Biosynthesis of cannabinoid precursors using novel aromatic prenyltransferases

Abstract

A method for producing cannabinoid precursors by contacting a substrate, geranyl pyrophosphate or farnesyl pyrophosphate, and an NphB ortholog. The NphB ortholog is derived from an organism other than Cannabis sativa, and the substrate can be 2,4-dihydroxy-6-pentylbenzoate or 2,4-dihydroxy-6-propylbenzoate. Also disclosed is a recombinant cell of Yarrowia lipolytica carrying in its genome a nucleic acid encoding an NphB ortholog derived from an organism other than Cannabis sativa, such that the NphB ortholog is expressed in the recombinant cell.

Description

[Technical field]

[0001] The structure and function of aromatic prenyltransferase (NphB) from Streptomyces sp. strain CL190 has been elucidated. See Kumano et al., Bioorg. Med. Chem. 2008, 16(17):8117-26. Previous work reported in Zirpel et al., J. Biotechnol. 2017, 259:204-212, showed that NphB can utilize the same substrate as membrane-bound geranyl pyrophosphate:olivetolic acid geranyltransferase from cannabis plants to form the cannabinoid precursor cannabigerolic acid (CBGA).

[0002] CBGA is commonly known as the first branch of the cannabinoid biosynthetic pathway found in cannabis plants, and wild-type NphB can make CBGA as well as its O-prenylation by-product, namely 2-O-geranylolivetolic acid.

[0003] Additional enzymes and methods are needed to synthesize known and novel cannabinoid precursors with specificity and high yield. Summary of the Invention [Problem to be solved by the invention]

[0004] A method for producing cannabinoid precursors is disclosed. The method includes contacting a substrate and geranyl pyrophosphate or farnesyl pyrophosphate with an NphB ortholog. The substrate can be, for example, 2,4-dihydroxy-6-pentylbenzoic acid or 2,4-dihydroxy-6-propylbenzoic acid, and the NphB ortholog is from an organism other than Cannabis sativa.

[0005] Also provided is a recombinant cell of Yarrowia lipolytica carrying within its genome a nucleic acid encoding an NphB ortholog, the NphB ortholog being from an organism other than Cannabis sativa and being expressed in the recombinant cell. 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.

[0006] The following description of the invention refers to the accompanying drawings. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 shows the structures of cannabinoid products using olivetolic acid and geranyl pyrophosphate as substrates. The molecular formulas and formula weights of all possible products are C22H31O4 and 359.22, respectively. Black: olivetolic acid moiety derived from three units of malonyl-CoA, dark grey: olivetolic acid moiety derived from hexanoyl-CoA, light grey: geranyl pyrophosphate moiety transferred by orthologues. [Diagram 2] Diagram of the pYLEX1 vector used to integrate genes encoding NphB orthologs into the genome of Yarrowia lipolytica. [Figure 3A] LC-MS spectra of the cannabinoid precursor with m / z=359.22 biosynthesized from three NphB orthologues. 1st row=CBGA standard, 2nd row=P3E2, 3rd row=negative control without enzyme, 4th row=P3A5, 5th row=P3E8, 6th row=NphB positive control, 7th row=1BF1, 8th row=Y1C5. Peaks are identified by retention time and relative area under the peak. [Figure 3B]LC-MS spectra of the cannabinoid precursor with m / z=359.22 biosynthesized from three additional NphB orthologs: 1st row=10 μg / ml CBGA standard, 2nd row=no enzyme negative control, 3rd row=NphB positive control, 4th row=P3F5, 5th row=P3A6, 6th row=1BC2. Peaks are identified by retention time and relative area under the peak. [Figure 4] FIG. 1 shows structures of potential cannabinoid products biosynthesized using divalinic acid (top row), which has two fewer carbon units compared to olivetolic acid, resulting in the production of CBGVA, which is two carbons shorter than CBGA, or using farnesyl pyrophosphate (bottom row) instead of geranyl pyrophosphate, resulting in the production of novel cannabinoid precursors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] Enzymes are disclosed that catalyze the biosynthesis of cannabinoid precursors by transferring an isoprene unit from certain pyrophosphates, e.g., geranyl pyrophosphate, to aromatic polyketides, e.g., 2,4-dihydroxy-6-pentylbenzoic acid, i.e., olivetolic acid, and 2,4-dihydroxy-6-propylbenzoic acid. These enzymes are derived from organisms other than Cannabis sativa and can be recombinantly expressed in Escherichia coli or Yarrowia lipolytica and then used for prenyltransferase activity.

