP pathway in suitable host cells. Therefore, in some embodiments, an expression cassette is provided comprising a promoter functionally ligated to a nucleic acid encoding a GPP synthase. This GPP synthase may also be included in an expression cassette that comprises a nucleic acid encoding a gene in the MEP pathway. Furthermore, or alternatively, this GPP synthase may also be included in an expression cassette that comprises a nucleic acid encoding a cannabinoid synthase. In some cases, the promoter of the expression cassette functionally ligated to the nucleic acid encoding the GPP synthase is also functionally ligated to the cannabinoid synthase. Furthermore, or alternatively, this GPP synthase may also be included in an expression cassette that comprises a nucleic acid encoding a GPP synthase. Furthermore, or alternatively, this GPP synthase may also be included in an expression cassette that comprises a nucleic acid encoding isoprene synthase.

[0098] The methods and compositions described herein can be used for the production of cannabinoids in host cells. Accordingly, in some embodiments, an expression cassette is provided comprising a promoter functionally linked to a nucleic acid encoding a cannabinoid synthase. The cannabinoid synthase may be an endogenous cannabinoid synthase for plants of the genus Cannabis, or an ortholog thereof. In some cases, the cannabinoid synthase is an endogenous cannabinoid synthase for Cannabis sativa or Cannabis indica, or an ortholog thereof. In some cases, the cannabinoid synthase is a CBGA synthase, THCA synthase, CBDA synthase, or CBCA synthase (for example, endogenous for plants of the genus Cannabis, or an ortholog thereof).

[0099] The cannabinoid synthase can be modified for expression in a host. For example, one or more transmembrane or signal peptide domains can be cleaved. Furthermore, or alternatively, one or more glycosylation sites can be deleted (for example, by mutations in the primary amino acid sequence). Similarly, one or more, or all, cysteines found in the intramolecular disulfide bonds of the native protein in its native host can be mutated to, for example, serine. Similarly, one or more, or all, cysteines found in the intermolecular disulfide bonds of the native protein in its native host can be mutated to, for example, serine.

[0100] host cell Any of the expression cassettes described above, or combinations thereof, can be introduced into a suitable host cell and used for the production of target terpenoids or cannabinoids. Suitable host cells include, but are not limited to, prokaryotes of the genera Escherichia, Panteoa, Corynebacterium, Bacillus, or Lactococcus. Preferred prokaryotic host cells include, but are not limited to, Escherichia coli (E. coli), Panteoa citrea, C. glutamicum, Bacillus subtilis, and Lactococcus lactis. In some embodiments, the expression cassettes described herein include a promoter (e.g., a heterologous promoter) functionally linked to a nucleic acid encoding one or more target genes (e.g., MEP pathway genes, cannabinoid synthase genes, ispA, ispS, ispDF, or GPP synthase), wherein the nucleic acid encoding one or more target genes is a codon optimized for the host cell containing the expression cassette.

[0101] In some cases, the host cells contain one or more products of the MEP pathway, such as DMAPP and / or IPP. For example, host cells containing the MEP pathway expression cassette described herein can increase the amount of MEP pathway products, such as DMAPP and / or IPP, compared to host cells that do not contain the MEP pathway expression cassette.

[0102] In some cases, the host cell may contain one or more products downstream of the MEP pathway. For example, a host cell containing a GPP synthase expression cassette may contain an increased amount of GPP compared to a host cell lacking the GPP synthase expression cassette. As another example, a host cell containing an isoprene synthase expression cassette may contain an increased amount of isoprene compared to a host cell lacking the isoprene synthase expression cassette.

[0103] As yet another example, host cells containing a cannabinoid synthase expression cassette may contain increased amounts of cannabinoids compared to host cells lacking the cannabinoid synthase expression cassette. In some cases, the cannabinoid is CBGA. In some cases, the cannabinoid is CBCA. In some cases, the cannabinoid is CBDA. In some cases, the cannabinoid is THCA. In some cases, the cannabinoid is CBN. In some cases, the cannabinoid is CBD. In some cases, the cannabinoid is THC. In some cases, the cannabinoid is CBC. In some cases, the cannabinoid is THCV. In some cases, the cannabinoid is CBDV. In some cases, the cannabinoid is CBCV.

[0104] Similarly, culturing the host cells under conditions suitable for inducing expression from the expression cassette, compared to non-inducible conditions, can increase the amount of one or more enzyme products encoded by the expression cassette in the host cells. For example, induction may result in increased DMAPP and / or IPP in the host cells compared to the same host cells cultured in the absence of an inducer (e.g., IPTG, arabinose, etc.). As another example, induction may result in increased GPP in the host cells compared to the same host cells cultured in the absence of an inducer (e.g., IPTG, arabinose, etc.). As yet another example, induction may result in increased isoprene in the host cells compared to the same host cells cultured in the absence of an inducer (e.g., IPTG, arabinose, etc.). As yet another example, induction may result in increased cannabinoids in the host cells compared to the same host cells cultured in the absence of an inducer (e.g., IPTG, arabinose, etc.).

[0105] In some embodiments, the host cells contain olivetolic acid (OA). OA can be introduced into the host cells by culturing them in a medium containing OA. In some embodiments, the host cells contain divalic acid (DVA). DVA can be introduced into the host cells by culturing them in a medium containing DVA.

[0106] In some embodiments, the host cell is genetically modified to remove or reduce the expression of one or more genes encoding endogenous enzymes that reduce the flux through the MEP pathway. In some embodiments, the host cell is genetically modified to reduce or decrease the amount or activity of endogenous enzymes that reduce the flux through the MEP pathway. For example, pyruvate and glyceraldehyde-3 phosphate (G3P) are substrates of the initial enzymes of the MEP pathway dxs. By modifying the endogenous pathway that consumes pyruvate and G3P, the amounts of pyruvate and G3P can be increased, and thus the flux through the MEP pathway can be increased. In some cases, one or more host intracellular genes or gene products selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and atoDA are modified to increase pyruvate or G3P levels.

[0107] Culture method The present invention further provides a process for culturing host cells of the present invention in a suitable medium under induction conditions to produce a target metabolite. The target metabolite may be a cannabinoid, a terpenoid, or a precursor thereof. This method may include concentrating the metabolite in the used medium and / or in the host cells.

[0108] The produced microorganisms can be cultured continuously, for example, as described in WO 05 / 021772, or discontinuously in a batch process (batch culture), fed-batch culture, or repeated fed-batch culture process, for the purpose of producing the desired organic compounds. An overview of the general properties of known culture methods can be found in Chmiel's text (BioprozeBtechnik. 1 : Einfiihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) or Storhas's text (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)).

[0109] The culture medium or fermentation medium used must be handled in an appropriate manner to satisfy the requirements of each strain. Descriptions of various microbial media are found in the "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981). The terminology for culture media and fermentation media is interchangeable.

[0110] As a carbon source, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, sucrose-containing solutions derived from sugar beets or sugarcane products, starch, starch hydrolysates, and cellulose; oils and fats such as soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol, methanol, and ethanol; and organic acids such as acetic acid or lactic acid can be used.

[0111] As a nitrogen source, organic nitrogen-containing compounds such as peptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour, urea, etc., or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, etc., can be used. These nitrogen sources can be used individually or as mixtures.

[0112] As a phosphorus source, phosphoric acid, potassium dihydrogen phosphate, or dipotassium hydrogen phosphate, or the corresponding sodium-containing salt can be used.

[0113] The culture medium may further contain salts in the form of chlorides or sulfates of metals such as sodium, potassium, magnesium, calcium, and iron, which are necessary for growth, such as magnesium sulfate or iron sulfate. Finally, essential growth factors such as amino acids, such as homoserine, and vitamins, such as thiamine, biotin, or pantothenic acid, may be used in addition to the above-mentioned substances.

[0114] The starting material can be added to the culture in a single-batch form, or supplied during cultivation by an appropriate method.

[0115] The pH of the culture can be controlled in an appropriate manner using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia; or acidic compounds such as phosphoric acid or sulfuric acid. The pH is generally adjusted to a value of 6.0 to 8.5, preferably 6.5 to 8. To control foaming, an antifoaming agent, such as fatty acid polyglycol ester, can be used. To maintain plasmid stability, a suitable selective substance, such as an antibiotic, can be added to the culture medium. This culture is preferably carried out under aerobic conditions. To maintain these conditions, oxygen, or an oxygen-containing gas mixture such as air, is introduced into the culture. Similarly, a hydrogen peroxide-rich liquid can be used. This culture is carried out under high pressure, for example, 0.03 to 0.2 MPa, if appropriate. The temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C, and particularly preferably 30°C to 37°C. In batch or fed-batch processes, the culture is preferably continued until a sufficient amount of the desired organic chemical compound to be recovered has formed. This objective is typically achieved within 10 to 160 hours. Continuous processes allow for even longer incubation periods. The activity of the microorganism concentrates (accumulates) organic chemical compounds in the fermentation medium and / or in the microbial cells.

[0116] Examples of suitable culture media are found, in particular, in patents US5,770,409, US5,990,350, US5,275,940, WO2007 / 012078, US5,827,698, WO2009 / 043803, US5,756,345, and US7,138,266.

[0117] Analysis of target metabolites to determine their concentration at one or more time points during culture can be performed by separating the metabolites using chromatography, preferably reverse-phase chromatography.

[0118] Detection can be performed photometrically (by absorption or fluorescence).

[0119] The implementation of the culture method using host cells containing one or more expression cassettes of the present invention can increase, with respect to one or more parameters selected from the group consisting of concentration (target metabolite formed per unit volume), yield (target metabolite formed per unit carbon source consumed), formation (target metabolite produced per unit volume and time), and specific formation (target metabolite per unit dry cell material or dry biomass and time, or unit cell protein and compound formed per time), or other process parameters, and combinations thereof, by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% with respect to the culture method using host cells without the expression cassettes of the present invention. This is considered to be of great value from the perspective of large-scale industrial processes.

[0120] Subsequently, a product containing the target metabolite can be provided, produced, or recovered in liquid or solid form.

[0121] Used culture medium refers to a medium in which host cells have been cultured for a specific time and at a specific temperature. The culture medium or medium used for culturing contains all the substances or components necessary to ensure the production of the desired target metabolite and, generally, to ensure growth and viability. When culturing is complete, the used culture medium obtained includes: a) the biomass (cell aggregate) of the microorganism, which is generated as a result of the growth of the microorganism's cells; b) the desired target metabolite formed during culturing; c) any organic by-products that may have been formed during culturing; and d) components of the culture medium used, or components of the starting material that were not consumed during culturing, such as vitamins like biotin or salts like magnesium sulfate.

[0122] The organic by-product includes, in addition to specific desired compounds, substances produced and optionally secreted by the microorganisms used in the culture. The used culture medium can be removed from the culture vessel or fermentation tank, recovered as needed, and used to provide a product containing the target metabolite in liquid or solid form. In the simplest case, the used culture medium containing the target metabolite, removed from the fermentation tank, constitutes the recovered product.

[0123] In some cases, the recovery of the target metabolite (e.g., terpenoids, cannabinoids, or their precursors) may involve: a) partial (>0% to <80%) to complete (100%) or substantially complete (>80%, >90%, >95%, >96%, >97%, >98%, or >99%) removal of water; b) partial (>0% to <80%) to complete (100%) or substantially complete (>80%, >90%, >95%, >96%, >97%, >98%, or >99%) removal of biomass, the latter optionally inactivated before removal; c) partial (>0% to <80%) to complete (100%) or There are one or more evaluation criteria selected from the group consisting of: d) the removal of substantially complete (>80%, >90%, >95%, >96%, >97%, >98%, >99%, >99.3%, or >99.7%) organic by-products formed during culture; and d) the removal of some (>0%) to complete (100%) or substantially complete (>80%, >90%, >95%, >96%, >97%, >98%, >99%, >99.3%, or >99.7%) components of the used fermentation medium, or the removal of components of starting materials from used medium not consumed in culture to achieve concentration or purification of the desired target metabolite. A composition containing the target metabolite in the desired content is thus isolated.

[0124] Partial (>0% to <80%) to complete (100%) or substantially complete (>80% to <100%) water removal (evaluation criterion a)) is also referred to as drying.

[0125] In some variations of the process, the complete or substantially complete removal of water, biomass, organic by-products, and unused components of the fermentation medium used results in a pure (>80% by weight, >90% by weight) or highly pure (>95% by weight, >97% by weight, or >99% by weight) product form of the desired target metabolite. Extensive technical guidance on means a), b), c), and d) can be obtained from the prior art.

[0126] If necessary, the biomass may be completely or partially removed from the spent culture medium, or left completely, by separation methods such as centrifugation, filtration, decantation, or a combination thereof. If appropriate, the biomass, or the spent culture medium containing the biomass, may be inactivated in an appropriate process step, for example, by heat treatment (heating) or by adding alkali or acid.

[0127] In some procedures, the biomass is completely or substantially removed, such that the prepared product contains no biomass at all (0%), or only a maximum of 30%, 20%, 10%, 5%, 1%, or 0.1% of it. In further procedures, the biomass is not removed, or only a small percentage is removed, such that the prepared product contains all of the biomass (100%), or more than 70%, 80%, 90%, 95%, 99%, or 99.9% of the biomass. Thus, in some processes of the present invention, biomass is removed in a range of >0% to <100%. Finally, the fermentation culture obtained after fermentation can be adjusted to an acidic pH with an inorganic acid, such as hydrochloric acid, sulfuric acid, or phosphoric acid, or with an organic acid, such as propionic acid, before or after the complete or partial removal of the biomass, in order to improve the handling properties of the final product (GB1,439,728, or EP1331220). Similarly, the fermentation culture can also be acidified with the entire contents of the biomass. Finally, the broth can be stabilized by adding sodium bicarbonate (NaHCO3, GB1,439,728) or another salt, such as ammonium sulfite, alkali metal, or alkaline earth metal salts.

[0128] During the removal of the biomass, any organic or inorganic solids present in the spent culture medium can be partially or completely removed. Organic by-products dissolved in the spent culture medium and non-consumed components (starting materials) dissolved in the fermentation medium can be left in at least a small amount (>0%), and in some cases, at least 25% of the product, in some cases at least 50%, and in some cases at least 75%. Where appropriate, they may remain in the product in whole (100%) or substantially whole, i.e., >95%, >98%, or >99%.

[0129] Next, water can be removed from the spent medium by reverse osmosis or by known methods such as nanofiltration, using, for example, a rotary evaporator, a thin-film evaporator, or a falling-film evaporator, or the spent medium can be concentrated. Then, this concentrated spent medium can be processed into a free-flowing product, particularly a fine powder or, preferably, a coarse granule, by freeze-drying, spray-drying, spray-granulation, or other processes such as a circulating fluidized bed as described in the examples in PCT / EP2004 / 006655.

[0130] Pharmaceutical composition The target metabolite can be formulated into a pharmaceutical composition. In some cases, used culture medium, concentrated used culture medium, or a target metabolite purified in part or in whole from said used culture medium, or biomass obtained by the culture method described herein, can be formulated into a pharmaceutical composition.

[0131] The pharmaceutical compositions of the present invention may contain one or more excipients. Such excipients suitable for use in topical compositions intended for application to the skin include, but are not limited to, preservatives; thickeners; buffers; liquid carriers; isotonic agents; wetting agents; solubilizers; and emulsifiers; acidifiers; antioxidants; alkalizing agents; carriers; chelating agents; complexing agents; solvents; suspending agents; or thickeners; oils and fats; penetration enhancers; polymers; curing agents; proteins; carbohydrates; and volume extenders.

[0132] As is generally known in the field of pharmaceutical formulations, depending on the concentration of the excipient, other excipients in the composition, the physical form in the composition, the concentration of the activator in the composition, the intended route of administration of the composition, and other factors, a particular excipient may satisfy one or more of these functions in a particular pharmaceutical composition. The descriptions of particular excipients in the following categories are not intended to exclude the possibility of using the excipient in other categories.

[0133] The liquid carrier may be selected from the group consisting of physiological saline, phosphate-buffered physiological saline, glycerol, and ethanol, but is not limited to these.

[0134] The thickening agent may be selected from the group consisting of glycerol and propylene glycol, but is not limited to these.

[0135] The isotonic agent may be, but is not limited to, a polyhydric alcohol selected from the group consisting of mannitol and sorbitol; sodium chloride; and potassium chloride.

[0136] Humectants, solubilizers, or emulsifiers are generally surfactants. Typically, surfactants include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, sodium docusate, nonoxynol 9, nonoxynol 10, octoxynol 9, poloxamer, polyoxyl 35 castor oil, polyoxyl 40, hydrogenated castor oil, polyoxyl 50 stearate, polyoxyl 10 oleyl ether, polyoxyl 20, cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, tyroxapol, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohol, lecithin, mono-, and Select from the group consisting of diglycerides, monoethanolamine (auxiliary agent), oleic acid (auxiliary agent), oleyl alcohol (stabilizer), poloxamer, polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propyl glycol diacetate, propylene glycol monostearate, sodium lauryl sulfate, sodium stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, stearic acid, triethanolamine, emulsifying wax, cetomacrogol, and cetyl alcohol.

[0137] Pharmaceutical compositions for topical application may contain emollients. As used herein, "emollient" refers to a hydrophobic substance that softens, smooths, and improves the lipid content of the skin or other mucous membranes. Examples of suitable emollients include isostearic acid derivatives, isopropyl palmitate, lanolin oil, diisopropyl dimerate, diisopropyl adipate, dimethyl isosorbide, maleate-derived soybean oil, octyl palmitate, isopropyl isostearate, cetyl alcohol, cetyl lactate, cetyl ricinoleate, tocopheryl acetate, acetylated lanolin alcohol, cetyl acetate, phenyl trimethicone, glyceryl oleate, tocopheryl linolenate, wheat germ glyceride, and propionic acid. These include arachidyl, myristyl lactate, decyl oleate, propylene glycol ricinoleate, isopropyl lanolinate, pentaerythrityl tetrastearate, neopentyl glycol dicaprylate / dicaprate, hydrogenated coco-glycerides, isononyl isononanoate, isotridecyl isononanoate, myristyl myristate, triisocetyl citrate, dodecanol octyl, octyl hydroxystearate, grape seed oil, one or more ceramides, cyclomethicone, and mixtures thereof. Other examples of suitable emollients can also be found in the Cosmetic Bench Reference, pp. 1.19 1.22 (1996). Those skilled in the art will understand that other emollients may be useful in the present invention.

