Engineered microorganism for production of cannabinoids

Abstract

To provide a genetically engineered microorganism capable of producing a cannabinoid.SOLUTION: Provided is a genetically engineered microorganism that is a photosynthetic microalgae or cyanobacterium and does not contain an exogenous nucleic acid molecule encoding an aromatic prenyltransferase. In one aspect, the genetically engineered microorganism is capable of producing tetrahydrocannabinolic acid or tetrahydrocannabinol and does not contain an exogenous nucleic acid molecule encoding a tetrahydrocannabinolic acid synthase.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001]

[0002] The present disclosure relates to genetically engineered microorganisms and cell cultures comprising same for the production of cannabinoids. The genetically engineered microorganisms comprise nucleic acid molecules having nucleic acid sequences encoding cannabinoid biosynthetic pathway enzymes to produce cannabinoid biosynthetic pathway products. [Background technology]

[0002]

[0003] Commercialization of various valuable plant natural products (PNPs) is often limited by the availability of PNP-producing plants, low accumulation of PNPs in plants, and / or time-consuming, often inefficient, extraction methods that are not always economically viable. Thus, commercialization of commercially interesting PNPs is often challenging. Recent advances in genetic engineering and synthetic biology have made it possible to produce heterologous PNPs in microorganisms, such as bacteria, yeast, and microalgae. For example, engineered microorganisms have been reported to produce the antimalarial drug artemisinin and the opiate (morphine, codeine) analgesic precursor reticuline (Keasling 2012; Fossati et al. 2014; DeLoache et al. 2015). However, genetically modified yeast have not yet successfully engineered the final metabolic reactions required to produce valuable end products, such as codeine and morphine, from reticuline. In some cases, bacterial or yeast platforms do not support the assembly of complex PNP pathways. In comparison, microalgal cells have been suggested to have advantages over other microorganisms, including the potential for post-translational protein modifications similar to those in plants and recombinant protein expression via the nuclear, mitochondrial, or chloroplast genomes (Singh et al. 2009).

[0003]

[0004] Δ9-Tetrahydrocannabinol and other cannabinoids (CBs) are polyketides responsible for the psychotropic and medicinal properties of cannabis (Cannabis sativa). Over 110 CBs have been identified to date, all derived from fatty acid and terpenoid precursors (ElSohly and Slade 2005). The first metabolic intermediate in the cannabis CB biosynthetic pathway is olivetolic acid, which forms the polyketide backbone of cannabinoids. Type III polyketide synthase (PKS; also known as tetraketide synthase (TKS) or olivetol synthase) enzymes condense hexanoyl-CoA with three malonyl-CoA units to form trioxododecanoyl-CoA in a multistep reaction. From there, olivetolate cyclase (OAC) (also known as 3,5,7-trioxododecanoyl-CoA CoA lyase) catalyzes an intramolecular aldol condensation to produce OA. In the next step, CB diversification occurs through the sequential action of "decorating" enzymes on the OA backbone. Gene sequences for PKSs and OACs have been identified and characterized in vitro (Lussier 2012; Gagne et al. 2012; Marks et al. 2009; Stout et al. 2012; Taura et al. 2009). Summary of the Invention

[0004]

[0005] The present disclosure describes engineered microorganisms, such as microalgae or cyanobacteria, for the production of plant natural products, such as cannabinoids.

[0005]

[0006] In one embodiment of the genetically engineered microorganism described herein, the genetically engineered microorganism does not include an exogenous nucleic acid molecule encoding an aromatic prenyltransferase.

[0006]

[0007] The present disclosure also provides a cell culture comprising a genetically engineered microorganism described herein and a substantially sugar-free medium.

[0007]

[0008] The present disclosure also provides a method for producing cannabinoids in a genetically engineered microorganism, comprising introducing into the microorganism at least one nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase, wherein the microorganism is a microalgae or a cyanobacterium.

[0008]

[0009] The present disclosure also provides a method for producing cannabinoids in a genetically engineered microorganism, the method comprising introducing into the microorganism at least one nucleic acid molecule encoding Steely1, Steely2 or a variant thereof, wherein the microorganism is a microalgae or a cyanobacterium.

[0009]

[0010] The present disclosure also provides a method for producing cannabinoids in a wild-type microorganism, the method comprising culturing the microorganism in a medium comprising 2,4-dihydroxy-6-alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoic acid ester, wherein the microorganism is a microalgae or a cyanobacterium.

[0010]

[0011] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0012] The present disclosure will now be described with reference to the drawings in which: [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows an exemplary cannabinoid biosynthetic pathway based on enzymes from cannabis. [Figure 2] FIG. 1 shows a portion of the cannabis cannabinoid biosynthetic pathway, terminating in the production of olivetolic acid. [Figure 3] FIG. 1 shows the concentration in the mobile phase versus time in an HPLC method for detecting cannabinoids in transformed cells. [Figure 4A] Figure 1 shows standard curves for (A) THC, (B) CBD, and (C) CBN, determined by diluting cannabinoids at concentrations of 0, 5, 10, 25, 50, 75, and 100 ppm in a solvent made with wild-type P. tricornutum extract. For each curve, the red line represents the standard curve calculated from the peak height, and the blue line represents the standard curve calculated from the peak area at each concentration. [Figure 4B] Figure 1 shows standard curves for (A) THC, (B) CBD, and (C) CBN, determined by diluting cannabinoids at concentrations of 0, 5, 10, 25, 50, 75, and 100 ppm in a solvent made with wild-type P. tricornutum extract. For each curve, the red line represents the standard curve calculated from the peak height, and the blue line represents the standard curve calculated from the peak area at each concentration. [Figure 4C] Figure 1 shows standard curves for (A) THC, (B) CBD, and (C) CBN, determined by diluting cannabinoids at concentrations of 0, 5, 10, 25, 50, 75, and 100 ppm in a solvent made with wild-type P. tricornutum extract. For each curve, the red line represents the standard curve calculated from the peak height, and the blue line represents the standard curve calculated from the peak area at each concentration. [Figure 5A] HPLC chromatograms (280 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, (C) Ptref3, and (D) Ptref7. (E) HPLC chromatogram of wild-type P. tricornutum control. Retention times (Rt) of authentic cannabinoid standards are: THC, 17.8-18.4 min; CBD, 15.09-15.4 min; and CBN, 17-17.5 min. [Figure 5B]HPLC chromatograms (280 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, (C) Ptref3, and (D) Ptref7. (E) HPLC chromatogram of wild-type P. tricornutum control. Retention times (Rt) of authentic cannabinoid standards are: THC, 17.8-18.4 min; CBD, 15.09-15.4 min; and CBN, 17-17.5 min. [Figure 5C] HPLC chromatograms (280 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, (C) Ptref3, and (D) Ptref7. (E) HPLC chromatogram of wild-type P. tricornutum control. Retention times (Rt) of authentic cannabinoid standards are: THC, 17.8-18.4 min; CBD, 15.09-15.4 min; and CBN, 17-17.5 min. [Figure 5D] HPLC chromatograms (280 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, (C) Ptref3, and (D) Ptref7. (E) HPLC chromatogram of wild-type P. tricornutum control. Retention times (Rt) of authentic cannabinoid standards are: THC, 17.8-18.4 min; CBD, 15.09-15.4 min; and CBN, 17-17.5 min. [Figure 5E] HPLC chromatograms (280 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, (C) Ptref3, and (D) Ptref7. (E) HPLC chromatogram of wild-type P. tricornutum control. Retention times (Rt) of authentic cannabinoid standards are: THC, 17.8-18.4 min; CBD, 15.09-15.4 min; and CBN, 17-17.5 min. [Figure 6A]Figure 1 shows UPLC chromatograms (220 nm) of (A) C. reinhardtii transformed with construct G1C1 and (B) a control sample containing cannabinoid standards. [Figure 6B] Figure 1 shows UPLC chromatograms (220 nm) of (A) C. reinhardtii transformed with construct G1C1 and (B) a control sample containing cannabinoid standards. [Figure 7A] Figure 1 shows UPLC chromatograms (220 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, and (C) wild-type P. tricornutum control. [Figure 7B] Figure 1 shows UPLC chromatograms (220 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, and (C) wild-type P. tricornutum control. [Figure 7C] Figure 1 shows UPLC chromatograms (220 nm) for the detection of cannabinoids in P. tricornutum transformed with constructs (A) Ptref1, (B) Ptref2, and (C) wild-type P. tricornutum control. DETAILED DESCRIPTION OF THE INVENTION

[0012] Detailed Description

[0020] The present inventors have surprisingly found that microalgae, such as Phaeodactylum tricornutum and Chlamydomonas reinhardtii, transformed with exogenous nucleic acid molecules encoding tetraketide synthase and olivetolic acid cyclase produced cannabinoids in the absence of other exogenous cannabinoid biosynthetic pathway enzymes.

[0013]

[0021] Without wishing to be bound by theory, it is predicted that microalgae genomes contain genes encoding enzymes with activity similar to that of enzymes found in cannabis (e.g., aromatic prenyltransferase (APT), tetrahydrocannabinolic acid synthase (THCAS), and / or cannabidiolic acid synthase (CBDAS)) that enable the production of cannabinoids in the presence of precursors, such as olivetol or olivetolic acid. For example, a search of the genome of P. tricornutum strain CCAP 1055 / 1 (NCBI BLAST) identifies a predicted protein (NCBI reference sequence XP 002182033.1) with 36% shared identity to an 81% query cover for a region of APT containing the active site (amino acids 108-383, APT). This predicted protein shares sequence identity with homogentisate solanesyltransferase enzymes capable of prenylation, hydroxybenzoate polyprenyltransferase, and contains a conserved magnesium-binding site similar to cannabis APT. Other potential candidates for APT activity in P. tricornutum include geranylgeranyltransferase and a predicted protein (NCBI Reference Sequence: XP 002180392.1). Furthermore, a search of the P. tricornutum genome for enzymes that generate HO identifies a violaxanthin de-epoxidase-like protein and spermine oxidase, which may have activity similar to CBDAS.

[0014]

[0022] Thus, the present disclosure provides a genetically engineered microorganism capable of producing cannabinoids, wherein the genetically engineered microorganism is a photosynthetic microalgae or a cyanobacterium, and the genetically engineered microorganism does not contain an exogenous nucleic acid molecule encoding an aromatic prenyltransferase.