[0009] As summarized above, a method for producing cannabinoid precursor is disclosed.In a specific example, the substrate is olivetolic acid, the pyrophosphate is geranyl pyrophosphate, and the cannabinoid precursor produced has a mass-to-charge ratio of 359.22 and a retention time of more than 6.1 minutes as determined by LC / MS analysis.The cannabinoid precursor described herein is also included within the scope of the present invention.

[0010] In the above methods, exemplary sources of NphB orthologs can be, but are not limited to, Streptomyces roseochromogenus subsp. oscitans, Streptomyces rubidus, Streptomyces cinnamonensis, Aspergillus calidoustus, Aspergillus terreus, Clostridium clariflavum, Nocardia brasiliensis, and the fastidious bacterium esnapd16.1.

[0011] In certain aspects of the method, the NphB ortholog has an amino acid sequence of any one of SEQ ID NOs: 1-8. Alternatively, the NphB ortholog can have an amino acid sequence at least 70% identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, and 99%) to any one of SEQ ID NOs: 1-8 and has aromatic prenyltransferase activity.

[0012] In an exemplary method, the NphB ortholog is a recombinant enzyme that can be produced, for example, in Escherichia coli and Yarrowia lipolytica.

[0013] Also provided is a recombinant Yarrowia lipolytica cell, the genome of which includes a nucleic acid encoding an NphB ortholog, the NphB ortholog being from an organism other than Cannabis sativa.

[0014] Exemplary sources of NphB orthologs are Streptomyces roseochromogenus subsp. oscitans, Streptomyces rubidus, Streptomyces cinnamonensis, Aspergillus calidoustus, Aspergillus terreus, Clostridium clariflavum, Nocardia brasiliensis, and the fastidious bacterium esnapd16.1.

[0015] In certain recombinant cells, the NphB ortholog has an amino acid sequence of any one of SEQ ID NOs: 1-8. In another example, the NphB ortholog can have an amino acid sequence at least 70% identical (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, and 99%) to any one of SEQ ID NOs: 1-8 and have aromatic prenyltransferase activity.

[0016] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent based on the disclosure herein. Accordingly, the following specific examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference in their entirety.

[0017] Working Example A search of available sequence databases identified 105 genes orthologs to NphB, which were also annotated in the UniProt database as potential aromatic prenyltransferases. The genes were synthesized and cloned into a modified pES2 vector containing a T7 promoter and terminator. Using a chemical treatment, the vectors were transformed into E. coli Acella™ cells and grown on LB + streptomycin selection plates at 37°C. Sequence-confirmed clones were picked and cultured at OD 600nm The cells were grown in LB + streptomycin medium until the affinity of the culture reached 0.8. Protein expression was induced by adding 0.1 mM IPTG to the culture and incubating at 25° C. for 24 hours. Cells were then harvested and stored at −20° C. until protein purification.

[0018] Pelleted cells were resuspended in 100 μL binding buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 5 mM imidazole. Cells were then lysed by sonication and cell debris was removed by centrifugation at 4°C for 30 min. The supernatant containing the His-tagged proteins was purified by centrifugation with Ni 2+ The recombinant proteins were purified using affinity chromatography and eluted with a buffer containing 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 100 mM L-histidine. The concentration of the His-tagged proteins was estimated using an ELISA detection kit, and the purified NphB orthologues were stored at 4°C.

[0019] NphB orthologues were tested for prenyltransferase activity using an in vitro prenyltransferase assay: In a reaction volume of 200 μL, 20 μL of 1 M Tris-HCl (pH 7.9), 2 μL of 1 M MgCl 2 , 4 μL of 50 mM aromatic polyketide substrates, such as olivetolic acid and divalinic acid, 20 μL of 10 mM geranyl pyrophosphate, and 50 μg of purified NphB orthologue were combined and incubated at 30° C. Control reactions without enzyme were also prepared.

[0020] After 24 h, the reaction mixture was acidified to pH 3.0 with 6 M HCl and extracted three times with ethyl acetate. Samples were dried under vacuum and redissolved in methanol for LC-MS analysis using the negative ion mode. Extracted ion chromatograms (EICs) of m / z = 359.22 (with olivetolic acid) and m / z = 331.19 (with divalinic acid) were generated for each sample to determine whether the biosynthesis of potential cannabinoid precursors is catalyzed by NphB orthologues. The structures of potential cannabinoid precursors that can be biosynthesized using olivetolic acid and geranyl pyrophosphate as substrates are shown in Figure 1.