[0138] The preservative can be selected from the group consisting of benzalkonium chloride, benzalkonium chloride solution, benzethonium chloride, benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, diazolidinyl urea, ethylparaben, methylparaben, sodium methylparaben, phenol, phenylethyl alcohol, phenylmercury acetate, phenylmercury nitrate, potassium benzoate, potassium sorbate, propylparaben, sodium propylparaben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimerosal, and thymol.

[0139] The composition contains acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, monopotassium hydrogen phosphate, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, sodium bicarbonate, tris(tris(hydroxymethyl)aminomethane), MOPS(3-(N-morpholino)propanesulfonic acid), HEPES(N-(2-hydroxyethyl) Piperazine-N'-(2-ethanesulfonic acid), ACES(2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid), ADA(N-(2-acetamide)2-iminodiacetic acid), AMPSO(3-[(1,1-dimethyl-2-hydroxyethylamino]-2-propanesulfonic acid), BES(N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), Bis(N,N-bis(2-hydroxyethylglycine), Bis-Tris(bis-(2-H) Droxyethyl)imino-tris(hydroxymethyl)methane, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO (3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), CHES (2-(N-cyclohexylamino)ethanesulfonic acid), DIPSO (3-[N,N-bis(2-hydroxyethylamino]-2-hydroxy-propanesulfonic acid), HEPPS (N-(2-hydroxyethylpiperazine) -N'-(3-propanesulfonic acid), HEPPSO(N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), triethanolamine, imidazole, glycine, ethanolamine, phosphate, MOPSO(3-(N-morpholino)-2-hydroxypropanesulfonic acid), PIPES(piperazine-N,N'-bis(2-ethanesulfonic acid), POPSO(piperazine-N,It may contain a buffer selected from the group consisting of N'-bis(2-hydroxypropanesulfonic acid), TAPS (N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid), TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxy-propanesulfonic acid), TES (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), Trisine (N-tris(hydroxymethyl)methylglycine), 2-amino-2-methyl-1,3-propanediol, and 2-amino-2-methyl-1-propanol.

[0140] Generally, the acidifying agent is selected from the group consisting of acetic acid, citric acid, fumaric acid, hydrochloric acid, dilute hydrochloric acid, malic acid, nitric acid, phosphoric acid, dilute phosphoric acid, sulfuric acid, and tartaric acid.

[0141] Generally, the antioxidant is selected from the group consisting of ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfate, sodium formaldehyde sulfoxylate, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, and tocopherol.

[0142] Generally, the alkalizing agent is selected from the group consisting of strong ammonia solution, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, and trolamine.

[0143] The carrier can be selected from the group consisting of corn oil, mineral oil, peanut oil, sesame oil, bacteriostatic sodium chloride, and bacteriostatic water.

[0144] The chelating agent can be selected from the group consisting of disodium edetate, ethylenediaminetetraacetic acid, citric acid, and salicylates.

[0145] The complexing agent can be selected from the group consisting of ethylenediaminetetraacetic acid, salts of ethylenediaminetetraacetic acid, gentisinic acid ethanolamide, and oxyquinoline sulfate.

[0146] The solvent can be selected from the group consisting of acetone, ethanol, dilution alcohol, amylene hydrate, benzyl benzoate, butyl alcohol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerol, hexylene glycol, isopropyl alcohol, methyl isobutyl ketone, mineral oil, oleic acid, peanut oil, polyethylene glycol, propylene carbonate, propylene glycol, sesame oil, water, sterile water, and purified water.

[0147] Generally, the suspending agent and / or thickening agent is selected from the group consisting of acacia, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magma bentonite, carbomer, carbomer 934p, calcium carboxymethylcellulose, sodium carboxymethylcellulose, sodium carboxymethylcellulose 12, carrageenan, microcrystals, and sodium carboxymethylcellulose cellulose, dextrin, gelatin, guar gum, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, aluminum magnesium silicate, methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, propylene glycol alginate, silicon dioxide, silicon dioxide colloid, sodium alginate, tragacanth, bee gum, and xanthan gum.

[0148] Generally, the oil in question is selected from the group consisting of peanut oil, mineral oil, olive oil, sesame oil, cottonseed oil, safflower oil, corn oil, and soybean oil.

[0149] Generally, the penetration enhancer is selected from the group consisting of monohydroxy or polyhydroxy alcohols, monohydric or polyhydric alcohols, saturated or unsaturated fatty alcohols, saturated or unsaturated fatty esters, saturated or unsaturated dicarboxylic acids, essential oils, phosphatidyl derivatives, cephalins, terpenes, amides, ethers, ketones, and urea.

[0150] Generally, the polymer is selected from the group consisting of cellulose acetate, alkylcellulose, hydroxyalkylcellulose, acrylic polymers, copolymers, polyesters, polycarbonates, and polyanhydrides.

[0151] Generally, the curing agent is selected from the group consisting of hydrogenated castor oil, cetostearyl alcohol, cetyl alcohol, cetyl ester wax, hard fat, paraffin, polyethylene excipients, stearyl alcohol, emulsifying wax, white wax, and yellow wax.

[0152] Generally, the protein in question is selected from the group consisting of bovine serum albumin, human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein.

[0153] Generally, the carbohydrate in question is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melegitose, maltodextrin, dextran, starch, mannitol, maltitol, lactitol, xylitol, sorbitol, and myo-inositol.

[0154] Generally, the bulking agent is selected from the group consisting of polypeptides and amino acids.

[0155] The composition may further contain a topical sedative for the skin, a topical anti-inflammatory agent, a topical antibacterial agent, a topical antifungal agent, a topical steroid, and a topical antioxidant.

[0156] Common topical sedatives for the skin include chamomile and aloe; other topical sedatives are known in the art and can be used.

[0157] Common topical anti-inflammatory agents include diclofenac, ketoprofen, ibuprofen, piroxicam, and indomethacin; other topical anti-inflammatory agents are known and can be used in the art.

[0158] Common topical antimicrobial agents include bacitracin, polymyxin B, erythromycin, sodium sulfacetamide, silver sulfadiazine, letapamulin, mupirocin, neomycin, and pramoxin; other topical antimicrobial agents are known and usable in the art.

[0159] Common topical antifungal agents include benzoic acid, salicylic acid, undecylenic acid, ketoconazole, nystatin, naphthifine, tolnaftate, miconazole, econazole, cyclopirox, oxiconazole, sertaconazole, efinaconazole, terbinafine, tababolol, clotrimazole, sulconazole, and butefinazole; other topical antifungal agents are known and can be used in the art.

[0160] Common topical steroids include hydrocortisone, triamcinolone, fluocinolone, prednicurbate, desonide, betamethasone, halcinonide, diflorasone, fluocinolone, clobetasol, desoximethasone, mometasone, crocoltolone, fluticasone, fluocinonide, flulandrenolide, acromethasone, and halobetazole; other topical steroids are known and can be used in the art.

[0161] Common topical antioxidants include vitamin C, vitamin E, and L-selenomethionine; other topical antioxidants are known in the art and can be used.

[0162] Other active agents may be included. Alternatively, one or more further pharmaceutical compositions containing one or more of the above-mentioned excipients, including numerous further active substances such as topical anti-inflammatory agents, topical antibacterial agents, topical antifungal agents, topical steroids, and topical antioxidants, may be administered individually.

[0163] In some alternatives, including the use of the prodrugs described above, the therapeutically active compounds used in the methods and compositions of the present invention, including but not limited to cannabinoids and terpenoids, are formed by covalently crosslinking the therapeutically active compound with one or more conjugation partners. Reagents suitable for crosslinking numerous combinations of functional groups are known in the art.

[0164] For example, electrophiles can react with numerous functional groups containing the same electrophile present in proteins or polypeptides. Various combinations of reactive amino acids and electrophiles are known and usable in the art. For example, N-terminal cysteine ​​containing a thiol group can react with halogens or maleimides. Thiol groups are known to react with numerous coupling agents, such as alkyl halides, haloacetyl derivatives, maleimides, aziridines, acryloyl derivatives, and aryl halides. These are described in GT Hermanson, “Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp. 146-150.

[0165] The reactivity of cysteine ​​residues can be optimized by appropriately selecting adjacent amino acid residues. For example, a histidine residue adjacent to a cysteine ​​residue enhances the reactivity of that cysteine ​​residue. Other combinations of reactive amino acids and electrophiles are known in the art. For example, maleimide can react with amino groups, such as the ε-amino group of the lysine side chain, particularly in a high pH range. Aryl halides can also react with such amino groups. Haloacetyl derivatives can react with the imidazolyl side chain nitrogen of histidine, the thioether group of the methionine side chain, and the epsilon-amino group of the lysine side chain. Numerous other electrophiles that react with the ε-amino group of the lysine side chain are known, including, but are not limited to, isothiocyanates, acyl azides, N-hydroxysuccinimide esters, sulfonyl chlorides, epoxides, oxiranes, carbonates, imide esters, carbodiimides, and anhydrides. These points are explained in G.T. Hermanson, “Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp. 137–146.

[0166] In addition, electrophiles that react with diazoalkanes, diazoacetyl compounds, aspartates such as carbonidylmidazole and carbodiimide, and carboxylate side chains such as glutamates are known. These are described in GT Hermanson, “Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp. 152-154. Furthermore, electrophiles that react with hydroxyl groups, such as the hydroxyl groups of serine and threonine side chains, including reactive haloalkane derivatives, are known. These are described in GT Hermanson, “Bioconjugate Techniques” (Academic Press, San Diego, 1996), pp. 154-158. In another alternative embodiment, the relative positions of the electrophile and nucleophile (i.e., the molecule that reacts with the electrophile) are reversed, and the protein has an amino acid residue with an electrophile that reacts with the nucleophile, and a target molecule that has a nucleophile therein. This includes the reaction between the aldehyde (electrophile) and hydroxylamine (nucleophile) described above, but is more general; other groups can be used as electrophiles and nucleophiles. Suitable groups are well known in organic chemistry, so further details are unnecessary.

[0167] All combinations of reactive groups for crosslinking are known in the art. For example, amino groups can react with isothiocyanates, isocyanates, acyl azides, N-hydroxysuccinimide (NHS) esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, alkylating agents, imide esters, carbodiimides, and anhydrides. Thiol groups can react with haloacetyl or alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, acylating agents, or other thiol groups by oxidation and the formation of mixed disulfides. Carboxylic groups can react with diazoalkanes, diazoacetyl compounds, carbonyldiimidazoles, and carbodiimides. Hydroxyl groups can react with epoxides, oxiranes, carbonyldiimidazoles, N,N'-disuccinimidyl carbonates, N-hydroxysuccinimidyl chloroformates, periodates (for oxidation), alkyl halogens, or isocyanates. Aldehydes and ketone groups can react with hydrazines, Schiff base-forming reagents, and other groups in reductive amination or Mannich condensation reactions. Further reactions suitable for crosslinking are known in the art. Such crosslinking reagents and reactions are described in G.T. Hermanson, “Bioconjugate Techniques” (Academic Press, San Diego, 1996).

[0168] The amount of a given therapeutic agent, such as, but not limited to, the cannabinoid or terpenoid described above, contained in a unit dose of the pharmaceutical composition of the present invention varies depending on factors such as the specific compound, the disease state and its severity, and the condition of the subject requiring treatment (e.g., body weight). Nevertheless, a person skilled in the art can determine it as usual. The selected dose level is based on various pharmacokinetic factors, including the activity of the specific therapeutic agent, the route of administration, the time of administration, the excretion rate of the specific compound used, the severity of the disease state, other health considerations affecting the patient, and the functional status of the liver and kidneys of the subject.

[0169] Furthermore, it is based on the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific therapeutic agent used, as well as the age, weight, condition, overall health status, and prior medical history of the person receiving treatment, and similar factors. Methods for determining the optimal dosage are described in the relevant technical field, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20 th This is explained in ed., 2000. The optimal dosage for a given set of pathological conditions can be determined by a person skilled in the art, taking into account experimental data of the active ingredient and using conventional dosage determination tests.

[0170] The compositions of the present invention, or compositions used in accordance with the present invention, can be manufactured using known techniques for preparing pharmaceutical compositions, such as conventional techniques including mixing, dissolving, granulation, sugar-coated tablet preparation, air levitation, emulsification, encapsulation, encapsulation, or freeze-drying. The pharmaceutical compositions can be formulated in conventional ways using one or more physiologically acceptable carriers, which can be selected from excipients and auxiliary agents that facilitate the processing of the active compound into a formulation.

[0171] The pharmaceutical composition according to the present invention is typically administered to a subject multiple times. The interval between single doses is once a week, once a month, or once a year. The interval may be irregular, as indicated by the therapeutic response or other parameters well known in the art. Alternatively, the pharmaceutical composition may be administered as a sustained-release formulation, in which case the frequency of administration must be reduced. The dosage and frequency of administration vary depending on the half-life of the pharmacologically active substance contained in the pharmaceutical composition in the subject. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic.

[0172] For prophylactic use, relatively low doses are administered over a long period at relatively infrequent intervals. Some subjects may continue treatment for life. For therapeutic use, relatively high doses are often required at relatively short intervals until the progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete improvement of the disease symptoms. A prophylactic regimen may then be administered to the subject.

[0173] Nardella's U.S. Patent No. 6,573,292, Nardella's U.S. Patent No. 6,921,722, Chao et al.'s U.S. Patent No. 7,314,886, and Chao et al.'s U.S. Patent No. 7,446,122, which constitute part of this Specified and draw upon its contents, disclose various pharmacological activators and methods for using pharmaceutical compositions in the treatment of numerous diseases and conditions, including cancer, and methods for determining the therapeutic efficacy of such pharmacological activators and pharmaceutical compositions.

[0174] References The following publications are incorporated herein by reference as forming part of this specification. These publications are referred to by the following numbers. The inclusion of any publication in this list does not constitute an endorsement that any publication mentioned herein is prior art. · JAMA. 2006;295(7): 761-775 ·Comput Struct Biotechnol J, 2012, 3, 1-11 ·Biotechnol.Bioeng.2004 88, 909-915. ·Science 2002, 298 (5599), 1790-3. [Examples]

[0175] Example 1 Genetic manipulation of the MEP pathway in E. coli The MEP pathway was selected due to its thermodynamic advantages and the availability of a suitable bacterial host system. This MEP pathway is specific to the host E. coli. In this experiment, E. coli BL21(DE3) was selected as the expression host. The gene encoding the rate-limiting enzyme of this MEP pathway was overexpressed to maximize the production of cannabinoid precursors.

[0176] The four steps of the MEP pathway are the slowest, and therefore have the lowest flux. Overexpression of the enzymes that catalyze these rate-limiting steps has been reported to improve the flux through the MEP pathway and increase downstream terpenoid biosynthesis. To maximize the production of cannabinoids (Figure 1), lycopene, monoterpenes, or isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are precursors of the isoprene pathway, the non-mevalonate pathway was genetically engineered to introduce extra copies of four different rate-limiting genes, dxs, ispD, ispF, and idi (Figure 2).

[0177] This process was carried out stepwise using polymerase chain reaction (PCR), and a gene cassette with the appropriate orientation was constructed by cloning to a vector backbone containing the T7 promoter and the p15A origin of replication via restriction digestion and ligation. Next, the entire gene cassette was subcloned into a pTrc-trGPPS(CO)-LS plasmid (Figure 3) containing the Trc promoter and the pBR322 origin of replication, obtaining a broad window for controlling gene expression and, consequently, the production of isoprenoid precursors. This gene cassette, along with the pTrc promoter and a selection marker gene, was then incorporated into the E. coli chromosome as an inducible extra copy to overproduce isoprenoid precursors. A schematic diagram of the MEP pathway expression cassette is shown in Figure 3. Overexpression of the rate-limiting enzyme was confirmed by SDS-PAGE analysis (Figure 4).

[0178] Similarly, GPP synthase can be cloned, for example, from a plant source and expressed in E. coli to produce GPP, a substrate for the cannabinoid synthase CBGA synthase, and substrates for monoterpene synthases such as carene, myrcene, or limonene synthase. Furthermore, a polyketide pathway for the synthesis of olivetolic acid, a substrate for CBGA synthase, can be cloned and expressed in E. coli, or olivetolic acid can be supplied externally. Thus, the pathway for CBGA production can be replicated in prokaryotic host cells.

[0179] Example 2 Cloning and expression of downstream cannabinoid synthase genes in E. coli Introduction: Cannabigerol acid (CBGA) is the parent compound for the synthesis of other cannabinoids. CBGA is produced by the enzymatic reaction of olivetolic acid (OA) and geranyl pyrophosphate (GPP), catalyzed by CBGA synthase (an enzyme of the aromatic prenyltransferase family). Cyclization of this prenylated product (CBGA) yields three different cannabinoid products, catalyzed by three different oxidocyclases. 9 -Tetrahydrocannabinolate (THCA) synthase, cannabidiol (CBDA) synthase, and cannabichromic acid (CBCA) synthase are Δ 9 - It catalyzes the formation of tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), and cannabichromic acid (CBCA). The biosynthesis of these cannabinoid products in microbial hosts (E. coli) involves the cloning, expression, and activity determination of THCA synthase, CBGA synthase, CBCA synthase, CBDA synthase, and combinations thereof. Generally, cannabinoid products are produced in microbial hosts that express at least CBGA synthase, and optionally in combination with one or more of THCA synthase, CBCA synthase, and CBDA synthase.