[0015]

[0023] The present disclosure further provides a cell culture comprising a genetically engineered microorganism and a substantially sugar-free medium for the production of cannabinoids, wherein the genetically engineered microorganism is a photosynthetic microalgae or a cyanobacterium, and the genetically engineered microorganism does not contain an exogenous nucleic acid molecule encoding an aromatic prenyltransferase.

[0016]

[0024] The present disclosure further provides a method for producing cannabinoids in a genetically engineered microorganism, comprising introducing into the microorganism at least one nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase, wherein the microorganism is a microalgae or a cyanobacterium.

[0017]

[0025] The present disclosure further provides a method for producing cannabinoids in a genetically engineered microorganism, comprising introducing into the microorganism at least one nucleic acid molecule encoding Steely1, Steely2 or a variant thereof, wherein the microorganism is a microalgae or a cyanobacterium.

[0018]

[0026] The present disclosure further provides a method for producing cannabinoids in a wild-type microorganism, comprising culturing the microorganism in a medium comprising 2,4-dihydroxy-6-alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoic acid ester, wherein the microorganism is a microalgae or a cyanobacterium.

[0019]

[0027] Unless otherwise indicated, the definitions and embodiments set forth in this and other sections are intended to be applicable to all embodiments and aspects of the disclosure described herein as appropriate as understood by one of skill in the art.

[0020]

[0028] For purposes of understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open-ended terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but not excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. The foregoing also applies to words of similar meaning, such as "including," "having," and their derivatives. The term "consisting of" and its derivatives, as used herein, are intended to be closed terms specifying the presence of stated features, elements, components, groups, integers, and / or steps, but excluding the presence of other unstated features, elements, components, groups, integers, and / or steps. The term "consisting essentially of," as used herein, is intended to specify the presence of stated features, elements, components, groups, integers, and / or steps, as well as things that do not materially affect the basic and novel characteristic(s) of the features, elements, components, groups, integers, and / or steps.

[0021]

[0029] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. In embodiments including "additional" or "second" components, the second component, as used herein, is different from the other component or the first component. A "third" component is different from the other, first, and second components, and further listed or "additional" components are similarly different.

[0022]

[0030] Unless any indication to the contrary is made, references made to "%" content throughout this specification shall be taken to mean w / v (weight / volume) %.

[0023]

[0031] As used herein, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) sequences or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. Determining the percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for comparing two sequences is the algorithm of Karlin and Altschul (1990), as modified by Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST nucleotide searches can be performed, for example, with the NBLAST nucleotide program parameters set at score=100 and wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present disclosure. BLAST protein searches can be performed, for example, with the XBLAST program parameters set at score=50 and wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. To obtain gapped alignments for comparison, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Altschul et al., 1997).When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., the default parameters of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps allowed. In calculating percent identity, typically, only exact matches are counted. In certain embodiments, nucleic acids are optimized for codon usage in a particular microalgae or cyanobacterial species.In particular, nucleic acid sequences encoding enzymes of the cannabinoid biosynthetic pathway are found in GC-rich microalgae such as Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta, and Haematococcus purpurea. plucialis; diatoms such as Phaeodactylum tricornutum and Thalassiosira pseudonana; or cyanobacteria such as Arthrospira platensis, Arthrospira maxima, Synechococcus elongatus, and Aphanizomenon flos-aquae.

[0024]

[0032] The sequences of the present disclosure may be at least 80% identical to the sequences described herein; in other examples, the sequences may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences described herein at the nucleic acid or amino acid level. Importantly, the proteins encoded by the variant sequences retain the activity and specificity of the proteins encoded by the reference sequences. Thus, the present disclosure also provides nucleic acid molecules comprising nucleic acid sequences encoding enzymes of the cannabinoid biosynthetic pathway having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 49-52. Additionally provided are amino acid sequences of enzymes in the cannabinoid biosynthetic pathway that have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-11.

[0025]

[0033] The nucleic acid and amino acid sequences described herein are presented in Table 1.

[0026] [Table 1] TIFF2025121918000003.tif204149 TIFF2025121918000004.tif31149

[0027]

[0034] As used herein, the term "genetically engineered" and its derivatives refer to microorganisms in which genetic material has been altered using molecular biological techniques, including, but not limited to, molecular cloning, recombinant DNA technology, transformation, and gene transfer. Genetically engineered microorganisms include living modified microorganisms, genetically altered microorganisms, or transgenic microorganisms. Genetic changes include addition, deletion, modification, and / or mutation of genetic material. In the present disclosure, such genetic engineering described herein increases the production of various plant natural products, such as cannabinoids, compared to the corresponding wild-type microorganism. The term "cannabinoid" is generally understood to include any compound that acts on a cannabinoid receptor. Examples of cannabinoids include cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabichromanone (CBCN), cannabielsoin (CBE), cannabifuran (CBF), tetrahydrocannabinol (THC), cannabinodiol (CBDL), cannabicyclol (CBL), cannabiditriol (CBT), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabinerolic acid, cannabidiolic acid (CBDA), cannabinodiol (CBND), cannabinol propyl Cannabinoids include tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), tetrahydrocannabivaric acid (THCVA), cannabigerovaric acid (CBGVA), cannabidivaric acid (CBDVA), cannabichromevaric acid (CBCVA), and derivatives thereof. Further examples of cannabinoids are described in PCT Patent Application Publication No. WO 2017 / 190249 and U.S. Patent Application Publication No. 2014 / 0271940.

[0028]

[0035] Cannabinoids may be in acid or non-acid form, and the non-acid form may also be referred to as decarboxylated, since the acid form can be decarboxylated to produce the non-acid form. Within the context of this disclosure, which refers to a particular cannabinoid, the cannabinoid may be in the acid or non-acid form, or a mixture of both the acid and non-acid forms.

[0029]

[0036] A product of the cannabinoid biosynthetic pathway is a product associated with the production of cannabinoids. Examples of products of the cannabinoid biosynthetic pathway include, but are not limited to, hexanoyl-CoA, butyryl-CoA, trioxododecanoyl-CoA, trioxodecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, and divalinol. In one embodiment, the product of the cannabinoid biosynthetic pathway is at least one, two, three, four, five, six, seven, or eight of hexanoyl-CoA, butyryl-CoA, trioxododecanoyl-CoA, trioxodecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, and divalinol.

[0030]

[0037] In one embodiment, the genetically engineered microorganism has increased production of at least one, two, three, four, five, six, seven, or eight cannabinoid biosynthetic pathway products relative to a corresponding wild-type microorganism. In another embodiment, the cannabinoid biosynthetic pathway products are at least one, two, three, four, five, six, seven, or eight of hexanoyl-CoA, butyryl-CoA, trioxododecanoyl-CoA, trioxodecanoyl-CoA, olivetolic acid, olivetol, divalinolic acid, and divalinol. For example, the genetically engineered microorganism may have increased production of olivetolic acid, or olivetolic acid and cannabigerolic acid, relative to a corresponding wild-type microorganism. In another example, the genetically engineered microorganism may have increased production of olivetol, or olivetol and cannabigerol, relative to a corresponding wild-type microorganism.

[0031]

[0038] The term "nucleic acid molecule" or its derivatives, as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, nucleic acid molecules of the present disclosure usefully comprise single-stranded and double-stranded DNA, mixed single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and mixed single-stranded and double-stranded RNA, and hybrid molecules include single-stranded, or more typically double-stranded, DNA and RNA, or mixed single-stranded and double-stranded DNA and RNA. Furthermore, nucleic acid molecules usefully comprise triple-stranded regions containing RNA or DNA, or both RNA and DNA. Nucleic acid molecules of the present disclosure may also contain one or more modified bases or DNA or RNA backbones modified for stability or other reasons. "Modified" bases include, for example, tritium-labeled bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA; thereby, "nucleic acid molecules" incorporate chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have the corresponding meaning. In some embodiments, the genetically engineered microorganism comprises at least one nucleic acid molecule described herein.

[0032]

[0039] As used herein, the term "exogenous" refers to an element introduced into a cell. An exogenous element can include a protein or a nucleic acid. An exogenous nucleic acid is a nucleic acid introduced into a cell, for example, by a method of transformation. An exogenous nucleic acid may encode the expression of an RNA and / or a protein. An exogenous nucleic acid may be derived from the same species (homologous) or a different species (heterologous). An exogenous nucleic acid may contain a homologous sequence that has been altered so that it is introduced into the cell in a form not normally found in the cell in nature. For example, compared to the endogenous version of the nucleic acid, a homologous exogenous nucleic acid may contain mutations, be operably linked to a different regulatory region, or be integrated into a different region of the genome. An exogenous nucleic acid may be integrated into the chromosome of the transformed cell in one or more copies, integrated into the plastid or mitochondrial DNA of the transformed cell, or maintained as an independent nucleic acid outside the genome of the transformed cell.

[0033]

[0040] As used herein, the term "nucleic acid sequence" refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, including cDNA. The term also encompasses modified or substituted sequences, including non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may contain naturally occurring bases, including adenine, guanine, cytosine, thymidine, and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, and uracil; and xanthine and hypoxanthine. Nucleic acids can be either double-stranded or single-stranded, representing either the sense or antisense strand. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences.

[0034]

[0041] The cannabinoids produced by the genetically engineered microorganisms provided herein may be the result of increasing the activity of one or more enzymes associated with the cannabinoid biosynthetic pathway. Increasing the activity of an enzyme in a microorganism can involve, for example, introducing a nucleic acid molecule comprising a nucleic acid sequence encoding the enzyme. In one embodiment, the introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding the enzyme can be achieved by transformation. Examples of enzymes in the cannabinoid biosynthetic pathway include, but are not limited to, hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steely 1 and Steely 2), olivetolic acid cyclase, geranyl pyrophosphate synthase, aromatic prenyltransferase (APT), geranyl pyrophosphate:olivetolic acid geranyltransferase, cannabichromene synthase, tetrahydrocannabinolic acid synthase (THCAS), and cannabidiolic acid synthase (CBDAS).

[0035]

[0042] Figure 1 shows an exemplary enzyme-based cannabinoid biosynthetic pathway from cannabis: tetraketide synthase (TKS) condenses hexanoyl-CoA with malonyl-CoA to form the intermediate trioxododecanoyl-CoA; olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation to produce olivetolic acid (OA); aromatic prenyltransferase transfers geranyl diphosphate (GPP) to OA to produce cannabigerolic acid (CBGA); and tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase catalyze the oxidative cyclization of CBGA to tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), respectively. Decarboxylation of THCA or CBDA to remove the carboxyl group results in the production of the decarboxylated cannabinoids, tetrahydrocannabinol (THC) or cannabidiol (CBD), respectively.