[0021] Certain orthologs of NphB produced novel products, identified as new peaks in LC-MS analysis, compared to the peak formed by wild-type NphB. We also demonstrated that certain orthologs produced higher yields of CBGA. Such orthologs are selected for subsequent cloning into Yarrowia lipolytica. They are subcloned into a modified pYLEX1 vector (Yeastern Biotech; see Figure 2) and transformed into Y. lipolytica using chemical treatment. Transformants are selected on YNB agar lacking leucine and used for subsequent yield optimization assays.

[0022] Of the 105 orthologs tested, eight orthologs showed good protein expression and novel prenyltransferase activity. The eight orthologs are listed in Table 1 below, along with their corresponding Uniprot IDs and source organisms. [Table 1]

[0023] P3E8 had comparable CBGA yields compared to wild-type NphB, and all eight orthologs showed at least one new peak in LC-MS analysis, which was C 22 H 31 O 4It also had a molecular formula of (FW=359.22).

[0024] As shown in Figure 3A and Figure 3B, all eight orthologs as well as wild-type NphB can produce CBGA (retention time: 6.4 min in Figure 3A and 6.1 min in Figure 3B). All orthologs except P3A5 also produced a by-product, namely 2-O-geranylolivetolic acid (retention time: 6.6 min in Figure 3A and 6.3 min in Figure 3B). Orthologs P3A5, Y1C5, and P3E2 each produced a significant amount of a new prenylated product with m / z = 359.2225 at retention times 7.23–7.24 min that was not observed in the reaction with wild-type NphB.

[0025] The novel products identified in Figures 3A and 3B, having m / z=359.22, are summarized below in Table 2. These products have retention times not seen in those produced with the wild-type NphB enzyme. [Table 2]

[0026] In one study, two orthologs corresponding to Uniprot IDs: C4PWA1 and Q9L9F1 were shown to produce only minor amounts of novel prenylation products, suggesting that these orthologs prenylated olivetolic acid at sites distinct from wild-type NphB.

[0027] Additionally, different substrates can be incubated with NphB orthologues to determine whether novel cannabinoid precursors can be generated. Figure 4 shows the products that can be formed when substituting either olivetolic acid or geranyl pyrophosphate or both. The novel cannabinoid precursors can then be tested with downstream cannabinoid synthases to diversify the cannabinoid compound library.

[0028] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is an example of a generic series of equivalent or similar features. 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 adapt the present invention to various applications and conditions without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. 1. A method for producing cannabinoid precursors, comprising: (a) contacting 2,4-dihydroxy-6-pentylbenzoic acid and geranyl pyrophosphate with an NphB ortholog to obtain 5-geranylolivetolic acid, 4-O-geranylolivetolic acid, or 2-O-geranylolivetolic acid; (b) contacting 2,4-dihydroxy-6-propylbenzoic acid and geranyl pyrophosphate with an NphB ortholog to obtain cannabigerovarinic acid; or (c) contacting 2,4-dihydroxy-6-pentylbenzoic acid and farnesyl pyrophosphate with an NphB ortholog; 【Chemistry 1】 To obtain a compound having the structure Including, The method, wherein the NphB ortholog has the amino acid sequence of any one of SEQ ID NOs: 1-8.

2. The method of claim 1, wherein the cannabinoid precursor has a mass-to-charge ratio of 359.22 and a retention time of greater than 6.1 minutes as determined by LC / MS analysis.

3. The NphB orthologues are selected from Streptomyces roseochromogenus subsp. oscitans, Streptomyces rubidus, Streptomyces cinnamonensis, Aspergillus calidoustus, Aspergillus terreus, Clostridium clariflavum, Nocardia braziliensis, and the like. The method according to claim 2, wherein the bacterium is derived from the fastidious bacterium esnapd16.1, or the fastidious bacterium esnapd16.

1.

4. 4. The method of claim 3, wherein the NphB ortholog is a recombinase enzyme produced in Yarrowia lipolytica.

5. The NphB orthologues are selected from Streptomyces roseochromogenus subsp. oscitans, Streptomyces rubidus, Streptomyces cinnamonensis, Aspergillus calidoustus, Aspergillus terreus, Clostridium clariflavum, Nocardia braziliensis, and the like. The method of claim 1, wherein the bacterium is derived from the fastidious bacterium esnapd16.1, or the fastidious bacterium esnapd16.

1.

6. 6. The method of claim 5, wherein the NphB ortholog is a recombinase enzyme produced in Yarrowia lipolytica.

7. A recombinant cell of Yarrowia lipolytica, comprising a nucleic acid encoding an NphB ortholog in its genome, said NphB ortholog having an amino acid sequence of any one of SEQ ID NOs: 1 to 8, and said NphB ortholog being expressed in said recombinant cell.

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