[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority from United States Provisional Patent Application No. 62/934,234 filed on November 12, 2019 and United States Provisional Patent Application No. 62/967,839 filed on January 30, 2020.

[0003] FIELD

[0004] The present disclosure relates to methods of culturing microorganisms or genetically engineered microorganisms which produce at least one cannabinoid biosynthetic pathway product.

[0005] BACKGROUND

[0006] The commercialization of valuable plant natural products (PNPs) is often limited by the availability of PNP producing-plants, by the low accumulation of PNPs in planta and/or the time-consuming and often inefficient extraction methods not always economically viable. Thus, commercialization of PNPs of commercial interest is often challenging. The recent progress in genetic engineering and synthetic biology makes it possible to produce heterologous PNPs or other biosynthetic products in microorganisms such as bacteria, yeast and microalgae. For example, engineered microorganisms have been reported to produce the antimalarial drug artemisinin and of the opiate (morphine, codeine) painkiller precursor reticuline (Keasling 2012; Fossati et al 2014; DeLoache et al 2015). However, the latest metabolic reactions to yield the valuable end-products such as codeine and morphine in genetically modified yeast-producing reticuline have yet to be successfully achieved. In some cases, bacterial or yeast platforms do not support the assembly of complex PNP or biosynthetic product pathways. In comparison, microalgal cells have been suggested to possess advantages over other microorganisms, including the likelihood to perform similar post-translational modifications of proteins as plant and recombinant protein expression through the nuclear, mitochondrial or chloroplastic genomes (Singh et al 2009).

[0007] Cannabinoid biosynthetic pathway products such as D9- tetrahydrocanannabinol and other cannabinoids (CBs) are polyketides responsible for the psychoactive and medicinal properties of Cannabis sativa. More than 110 CBs have been identified so far and are all derived from fatty acid and terpenoid precursors (ElSohly and

Slade 2005). The first metabolite intermediate in the CB biosynthetic pathway in Cannabis sativa is olivetolic acid that forms the polyketide skeleton of cannabinoids. A type III polyketide synthase (PKS; also known as tetraketide synthase (TKS) orolivetol synthase) enzyme condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form trioxododecanoyl-CoA. From there, olivetolic acid cyclase (OAC) (OAC; also known as 3,5,7-trioxododecanoyl-CoA CoA-lyase) catalyzes an intramolecular aldol condensation to yield OA. In subsequent steps, CB diversification is generated by the sequential action of “decorating” enzymes on the OA backbone. The gene sequence for PKS and OAC have been identified and characterized in vitro (Lussier2012; Gagne ef a/2012; Marks et al 2009; Stout et a/ 2012; Taura et al 2009).

[0008] Microorganisms, such as microalgae and cyanobacteria, have industrial potential for the production of PNPs or other biosynthetic products. However, the commercial use of microorganisms is dependent on input energy and nutrients cost. Light is the energy source harvested by the photosynthetic machinery of autotrophic (such as photoautotrophic or photosynthetic) microorganisms. Studies have reported that microalgae can adapt to variations in light spectrum and intensity. It has been shown that light quality has an impact on chloroplast migration, zygote germination, and light acclimation (Furukawa et al 1998; Shikata et al 2011 ; Holdsworth et al 1985). Recent discoveries have revealed photoreceptors such as red light sensing phytochromes, blue- light sensing cryptochromes and aureochromes (Armbrust et al 2004; Bowler et al 2008).

[0009] SUMMARY

[0010] The present disclosure describes a method for culturing a microorganism under a first light spectrum for a first period of time and subsequently in a second light spectrum for a second period of time.

[0011] Accordingly, the present disclosure provides a method of culturing a photosynthetic microorganism, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.

[0012] The present disclosure further provides a method of culturing a photosynthetic microorganism capable of producing at least one biosynthetic product in a culture medium, comprising (a) culturing the microorganism under a first light spectrum for a first period of time; and (b) culturing the microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6- alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.

