Patent Application
20220298533
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 22, 2022, is named CBTH-12-US_SL.txt and is 339,457 bytes in size.
The present application generally relates to manipulation of cannabinoids. More specifically, the application provides methods and compositions for the enzymatic modification or degradation of cannabinoids.
Cannabinoids are a class of organic small molecules of meroterpenoid structures found in the plant genus Cannabis. The small molecules are currently under investigation as therapeutic agents for a wide variety of health issues, including epilepsy, pain, and other neurological problems, and mental health conditions such as depression, PTSD, opioid addiction, and alcoholism (Committee on the Health Effects of Marijuana, 2017).
Numerous cannabinoids of varying structure are produced in Cannabis spp., each with their own therapeutic profile. However, since some cannabinoids are made in very small quantities in Cannabis spp. and are challenging to separate from other cannabinoids in Cannabis extracts, it is difficult to evaluate the therapeutic and psychotropic effect of each particular cannabinoid.
Rare cannabinoids from Cannabis spp. or from microbial bioproduction are gaining intense interest in the nutraceutical and clinical markets.
In one example, conversion of the abundant cannabinoid, tetrahydrocannabinol (THC) to a rare cannabinoid, cannabinol (CBN) is desirable for many reasons. THC is lower value, has intoxicating psychoactive side effects and is illegal in many jurisdictions. CBN is a high value, legal molecule that shows great clinical promise in treating sleep and skin disorders, and it has shown potential as a therapeutic for amyotrophic lateral sclerosis (Lou Gehrig's disease) (Carter, 2010; reviewed in Giacoppo, 2016). CBN is naturally formed by slow and inefficient non-enzymatic oxidation of THC in Cannabis spp. However, there is no known enzymatic route to produce CBN from THC. CBN can also be synthesized in small batches using organic chemistry (Caprioglio, 2019). Other approaches to make CBN include non-enzymatic oxidation methods applied to purified plant derived cannabinoids, such as heating and exposure to UV light or sunlight (PCT Patent Application Publication WO2014/159688A1 and US Patent Application Publication 2017/0020943A1) These routes are expensive, slow and environmentally unfriendly. An enzymatic route to CBN would greatly aid efforts to produce larger, cheaper and more consistent batches of this highly valuable compound.
There is thus a need to (a) synthesize individual cannabinoids, (b) convert one cannabinoid into another cannabinoid, or (c) convert a particular cannabinoid into a non-cannabinoid. The present invention addresses that need.
The present invention provides enzymes and methods using those enzymes to modify or degrade cannabinoids. Thus, in some embodiments, a method of modifying a first cannabinoid into a second cannabinoid or a non-cannabinoid is provided. The method comprises combining the first cannabinoid with an enzyme that can modify the first cannabinoid into the second cannabinoid or non-cannabinoid under conditions where the first cannabinoid is modified into the second cannabinoid or non-cannabinoid.
Also provided is a non-naturally occurring enzyme that can modify a first cannabinoid into a second cannabinoid or a non-cannabinoid. A nucleic acid encoding that enzyme is additionally provided.
Further provided is a non-naturally occurring nucleic acid that encodes an enzyme having the enzymatic activity of the above non-naturally occurring enzyme. An expression cassette comprising that nucleic acid is additionally provided.
In other embodiments, a cell comprising the above expression cassette is provided. In these embodiments, the cell is capable of expressing the enzyme provided above, or a naturally occurring equivalent thereof.
Also provided is a plant expression cassette comprising the above-identified nucleic acid, as is a plant comprising the expression cassette, where the plant is capable of expressing the above-identified enzyme, or a naturally occurring equivalent thereof.
To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:
Conservative amino acid substitutions: As used herein, when referring to mutations in a protein, “conservative amino acid substitutions” are those in which at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gln for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gln; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.
Functional variant: The term “functional variant,” as used herein, refers to a recombinant enzyme such as a CBN synthase that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent protein and that is still capable of performing an enzymatic function (e.g., synthesis of CBN) of the parent enzyme. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent enzyme may cause desirable changes in reaction parameters without altering fundamental enzymatic function encoded by the nucleotide sequence or containing the amino acid sequence. The functional variant may have conservative change including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids. Also envisioned is the use of amino acid analogs, e.g. amino acids not DNA or RNA encoded in biological systems, and labels such as fluorescent dyes, radioactive elements, electron dense agents, or any other protein modification, now known or later discovered.
Recombinant nucleic acid and recombinant protein: As used herein, a recombinant nucleic acid or protein is a nucleic acid or protein produced by recombinant DNA technology, e.g., as described in Green and Sambrook (2012).
Polypeptide, protein, and peptide: The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof. Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include protein cyclization, branching of the amino acid chain, and cross-linking of the protein. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.
The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).
Primer, probe and oligonucleotide: The terms “primer,” “probe,” and “oligonucleotide” may be used herein interchangeably to refer to a relatively short nucleic acid fragment or sequence. They can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands that can be separated apart by denaturation. In certain aspects, they are of a length of from about 8 nucleotides to about 200 nucleotides. In other aspects, they are from about 12 nucleotides to about 100 nucleotides. In additional aspects, they are about 18 to about 50 nucleotides. They can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications.
Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Various vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
Linker: The term “linker” refers to a short amino acid sequence that separates multiple domains of a polypeptide. In some embodiments, the linker prohibits energetically or structurally unfavorable interactions between the discrete domains.
Cannabinoid: As used herein, the term “cannabinoid” refers to a family of structurally related aromatic meroterpenoid molecules. Cannabinoids are generally formed by the enzymatic fusion, by a cannabinoid synthase (having geranylpyrophosphate:olivetolate geranyltransferase activity), of an alkylresorcylic acid
where R1═CH3, (CH2)2CH3 (divarinolic acid), (CH2)4CH3 (olivetolic acid), or (CH2)6CH3, with a polyprenyl pyrophosphate such as geranyl pyrophosphate, neryl pyrophosphate, geranylgeranyl pyrophosphate, of farnesyl pyrophosphate (
Codon optimized: As used herein, a recombinant gene is “codon optimized” when its nucleotide sequence is modified to accommodate codon bias of the host organism to improve gene expression and increase translational efficiency of the gene.
Expression cassette: As used herein, an “expression cassette” is a nucleic acid that comprises a gene and a regulatory sequence operatively coupled to the gene such that the promoter drives the expression of the gene in a cell. An example is a gene for an enzyme with a promoter functional in yeast, where the promoter is situated such that the promoter drives the expression of the enzyme in a yeast cell.