[0180] CBGA Synthase We successfully cloned a Cannabis sativa-derived CBGA synthase gene, codon-optimized for E. coli, into a plasmid vector functionally linked to a strong IPTG-inducible T5 promoter. This plasmid contains a high-copy pUC replication origin and is kanamycin-resistant. The plasmid construct for CBGA synthase is shown in Figure 5. Expression of the CBGA synthase in E. coli was confirmed by SDS-PAGE analysis (Figure 6). After confirming CBGAS expression, the enzyme activity was determined by adding the substrates (OA and GPP) to the enzyme solution, and product profiling was performed using our proprietary HPLC method. The clarified cell lysate was added to a mixture of OA and GPP at 37°C to carry out the prenylation reaction. The reaction mixture was extracted with ethyl acetate, and the formation of the product was measured by HPLC (Figure 7). These results demonstrate that CBGAS can be expressed in E. coli and can catalyze the prenylation reaction from the substrates OA and GPP to CBGA.

[0181] In another experiment, the CBGA activity was tested in vivo. In this experiment, host cells containing a CBGAS expression plasmid were cultured, and then IPTG was added to promote logarithmic growth (OD). 600 Induction was initiated as soon as the irradiance (=0.6) was reached. During the induction phase, the cells were given GPP and OA and grown overnight. Next, the cell suspension was centrifuged, and the supernatant was injected into HPLC to confirm CBGA formation. Figure 8 shows the HPLC chromatogram for detecting CBGA produced in E. coli (using the low-copy plasmid, pBAD33). The concentration of CBGA produced was calculated to be approximately 1.2 μg / ml in a 5 mL culture.

[0182] THCA synthase (THCAS) The THCA synthase gene was also cloned into a high-copy kanamycin-resistant plasmid under the control of a strong IPTG-inducible T5 promoter. This gene insertion was confirmed by PCR and gene sequencing. A schematic diagram of the resulting THCA synthase gene expression cassette is shown in Figure 9. THCA synthase expression was induced by IPTG, and cell lysates were analyzed by SDS-PAGE to confirm expression. However, the THCA synthase was not successfully expressed in E. coli.

[0183] The THCA synthase gene encodes a flavinyl oxidase class protein with eight glycosylation sites and a 28-amino acid signal peptide at the N-terminus. These limitations of E. coli regarding the production of glycosylated transmembrane proteins make it difficult to express the active form of THCA synthase class proteins. To overcome this limitation, a multifactor strategy has been designed: the gene without the signal peptide is expressed to ensure protein expression in the cytosol; the gene is co-expressed with a modular chaperonin to assist protein folding; the production of the active protein is increased (e.g., Figure 10); and then co-expressed with the glycosylation mechanism to assist protein folding (e.g., Figure 11). Since CBDA synthase (CBDAS) belongs to the same protein family, the same strategy is applied to the expression of CBDA synthase. Similarly, since CBCA synthase (CBCAS) is a glycosylated protein with a native signal sequence, the same strategy is applied to the expression of CBCA synthase. This part of the research is still ongoing, and the following steps are being considered for THCAS, and those skilled in the art will understand that these steps are also applicable to CBDAS and CBCAS.

[0184] Disconnected THCAS THCAS is cleaved by removing 28 amino acids from the encoded N-terminus (84 bp from the 5' end of the gene sequence).

[0185] Co-expression with chaperonin plasmids The THCA synthase gene was cloned into an ampicillin-resistant plasmid with a pBR322 origin of replication under the control of the pTRC promoter, and it was confirmed that it has different and compatible origins of replication for co-expression in plasmids containing both chaperonin and glycosyl systems (Figure 11). THCA co-expresses with DnaK-DnaJ-GrpE and GroEl-GroES, two major chaperone pathway components of E. coli, which play distinct but cooperative roles in protein folding in E. coli, regardless of the presence or absence of signal peptides. Although E. coli possesses its native chaperone system, high levels of recombinant protein production can saturate the endogenous chaperonin system and cause unproductive aggregation. Under these conditions, increasing the intracellular concentration of molecular chaperones that restrict folding can suppress inclusion body formation. A group led by Yura Takahashi in Japan constructed a pACYC184-based plasmid with two chaperone pathways under different promoters. This plasmid (pG-KJE8), or its derivatives, can be used to identify the conditions for the production of active THCA synthase in host cells.

[0186] Co-expression using a glycosylation system Glycosylation may be required for the expression, folding, processing, solubility, and / or increased activity of THCA synthase in E. coli. Unfortunately, E. coli lacks a native protein glycosylation system. The N-glycosylation system from Campylobacter jejuni, also known as the pgl system, is successfully expressed in E. coli. We provide a plasmid containing the C. jejuni pgl system gene. This plasmid, or its derivatives, can be used to identify the conditions for the production of active THCA synthase in host cells (Figure 11).

[0187] Example 3 Optimization of cannabinoid pathway gene expression To optimize the endogenous production of CBGA in E. coli, CBGA synthase is co-expressed with components of the MEP pathway, GPP synthase, and / or polyketide pathway to provide the substrates GPP and OA necessary for CBGA production.

[0188] As potential co-expression candidates: • Co-expression with the MEP pathway that produces IPP and DMAPP • Co-expression with GPP synthase that endogenously supplies GPP from IPP and DMAPP. • Co-expression with the polyketide pathway, which provides endogenous olivetolic acid (OA), is one possibility.

[0189] To co-express two different plasmids in E. coli, low-copy-number or medium-copy-number plasmids were used to minimize adverse effects on cell proliferation, and two or more plasmids were maintained using interchangeable replication start sites. Independent antibiotic selection was also performed for stringent selection of each plasmid. Therefore, the CBGA synthase gene was cloned into the low-copy plasmid pBAD33. A schematic diagram of the low-copy CBGA synthase expression plasmid is shown in (Figure 12). The cloned CBGAS gene was validated by restriction digestion and sequencing. For co-expression of CBGAS with the MEP pathway and GPP synthase, GPP synthase was cloned upstream of CBGAS in pBAD33 and co-transformed with pTRC_RDE (Figure 13). After co-transformation, cells were grown with antibiotic selection, and then OD 600 Cells were induced when the concentration reached 0.6. The concentration of the inducing substance is shown in Figure 14.

[0190] After adding the inducing substance, the cells were grown overnight. To check the cell viability during protein production, OD (Oral Production) was performed. 600 The OD was measured for different cultures. 600A comparison is shown in Figure 15. Strains containing pBAD33_GPPS_CBGAS were induced with arabinose at concentrations of 10, 15, and 20 mM. Strains containing pBAD33_GPPS_CBGAS and pTRC_RDE were induced with different combinations of arabinose and IPTG concentrations. The proteins were extracted after cell lysis, and the protein concentration was measured using the Bradford method. Protein induction and expression were confirmed by SDS-PAGE (Figure 16). The extracted total protein concentration was plotted (Figure 17) and compared with the expression data from SDS-PAGE. The total protein concentration was found to increase with increasing inducer concentration and was inversely proportional with respect to cell population and growth.

[0191] Lee et al. discovered IPTG interference with arabinose induction. CBGAS expression was confirmed with different combinations of arabinose and IPTG concentrations. The total protein concentration was found to be even higher when cells were induced with 10 mM arabinose and 0.5 mM IPTG. Maintaining multiple plasmids increases the metabolic burden on cells from DNA, RNA, and protein synthesis, as well as the total number of antibiotic resistance proteins that the cells must produce, leading to low production of the desired end product. Furthermore, maintaining a balance between different inducers results in inconsistent production levels of the end product. To overcome these limitations, the entire gene cassette is constructed into a single plasmid containing the MEP pathway and the GPPS and CBGAS operons together. Alternatively, the MEP pathway, GPPS, and / or CBGAS are integrated into the host cell genome.

[0192] Example 4 Metagenomic screening and cloning of the bifunctional ispDF gene in the MEP pathway We screened the environmental metagenomics of soil bacteria to identify alternative MEP pathway genes. We identified a novel bifunctional enzyme in the non-mevalonate pathway that was significantly more active than the corresponding E. coli orthologue. This novel bifunctional enzyme colocalized the active sites of IspD and IspF onto a single polypeptide scaffold. The activity of the bifunctional gene was evaluated for lycopene production, surrogating cannabinoid synthesis. This gene was named ispDF, and was cloned using the same potent RBS in a pTrc-RDE operon in which ispD and ispF were replaced with ispDF. This genetically engineered plasmid was then processed using the pTrc-RDE operon. * It was named and then transformed with E. coli (DE3). Two further metagenomic bifunctional enzymes containing the active sites of ispD and ispF were named ispDF2 and ispDF3, respectively.

[0193] Protein expression research Plasmid pTrc-RDE, and pTrc-RDE * Strains containing [the specified ingredient] were tested for protein expression induced using IPTG. Cells were heated at 37°C in LB broth and then induced with [the specified ingredient]. 600 The cells were grown until the ratio reached 0.6, then IPTG was added to the culture medium to induce growth, and the culture was incubated overnight at 30°C. The cells were harvested from the culture and lysed, and total protein was extracted. The total protein concentration was estimated using the Bradford method and analyzed by SDS-PAGE gel. These gels were stained with Coomassie Brilliant Blue G-250 dye and visualized. pTrc-RDE and pTrc-RDE during expression * Both plasmids showed bands corresponding to the pathway enzymes on SDS-PAGE. ispDF expression was demonstrated by SDS-PAGE analysis of induced cell lysates (Figure 18).

[0194] ispDF modeling: The bifunctional ispDF derived from Campylobacter jejuni has been reported as CJ-ispDF. Our reported ispDF (ispDFl) was modeled using the Swiss model with CJ-ispDF. There is approximately 31% sequence similarity. The alignment of CJ-ispDF with native ispD and ispF shows different amino acids (Figure 19). This suggests that ispDF is novel and has not yet been reported. Further analysis regarding the co-localization of the active site is underway.

[0195] Functional analysis of platform stocks: Genetically modified E. coli strains were transformed with plasmids containing downstream genes for the conversion of C5 isoprenoid precursors to lycopene. Lycopene biosynthesis genes were cloned under the control of a constitutive promoter. The lycopene expression plasmid was low-copy and compatible with origins of replication for co-expression.

[0196] It has been reported that the ispD, ispF, and ispE enzymes form a multicomponent complex that leads to channel metabolites via three consecutive catalytic steps. To further investigate this, native ispE genes amplified from the genomes of both operons were cloned. Mutant plasmids were then created using pTrc-RDE and pTrc-RDE, respectively. * pTrc-RDEE and pTrc-RDE * This mutant was named E (Figure 20). Both of these mutants were also tested for functional aspects, including lycopene production, as described below.

[0197] Example 5 Increased terpenoid biosynthesis via heterologous expression of one or more components of the MEP pathway Various constructs were tested to assess their ability to support increased terpenoid biosynthesis (Figure 21).

[0198] Isoprene synthesis The following strains of E. coli were prepared: parental control strain (BL21); control strain SA01 containing an expression cassette encoding isoprene synthase (ispS) functionally linked to the arabinose promoter; strain SA02 containing the expression cassette from SA01 and an RDE expression cassette encoding dxs, ispD, ispF, and idi functionally linked to the Trc promoter; and the expression cassette from SA01 and an RDE encoding dxs, ispDF1, and idi functionally linked to the Trc promoter. * SA03 strain (Figure 22 left) including the expression cassette. In SA03, the designation ispDFl- indicates that the nucleic acid encoding ispDF1 is not codon-optimized for increased expression in heterologous host cells, while ispDF+ (not shown in Figure 22) indicates that the nucleic acid encoding the enzyme is codon-optimized. The culture was grown in a sealed glass culture tube at 230 rpm and 30°C. After the OD600 reached 0.6, the culture was induced and incubated for a further 20 hours. 0.05 mM IPTG and 10 mM arabinose were used as inducers. The headspace of the culture was analyzed by GC-MS. The culture was heated to 70°C before sample injection. Isoprene was quantified using a calibration curve. Isoprene production by the strains described herein is shown on the right in Figure 22.

[0199] Lycopene synthesis Plasmid pAC-LYC (Addgene plasmid #53270) was obtained. This plasmid contains an expression cassette with constitutive promoters functionally linked to the lycopene synthesis genes crtE, crtI, and crtB. See Cunningham FX Jr, et al., Plant Cell. 1994 Aug;6(8):1107-21. The following strains of E. coli were prepared: parental control strain (BL21(DE3)); strain RDE containing plasmid pAC-LYC and the above-mentioned RDE expression cassette; plasmid pAC-LYC and the above-mentioned RDE * Plasmid containing an expression cassette, and strain RDE containing an expression cassette *(DF1-); RDE containing plasmid pAC-LYC and nucleic acid encoding codon-optimized ispDF1 * RDE strain containing a plasmid with an expression cassette * (DF1+); RDE containing plasmid pAC-LYC and nucleic acid encoding codon-optimized ispDF2 * Plasmid containing an expression cassette, and strain RDE containing an expression cassette * (DF2+); and RDE having plasmid pAC-LYC and nucleic acid encoding codon-optimized ispDF3 * Plasmid containing an expression cassette, and strain RDE containing an expression cassette * (DF3+). Further strains produced include SA01;pAC-LYC containing pAC-LYC, SA02;pAC-LYC containing pTrc-RDE, SA03;pAC-LYC containing pTrc-RDEE, and pTrc-RDE. * SA04, which includes pAC-LYC, and pTrc-RDE * SA05 contains E. The sequences of ispDF1, ispDF2, and ispDF3 are shown in Figure 26.

[0200] For lycopene production, seed cultures of SA01-SA05 were grown overnight in LB medium at 30°C. These cultures were then diluted in fresh medium until the optical density reached 0.2, and subsequently induced with IPTG. The resulting cultures were incubated at 30°C for 20 hours with shaking at 250 rpm in the dark. Lycopene extraction was performed by extracting 4 mL of culture with 2 mL of acetone. This acetone extract was analyzed by HPLC. HPLC method: Column - C18, Mobile phase - methanol:tetrahydrofuran:water (66:30:4), Flow rate - 1 mL / min, Detection at 472 nm wavelength using a photodiode array detector and lycopene peak was confirmed with the in-house lycopene standard. Figure 23 shows that IPTG induction increases lycopene production. The optimal induction level varies depending on the strain.

[0201] In the second experiment, the cultures were grown overnight in LB at 30°C. These cultures were then diluted in LB until the OD600 reached 0.2. They were induced with IPTG and grown for 24 hours at 30°C. 2 mL of the culture was centrifuged at 8000 rpm for 5 minutes to produce a cell pellet. Lycopene from the cell pellet was extracted with 1 mL of acetone at 55°C for 15 minutes. The mixture was then centrifuged at 14000 rpm. The supernatant was analyzed on a C-18 column by HPLC using isocratic elution with a methanol, tetrahydrofuran, and water mixture (66:30:4) at a flow rate of 1 mL / min. Lycopene yield was measured as the area under the HPLC curve for the lycopene peak. The peak was validated against an in-house standard. This area was normalized for the RDE strain using 0 μM IPTG. The results are shown in Figure 24.

[0202] Monoterpene synthesis The upper part of Figure 25 shows a construct for producing monoterpenes in host cells. This construct includes an MEP pathway platform operon encoding dxs, codon-optimized ispDF1(DF1+), and idi, functionally ligated to the T7 promoter, and a monoterpene operon encoding GPP synthase and monoterpene synthase, functionally ligated to the Trc promoter. In this experiment, the monoterpene synthase was kalen synthase.

[0203] The cultures were grown overnight at 30°C in LB + 0.5% yeast extract. These cultures were then diluted in medium with 2 mM magnesium chloride until the OD600 reached 0.2, incubated at 37°C, and subsequently induced with IPTG until the OD600 reached 0.8. 10% dodecane was overlaid on the cultures. Monoterpene concentrations were analyzed by GC-MS using a standard curve. The results are shown at the bottom of Figure 25. The production of the monoterpenes limonene and myrcene was successfully achieved using limonene synthase and myrcene synthase, respectively. IspDF2+; ispDF3+; and dxs, ispD, ispF, and idi were also able to assist in the increased production of monoterpenes.

[0204] Exemplary arrangement Examples of arrangements mentioned in this application include, but are not limited to, those listed in the table below:

[0205] [Table 1]

[0206] Some genes were obtained from the GenBank database, while others were obtained from the Cannabis genome browser (genome.ccbr.utoronto.ca / cgi-bin / hgGateway). IM00001.1 is a sequence identifier for desaturase generated from publicly available Cannabis mRNA and EST sequences.