[0013] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description in conjunction with the accompanying figures.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying figures illustrate embodiments of the invention by way of example.

[0016] Figure 1. An exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa

[0017] Figure 2. Gravimetric biomass analysis in mixotrophic and autotrophic conditions under different colours of light (White Light [C], Red Light [R], and Yellow Light [Y]). C, R, and Y refer to autotrophic cultures; C1 , R1 , and Y1 refers to mixotrophic cultures in L1 media supplemented with 1 % glucose; C2, R2, and Y2 refers to mixotrophic cultures in L1 media supplemented with 1 % Glycine. Statistical analysis by one-way ANOVA indicated statistical significance for pairings C vs C2, C vs R, C vs R2 C vs Y2, C1 vs R, C1 vs Y2, C1 vs R2, C2 vs R2, R2 Vs Y, and R1 vs Y1.

[0018] Figure 3. Gravimetric total lipid analysis in mixotrophic and autotrophic conditions under different colours of light (White Light [C], Red Light [R] and Yellow Light [Y]). C, R, and Y refer to autotrophic cultures; C1 , R1 , and Y1 refers to mixotrophic cultures in L1 media supplemented with 1 % glucose; C2, R2, and Y2 refers to mixotrophic cultures in L1 media supplemented with 1 % Glycine. Statistical analysis by one-way ANOVA indicated statistical significance for pairings C1 vs R2, R1 vs R2, R2 vs Y, and R2 vs Y1.

[0019] Figure 4. Cell growth analysis of P.tricornutum measured by optical density at 680 nm at different days during autotrophic culture. White light; Red light; (RS) grown under red light during lag phase and exponential phase; (RT1) grown under red light during lag phase; (RT2) grown under red light during exponential phase; (RT3) grown under red light during stationary phase; Yellow light. Absorbance units (a.u.) are reported.

[0020] Figure 5. Growth curve of P.tricornutum in autotrophic culture in different light conditions as measured by optical density at 680 nm: white light (C); red light (R), red light during lag and exponential phases (Rs); red light during lag phase (RT1); red light during exponential phase (RT2); red light during stationary phase (RT3). Absorbance units (a.u.) are reported.

[0021] Figure 6. Biomass analysis of autotrophic cultures under white light (C), red light (R), yellow light (Y), and red light under the lag and exponential phases (RS). Statistical analysis by one-way ANOVA indicated statistical significance for pairing C vs RS.

[0022] Figure 7. Total lipid analysis of autotrophic cultures under white light (C), red light (R), yellow light (Y), and red light under the lag and exponential phases (RS). Statistical analysis by one-way ANOVA indicated statistical significance for pairing C vs RS.

[0023] Figure 8. Visual analysis of the lipid droplets in P.tricornutum grown in white light (left images) and red light (right images). Bright field (top images) and fluorescent (bottom images).

[0024] Figure 9. Transmittance spectrum of red light colour filter from 400nm to 720nm.

[0025] Figure 10. Biomass of analysis of autotrophic cultures under white light (C), blue light (B), blue light during lag and exponential phases (Bs), yellow light (Y), yellow light during lag and exponential phases (Ys), red light (r), and red light during lag and exponential phases (rs).

[0026] DETAILED DESCRIPTION

[0027] The present disclosure provides the discovery that culturing a photosynthetic microorganism under a first light spectrum for a period of time and then subsequently growing said microorganism under second light spectrum may increase biomass and/or lipid production, which may in turn improve the production of a biosynthetic product in said microorganism. For example, the microorganism may produce a cannabinoid biosynthetic pathway product, the production of which is improved by the methods of the present invention.

