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JP-7856349-B2 - Cannabinoid biosynthesis from cannabigerol acid using a novel cannabinoid synthase

JP7856349B2JP 7856349 B2JP7856349 B2JP 7856349B2JP-7856349-B2

Inventors

  • ゴー，メイベル ダーリーン コー
  • ユー，ウェン シャン

Assignees

  • ナショナル ユニヴァーシティ オブ シンガポール

Dates

Publication Date
20260511
Application Date
20250611
Priority Date
20191011

Claims (6)

  1. A method for producing one or more cannabinoids, comprising the step of contacting cannabigerol acid (CBGA) with a cannabinoid synthase ortholog, wherein the cannabinoid synthase ortholog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 23, 47, 57, 87, 107, and 117, and the resulting cannabinoid has a formula weight of 358.5 g/mol, 374.5 g/mol, or 330.5 g/mol.
  2. The method according to claim 1, wherein the cannabinoid produced comprises cannabiersonic acid and cannabiersoin.
  3. The method according to claim 1, wherein the cannabinoid synthase ortholog is a recombinant enzyme produced in Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, or Pichia pastoris.
  4. Recombinant cells of Saccharomyces cerevisiae or Pichia pastoris, wherein the genome contains a nucleic acid encoding a cannabinoid synthase ortholog, the cannabinoid synthase ortholog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 23, 47, 57, 87, 107, and 117, and the cannabinoid synthase ortholog is expressed in an active form in the recombinant cells.
  5. The recombinant cell according to claim 4, wherein the cannabinoid synthase ortholog, upon contact with cannabigerol acid (CBGA), produces a cannabinoid having a formula weight of 358.5 g/mol, 374.5 g/mol, or 330.5 g/mol.
  6. The recombinant cell according to claim 4, wherein the cannabinoid synthase ortholog generates cannabinoids containing cannabiersonic acid and cannabiersoin when it comes into contact with cannabigerolic acid (CBGA).

Description

Background Technology: The cannabinoid biosynthesis pathway has been the subject of extensive investigation. Cloning and expression of various enzymes in the cannabinoid biosynthesis pathway have been achieved by several research groups. For example, the structure and function of Δ1 -tetrahydrocannabinolate synthase, i.e., cannabinoid synthase, have been elucidated. See Shoyama et al., J. Mol. Biol. 2012, 423(1):96-105. The biosynthesis of two cannabinoids, cannabidiolic acid and Δ9 -tetrahydrocannabinolic acid, has been achieved in Saccharomyces cerevisiae by heterologous expression of enzymes from the cannabinoid biosynthesis pathway from Cannabis sativa and other organisms. See, for example, Luo et al., Nature 2019, 567(7746):123-126 and Zirpel et al., J. Biotechnol. 2017, 259:204-212. These studies have established that it is possible to construct cannabinoid biosynthesis pathways in heterologous systems. Further enzymes and methods are needed for the specific and high-yield synthesis of cannabinoids. A method for producing cannabinoids is disclosed. This method includes the step of contacting cannabigerol acid with a cannabinoid synthase ortholog. Cannabinoid synthase orthollogs are found in organisms other than hemp (Cannabis sativa), such as oranges (Citrus sinensis), melons (Cucumis melo), chili peppers (Capsicum annuum), rapeseed (Brassica napus), Nicotiana attenuata, tobacco (Nicotiana tabacum), Noccaea caerulescens (Thlaspi caerulescens), cotton (Gossypium hirsutum) (Gossypium mexicanum), and rice (Indica variety) (Oryza). Sativa subsp. indica), Japonica rice (Oryza sativa subsp. japonica), Arabidopsis lyrata subsp. lyrata, Paenibacillus sp. It is derived from Aloe-11 (Paenibacillus sp. Aloe-11), Streptomyces ipomoeae 91-03, Chinese cabbage (Brassica rapa subsp. pekinensis), peach (Prunus persica), Bacillus subtilis 168, Arabidopsis thaliana, poppy (Papaver somniferum), and Phytophthora parasitica P1569. Furthermore, recombinant cells of Saccharomyces cerevisiae and Pichia pastoris are provided, each containing nucleic acids encoding cannabinoid synthase orthologues in its genome, wherein the cannabinoid synthase orthologues are derived from one of the organisms listed in the preceding paragraph, and the cannabinoid synthase orthologues are expressed in their active form in the recombinant cells. Details of one or more embodiments are described below in the description and examples. Other characteristics, purposes, and advantages will be apparent from the detailed description, drawings, and appended claims. The following description of the present invention is accompanied by the accompanying drawings. Figure 1 shows the cannabinoid products that can be formed from cannabigerol acid ("CBGA") as a substrate. The molecular formula and molecular weight of cannabiersonic acid ("CBEA") are C22H30O5 and 374.5 , respectively. The molecular formula and molecular weight of cannabiersoin (decarboxylated CBEA) are C21H30O3 and 330.5 , respectively. The molecular formulas and molecular weights of all other compounds shown are C22H30O4 and 358.5, respectively. CBCA = cannabichromenic acid, CBTA = cannabicitranic acid, CBDA = cannabidiolic acid, THCA = tetrahydroisocannabinolic acid, THCA = tetrahydrocannabinolic acid, and CBLA = cannabicyclolic acid.Figure 2A shows representative total ion chromatograms for 18 reaction sets of cannabinoid synthase orthologs. Each reaction set was analyzed for 1.7 minutes.Figure 2B shows representative extracted ion chromatograms (EICs) of 18 reaction sets of cannabinoid synthase orthologues obtained with a mass-to-charge ratio "(m/z)" = 359.5, indicating the presence of CBGA, in nine of the 18 reaction sets, namely sets 1-3, 7-9, and 13-15.Figure 2C shows representative EICs for 18 reaction sets that demonstrated the production of cannabinoids with m/z = 357.5.Figure 3A shows the total ion chromatographs of a group of 18 cannabinoid synthase orthologues. Each reaction set was administered for 1.7 minutes.Figure 3B shows representative EICs for 18 reaction sets of cannabinoid synthase orthologues obtained at m/z = 359.5, indicating the presence of CBGA, specifically in sets 1-3, 7-9, and 13-15.Figure 3C shows representative EICs from 18 reaction sets that produced cannabinoids with m/z = 357.5.Figure 4 shows representative chromatograms of cannabinoid synthase orthologues that produced cannabinoids with m/z = 357.2071. Each reaction set was administered for 15.70 minutes for analysis. The chromatograms in columns 1, 2, and 4 show the presence of peaks when CBGA was incubated with positive cannabinoid synthase orthologues, which are 65 times larger than the peaks found in the control experiment (column 3) performed in the absence of cannabinoid synthase orthologues.Figure 5 shows representative chromatograms of cannabinoid synthase orthologues that produced cannabinoids with m/z = 373.2020. Each reaction set was administered for 15.70 minutes for analysis. The chromatograms in c