[0036]

[0043] In addition to the exemplary cannabinoid biosynthetic pathway in cannabis shown in Figure 1, alternative biosynthetic intermediates can be used in cannabinoid biosynthetic pathways in genetically engineered microorganisms. For example, olivetol is an intermediate that lacks the carboxyl group of olivetolic acid. Using olivetol instead of olivetolic acid in a cannabinoid biosynthetic pathway results in the production of cannabinoids that also lack the carboxyl group, such as cannabigerol (CBG), tetrahydrocannabinol (THC), or cannabidiol (CBD). In another example, a tetraketide synthase (TKS) condenses butyryl-CoA with malonyl-CoA to form the intermediate trioxodecanoyl-CoA, and olivetolic acid cyclase (OAC) catalyzes the intramolecular aldol condensation of trioxodecanoyl-CoA to produce divalinolic acid. Divalinolic acid is an intermediate that contains an n-propyl group instead of the n-pentyl group found in olivetolic acid. Substituting divalinolic acid for olivetolic acid in the cannabinoid biosynthesis pathway will produce cannabinoids that also contain an n-propyl group, such as cannabigerovaric acid (CBGVA), tetrahydrocannabivaric acid (THCVA), cannabidivaric acid (CBDVA), or cannabichromevaric acid (CBCVA). In another example, divalinol is an intermediate of divalinolic acid that lacks a carboxyl group and contains an n-propyl group instead of the n-pentyl group found in olivetol. Substituting divalinol for divalinolic acid in the cannabinoid biosynthesis pathway will produce cannabinoids that also contain an n-propyl group and lack a carboxyl group, such as cannabigerovarin (CBGV), tetrahydrocannabivarin (THCV), cannabidivaric acid (CBDV), or cannabichromevaric acid (CBCV).

[0037]

[0044] In addition to the exemplary cannabinoid biosynthetic pathway of cannabis shown in Figure 1, alternative enzymes can be used in the cannabinoid biosynthetic pathway in genetically engineered microorganisms. For example, in addition to the enzymes found in cannabis, alternative cannabinoid biosynthetic pathway enzymes can be found in other plants (e.g., hops (Humulus lupulus)), bacteria (e.g., Streptomyces), or protists (e.g., Dictyostelium discoideum). Enzymes that differ in structure but perform the same function can be used interchangeably in the cannabinoid biosynthetic pathway in genetically engineered microorganisms. For example, aromatic prenyltransferases CsPT1 (SEQ ID NO: 3) and CsPT4 (SEQ ID NO: 10) from cannabis, HlPT1 (SEQ ID NO: 11) from hops, and Orf2 (SEQ ID NO: 9) from Streptomyces sp. strain Cl190 are all aromatic prenyltransferases that catalyze the synthesis of CBGA from GPP and OA. In a further example, Steely1 (SEQ ID NO: 7) or Steely2 (SEQ ID NO: 8) polyketide synthase from Dictyostelium discoideum, or a variant thereof, can be used to condense malonyl-CoA to olivetol and may be used in place of TKS to produce olivetol in the absence of OAC.

[0038]

[0045] In addition to the wild-type enzymes found in the organisms described herein, engineered variants of these enzymes can be used in cannabinoid biosynthetic pathways in genetically engineered microorganisms. Enzyme variants for use in cannabinoid biosynthetic pathways can be generated by altering the nucleic acid sequence encoding the enzyme to, for example, increase / decrease the activity of a domain, add / remove a domain, add / remove a signaling sequence, or otherwise alter the activity or specificity of the enzyme. For example, to produce unmethylated cannabinoids, the Steely1 sequence can be modified to reduce the activity of the methyltransferase domain. For example, this can be done by mutating amino acids G1516D+G1518A or G1516R relative to SEQ ID NO: 7, as disclosed in WO 2018 / 148849, incorporated herein by reference. In a further example, the sequence of tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase can be modified to remove the N-terminal secretory peptide. By way of example, this can be done by removing amino acids 1-28 of SEQ ID NO: 5 or 6 to generate a truncated enzyme, as disclosed in WO 2018 / 200888, which is incorporated herein by reference.

[0039]

[0046] Acyl-CoA synthetase is an acyl-activating enzyme that links CoA with a straight-chain alkanoic acid or alkanoate containing 2 to 6 carbon atoms to produce alkanoyl-CoA, which is a thioester of coenzyme A containing an alkanoyl group of 2 to 6 carbon atoms. In one embodiment, the acyl-CoA synthetase is a hexanoyl-CoA synthetase, which links CoA with hexanoic acid or hexanoate to produce hexanoyl-CoA. The hexanoyl-CoA synthetase may have the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% identity to SEQ ID NO: 4. In another embodiment, the acyl-CoA synthetase links CoA with butyric acid or butyrate to produce butyryl-CoA.

[0040]

[0047] Type III polyketide synthases are enzymes that produce polyketides by catalyzing the condensation of acetyl units onto thioester-linked starter molecules. The type III polyketide synthase may have the amino acid sequence of SEQ ID NO: 1, 7, or 8, or an amino acid sequence having at least 90% identity to SEQ ID NO: 1, 7, or 8. In one embodiment, the type III polyketide synthase condenses an alkanoyl-CoA with three malonyl-CoAs in a multi-step reaction to form 3,5,7-trioxoalkanoyl-CoA, where the 3,5,7-trioxoalkanoyl-CoA contains 8 to 12 carbon atoms. In another embodiment, the type III polyketide synthase is a tetraketide synthase from cannabis, also known in the art as olivetol synthase and 3,5,7-trioxododecanoyl-CoA synthase. In one embodiment, the tetraketide synthase condenses hexanoyl-CoA with three malonyl-CoAs to form 3,5,7-trioxododecanoyl-CoA in a multi-step reaction. In another embodiment, the tetraketide synthase condenses butyryl-CoA with three malonyl-CoAs to form 3,5,7-trioxodecanoyl-CoA in a multi-step reaction. In another embodiment, the type III polyketide synthase is Steely1 or Steely2 from Dictyostelium discoideum, or a variant thereof, comprising a domain having type III polyketide synthase activity (e.g., Steely1(G1516D+G1518A) or Steely1(G1516R) disclosed in WO 2018 / 148849). Steely1 is also known in the art as DiPKS or DiPKS1, and Steely2 is also known in the art as DiPKS37.

[0041]

[0048] Olivetolic acid cyclase, as used herein, refers to an enzyme that catalyzes the intramolecular aldol condensation of 3,5,7-trioxoalkanoyl-CoA to form 2,4-dihydroxy-6-alkylbenzoic acid, where the alkyl group of the benzoic acid contains 1 to 5 carbons. In one embodiment, olivetolic acid cyclase catalyzes the formation of olivetolic acid from 3,5,7-trioxododecanoyl-CoA. In another embodiment, olivetolic acid cyclase catalyzes the formation of divalinolic acid from 3,5,7-trioxodecanoyl-CoA. Olivetolic acid cyclase may have the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity to SEQ ID NO: 2. Olivetolic acid cyclase from cannabis is also known in the art as olivetolic acid synthase and 3,5,7-trioxododecanoyl-CoA CoA-lyase.

[0042]

[0049] As used herein, aromatic prenyltransferase refers to an enzyme capable of transferring geranyl diphosphate to 5-alkylbenzene-1,3-diol to synthesize 2-geranyl-5-alkylbenzene-1,3-diol, where the alkyl group of the product contains 1 to 5 carbons. In one embodiment, the aromatic prenyltransferase transfers geranyl diphosphate to olivetol to synthesize cannabigerol (CBG). In another embodiment, the aromatic prenyltransferase transfers geranyl diphosphate to olivetolic acid (OA) to synthesize cannabigerolic acid (CBGA). In another embodiment, the aromatic prenyltransferase transfers geranyl diphosphate to divalinolic acid to synthesize cannabigerovarin (CBGV). In another embodiment, the aromatic prenyltransferase transfers geranyl diphosphate to divalinolic acid to synthesize cannabigerovarin (CBGVA). Examples of aromatic prenyltransferases are aromatic prenyltransferases from cannabis, also known in the art as CsPT1, prenyltransferase 1, geranyl pyrophosphate-olivetolic acid geranyltransferase, and geranyl diphosphate:olivetolate geranyltransferase. Further examples of aromatic prenyltransferases include HIPT1 from hops, CsPT4 from cannabis, and Orf2 (NphB) from Streptomyces sp. strain Cl190. The aromatic prenyltransferase may have the amino acid sequence of SEQ ID NO: 3, 9, 10, or 11, or an amino acid sequence having at least 90% identity to SEQ ID NO: 3, 9, 10, or 11.

[0043]

[0050] Tetrahydrocannabinolic acid synthase, also known in the art as Δ9-tetrahydrocannabinolic acid synthase, synthesizes Δ9-tetrahydrocannabinolic acid by catalyzing the cyclization of the monoterpene moiety of cannabigerolic acid. The tetrahydrocannabinolic acid synthase may have the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 90% identity to SEQ ID NO:5.

[0044]

[0051] Cannabidiolic acid synthase synthesizes cannabidiolic acid by catalyzing the stereoselective oxidative cyclization of the monoterpene moiety of cannabigerolic acid. The cannabidiolic acid synthase may have the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 90% identity to SEQ ID NO:6.

[0045]

[0052] In one embodiment, the genetically modified microorganism provided herein encodes one, two, or three or less of a hexanoyl-CoA synthetase, a type III polyketide synthase (e.g., the tetraketide synthases Steely1 and Steely2), and an olivetolic acid cyclase; or comprises an exogenous nucleic acid molecule encoding one or two or less of a type III polyketide synthase (e.g., the tetraketide synthases Steely1 and Steely2) and an olivetolic acid cyclase, but does not comprise an exogenous nucleic acid molecule encoding an aromatic prenyltransferase, and optionally does not comprise an exogenous nucleic acid molecule encoding a tetrahydrocannabinolic acid synthase or a cannabidiolic acid synthase.