[0028] For example, without being bound by theory, it is expected that the improvement in the production of at least one cannabinoid biosynthetic pathway product results from increased cell growth, biomass, and/or lipid production of said cultured microorganism cultured using methods provided herein. Increasing biomass and cell growth can increase the total yield of the desired product, and can increase the number of culture cycles in a given period of time. Increasing lipid production, particularly of short fatty acids, such as hexanoic acid and malonyl-CoA, may increase precursor substrates for cannabinoid biosynthesis and thus may increase the total yield of desired products. Increasing lipid production may also facilitate the downstream extraction of hydrophobic cannabinoid biosynthetic pathway products from the biomass, and may further improve the viability of microorganisms producing cannabinoid biosynthetic pathway products by allowing the microorganisms to sequester toxic cannabinoid biosynthetic pathway products in intracellular lipid bodies.

[0029] Accordingly, the present disclosure provides a method of culturing a photosynthetic microorganism, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.

[0030] The present disclosure further provides a method of culturing a photosynthetic microorganism capable of producing at least one biosynthetic product in a culture medium, comprising (a) culturing the microorganism under a first light spectrum for a first period of time; and (b) culturing the microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6- alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.

[0031] Methods provided herein may be carried out under mixotrophic or autotrophic growth conditions. Photosynthetic microorganisms are capable of carbon fixation wherein carbon dioxide (which is not a fixed carbon source) is fixed 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. Suitable sources of light for the provision of energy in photosynthesis include sunlight and artificial lights. Photosynthetic microorganisms are capable of growth and/or metabolism without a fixed carbon source. For example, microalgae can fix carbon dioxide from a variety of sources, including atmospheric carbon dioxide, industrially-discharged carbon dioxide (e.g. flue gas and flaring gas), and from soluble carbonates (e.g. NaHC03 and Na2C03) (Singh et al 2014). A non-fixed carbon source such as carbon dioxide can be added to a culture of microalgae by injection or by bubbling of a carbon dioxide gas mixture into the culture medium. Photosynthetic growth is a form of autotrophic growth, wherein a microorganism is able to produce organic molecules on its own using an external energy source such as light. This is in contrast to heterotrophic growth, wherein a microorganism must consume reduced organic molecules for growth and/or metabolism. Heterotrophic microorganisms therefore require a fixed carbon source for growth and/or metabolism.

[0032] Some photosynthetic microorganisms are capable of mixotrophic growth, wherein the microorganism fixes carbon by photosynthesis while also consuming fixed carbon sources. In mixotrophic growth, the autotrophic metabolism is integrated with a heterotrophic metabolism that oxidizes reduced carbon sources available in the culture medium. Photosynthetic microalgae are commonly cultivated in mixotrophic conditions by adding fixed carbon sources as described herein to the culture medium. 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 cellulosic material, plant material (e.g. sugar cane, sugar beet), and waste products from industry such as acetate or glycerol (see Cecchin et al 2018). Microorganisms such as microalgae and cyanobacteria may be cultured using methods and conditions known in the art (see, e.g., Biofuels from Algae, eds. Pandey et al., 2014, Elsevier, ISBN 978-0- 444-59558-4). Some microorganisms are capable of chemoautotrophic growth. Similar to photosynthetic microorganisms, chemoautotrophic organisms are capable of carbon dioxide fixation but using energy derived from chemical sources (e.g. hydrogen sulfide, ferrous iron, molecular hydrogen, ammonia) rather than light.

