Cannabinoid biosynthesis from cannabigerol acid using a novel cannabinoid synthase

Abstract

This invention provides enzymes and methods for synthesizing cannabinoids specifically and in high yield. [Solution] A method is provided for producing one or more cannabinoids, comprising the step of contacting cannabigerol acid with a cannabinoid synthase ortholog. The cannabinoid synthase ortholog is derived from an organism other than hemp (Cannabis sativa). Also disclosed are recombinant cells of Saccharomyces cerevisiae or Pichia pastoris, which contain nucleic acids encoding the cannabinoid synthase ortholog in their genome. The cannabinoid synthase ortholog is expressed in its active form in the recombinant cells.

Description

[Technical Field]

[0001] Background technology The cannabinoid biosynthesis pathway has been the subject of thorough investigation. Cloning and expression of various enzymes in the cannabinoid biosynthesis pathway have been achieved by several research groups. For example, Δ 1 -The structure and function of tetrahydrocannabinolate synthase, or cannabinoid synthase, have been elucidated. See Shoyama et al., J. Mol. Biol. 2012, 423(1):96-105.

[0002] Two cannabinoids, cannabidiolic acid and Δ 9 - The biosynthesis of 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.

[0003] Further enzymes and methods are needed for the specific and high-yield synthesis of cannabinoids. [Overview of the Initiative]

[0004] A method for producing cannabinoids is disclosed. The 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), rice (Indica variety) (Oryza sativa subsp. indica), Japonica rice (Oryza sativa subsp. japonica), Arabidopsis lyrata subsp. lyrata, and 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.

[0005] 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 any of the organisms listed in the preceding paragraph, and the cannabinoid synthase orthologues are expressed in their active form in the recombinant cells.

[0006] 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.

[0007] The following description of the present invention is accompanied by reference to the attached drawings. [Brief explanation of the drawing]

[0008] [Figure 1] 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, THICA = tetrahydroisocannabinolic acid, THCA = tetrahydrocannabinolic acid, and CBLA = cannabicyclolic acid. [Figure 2A] 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]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, added to 9 of the 18 reaction sets, namely sets 1-3, 7-9, and 13-15. [Figure 2C] Figure 2C shows representative EICs for 18 reaction sets that demonstrated the production of cannabinoids with m / z = 357.5. [Figure 3A] 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] 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] Figure 3C shows representative EICs from 18 reaction sets that produced cannabinoids with m / z = 357.5. [Figure 4] 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. 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] 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. Chromatograms in columns 2–4 show the presence of a peak when CBGA was incubated with a positive cannabinoid synthase orthologue, which is 120 times larger than the peak found in the control experiment (column 1) performed in the absence of the cannabinoid synthase orthologue. [Figure 6] Figure 6 shows representative chromatograms of cannabinoid synthase orthologues that produced cannabinoids with m / z = 329.2122. Each reaction set was administered for 15.70 minutes for analysis. Chromatograms in columns 1, 2, and 4 show the presence of peaks when CBGA was incubated with positive cannabinoid synthase orthologues, which are 300 times larger than the peaks found in the control experiment (column 3) performed in the absence of cannabinoid synthase orthologues. [Modes for carrying out the invention]

[0009] Enzymes that catalyze the biosynthesis of cannabinoids from CBGA are disclosed. These enzymes, previously unknown as cannabinoid synthases, originate from organisms other than Cannabis sativa. These enzymes can be expressed in their active form by recombinant technology in Saccharomyces cerevisiae and Pichia pastoris.

[0010] The method summarized above for producing cannabinoids requires the step of contacting CBGA with a cannabinoid synthase orthologue not derived from hemp (Cannabis sativa). The sources of cannabinoid synthase orthologs are not limited to oranges (Citrus sinensis), rapeseed (Brassica napus), Nicotiana attenuate, cotton (Gossypium hirsutum), rice (Indica variety) (Oryza sativa subsp. indica), japonica rice (Oryza sativa subsp. japonica), Arabidopsis lyrata subsp. lyrata, Paenibacillus sp. Aloe-11, Streptomyces ipomoeae 91-03, Chinese cabbage (Brassica rapa subsp. pekinensis), and peaches (Prunus serrulata). It could be persica), Bacillus subtilis 168, Arabidopsis thaliana, poppy (Papaver somniferum), and Phytophthora parasitica P1569.