[0046]

[0053] In one embodiment, the nucleic acid molecule comprising a nucleic acid sequence encoding at least one hexanoyl-CoA synthetase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:4, the type III polyketide synthase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:1, 7 or 8, and the olivetolic acid cyclase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2. In another embodiment, the nucleic acid molecule does not comprise a nucleic acid sequence encoding a hexanoyl-CoA synthetase. In another embodiment, the nucleic acid molecule is comprised in a genetically engineered microorganism.

[0047]

[0054] In one embodiment, the nucleic acid molecule comprising a nucleic acid sequence encoding a type III polyketide synthase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:1, and the olivetolic acid cyclase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO:2.

[0048]

[0055] In one embodiment, the nucleic acid molecule comprising a nucleic acid sequence encoding a type III polyketide synthase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence set forth in SEQ ID NO: 7 or 8.

[0049]

[0056] As used herein, the term "vector" or "nucleic acid vector" refers to a nucleic acid molecule, such as a plasmid, containing control elements and a site for introducing transgenic DNA, and is used to introduce the transgenic DNA into a microorganism. The transgenic DNA can encode a heterologous protein, which can be expressed in the microorganism and isolated from the microorganism. The transgenic DNA can be integrated into the nuclear, mitochondrial, or chloroplast genome by homologous or non-homologous recombination. The transgenic DNA can also autonomously replicate in an extrachromosomal vector without being integrated into the nuclear, mitochondrial, or chloroplast genome. The vector can contain a single, operably linked set of control elements, including a promoter, a 5' untranslated region (5' UTR), an insertion site for the transgenic DNA, a 3' untranslated region (3' UTR), and a terminator sequence. Vectors useful in this method are well known in the art. In one embodiment, the nucleic acid molecule is an episomal vector.

[0050]

[0057] As used herein, the term "episomal vector" refers to a DNA vector based on a bacterial episome that can be expressed in a transformed cell without being integrated into the genome of the transformed cell. Episomal vectors can be transferred from a bacterium (e.g., Escherichia coli) to another target microorganism (e.g., microalgae) via conjugation.

[0051]

[0058] In another embodiment, the vector is a commercially available vector. As used herein, the term "expression cassette" refers to a single, operably linked set of control elements comprising a promoter, a 5' untranslated region (5' UTR), an insertion site for transgenic DNA, a 3' untranslated region (3' UTR), and a terminator sequence. In one embodiment, at least one nucleic acid molecule is an episomal vector.

[0052]

[0059] The term "operably linked," as used herein, refers to the arrangement of two or more components, wherein the components so described are in a relationship permitting them to function cooperatively. For example, a transcriptional control sequence or promoter is operably linked to a coding sequence if it promotes that aspect of transcription of the coding sequence. Those of skill in the art can readily recognize aspects of the transcription process, including, but not limited to, initiation, elongation, attenuation, and termination. Generally, an operably linked transcriptional control sequence is connected in cis with the coding sequence, but is not necessarily directly adjacent to the coding sequence.

[0053]

[0060] Thus, nucleic acid vectors encoding enzymes of the cannabinoid biosynthetic pathway contain elements suitable for proper expression of the enzymes in a microorganism. In particular, each expression vector contains a promoter that promotes transcription in a microorganism. The term "promoter," as used herein, refers to a nucleotide sequence that directs transcription of an operably linked gene, i.e., coding sequence. Suitable promoters include, but are not limited to, pEF-1α, p40SRPS8, pH4-1B, pγ-Tubulin, pRBCMT, pFcpA, pFcpB, pFcpC, pFcpD, HSP70A-RbcS2 (shown in Table 1 as SEQ ID NOS: 21-28, 53, and 55; see Slattery et al., 2018), and RbcS2. Those skilled in the art can readily appreciate that inducible promoters, including chemically inducible promoters, alcohol-inducible promoters, and estrogen-inducible promoters, can also be used. Predicted promoters, such as those found through genome database mining, may also be used. Additionally, the nucleic acid molecule or vector may contain one or more introns before the cloning site or within the gene sequence to drive strong expression of the gene of interest. The one or more introns include the FBAC2-1, TUFA-1, EIF6-1, RPS4-1, RbcS2-1, and RbcS2-2 introns (shown in Table 1 as SEQ ID NOS: 15-20). The nucleic acid molecule may contain more than one intron or more than one copy of the same intron. The nucleic acid molecule or vector may also contain a suitable terminator, such as tEF-1α, t40SRPS8, tH4-1B, tγ-Tubulin, tRBCMT, tFcpB, tFcpC, tFcpD, tFcpA, or tRbcS2 (shown in Table 1 as SEQ ID NOS: 29-36, 54, and 56). A selectable marker gene, such as the kanamycin resistance gene (also known as the neomycin phosphotransferase II gene or nptII), zeocin resistance gene, hygromycin resistance gene, basta resistance gene, hygromycin resistance gene, or other genes, can also be ligated into the vector.As used herein, the term "tag" refers to an amino acid sequence that is recognized by an antibody. The amino acid sequence of the tag is linked to, for example, the sequence of an enzyme, thereby allowing detection or isolation of the enzyme by binding between the tag and a tag-specific antibody. For example, common tags known in the art include 6His, MYC, FLAG, V5, HA, and HSV. These tags are useful when placed at the N- or C-terminus.

[0054]

[0061] In one embodiment, the nucleic acid molecule comprises a sequence encoding the Rubisco small subunit. The Rubisco small subunit may enable the polypeptide to which it binds to be targeted for transport to the chloroplast via an internal plastid targeting signal (Hirakawa and Ishida 2010). Without being bound by theory, it is believed that because substrates containing acetyl-CoA and malonyl-CoA are available in chloroplasts, transporting cannabinoid biosynthetic enzymes to the chloroplast compartment may enhance an exogenous cannabinoid biosynthetic pathway in microalgae. In some embodiments, at least one nucleic acid molecule comprises a sequence encoding the Rubisco small subunit having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12.

[0055]

[0062] As used herein, the term "reporter" refers to a molecule that allows for the detection of another molecule to which the reporter binds or associates, or the detection of an organism containing the reporter. Reporters can include fluorescent molecules, including fluorescent proteins, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP). In some embodiments, at least one nucleic acid molecule comprises one or more reporter sequences encoding a reporter having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NOs: 13-14.

[0056]

[0063] In one embodiment, a nucleic acid molecule or vector encoding at least one cannabinoid biosynthetic pathway enzyme comprises a promoter nucleic acid sequence selected from SEQ ID NOs: 21-28, 53, and 55. In another embodiment, the nucleic acid molecule comprises at least one intron sequence selected from SEQ ID NOs: 15-20. In another embodiment, the nucleic acid molecule comprises a terminator nucleic acid sequence selected from SEQ ID NOs: 29-36, 54, and 56. In another embodiment, the genetically engineered microorganism comprises a nucleic acid molecule comprising at least one sequence encoding a tag having an amino acid sequence selected from SEQ ID NOs: 37-42.

[0057]

[0064] Nucleic acid molecules can be constructed to express one, two, or up to three enzymes associated with the cannabinoid biosynthetic pathway. In one embodiment, the nucleic acid molecule comprises two or more polynucleotide sequences, each encoding an enzyme of a cannabinoid biosynthetic pathway, operably linked to the same promoter. When two or three enzymes are encoded in the construct, the construct may contain a nucleotide sequence encoding a self-cleaving peptide linker, such as FMDV2a, extFMDV2a, or T2A, thereby resulting in the enzymes being produced as independent proteins; or the construct may contain a peptide linker sequence, such as 3(GGGGS) and FPL1 peptide linkers, that links the enzymes as a fusion protein, allowing substrate channeling to pass intermediate metabolites of one enzyme directly to another enzyme or active site without releasing them into solution; or the construct may contain a combination of self-cleaving and non-self-cleaving sequences. In one embodiment, the nucleic acid molecule comprises at least one linker sequence between at least two polynucleotide sequences. In another embodiment, the linker sequence encodes a self-cleaving peptide linker, optionally a self-cleaving peptide linker having an amino acid sequence set forth in SEQ ID NOs: 43-45. In some embodiments, at least one nucleic acid molecule comprises one or more linker sequences encoding a peptide linker having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in SEQ ID NOs: 43-48.

[0058]

[0065] In another embodiment, a vector comprises a nucleic acid sequence described herein. In another embodiment, a host cell is transformed with a vector or nucleic acid molecule comprising a nucleic acid sequence described herein. In another embodiment, the host cell is any microorganism described herein.

[0059]

[0066] The nucleic acid sequences described herein can be provided in vectors in various configurations or combinations. Each individual sequence encoding an enzyme in the cannabinoid biosynthetic pathway can be provided on a separate vector. Alternatively, multiple sequences can be provided together in the same vector. For example, nucleic acid sequences encoding type III polyketide synthase and oberitol acid cyclase can be provided together in one vector, and a nucleic acid sequence encoding hexanoyl-CoA synthetase can be provided in a second vector. Alternatively, sequences encoding all enzymes can be provided together in the same vector. When more than one enzyme-encoding sequence is provided in the same vector, the sequences can be provided in separate expression cassettes or together in the same expression cassette. When two or more sequences are in the same expression cassette, they can be provided in the same open reading frame to generate a fusion protein. Two or more sequences encoding a fusion protein can be separated by a linker sequence encoding a restriction enzyme recognition site or a self-cleaving peptide linker. Thus, genetically modified microorganisms for the production of cannabinoids can be engineered by stepwise transfection with multiple vectors, each containing nucleic acid sequences encoding one or more enzymes of the cannabinoid biosynthetic pathway, or by stepwise transfection with a single vector containing nucleic acid sequences encoding all of the enzymes.

[0060]

[0067] As used herein, the term "microalgae" and its derivatives include eukaryotic photosynthetic and non-photosynthetic microorganisms. As used herein, the term "cyanobacteria" and its derivatives include prokaryotic photosynthetic microorganisms. In one embodiment, the microalgae are GC-rich microalgae. As used herein, "GC-rich microalgae" refers to microalgae in which the DNA of the nuclear genome and / or plastid genome contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% GC content. In one embodiment, the microalgae are oleaginous microalgae. As used herein, "oleaginous" refers to microalgae that contain at least 35%, at least 40%, at least 45%, or at least 50% lipid content by weight. In one embodiment, the microalgae are cold-adapted microalgae. As used herein, "cold-adapted" refers to microalgae that grow naturally in temperate, subarctic, or polar regions, or microalgae that have adapted to artificial growth conditions where they are grown at temperatures found in temperate, subarctic, or polar regions. In some embodiments, cold-adapted microalgae grow at temperatures below 24°C, below 20°C, below 16°C, or below 12°C. In one embodiment, the microalgae are cold-adapted microalgae that exhibit increased lipid content when grown at temperatures below 24°C, below 20°C, below 16°C, or below 12°C.