[0033] Photosynthetic culture of a microorganism requires the provision of light. Various sources of light may be used, including solar light and artificial light. Artificial light may be provided from a variety of sources known in the art, including but not limited to, fluorescent lights, high intensity discharge lights, halide lights, and light-emitting diodes (LED). The use of artificial light sources allows for the control of the light spectrum provided to the culture. The light spectrum provided to a culture can also be controlled by passing white light through a colour filter. “Light spectrum”, as used herein, refers to a defined wavelength, a plurality of defined wavelengths, or range of wavelengths of light. A light spectrum can therefore comprise a single wavelength of light, multiple wavelengths of light, or an entire range of wavelengths of light. The wavelength of visible and ultraviolet light is commonly measured in nanometers (nm). Photosynthetic microorganisms are able to perform photosynthesis in response to a range of light spectra. Visible light is typically defined in the art as light spectrum comprising about 400 nm to about 700 nm. Within visible light are colour spectra typically defined in the art as violet light (e.g. about 400 nm to about 450 nm), blue light (e.g. about 450 nm to about 485 nm), cyan light (e.g. about 485 nm to about 500 nm), green light (e.g. about 500 nm to about 565 nm), yellow light (e.g. about 565 nm to about 590 nm), orange light (e.g. about 590 nm to about 625 nm), red light (e.g. about 625 nm to about 740 nm), white light (e.g. about 400 nm to about 700 nm). Solar light comprises both white light as well as ultraviolet light (about 10 nm to about 400 nm) and infrared light (about 700 nm to about 1 mm).

[0034] In some embodiments, the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 590 nm to about 740 nm, about 625 nm to about 740 nm, about 625 nm to about 720 nm, about 625 nm to about 700 nm, about 600 nm to about 730 nm, about 610 nm to about 720 nm, about 620 nm to about 710 nm, about 630 nm to about 700 nm, about 640 nm to about 690 nm, about 650 nm to about 680 nm, about 660 nm to about 670 nm, about 590 nm to about 625 nm, about 625 nm to about 660 nm, about 660 nm to about 695 nm, about 680 nm to about 700 nm, or about 695 nm to about 740 nm in wavelength. By percent (%) of light, it is meant the portion of the light within a defined range of wavelengths as a percentage of the total light emitted. The percent (%) of light may calculated as a percent of total light by units of measuring light known in the art including lux (lx), lumen (Im), pmol nr ² s ¹ , and/or as a percent of the total transmitted light.

[0035] In some embodiments, the first light spectrum comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm, about 625 nm to about 740 nm, about 625 nm to about 720 nm, about 625 nm to about 700 nm, about 600 nm to about 730 nm, about 610 nm to about 720 nm, about 620 nm to about 710 nm, about 630 nm to about 700 nm, about 640 nm to about 690 nm, about 650 nm to about 680 nm, about 660 nm to about 670 nm, about 590 nm to about 625 nm, about 625 nm to about 660 nm, about 660 nm to about 695 nm, about 680 nm to about 700 nm, or about 695 nm to about 740 nm in wavelength.

[0036] In some embodiments, the first light spectrum comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about

90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about

80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm, about 625 nm to about 740 nm, about 625 nm to about 720 nm, about 625 nm to about 700 nm, about 600 nm to about 730 nm, about 610 nm to about 720 nm, about 620 nm to about 710 nm, about 630 nm to about 700 nm, about 640 nm to about 690 nm, about 650 nm to about 680 nm, about 660 nm to about 670 nm, about 590 nm to about 625 nm, about 625 nm to about 660 nm, about 660 nm to about 695 nm, about 680 nm to about 700 nm, or about 695 nm to about 740 nm in wavelength.

[0037] In some embodiments, the second light spectrum comprises or consists essentially of light within about 400 nm to about 700 nm, about 400 nm to about 740 nm, about 400 nm to about 450 nm, about 450 nm to about 485 nm, about 485 nm to about 500 nm, about 500 nm to about 565 nm, about 565 nm to about 590 nm, about 410 nm to about 690 nm, about 420 nm to about 680 nm, about 430 nm to about 670 nm, about 440 nm to about 660 nm, or about 450 nm to about 650 nm. In an embodiment, the second light spectrum comprises or consists essentially of solar light.

[0038] Methods provided herein involve culturing a microorganism under at least two different light spectra. In one embodiment, the first light spectrum comprises at least 65% of light within about 590nm to 740nm. Light perceived by red-light receptors (phytochromes) can regulate nutrient metabolism, regulation of cellular events and signalling cascades (Wang et al 2015). In one embodiment, the second light spectrum comprises or consists essentially of blue light, white light, or solar light.

