Biosynthesis of cannabinoids from cannabinoid phenolic acids using cannabinoid synthetases

Abstract

A method of producing cannabinoids by contacting a cannabinoid phenolic acid with a cannabinoid synthase ortholog. The cannabinoid synthase ortholog is derived from organisms other than cannabis sativa. Also disclosed herein is a recombinant cell of Saccharomyces cerevisiae or Pichia pastoris comprising in its genome a nucleic acid encoding the above cannabinoid synthase ortholog. The cannabinoid synthase ortholog is expressed in an active form in a recombinant cell.

Description

[0001] This application is a divisional application of a patent application filed on October 12, 2020, with international application number: PCT / SG2020 / 050583, entering the Chinese national phase date of April 11, 2022, national application number 202080071171.7, and titled "Biosynthesis of cannabinoids from cannabinoid acid using new cannabinoid synthases". Background Art

[0002] The cannabinoid biosynthetic pathway has been the subject of intensive research. Several research groups have completed the cloning and expression of various enzymes in the cannabinoid biosynthetic pathway. For example, Δ 1 - Structure and function of tetrahydrocannabinolic acid synthase (ie, cannabinoid synthase). See Shoyama et al., J. Mol. Biol. 2012, 423(1):96-105.

[0003] Cannabidiolic acid and Δ 9 -tetrahydrocannabinolic acid The biosynthesis of these two cannabinoids is achieved by heterologously expressing cannabinoid biosynthetic pathway enzymes from cannabis 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 have demonstrated that it is possible to construct cannabinoid biosynthetic pathways in heterologous systems.

[0004] New enzymes and methods for synthesizing cannabinoids with specificity and high yield are urgently needed. Summary of the invention

[0005] Disclosed herein is a method for producing cannabinoids. The method comprises the step of contacting cannabinoid phenolic acid with a cannabinoid synthase ortholog. The cannabinoid synthase ortholog is from an organism other than cannabis, such as orange (Citrussinensis), melon (Cucumis melo), pepper (Capsicum annuum), Brassicanapus, tobacco (Nicotiana attenuata), red tobacco (Nicotiana tabacum), Noccaea caerulescens (Thlaspi caerulescens), upland cotton (Gossypium hirsutum, Gossypium mexicanum), rice subspecies indica (Oryza sativa subsp. Indica), rice subspecies japonica (Oryza sativa japonica), Arabidopsis lyrata subsp. lyrata, Paenibacillus sp. Aloe-11, Streptomyces ipomoeae91-03, Brassica rapa subsp. pekinensis, Prunus persica, Bacillus subtilis 168, Arabidopsis thaliana, Papaver somniferum, and Phytophthora parasitica P1569.

[0006] Also provided herein are recombinant cells of Saccharomyces cerevisiae and Pichia pastoris, each of which comprises in its genome a nucleic acid encoding a cannabinoid synthase ortholog, wherein the cannabinoid synthase ortholog is from any of the organisms listed in the preceding paragraph and the cannabinoid synthase ortholog is expressed in an active form in the recombinant cell.

[0007] One or more embodiments are described in detail in the following description and examples. Other features, objects, and advantages will become apparent from the detailed description, drawings, and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The present invention is described below with reference to the accompanying drawings, in which:

[0009] Figure 1The cannabinoid products that can be formed from cannabigerolic acid ("CBGA") as a substrate are shown. The molecular formula and molecular weight of hexahydrocannabifuroic acid ("CBEA") are C 22 H 30 The molecular formula and molecular weight of cannabielsoin (decarboxylated CBEA) are C 21 H 30 O3 and 330.5. The molecular formulas and molecular weights of the other compounds shown are C 22 H 30 O4 and 358.5. CBCA = cannabidiolic acid, CBTA = cannabicitranic acid, CBDA = cannabidiolic acid, THICA = tetrahydrocannabinolic acid, THCA = tetrahydrocannabinolic acid, CBLA = cannabicyclolic acid.

[0010] Figure 2A Representative total ion chromatograms of 18 reaction sets of cannabinoid synthase orthologs are shown. Each set of reactions took 1.7 min to analyze.

[0011] Figure 2B Representative extracted ion chromatograms ("EICs") of 18 reaction sets of cannabinoid synthase orthologs obtained at mass-to-charge ratio ("m / z") = 359.5 are shown, indicating the presence of CBGA in 9 of the 18 reaction sets added (i.e., Sets 1-3, 7-9, 13-15).