[0207] An example sequence is shown below. >AB057805.1| Cannabis sativa mRNA for tetrahydrocannabinolate synthase (THCAS) and complete cds ATGAATTGCTCAGCATTTTCCTTTTGGTTTGTTTGCAAAATAAT ATTTTTCTTTCTCTCATTCCATATCCAAATTTCAATAGCTAATCCTCGAGAAAACTTCCTTAAATGCTTC TCAAAACATATTCCCAACAATGTAGCAAATCCAAAACTCGTATACACTCAACACGACCAATTGTATATGT CTATCCTGAATTCGACAATACAAAATCTTAGATTCATCTCTGATACAACCCCAAAACCACTCGTTATTGT CACTCCTTCAAATAACTCCCATATCCAAGCAACTATTTTATGCTCTAAGAAAGTTGGCTTGCAGATTCGA ACTCGAAGCGGTGGCCATGATGCTGAGGGTATGTCCTACATATCTCAAGTCCCATTTGTTGTAGTAGACT TGAGAAACATGCATTCGATCAAAATAGATGTTCATAGCCAAACTGCGTGGGTTGAAGCCGGAGCTACCCT TGGAGAAGTTTATTATTGGATCAATGAGAAGAATGAGAATCTTAGTTTTCCTGGTGGGTATTGCCCTACT GTTGGCGTAGGTGGACACTTTAGTGGAGGAGGCTATGGAGCATTGATGCGAAATTATGGCCTTGCGGCTG ATAATATTATTGATGCACACTTAGTCAATGTTGATGGAAAAGTTCTAGATCGAAAATCCATGGGAGAAGA TCTGTTTTGGGCTATACGTGGTGGTGGAGGAGAAAACTTTGGAATCATTGCAGCATGGAAAATCAAACTG GTTGCTGTCCCATCAAAGTCTACTATATTCAGTGTTAAAAAGAACATGGAGATACATGGGCTTGTCAAGT TATTTAACAAATGGCAAAATATTGCTTACAAGTATGACAAAGATTTAGTACTCATGACTCACTTCATAAC AAAGAATATTACAGATAATCATGGGAAGAATAAGACTACAGTACATGGTTACTTCTCTTCAATTTTTCAT GGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGCTTTCCTGAGTTGGGTATTAAAAAAACTGATT GCAAAGAATTTAGCTGGATTGATACAACCATCTTCTACAGTGGTGTTGTAAATTTTAACACTGCTAATTT TAAAAAGGAAATTTTGCTTGATAGATCAGCTGGGAAGAAGACGGCTTTCTCAATTAAGTTAGACTATGTT AAGAAACCAATTCCAGAAACTGCAATGGTCAAAATTTTGGAAAAATTATATGAAGAAGATGTAGGAGCTG GGATGTATGTGTTGTACCCTTACGGTGGTATAATGGAGGAGATTTCAGAATCAGCAATTCCATTCCCTCA TCGAGCTGGAATAATGTATGAACTTTGGTACACTGCTTCCTGGGAGAAGCAAGAAGATAATGAAAAGCAT ATAAACTGGGTTCGAAGTGTTTATAATTTTACGACTCCTTATGTGTCCCAAAATCCAAGATTGGCGTATC TCAATTATAGGGACCTTGATTTAGGAAAAACTAATCATGCGAGTCCTAATAATTACACACAAGCACGTAT TTGGGGTGAAAAGTATTTTGGTAAAAATTTTAACAGGTTAGTTAAGGTGAAAACTAAAGTTGATCCCAAT AATTTTTTTAGAAACGAACAAAGTATCCCACCTCTTCCACCGCATCATCATTAA(SEQ ID NO: 4)

[0208] > AB292682.1 | Cannabis sativa CBDAS mRNA for cannabidiolic acid synthase (CBDAS) with transport peptides ATGAAGTGCTCAACATTCTCCTTTTGGTTTGTTTGCAAGATAATATTTTTCTTTTTCTCATTCAATATCC AAACTTCCATTGCTAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAAC AAATCTAAAACTCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAAT CTTAGATTCACCTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCTCATATCC AAGGCACTATTCTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTGGTCATGATTCTGA GGGCATGTCCTACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAACATGCGTTCAATCAAAATA GATGTTCATAGCCAAACTGCATGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATG AGAAAAAATGAGAATCTTAGTTTGGCGGCTGGGTATTGCCCTACTGTTTGCGCAGGTGGACACTTTGGTGG AGGAGGCTATGGACCATTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTC AACGTTCATGGAAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTG GAGCAGAAAGCTTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTT TAGTGTTAAAAAGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTAC AAGTATGACAAAGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGA ATAAGACAGCAATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTTGAT GAACAAGAGTTTTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATTGATACTATC ATCTTCTATAGTGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTTTGCTTGATAGATCCG CTGGGCAGAACGGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGT CCAAATTTTGGAAAAATTATATGAAGAAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGT ATAATGGATGAGATTTCAGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGT ACATATGTAGTTGGGAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTT CATGACTCCTTATGTGTCCAAAAATCCAAGATTGGCATATCTCAATTATAGAGACCTTGATATAGGAATA AATGATCCCAAGAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATT TTGACAGGCTAGTAAAAGTGAAAACCCTGGTTGATCCCAATAACTTTTTTAGAAACGAACAAAGCATCCC ACCTCTTCCACGGCATCGTCATTAA (Sequence ID 5)

[0209] >IM00002.1| Cannabis sativa CBDAS mRNA for cannabidiolic acid synthase II (CBDASII) that lacks a transport peptide ATGAATCCTCGAGAAAACTTCCTTAAATGCTTCTCGCAATATATTCCCAATAATGCAACAAATCTAAAAC TCGTATACACTCAAAACAACCCATTGTATATGTCTGTCCTAAATTCGACAATACACAATCTTAGATTCAC CTCTGACACAACCCCAAAACCACTTGTTATCGTCACTCCTTCACATGTCTCTCATATCCAAGGCACTATT CTATGCTCCAAGAAAGTTGGCTTGCAGATTCGAACTCGAAGTGGTGGTCATGATTCTGAGGGCATGTCCT ACATATCTCAAGTCCCATTTGTTATAGTAGACTTGAGAAACATGCGTTCAATCAAAATAGATGTTCATAG CCAAACTGCATGGGTTGAAGCCGGAGCTACCCTTGGAGAAGTTTATTATTGGGTTAATGAGAAAAATGAG AATCTTAGTTTGGGCGGCTGGGTATTGCCCTACTGTTTGCGCAGGTGGACACTTTGGTGGAGGAGGCTATG GACCATTGATGAGAAACTATGGCCTCGCGGCTGATAATATCATTGATGCACACTTAGTCAACGTTCATGG AAAAGTGCTAGATCGAAAATCTATGGGGGAAGATCTCTTTTGGGCTTTACGTGGTGGTGGAGCAGAAAGC TTCGGAATCATTGTAGCATGGAAAATTAGACTGGTTGCTGTCCCAAAGTCTACTATGTTTAGTGTTAAAA AGATCATGGAGATACATGAGCTTGTCAAGTTAGTTAACAAATGGCAAAATATTGCTTACAAGTATGACAA AGATTTATTACTCATGACTCACTTCATAACTAGGAACATTACAGATAATCAAGGGAAGAATAAGACAGCA ATACACACTTACTTCTCTTCAGTTTTCCTTGGTGGAGTGGATAGTCTAGTCGACTTGATGAACAAGAGTT TTCCTGAGTTGGGTATTAAAAAAACGGATTGCAGACAATTGAGCTGGATTGATACTATCATCTTCTATAG TGGTGTTGTAAATTACGACACTGATAATTTTAACAAGGAAATTTTGCTTGATAGATCCGCTGGGCAGAAC GGTGCTTTCAAGATTAAGTTAGACTACGTTAAGAAACCAATTCCAGAATCTGTATTTGTCCAAATTTTGG AAAAATTATATGAAGAAGATATAGGAGCTGGGATGTATGCGTTGTACCCTTACGGTGGTATAATGGATGA GATTTCAGAATCAGCAATTCCATTCCCTCATCGAGCTGGAATCTTGTATGAGTTATGGTACATATGTAGT TGGGAGAAGCAAGAAGATAACGAAAAGCATCTAAACTGGATTAGAAATATTTATAACTTCATGACTCCTT ATGTGTCCAAAAATCCAAGATTGGCATATCTCAATTATAGAGACCTTGATATAGGAATAAATGATCCCAA GAATCCAAATAATTACACACAAGCACGTATTTGGGGTGAGAAGTATTTTGGTAAAAATTTTGACAGGCTA GTAAAAGTGAAAACCCTGGTTGATCCCAATAACTTTTTTAGAAACGAACAAAGCATCCCACCTCTTCCAC GGCATCGTCATTAA (SEQ ID NO: 6)

[0210] > PK28436.1 | Aromatic prenyltransferase (PT), or geranylpyrophosphate olivetolate geranyltransferase ATGGGACTCTCATCAGTTTGTACCTTTTCATTTCAAACTAATTACCATACTTTATTAAATCCTCACAATA ATAATCCCAAAACCTCATTATTATGTTATCGACACCCCAAAACACCAATTAAATACTCTTACAATAATTT TCCCTCTAAACATTGCTCCACCAAGAGTTTTCATCTACAAAACAAATGCTCAGAATCATTATCAATCGCA AAAAATTCCATTAGGGCAGCTACTACAAATCAAACTGAGCCTCCAGAATCTGATAATCATTCAGTAGCAA CTAAAATTTTAAACTTTGGGAAGGCATGTTGGAAACTTCAAAGACCATATACAATCATAGCATTTACTTC ATGCGCTTGTGGATTGTTTGGGAAAGAGTTGTTGCATAACACAAATTTAATAAGTTGGTCTCTGATGTTC AAGGCATTCTTTTTTTTGGTGGCTATATTATGCATTGCTTCTTTTACAACTACCATCAATCAGATTTACG ATCTTCACATTGACAGAATAAACAAGCCTGATCTACCACTAGCTTCAGGGGAAATATCAGTAAACACAGC TTGGATTATGAGCATAATTGTGGCACTGTTTGGATTGATAATAACTATAAAAATGAAGGGTGGACCACTC TATATATTTGGCTACTGTTTTGGTATTTTTGGTGGGATTGTCTATTCTGTTCCACCATTTAGATGGAAGC​AAAATCCTTCCACTGCATTTCTTCTCAATTTCCTGGCCCATATTATTACAAATTTCACATTTTATTATGC CAGCAGAGCAGCTCTTGGCCTACCATTTGAGTTGAGGCCTTCTTTTACTTTCCTGCTAGCATTTATGAAA TCAATGGGTTCAGCTTTGGCTTTAATCAAAGATGCTTCAGACGTTGAAGGCGACACTAAATTTGGCATAT CAACCTTGGCAAGTAAATATGGTTCCAGAAACTTGACATTATTTTGTTCTGGAATTGTTCTCCTATCCTA TGTGGCTGCTATACTTGCTGGGATTATCTGGCCCCAGGCTTTCAACAGTAACGTAATGTTACTTTCTCAT GCAATCTTAGCATTTTGGTTAATCCTCCAGACTCGAGATTTTGCGTTAACAAATTACGACCCGGAAGCAG GCAGAAGATTTTACGAGTTCATGTGGAAGCTTTATTATGCTGAATATTTAGTATATGTTTTCATATAA(SEQ ID NO: 7)

[0211] > JN679224.1| Cannabis sativa Olivetolic acid cyclase (OAC) mRNA, complete cds ATGGCAGTGAAGCATTTGATTGTATTGAAGTTCAAAGATGAAATCACAGAAGCCCAAAAGGAAGAATTTT TCAAGACGTATGTGAATCTTGTGAATATCATCCCAGCCATGAAAGATGTATACTGGGGTAAAGATGTGAC TCAAAAGAATAAGGAAGAAGGGTACACTCACATAGTTGAGGTAACATTTGAGAGTGTGGAGACTATTCAG GACTACATTATTCATCCTGCCCATGTTGGATTTGGAGATGTCTATCGTTCTTTCTGGGAAAAACTTCTCA TTTTTGACTACACACCACGAAAGTAG (SEQ ID NO: 8)

[0212] > AB164375.1| Cannabis sativa OLS mRNA for olivetol synthase (OLS), complete cds ATGAATCATCTTCGTGCTGAGGGTCCGGCCTCCGTTCTCGCCATTGGCACCGCCAATCCGGAGAACATTT TATTACAAGATGAGTTTCCTGACTACTATTTTCGCGTCACCAAAAGTGAACACATGACTCAACTCAAAGA AAAGTTTCGAAAAATATGTGACAAAAGTATGATAAGGAAACGTAACTGTTTCTTAAATGAAGAACACCTA AAGCAAAACCCAAGATTGGTGGAGCACGAGATGCAAACTCTGGATGCACGTCAAGACATGTTGGTAGTTG AGGTTCCAAAACTTGGGAAGGATGCTTGTGCAAAGGCCATCAAAGAATGGGGTCAACCCAAGTCTAAAAT CACTCATTTAATCTTCACTAGCGCATCAACCACTGACATGCCCGGTGCAGACTACCATTGCGCTAAGCTT CTCGGACTGAGTCCCTCAGTGAAGCGTGTGATGATGTATCAACTAGGCTGTTATGGTGGTGGAACCGTTC TACGCATTGCCAAGGACATAGCAGAGAATAACAAAGGCGCACGAGTTCTCGCCGTGTGTTGTGACATAAT GGCTTGCTTGTTTCGTGGGCCTTCAGAGTCTGACCTCGAATTACTAGTGGGACAAGCTATCTTTGGTGAT GGGGCTGCTGCGGTGATTGTTGGAGCTGAACCCGATGAGTCAGTTGGGGAAAGGCCGATATTTGAGTTGG TGTCAACTGGGCAAACAATCTTACCAAACTCGGAAGGAACTATTGGGGGACATATAAGGGAAGCAGGACT GATATTTGATTTACATAAGGATGTGCCTATGTTGATCTCTAATAATATTGAGAAATGTTTGATTGAGGCA TTTACTCCTATTGGGATTAGTGATTGGAACTCCATATTTTGGATTACACACCCAGGTGGGAAAGCTATTT TGGACAAAGTGGAGGAGAAGTTGCATCTAAAGAGTGATAAGTTTGTGGATTCACGTCATGTGCTGAGTGA GCATGGGAATATGTCTAGCTCAACTGTCTTGTTTGTTATGGATGAGTTGAGGAAGAGGTCGTTGGAGGAA GGGAAGTCTACCACTGGAGATGGATTTGAGTGGGGTGTTCTTTTTGGGTTTGGACCAGGTTTGACTGTCG AAAGAGTGGTCGTGCGTAGTGTTCCCATCAAATATTAA(SEQ ID NO: 9)

[0213] > JN717233.1 | PK04797.1 | Acyl-activating enzyme 1 (AAE1) ATGGGTAAGAATTACAAGTCCCTGGACTCTGTTGTGGCCTCTGACTTCATAGCCCTAGGTATCACCTCTG AAGTTGCTGAGACACTCCATGGTAGACTGGCCGAGATCGTGTGTAATTATGGCGCTGCCACTCCCCAAAC ATGGATCAATATTGCCAACCATATTCTGTCGCCTGACCTCCCCTTCTCCCTGCACCAGATGCTCTTCTAT GGTTGCTATAAAGACTTTGGACCTGCCCCTCCTGCTTGGATACCCGACCCGGAGAAAGTAAAGTCCACCA ATCTGGGCGCACTTTTGGAGAAGCGAGGAAGAGGTTTTTGGGAGTCAAGTATAAGGATCCCATTTCAAG CTTTTCTCATTTCCAAGAATTTTCTGTAAGAAACCCTGAGGTGTATTGGAGAACAGTACTAATGGATGAG ATGAAGATAAGTTTTTCAAAGGATCCAGAATGTATATTGCGTAGAGATGATGACATTAATAATCCAGGGG GTAGTGAATGGCTTCCAGGAGGTTATCTTAACTCAGCAAAGAATTGCTTGAATGTAAATAGTAACAAGAA ATTGAATGATACAATGATTGTATGGCGTGATGAAGGAAATGATGATTTGCCTCTAAACAAATTGACACTT GACCAATTGCGTAAACGTGTTTGGTTAGTTGGTTATGCACTTGAAGAAATGGGTTTGGAGAAGGGTTGTG CAATTGCAATTGATATGCCAATGCATGTGATGCTGTGGTTATCTATCTAGCTATTGTTCTTGCGGGATA TGTAGTTGTTTCTATTGCTGATAGTTTTTCTGCTCCTGAAATATCAACAAGACTTCGACTATCAAAAGCA AAAGCCATTTTTACACAGGATCATATTATTCGTGGGAAGAAGCGTATTCCCTTATACAGTAGAGTTGTGG AAGCCAAGTCTCCCATGGCCATTGTTATTCCTTGTAGTGGCTCTAATATTGGTGCAGAATTGCGTGATGG CGATATTTCTTGGGATTACTTTCTAGAAAGAGCAAAAGAGTTTAAAAATTGTGAATTTACTGCTAGAGAA CAACCAGTTGATGCCTATACAAACATCCTCTTCTCATCTGGAACAACAGGGGAGCCAAAGGCAATTCCAT GGACTCAAGCAACTCCTTTAAAAGCAGCTGCAGATGGGTGGAGCCATTTGGACATTAGGAAAGGTGATGT CATTGTTTGGCCCACTAATCTTGGTTGGATGATGGGTCCTTGGCTGGTCTATGCTTCACTCCTTAATGGG GCTTCTATTGCCTTGTATAATGGATCACCACTTGTTTCTGGCTTTGCCAAATTTGTGCAGGATGCTAAAG TAACAATGCTAGGTGTGGTCCCTAGTATTGTTCGATCATGGAAAAGTACCAATTGTGTTAGTGGCTATGA TTGGTCCACCATCCGTTGCTTTTCCTCTTCTGGTGAAGCATCTAATGTAGATGAATACCTATGGTTGATG GGGAGAGCAAACTACAAGCCTGTTATCGAAATGTGTGGTGGCACAGAAATTGGTGGTGCATTTTCTGCTG GCTCTTTCTTACAAGCTCAATCATTATCTTCATTTAGTTCACAATGTATGGGTTGCACTTTATACATACT TGACAAGAATGGTTATCCAATGCCTAAAAACAAACCAGGAATTGGTGAATTAGCGCTTGGTCCAGTCATG TTTGGAGCATCGAAGACTCTGTTGAATGGTAATCACCATGATGTTTATTTTAAGGGAATGCCTACATTGA ATGGAGAGGTTTTAAGGAGGCATGGGGACATTTTTGAGCTTACATCTAATGGTTATTATCATGCACATGG TCGTGCAGATGATACAATGAATATTGGAGGCATCAAGATTAGTTCCATAGAGATTGAACGAGTTTGTAAT GAAGTTGATGACAGAGTTTTCGAGACAACTGCTATTGGAGTGCCACCTTTGGGCGGTGGACCTGAGCAAT TAGTAATTTTCTTTGTATTAAAAGATTCAAATGATACAACTATTGACTTAAATCAATTGAGGTTATCTTT CAACTTGGGTTTACAGAAGAAACTAAATCCTCTGTTCAAGGTCACTCGTGTTGTGCCTCTTTCATCACTT CCGAGAACAGCAACCAACAAGATCATGAGAAGGGTTTTGCGCCAACAATTTTCTCACTTTGAATGA(SEQ ID NO: 10)

