Biosynthesis of cannabinoids from cannabigerolic acid using a novel cannabinoid synthase

Abstract

A method for producing cannabinoids by contacting cannabigerolic acid with a cannabinoid synthase ortholog, wherein the cannabinoid synthase ortholog is derived from an organism other than Cannabis sativa. Also disclosed is a recombinant cell of Saccharomyces cerevisiae or Pichia pastoris, the recombinant cell comprising a nucleic acid encoding the cannabinoid synthase ortholog in its genome. The cannabinoid synthase ortholog is expressed in its active form in the recombinant cell.

Description

Technical Field

[0001] Background Art The biosynthetic pathway of cannabinoids has been the subject of intensive investigation. The cloning and expression of various enzymes in the cannabinoid biosynthetic pathway have been achieved by several research groups. For example, the structure and function of Δ 1 -tetrahydrocannabinolic acid synthase, i.e., cannabinoid synthase, have been elucidated. See Shoyama et al., J. Mol. Biol. 2012, 423(1):96-105.

[0002] The biosynthesis of two cannabinoids, cannabidiolic acid and Δ 9 -tetrahydrocannabinolic acid, has been achieved by heterologous expression in Saccharomyces cerevisiae of enzymes of the cannabinoid biosynthetic 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. Such studies have established that it is possible to construct the cannabinoid biosynthetic pathway in a heterologous system.

[0003] There is a need for additional enzymes and methods for synthesizing cannabinoids specifically and in high yields.

Summary of the Invention

[0004] A method for producing cannabinoids is disclosed. The method includes the step of contacting cannabigerolic acid with a cannabinoid synthase ortholog. The cannabinoid synthase ortholog is derived from an organism other than Cannabis sativa, such as Citrus sinensis, Cucumis melo, Capsicum annuum, Brassica napus, Nicotiana attenuata, Nicotiana tabacum, Noccaea caerulescens (Thlaspi caerulescens), Gossypium hirsutum (Gossypium mexicanum), Oryza sativa subsp. indica, Oryza sativa subsp. japonica, Arabidopsis lyrata subsp. lyrata, Paenibacillus sp. Aloe-11, Streptomyces ipomoeae 91-03, Brassica rapa subsp. pekinensis, Prunus persica, Bacillus subtilis 168, Arabidopsis thaliana, Papaver somniferum, and Phytophthora parasitica P1569.

[0005] Also provided are recombinant cells of Saccharomyces cerevisiae and Pichia pastoris, each containing a nucleic acid encoding a cannabinoid synthase ortholog in their genomes, wherein the cannabinoid synthase ortholog is derived from any of the organisms listed in the preceding paragraph and is expressed in its active form in the recombinant cells.

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

[0007] The following description of the invention is presented with reference to the accompanying drawings.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 3A

Figure 3B

Figure 3C

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

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

[0010] The method summarized above for generating cannabinoids requires contacting CBGA with a cannabinoid synthase ortholog that is not derived from Cannabis sativa. Sources of the cannabinoid synthase ortholog can include, but are not limited to, Citrus sinensis, Brassica napus, Nicotiana attenuata, Gossypium hirsutum, Oryza sativa subsp. indica, Oryza sativa subsp. japonica, Arabidopsis lyrata subsp. lyrata, Paenibacillus sp. Aloe-11, Streptomyces ipomoeae 91-03, Brassica rapa subsp. pekinensis, Prunus persica, Bacillus subtilis 168, Arabidopsis thaliana, Papaver somniferum, and Phytophthora parasitica P1569.

[0011] The cannabinoid synthase ortholog can include, but is not limited to, those shown in Tables 1 and 2 below. Exemplary cannabinoid synthase orthologs can have the 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 to SEQ ID NOs: 7, 16, 23, 31, 37, 47, 57, 67, 77, 87, 97, 107, 117, and 127.

[0012] The above method can generate cannabinoids having a formula weight of 358.5 g / mol, 374.5 g / mol, or 330.5 g / mol. In a specific method, cannabinoids having formula weights of 358.5 g / mol, 374.5 g / mol, and 330.5 g / mol are each generated.