[0011] Cannabinoid synthase orthologs may include, but are not limited to, those shown in Tables 1 and 2 below. Exemplary cannabinoid synthase orthologs may have amino acid sequences of SEQ ID NOs. 7, 16, 23, 31, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127, or sequences having 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, and 99%) identity with SEQ ID NOs.

[0012] The methods described above can produce cannabinoids with a formula weight of 358.5 g / mol, 374.5 g / mol, or 330.5 g / mol. In particular, the methods produce cannabinoids with formula weights of 358.5 g / mol, 374.5 g / mol, and 330.5 g / mol, respectively.

[0013] Another method produces cannabiellsonic acid and cannabiellsoin. One example of this method produces a cannabinoid containing both cannabiellsonic acid and cannabiellsoin, but the product contains no cannabinoid at all with a formula weight of 358.5 g / mol. In this exemplary method, the cannabinoid synthase ortholog contains the amino acid sequence of SEQ ID NOs. 67, 77, 97, or 127, or a sequence having 70% or more identity with SEQ ID NOs. 67, 77, 97, or 127.

[0014] In the method described above, the cannabinoid synthase ortholog may be a recombinant enzyme. Recombinant enzymes can be produced, for example, in Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, and Pichia pastoris. The specific method is characterized by a recombinant enzyme produced in Saccharomyces cerevisiae or Pichia pastoris.

[0015] Furthermore, recombinant cells of Saccharomyces cerevisiae or Pichia pastoris have been shown to contain nucleic acids encoding cannabinoid synthase orthologues in their genomes. These orthologues originate from organisms other than Cannabis sativa. Exemplary sources of cannabinoid synthase orthologues are listed above and are shown in Tables 1 and 2 below.

[0016] The recombinant cell may contain a nucleic acid encoding a cannabinoid synthase ortholog that includes an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 16, 23, 31, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127, or a sequence having 70% or more (e.g., 70%, 75%, 80%, 85%, 90%, 95%, and 99%) identity to SEQ ID NO: 7, 16, 23, 31, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127.

[0017] Without further elaboration, it is believed that one of ordinary skill in the art can, based on the disclosure herein, utilize the present disclosure to its fullest extent. The following specific examples are, therefore, to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way. All publications recited herein are hereby incorporated by reference in their entirety.

[0018] Examples Example 1: Identification and Preparation of Prospective Cannabinoid Synthase Based on its sequence, THCA synthase can be classified as a berberine-bridged FAD-dependent enzyme. Through a search of available sequence databases, 232 related genes annotated in the UniProt database as berberine-bridged FAD-dependent enzymes were identified. In other words, the 232 genes were prospective cannabinoid synthase orthologs.

[0019] Each gene sequence was codon-optimized for S. cerevisiae protein expression, synthesized, and cloned into a pYES2-CT vector (Thermo Fisher). These vectors were used to separately transform S. cerevisiae BY4741 cells using chemical treatment, and the cells were grown on SC-URA glucose-selective plates at 30°C for 2 days. Three single colonies were taken from each plate, replicated on SC-URA glucose-selective plates, and incubated at 30°C for 2 days. The replicated colonies were grown in 10 mL of SC-URA glucose medium at 30°C for 16 hours. The cells were then harvested, resuspended in 5 mL of SC-URA galactose medium, and grown at 30°C for 16 hours for protein expression. After 16 hours, the cells were harvested again and stored at -20°C. Cells completely free of cannabinoid synthase orthologues were prepared as described above and used as a negative control.

[0020] The cells were resuspended in 400 μL of 100 mM citrate buffer (pH 5.5) with 1 mM MgCl2 and 5 units of lithicase. The cells were incubated at 37°C for 1 hour with shaking. 1 gram of glass beads was added to the suspension, and the cells were cracked open using an MP Biomedical FastPrep 24 Tissue Homogenizer. The cell fragments were removed by centrifugation at 14,800 rpm at 4°C for 30 minutes.