[0214] > JN717235.1 | PK13710.1| Acyl-activating enzyme 3 (AAE3) ATGGAGAAATCTGGGTATGGAAGAGACGGTATTTACAGGTCTCTGAGACCACCTCTACACCTCCCCAACA ACAACAACCTCTCAATGGTTTCATTCCTTTTCAGAAACTCATCTTCATACCCACAAAAGCCAGCTCTCAT TGATTCCGAAACCAACCAAATACTCTCCTTTTCCCACTTCAAATCTACGGTTATCAAGGTCTCCCATGGC [[ID=!16]]TTTCTCAATCTGGGTATCAAGAAAAACGACGTCGTTCTCATCTACGCCCCTAATTCTATCCACTTCCCTG TTTGTTTCCTTGGAATTATAGCCTCTGGAGCCATTGCCACTACCTCAAATCCTCTCTACACAGTTTCCGA [[ID=2!0]]GCTTTCCAAACAGGTCAAGGATTCCAATCCCAAACTCATTATCACCGTTCCTCAACTCTTGGAAAAAGTA AAGGGTTTCAATCTCCCCACGATTCTAATTGGTCCTGATTCTGAACAAGAATCTTCTAGTGATAAAGTAA TGACCTTTAACGATTTGGTCAACTTAGGTGGGTCGTCTGGCTCAGAATTTCCAATTGTTGATGATTTTAA Note: There seems to be a formatting issue in the original text where "配列番号10" should be "SEQ ID NO: 10" in English. Also, there might be a typo in the tag "!16" and "2!0" which are likely incorrect tags in the original context. I've translated them as they are but they might need to be corrected in the source for proper representation.GCAGGTGACACTGCTGCGCTATTGTACTCATCTGGCACAACGGGAATGAGTAAAGGTGTGGTTTTGACT CACAAAAACTTCATTGCCTCTTCTTTAATGGTGACAATGGAGCAAGACCTAGTTGGAGAGATGGATAATG TGTTCTATGCTTTTTGCCAATGTTTCATGTATTTGGTTTGGCTATCATCACCTATGCTCAGTTGCAGAG AGGAAACACTGTTATTTCAATGGCGAGATTTGACCTTGAGAAGATGTTAAAAGATGTGGAAAAGTATAAA GTTACCCATTTGTGGGTTGTGCCTCCTGTGATACTGGCTCTGAGTAAGAACAGTTTGGTGAAGAAGTTTA ATTCTTTCTTCTATAAAGTATATTGGCTCTGGTGCAGCTCCTTTGGGCAAAGATTTAATGGAGGAGTGCTC TAAGGTTGTTCCTTATGGTATTGTTGCTCAGGGATATGGTATGACAGAAACTTGTGGGATTGTATCCATG GAGGATATAAGAGGAGGTAAACGAAATAGTGGTTCAGCTGGAATGCTGGCATCTGGAGTAGAAGCCCAGA TAGTAGTGTAGATACACTGAAGCCCTTACCTCCTAATCAATTGGGGGAGATATGGGTGAAGGGGCCTAA TATGATGCAAGGTTACTTCAATAACCCACAGGCAACCAAGTTGACTATAGATAAGAAAGGTTGGGTACAT ACTGGTGATCTTGGATATTTTGATGAAGATGGACATCTTTATGTTGTTGACCGTATAAAAGAGCTCATCA AATATAAAGGATTTCAGGTTGCTCCTGCTGAGCTTGAAGGATTGCTTGTTTCTCACCCTGAAATACTCGA TGCTGTTGTGATCCCATTTCCTGATGCTGAAGCGGGTGAAGTCCCAGTTGCTTATGTTGTGCGCTCTCCC AACAGTTCATTAACCGAAAATGATGTGAAGAAATTTATCGCGGGCCAGGTTGCATCTTTCAAAAGATTGA GAAAAGTAACATTTATAAACAGTGTCCCGAAATCTGCTTCGGGGAAAATCCTCAGAAGAGAACTCATTCA GAAAGTACGCTCCAACATGTGA (SEQ ID NO: 11)

[0215] > PK04410.1 | Hydroperoxidase (HPL) ATGTCTTTTATGATGAGCATGAATCCTTCTCCCTCCTCGCCACCGCCACCGTTATCGTCGCCGTCGGAAT CTTCCTCAACGCCGTCAACACTGCCAGTCCGTACGATCCCGGGAAGCTACGGATGGCCGTTACTGGGGCC CATCTCGGACCGGTTAGACTACTTCTGGTTCCAAGGCCCAGATACGTTTTTCAGAAAAAGAGTAGAGAAA TACAAGAGCACAGTGTTCCGTACCAACATACCCCCGACCTTTCCTTTCTTCAGCGTTAATCCGAACATTG TGGCCGTGCTGGACTGTAAATCATTTTCTCATCTTTTCGACATGGAAATTGTCGAGAAAAAGAATGTTCT TGTTGGAGATTTCATGCCCAGTGTCAATTACACTGGTGATATTAGGGTTGGAGCTTATCTCGACACTTCT GAACCACAACACGCTAAGGTTAAGAACTTCGCAATGGATGTACTAAAACAAAGCTCGAAGATATGGGTGG GAGAACTGACATCAAATCTGTCGACGATGTGGGACACAATAGAAAAAGACGTATCTGAGAAATCATCCTC ATCCTACTTAGCCCCACTTCAAAAGTTCTTGTTCAACTTCCTGGTCAAGTGTCTAATTGGTGCTGACCCT TCCAACTCCCCCAAGATTGCAGAGTCTGGCTACATCATGCTCGACCGATGGTTAGCCTTCCAGCTCCTTC CCACTATCAAGATTGGGATCCTTCAGCCTCTTGAGGAGCTTTTCATTCACTCTTTTGCCTATCCTTTTTT CTTGGTCAGTGGTGACTACAATAACCTCTCCAGTTTTGTAGAGGAATATGGTAAAGAAATAGTAGCGAGA GGTGAAACCGAGTTCGGGCTGAGTAAACAAGAAGCGATTCACAACCTTCTCTTCATTTTGGGTTTCAACG CCTTCGGGGGATTCTCTATATTTCTACCGAGCCTACTGGGCACCGTGGCGAGTGACACAACCGATCTACA ACAAAGACTGGTCAAAGAAGTCAGACAAAATGGCGGGTCAACTCTGACGTTTGACTCGATCAAAGAAATG CCACTCGTTCAATCGGTCGTGTACGAGACTCTCCGGCTCAATCCACCTGTTCCGCTCCAATTCGCCAGGG CCAGGAAGGACTTCCGGCTCAGCTCGCACGACGCGGCCTTCGAGGTGAAGAAAGGCGAGCTCCTATGCGG GTTTCAAAGCCTTGTTATGAGGGACCCAAAAATATTCTCGGAACCGGAGTCGTTCATTGGGGACCGGTTC ATGAAAGATAAAGGTCTCTTAGATTATCTTTACTGGTCCAATGGACCTCAAACCGGTGTGCCCAGCGTCA CCAATAAGCAATGCGCGGGAAAAGATATCGTCACGCTTACGGCTTGTTTGATCTTGGCTTACACCTTCCG TCGTTATGACTCCATCAGCGGGAGCTCAAGTTCAATCACAGCCCTTAAAAAGGCTTAA(SEQ ID NO: 12)

[0216] > PK08276.1 | Lipoxygenase (LOX1) ATGTTGAAGCCTCCTCATCAAGTAGTTCAAAATTTGAAATATGAGAAAACCCTAGTTCTTTTGAACAAGC CATTCATCCATGGCTACAACGGGGCTATTATCGGTGTCAACTCTCGGCTATTTCCAGTAAAACCTAAAAC CAAAAGACGAGTCGCTTCATCATCATCATCATCATCTCCCGGAACCAAAAACATTATTAAAGCTTCTTTA TTTTCTCCAATGGAGAAGAAGAATACAGCTAGGGTTTCGGTTAGTGTGGCGGTACAACGTGTGACTCCAA AGTTTTGGAGATTTGAATTGTCTGAGAAAATCCAAGATGGACGTGATAGGCTTGAGGATCTTCTAGGGCT AAACTCTTTAAGTATTGAGCTTGTTAGTACTCAAAAAGATCCAGTAACGGGGAAAGAGCGAACGGTTAAA GGTTTTCCAAAAAGGCCCAACTTTAACATATTTTCATCAAGTGATGTAAAATACGAAGCGAAATTTGACA TACCAAAAGATTTTGGAGAAGTGGGTGCTATAATCGTCGAAAATGATTTTGAAAGAGAAATATTTTTAAA GAATATTATACTCGAAGACTTGCCCTCCGAACCAAGCACCCTTGAATTCTCTTGCAACTCGTGGGTTCAG TCCAAACATGATGTCCCTACTGATCAACACAAGAGAGTCTTCTTCTCTAATAAGTGTTACCTACCATCAC AAACACCAAGTGGGATAAAAGAATTGAGAAAAATTGCATTGGAAAATTTGAGAGGAGATGGAAAAGGAGA GAGGAAGAAGAATGAAAGAGTTTATGATTATGATGTGTATAATGATCTTGGACAACCGGACAACAATGAT GACCTAAAAAGACCTATTCTTGGCGGATCAAAAGAATTCCCTTATCCTAGGCGTTGTAGAACCGGACGGC CTCCAACTGAAACTGATCCATTATCTGAGTCAAGGATTAGTGATTTTTATGTACCAAGAGATGAAGAATT TGCAGAAGTGAAGCAAAGTAATTTTAGTTTGAAGACTGTATACTCAGTAATACATGCAGTGATTCCCATA CTCAGACAAGTCTTAATTGATGAAAATTTCCCATACTTCACTGCCATTGATGTTCTCTATGATGAAGGCA TTAAAATCCCTTCTAATGCTGAAAAGACCTTAATTCAAACCATCAAAAATGTCAATGCAAGAATATACAA AACTGTTTCTGATGCTGATGATTTTTTACAGTTTCAGCAGCCTCCAACCATGGACAAGGACAAATTCTTC TGGTTTAGAGATGAAGAGTTTTGTAGACAAACTATTGCCGGTCTCAACCCTTGCTGCATTGAATTGGTTA AGGAGTGGCCTTTGAAAAGTGAACTTGACCCCACAATCTATGGCCCACCAGAGTCAAAAATCACCACAGA ATTGGTTGAGAAATTCATCAAAGTATATGGCTACAATAATATTAATGAGGCTTTAAAAGAAAAAAAATTG TTCATGTTGGATTACCATGATGTATTATTACCATATGTTAGCAAAGTAAGGGAACTGGAAAATAAAACCT TGTATGGATCAAGAACACTTTTTTTCTTGACTCCTTATGGTACATTGTTGCCTTTGGCCATTGAATTGAT TCGGCCACCGATGGATGGTAAGCCGCAATGGAAGGAAGTCTACACCCCGATGAATTGGCATTCTACCGAT CTTTGGCTTTGGAGACTCGCAAAAGCTCATGTCCTTGCTCATGATTCCGGTGTTCATCAACTCGTTAGTC ACTGGCTAAGAACACATTGTGCAGTTGAGCCATATATAATTGCAACAAATAGACAATTGAGTGCAATGCA TCCTATCCATAGATTATTGAAGCCACATTTTAGATACACAATGGAGATTAATGCTCTTGCTCGAGAAAGT TTGATCAATGCAGGTGGTATCATCGAAACAGCATTTGCACCTGGAAAATATTCTATGGAGTTAAGCTCCG TCATGTACGACAAACAATGGCGATTCGATCTACAAGCATTGCCAGCTGACCTAATTCATAGAGGAATGGC TGTTGAGGACAAGGATAGTGAACATGGTGTAAGAGTAATAATTGAAGATTACCCTTACGCCAACGACGGT CTTCTCATATGGAGCTCCATCAAACAATGGGTTACTGACTACGTCAACCACTACTACCCTACCTCCAGTG AGGTAGAGCGCGACGAAGAATTACAAGCATGGTGGACAGAGATCAGAACTGTAGGTCACGCTGACAAGAA AGACGCACCTGGGTGGCCTGACTTAAAAACGAAACAAGATCTCATAGACATTGTCACAAACATGGCATGG ACAGCATCAGCTCACCATGCAGCTGTCAACTTTGGACAATATGCTTACGCTGGCTATTTCCCTAACCGAC CAACCATAACAAGAACTGTTATGCCGTCAGAAGAGAAGGAGTATAACCTAGATGCGTGGAAACACTTCAA AAATAGTCCTGAAGACGCCCTTTTGAAGTGCTTACCTACGCAATTACAAGCAGGCCTAGTTGTGGCCGTG TTAGACGTGTTGTCTACTCACTCGCCAGACGAAGAGTATCTTGGAGACAAGATGGAACCCTCGTGGGGCT CGAATCTTGTTATAGCGGAAGCTTTTAATCGGTTCAATAAGAGGATGAACGAGATTGAAAGTATCATTAA TGAAAAGAATGATAATGAGAATTTAAGGAATAGACATGGAGCTGGAATTTTGTCTTATGAACTTCTCAAG CCCTTTTCTGAGCCTGGTGTCACTAACAAGGGTATTCCATATAGCATATCTATTTGA(SEQ ID NO: 13)

[0217] > IM00001.1 | Desaturase (DS) coding sequence ATGGGAGCCGGTGGCAAAAATAGTAGACTTGAGCGAGCACCACACACCACACCACCATTCACACTAAGCC AACTCAAGAAAGCCATTCCACCCCATTGCTTCAACCATTCTCTTCTTCGTTCCTTCTCTCATGTCCTTCA AGACCTTTTTTTCTCCTTTTTGTTCTACTACATAGCAACCTCTTACTTCCATCTTCTCCCACACCCGCTC CAATACTTAGCTTGGCCACTTTATTGGATCTTCCAAGGCAGCATTTTTGCTGGTATTTGGGTCCTTGGTC ATGATTGTGGTCACCAAGCTTTCAGTGACCACCAATGGGTGGATGACACcGTTGGCTTGTCCTCCCACTC CGCTCTTCTCTTCCCATACTTCTCTTTTAAGTATAGTCATCGTCGCCATCATTCAAACATCGGCTCCCTT GAACATGATCAATTGTTTGTTCCAGTCCCCGAATCTCAAATCGCATGGCTCTACAAACgTTACTTGGACA ATCCACTAGGAAGAGCCCTAAAGCTTTCCACTATAGTGTTCCTTGGTTtTCCTTTGTACTTAGGTTTCAA TCTTACAGGCAAACcATATGATCGTTaTGCATGTCATTATGATCCTTACTCTCCACTCTACTCAAAAAGT GAAAGGCTTCATATATTGATTTCAGATATCGGTGTTTTCATCACCACATTaGTGTTATACCAGCTTGGCT CGACTAAAGGgTTGAGTTGGCTTGTGTTCATGTATGGGGTGCCATTGTTTACAGGGAATAGCATCCTTGT GACAATCGCATACTTGAATCATACTCACCCTTCATTGCCTATTATGACTCGTCaGAGTGGGATTGGTTG AAAGGAGCATTGTCAACAACTGATCGAAACTATGGATCAATTCTCAATAGGGTTTTCCATCACCTTACAG ATGCTCATATGGCACACCATTTATTCGCAACAATACCTCACTACCATGCAAATGAAGCCACCAAAGTTAT CAAATCCATATTGGGAGAATACTACTCTTTTGATGATACTCCAATAATTAAAGCTCTTTGGAGAGAGACT AAGGAGTGTGTCTATATTGAGCCAAATCATGAATCTTCTCCTAATAATAACAAAGGTGTTTTTCTGGTACA ACAACAAGTTCTGA (SEQ ID NO: 14)

[0218] > PK10442.1 | Geranyl pyrophosphate (GPP) synthase large subunit | GPP synthase lsu ATGAGCACTGTAAATCTCACATGGGTTCAAACCTGTTCCATGTTCAACCAAGGAGGTAGATCCAGATCCT TATCAACTTTCAATCTCAATCTCTACCACCCTTTGAAAAAAACACCCTTTTCAATCCAAACCCCAAAACA AAAACGACCCACTTCACCATTTTCATCAATCTCAGCTGTTCTAACCGAGCAAGAAGCCGTTAAAGAAGGC GATGAAGAAAAATCCATCTTCAATTTCAAGTCTTACATGGTCCAAAAAGCCAACTCAGTCAACCAAGCTT TAGACTCAGCCGTTTTGCTCAGAGATCCCATTATGATACACGAGTCCATGCGTTACTCACTCCTCGCCGG AGGAAAACGAGTCAGACCCATGCTCTGTCTCTCAGCCTGTGAACTCGTAGGCGGAAAAGAATCCGTAGCC ATGCCGGCTGCCTGCGCCGTCGAAATGATCCACACCATGTCTCTAATCCACGACGACCTCCCTTGTATGG ACAACGATGACCTCCGCCGTGGAAAGCCCACAAACCACAAAGTCTTCGGAGAAGACGTGGCCGTTTTAGC CGGCGATGCACTTTTAGCCTTTGCTTTTGAGCACATGGCGGTCTCTACCGTTGGTGTTCCGGCAGCCAAG ATTGTCAGGGCGATTGGTGAGCTTGCTAAGTCAATTGGGTCAGAAGGATTAGTGGCTGGTCAAGTGGTTG ATATTGATTCAGAGGGTTTGGCTAATGTTGGGCTTGAACAACTTGAGTTCATTCATCTCCATAAGACTGG GGCTCTTCTAGAAGCTTCTGTTGTTTTGGGGGCTATTCTTGGTGGTGGTACAGATGAAGAAGTTGAAAAA CTTAGGAGCTTTGCTAGGTGTATTGGCTTGCTTTTTCAGGTTGTTGATGACATTCTTGATGTGACTAAAT CTTCTCAAGAATTGGGTAAAACTGCTGGGAAAGATTTGGTGGCTGATAAGGTTACTTATCCAAGGCTAAT GGGTATTGACAAATCAAGAGAATTTGCTGAGCAATTGAACACAGAAGCCAAACAGCATCTTTCTGGTTTT GATCCCATAAAGGCTGCTCCTTTAATTGCTTTGGCTAATTATATTGCTTATAGGCAAAATTGA (Sequence No. 15)

