Biosynthesis of cannabinoids from cannabigerolic acid using novel cannabinoid synthases

Abstract

To provide enzymes and methods for synthesizing cannabinoids with specificity and a high yield.SOLUTION: Provided herein is a method for generating one or more cannabinoids, comprising a step of contacting cannabigerolic acid to a cannabinoid synthase ortholog. The cannabinoid synthase ortholog derives from organisms other than Cannabis sativa. Also disclosed is a recombinant cell of Saccharomyces cerevisiae or Pichia pastoris comprising a nucleic acid encoding the cannabinoid synthase ortholog in the genome. The cannabinoid synthase ortholog is expressed in the recombinant cell in an active form.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Background technology The cannabinoid biosynthetic pathway has been the subject of intensive investigation. Cloning and expression of various enzymes in the cannabinoid biosynthetic pathway has been achieved by several research groups. For example, Δ 1 The structure and function of -tetrahydrocannabinolic acid synthase, a 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 Biosynthesis of α-tetrahydrocannabinolic acid has been achieved by heterologously expressing cannabinoid biosynthetic pathway enzymes from Cannabis sativa and other organisms in Saccharomyces cerevisiae. See, for example, Luo et al., Nature 2019, 567(7746):123-126 and Zirpel et al., J. Biotechnol. 2017, 259:204-212. Studies such as these establish that it is possible to engineer cannabinoid biosynthetic pathways in heterologous systems.

[0003] There is a need for additional enzymes and methods for synthesizing cannabinoids specifically and in high yield. Summary of the Invention

[0004] A method for producing cannabinoids is disclosed, the method comprising contacting cannabigerolic acid with a cannabinoid synthase ortholog. Cannabinoid synthase orthologs have been found in organisms other than hemp (Cannabis sativa), such as orange (Citrus sinensis), melon (Cucumis melo), chili pepper (Capsicum annuum), rapeseed (Brassica napus), Nicotiana attenuata, tobacco (Nicotiana tabacum), Noccaea caerulescens (Thlaspi caerulescens), cotton (Gossypium hirsutum), cotton (Gossypium mexicanum), rice (Indica species) (Oryza sativa subsp. indica), rice (Oryza sativa subsp. japonica), Arabidopsis lyrata subsp. lyrata, and Paenibacillus sp. The isolates were 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] Also provided are recombinant cells of Saccharomyces cerevisiae and Pichia pastoris, each comprising in their genome a nucleic acid encoding a cannabinoid synthase ortholog, wherein the cannabinoid synthase ortholog is derived from any of the organisms listed in the preceding paragraph, and wherein the cannabinoid synthase ortholog is expressed in its active form in the recombinant cell.

[0006] The details of one or more embodiments are set forth in the description and examples that follow. Other features, objects, and advantages will be apparent from the detailed description, drawings, and appended claims.

[0007] The following description of the invention refers to the accompanying drawings. [Brief explanation of the drawings]

[0008] [Figure 1] Figure 1 shows the cannabinoid products that can be formed from cannabigerolic acid ("CBGA") as a substrate. The molecular formula and formula weight of cannabielsoic acid ("CBEA") are C22H30O5 and 374.5, respectively. The molecular formula and formula weight of cannabielsoin (decarboxylated CBEA) are C21H30O3 and 330.5, respectively. The molecular formulas and formula 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 a representative total ion chromatogram of 18 reaction sets of cannabinoid synthase orthologs, each of which took 1.7 minutes to analyze. [Figure 2B]Figure 2B shows representative extracted ion chromatograms (EICs) of 18 reaction sets of cannabinoid synthase orthologs 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 a representative EIC of a set of 18 reactions that showed production of a cannabinoid with m / z = 357.5. [Figure 3A] Figure 3A shows the total ion chromatogram of a group of 18 cannabinoid synthase orthologs. Each reaction set took 1.7 minutes. [Figure 3B] Figure 3B shows representative EICs of 18 reaction sets of cannabinoid synthase orthologs obtained at m / z = 359.5 showing the presence of CBGA in nine of the 18 reaction sets, namely sets 1-3, 7-9, and 13-15. [Figure 3C] FIG. 3C shows a representative EIC of a set of 18 reactions that exhibited production of a cannabinoid with m / z=357.5. [Figure 4] Figure 4 shows a representative chromatogram of a cannabinoid synthase ortholog that produced a cannabinoid with m / z = 357.2071. Each reaction set took 15.70 min to analyze. The chromatograms in columns 1, 2, and 4 show the presence of a peak when CBGA was incubated with the positive cannabinoid synthase ortholog, which is 65-fold larger than the peak found in the control experiment (column 3) performed in the absence of the cannabinoid synthase ortholog. [Figure 5] Figure 5 shows a representative chromatogram of a cannabinoid synthase ortholog that produced a cannabinoid with m / z = 373.2020. Each reaction set took 15.70 min to analyze. The chromatograms in columns 2-4 show the presence of a peak when CBGA was incubated with a positive cannabinoid synthase ortholog; this peak is 120-fold larger than the peak found in the control experiment (column 1) performed in the absence of the cannabinoid synthase ortholog. [Figure 6] Figure 6 shows a representative chromatogram of a cannabinoid synthase ortholog that produced a cannabinoid with m / z = 329.2122. Each reaction set took 15.70 min to analyze. The chromatograms in columns 1, 2, and 4 show the presence of a peak when CBGA was incubated with the positive cannabinoid synthase ortholog, which is 300-fold larger than the peak found in the control experiment (column 3) performed in the absence of the cannabinoid synthase ortholog. DETAILED DESCRIPTION OF THE INVENTION