The present invention is directed to methods and compositions for modifying a first cannabinoid into a second cannabinoid or a non-cannabinoid using recombinant enzymes in microorganisms.
In some embodiments, a method of modifying a first cannabinoid into a second cannabinoid or a non-cannabinoid is provided. The method comprises combining the first cannabinoid with an enzyme that can modify the first cannabinoid into the second cannabinoid or non-cannabinoid under conditions where the first cannabinoid is modified into the second cannabinoid or non-cannabinoid.
In these embodiments, the first cannabinoid and the second cannabinoid can be any cannabinoid now known or later discovered. In some of these embodiments, the first and/or second cannabinoid comprises the structure
wherein R1═CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, or (CH2)6CH3; R2═H or COOH; and R3═CH3 or CH2OH.
Non-limiting examples of the first cannabinoid or the second cannabinoid are cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), cannabichromene (CBC), cannabidivarin (CBCV), cannabichromenic acid (CBCA), cannabichromevarinic acid (CBCVA) cannabinol (CBN), cannabinerolic acid (CBNA), cannabivarin (CBV), cannabigerolic acid (CBGA), cannabinerovarinic acid (CBNVA), cannabigerophorolic acid (CBGPA), cannabigerovarinic acid (CBGVA), cannabigerogerovarinic acid (CB GGVA), tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), tetrahydrocannabivarin (THCV), tetrahydrocannabivarin acid (THCVA), cannabinerovarinic acid (CBNVA), sesquicannabigerol (CBF), cannabigerogerol (CBGG), sesqui-cannabigerolic acid (CBFA), cannabigerogerolic acid (CBGGA), sesquicannabigerolic acid (CBFA), sesquicannabidiolic acid (CBDFA), sesquiTHCA (THCFA), sesqui-cannabigerovarinic acid (CBFVA), sesquiCBCA (CBCFA), sesquiCBGPA (CBFPA), tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabidivarinic acid (CBDVA), or cannabidivarin (CBDV) (
These methods can use any enzyme, now known or later discovered, that can carry out the conversion of the first cannabinoid into the second cannabinoid or degrade the first cannabinoid. In some embodiments, the enzyme is an aromatase, a dehydrogenase, an oxidase or a desaturase.
In some embodiments, the first cannabinoid is tetrahydrocannabinol (THC) or tetrahydrocannabinolic acid (THCA) and the second cannabinoid is cannabinol (CBN) or cannabinolic acid (CBNA). In other embodiments, the first cannabinoid is tetrahydrocannabivarinic acid (THCVA), tetrahydrocannabiphorolic acid (TCHPA), tetrahydrocannabiorcinic acid (THCOA) or sesquiTHCA (THCFA) and the second cannabinoid is cannabinerolic acid (CBNA), cannabinerovarinic acid (CBNVA), cannabiphorolic acid (CBNPA), cannabinorcinic acid (CBNOA) or sesqui cannabinerolic acid (sesqui-CBNA), respectively. In additional embodiments, the first cannabinoid is tetrahydrocannabivarinol (THCV), tetrahydrocannabiphorol (TCHP), tetrahydrocannabiorcinol (THCO) or sesquitetrahydrocannabinolic acid (sesquiTHCA) and the second cannabinoid is cannabinerovarinol (CBNV), cannabiphorol (CBNP), cannabinorcinol (CBNO) or sesqui cannabinerol (sesqui-CBN), respectively.
Exemplary enzymatic reactions are shown in
In various embodiments, the first cannabinoid is converted into a second cannabinoid that is an 11-hydroxy derivative of the first cannabinoid. In some of these embodiments, the conversion is carried out by the combination of a cytochrome P450 (CYP-450) and a cytochrome P450 reductase (CPR).
In various embodiments, a cannabinoid is oxidized by an oxidase into a polymeric state, such as a dimer of cannabinoids. This can occur between oxidized cannabinoids of the same species, such as THC or CBN, respectively, to form homopolymers, or a mixture of cannabinoid species, such as THC and CBN, which are oxidized to a heteropolymer of cannabinoids, as show in
The enzyme utilized in these methods can have any activity that can modify the first cannabinoid into the second cannabinoid. For example,
In some embodiments, the enzymes utilized in these methods additionally enable reduction of cannabinoid, e.g., THC, levels in pure cannabinoid preparations while not affecting other cannabinoid molecules. Cannabidiol (CBD) products often contain unwanted THC. Federal law bans any product containing more than 0.3% THC, so even small reductions in THC are critical to maintenance of cannabis products under this legal limit. Enzymes that destroy THC completely or convert THC to a molecule besides CBN are useful for certain applications and are commercially valuable.
The invention methods can be part of a complete biosynthesis pathway for cannabinoids such as CBN, including production of its acidic cannabinoid variant, cannabinolic acid (CBNA). The complete biosynthesis pathway for any cannabinoid is amenable to integration in a cannabinoid producing host cell. If the pathway includes a functional CBN synthase, accumulation of THC during an industrial fermentation is avoided.
The microorganism, e.g., yeast or bacterium, in which the methods are carried out can further comprise other enzymes, e.g., recombinantly transformed enzymes, that can affect the cannabinoid pathway, for example an enzyme that synthesizes the first cannabinoid from a non-cannabinoid or from another cannabinoid. This is illustrated in
To execute a CYP reaction, a CPR (cytochrome P450 reductase) is necessary to supply the P450 enzyme with reducing equivalents in the form of NADPH. The combination of the recombinant P450 and CPR genes and enzymes results in an 11-OH hydroxylase capable of acting on various cannabinoid substrates. In some embodiments, the hydroxyl group at the 11-position is added by recombinant CYP-450+CPR before the conversion of tetrahydrocannabinol or tetrahydrocannabinolic acid (THC/A) to CBN/A, yielding a conversion from 11-hydroxy tetrahydrocannabinol (11-OH THC) to 11-OH CBN.
The recombinant hydroxylation enzymes herein described may also hydroxylate other cannabinoid substrates, such as CBD, when expressed in a recombinant host capable of cannabinoid bioproduction. Additional reactions, substrates, and products for the above reconstituted biosynthetic pathways in a modified organism are depicted in
The enzymes used in these methods can be recombinantly expressed in a microorganism such as a yeast or bacterium, or a plant such as a Cannabis sp. In those systems, the gene for those enzymes can be modified, e.g., by codon optimizing the gene for the recombinant microorganism or plant.
In other embodiments, the enzyme is not naturally occurring. Such enzymes can be modified from a naturally occurring enzyme by, e.g., having conservative amino acid substitutions or substitutions that alter the enzymatic activity. Those enzymes can also be derived from a naturally occurring gene that has been codon optimized for expression in a recombinant host such as bacteria, yeast or plants.