[0061]

[0068] In one embodiment, the microalgae is a green algae. In one embodiment, the microalgae is from the Chlorophyta division. In one embodiment, the microalgae is from the genus Ankistrodesmus, Asteromonas, Auxenochlorella, Basichlamys, Botryococcus, Botryokoryne, Borodinella, Brachiomonas, Catena, Carteria, Chaetofae, or the like. Chaetophora, Characiochloris, Characiosiphon, Chlainomonas, Chlamydomonas, Chlorella, Chlorochytrium, Chlorococcum, Chlorogonium, Chloromonas, Closteriopsis Closteriopsis, Dictyochloropsis, Dunaliella, Ellipsoidon, Eremosphaera, Eudorina, Floydiella, Friedmania, Haematococcus, Hafniomonas, Heterochlorella a), Gonium, Halosarcinochlamys, Koliella, Lobocharacium, Lobochlamys, Lobomonas, Lobosphaera, Lobosphaeropsis, Marvania, Monoraphidium, Myrmecia,Nannochloris, Oocystis, Oogamochlamys, Pabia, Pandorina, Parietochloris, Phacotus, Platydorina, Platymonas, Pleodorina, Polulichlori s), Polytoma, Polytomella, Prasiola, Prasiolopsis, Prasiococcus, Prototheca, Pseudochlorella, Pseudocarteria, Pseudotrebouxia, Pteromonas onas, Pyrobotrys, Rosenvingiella, Scenedesmus, Spirogyra, Stephanosphaera, Tetrabaena, Tetraedron, Tetraselmis, Trebouxia, Trochisciopsis In one embodiment, the microalgae is from the genera Chlamydia reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chei, Nannochloropsis ocrate, Scenedesmus obliquus, Actodesmus dimorphus,In another embodiment, the microalga is a diatom, optionally Phaeodactylum tricornutum or Thalassiosira pseudonana.

[0062]

[0069] In another embodiment, the cyanobacterium is from the family Spirulinaceae, Phormidiaceae, Synechococcaceae, or Nostocaceae. In one embodiment, the cyanobacterium is Arthrospira plantensis, Arthrospira maxima, Synechococcus elongatus, or Aphanizomenon flos aquae.

[0063]

[0070] The present disclosure also provides a cell culture for the production of cannabinoids, comprising the genetically engineered microorganism described herein and a medium for culturing the genetically engineered microorganism. In one embodiment, the medium is substantially sugar-free, i.e., the sugar concentration is less than 2% by weight, less than 1.5% by weight, less than 1% by weight, less than 0.5% by weight, or less than 0.1% by weight. In another embodiment, the medium contains no more than a trace amount of sugar, where a trace amount is generally understood in the art to mean an amount that is insignificant or close to the limit of detection. Sugars known to be necessary for culturing microorganisms that are not capable of photosynthesis include, but are not limited to, monosaccharides (e.g., glucose, fructose, ribose, xylose, mannose, and galactose) and disaccharides (e.g., sucrose, lactose, maltose, lactulose, trehalose, and cellobiose).

[0064]

[0071] In another embodiment, the medium is substantially free of a fixed carbon source, i.e., the concentration of the fixed carbon source is less than 2%, less than 1.5%, less than 1%, less than 0.5%, or less than 0.1% by weight. In another embodiment, the medium contains no more than a trace amount of a fixed carbon source. The term "fixed carbon source," as used herein, refers to an organic carbon molecule that is liquid or solid at ambient temperature and pressure and provides a source of carbon for growth, biosynthesis, and / or metabolism. Examples of fixed carbon sources include, but are not limited to, sugars (e.g., glucose, galactose, mannose, fructose, sucrose, lactose), amino acids or amino acid derivatives (e.g., glycine, N-acetylglucosamine, glycerol, floridoside, glucuronic acid, corn starch, depolymerized cellulose material, plant material (e.g., sugar cane, sugar beet)), and carboxylic acids (e.g., hexanoic acid, butyric acid, and their respective salts). Sources of fixed carbon are disclosed in WO 2015 / 168458, the contents of which are incorporated herein by reference.

[0065]

[0072] Microorganisms can be cultured under conditions permissive for their growth. Photosynthetic microorganisms are known to be capable of carbon fixation, which involves fixing carbon dioxide (not a fixed carbon source) into organic molecules, such as sugars, using energy from a light source. The fixation of carbon dioxide using energy from a light source is photosynthesis. Light sources suitable for providing energy in photosynthesis include sunlight and artificial light. Photosynthetic microorganisms can grow and / or metabolize without a fixed carbon source. Microalgae can fix carbon dioxide from various sources, including atmospheric carbon dioxide, industrially emitted carbon dioxide (e.g., flue gas and flaring gas), and soluble carbonate (e.g., NaHCO3 and Na2CO3) (see Singh et al. 2014, the contents of which are incorporated herein by reference). Non-fixed carbon sources, such as carbon dioxide, can be added to microalgae cultures by injecting or bubbling a carbon dioxide gas mixture into the culture medium. Photosynthetic growth is a type of autotrophic growth, in which microorganisms can produce organic molecules themselves using an external energy source, such as light. This contrasts with heterotrophic growth, in which microorganisms must consume organic molecules for growth and / or metabolism. Heterotrophic organisms therefore require a fixed carbon source for growth and / or metabolism. Some photosynthetic organisms are capable of mixotrophic growth, in which microorganisms fix carbon through photosynthesis while also consuming a fixed carbon source. In mixotrophic growth, autotrophic metabolism is integrated with heterotrophic metabolism, which oxidizes a reduced carbon source available in the culture medium. Photosynthetic microalgae are typically cultured under mixotrophic conditions by adding a fixed carbon source, as described herein, to the culture medium. Common sources of fixed carbon used include glucose, ethanol, or industrial waste products, such as acetate or glycerol (see Cecchin et al. 2018, the contents of which are incorporated herein by reference).Microorganisms, such as microalgae and cyanobacteria, can be cultured using methods and conditions known in the art (see, for example, Biofuels from Algae, ed., Pandey et al., 2014, Elsevier, ISBN 978-0-444-59558-4, the contents of which are incorporated herein by reference). Some microorganisms are capable of chemoautotrophic growth. Like photosynthetic microorganisms, chemoautotrophs are capable of carbon dioxide fixation, but use energy derived from chemical sources (e.g., hydrogen sulfide, ferrous iron, molecular hydrogen, ammonia) instead of light.

[0066]

[0073] Microalgae can be grown organically without the use of chemicals or additives that violate standards for organically produced products. Microalgae can be grown organically, for example, by growing them in conditions that comply with jurisdictional standards, such as those established by the United States (US Organic Food Production Act; USDA National Organic Program Certification; USDA Organic Regulations), the European Union (Regulation No. 834 / 2007 prior to January 1, 2021; Regulation 2018 / 848 from January 1, 2021), and Canada (Canadian Food Inspection Agency Canadian Organic Standards). Growing microalgae in organic conditions allows for the production of organic plant natural products in the microalgae.

[0067]

[0074] The present disclosure also provides nucleic acid molecules comprising nucleotide sequences encoding one, two, or no more than three enzymes of the cannabinoid biosynthetic pathway. In one embodiment, the nucleic acid molecule comprises nucleic acid sequences encoding one, two, or no more than three of a hexanoyl-CoA synthetase, a type III polyketide synthase (e.g., the tetraketide synthases Steely 1 and Steely 2), and an olivetolic acid cyclase. In another embodiment, the nucleic acid molecule comprises nucleic acid sequences encoding a type III polyketide synthase (e.g., the tetraketide synthases Steely 1 and Steely 2), an olivetolic acid cyclase, or both, but not a hexanoyl-CoA synthetase.

[0068]

[0075] The phrase "introducing a nucleic acid molecule into a microorganism" includes both stable integration of a nucleic acid molecule into the genome of a microorganism as well as transient integration of a nucleic acid into a microorganism to prepare a genetically engineered microorganism. Introduction of a nucleic acid into a cell is also known in the art as transformation. Nucleic acid vectors can be introduced into a microorganism using techniques known in the art, including, but not limited to, glass bead agitation, electroporation, Agrobacterium-mediated transformation, accelerated particle delivery, i.e., biolistics, cell fusion, or any other method for delivering a nucleic acid vector into a microorganism.

[0069]