[0219] > PK15935.1 | Geranyl pyrophosphate (GPP) synthase small subunit | GPP synthase ssu ATGGCGGTTTATAATCTATCAATTAATTGCAGTCCAAGATTTGTTCATCATGTTTACGTTCCACATTTCA CATGTAAATCCAATAAGTCGTTAAGTCACGTACCCATGAGAATAACCATGTCCAAACAGCATCATCATTC TTATTTTGCCTCCACAACAGCCGATGTAGATGCCCATCTCAAGCAATCCATCACTATCAAGCCACCACTC TCAGTTCACGAGGCCATGTACAATTTCATCTTTTCCAACCTCCGAATTTAGCACCGTCATTGTGCGTGG CGGCGTGTGAGCTTGTCGGGGGCCACCAGGACCAGGCCATGGCAGCAGCCTCCGCCTTGCGCGTCATCCA CGCAGCCATCTTCACTCATGACCACCTCCCTTTAACGGGCAGGCCCAATCCAACAAGTCCTGAGGCAGCG ACCCACAATTCTTACAACCCAAATATTCAGCTCCTTCTCCCGGACGCAATTGTACCTTTTGGGTTCGAAT TGTTGGCCAATTCTGATGACCTTACCCATAATAAATCAGATCGGATTTTGCGGGTCATTGTAGAGTTCAC ACGCACCTTTGGATCACGAGGAACTATTGATGCTCAATACCATGAGAAGCTAGCCAGTAGATTTGACGTT<松本 GATAGTCATGAAGCCAAAACTGTCGGGTGGGGCCATTATCCCTCTTTGAAGAAGGAAGGTGCGATGCATG CATGCGCTGCTGCATGTGGGGCCATTCTTGGAGAGGCACATGAAGAAGAGGTTGAGAAGTTGAGAACTTT TGGTCTTTATGTGGGCATGATTCAAGGATATGCCAATAGATTTATAATGAGCAGCACAGAAGAAAAGAAA GAAGCAGATAGAATCATCGAGGAGTTAACCAATTTGGCTCGCCAGGAACTAAAATATTTCGATGGGAGAA ACTTAGAGCCATTTTCAACCTTTCTTTTTCGTCTATAG(SEQ ID NO:16)

[0220] > PK17903.1 | Geranyl pyrophosphate (IPP) isomerase ATGGGAGACTCTGCCGACGCTGGAATGGACGCTGTCCAGAGACGCCTTATGTTTGATGATGAATGCATTC TAGTGGATGAGAATGACCGAGTTGTTGGTCATGATACAAAATATAACTGTCACTTGATGGAAAAGATTGA AAAGGATAATTTGCTACACAGGGCTTTCAGTGTGTTCTTGTTCAACTCAAAATATGAGTTGCTTCTTCAG CAACGTTCTGCAACAAAGGTAACATTCCCTCTTGTGTGGACAAACACCTGTTGTAGCCACCCGCTCTACC GTGAATCTGAGCTTATCGATGAGGAGTCCCTTGGAGCAAGGAATGCAGCACAGAGAAAGCTTTTAGATGA GCTGGGTATTCCTGCTGAAGATGTGCCAGTTGATCAATTTACCCCACTAGGCAGGATGCTGTACAAAGCT CCTTCTGATGGCAAATGGGGCGAGCATGAACTTGATTACCTGCTCTTCATCGTCCGGGATGTTAGTGTCA ATCCAAATCCAGATGAAGTAGCTGATATCAAGTATGTAAACCGGGACGAGTTGAAAGAGTTGTTGAGGAA AGCAGATGCTGGGGAAGGAGGCTTGAAGCTATCCCCTTGGTTCAGACTGGTTGTGGATAATTTCTTGTTC AAGTGGTGGGACCATGTTGAGAAAGGCACACTTAAGGAAGTTGCTGATATGAAAACCATTCACAAGTTGA CTTAA(SEQ ID NO: 17)

[0221] > PK16122.1 | 1-Deoxyxylulose-5-phosphate synthase 1 (DXS1) ATGGCGTTTTGTGCATTATCATTTCCTGCTCATATTAGCCGGGCAACTACACCAGCACCTTCAGATCTTC ACAAATCTAGTTCTTTCTCTTCTCGGTTTTATTGGGGAGCAGATCTGCTGAGGCCATCTCAATACAAGGT CAGGAAAATACAAAGTGGGGTTTATGCATCACTGTCAGAAAGTGGGAGAATATCACTCAAGGGAGACCACCA ACTCCTCTCTTGGACACCATAAATTATCCAATTCATATGAAAAATCTCTCTGTTAAGGAGCTTAAAAAAC TATCAGATGAACTAAGGTCTGATGTCATCTTCAACGTTTCTAACACCGGGGTCACCTGGGCTCAAGCCT TGGTGTTGTTGAGCTTACTGTGGCTCTTCATTTTGTCTTCAATACTCCTCAGGATAGGATACTATGGGAT GTTGGTCATCAGTCTTACCCTCATAAAATTCTGACTGGAAAGAGATAAGATGCACACCATGAGGCAGA CCAACGGGTTAGCCGGATTCACTAAGCGGTCTGAGAGGTGAATATGATTGTTTTGGGACTGGTCATAGTTC TACCACCATCTCAGCTGGCTTGGGAATGGCTGTTGGAAGGGATCTTAAAGGAAGAAAGAATAATGTTGTG GCTGTCATAGGTGATGGTGCCATGACAGCAGGTCAAGCTTATGAAGCCATGAATAATGCCGGGTACCTTG ATTCCGACATGATTATTATTCTTAACGACAATAAACAGGTTTCTTTACCTACTGCCTCTCTTGATGGGCC CATACCACCTGTTGGAGCTTTGAGTAGTGCTCTCAGTAGGCTGCAATCAAACAGGCCTCTTAGAGAACTA AGAGAAGTAGCCAAGGGAGTTACTAAACAAATAGGTGGATCAGTACATGAATTGGCTGCAAAAGTTGATG AATATGCTCGTGGAATGATAAGTGGTTCTGGCTCAACATTGTTTGAGGAGCTTGGACTCTATTATTATTGG TCCAGTTGATGGTCACAATATAGATGATCTTGTTTCCATACTAGAGGAGGTTAAGAGCACTAAAACAACA GGTCCAGTCTTGATCCATTGCATCACTGAGAAAGGAAGAGGATATCCATATGCAGAGAAAGCTGCTGATA AGTATCATGGGGTGGCCAAGTTTGATCCAGCAACTGGAAAGCAATTCAAAGGCACTTCTAACACACAGTC ATACACTACATACTTTGCTGAGGCTTTGGTTGCAGAAGCAGAGGCAGACAAAGATGTTGTGGCCATCCAT GCTGCAATGGGTGGTGGAACAGGCTTGAATCTCTTCCTTCGCCGTTTTCCAACAAGATGTTTTGATGTTG GGATAGCAGAACAGCATGCTGTTACTTTCGCTGCTGGTTTGGCTTGCGAGGGCCTTAAGCCGTTTTGTGC AATTTACTCATCTTTCATGCAGCGAGCCTATGATCAGGTAGTACATGATGTTGATTTGCAGAAGTTGCCG GTGAGATTTGCAATGGACAGAGCTGGACTTGTTGGGGCCGACGGCCCTACACATTGTGGTGCTTTTGATG TTACTTTCATGGCATGCCTCCCAAACATGGTTGTGATGGCTCCTTCCGATGAGGCAGAGCTCTTCCACAT GGTTGCCACCGCTGCTGCCATAGATGACAGACCAAGTTGTTTCCGTTACCCCAGAGGAAATGGAATTGGT GTTCCATTACCTCAAGGGAATAAAGGAACTCCTCTTGAGATCGGAAAAGGCAGGGTATTGGTTGAAGGGG AAAGAGTAGCACTTCTAGGCTATGGAACAGCAGTTCAGAGTTGTTTGGCTGCTGCAGCCTTAGTAGAACC TCACGGTCTACGGCTAACAGTTGCTGATGCACGATTTTGCAAGCCTTTGGATCATGCCCTCATTCGCGAA CTAGCGAAAAATCACGAGGTTTTGATTACAGTGGAAGAAGGATCTATAGGAGGTTTTGGATCTCATGTTG CTCAGTTTATGGCCCTTGATGGCCTTCTTGATGGAAAAACAAAGTGGAGACCAATTGTTCTTCCTGACCG ATACATCGACCACGGTTCGCCTGCTGATCAATATGTCGACGCGGGTCTCACGCCACCTCACATTGCAGCC ACAGTTTTCAATGTACTAGGACAAACAAGAGAGGCCTTGAAGGTTATGACAACATGA(SEQ ID NO: 18)

[0222] > PK26473.1 | 1-Deoxyxylulose-5-phosphate synthase 二 (DXS2) ATGGCGGTTTCTGGTTCATTCATTGTACCAAATCATTCATTCCTTTCACAACTTAAATCTCCACAGCCAT ATTACAGTTCCAACAAACAGTTGAGTTTAAGGGTGAGAGGATCTCTTTGTAGCTCAGATGATGGGGAAGG AAAATTCATCAGCAAAGAAAAAGATGAATGGAAAATCAAGTATTCCAGTGAAAAACCAATCACTCCATTG CTTGATACAGTCAATTACCCAGTTCACATGAAGAATTTATCCACACAGGATCTTGAACAGCTAGCAGCAG AGCTTAGAGCAGATGTAGTCCATACAGTATCAAAAACAGGTGGTCATCTGAGTGCAAGCTTGGGAGTTGT GGAGCTCACTGTAGCACTGCATCATGTTTTCAATACCCCTGATGATAAAATCATATGGGATGTTGGACAT CAGACATACCCGCATAAGATTCTTACAGGAAGGAGGTCTCAAATGCATACCATTAGAAAGACTTCTGGTC TAGCAGGGTTTCCCAAAAGAGATGAGAGTGTTTACGATGCTTTCGGTGCAGGTCACAGTTCTACAAGCAT ATCAGCAGGCCTTGGCATGGCAGTTGCCAGGGATCTTCTGGGAAAGAAGAACAGTGTTGTTTCTGTGATT GGAGATGGGGCCATGACTGCAGGAATGGCATATGAAGCCATGAATAATGCCGGCTACTTGGACGCCAACT TGATTGTTGTATTAAACGACAATAAACAAGTTTCTTTACCAACTGCTACTCTTGATGGTCCTGCAACCCC AGTGGGAGCTCTAAGTGGTGCTTTGACTAAGCTTCAAGCAAGCACCAAGTTCAGAAAACTTCGCGAAGCT GCGAAAACCATCACAAAACAAATTGGAGGGCCAGCACATGAAGTTGCAGCTAAAGTAGATGAGTATGCTA GAGGAATGATAAGTGCTTCTGGGTCAACACTCTTTGAGGAGCTTGGGTTGTATTATATTGGTCCGGTGGA TGGACATAATGTTGGAGATTTAGTCACCATTTTTGAGAAAGTGAAATCAATGCCAGCGCCAGGACCAGTC TTGATCCACATCGTCACAGAGAAAGGGAAAGGCTATCCCCCAGCTGAAGTAGCACCTGATAAAATGCATG GAGTTGTAAAGTTTGACCCAACAACAGGAAAGCAATTTAAGTCCAAATCGTCGACACTTTCATATACTCA ATACTTTGCTGAATCTCTAATAAAAGAAGCTGAAGAAGATGACAAGATTGTTGCCATACACGCAGCAATG GGTGGTGGCACTGGTCTCAATTATTTCCAGAAGAAATTTCCTGATCGTTGCTTTGATGTGGGGATTGCTG AGCAACATGCTGTCACGTTTGCAGCTGGATTAGCTGCAGAGGGTCTCAAACCATTCTGTGCCATATACTC ATCATTCCTGCAACGAGGATATGATCAGGTTGCACATGATGTAGACCTTCAAAAATTACCTGTCCGTTTT GCATTGGATAGAGCTGGCATGGTTGGCGCAGATGGGCCTACCCACTGCGGTGCATTTGATATCACCTACA TGGCCTGCTTGCCCAACATGGTTGTCATGGCTCCATCAGATGAGGCTGAACTTATGCACATGGTGGCCAC AGCAACAGCTATAGATGACAGACCCAGTTGCTTCAGGTTTCCAAGGGGCAATGGAATTGGAGCAAAGCTT CCAGCTAATAATAAAGGAACTATACTTGAGATTGGAAAAGGCAGAATATTAATGGAAGGCAGCAGAGTAG CTATTTTGGGTTATGGTTCTATTGTTCAGCAATGTGTGGAAGCTGCAAGCATATTAAAGAAACAAGACAT TTCAGTGACAGTAGCTGATGCAAGATTTTGCAAACCATTGGATACAAATCTCATAAGACGGTTAGCCAAC GAGCATGAAATCCTAATCACTGCCGAAGAAGGTTCTATTGGAGGCTTTGGGTCTCATGTGTCACACTTTC TAAGCTTAAGTGGACTTCTTGATGGGTCTTTAAAGTTGAGAGCAATGGTTCTTCCTGATAGATACATTGA CCATGGATCACCCCAAGATCAGACTGAAACAGCCGGGCTCTCCTCGAGGCATATATCTGCAACAGTCTTA TCTCTCTTGGGGAAGCCCAAGGAAGCACTTCAGTTCATGTAA (SEQ ID NO: 19)

[0223] > PK04218.1 | 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (XDR) ATGGCTCTGAACTTGTTATCCCCAGCTGAAGTGAAGGCTCTATCCTTTTTGGACTCCACCAAGTCCACCC GCTTCCCTAAGCTGTGTCCAGGTGGAATTACTTTGCATAGAAAGGATTGCAGAGTACCACTTAGAAGAAG AGTTCATTGTTCGTTGCAGCAACCTCCTCCAGCTTGGCCAGGAAGAGCTATTCCAGAGCAAGATCTTTGT AATTGGAATGTCCCAAAGCCTATATCTATTATTGGCTCTACTGGCTCTATAGGAACTCAGACACTGGACA TTGTGGCAGAGAATCCAGATAAATTCAGAATAGTGGGACTTGCAGCTGGTTCGAATGTGACACTTCTTGC AGACCAGGTGAAGAGATTCAAGCCTCAAATAGTTGCTCTTAGAAATGAATCATTAATTGGTGAACTAAAA GAGGCCTTAGCTGATGTGGAAGAAATGCCCGAAATTATTCCTGGGGAACAAGGAGTAATTGAGGTTGCCC GGCACCCAGATGCAGTCACAGTGGTTACAGGAATAGTAGGTTGTGCTGGATTACAGCCTACAGTTGCTGC AATTGAGGCAGGTAAACACATAGCTTTAGCCAATAAAGAGACCCTGATTGCTGGAGGTCCATTCATCCTT CCTCTAGCTCACAAGCATAACATAAAAATTCTTCCTGCCGATTCAGAACATTCGGCAATATTCCAGTGTA TCCAGGGCTTGCCTGATGGTGCACTACGGCGTATCATTTTGACAGCATCTGGGGGAGCTTTCAGAGATTG GCCGGTTGAAAAGCTAAAAGATGTTAAGGTTGCTGATGCTCTGAAACATCCAAACTGGCCGGGTATGGGA AAGAAAGTCACTATTGATTCTGCTACCCTTTTCAACAAGGGTCTGGAAGTCATTGAAGCCCATTATCTAT TCGGAGCAGACTATGACGATATTGACATTGTGATTCACCCAGAAGCTATTATACACTCTATGATTGAAAC ACAGGATTCTTCTGTTCTGGCTCAGTTAGGGTGGCCTGACATGCATATACCGATTCTCTATACTATGTCA TGGCCAGACAGAATATACTGTTCTGAAGTAACTTGGCCTCGACTTGATCTTTGCAAGCTTGGTTCGCTGA CCTTTAGGAGTCCTGACAACCAGAAGTACCCATCCATAGATCTTGCCTATTCTGCTGGACGTGCTGGGGG CACCATGACTGGAGTTCTCAGTGCAGCCAATGAGAAAGCTGTAGAGATGTTTATTGATGAGAAGATAAGT TATCTTGAAATCTTCAAAGTTGTTGAGCTAACATGCGACAAGCATCGATCAGAGATGGTGACTTCACCTT CTCTTGATGAAATTATCCACTATGACTCGTGGGCACGAGAGTATGCAACTACTAGTTTGAAGAGTTCTTC CAGTCCAAGACCTGTTACAGCATGA (Sequence ID 20)