In some of these methods, the first cannabinoid is converted (degraded) into a non-cannabinoid, for example by eliminating the cannabinoid aromatic ring that is derived from an alkylresorcylic acid in the naturally occurring cannabinoid pathway in Cannabis spp. Acetyl-CoA can also be produced as a result of this conversion.
These methods can be carried out in vivo or in vitro. When in vitro, the enzyme can be synthesized in a recombinant microorganism or plant and extracts of the microorganism or plant can be combined with the first cannabinoid. In various embodiments, the enzyme can be at least partially purified from the extract.
In these in vitro methods, the first cannabinoid can be present in a crude extract of a Cannabis sp. plant or a microorganism from which the first cannabinoid was synthesized. Alternatively, the first cannabinoid can be substantially purified when combined with the enzyme.
Exemplary in vitro methods are illustrated in
In other embodiments, bioconversion of THC to CBN takes place using lysate of a microbe containing the CBN synthase while the THC precursor is produced in a second microorganism. The first microbe could express the CBN synthase natively or recombinantly.
In additional embodiments, bioconversion of THC to CBN takes place using lysate of a microorganism containing the CBN synthase while the THC precursor is supplied as lysate from a second, cannabinoid producing microorganism. The first microbe could express the THC-to-CBN synthase natively or recombinantly.
In further embodiments, the CBN synthase is expressed recombinantly in a microbial host and the enzyme purified. The purified enzyme can then be used on purified plant derived THC to do an enzymatic conversion of THC to CBN in vitro.
The methods provided herein can facilitate development of industrial processes to eliminate THC and/or produce CBN in crude cannabinoid preparations, including plant material and microbial cell mass.
In the above exemplary embodiments, THC/A can be selectively degraded instead of being converted to CBN.
When the method is carried out in vivo, the method can be carried out by a living organism that synthesizes the enzyme. Any living organism can be utilized to carry out the method. In some embodiments, the method is carried out in a plant, e.g., a tobacco or Cannabis sp. plant.
In other embodiments, the method is carried out in a microorganism, as illustrated in
The microorganism can also comprise a recombinant enzyme “upstream” from cannabinoid synthase, e.g., a recombinant geranyl pyrophosphate synthase (GPPS) (see U.S. Provisional Patent Application 63/141,486). In various embodiments, the microorganism further comprises a recombinant GPPS and cannabinoid synthase, where the cannabinoid synthase can combine a polyprenyl pyrophosphate with alkylresorcylic acid to create a cannabinoid.
In some in vivo embodiments of these methods where the enzyme is in a first microorganism (yeast or bacteria), the first cannabinoid is synthesized in a second microorganism, wherein the method further comprises incubating the first microorganism, or an extract thereof, with the second microorganism. This is illustrated in
In other embodiments, the first cannabinoid is synthesized in a Cannabis sp. plant and matter from the Cannabis sp. plant is incubated with the first microorganism. This is illustrated in
In embodiments described above where the first cannabinoid is extracted from the second microorganism or a plant (e.g., a Cannabis sp. plant or tobacco), the first cannabinoid can be in a crude extract or can be partially or substantially purified from the second microorganism.
Various additional in vivo scenarios are illustrated in
Nonlimiting examples of enzymes that can be utilized in these reactions are provided in Table 1, where SEQ ID NOs:1-50 provide nucleic acid sequences for the enzymes, codon optimized for expression in yeast, and SEQ ID NOs:51-100 provide corresponding amino acid sequences. SEQ ID NOs:1-12 and 51-62 are P450 nucleic acid and amino acid sequences, respectively; SEQ ID NOs:13-20 and 63-70 are CPR nucleic acid and amino acid sequences, respectively; SEQ ID NOs:21-28 and 71-78 are CBN synthase nucleic acid and amino acid sequences, respectively; SEQ ID NOs:29-38 and 79-88 are THC degradase nucleic acid and amino acid sequences, respectively; and SEQ ID NOs:39-50 and 89-100 are oxidase nucleic acid and amino acid sequences, respectively. Of the oxidase enzymes provided, those comprising nucleic acid sequences SEQ ID NOs:42-50 and amino acid sequences SEQ ID NO:92-100 are laccases.
Also provided is a non-naturally occurring enzyme that can modify a first cannabinoid into a second cannabinoid or a non-cannabinoid.
The non-naturally occurring enzyme in these embodiments can have any alterations from a naturally occurring counterpart. In some embodiments, the enzyme comprises at least one amino acid that is not in a naturally occurring enzyme that has the same enzymatic activity. In some of those embodiments, the enzyme comprises a conservative substitution of an amino acid in a naturally occurring enzyme that has the same enzymatic activity. In various embodiments, the naturally occurring enzyme comprises any of SEQ ID NOs:51-100.
These enzymes can be utilized in the above-described methods. As such, in some embodiments, the first and/or second cannabinoid comprises the structure
where R1═CH3, CH2CH3, (CH2)2CH3, (CH2)3CH3, (CH2)3CH3, (CH2)4CH3, (CH2)5CH3, or (CH2)6CH3; R2═H or COOH; and R3═CH3 or CH2OH. In other embodiments, the enzyme is an aromatase, a dehydrogenase, an oxidase or a desaturase. In additional embodiments, the first cannabinoid is tetrahydrocannabinol (THC) or tetrahydrocannabinolic acid (THCA) and the second cannabinoid is cannabinol (CBN) or cannabinolic acid (CBNA) and the enzyme is an aromatase, a dehydrogenase, an oxidase or a desaturase. In further embodiments, the first cannabinoid is tetrahydrocannabivarinic acid (THCVA), tetrahydrocannabiphorolic acid (TCHPA), tetrahydrocannabiorcinic acid (THCOA) or sesquiTHCA (THCFA) and the second cannabinoid is cannabinerolic acid (CBNA), cannabinerovarinic acid (CBNVA), cannabiphorolic acid (CBNPA), cannabinorcinic acid (CBNOA) or sesqui cannabinerolic acid (sesqui-CBNA), respectively. Also, the first cannabinoid can be tetrahydrocannabivarinol (THCV), tetrahydrocannabiphorol (TCHP), tetrahydrocannabiorcinol (THCO) or sesquitetrahydrocannabinolic acid (sesquiTHCA) and the second cannabinoid can be cannabinerovarinol (CBNV), cannabiphorol (CBNP), cannabinorcinol (CBNO) or sesqui cannabinerol (sesqui-CBN), respectively.