[0021] Example 2: Enzyme assay Promising cannabinoid synthase orthologues were tested for enzymatic activity as follows: 2.5 μL of 1 mg / L CBGA, 2.5 μL of 20 mM FAD, and 50 μL of the supernatant of yeast cells prepared as described above were combined in a 55 μL reaction volume and incubated under atmospheric conditions. A corresponding control reaction lacking CBGA was also performed. After 24 hours, the reaction product was extracted three times with ethyl acetate. The extracted samples were vacuum-dried and redissolved in acetonitrile for mass spectrometry using the negative ion mode. EICs were prepared for each sample at m / z=357.5 and m / z=373.5 to determine whether cannabinoid biosynthesis was catalyzed by these orthologues. Promising reaction products are shown in Figure 1.

[0022] Three single yeast colonies were collected from each promising cannabinoid synthase orthologue and grown as described above. The supernatant prepared from each colony was divided into six parts. Three of these parts were incubated with CBGA, and the remaining three parts were not incubated with CBGA as a negative control. In total, nine reactions were carried out in the presence of CBGA for each promising orthologue, and nine reaction products were not incubated with CBGA.

[0023] Samples incubated with CBGA were considered positive for cannabinoid synthesis if the extracted substance had a peak region in the EIC chromatograph with an abundance of at least 800,000 at m / z=357.5 and / or m / z=373.5. A promising cannabinoid synthase orthologue was considered positive if seven out of a total of nine reaction products with CBGA were positive using the above criteria. These results are shown in Table 1 below, as well as in Figures 2A-2C and 3A-3C.

[0024] Figures 2A-2C and 3A-3C are representative chromatograms of Rapid-Fire / Triple Quad mass spectrometry. Figure 2C shows a representative chromatogram that shows the presence of a peak at m / z 357.5. Figure 3C shows a representative chromatogram comparing the presence of a peak at m / z 373.5.

[0025] [Table 1] TIFF2026123064000003.tif123170

[0026] Of the 72 cannabinoid synthase orthologues tested, 20 were C 22 H 30 It shows the production of cannabinoids with the molecular formula O4 (MW=358.5; m / z 357.5), and 16 orthologues, C 22 H 30 The synthesis of a cannabinoid with the molecular formula O4 (MW=374.5; m / z 373.5) was demonstrated. Five orthologues demonstrated the synthesis of both types of cannabinoids. See Table 1 above.

[0027] Example 3: Expression of cannabinoid synthase orthologues in Pichia pastoris Genes encoding orthologues of cannabinoid synthases that exhibited activity using CBGA as a substrate were cloned into a P. pastoris expression system for larger-scale protein expression. Expression of THCA synthase in P. pastoris had been previously demonstrated. See, for example, Zirpel et al., Biotechnology Lett. 2015, 37(9):1869-1875 and Lange et al., J. Biotechnol. 2015, 211:68-76.

[0028] These genes were cloned into the pPICZA plasmid (Invitrogen) and then incorporated into the P. pastoris genome using standard techniques. The resulting P. pastoris constructs were inoculated from agar plates and incubated in a baffled flask in 20 mL of buffered complex glycerol medium at 30°C for 48 hours with shaking at 190 rpm. The cells were harvested by centrifugation and resuspended in 200 mL of buffered methanol complex medium containing 0.5 (w / v)% casamino acid and 0.01 (w / v)% riboflavin. Orthologous protein expression was induced by adding 1% methanol every 24 hours for a total of 120 hours.

[0029] Cells were collected by centrifugation and resuspended in Buffer A (100 mM Tris, pH 8.0, and 150 mM NaCl). Cells were lysed using an M110P Microfluidizer® (Microfluidics International Corp.). Cell fragments were removed by centrifugation, and the cell lysate was packed onto a 5 mL StrepTrap® HP column (Cytiva Life Sciences) at a flow rate of 1 mL / min. The column was washed with 25 mL, i.e., 5 column volumes ("CV") of Buffer A, and eluted with 6 CV of Buffer B (100 mM Tris, pH 8.0, 150 mM NaCl, and 2.5 mM desthiobiotin). Fractions containing proteins identified by increased absorbance at 280 nm were collected, concentrated, and frozen at -80°C.