[0076] Specific embodiments of the present disclosure include, but are not limited to, the following: 1. A genetically engineered microorganism capable of producing cannabinoids, wherein the microorganism is a photosynthetic microalgae or a cyanobacterium, and the genetically engineered microorganism does not contain an exogenous nucleic acid molecule encoding an aromatic prenyltransferase. 2. The genetically engineered microorganism of embodiment 1, wherein the microorganism is capable of producing tetrahydrocannabinolic acid or tetrahydrocannabinol and does not contain an exogenous nucleic acid molecule encoding tetrahydrocannabinolic acid synthase. 3. A genetically engineered microorganism of embodiment 1 or 2, capable of producing cannabidiolic acid or cannabidiol, wherein the microorganism does not contain an exogenous nucleic acid molecule encoding cannabidiolic acid synthase. 4. A genetically engineered microorganism according to any one of embodiments 1 to 3, comprising at least one exogenous nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase. 5. The genetically engineered microorganism of embodiment 4, wherein the tetraketide synthase comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1, and the olivetolic acid cyclase comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 2. 6. The genetically engineered microorganism of embodiment 4 or 5, wherein the at least one exogenous nucleic acid molecule comprises a first polynucleotide sequence encoding a tetraketide synthase and a second polynucleotide sequence encoding an olivetolic acid cyclase. 7. The genetically engineered microorganism of embodiment 6, wherein the first polynucleotide sequence is 5' to the second polynucleotide sequence. 8. The genetically engineered microorganism of embodiment 6 or 7, wherein the at least one exogenous nucleic acid molecule further comprises at least one linker sequence between the first polynucleotide sequence and the second polynucleotide sequence. 9. The genetically engineered microorganism of embodiment 8, wherein the linker sequence encodes a self-cleaving linker sequence (e.g., amino acid sequences SEQ ID NOs: 43-45) or a fusion linker sequence (e.g., amino acid sequences SEQ ID NOs: 46-48). 10. The genetically engineered microorganism of any one of embodiments 6 to 9, wherein the first polynucleotide sequence comprises at least one intron sequence (e.g., SEQ ID NOs: 15 to 20). 11. The genetically engineered microorganism of embodiment 4 or 5, wherein the at least one exogenous nucleic acid molecule comprises a first nucleic acid molecule encoding a tetraketide synthase and a second nucleic acid molecule encoding an olivetolic acid cyclase. 12. The genetically engineered microorganism of any one of embodiments 4 to 11, wherein the at least one exogenous nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NOs: 21 to 28, 53, and 55), a sequence encoding a tag (e.g., amino acid sequences SEQ ID NOs: 37 to 42), a sequence encoding a reporter (e.g., amino acid sequences SEQ ID NOs: 13 to 14), a sequence encoding a Rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 12), and a terminator nucleic acid sequence (e.g., SEQ ID NOs: 29 to 36, 54, and 56). 13. The genetically engineered microorganism of any one of embodiments 4 to 12, wherein at least one exogenous nucleic acid molecule is an episomal vector. 14. A genetically engineered microorganism according to any one of embodiments 4 to 13, comprising at least one exogenous nucleic acid molecule. 15. A genetically engineered microorganism according to any one of embodiments 1 to 3, comprising at least one exogenous nucleic acid molecule encoding Steely1, Steely2 or a variant thereof. 16. The genetically engineered microorganism of embodiment 15, wherein the variant of Steely1 or Steely2 comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:7 or SEQ ID NO:8, respectively. 17. The genetically engineered microorganism of embodiment 15 or 16, wherein at least one exogenous nucleic acid molecule comprises at least one intron sequence (e.g., SEQ ID NOs: 15-20). 18. The genetically engineered microorganism of any one of embodiments 15 to 17, wherein the at least one exogenous nucleic acid molecule further comprises one or more of a promoter nucleic acid sequence (e.g., SEQ ID NOs: 21 to 28, 53, and 55), a sequence encoding a tag (e.g., amino acid sequences SEQ ID NOs: 37 to 42), a sequence encoding a reporter (e.g., amino acid sequences SEQ ID NOs: 13 to 14), a sequence encoding a Rubisco small subunit (e.g., amino acid sequence SEQ ID NO: 12), and a terminator nucleic acid sequence (e.g., SEQ ID NOs: 29 to 36, 54, and 56). 19. A genetically engineered microorganism according to any one of embodiments 15 to 18, wherein at least one exogenous nucleic acid molecule is an episomal vector. 20. A genetically engineered microorganism according to any one of embodiments 15 to 19, comprising at least one exogenous nucleic acid molecule. 21. The genetically engineered microorganism of any one of embodiments 1 to 20, which does not comprise an exogenous nucleic acid molecule encoding a hexanoyl-CoA synthetase. 22. The genetically engineered microorganism of any one of embodiments 1 to 21, wherein the microalgae is a diatom or a chlorophyte. 23. The genetically engineered microorganism of embodiment 22, wherein the microalgae is Phaeodactylum tricornutum or Thalassiosira pseudonana. 24. The genetically engineered microorganism of embodiment 23, wherein the microalgae is Phaeodactylum tricornutum. 25. The genetically engineered microorganism of embodiment 22, wherein the microalgae is Chlamydomonas reinhardtii or Chlorella vulgaris. 26. The genetically engineered microorganism of embodiment 25, wherein the microalgae is Chlamydomonas reinhardtii. 27. The genetically engineered microorganism of any one of embodiments 1-21, wherein the cyanobacterium is a species of the family Spirulinaceae, Phormidiaceae, Synechococcaceae or Nostocaceae, optionally Arthrospira platensis, Arthrospira maxima, Synechococcus elongatus or Aphanizomenon flos aquae. 28. A cell culture comprising a genetically engineered microorganism according to any one of embodiments 1 to 27 and a substantially sugar-free medium. 29. The cell culture of embodiment 28, wherein sugars are present in the medium at a concentration of less than 2% by weight. 30. The cell culture of embodiment 29, wherein sugars are present in the medium at a concentration of less than 1% by weight.

[0070] 31. The cell culture of embodiment 30, wherein sugars are present in the medium at a concentration of less than 0.5% by weight. 32. The cell culture of embodiment 31, wherein sugars are present in the medium at a concentration of less than 0.1% by weight. 33. The cell culture of embodiment 32, wherein sugars are present in the medium in trace amounts or less. 34. The cell culture of any one of embodiments 28-33, wherein the sugar is a monosaccharide. 35. The cell culture of embodiment 34, wherein the monosaccharide is at least one of glucose, fructose, ribose, xylose, mannose, and galactose. 36. The cell culture of any one of embodiments 28-33, wherein the sugar is a disaccharide. 37. The cell culture of embodiment 36, wherein the disaccharide is at least one of sucrose, lactose, maltose, lactulose, trehalose, and cellobiose. 38. The cell culture of any one of embodiments 28-37, wherein the medium is substantially free of a fixed carbon source. 39. The cell culture of embodiment 38, wherein the fixed carbon source is at least one of a carboxylic acid and glycerol. 40. The cell culture of embodiment 39, wherein the carboxylic acid is hexanoic acid. 41. The cell culture of any one of embodiments 28 to 40, which undergoes autotrophic growth. 42. The cell culture of embodiment 41, wherein the autotrophic growth is photosynthetic growth. 43. The cell culture of embodiment 42, wherein photosynthetic growth occurs in the presence of a solar light source. 44. The cell culture of embodiment 42, wherein photosynthetic growth occurs in the presence of an artificial light source. 45. A cell culture according to any one of embodiments 28 to 44, which undergoes growth under organic conditions. 46. A method for producing cannabinoids in a genetically engineered microorganism, comprising the step of introducing into the microorganism at least one nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase, wherein the microorganism is a microalga or a cyanobacterium. 47. A method for producing cannabinoids in a genetically engineered microorganism, comprising the step of introducing into the microorganism at least one nucleic acid molecule encoding Steely1, Steely2 or a variant thereof, wherein the microorganism is a microalgae or a cyanobacterium. 48. A method for producing cannabinoids in a wild-type microorganism, comprising culturing the microorganism in a medium containing 2,4-dihydroxy-6-alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoic acid ester, wherein the microorganism is a microalgae or a cyanobacterium. [Example]

[0071]

[0077] The following non-limiting examples are illustrative of the present disclosure: Example 1

[0078] Construction of episomal vectors and transformation of diatom Phaeodactylum tricornutum cells Example 1.1 Gene Sequence

[0079] Early in the CB biosynthetic pathway, hexanoyl-CoA synthetase has been suggested to convert hexanoic acid to hexanoyl-CoA (Figure 1; modified from Gagne et al. 2012). Another early metabolic intermediate in the CB biosynthetic pathway is olivetolic acid (OA), which forms the polyketide backbone of cannabinoids. Without wishing to be bound by theory, OA is produced as follows (Figure 2): First, a type III tetra / polyketide synthase (TKS) enzyme condenses hexanoyl-CoA with three malonyl-CoA units in a multistep reaction to form trioxododecanoyl-CoA. Olivetolic acid cyclase (OAC) then catalyzes an intramolecular aldol condensation to produce OA. In the next step, CB diversification occurs through the sequential action of “decorating” enzymes on the OA backbone, resulting in the cannabinoids Δ9-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA), each of which decarboxylates to yield Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively (Figure 1).

[0072]

[0080] The gene sequences for TKS and OAC have been identified and characterized in vitro (Lussier 2012; Gagne et al. 2012; Marks et al. 2009; Stout et al. 2012; Taura et al. 2009). The complete coding sequences of unoptimized TKS (GenBank: AB164375.1) and OAC (GenBank: JN679224.1) were obtained from public databases. The TKS open reading frame (1158 bp) encodes a 385-amino acid protein with a calculated MW of 42 kDa (Taura et al. 2009; Flores-Sanchez et al. 2010). On the other hand, OAC is a relatively short sequence (485 bp) encoding a small protein of 101 amino acids with a MW of 12 kDa (Marks et al. 2009). Without wishing to be bound by theory, codon optimization has been proposed to improve protein expression in host organisms by replacing nucleic acid coding for specific amino acids (i.e., codons) with alternative codons that are believed to result in better expression in the host organism. This effect can occur because different organisms exhibit different codon preferences. In particular, microalgae and cyanobacteria may prefer different codons than plants and animals. The process of altering nucleic acid sequences to achieve better expression based on codon preferences is called codon optimization. Statistical methods have been developed to analyze codon usage biases in various organisms, and many computer algorithms have been developed to perform these statistical analyses in the design of codon-optimized gene sequences (Lithwick and Margalit 2003). Other modifications in codon usage to increase protein expression that are independent of codon bias have also been described (Welch et al. 2009). The open reading frame of the construct, including the gene and other elements (eg, reporter, tag, peptide linker), was codon-optimized (eg, SEQ ID NOs: 49-52), synthesized, and inserted into a transformation vector.

[0073] Example 1.2 Engineered Diatoms

[0081] Microalgae offer a promising yet challenging platform for the bioproduction of high-value chemicals. Compared to model organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement have been hindered by inefficient molecular tools. To date, many microalgae are transformable, but introduced DNA is randomly integrated into the nuclear genome by mechanisms involving non-homologous recombination, resulting in a high probability of gene silencing. Therefore, molecular tools to circumvent these challenges are needed to facilitate efficient genetic engineering. Recently, an episomal vector system for diatoms has been developed and shown to be highly stable (Karas et al. 2015). Because episomes should be unaffected by gene silencing mechanisms, diatom strains were engineered with constructs containing TKS and OAC transgenes. Constructs optimized for the codon usage of Phaeodactylum tricornutum are shown in SEQ ID NOs: 49–52. These optimized sequences can also be used for other diatoms, such as Thalassiosira pseudonana.

[0074] Example 1.3 Extraction for HPLC Analysis

[0082] HPLC analysis was performed on cell extracts prepared by the exemplary methods described herein. Approximately 100 mg of algal culture was centrifuged, and the supernatant was discarded. 5 mL of 100% ethanol was added to the precipitate and placed at -20°C overnight. The precipitate was centrifuged at 4000 g for 10 minutes at 4°C. 1 mL of the supernatant was transferred to a 1.7 mL Eppendorf tube, and the ethanol was evaporated in a Speedvac at maximum vacuum without heating. 250 μL of mobile phase solution (water:formic acid:acetonitrile at 59.9%, 0.1%, and 40%) was used to resuspend the precipitate. The suspension was homogenized by vortexing each tube at high speed for 2 minutes, followed by centrifugation at maximum speed for 10 minutes at 4°C. 200 μL of the supernatant was collected in an HPLC vial.