Where the enzyme activity is the conversion of the first cannabinoid, e.g., THC, THCA, CBN or CBNA, into a 11-hydroxy analog, the enzyme can be a combination of a cytochrome P450 (CYP-450) and a cytochrome P450 reductase (CPR). In some of these embodiments, the CYP-450 is a CYP2C9 or a CYP3A4 or a CYP76AH22-24 or a CYP76AH1 (ferruginol synthases).
In various embodiments, the enzyme is expressed from a codon optimized gene sequence in a yeast or a bacterium, e.g. E. coli.
The enzyme can be in vivo (e.g., in a yeast, bacterium or plant), or in vitro. Nonlimiting examples of transgenic plants in which the enzyme can be expressed are a Cannabis sp. or a tobacco plant. Nonlimiting examples of transgenic yeast in which the enzyme can be expressed are species of Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, or Yarrowia. In some embodiments, the enzyme is in a yeast that further comprises enzymes to synthesize the first cannabinoid.
Chemically, the conversion of THC to CBN requires creation of 2 double bonds in a cyclohexene ring resulting in formation of an aromatic ring. See
Classes of enzymes that are capable of derivatizing cannabinoids and species that contain such enzymes are provided herewith. Multiple CBN synthase enzymes and enzymes specific for THC catabolism without production of CBN can be provided. Different enzymatic specificity is also envisioned, e.g. conversion of the acid derivative of THC (THCA) to CBNA. Derivatives of THC can also be converted to the appropriate derivatives of CBN, e.g. THCVA to CBVA. See
Also envisioned are enzymes of these classes that selectively degrade THC by converting it to molecules other than CBN but leave other cannabinoids untouched.
The conversion of THC/A to CBN/A is an oxidation reaction, so it may be catalyzed by oxidases. CYP-450s are examples of enzymes of this reaction. Some oxygenases may add hydroxyl or ketone groups to the structure as they form the aromatic ring of CBN/A. This would generate a hydroxylated variant of CBN/A, a novel molecule. Oxidases may also include non P450s such as flavin-dependent monooxygenases, copper-dependent monooxygenases, bacterial polysaccharide monooxygenases, non-heme iron-dependent monooxygenases, pterin-dependent monooxygenases, diiron hydroxylases, alpha-ketoglutarate-dependent hydroxylases, other cofactor-dependent monooxygenases, cofactor-independent monooxygenases, and/or laccases (reviewed in Tones Pazmino, 2010).
An aromatic ring is formed by the CBN synthase, so it may also be catalyzed by aromatases (
As hydrogen atoms are abstracted to make the double bonds in CBN/A, a dehydrogenase may be able to catalyze the reaction. An example of a dehydrogenase that catalyzes a similar reaction would be arogenate dehydrogenase, as described here: https://www.uniprot.org/uniprot/Q944B6. Since double bonds are formed in creation of CBN/A, a desaturase may be responsible. An example of a desaturase that catalyzes a similar reaction would be arogenate dehydratase/prephenate dehydratase, as described at https://www.uniprot.org/uniprot/Q9LMR3
Some enzymes of these classes will also degrade THC by converting it to molecules other than CBN. A non-limiting example is reversing THCA synthase to generate CBGA.
In some embodiments, the CBN synthase can use any variant of tetrahydrocannabinolic acid THCA, as starting material, including: tetrahydrocannabivarinic acid (THCVA), tetrahydrocannabiphorolic acid (TCHPA), tetrahydrocannabiorcinic acid (THCOA), sesquiTHCA (THCFA) and produce, respectively, cannabinerolic acid (CBNA), cannabinerovarinic acid (CBNVA), cannabiphorolic acid (CBNPA), cannabinorcinic acid (CBNOA) sesqui cannabinerolic acid (sesqui-CBNA). Decarboxylation of any of these products, either enzymatically or by non-enzymatic methods such as heat, will produce the respective decarboxylated derivatives and is an optional last step of the pathway.
The enzyme can be a naturally occurring enzyme, or an enzyme derived from a naturally occurring enzyme, now known or later discovered, that occurs in any living organism, for example a bacterium, an archaeon, a protist, a fungus, an algae, an animal or a plant.
Many microbial enzymes catalyze reactions of these classes using similar substrates, but have never been tested for activity on cannabinoids. To determine a source of a CBN synthase, microbes can be screened for bioconversion activity of appropriate cannabinoids, after the methods of Abbott (1977). Microbes possessing this activity should have their genomes sequenced if there is no publicly available genome. Enzymes from the above listed enzyme classes should be found from the sequenced genomes and thereby identified as good candidates for the CBN synthase activity. Organisms that make molecules similar to desired cannabinoids can be identified from literature and those genomes searched as well to identify additional candidate enzymes. Bioinformatics methods to do this are in U.S. Pat. No. 10,671,632
Some microbes screened will contain a THC degradase instead of a CBN synthase. This is detectable as a reduction in a THC containing starting material relative to a negative control (
In some embodiments, the gene for the enzyme is derived from a bacterium. It is envisioned that an enzyme derived from any bacterium now known or later discovered can be utilized in the present invention. For example, the bacterium can be from phylum Abditibacteriota, including class Abditibacteria, including order Abditibacteriales; phylum Abyssubacteria or Acidobacteria, including class Acidobacteriia, Blastocatellia, Holophagae, Thermoanaerobaculia, or Vicinamibacteria, including order Acidobacteriales, Bryobacterales, Blastocatellales, Acanthopleuribacterales, Holophagales, Thermotomaculales, Thermoanaerobaculales, or Vicinamibacteraceae; phylum Actinobacteria, including class Acidimicrobiia, Actinobacteria, Actinomarinidae, Coriobacteriia, Nitriliruptoria, Rubrobacteria, or Thermoleophilia, including orders