[0030] With a reaction volume of 50 μL, the purified cannabinoid synthase was incubated with 50 mM citrate (pH 8.0) containing 150 μM CBGA and 0.2 mM FAD. The reaction was incubated at 30 °C for 24 - 48 h, then 又に160 μL of acetonitrile was added to stop the reaction and precipitate the protein. 120 μL of this mixture was injected onto an Agilent InfinityLab Poroshell 120 EC-C18 column for mass spectrometry using an Agilent 1290 Infinity HPLC combined with an Agilent 6550 iFunnel Q-TOF high-resolution mass spectrometer in negative ion mode. Extracted ion chromatograms (EICs) were generated for m / z = 357.2071, m / z = 373.2020, and m / z = 329.2122 for each sample to determine whether the biosynthesis of the putative cannabinoids was catalyzed by the orthologs.

[0031] The results are shown in Figures 4 - 6 and Table 2 below.

[0032]

Table 2

[0033] Of the 232 putative CBS orthologs screened, 7 orthologs showed the production of two types of cannabinoids: (i) the "cannabidiolic acid group" type (FW = 358.5; m / z = 357.2071, see Figure 4) with the molecular formula of C 22 H 30 O4, and (ii) cannabielsoic acid (FW = 374.5; m / z = 373.2020, see Figure 5) with the molecular formula of C 22 H[[ID=2,3]] 30 O4. The decarboxylation product of cannabielsoic acid, i.e., C 21 H 30 It should be noted that there seems to be some garbled or incomplete information in the original text, especially in the part "160 μL of acetonitrile was added to stop the reaction and precipitate the protein. 120 μL of this mixture was injected onto an Agilent InfinityLab Poroshell 120 EC-C18 column for mass spectrometry using an Agilent 1290 Infinity HPLC combined with an Agilent 6550 iFunnel Q-TOF high-resolution mass spectrometer in negative ion mode." where "又に" might be a typo. The translation is done based on the best understanding of the overall context.Cannabiersoin with the molecular formula O2 (FW=330.5; m / z=329.2122, see Figure 6) was also observed. Three orthologues produced only cannabinoids with a cannabidiolic acid group. Four orthologues produced only cannabiersonic acid and cannabiersoin.

[0034] Other Embodiments All properties disclosed herein can be combined in any combination. Each property disclosed herein may be substituted for another property that serves the same, equivalent, or similar purpose. Thus, unless otherwise expressly stated, each disclosed property is merely an example of a general series of equivalent or similar properties.

[0035] From the above description, those skilled in the art will readily be able to clarify the essential features of the present invention and make various changes and modifications to the invention to suit various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the following claims.

Claims

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: 7, 31, 37, 67, 77, 97, and 127, and the produced 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 is selected from the group consisting of cannabidiolic acid, cannabiersonic acid, and cannabiersoin.

3. The method according to claim 2, wherein the cannabinoid produced comprises cannabiersonic acid and cannabiersoin.

4. The method according to claim 3, wherein the cannabinoid produced does not contain cannabidiolic acid, and the cannabinoid synthase ortholog comprises the amino acid sequence of SEQ ID NO: 67, 77, 97, or 127.

5. 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.

6. Recombinant cells of Saccharomyces cerevisiae or Pichia pastoris, wherein the genome contains a nucleic acid encoding a cannabinoid synthase ortholog containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 31, 37, 67, 77, 97, and 127, and the cannabinoid synthase ortholog is expressed in an active form in the recombinant cells.

7. The recombinant cell according to claim 6, wherein the nucleic acid encodes a cannabinoid synthase ortholog comprising the amino acid sequence of SEQ ID NO: 67, 77, 97, or 127.

Images