[0012] Figure 2C Representative EICs of 18 reaction sets are shown, demonstrating the production of the cannabinoid m / z = 357.5.

[0013] Figure 3A Total ion chromatograms for a panel of 18 cannabinoid synthase orthologs are shown. Each reaction took 1.7 min.

[0014] Figure 3B A representative EIC of 18 reaction sets of cannabinoid synthase orthologs obtained at m / z = 359.5 is shown, indicating the presence of CBGA added to 9 of the 18 reaction sets (ie, Sets 1-3, 7-9, 13-15).

[0015] Figure 3C Representative EICs from 18 reaction sets are shown, demonstrating the production of the cannabinoid m / z = 357.5.

[0016] Figure 4Representative chromatograms of cannabinoid synthase orthologs producing the cannabinoid m / z = 357.2071 are shown. Each set of reactions took 15.70 min to analyze. The chromatograms in rows 1, 2, and 4 show that when CBGA was incubated with the positive cannabinoid synthase ortholog, there was a peak that was 65 times larger than the peak found in the control experiment (row 3) performed in the absence of the cannabinoid synthase ortholog.

[0017] Figure 5 Representative chromatograms of cannabinoid synthase orthologs that produced the cannabinoid m / z = 373.2020 are shown. Each set of reactions took 15.70 min to analyze. The chromatograms in rows 2-4 show that when CBGA was incubated with the positive cannabinoid synthase ortholog, there was a peak that was 120 times larger than the peak found in the control experiment (row 1) performed in the absence of the cannabinoid synthase ortholog.

[0018] Figure 6 Representative chromatograms of cannabinoid synthase orthologs that produced the cannabinoid of m / z = 329.2122 are shown. Each set of reactions took 15.70 min to analyze. The chromatograms in rows 1, 2, and 4 show that when CBGA was incubated with the positive cannabinoid synthase ortholog, there was a peak that was 300 times larger than the peak found in the control experiment (row 3) performed in the absence of the cannabinoid synthase ortholog. DETAILED DESCRIPTION

[0019] Disclosed herein are enzymes that catalyze the biosynthesis of cannabinoids from CBGA. These enzymes, which were not previously known as cannabinoid synthases, are from organisms other than cannabis. These enzymes can be recombinantly expressed in active form in Saccharomyces cerevisiae and Pichia pastoris.

[0020] The methods for producing cannabinoids outlined above require contacting CBGA with a cannabinoid synthase ortholog from a non-cannabis source. The sources of cannabinoid synthase orthologs can be, but are not limited to, orange, Brassica napus, tobacco, cotton, rice subspecies indica, rice subspecies japonica, Arabidopsis thaliana subspecies lyrata, Paenibacillus Aloe-11, Streptomyces ipomoeae 91-03, turnip, peach, Bacillus subtilis 168, Arabidopsis thaliana, opium poppy, and Phytophthora parasiticus P1569.

[0021] Cannabinoid synthase orthologs may be, but are not limited to, those shown in Tables 1 and 2 below. Exemplary cannabinoid synthase orthologs may have an 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 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.

[0022] The above method can produce cannabinoids with a formulation weight of 358.5 g / mol, 374.5 g / mol or 330.5 g / mol. In a specific method, cannabinoids with a formulation weight of 358.5 g / mol, 374.5 g / mol and 330.5 g / mol are produced respectively.

[0023] Another method produces hexahydrocannabinofuric acid and cannabinoids. An example of this method is to produce cannabinoids containing hexahydrocannabinofuric acid and cannabinoids, but the product does not contain any cannabinoids at a formula weight of 358.5 g / mol. In this exemplary method, the cannabinoid synthase ortholog includes an amino acid sequence of SEQ ID NO: 67, 77, 97 or 127 or a sequence with 70% or greater identity to SEQ ID NO: 67, 77, 97 or 127.

[0024] In the above 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. A specific method features a recombinant enzyme produced in Saccharomyces cerevisiae or Pichia pastoris.

[0025] Also mentioned above are recombinant cells of Saccharomyces cerevisiae or Pichia pastoris containing in their genome a nucleic acid encoding a cannabinoid synthase ortholog. The ortholog is from an organism other than cannabis. Exemplary sources of cannabinoid synthase orthologs have been listed above and are shown in Tables 1 and 2 below.

[0026] The recombinant cell may contain a nucleic acid encoding a cannabinoid synthase ortholog, the ortholog comprising an amino acid sequence selected from 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.