Acidimicrobiales, Acidothermales, Actinomycetales, Actinopolysporales, Bifidobacteriales, Nanopelagicales, Catenulisporales, Corunebacteriales, Cryptosporangiales, Frankiales, Geodermatophilales, Glycomycetales, Jiangellales, Micrococcales, Micromonosporales, Nakamurellales, Propionibacteriales, Pseudonocardiales, Sporichthyales, Streptomycetales, Streptosporangiales, Actinomarinales, Coriobacteriales, Eggerthellales, Egibacterales, Egicoccales, Euzebyales, Nitriliruptorales, Gaiellales, Rubrobacterales, Solirubrobacterales, or Thermoleophilales; phylum Aquificae, including class Aquificae, including order Aquificales or Desulfurobacteriales; phylum Armatimonadetes, including class Armatimonadia, including order Armatimonadales, Capsulimonadales, Chthonomonadetes, Chthonomonadales, Fimbriimonadia, or Fimbriimonadales; phylum Aureabacteria or Bacteroidetes, including class Armatimonadia, Bacteroidia, Chitinophagia, Cytophagia, Flavobacteria, Saprospiria or Sphingobacteriia, including order Bacteroidales, Marinilabiliales, Chitinophagales, Cytophagales, Flavobacteriales, Saprospirales, or Sphingopacteriales; phylum Balneolaeota, Caldiserica, Calditrichaeota, or Chlamydiae, including class Balneolia, Caldisericia, Calditrichae, or Chlamydia, including order Balneolales, Caldisericales, Calditrichales, Anoxychlamydiales, Chlamydiales, or Parachlamydiales; phylum Chlorobi or Chloroflexi, including class Chlorobia, Anaerolineae, Ardenticatenia, Caldilineae, Thermofonsia, Chloroflexia, Dehalococcoidia, Ktedonobacteria, Tepidiformia, Thermoflexia, Thermomicrobia, or Sphaerobacteridae, including order Chlorobiales, Anaerolineales, Ardenticatenales, Caldilineales, Chloroflexales, Herpetosiphonales, Kallotenuales, Dehalococcoidales, Dehalogenimonas, Kte donob acteral es, Thermogemmatisporales, Tepidiformales, Thermoflexales, Thermomicrobiales, or Sphaerobacterales; phylum Chrysiogenetes, Cloacimonetes, Coprothermobacterota, Cryosericota, or Cyanobacteria, including class Chrysiogenetes, Coprothermobacteria, Gloeobacteria, or Oscillatoriophycideae, including order Chrysiogenales, Coprothermobacterales, Chroococcidiopsidales, Gloeoemargaritales, Nostocales, Pleurocapsales, Spirulinales, Synechococcales, Gloeobacterales, Chroococcales, or Oscillatoriales; phyla: Eferribacteres, Deinococcus-thermus, Dictyoglomi, Dormibacteraeota, Elusimicrobia, Eremiobacteraeota, Fermentibacteria, or Fibrobacteres, including class Deferribacteres, Deinococci, Dictyoglomia, Elusimicrobia, Endomicrobia, Chitinispirillia, Chitinivibrionia, or Fibrobacteria, including order Deferribacterales, Deinococcales, Thermales, Dictyoglomales, Elusimicrobiales, Endomicrobiales, Chitinspirillales, Chitinvibrionales, Fibrobacterales, or Fibromonadales; phylum Firmicutes, Fusobacteria, Gemmatimonadetes, or Hydrogenedentes, including class Bacilli, Clostridia, Erysipelotrichia, Limnochordia, Negativicutes, Thermolithobacteria, Tissierellia, Fusobacteriia, Gemmatimonadetes, Longimicrobia, including order Bacillales, Lactobacillales, Borkfalkiales, Clostridiales, Halanaerobiales, Natranaerobiales, Thermoanaerobacterales, Erysipelotrichales, Limnochordales, Acidaminococcales, Selenomonadales, Veillonellales, Thermolithobacterales, Tissierellales, Fusobacteriales, Gemmatimonadales, or Longimicrobia; phylum Hydrogenedentes, Ignavibacteriae, Kapabacteria, Kiritimatiellaeota, Krumholzibacteriota, Kryptonia, Latescibacteria, LCP-89, Lentisphaerae, Margulisbacteria, Marinimicrobia, Melainabacteria, Nitrospinae, or Omnitrophica, including class Ignavibacteria, Kiritimatiellae, Krumholzibacteria, Lentisphaeria, Oligosphaeria, or Nitrospinae, including order Ignavibacteriales, Kiritimatiellales, Krumholzibacteriales, Lentisphaerales, Victivallales, Oligosphaerales, or Nitrospinia; phylum Omnitrophica or Planctomycetes, including class Brocadiae, Phycisphaerae, Planctomycetia, or Phycisphaerales, including order Sedimentisphaerales, Tepidisphaerales, Gemmatales, Isosphaerales, Pirellulales, or Planctomycetales; phylum Proteobacteria including class Acidithiobacillia, Alphaproteobacteria, Betaproteobacteria, Lambdaproteobacteria, Muproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Hydrogenophilalia, Oligoflexia, or Zetaproteobacteria, including order Acidithiobacillales, Caulobacterales, Emcibacterales, Holosporales, Iodidimonadales, Kiloniellales, Kopriimonadales, Kordiimonadales, Magnetococcales, Micropepsales, Minwuiales, Parvularculales, Pelagibacterales, Rhizobiales, Rhodobacterales, Rhodospirillales, Rhodothalassiales, Rickettsiales, Sneathiellales, Sphingomonadales, Burkholderiales, Ferritrophicales, Ferrovales, Neisseriales, Nitrosomonadales, Procabacteriales, Rhodocyclales, Bradymonadales, Acidulodesulfobacterales, Desulfarculales, Desulfobacterales, Desulfovibrionales, Desulfurellales, Desulfuromonadales, Myxococcales, Syntrophobacterales, Campylobacterales, Nautiliales, Acidiferrobacterales, Aeromonadales, Alteromonadales, Arenicellales, Cardiobacteriales, Cellvibrionales, Chromatiales, Enterobacterales, Immundisolibacterales, Legionellales, Methylococcales, Nevskiales, Oceanospirillales, Orbales, Pasteurellales Pseudomonadales, Salinisphaerales, Thiotrichales, Vibrionales, Xanthomonadales, Hydrogenophilales, Bacteriovoracales, Bdellovibrionales, Oligoflexales, Silvanigrellales, or Mariprofundales; phylum Rhodothermaeota, Saganbacteria, Sericytochromatia, Spirochaetes, Synergistetes, Tectomicrobia, or Tenericutes, including class Rhodothermia, Spirochaetia, Synergistia, Izimaplasma, or Mollicutes, including order Rhodothermales, Brachyspirales, Brevinematales, Leptospirales, Spirochaetales, Synergistales, Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, or Mycoplasmatales; phylum Thermodesulfobacteria, Thermotogae, Verrucomicrobia, or Zixibacteria, including class Thermodesulfobacteria, Thermotogae, Methylacidiphilae, Opitutae, Spartobacteria, or Verrucomicrobiae, including order Thermodesulfobacteriales, Kosmotogales, Mesoaciditogales, Petrotogales, Thermotogales, Methylacidiphilales, Opitutales, Puniceicoccales, Xiphinematobacter, Chthoniobacterales, Terrimicrobium, or Verrucomicrobiales.