[0039] In one embodiment, methods described herein involve withdrawal of the first light spectrum and introduction of the second light spectrum. In another embodiment, methods described herein involve maintaining the first light spectrum while introducing a different light spectrum to form the second light spectrum. In another embodiment, methods described herein involve adjusting the first light spectrum to form the second light spectrum, for example, by removing light of certain wavelengths within the spectrum and/or adding light within the same spectrum but having different wavelengths.

[0040] The culture of microorganisms comprises distinct phases. Initially, a new culture of microorganisms exists in a lag phase, wherein the microorganisms are adapting to the culture conditions. In the lag phase, the microorganisms are active but cell division is low or is not occurring. Subsequent to the lag phase, the rate of cell division accelerates as the culture enters the exponential phase, wherein nutrients are typically in excess and the microorganisms multiply exponentially at a constant rate. When a culture of microorganisms reaches a high density, the availability of nutrients begins to decline, and/or metabolic waste products accumulate in the culture environment, causing the culture to enter a stationary phase wherein the rate of cell division begins to decline. In the stationary phase the rate of cell division will slow down and the population of the culture reaches a plateau. If a culture is allowed to continue for long enough in the stationary phase then a death phase will begin wherein microorganisms die due to nutrient starvation and/or the toxicity of metabolic waste products, and the population of the culture declines. It is within the ability of the skilled person to determine the phase of a culture of microorganisms by methods known in the art, for example by using optical density to determine the cell density within the culture.

[0041] Methods provided herein involve changing the first light spectrum to the second light spectrum at a selected time point during a phase or as one phase ends and the next phase starts. In an embodiment, the first light spectrum is changed to the second light spectrum at the end of the lag phase, at the beginning of the exponential phase, during the exponential phase, at the end of the exponential phase, or at the beginning of the stationary phase.

[0042] In some embodiments, microorganisms for use in the present invention can be genetically engineered to express one or more proteins or enzymes to produce a biosynthetic product. As used herein, a “biosynthetic product” is a desired product produced by a cultured microorganism including, but not limited to, a polypeptide, a lipid, a nucleic acid, a polysaccharide, a glycoprotein, or an organic molecule. A biosynthetic product may be produced naturally by a wild-type microorganism in culture, produced by the conversion of a substrate or precursor in the culture medium by a microorganism in culture, or produced by the expression of exogenous genes in a microorganism in culture. In some embodiments, the biosynthetic product is a cannabinoid biosynthetic pathway product. In some embodiments, the microorganism is engineered to express enzymes of a cannabinoid biosynthetic pathway to produce a cannabinoid biosynthetic pathway product. As used herein, the term “genetically engineered” refers to the alteration of genetic material of an organism using molecular biology techniques known in the art, including but not limited to, introducing exogenous genes into a microorganism via episomes, integrative vectors, and homologous recombination vectors; DNA editing by zinc fingers or CRISPR-Cas; and/or by transformation methods known in the art. The genetically engineered microorganism includes a living modified microorganism, genetically modified microorganism or a transgenic microorganism. Genetic engineering includes addition, deletion, modification and/or mutation of genetic material.

[0043] The term "nucleic acid molecule", as used herein, is intended to include unmodified DNA or RNA or modified DNA or RNA. For example, nucleic acid molecules of the disclosure may be composed of single- or double-stranded DNA; DNA that is a mixture of single- and double-stranded regions; single- or double-stranded RNA; RNA that is a mixture of single- and double-stranded regions; hybrid molecules comprising DNA and RNA that may be single-stranded, double-stranded, or a mixture of single- and double-stranded; or circular or linearized DNA-based vectors. The nucleic acid molecules of the disclosure may contain one or more modified bases. Modified bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule" embraces chemically, enzymatically, or metabolically modified forms. The term "polynucleotide" shall have a corresponding meaning. In some embodiments, the genetically engineered microorganism comprises at least one nucleic acid molecule encoding a polypeptide disclosed herein.