[0027] Without further elaboration, it is believed that those skilled in the art will be able to utilize the disclosure to the greatest extent possible based on the disclosure herein. Therefore, the following specific examples should be interpreted as merely illustrative and not limiting the remainder of the disclosure in any way. All publications cited herein are incorporated herein by reference in their entirety.

[0028] Example

[0029] Example 1: Identification and preparation of potential cannabinoid synthases

[0030] Based on its sequence, THCA synthase can be classified as a berberine-bridged FAD-dependent enzyme. By searching available sequence databases, 232 related genes were identified, which were annotated as berberine-bridged FAD-dependent enzymes in the UniProt database. In other words, these 232 genes are potential cannabinoid synthase orthologs.

[0031] Each gene sequence was codon optimized for Saccharomyces cerevisiae protein expression, synthesized and cloned into the pYES2-CT vector (Thermo Fisher). The vectors were transformed into Saccharomyces cerevisiae BY4741 cells using chemical treatment and grown on SC-URA-glucose selection plates at 30°C for two days. Three single colonies were picked from each plate, replicated onto SC-URA-glucose selection plates, and incubated at 30°C for two days. The replicated colonies were cultured in 10mL SC-URA-glucose medium at 30°C for 16h. The cells were then harvested and resuspended in 5mL SC-URA-galactose medium and grown at 30°C for 16h for protein expression. After 16 hours, the cells were harvested again and stored at -20°C. Cells that did not carry any cannabinoid synthase orthologs were also prepared as described above and used as negative controls.

[0032] Resuspend the cells in 400 μL of 100 mM citrate buffer (pH 5.5), add 1 mM MgCl2 and 5 units of lyticase. Incubate the cells at 37°C with shaking for 1 h. Add 1 g of glass beads to the suspension and lyse the cells using an MPBiomedical FastPrep 24 tissue homogenizer. Centrifuge for 30 min to remove cell debris. The speed is 14,800 rpm and the temperature is 4°C.

[0033] Example 2: Enzyme activity assay

[0034] The enzymatic activity of potential cannabinoid synthase orthologs was tested as follows. In a reaction volume of 55 μL, 2.5 μL of 1 mg / mL CBGA, 2.5 μL of 20 mM FAD and 50 μL of yeast cell supernatant prepared as above were combined and incubated under ambient conditions. Corresponding control reactions without CBGA were also performed. After 24 h, extractions were performed three times with ethyl acetate. Samples of the extracted material were dried under vacuum and redissolved in acetonitrile for mass analysis using negative ion mode. EICs of m / z = 357.5 and m / z = 373.5 were generated for each sample to determine whether the biosynthesis of cannabinoids was catalyzed by the orthologs. Potential reaction products such as Figure 1 shown.

[0035] For each potential cannabinoid synthase ortholog, three single yeast colonies were picked and cultured as described above. Cell supernatants prepared from each colony were divided into six portions. Three portions were incubated with CBGA and 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 performed without CBGA.

[0036] Samples incubated with CBGA were considered positive for cannabinoid synthesis if the extracted material had an abundance of at least 800,000 in the chromatographic peak area of ​​the EIC at m / z = 357.5 and / or m / z = 373.5. Potential cannabinoid synthase orthologs were considered positive if 7 out of 9 total reactions with CBGA were positive using the above criteria. The results are shown in Table 1 below, and Figure 2A-2C and Figure 3A-3C .

[0037] Figure 2A-2C and 3A-3C are representative chromatograms of Rapid-Fire / Triple Quad mass analysis. Figure 2C A representative chromatogram showing the presence of a peak at m / z 357.5 is shown. Figure 3C Representative chromatograms comparing the presence of the peak at m / z 373.5 are shown.

[0038] Table 1. Activities of cannabinoid synthase orthologs expressed in Saccharomyces cerevisiae.

[0039]

[0040]

[0041] Of the 72 cannabinoid synthase orthologs tested, 20 were shown to produce a molecule with the formula C 22 H 30 The cannabinoid O4 (MW = 358.5; m / z 357.5) and 16 orthologs showed the generation of a molecular formula of C 22 H 30 Cannabinoid of O4 (MW = 374.5; m / z 373.5). Five orthologs were shown to produce two cannabinoids. See Table 1 above.