In other embodiments, the gene for the enzyme is derived from an archaeon. It is envisioned that an enzyme derived from any archaeon now known or later discovered can be utilized in the present invention. For example, the archaeon can be from phylum Euryarchaeota, including class Archaeoglobi, Hadesarchaea, Halobacteria, Methanobacteria, Methanococci, Methanofastidiosa, Methanomicrobia, Methanopyri, Nanohaloarchaea, Theionarchaea, Thermococci, or Thermoplasmata, including order Archaeoglobales, Hadesarchaeales, Halobacteriales, Methanobacteriales, Methanococcales, Methanocellales, Methanomicrobiales, Methanophagales, Methanosarcinales, Methanopyrales, Thermococcales, Methanomassiliicoccales, Thermoplasmatales, or Nanoarchaeales; DPANN superphylum, including subphyla Aenigmarcheota, Altiarchaeota, Diapherotrites, Micrarchaeota, Nanoarchaeota, Pacearchaeota, Parvarchaeota, or Woesearchaeota; TACK superphylum, including subphylum Korarchaeota, Crenarchaeota, Aigarchaeota, Geoarchaeota, Thaumarchaeota, or Bathyarchaeota; Asgard superphylum including subphylium Odinarchaeota, Thorarchaeota, Lokiarchaeota, Helarchaeota, or Heimdallarchaeota.
In additional embodiments, the gene for the enzyme is derived from a fungus. It is envisioned that a CBN synthase or THC degradase from any fungus now known or later discovered can be utilized in the present invention. This includes but is not limited to the phyla Chytridiomycota, Basidiomycota, Ascomycota, Blastocladiomycota, Ascomycota, Microsporidia, Basidiomycota, Glomeromycota, Symbiomycota, and Neocallimastigomycota. For example, the fungus can be from the phylum Ascomycota, including classes and orders Pezizomycotina, Arthoniomycetes, Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Geoglossomycetes, Laboulbeniomycetes, Lecanoromycetes, Leotiomycetes, Lichinomycetes, Orbiliomycetes, Pezizomycetes, Sordariomycetes, Xylonomycetes, Lahmiales, Itchiclahmadion, Triblidiales, Saccharomycotina, Saccharomycetes, Taphrinomycotina, Archaeorhizomyces, Neolectomycetes, Pneumocystidomycetes, Schizosaccharomycetes, Taphrinomycetes; phylum Basidiomycota including subphyla or classes Pucciniomycotina, Ustilaginomycotina, Wallemiomycetes, and Entorrhizomycetes; subphylum Agaricomycotina including classes Tremellomycetes, Dacrymycetes, and Agaricomycetes; phylum Symbiomycota, including class Entorrhizomycota; subphylum Ustilaginomycotina including classes Ustilaginomycetes and Exobasidiomycetes; phylum Glomeromycota including classes Archaeosporomycetes, Glomeromycetes, and Paraglomeromycetes; subphylum Pucciniomycotina including orders and classes: Pucciniomycotina, Cystobasidiomycetes, Agaricostilbomycetes, Microbotryomycetes, Atractiellomycetes, Classiculomycetes, Mixiomycetes, and Cryptomycocolacomycetes; subphylum incertae sedis Mucoromyceta including orders Calcarisporiellomycota and Mucoromycota; phylum Mortierellomyceta including class Mortierellomycota; subphylum incertae sedis Entomophthoromycotina including order Entomophthorales; phylum Zoopagomyceta including classes Basidiobolomycota, Entomophthoromycota, Kickxellomycota, and Zoopagomycotina; subphylum incertae sedis Mucoromycotina including orders Mucorales, Endogonales, and Mortierellales; phylum Neocallimastigomycota including class Neocallimastigomycetes; phylum Blastocladiomycota including classes Physodermatomycetes and Blastocladiomycetes; phylum Rozellomyceta including classes Rozellomycota and Microsporidia; phylum Aphelidiomyceta including class Aphelidiomycota; Chytridiomyceta including classes Chytridiomycetes and Monoblepharidomycetes; and phylum Oomycota including classes or orders Leptomitales, Myzocytiopsidales, Olpidiopsidales, Peronosporales, Pythiales, Rhipidiales, Salilagenidiales, Saprolegniales, Sclerosporales, Anisolpidiales, Lagenismatales, Rozellopsidales, and Haptoglossales.
The present invention is additionally directed to nucleic acids encoding any of the above-identified enzymes. In some embodiments, the nucleic acids are codon optimized to improve expression, e.g., using techniques as disclosed in U.S. Pat. No. 10,435,727. In some of these embodiments, the codon optimized nucleic acids comprise any of SEQ ID NOs:1-50.
More specifically, optimized nucleotide sequences are generated based on a number of considerations: (1) For each amino acid of the recombinant polypeptide to be expressed, a codon (triplet of nucleotide bases) is selected based on the frequency of each codon in the Saccharomyces cerevisiae genome; the codon can be chosen to be the most frequent codon or can be selected probabilistically based on the frequencies of all possible codons. (2) In order to prevent DNA cleavage due to a restriction enzyme, certain restriction sites are removed by changing codons that cover those sites. (3) To prevent low-complexity regions, long repeats (sequences of any single base longer than five bases) are modified. (2) and (3) are performed recursively to ensure that codon modification does not lead to additional undesirable sequences. (4) A ribosome binding site is added to the N-terminus. (5) A stop codon is added.
In various embodiments, the nucleic acids further comprise additional nucleic acids encoding amino acids that are not part of the enzyme. In some of these embodiments, the additional sequences encode additional amino acids present when the nucleic acid is translated, encoding, for example, an additional protein domain, with or without a linker sequence, creating a fusion protein. Other examples are localization sequences, i.e., signals directing the localization of the folded protein to a specific subcellular compartment or membrane.
In some embodiments, the nucleic acids have, at the 5′ end, a nucleic acid encoding codon optimized cofolding peptides to create a fusion protein, e.g., having SEQ ID NOs:69-73 (Table 2), joining the sequences together to form a fusion polypeptide, e.g., having the amino acid sequence of SEQ ID NO:74-78 fused at the N terminus of the enzyme polypeptide, generating recombinant fusion polypeptides.
Further provided is a non-naturally occurring nucleic acids that encode an enzyme having the enzymatic activity of any of the non-naturally occurring enzymes described above, or a naturally occurring enzyme having any of the enzyme activities described above. The nucleic acids may be codon optimized, e.g., for production in yeast.
In some embodiments, the nucleic acid comprises additional nucleotide sequences that are not translated. Examples include promoters, terminators, barcodes, Kozak sequences, targeting sequences, and enhancer elements. Particularly useful here are promoters that are functional in yeast.