[0009] Enzymes that catalyze the biosynthesis of cannabinoids from CBGA are disclosed. These enzymes, not previously known as cannabinoid synthases, are derived from organisms other than Cannabis sativa. These enzymes can be recombinantly expressed in their active form in Saccharomyces cerevisiae and Pichia pastoris.

[0010] The methods summarized above for producing cannabinoids require contacting CBGA with a cannabinoid synthase ortholog that is not derived from cannabis (Cannabis sativa). Sources of cannabinoid synthase orthologs include, but are not limited to, orange (Citrus sinensis), rapeseed (Brassica napus), Nicotiana attenuate, cotton (Gossypium hirsutum), rice (Indica species) (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), peach (Prunus persica, Bacillus subtilis 168, Arabidopsis thaliana, Papaver somniferum, and Phytophthora parasitica P1569.

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

[0012] The above-described methods may produce cannabinoids with formula weights of 358.5 g / mol, 374.5 g / mol, or 330.5 g / mol. In particular methods, cannabinoids with formula weights of 358.5 g / mol, 374.5 g / mol, and 330.5 g / mol are produced, respectively.

[0013] Another method produces cannabielsoic acid and cannabielsoin. One example of this method produces cannabinoids that include both cannabielsoic acid and cannabielsoin, but the product does not contain any cannabinoids having a formula weight of 358.5 g / mol. In this exemplary method, the cannabinoid synthase ortholog comprises the amino acid sequence of SEQ ID NO: 67, 77, 97, or 127, or a sequence having 70% or more identity to SEQ ID NO: 67, 77, 97, or 127.

[0014] In the above-described methods, the cannabinoid synthase ortholog can be a recombinant enzyme. The recombinant enzyme can be produced in, for example, Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, and Pichia pastoris. Particular methods feature recombinant enzymes produced in Saccharomyces cerevisiae or Pichia pastoris.

[0015] Also described above are recombinant Saccharomyces cerevisiae or Pichia pastoris cells that contain a nucleic acid encoding a cannabinoid synthase ortholog in their genome, where the ortholog is derived from an organism other than Cannabis sativa. Exemplary sources of cannabinoid synthase orthologs are listed above and provided in Tables 1 and 2 below.

[0016] The recombinant cell may comprise a nucleic acid encoding a cannabinoid synthase ortholog comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 NOs: 7, 16, 23, 31, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127.

[0017] Without further elaboration, it is believed that one skilled 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 whatsoever. All publications mentioned herein are incorporated by reference in their entirety.

[0018] Example Example 1: Identification and preparation of potential cannabinoid synthases Based on its sequence, THCA synthase can be classified as a berberine-linked FAD-dependent enzyme. Through a search of available sequence databases, we identified 232 related genes that are annotated in the UniProt database as berberine-linked FAD-dependent enzymes. In other words, these 232 genes are potential cannabinoid synthase orthologs.

[0019] Each gene sequence was codon-optimized for S. cerevisiae protein expression, synthesized, and cloned into the pYES2-CT vector (Thermo Fisher). These vectors were separately transformed into S. cerevisiae BY4741 cells using chemical treatment and grown on SC-URA glucose selection plates at 30°C for 2 days. Three single colonies from each plate were picked, replicated on SC-URA glucose selection 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 lacking any cannabinoid synthase orthologs were also prepared as described above and served 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 lyticase. The cells were incubated at 37°C for 1 hour with shaking. One gram of glass beads was added to the suspension, and the cells were cracked open using an MP Biomedical FastPrep 24 Tissue Homogenizer. Cell debris was removed by centrifugation at 14,800 rpm at 4°C for 30 minutes.

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

[0022] For each potential cannabinoid synthase ortholog, three single yeast colonies were picked and grown as described above. Cell supernatant prepared from each colony was divided into six portions. Three of these portions were incubated with CBGA, and the remaining three portions were not incubated with CBGA as negative control reactions. In total, for each potential ortholog, nine reactions were performed in the presence of CBGA and nine reactions were not incubated with CBGA.