[0224] > PK03569.1 | 4-Diphosphocytidylyl-methylerythritol 2-phosphate synthase (MCT) ATGGCGTTACTTGCAATGGACCTTACTTTCTCTTCTGCTTCTCTTTCTTCTTCTTCCTACAATGCTGCTC CTCTACTATTTCCTTCTATTCGCCCATCCTCTCAATCCATTGTTCGATTCCCAGTCCATGAGGTGGGATT CAGGGGGAAATGCAGAATTTCCAAGATAAGGTTCGCTCGCTGCTCTGCAAATGTTGGCCAAAAGCCTGGT GTTGTGGAAAAGAAAAGCGTTTCGGTGGTTCTTCTGGCAGGTGGGAAGGGTAAACGGATGGGGGCCAACA TGCCAAAGCAGTATCTTCCACTTTTAGGGCAACCAATTGCACTGTATAGCTTCTACACTTTTTCTAAAAT GATTGAAGTGACTGAAATTGTTGTAGTTTGTGATCCCTCTTACGAGGATATCTTTGAAGATTCCAAAGTC AAGATCCATGTTGGACTTAAATTCGCTCTGCCTGGAAAGGAAAGACAGGATTCAGTTTATAGTGGACTTC AGGCAATTGATCCAAACTCTAAGCTTGTGTGCATTCACGATTCTGCTAGACCTTTGGTAACAACAGAAGA AGTTAAAAAGGTCATTGAAGATGGTTGGTTGCATGGAGCAGCTGTACTTGGTGTTCCTGTCAAAGCTACA ATCAAAGAGGCAAACAATGCATCTTTTGTAACTAAAACGTTGGAGAGGAAAAAACTTTGGGAAATGCAGA CACCCCAGGTGATCAAACCCGAGTTGCTCAAGGAAGGATTTGAGCTTGTAAATAGGGAAAATCTGGAAGT GACTGATGATGTGTCTATAGTGGAACACCTTGGACATCCTGTATATATAACTGAAGGTGCTTACACCAAC ATCAAGGTTACTACTCCAGATGATTTATTGCTTGCGGAGAGAGTATTGAGTATGAACTCTGTGAAGGCTG TTGCATAA(SEQ ID NO: 21)

[0225] > PK19074.1 | 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK) ATGGCTTCCTCTCATATTCTCTGCCACAACAACGTTTTTAATCTTTCTCCCAATCCTTTTAGGAACAGGG GTCTCTCTTCCTTAAACTCAAATGGGTTTTGTTTTTTTGGTTCGAAATCTAGAATTTCGAGGCCTTCATC TCTCAAAATTGTGGTTTCTGAAAGAAGACAAGTTGAGATAGTTTATGATGCTGATGAAAGGATAAACAAA TTGGCTGATGTAGTGGACAAGGAAGCGCCTCTTTCTAGGCTCACTCTTTTCTCACCTTGCAAGATTAATG TTTTCTTGAGAATAACTAGCAAAAGGGAAGATGGGTATCATGATTTGGCATCCCTCTTTCACGTGATAAG TCTTGGAGATGTGCTTAAGTTCTCTTTGTCTCCTTCAACAAAGAAAGATTCTTTGTCAACGAATGCCTCT GGGGTACCACTTGATGATAGAAATTTGATTATCAAGGCCCTTAATCTTTACCGAAAGAAAACTGGTACAA ACAAATACTTTTGGATTCATCTTGACAAGAAAGTGCCCACTGGAGCAGGGCTAGGTGGTGGGAGCAGCAA TGCTGCAACAGCCCTATGGGCAGCAAATCAGTTCAATGGTTGTCTTGTTACTGAAAAGGAATTGCAAGAA TGGTCAAGTGAGATTGGTTCAGATGTTCCTTTCTTTTTCTCCCAAGGGGCAGCCTATTGTACAGGTCGAG GTGAGGTTGTTCAGGATATTCTACCACCTGTACCATTAAACATTCCCATGGTTCTCATAAAGCCCCCAGA AGCATGTTCAACAGCCGAAGTTTATAAGCGTTTTCGGTTGGATAAAACCAGTAATAGTGATCCTTTACAA TTGCTCCACAAGATCTCAAGTGATGGAATAAGTCAAGATGTCTGCATCAATGACTTAGAACCTCCTGCCT TTGAAGTTCTTCCATCTCTTAAGAGATTGAAACAGCGTATAATTGCAGCTAGTCGTGGACAATATGATGC TGTTTTTATGTCTGGGAGTGGAAGCACCATTGTCGGGGTCGGTTCCCCAGATCCACCTCAGTTTATATAT GATGATGAGGACTACAAGGATGTGTTTTTGTCAGAGGCCAACTTTCTGACTCGAGAAGCAAATGAATGGT ACAAAGAACCTGCTTCAGCTAGCGCTTGTAGCCCTTCAGATGATTTCTCTCGTAATTTTTCCTCCTCTGT CGAGTAA (SEQ ID NO: 22)

[0226] > PK25433.1 | 2-C-methyl-D-erythritol 2:4-cyclodiphosphate synthase (MDS) ATGGCGGCGGCGACGGCAACACCACTCTGTGCTTCAACTCTTCCACCACACTACTCCAATACCTCCCCCA AATCATTCAATCACTCCCATTTCACAGTCGCAGTTCCCAGAAATCTCTTCTCATCGTCCTCAATTTCATC TCTAAGACAATCGAAAACGACGCCGCTTTCGGCTCTGCCTTCTGTATCGGCCGCCGCGACCACCGCTTTG AACGCTGAGCAAGCTCCGTCTGAGGTATCTGCTACTCCCTCAAAGGCTCTTCCTTTTCGGGTTGGTCATG GGTTTGACCTTCATCGATTGGAGCCTGGGTATCCTTTGATAATTGGAGGTATTAATATACCTCATGAGAA AGGTTGCGAAGCTCATTCTGATGGGGATGTTTTGCTTCATTGTGTAGTTGATGCTATTTTGGGTGCTTTG GGGCTTCCTGATATTGGTCAAATTTTCCTGATTCTGATCCCAAATGGAAAGGGCTGCATCATCAGTTT TCATCAAAGAAGCTGTGAGACTGATGCATGAGGCAGGTTATGAGCTTGGAAATTTAGATGCAACATTAAT TCTTCAAAGACCAAAGTTAAGTCCACAAGGAAGCTATCAGAGCCAACTTGTCTGAACTTCTAGGAGCT GACCCTGCAGTTGTTAATCTGAAAGCGAAAACTCACGAAAAGGTCGATAGTCTCGGGGAAAATCGAAGCA TCGCTGCTCACACTGTGGTTCTTCTTATGAAAAAATAG (Sequence No. 23)

[0227] > PK23068.1 | 4-Hydroxy-3-methylbuto-2-en-1-yl diphosphate synthase (HDS) ATGGCTTCTGGAGCTGTACCAGCATCAATTTCATGTCTGAAAAGCAGAGACTCTGGCTTGAGCTTTGCTA AAAGTTCTGATTTTGTGAGGCTTTCTGATTTAAAGAGGGTTGGTTCATCTAGAACAAGAGTTTCAGTTAT CCGAAATTCGAATCCTGGTTCAGATATTGCTGAACTTCAGCCTGCATCAAAAGGAAGCCCTCTATTAGTT CCTAGACAGAAGTACTGTGAATCCTTACACAAAACTGTTAGGAGGAAAACGCGAACTGTGATGGTGGGAA ATGTGGCTCTTGGCAGTGAGCATCCCATAAGAATTCAAACGATGACGACAAATGATACCAAGGATGTTGC TGGAACAGTTGAAGAGGTGATGAGAATAGCTGATAAGGGAGCTGATATTGTTCGGATAACAGTTCAGGGA AGAAAAAGAAGCAGATGCTTGTTTCGAAATAAAAAATTCACTTGTGCAGAAGAATTATAATATACCTCTTG TGGCAGATATTCATTTTGCTCCCCCAGTTGCATTAAGAGTTGCTGAATGCTTCGATAAAATTCGTGTCAA TCCTGGAAATTTCGCTGACAGACGGGCTCAGTTTGAGACGCTCGAGTACACAGACGACGACTATCAGAAA GAACTTGAGCATATTGAGCAGGTTTTTTCTCCATTGGTTGAGAAATGTAAGAAATATGGTAGAGCAATGC GTATCGGGACAAACCATGGGAGTCTTTCAGATCGTATCATGAGCTACTATGGAGATTCTCCAAGGGGAAT GGTTGAATCTGCATTTGAGTTTGCAAGGATTTGCCGGAAGTTGGATTTCCATAATTTTGTGTTTTCGATG AAAGCAAGCAACCCAGTTGTCATGGTTCAGGCGTATCGTCTACTTGTTGCTGAAATGTATGTCCAGGGCT GGGACTATCCACTTCACTTGGGAGTTACTGAAGCGGGGAAGGACGAATGAAATCTGCAAT TGGCATCGGGACCCTTCTTCAGGATGGTTTGGGTGATACTATCAGGGTTTCACTCACCGAACCGCCCGAG GAGGAGATTGATCCCTGCAGAAGGTTGGCCAATTTGGGTACAAAAGCAGCTGATCTTCAGCAAGGAGTGG CTCCATTTGAAGAGAAGCACAGGCATTATTTTGATTTTCAACGACGAACTGGTCAACTGCCTCTACAGAA GGAGGGCGATGAGGTTGACTATAGAGGTGCTCTGCACCGTGATGGTTCTGTTCTCATGTCAGTGTCTCTC AATAACTTAAAGATGCCCGAGCTCCTATACAGGTCACTAGCAGCAAAGCTTGTCGTCGGGATGCCATTTA AGGATCTGGCAACAGTAGACTCCATCTTATTGAGACAACTTCCACCTATTGACGATGACAACGCTCGATT AGCTCTCAAAAGATTGATAGACATAAGTATGGGGGTCATAACTCCTTTGTCGGAGCAGCTAACAAAGCCA TTGCCAAATGCTATGGTTTTGGTAAATCTTAAGGAGTTATCATCTGGTGCACACAAGCTTTTGCCAGAAG GCACGCGTTTGGTTGTATCCTTGCGCGGTGATGAACCTTACGAAGAACTGGAGATTCTCAAAGGGGTTGA TGATGTTGTTATGATTCTTCATGATCTTCCGTTCGATGAACATAAAATTAGCAGAGTCCACTCAGCAAGA AGATTATTTGAGTATCTATCAGATAATTCTCTTAACTTTCCTGTAATACACCACATTCAATTTCCAAATG GAATCCACAGGGATGACTTAGTCATCGGTGCAGGTAGCAACGCTGGTGCCCTTTTAGTAGATGGACTCGG GGACGGTATCCTCTTAGAAGCCCCAGATCAGGATTTCGATTTTCTTAGAAATACTTCTTTCAACCTACTT CAAGGTTGTAGAATGCGAAATACAAAGACGGAGTATGTCTCGTGCCCATCCTGCGGTAGAACTTTGTTTG ACCTTCAAGAAATCAGCGCAGAGATTCGAGAGAAGACATCACACCTGCCCGGTGTCTCAATTGCAATCAT GGGTTGCATTGTTAATGGACCCGGAGAGATGGCTGATGCAGACTTCGGTTATGTCGGTGGTGCTCCCGGA AAGATTGACCTTTATGTTGGAAAGACGGTAGTGCAGCGTGGAATCGCAATGGAACAAGCGACCGATGCAT TGATTCAGCTAATAAAAGATCATGGCCGATGGGTTGAACCACCCTCGGACGAAGAATGA(SEQ ID NO: 24)

[0228] > PK13726.1 | 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) ATGTCGATCACTTTCCAGCTCTGCCGGATTCCAATCCGTACCGACCTCGCCTTGGCGGAGCCTCTCTCCG TAACCGGAACCCTCCGCTGCCGGAAACCTTTCGTCATCCGATGCGCCGGCGAGTCATCTTCAACGGCAGC AGATTCTGATTTCGATGCGAAAGTGTTCCGTAAGAACTTGGTCCGAAGCAAGAACTACAATCGGAAAGGT TTTGGCCATAAGGAAGAGACCCTTCAACTCATGGACAGCGAGTACACCAGTGATATTATAAAGACTTTGA AGGATAATGGAAATGAGTACAGGTGGGGGAACGTGACGGTAAAATTGGCCGAAGCATATGGGTTTTGCTG GGGTGTGGAGCGAGCTGTCCAAATTGCTTACGAAGCAAGGAAACAGTTCCCCGAAGAAAAGATTTGGATT ACAAACGAAATTATTCATAATCCGACAGTCAACAAGAGACTAGAGGAAATGAAAGTGGAAAATATTCCAA TTGATGAAGGGAGGAAACAATTTGAGATTGTAAACAAGGGTGATGTTGTGATATTGCCTGCTTTTGGTGC TGGAGTGGATGAGATGTTGGCTTTGAGTGATAGGAATGTTCAAATTGTTGATACCACATGCCCATGGGTT TCCAAGGTTTGGAATACAGTCGAGAAACATAAGAAAGGTGAATACACTTCCATTATTCATGGTAAATATG CTCATGAGGAGACTATAGCTACTGCATCTTTTGCTGGAACTTACATTATTGTAAAGAACATGAAAGAGGC AATGTATGTCTGTGATTATATTCTTGGCGGTCAACTTGATGGATCCAGCTCAACAAGAGAGGAGTTTATG GAGAAATTTAAGAATGCAGTTTCTAAGGGATTTGATCCTGACAAACATCTTGTGAAGGCTGGTATTGCAA ATCAGACTACAATGCTCAAGGGGGAAACCGAAGAGATTGGGAAACTGGTTGAGAGGACTATGATGCAAAA GTACGGAGTTGAAAACATTAATGAACACTTCCAAAGCTTTAACACAATTTGCGATGCAACCCAAGAGCGT CAAGATGCAATGTACAAGATGGTGGAGGAACGTATTGACCTTATGTTAGTTGTTGGAGGATGGAACTCTA GTAACACTTCTCATCTACAAGAGATTGCAGAGGAACGAGGTATTCCCTCGTATTGGATTGACAGTGAACA GAGAATAGGTCCTGGAAACAAGATAGCCTACAAGCTAAATCATGGAGAGTTGGTTGAGAAAGAGAACTGG TTACCAGAGGGTCCCATCACGGTCGGTGTAACATCAGGTGCTTCTACTCCAGATAAGGTTGTGGAAGATG TTCTCATCAAGGTGTTTGACCTTAAGAGCGAAGAAGCTTTGCAAGTTGCTTAG(Sequence No. 25)

[0229] > AAO73863 | Calen Synthase MonoTS MSVISILPLASKSCLYKSLMSSTHELKALCRPIATLGMCRRGKSVMASKSTSLTTAVSDDGVQRRIGDHH SNLWDDNFIQSLSSPYGASSYGERAERLIGEVKEIFNSLSRTDGELVSHVDDLLQHLSMVDNVERLGIDR HFQTEIKVSLDYVYSYWSEKGIGSGRDIVCDTDLNTTALGFRILRLHGYTVFPDVFEHFKDQMGRIACSDN HTERQISSILNLFRASLIAFPGEKVMEEAEIFSATYLKEALQTIPVSSLSQEIQYVLQYRWHSNLPRLEA RTYIDILQENTKNQMLDVNTKKVLELAKLEFNIFHSLQQNELKSVSRWWKESGFPDLNFIRHRHVEFYTL VSGIDMEPKHCTFRLSFVKMCHLITVLDDMYDTFGTIDELRLFTAAVKRWDPSTTECLPEYMKGVYTVLY ETVNEMAQEAQKSQGRDTLSYVRQALEAYIGAYHKEAEWISSGYLPTFDEYFENGKVSSGHRIATLQPTF MLDIPFPHHVLQEIDFPSKFNDFACSILRLRGDTRCYQADRARGEEASCISCYMKDNPGSTQEDALNHIN NMIEETIKKLNWELLKPDNNVPISSKKHAFDINRGLHHFYNYRDGYTVASNETKNLVIKTVLEPVPM (Sequence ID 26)

[0230] >EFF14228 1-Deoxy-D-xylulose-5-phosphate synthase Dxs [Escherichia coli B354] 1 mmsfdiakyp tlalvdstqe lrllpkeslp klcdelrryl ldsvsrssgh fasglgtvel 61 tvalhyvynt pfdqliwdvg hqayphkilt grrdkigtir qkgglhpfpw rgeseydvls 121 vghsstsisa gigiavaaek egknrrtvcv igdgaitagm afeamnhagd irpdmlvvln 181 dnemsisenv galnnhlaql lsgklysslr eggkkvfsgv ppikellkrt eehikgmvvp 241 gtlfeelgfn yigpvdghdv lglittlknm rdlkgpqflh imtkkgrgye paekdpitfh 301 avpkfdpssg clpkssgglp syskifgdwl cetaakdnkl maitpamreg sgmvefsrkf 361 pdryfdvaia eqhavtfaag laiggykpiv aiystflqra ydqvlhdvai qklpvlfaid 421 ragivgadgq thqgafdlsy lrcipemvim tpsdenecrq mlytgyhynd gpsavryprg 481 navgveltpl eklpigkgiv krrgeklail nfgtlmpeaa kvaeslnatl vdmrfvkpld 541 ealilemaas healvtveen aimggagsgv nevlmahrkp vpvlniglpd ffipqgtqee 601 mraelgldaa gmeakikawl a (Sequence ID 27)

[0231] >WP_072972099 2-C-methyl-D-erythritol 4-cytididylyl phosphate transferase IspD / MCT 1 matthldvca vvpaagfgrr mqtecpkqyl signqtileh svhallahpr vkrvviaisp 61 gdsrfaqlpl anhpqitvvd ggderadsvl aglkaagdaq wvlvhdaarp clhqddlarl 121 lalsetsrtg gilaapvrdt mkraepgkna iahtvdrngl whaltpqffp rellhdcltr 181 alnegatitd easaleycgf hpqlvegrad nikitrpedl alaefyltrt ihqent(Sequence ID 28)

[0232] >WP_086589482 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF / MDS 1 mrighgfdvh afggegpiii ggvripyekg llahsdgdva lhaltdallg aaalgdigkl 61 fpdtdpafkg adsrellrea wrriqakgyt lgnidvtiia qapkmlphip qmrvfiaedl 121 gchmddvnvk attteklgft grgegiacea vallikatk(Sequence ID 29)