Expression of a gene encoding an enzyme is determined by the promoter controlling the gene. In order for a gene to be expressed, a promoter must be present within 1,000 nucleotides upstream of the gene. A gene is generally cloned under the control of a desired promoter. The promoter regulates the amount of enzyme expressed in the cell and also the timing of expression, or expression in response to external factors such as sugar source.
Any promoter now known or later discovered can be utilized to drive the expression of the various genes (e.g., 11-OH hydroxylase, CBN synthase, THC degradase) described herein. See e.g. http://parts.igem.org/Yeast for a listing of various yeast promoters. Exemplary promoters listed in Table 3 below drive strong expression, constant gene expression, medium or weak gene expression, or inducible gene expression. Inducible or repressible gene expression is dependent on the presence or absence of a certain molecule. For example, the GAL1, GAL 7, and GAL10 promoters are activated by the presence of the sugar galactose and repressed by the presence of the sugar glucose. The HO promoter is active and drives gene expression only in the presence of the alpha factor peptide. The HXT1 promoter is activated by the presence of glucose while the ADH2 promoter is repressed by the presence of glucose.
In various embodiments, the nucleic acid is in a yeast expression cassette. Any yeast expression cassette capable of expressing the enzyme in a yeast cell can be utilized. In some embodiments, the expression cassette consists of a nucleic acid encoding a CBN synthase or THC degradase with a promoter.
Additional regulatory elements can also be present in the expression cassette, including restriction enzyme cleavage sites, antibiotic resistance genes, integration sites, auxotrophic selection markers, origins of replication, and degrons.
The expression cassette can be present in a vector that, when transformed into a host cell, either integrates into chromosomal DNA or remains episomal in the host cell. Such vectors are well-known in the art. See e.g. http://parts.igem.org/Yeast for a listing of various yeast vectors.
A nonlimiting example of a yeast vector is a yeast episomal plasmid (YEp) that contains the pBluescript II SK(+) phagemid backbone, an auxotrophic selectable marker, yeast and bacterial origins of replication and multiple cloning sites enabling gene cloning under a suitable promoter (see Table 3). Other exemplary vectors include pRS series plasmids.
The present invention is also directed to genetically engineered host cells that comprise the above-described nucleic acids. Such cells may be, e.g., any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules, intermediate molecules, or cannabinoid molecules. Host cells may also be any species of bacteria, including but not limited to Escherichia, Corynebacterium, Caulobacter, Pseudomonas, Streptomyces, Bacillus, or Lactobacillus.
In some embodiments, the genetically engineered host cell is a yeast cell, which may comprise any of the above-described expression cassettes, and capable of expressing the recombinant enzyme encoded therein.
Any yeast cell capable of being genetically engineered can be utilized in these embodiments. Nonlimiting examples of such yeast cells include species of Saccharomyces, Candida, Pichia, Schizosaccharomyces, Scheffersomyces, Blakeslea, Rhodotorula, or Yarrowia.
These cells can achieve gene expression controlled by inducible promoter systems; natural or induced mutagenesis, recombination, and/or shuffling of genes, pathways, and whole cells performed sequentially or in cycles; overexpression and/or deletion of single or multiple genes and reducing or eliminating parasitic side pathways that reduce precursor concentration.
The host cells of the recombinant organism may also be engineered to produce any or all precursor molecules necessary for the biosynthesis of cannabinoids, including but not limited to olivetolic acid (OA), olivetol (OL), FPP and GPP, hexanoic acid and hexanoyl-CoA, malonic acid and malonyl-CoA, dimethylallylpyrophosphate (DMAPP) and isopentenylpyrophosphate (IPP) as disclosed in U.S. Pat. No. 10,435,727.
Construction of Saccharomyces cerevisiae strains expressing a cannabinoid modifying or degrading enzyme such as CBN synthase or THC degradase is carried out via expression of a gene which encodes for the enzyme. The gene encoding the enzyme can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the gene encoding the enzyme may be inserted into the recombinant host genome. Integration may be achieved by a single or double cross-over insertion event of a plasmid, or by nuclease-based genome editing methods, as are known in the art e.g. CRISPR, TALEN and ZFR. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing. See, e.g., Green and Sambrook (2012).
To produce the desired cannabinoid, each candidate polypeptide may be introduced into a host cell genetically modified to contain all necessary components for cannabinoid biosynthesis using standard yeast cell transformation techniques (Green and Sambrook, 2012). Cells are subjected to fermentation under conditions that activate the promoter controlling the candidate polypeptide (see, e.g., Table 3). The broth may be subsequently subjected to HPLC analysis (
In some embodiments, for recombinant enzyme purification, the gene encoding the enzyme is cloned into an expression vector such as the pET expression vectors from Novagen, transformed into a protease deficient strain of E. coli such as BL21 and expressed by induction with IPTG. The protein of interest may be tagged with a common tag to facilitate purification, e.g. hexahistidine, GST, calmodulin, TAP, AP, CAT, HA, FLAG, MBP etc. Coexpression of a bacterial chaperone such as dnaK, GroES/GroEL or SecY may help facilitate protein folding. See Green and Sambrook (2012).
Any of the enzymes described above can also be produced in transgenic plants, using techniques known in the art (see, e.g., Keshavareddy et al., 2018). In these embodiments, the above-described nucleic acid encoding the enzyme further comprises a promoter functional in a plant. In various embodiments, the nucleic acid is in a plant expression cassette. Any plant capable of being transformed with the nucleic acid can be utilized here. In some embodiments, the plant is a tobacco or a Cannabis sp. plant. Cannabis sp. that are transformed with a THC degradase are particularly useful, since such an enzyme expressed in Cannabis sp. plants grown for fiber could reduce the THC content to below the 0.3% current legal THC limit.
Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.
Various methods and compositions provided in U.S. patent applications Ser. Nos. 16/553,103, 16/553,120, 16/558,973, 17/068,636 and 63/053,539; U.S. Pat. No. 10,435,727; and US Patent Publications 2020/0063170 and 2020/0063171 are utilized in the examples.
Construction of Saccharomyces cerevisiae strains expressing CBN synthase, THC degradase, P450, and/or CPR enzymes fused with N terminal cofolding peptides from Table 1, having SEQ ID NOs:106-110 to produce CBN/A from THC/A, and 11-hydroxy variants such as 11-OH CBN, is carried out via expression of a fusion gene of any codon optimized nucleic acid sequence SEQ ID NOs:101-105 combined at the 5′ end of a nucleic acid sequence encoding an enzyme that modifies a first cannabinoid into a second cannabinoid or non-cannabinoid. The fusion genes were cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid was confirmed by DNA sequencing. The fusion genes were also inserted into the recombinant host genome. Integration was achieved by a single or double cross-over insertion event of the plasmid. Strains with the integrated gene were screened by rescue of auxotrophy and genome sequencing.