[0075] Example 1.4 HPLC Analysis

[0083] Instrument: Prominence-i LC-2030 C 3D; Detector: UV-DAD / PDA; Column: P / No: 00F-4633-EVO, Model Kinetex 5 μm EVO C18 100 Å, LC Column 150 × 4.6 mm, Serial Number: H15010692, B / No: 5720-050; Oven Temperature: 30 °C; Flow Rate: 0.5 mL / min; Mobile Phase: A = water with 1% formic acid, B = 100% acetonitrile; Gradient Phase: T0: 40% B, T16: 90% B, T18: 90% B, T20: 99% B, T23: 99% B, T25: 40% B. The mobile phase concentration versus time is shown in Figure 3.

[0076]

[0084] The analytical wavelengths were selected based on the peak maxima of each standard in wild-type algal matrix on a Cary 60 UV-Vis precision spectrophotometer: THC: 280 nm, 17.8–18.4 min depending on whether it was neutral or acidic (THCA appears further); CBD: 275 nm, 15.09–15.4 min; CBN: 285 nm, 17–17.5 min.

[0077]

[0085] To determine the THC, CBD, or CBN peaks above the sample background, each standard was diluted to concentrations of 0, 5, 10, 25, 50, 75, and 100 ppm in a solvent made from wild-type P. tricornutum extract as a matrix. Peaks in the samples were identified after normalization using the standard curves and blanks. The standard curves for THC, CBD, and CBN are shown in Figure 4.

[0078] Example 2 Example 2.1 (AC_1 / Ptref1)

[0086] The construct containing the sequences encoding the TKS and OAC enzymes was transformed into P. tricornutum.

[0079]

[0087] The construct (Ptref1, SEQ ID NO: 49) containing the 5' to 3' sequences: the sequence encoding TKS (positions 1 to 1155); the T2A self-cleaving peptide linker sequence (positions 1156 to 1218); and the sequence encoding OAC (positions 1219 to 1521) was inserted into a modified pPtGE30 plasmid (Slattery et al. 2018) containing the zeocin resistance gene for algae and the chloramphenicol resistance gene for E. coli and His selection in yeast.

[0080]

[0088] The construct was operably linked to the 40SRPS8 promoter (SEQ ID NO: 22) and FcpA terminator (SEQ ID NO: 54).

[0081]

[0089] The PtGE30 episomal vector was conjugatively transferred from E. coli to P. tricornutum.

[0082]

[0090] Zeocin-resistant clones of P. tricornutum were confirmed by PCR and whole-episome sequencing and selected for analysis by HPLC. Example 2.2 (AC_2 / Ptref2)

[0091] The construct containing the sequences encoding the TKS and OAC enzymes was transformed into P. tricornutum.

[0083]

[0092] The construct (Ptref2, SEQ ID NO: 50) containing the 5' to 3' sequences: the sequence encoding TKS (positions 1-1155); the 3(GGGGS) peptide linker sequence (positions 1156-1200); and the sequence encoding OAC (positions 1201-1503) was inserted into a modified pPtGE30 plasmid (Slattery et al. 2018) containing a zeocin resistance gene for algae and a chloramphenicol resistance gene for E. coli and His selection in yeast.

[0084]

[0093] The construct was operably linked to the 40SRPS8 promoter (SEQ ID NO: 22) and FcpA terminator (SEQ ID NO: 54).

[0085]

[0094] The PtGE30 episomal vector was conjugatively transferred from E. coli to P. tricornutum.

[0086]

[0095] Zeocin-resistant clones of P. tricornutum were confirmed by PCR and whole-episome sequencing and selected for analysis by HPLC.

[0087] Example 2.3 (AC_3 / Ptref3)

[0096] The construct containing the sequences encoding the TKS and OAC enzymes was transformed into P. tricornutum.

[0088]

[0097] The construct (Ptref3, SEQ ID NO: 51) containing the 5' to 3' sequences: TKS coding sequence (positions 1-1155); 6His tag (positions 1156-1173); T2A self-cleaving peptide linker sequence (positions 1174-1236); OAC coding sequence (positions 1237-1539); and Myc tag sequence (positions 1540-1569) was inserted into a modified pPtGE30 plasmid (Slattery et al. 2018) containing a zeocin resistance gene for algae and a chloramphenicol resistance gene for E. coli and His selection in yeast.

[0089]

[0098] The construct was operably linked to the 40SRPS8 promoter (SEQ ID NO: 22) and FcpA terminator (SEQ ID NO: 54).

[0090]

[0099] The PtGE30 episomal vector was conjugatively transferred from E. coli to P. tricornutum.

[0091]

[0100] Zeocin-resistant clones of P. tricornutum were confirmed by PCR and whole-episome sequencing and selected for analysis by HPLC.

[0092] Example 2.4 (AC_7 / Ptref7)

[0101] The construct containing the sequences encoding the TKS and OAC enzymes was transformed into P. tricornutum.

[0093]

[0102] The construct (Ptref7, SEQ ID NO: 52) containing the 5' to 3' sequences: YFP reporter sequence (positions 1-753); glycine codon (positions 754-756); sequence encoding TKS (positions 757-1911); 3(GGGGS) peptide linker sequence (positions 1912-1956); sequence encoding OAC (positions 1957-2259); and Myc tag sequence (positions 2260-2289) was inserted into a modified pPtGE30 plasmid (Slattery et al. 2018) containing a zeocin resistance gene for algae and a chloramphenicol resistance gene for E. coli and a His selection gene for yeast.

[0094]

[0103] The construct was operably linked to the 40SRPS8 promoter (SEQ ID NO: 22) and FcpA terminator (SEQ ID NO: 54).

[0095]

[0104] The PtGE30 episomal vector was conjugatively transferred from E. coli to P. tricornutum.

[0096]

[0105] Zeocin-resistant clones of P. tricornutum were confirmed by PCR and whole-episome sequencing and selected for analysis by HPLC.

[0097] Example 2.5

[0106] HPLC curves showed the presence of CBD, THC, and other cannabinoids in P. tricornutum clones transformed with constructs Ptref1 (Figure 5A), Ptref2 (Figure 5B), Ptref3 (Figure 5C), and Ptref7 (Figure 5D), which contain the TKS and OAC transgenes, compared to the wild-type control (Figure 5E). This result was unexpected, as it was thought that additional exogenous enzymes, including APT and CBDAS / THCAS, would be required to transform into microorganisms to complete the cannabinoid biosynthetic pathway.

[0098]

[0107] The amount of CBD in each sample was calculated based on the standard curve for CBD detected by HPLC (Figure 4B) and is shown in Table 2.

[0108]

[0099] [Table 2]

[0100]

[0109] Analysis by UPLC further demonstrated the presence of CBD and THC in P. tricornutum clones transformed with constructs Ptref1 (Figure 7A) and Ptref2 (Figure 7B) compared to the wild-type control (Figure 7C).

[0101]

[0110] These results demonstrate that microalgae can produce cannabinoids when transformed with only genes encoding the TKS and OAC enzymes, indicating that microalgae may possess enzymes that utilize olivetolic acid (OA) and / or derivatives as substrates and that can synthesize CBD and other cannabinoids.

[0102] Example 3

[0111] Vector construction and transformation of the green alga Chlamydomonas reinhardtii. Example 3.1 Plasmid propagation and extraction from E. coli

[0112] The synthetic construct for transformation into Chlamydomonas reinhardtii was first inserted into a default vector (KanR, high copy number) and then transformed into E. coli by electroporation. The transformed E. coli was grown to amplify the plasmid containing the construct. Positive E. coli were confirmed by colony PCR. The plasmid was then extracted and prepared for Gibson assembly into pChlamy3, which contains the strong hybrid promoter HSP70-RbcS2 and intron 1 of RbcS2, preceded by a cloning site.

[0103] Example 3.2 Gibson assembly and transformation in E. coli

[0113] The assembled pChlamy3 vector was used to transform E. coli by heat shock. Positive colonies were grown on ampicillin plates and confirmed by colony PCR. The transformed E. coli was then grown in liquid medium LB-amp100 to amplify the vector before extraction and purification (Biobasic, Miniprep Kit). After 3 hours of linearization (digestion with ScaI), the linearized vector was visualized on a 1% agarose gel and purified (Biobasic, PCR Cleanup Kit). The purified vector was used to transform Chlamydomonas reinhardtii cells.

[0104] Example 3.3 Transformation of Chlamydomonas reinhardtii by electroporation

[0114] The cells were exposed to 50 μmol photons m -2 s -1 The strains were cultured mixotrophically at 25°C in Tris-acetate phosphate (TAP) medium in shake flasks or on agar plates with 50% relative humidity (Rh) under moderate and continuous white fluorescent light of intensity 1000 Hz.

[0105]

[0115] Electroporation for transformation was performed as previously described (Shimogawara et al. 1998; Wittkopp 2018; Wang et al. 2019) with minor modifications. Chlamydomonas reinhardtii cells were transformed using a Bio-Rad Genepulser Xcell™ electroporation device and a 4 mm cuvette under the following parameters: voltage 0.5 kV; capacitance 50 μF; resistance 800 Ω.

[0106]

[0116] Briefly, cells in liquid state were cultured in 30 mL of TAP culture medium in a 125 mL Erlenmeyer flask until the initial OD 750nm is 0.1(1×10 5 cells / mL) and measure the final OD while gently shaking (100 rpm). 750nm is 0.7(7×10 6The cells were grown to a density of 1000 cells / mL. The cells were harvested by centrifugation at 7000 × g for 5 minutes, and the pellet was then washed three times by resuspending in 5 mL of Max Efficiency™ Transformation Reagent for Algae (Invitrogen, Catalog No. A24229) and centrifuging under the same conditions as in the harvesting step. After incubating the sample on ice for 10 minutes, electroporation was performed by applying an electric pulse using 250 μL of Chlamydomonas reinhardtii cells and 500 ng of linearized purified plasmid. The transgenic strain was resuspended in 5 mL of TAP liquid medium supplemented with 40 mM sucrose (TAP / sucrose) and then incubated at 25°C with gentle shaking (100 rpm) under continuous light for 22 hours. After incubation, the transformed cells were harvested by centrifugation at 7000 × g for 5 minutes, resuspended in 250 μl of Max Efficiency, and then spread onto TAP agar medium supplemented with hygromycin (10 μg / mL) and incubated in a growth chamber for approximately 5–7 days.