[0023] Samples incubated with CBGA were considered positive for cannabinoid synthesis if the extracted material had a chromatographic peak area of ​​the EIC with an abundance of at least 800,000 at m / z = 357.5 and / or m / z = 373.5. Potential cannabinoid synthase orthologs were considered positive if seven of the nine total CBGA reactions were positive using the above criteria. The results are shown in Table 1 below, as well as Figures 2A-2C and 3A-3C.

[0024] Figures 2A-2C and 3A-3C are representative chromatograms from Rapid-Fire / Triple Quad mass spectrometry. Figure 2C shows a representative chromatogram demonstrating 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] TIFF2025131805000003.tif123170

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

[0027] Example 3: Expression of cannabinoid synthase orthologs in Pichia pastoris Genes encoding orthologs that showed cannabinoid synthase activity using CBGA as a substrate were cloned into the P. pastoris expression system for larger-scale protein expression. Expression of THCA synthase in P. pastoris has 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 integrated into the P. pastoris genome using standard techniques. The resulting P. pastoris integrants were inoculated from agar plates and incubated in 20 mL of buffered complex glycerol medium in a baffled flask at 30°C for 48 hours with shaking at 190 rpm. Cells were harvested by centrifugation and resuspended in 200 mL of buffered methanol complex medium containing 0.5% (w / v) casamino acids and 0.01% (w / v) riboflavin. Expression of the orthologous proteins was induced by adding 1% methanol every 24 hours for a total of 120 hours.

[0029] Cells were harvested 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 debris was removed by centrifugation, and the cell lysate was loaded 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, or 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 protein, identified by increased absorbance at 280 nm, were collected, concentrated, and frozen at −80°C.

[0030] In a 50 μL reaction volume, 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, followed by the addition of 160 μL of acetonitrile to terminate the reaction and precipitate the protein. This mixture (120 μL) was injected onto an Agilent InfinityLab Poroshell 120 EC-C18 column for mass spectrometry analysis using an Agilent 1290 Infinity HPLC coupled to an Agilent 6550 iFunnel Q-TOF high-resolution mass spectrometer in negative ion mode. EICs at m / z = 357.2071, m / z = 373.2020, and m / z = 329.2122 for each sample were generated to determine whether biosynthesis of the potential cannabinoids was catalyzed by the orthologs.

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

[0032] [Table 2]

[0033] Of the 232 potential CBS orthologs screened, seven orthologs were identified as orthologs of two cannabinoids: (i) C 22 H 30 (ii) the “cannabidiolic acid group” type with the molecular formula of O4 (FW=358.5; m / z=357.2071, see Figure 4), and (ii) C 22 H 30 The production of cannabielonic acid (FW = 374.5; m / z = 373.2020, see Figure 5) with the molecular formula of O4 was shown. The decarboxylation product of cannabielonic acid, i.e., C 21 H 30Cannabielsoin (FW = 330.5; m / z = 329.2122, see Figure 6), with the molecular formula O2, was also observed. Three orthologs produced only cannabinoids of the cannabidiolic acid group. Four orthologs produced only cannabielsoin and cannabielsoin.

[0034] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by another feature serving the same, equivalent, or similar purpose. Thus, unless expressly indicated otherwise, each feature disclosed is merely an example in a generic series of equivalent or similar features.

[0035] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various uses and conditions without departing from the spirit and scope of the present invention. Accordingly, other embodiments are also within the scope of the following claims.

Claims

1. 1. A method for producing one or more cannabinoids, comprising the step of contacting cannabigerolic 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 wherein the cannabinoids produced have a formula weight of 358.5 g / mol, 374.5 g / mol, or 330.5 g / mol.

2. 10. The method of claim 1, wherein the cannabinoids produced include cannabielsoic acid and cannabielsoin.

3. 2. The method of claim 1, wherein the cannabinoid synthase ortholog is a recombinant enzyme produced in Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, or Pichia pastoris.

4. 1. A recombinant cell of Saccharomyces cerevisiae or Pichia pastoris, comprising in its genome a nucleic acid encoding a cannabinoid synthase ortholog, wherein said 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 wherein said cannabinoid synthase ortholog is expressed in an active form in said recombinant cell.

5. 5. The recombinant cell of claim 4, wherein the cannabinoid synthase ortholog produces a cannabinoid having a formula weight of 358.5 g / mol, 374.5 g / mol, or 330.5 g / mol when contacted with cannabigerolic acid (CBGA).

6. 5. The recombinant cell of claim 4, wherein the cannabinoid synthase ortholog produces cannabinoids including cannabielsoic acid and cannabielsoin when contacted with cannabigerolic acid (CBGA).

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