[0042] Example 3: Homologous expression of cannabinoid synthase in Pichia pastoris

[0043] Genes encoding orthologs that exhibit cannabinoid synthase activity with CBGA as a substrate were cloned into the Pichia pastoris expression system for larger-scale protein expression. Expression of THCA synthase in Pichia pastoris has been demonstrated. See, for example, Zirpel et al., Biotechnology Lett. 2015, 37(9): 1869-1875 and Lange et al., J. Biotechnol. 2015, 211: 68-76.

[0044] The gene was cloned into the pPICZA plasmid (Invitrogen) and then integrated into the Pichia genome using conventional techniques. The resulting Pichia integrant was inoculated from an agar plate and cultured in a shaking culture flask containing 20 mL of buffered complex glycerol medium at 30°C and 190 rpm for 48 h. The cells were collected 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. 1% methanol was added every 24 h for a total of 120 h to induce expression of the orthologous protein.

[0045] The cells were collected by centrifugation and resuspended in buffer A (100 mM Tris, pH 8.0 and 150 mM NaCl). (Microfluidics International Corp.). Cell debris was removed by centrifugation, and the cell lysate was loaded onto a 5 mL StrepTrap TMHP chromatography column (Cytiva Life Sciences) at a flow rate of 1 mL / min. The column was washed with 25 mL of buffer A, i.e., 5 column volumes ("CV"), and eluted with 6 CV of buffer B (100 mM Tris, pH 8.0, 150 mM NaCl, and 2.5 mM desthiobiotin). Protein-containing fractions identified by an increase in absorbance at 280 nm were pooled, concentrated, and frozen at -80°C.

[0046] In a reaction volume of 50 μL, the cannabinoid synthase purified therefrom was incubated with 150 μM CBGA and 0.2 mM FAD in 50 mM citrate (pH 8.0). The reaction was incubated at 30 ° C for 24-48 h, and then 160 μL acetonitrile was added to stop the reaction and precipitate the protein. The mixture (120 μL) was injected into an Agilent InfinityLab Poroshell 120EC-C18 column and mass analyzed in negative ion mode using an Agilent 1290 Infinity HPLC coupled to an Agilent 6550iFunnel Q-TOF high-resolution mass spectrometer. EICs of m / z = 357.2071, m / z = 373.2020, and m / z = 329.2122 were generated for each sample to determine whether the biosynthesis of potential cannabinoids is catalyzed by orthologs.

[0047] Results Figure 4-Figure 6 and Table 2 below.

[0048] Table 2 Activity of cannabinoid synthase orthologs expressed in Pichia pastoris

[0049]

[0050] Among the 232 potential CBS orthologs screened, 7 orthologs were shown to produce two types of cannabinoids, namely (i) 22 H 30 O4 (FW = 358.5; m / z = 357.2071, see Figure 4 ) of the "cannabidiol acid group" type and (ii) hexahydrocannabinofuric acid, the molecular formula is C 22 H 30 O4 (FW = 374.5; m / z = 373.2020, see Figure 5 The decarboxylation product of hexahydrocannabinol acid, cannabinoid, was also observed, with the molecular formula C 21 H 30 O2 (FW = 330.5; m / z = 329.2122, see Figure 6). Three orthologs produce only cannabidiol acid cannabinoids. Four orthologs produce only hexahydrocannabinofuric acid and cannabinoids.

[0051] Other embodiments

[0052] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a series of equivalent or similar features.

[0053] Through the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the appended claims.

Claims

1. A method for producing one or more cannabinoids, the method comprising contacting cannabigerolic acid (CBGA) with a cannabinoid synthase ortholog, wherein the ortholog comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 31, 37, 67, 77, 97, 117 and 127.

2. The method according to claim 1, wherein: The prepared cannabinoids are selected from cannabidiolic acid, hexahydrocannabifurconic acid and cannabinoids.

3. The method of claim 2, wherein the prepared cannabinoids include hexahydrocannabinofuric acid and cannabinoids.

4. A method according to claim 3, wherein the prepared cannabinoids do 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 of claim 1, wherein the cannabinoid synthase ortholog is a recombinant enzyme produced in Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus or Pichia pastoris.

6. A recombinant cell of Saccharomyces cerevisiae or Pichia pastoris, comprising in its genome a nucleic acid encoding a cannabinoid synthase ortholog, wherein the cannabinoid synthase ortholog is selected from the amino acid sequences of SEQ ID NO: 7, 31, 37, 67, 77, 97, 117 and 127, and the cannabinoid synthase ortholog is expressed in an active form in the recombinant cell.

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

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