Modified host cells which yield cannabinoids such as THC/A, express recombinant (i) CBN synthase for THC/A conversion to CBN/A, (ii) p450 and CPR protein combinations (11-OH hydroxylases) for 11-OH hydroxy variants of cannabinoids such as 11-OH-THC, or (iii) a combination of CBN synthase and 11-OH hydroxylases for production of cannabinoids such as 11-OH-CBN. More specifically, the cannabinoid-producing strain expressing CBN synthases and/or 11-OH hydroxylases herein is grown in a feedstock as described in U.S. patent application Ser. No. 17/068,636. An example feedstock used for a modified host expressing the recombinant CBN synthase is growing the strain in a minimal-complete or rich culture media containing yeast nitrogen base, amino acids, vitamins, ammonium sulfate, and a carbon source, such as glucose or molasses. The feedstock is consumed by the modified host which expresses the recombinant CBN synthase with a cannabinoid biosynthesis pathway to convert the feedstock into (i) biomass, (ii) THC/A and 11-OH-THC variants thereof, (iii) CBN/A and 11-OH CBN, and variants thereof, or (iv) biomass and the cannabinoids products in (ii) and (iii). Strains expressing the recombinant CBN synthase genes can be grown on feedstock for 12 to 160 hours at 25-37° C. for isolation of products.
Cells are genetically engineered to contain one or more laccase enzymes. Integration is achieved by a single or double cross-over insertion event of the plasmid. Strains with the integrated gene are screened by rescue of auxotrophy and genome sequencing. The laccase gene can be under the control of an inducible promoter. When polymerization of THC/A is desired, inducer is added to the culture along with supplemental copper at a final concentration of 100 μM-100 mM. Polymerized cannabinoids can be separated from the culture by filtration, centrifugation or dialysis. Membranes for filtration and dialysis should be selected such that molecules corresponding to the size of a monomeric cannabinoid pass through the pores of the membrane, but larger molecules such as polymers are retained on the other side of the membrane.
The CBN synthase or THC degradase enzyme is cloned into a high-copy vector with key features that allow 1) tight induction by the lactose analog, β-D-thiogalactoside (IPTG), 2) an N-terminal secretory signal peptide (e.g., MKKTAIAIAVALAGFATVAQA), and 3) C-terminal fusion to a HIS tag for purification. E. coli cells harboring the CBN synthase or THC degradase expression vector are grown in M9 minimal media with 1% glucose for 18 h at 37° C. and shaking at 300 rpm. Concentrated cell culture is diluted to an OD600=1 in fresh M9 minimal media with 1% glucose and 0.2 mM IPTG and grown for 48 h.
The supernatant containing the recombinant proteins is equilibrated in binding buffer (50 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 1 mM MgCl2, 10% glycerol, 10 mM 2-mercaptoethanol, 1 mM PMSF, Complete EDTA-free (1 tablet/100 ml), 20 mM 1-phenyl-2-thiourea; pH 7.4) and centrifuged at 2,500 g for 5 min to remove insoluble matter. Then the supernatant is filtered through a 0.45 μm filter (Millipore, MA, USA) and applied onto a HisTrap HP column (GE Healthcare Bioscience). The recombinant proteins are eluted with a step gradient of imidazole (concentrations of 5, 20, 40 and 300 mM). Fractions are analyzed by SDS-PAGE.
Purified CBN synthase or THC degradase protein is resuspended in activity buffer [100 mM sodium phosphate buffer, pH 6.55, 1 mM PMSF, EDTA-free protease inhibitor cocktail at working concentration (Roche, Meylan, France)] for use in converting or degrading THC/A in crude plant matter or THC/A in cannabinoid isolate via incubation and continuous shaking for 6-12 hrs at 30° C.
Host cells expressing recombinant CBN synthase or THC degradase are resuspended in lysis buffer consisting of 50 mM Tris-HCl pH7.5, 200 mM NaCl, 1 mM MgCl2, 5 mM DTT, 1 mM PMSF, and DNAse. Resuspended host cells are then lysed by sonication/French press/homogenization or enzymatic lysis such as zymolyase or lysozyme. Lysate is cleared by centrifugation at 16000 rpm for 15 min at 4° C. Cleared lysate is added to crude or purified cannabinoid preparations at concentrations ranging from 1 mg/gram to 1 g/g. The mixture is incubated with continuous shaking for 6-12 hrs at 30° C. Cannabinoids are then extracted.
To identify cannabinoid conversion products from CBN synthase, the degradation of THC via THC degradase, 11-hydroxy variants of cannabinoids, and all other products of converted plant matter, cannabinoid isolate, or from a host cell expressing an engineered biosynthetic pathway for cannabinoids, an Agilent 1100 series liquid chromatography (LC) system equipped with a reverse phase C18 column (Agilent Eclipse Plus C18, Santa Clara, Calif., USA) was used. A gradient was used of mobile phase A (ultraviolet (UV) grade H2O+0.1% formic acid) and mobile phase B (UV grade acetonitrile+0.1% formic acid). Column temperature was set at 30° C. Compound absorbance was measured at 210 nm and 305 nm using a diode array detector (DAD) and spectral analysis from 200 nm to 400 nm wavelengths. A 0.1 milligram (mg)/milliliter (mL) analytical standard was made from certified reference material for each terpene and cannabinoid (Cayman Chemical Company, USA). Each sample was prepared by diluting 1) fermentation biomass from a recombinant host expressing the engineered cannabinoid and CBN synthase biosynthesis pathway or 2) a conversion or degradation reaction containing CBN synthase or THC degradase by 1:3 or 1:20 in 100% acetonitrile and filtered in 0.2 um nanofilter vials. The retention time and UV-visible absorption spectrum (i.e., spectral fingerprint) of the samples were compared to the analytical standard retention time and UV-visible spectra (i.e. spectral fingerprint) when identifying the terpene and cannabinoid compounds.
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In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.
As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
All references cited in this specification, including but not limited to patent publications and non-patent literature, and references cited therein, are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.
As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of” “only one of” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.
As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements can optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc
This application claims the benefit of U.S. Provisional Application No. 63/164,126, filed Mar. 22, 2021, and incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63164126 | Mar 2021 | US |