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

[0014] In the above method, the cannabinoid synthase ortholog can be a recombinant enzyme. The recombinant enzyme can be produced, for example, in Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, and Pichia pastoris. A specific method features a recombinant enzyme produced in Saccharomyces cerevisiae or Pichia pastoris.

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

[0016] The recombinant cell may contain 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 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 Synthases 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 the pYES2-CT vector (Thermo Fisher). These vectors were separately transformed into S. cerevisiae BY4741 cells using chemical treatment and grown at 30 °C for 2 days on SC-URA glucose selection plates. 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 at 30 °C for 16 hours in 10 mL of SC-URA glucose medium. Next, the cells were 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. Also, cells carrying no cannabinoid synthase ortholog 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 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 lysed 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: Enzymatic assay Putative cannabioid synthase orthologs were tested for enzyme activity as follows. In a reaction volume of 55 μL, 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 and incubated under atmospheric conditions. A corresponding control reaction lacking CBGA was also performed. After 24 hours, the reaction was extracted three times with ethyl acetate. Samples of the extracted material were vacuum dried and redissolved in acetonitrile for mass spectrometry using the negative ion mode. Extracted ion chromatograms (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. Putative reaction products are shown in Figure 1.

[0022] For each putative cannabioid synthase ortholog, three single yeast colonies were picked and grown as described above. The 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 a negative control reaction. In total, for each putative 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 chromatographic peak region of the EIC of the extracted material had an abundance of at least 800,000 at m / z = 357.5 and / or m / z = 373.5. Putative cannabioid synthase orthologs were considered positive if seven out of a total of nine reactions with CBGA met 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 indicating 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] TIFF0007699832000002.tif123170

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

[0027] Example 3: Expression of Cannabinoid Synthase Orthologs in Pichia pastoris The gene encoding the ortholog that showed the activity of cannabinoid synthase using CBGA as a substrate was cloned into the P. pastoris expression system for larger - scale protein expression. The expression of THCA synthase in P. pastoris has been shown previously. 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 genome of P. pastoris using standard techniques. The resulting P. pastoris integrants were inoculated from agar plates and incubated in baffled flasks with 20 mL of buffered complex glycerol medium at 30 °C for 48 h 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 acids and 0.01 (w / v)% riboflavin. Expression of the orthologous protein was induced by adding 1% methanol every 24 h for a total of 120 h.

[0029] The cells were harvested by centrifugation and resuspended in Buffer A (100 mM Tris, pH 8.0 and 150 mM NaCl). The 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, 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 the protein identified by an increase in 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 160 μL of acetonitrile was added to stop the reaction and precipitate the protein. This mixture (120 μL) 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 30 O4. The decarboxylation product of cannabielsoic acid, i.e., C 21 H 30Cannabielsoin having the molecular formula of O2 (FW = 330.5; m / z = 329.2122, see Figure 6) was also observed. Three orthologs produced only cannabinoids of the cannabidiolic acid group. Four orthologs produced only cannabielsoic acid and cannabielsoin.

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

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

Claims

**Claim 1** A method for generating one or more cannabinoids, comprising the step of contacting cannabigerolic acid (CBGA) with a cannabinoid synthase ortholog derived from Citrus sinensis, wherein the cannabinoid synthase ortholog has the amino acid sequence of SEQ ID NO: 136, and wherein the cannabinoid synthase ortholog is a recombinant enzyme produced in Saccharomyces cerevisiae or Pichia pastoris. A method. **Claim 2** The method according to claim 1, wherein the generated cannabinoid has a formula weight of 358.5 g / mol and m / z = 357.

5. **Claim 3** The method according to claim 2, wherein the generated cannabinoid is cannabidiolic acid (CBDA) or tetrahydrocannabinolic acid (THCA). **Claim 4** A recombinant cell of Saccharomyces cerevisiae or Pichia pastoris, comprising a nucleic acid encoding a cannabinoid synthase ortholog in its genome, wherein the cannabinoid synthase ortholog is derived from Citrus sinensis, the cannabinoid synthase ortholog has the amino acid sequence of SEQ ID NO: 136, and the cannabinoid synthase ortholog is expressed in an active form in the recombinant cell.

Images