[0107]

[0117] Once single clones appeared on the agar Petri plates, the total number of transformants on each plate was counted using OpenCFU software to determine transformation efficiency. Example 3.3 Transgenic constructs and HPLC analysis

[0118] A construct containing sequences encoding the TKS and OAC enzymes was transformed into Chlamydomonas reinhardtii. The construct (G1C1, SEQ ID NO: 57) containing the 5' to 3' sequences: the TKS coding sequence (positions 1 to 1155); the FMDV2A self-cleaving peptide linker sequence (positions 1156 to 1227); and the OAC coding sequence (positions 1228 to 1530) was inserted into the pChlamy3 plasmid. The construct was operably linked to the HSP70A-RbcS2 hybrid promoter (SEQ ID NO: 55) and RbcS2 terminator (SEQ ID NO: 56). The vector was transfected into Chlamydomonas reinhardtii strain C-137 by electroporation.

[0108]

[0119] Positive transformants were selected by hygromycin resistance and PCR, grown in TAP medium, and then harvested and extracted for analysis by UPLC. UPLC analysis at 220 nm revealed the presence of a peak at 25.023 minutes (Figure 6A), corresponding to cannabinol (CBN) at 25.406 minutes (Figure 6B) in the control sample containing the cannabinoid standards. The control sample containing the cannabinoid standards (Figure 6B) exhibits a THCA peak at 68.240 minutes, a THC peak at 31.587 minutes, a CBN peak at 25.406 minutes, a CBGA peak at 20.130 minutes, a CBDA peak at 16.292 minutes, and a CBD peak at 14.628 minutes.

[0109]

[0120] While the present disclosure has been described with respect to what are presently considered to be the preferred embodiments, it is to be understood that the disclosure is not limited to the disclosed embodiments. On the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[0110]

[0121] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0111] References Altschul, SF et al (1990) Basic local alignment search tool." Journal of molecular biology 215, 403-410. Altschul, SF et al (1997) Gapped BLAST andPSI-BLAST: a new generation of protein database search programs. Nucleic acidsresearch 25, 3389-3402. Cecchin, M. et al. (2018) Molecular basisof autotrophic vs mixotrophic growth in Chlorella sorokiniana. Sci Rep,8(1):6465. DeLoache, W.C., et al. (2015) An enzyme-coupled biosensor enables (S)-reticuline productionin yeast from glucose. Nat Chem Biol, 11(7):465. Diaz-Santos, E., et al. (2013) Efficiency of different heterologouspromoters in the unicellular microalga Chlamydomonas reinhardtii. BiotechnolProg, 29(2):319-328. ElSohly, M.A. and Slade D. (2005) Chemicalconstituents of marijuana: The complex mixture of natural cannabinoids. LifeSciences, 2005. 78(5):539-548. Flores-Sanchez, I.J., et al. (2010) In silicio expression analysis of PKS genes isolated fromCannabis sativa L. Genet Mol Biol, 33(4): 703-713. Fossati, E., et al. (2014) Reconstitutionof a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine inSaccharomyces cerevisiae. Nat Commun, (5):3283. Gagne, S.J., et al. (2012) Identification of olivetolic acidcyclase from Cannabis sativa reveals a unique catalytic route to plantpolyketides. Proc Natl Acad Sci USA109(31):12811-12816. Gibson, D.G., et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Meth, 6(5):343-345. Hirakawa, Y., and Ishida, K. (2010) Internal plastid-targeting signal found in a RubisCO small subunitprotein of a chlorarachniophyte alga. Plant J, 64(3);402-410. Karas, B.J., et al., Designer diatomepisomes delivered by bacterial conjugation. Nat Commun, 2015. 6: p. 6925. Karlin, S. and Altschul, S.F. (1990)Methods for assessing the statistical significance of molecular sequencefeatures by using general scoring schemes. Proceedings of the National Academyof Sciences 87, 2264-2268. Karlin, S. and Altschul, S.F. (1993)Applications and statistics for multiple high-scoring segments in molecularsequences. Proceedings of the National Academy of Sciences 90, 5873-5877. Keasling, J.D. (2012) Synthetic biology andthe development of tools for metabolic engineering. Metab Eng, 14(3):189-195. Lithwick G, and Margalit H. (2003)Hierarchy of sequence-dependent features associated with prokaryotictranslation. Genome Research 13:2665-2673. Lussier, F.X., et al. (2012)Engineering microbes for plantpolyketide biosynthesis. Comput Struct Biotechnol J 3:e201210020. Marks, M.D., et al. (2009) Identification of candidate genes affectingDelta9-tetrahydrocannabinol biosynthesis in Cannabis sativa. J Exp Bot,60(13):3715-3726. Pandey, A. et al. (2013) Biofuels fromAlgae 1 st Ed. , Elsevier, ISBN 9780444595584. Plecenikova, A., et al. (2013) Studies onrecombination processes in two Chlamydomonas reinhardtii endogenous genes, NIT1and ARG7. Protist, 164(4):570-582. Schroda, M., et al. (2000) The HSP70Apromoter as a tool for the improved expression of transgenes in Chlamydomonas.Plant J, 2000. 21(2): 121-131. Shimogawara, K., et al. (1998)High-efficiency transformation of Chlamydomonas reinhardtii by electroporation.Genetics, 148: 1821-1828. Singh, N.D., et al. (2009)Chloroplast-derived vaccine antigens and biopharmaceuticals: protocols forexpression, purification, or oral delivery and functional evaluation. MethodsMol Biol, 483:163-192. Singh, S.P., and Singh, P. (2014) Effect ofCO2 concentration on algal growth: A review. Ren Sus En Rev, 38:172-179. Slattery, S.S., et al. (2018) An expandedplasmid-based genetic toolbox enables Cas9 genome editing and stablemaintenance of synthetic pathways in Phaeodactylum tricornutum. ACS SyntheticBiology, 7(2):328-338. Stout, J.M., et al. (2012) The hexanoyl-CoA precursor for cannabinoid biosynthesis isformed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J,71(3):353-365. Taura, F., et al. (2009) Characterizationof olivetol synthase, a polyketide synthase putatively involved in cannabinoidbiosynthetic pathway. FEBS Lett, 583(12):2061-2066. Wang, L., et al. (2019) Rapid and highefficiency transformation of Chlamydomonas reinhardtii by square-waveelectroporation. Bioscience Reports, 39. Welch et al. (2009) Design parameters tocontrol synthetic gene expression in Escherichia coli. PLoS ONE 4: e7002. Wittkopp, T.M. (2018) NuclearTransformation of Chlamydomonas reinhardtii by electroporation, Bio-Protocol,8.

[0112] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority from U.S. Provisional Patent Application No. 62 / 927,321, filed October 29, 2019.

Claims

1. A genetically engineered microorganism capable of producing cannabinoids, the microorganism being a photosynthetic microalga or a cyanobacterium, and the genetically engineered microorganism does not contain an exogenous nucleic acid molecule encoding an aromatic prenyltransferase.

2. 10. The genetically engineered microorganism of claim 1, which is capable of producing tetrahydrocannabinolic acid or tetrahydrocannabinol and does not contain an exogenous nucleic acid molecule encoding tetrahydrocannabinolic acid synthase.

3. 3. The genetically engineered microorganism of claim 1 or 2, which is capable of producing cannabidiolic acid or cannabidiol and does not contain an exogenous nucleic acid molecule encoding a cannabidiolic acid synthase.

4. 4. The genetically engineered microorganism of claim 1, comprising at least one exogenous nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase.

5. The genetically engineered microorganism of claim 4, wherein the tetraketide synthase comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1, and the olivetolic acid cyclase comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

2.

6. 6. The genetically engineered microorganism of claim 4 or 5, wherein the at least one exogenous nucleic acid molecule comprises a first polynucleotide sequence encoding a tetraketide synthase and a second polynucleotide sequence encoding an olivetolic acid cyclase.

7. 7. The genetically engineered microorganism of claim 6, wherein the first polynucleotide sequence is 5' to the second polynucleotide sequence.

8. 8. The genetically engineered microorganism of claim 6 or 7, wherein the at least one exogenous nucleic acid molecule further comprises at least one linker sequence between the first polynucleotide sequence and the second polynucleotide sequence.

9. 6. The genetically engineered microorganism of claim 4 or 5, wherein the at least one exogenous nucleic acid molecule comprises a first nucleic acid molecule encoding a tetraketide synthase and a second nucleic acid molecule encoding an olivetolic acid cyclase.

10. 4. The genetically engineered microorganism of claim 1, comprising at least one exogenous nucleic acid molecule encoding Steely 1, Steely 2 or a variant thereof.

11. 11. The genetically engineered microorganism of claim 10, wherein the variant of Steely 1 or Steely 2 comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 8, respectively.

12. The genetically engineered microorganism of any one of claims 4 to 11, wherein at least one exogenous nucleic acid molecule is an episomal vector.

13. A genetically engineered microorganism according to any one of claims 4 to 12, comprising at least one exogenous nucleic acid molecule.

14. 13. The genetically engineered microorganism of any one of claims 1 to 12, which does not contain an exogenous nucleic acid molecule encoding a hexanoyl-CoA synthetase.

15. 15. The genetically engineered microorganism of any one of claims 1 to 14, wherein the microalgae is a diatom or a chlorophyte.

16. 16. The genetically engineered microorganism of claim 15, wherein the microalgae is Phaeodactylum tricornutum or Chlamydomonas reinhardtii.

17. 17. A cell culture comprising the genetically engineered microorganism of any one of claims 1 to 16 and a substantially sugar-free medium.

18. 18. The cell culture of claim 17, wherein the medium is substantially free of a fixed carbon source.

19. 19. The cell culture of claim 17 or 18, which undergoes autotrophic growth.

20. 1. A method for producing cannabinoids in a genetically engineered microorganism, comprising the step of introducing into the microorganism at least one nucleic acid molecule encoding a tetraketide synthase and an olivetolic acid cyclase, wherein the microorganism is a microalga or a cyanobacterium.

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