[0233] >WP_115903881 Isopentenyl diphosphate synthase delta-isomerase Idi 1 mqtehvilln aqgvptgtle kyaahtadtr lhlafsswlf nakgqllvtr ralskkawpg 61 vwtnsvcghp qlgesnedav irrcryelgv eitppesiyp dfryratdps givenevcpv 121 faarttsalq inddevmdyq wcdladilhg idatpwafsp wmvmqatnre arkrlsaftq 181 lk (Sequence ID 30)

[0234] >AF513112.1 Geranyl diphosphate synthase GPPS MAYSAMATMGYNGMAASCHTLHPTSPLKPFHGASTSLEAFNGEHMGLLRGYSKRKLSSYKNPASRSSNATVAQLLNPPQKGKKAVEFDFNKYMDSKAMTVNEALNKAIPLRYPQKIYESMRYSLLAGGKRVRPVLCIAACELVGGTEELAIPTACAIEMIHTMSLMHDDLPCIDNDDLRRGKPTNHKIFGEDTAVTAGNALHSYAFEHIAVSTSKTVGADRILRMVSELGRATGSEGVMGGQMVDIASEGDPSIDLQTLEWIHIHKTAMLLECSVVCGAIIGGASEIVIERARRYARCVGLLFQVVDDILDVTKSSDELGKTAGKDLISDKATYPKLMGLEKAKEFSDELLNRAKGELSCFDPVKAAPLLGLADYVAFRQN (Sequence ID 31)

[0235] >WP_053287215 Geranyltransferase IspA 1 mdfpqqleac vkqanqalsr fiaplpfqnt pvvetmqyga llggkrlrpf lvyatghmfg 61 istntldapa aavecihays lihddlpamd dddlrrglpt chvkfgeana ilagdalqtl 121 afsilsdadm pevsdrdris miselasasg iagmcggqal dldaegkhvp ldalerihrh 181 ktgaliraav rlgalsagdk grralpvldk yaesiglafq vqddildvvg dtatlgkrqg 241 adqqlgksty pallgleqar kkardlidda rqslkqlaeq sldtsaleal adyiiqrnk(Sequence ID 32)

[0236] >NP_414715 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomer IspC / DXR 1 mkqltilgst gsigcstldv vrhnpehfrv valvagknvt rmveqclefs pryavmddea 61 sakllktmlq qqgsrtevls gqqaacdmaa ledvdqvmaa ivgaagllpt laairagkti 121 llankeslvt cgrlfmdavk qskaqllpvd sehnaifqsl pqpiqhnlgy adleqngvvs 181 illtgsggpf retplrdlat mtpdqacrhp nwsmgrkisv dsatmmnkgl eyiearwlfn 241 asasqmevli hpqsvihsmv ryqdgsvlaq lgepdmrtpi ahtmawpnrv nsgvkpldfc 301 klsaltfaap dydrypclkl ameafeqgqa attalnaane itvaaflaqq irftdiaaln 361 lsvlekmdmr epqcvddvls vdanarevar kevmrlas (Sequence ID 33)

[0237] >EGT67781 4-diphosphocytidyl-2-C-methylerythritol kinase IspE / CMK 1 mrtqwpspak lnlflyitgq radgyhtlqt lfqfldygdt isielrddgd irlltpvegv 61 ehednlivra arlliktaad sgrlptgsga nisidkrlpm ggglgggssn aatvlvalny 121 lwqcglsmde laemgltlga dvpvfvrgha afaegvgeil tpvdppekwy lvahpgvsip 181 tpvifkdpel prntpkrsie tllkcefsnd ceviarkrfr evdavlswll eyapsrltgt 241 gacvfaefdt esearqvleq apewlngfva kgvnlsplhr aml(SEQ ID NO: 34)

[0238] >ANK02812 4-Hydroxy-3-methylbuto-2-en-1-yl diphosphate sinter IspG / HDS 1 mhnqapiqrr kstriyvgnv pigdgapiav qsmtntrttd veatvnqika lervgadivr 61 vsvptmdaae afklikqqvn vplvadihfd yrialkvaey gvdclrinpg nigneerrm 121 vvdcardkni pirigvnags lekdlqekyg eptpqalles amrhvdhldr lnfdqfkvsv 181 kasdvflave syrllakqid qplhlgitea ggarsgavks aiglglllse gigdtlrvsl 241 aadpveeikv gfdilkslri rsrginfiac ptcsrqefdv igtvnaleqr lediitpmdv 301 siigcvvngp gealvstlgv tggnkksgly edgvrkdrld nndmidqlea rirakasqld 361 earridvqqv ek (Sequence ID 35)

[0239] >AAL38655 4-Hydroxy-3-methylbuto-2-enyl diphosphate reductase IspH / HDR 1 mqillanprg fcagvdrais ivenalaiyg apiyvrhevv hnryvvdslr ergaifieqi 61 sevpdgaili fsahgvsqav rneaksrdlt vfdatcplvt kvhmevaras rrgeesilig 121 haghpevegt mgqysnpegg mylvespddv wkltvkneek lsfmtqttls vddtsdvida 181 lrkrfpkivg prkddicyat tnrqeavral aeqaevvlvv gsknssnsnr laelaqrmgk 241 rafliddakd iqeewvkevk cvgvtagasa pdilvqnvva rlqqlgggea iplegreeni 301 vfevpkelrv direvd(Sequence ID 36)

[0240] >AAA24819.1 Phytoen Synthase CrtE 1 mvsgskagvs phreievmrq siddhlagll petdsqdivs lamregvmap gkrirpllml 61 laardlryqg smptlldlac avelthtasl mlddmpcmdn aelrrgqptt hkkfgesvai 121 lasvgllska fgliaatgdl pgerraqavn elstavgvqg lvlgqfrdln daaldrtpda 181 ilstnhlktg ilfsamlqiv aiasasspst retlhafald fgqafqlldd lrddhpetgk 241 drnkdagkst lvnrlgadaa rqklrehids adkhltfacp qggairqfmh lwfghhladw 301 spvmkia (Sequence ID 37)

[0241] >AAA24820.1 Phytoendehydrogenase CrtI 1 mkktvvigag fgglalairl qaagiptvll eqrdkpggra yvwhdqgftf dagptvitdp 61 talealftla grrmedyvrl lpvkpfyrlc wesgktldya ndsaeleaqi tqfnprdveg 121 yrrflaysqa vfqegylrlg svpflsfrdm lragpqllkl qawqsvyqsv srfiedehlr 181 qafsfhsllv ggnpfttssi ytlihalere wgvwfpeggt galvngmvkl ftdlggeiel 241 narveelvva dnrvsqvrla dgrifdtdav asnadvvnty kkllghhpvg qkraaalerk 301 smsnslfvly fglnqphsql ahhticfgpr yrelideift gsaladdfsl ylhspcvtdp 361 slappgcasf yvlapvphlg napldwaqeg pklrdrifdy leerympglr sqlvtqrift 421 padfhdtlda hlgsafsiep lltqsawfrp hnrdsdianl ylvgagthpg agipgvvasa 481 kataslmied lq (sequence number 38)

[0242] >AAA24821.1 Prephytoempyrophosphate synthase [Pantoea agglomerans]CrtB MSQPPLLDHATQTMANGSKSFATAAKLFDPATRRSVLMLYTWCRHCDDVIDDQTHGFASEAAAEEEATQRLARLRTL TLAAFEGAEMQDPAFAAFQEVALTHGITPRMALDHLDGFAMDVAQTRYVTFEDTLRYCYHVAGVVGLMMARVMGVRD ERVLDRACDLGLAFQLTNIARDIIDDAAIDRCYLPAEWLQDAGLTPENYAARENRAALARVAERLIDAAEPYYISSQ AGLHDLPPRCAWAIATARSVYREIGIKVKAAGGSAWDRRQHTSKGEKIAMLMAAPGQVIRAKTTRVTPRPAGLWQRP V (Accession No. 39)

[0243] >sp|Q50L36.1|ISPS_POPAL Name: Official name = Isoprene synthase, chloroplast; Abbreviation = PaIspS; IspS. MATELLCLHRPISLTHKLFRNPLPKVIQATPLTLKLRCSVSTENVSFTETETEARRSANYEPNSWDYDYLLSSDTDE SIEVYKDKAKKLEAEVRREINNEKAEFLTLLELIDNVQRLGLGYRFESDIRGALDRFVSSGGFDAVTKTSLHGTALS FRLLRQHGFEVSQEAFSGFKDQNGNFLENLKEDIKAILSLYEASFLALEGENILDEAKVFAISHLKELSEEKIGKEL AEQVNHALELPLHRRTQRLEAVWSIEAYRKKEDANQVLLELAILDYNMIQSVYQRDLRETSRWWRRVGLATKLHFAR DRLIESFYWAVGVAFEPQYSDCRNSVAKMFSFVTIIDDIYDVYGTLDELELFTDAVERWDVNAINDLPDYMKLCFLA LYNTINEIAYDNLKDKGENILPYLTKAWADLCNAFLQEAKWLYNKSTPTFDDYFGNAWKSSSGPLQLVFAYFAVVQN IKKEEIENLQKYHDTISRPSHIFRLCNDLASASAEIARGETANSVSCYMRTKGISEELATESVMNLIDETWKKMNKE KLGGSLFAKPFVETAINLARQSHCTYHNGDAHTSPDELTRKRVLSVITEPILPFER (Sequence ID 40)

[0244] The present invention, as described herein as illustrative examples, can be adequately implemented without any elements(s) or limitations(s) not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and not as limiting. Furthermore, the terms and expressions used herein are for illustrative purposes only, not for limitation, and their use is not intended to exclude any future equivalents or parts thereof of what is expressed or described, and it is recognized that various modifications are possible within the scope of the claimed invention.

[0245] Therefore, although the present invention is specifically disclosed in preferred embodiments and optional features, those skilled in the art will be able to modify and alter the invention disclosed herein, and such modifications and alterations should be understood to be within the scope of the invention disclosed herein. This specification provides a broad and general description of the invention. The groupings of species and subgenerals in the narrow sense, which are within the scope of the general disclosure, also form part of these inventions. Therein, the general description of each invention includes the condition, or negative limitation, of excluding all objects from the genus, regardless of the presence or absence of specific materials extracted.

[0246] In addition, where features or embodiments of the present invention are described in relation to the Markush Group, a person skilled in the art will recognize that the present invention also describes individual members or subgroups of members of the Markush Group. It should also be understood that the above descriptions are illustrative and not limiting. Numerous embodiments will be obvious to a person skilled in the art in consideration of the above descriptions. Therefore, the scope of the present invention should not be determined by reference to the above descriptions, but rather by reference to the appended claims and the entire scope of equivalents to which such claims apply. All publications and references, including those accessible by patent publications and database (e.g., Genbank) accession numbers, are incorporated herein by reference as constituting part of this specification.

Claims

1. An expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme, wherein the bifunctional ispDF enzyme comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the bifunctional ispDF enzyme has 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase activity and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity.

2. The aforementioned bifunctional ispDF enzymes are the following ispDF enzymes: H. pylori HP1020, H. pylori J99 jhp0404, H. pylori HPAG1 HPAG1_0427, H. hepaticus HH1582, H. acinonychis st. Sheeba Hac_1124, W. succinogenes DSM 1740 WS1940, S. denitrificans DSM 1251 Suden_1487, C. jejuni subsp. jejuni NCTC 11168Cj1607, C. The expression cassette according to claim 1, wherein at least one amino acid is different from jejuni RM1221 CJE1779, C. jejuni subsp. jejuni 81-176 CJJ81176_1594, and C. fetus subsp. fetus 82-40 CFF8240_0409.

3. The expression cassette according to claim 1, wherein the promoter functionally linked to the nucleic acid encoding the bifunctional ispDF enzyme is selected from a constitutive promoter and an inducible promoter.

4. The expression cassette according to claim 1, wherein the nucleic acid encoding the bifunctional ispDF enzyme is codon-optimized; or the bifunctional ispDF is 31% or less similar to CJ-ispDF.

5. The expression cassette according to claim 1, wherein the expression cassette further comprises nucleic acids encoding one or more, two or more, or all of the enzymes selected from the group consisting of dxs, idi, and ispE; or the expression cassette further comprises nucleic acids encoding dxs and idi; or the expression cassette further comprises nucleic acids encoding dxs, idi, and ispE.

6. A plasmid comprising an expression cassette according to any one of claims 1 to 5.

7. The plasmid according to claim 6, wherein the plasmid further comprises: (1) an expression cassette containing a nucleic acid encoding isoprene synthase (ispS); (2) an expression cassette containing a nucleic acid encoding GPP synthase; (3) an expression cassette containing a nucleic acid encoding eukaryote-derived GPP synthase; or (4) an expression cassette containing a nucleic acid encoding plant-derived GPP synthase.

8. The plasmid according to claim 7, wherein the expression cassette containing the nucleic acid encoding the GPP synthase is of eukaryote origin, and the nucleic acid encoding the GPP synthase is codon-optimized.

9. The plasmid according to claim 7, wherein the expression cassette comprising the nucleic acid encoding GPP synthase further comprises: (1) a nucleic acid encoding one or more components of the lycopene synthesis pathway (e.g., crtE, crt1, and / or crtB) or a monoterpene synthase; (2) a nucleic acid encoding a calen synthase, myrcene synthase, or limonene synthase; or (3) a nucleic acid encoding a cannabinoid synthase.

10. The plasmid according to claim 9, wherein the cannabinoid synthase is selected from the group consisting of Cannabis CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase.

11. (1) Any one of the expression cassettes described in any one of claims 1 to 5; (2) Any one of the plasmids described in any one of claims 6 to 10; or (3) A host cell comprising any one of the expression cassettes described in any one of claims 1 to 5 and any one of the plasmids described in any one of claims 6 to 10.

12. It is a host cell: a. An expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding a bifunctional ispDF enzyme, wherein the bifunctional ispDF enzyme comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the bifunctional ispDF enzyme has 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase activity and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity; and b. The host cell comprising an expression cassette containing a heterologous promoter functionally linked to a nucleic acid encoding a terpenoid synthase.

13. The host cell according to claim 12, wherein the terpenoid synthase is isoprene synthase; or a component of the lycopene synthesis pathway; or cannabinoid synthase.

14. The host cell according to claim 13, wherein the terpenoid synthase is a cannabinoid synthase, and the cannabinoid synthase is selected from the group consisting of CBGA synthase, THCA synthase, CBDA synthase, and CBCA synthase, preferably a synthase of the genus Cannabis; or the cannabinoid synthase is a cleaved cannabinoid synthase selected from the group consisting of THCA synthase, CBDA synthase, and CBCA synthase, and the cleavage is the deletion of all or part of the signal peptide.

15. The host cell according to claim 12, wherein the host cell comprises an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding GPP synthase; or the expression cassette in a) or b) further comprises a nucleic acid encoding GPP synthase.

16. The host cell according to claim 12, wherein the host cell exhibits increased flux through the MEP pathway compared to a control cell that does not contain at least one of the one or more expression cassettes.

17. The host cell according to claim 12, wherein the host cell does not include heterogeneous nucleic acids encoding ispC, ispE, ispG, or ispH; combinations thereof; or all thereof.

18. The host cell according to any one of claims 12 to 17, wherein the host cell is a prokaryote; or the expression cassette of a) and / or b) is incorporated into the genome of the host cell; or the expression cassette of a) and / or b) is incorporated into the genome of the host cell and the host cell is a prokaryote.

19. The host cell according to any one of claims 12 to 18, wherein the host cell comprises a nucleic acid encoding a cannabinoid synthase functionally linked to a promoter, and the promoter functionally linked to the nucleic acid encoding the cannabinoid synthase is selected from a constitutive promoter and an inducible promoter.

20. A host cell according to any one of claims 12 to 19, wherein the expression cassette of a) and the expression cassette of b) are present in a single plasmid or are inserted into the genome of the host cell at a single locus; or the expression cassette of a) and the expression cassette of b) are present in different plasmids or are inserted into the genome of the host cell at different loci.

21. The host cell according to any one of claims 12 to 20, wherein the host cell further comprises olivetolic acid (OA).

22. The host cell according to claim 21, wherein the olivetolic acid is exogenous to the host cell.

23. The host cell according to any one of claims 12 to 22, wherein the host cell comprises (1) an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding one or more glycosylation pathway genes; (2) deletions in one, two, three, four, five, six, or all genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and totoDA; or (3) an expression cassette comprising a heterologous promoter functionally linked to a nucleic acid encoding one or more glycosylation pathway genes, and deletions in one, two, three, four, five, six, or all genes selected from the group consisting of ackA-pta, poxB, ldhA, dld, adhE, pps, and totoDA.

24. A method for obtaining a target metabolite, comprising culturing a host cell according to any one of claims 12 to 23 in a suitable medium under conditions suitable for inducing expression with one or more host cell expression cassettes, then recovering the cultured cells or used medium, thereby obtaining the target metabolite.

25. The method according to claim 24, wherein the metabolite is a cannabinoid; or a cannabinoid selected from THCA, CBDA, CBCA, CBN, THC, CBD, or CBC, or a mixture of one or more thereof; or a terpenoid or isoprene.

26. The method according to any one of claims 24 or 25, wherein the method comprises recovering and lysing the cultured cells and thereby producing a cell lysate.

27. The method according to claim 26, wherein the method comprises purifying the target metabolite from the cell lysate, or the used culture medium, or from the cell lysate and the used culture medium, thereby producing the purified target metabolite.

28. The method according to claim 27, wherein the purified target metabolite is a cannabinoid, and the method comprises (1) formulating a cannabinoid in a pharmaceutical composition; or (2) forming a salt, prodrug, or solvate of the purified cannabinoid; or (3) forming a salt, prodrug, or solvate of the purified cannabinoid and formulating a cannabinoid in a pharmaceutical composition.

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