[0044] As used herein, the term “exogenous” refers to an element that has been introduced into a cell. An exogenous element can include a polypeptide or a nucleic acid. An exogenous nucleic acid is a nucleic acid that has been introduced into a cell, such as by a method of transformation. An exogenous nucleic acid may code for the expression of an RNA and/or a polypeptide. An exogenous nucleic acid may have been derived from the same species (homologous) orfrom a different species (heterologous). An exogenous nucleic acid may comprise a homologous sequence that is altered such that it is introduced into the cell in a form that is not normally found in the cell in nature. For example, an exogenous nucleic acid that is homologous may contain mutations, be operably linked to a different control region, or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid. An exogenous nucleic acid may be incorporated into the chromosomes of the transformed cell in one or more copies, into the plastid or mitochondrial DNA of the transformed cell, or be maintained as a separate nucleic acid outside of the transformed cell genome.

[0045] As used herein, the term “vector” or “nucleic acid vector” means a nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a microorganism. The transgenic DNA can encode a heterologous polypeptide, which can be expressed in and isolated from a microorganism. The transgenic DNA can be integrated into nuclear, mitochondrial or chloroplastic genomes through homologous or non-homologous recombination. The transgenic DNA can also replicate without integrating into nuclear, mitochondrial or chloroplastic genomes, such as in an episomal vector. The vector can contain a single, operably-linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence. Vectors useful in the present methods are well known in the art. 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 integration into the transformed cell genome (Karas et al 2015). Episomal vectors can be transferred from a bacteria (e,g, Escherichia coli) to another target microorganism (e.g. a microalgae) via conjugation. As used herein, the term “expression cassette” means a single, operably-linked set of regulatory elements that includes 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 an embodiment, the at least one nucleic acid molecule is an episomal vector. The term “operably-linked”, as used herein, refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner. For example, a transcriptional regulatory sequence or a promoter is operably- linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence. The skilled person can readily recognize aspects of the transcription process, which include, but not limited to, initiation, elongation, attenuation and termination. In general, an operably-linked transcriptional regulatory sequence joined in cis with the coding sequence, but it is not necessarily directly adjacent to it.

[0046] The phrase “introducing a nucleic acid molecule into a microorganism” includes the stable integration of the nucleic acid molecule into the genome of a microorganism or the introduction of a replicating vector into a microorganism, or to the transient integration of the nucleic acid molecule into the microorganism. The introduction of a nucleic acid into a cell is also known in the art as transformation. The nucleic acid vectors may be introduced into the microorganism using techniques known in the art including, without limitation, agitation with glass beads, electroporation, agrobacterium- mediated transformation, an accelerated particle delivery method (i.e. particle bombardment), a cell fusion method or by any other method to deliver the nucleic acid molecule into a microorganism.

[0047] As used here, the term "sequence identity" refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/.e., % identity=number of identical overlapping positions/total number of positions multiplied by 100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in 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 with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present disclosure. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score=50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997). Alternatively, PSI-BLAST can be used to perform an iterated search which 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., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing 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 allowing gaps. In calculating percent identity, typically only exact matches are counted. In a specific embodiment, the nucleic acids are optimized for codon usage in a specific microalgal or cyanobacterial species.

[0048] The production of cannabinoid biosynthetic pathway products by a genetically engineered microorganism provided herein can be the result of introducing or increasing the activity of one or more enzymes associated with the cannabinoid biosynthetic pathway. This can include, for example, the introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding an enzyme of a cannabinoid biosynthetic pathway. Examples of cannabinoid biosynthetic pathway enzymes 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 geranyltransferasecannabichromene synthase, tetrahydrocannabinolic acid synthase (THCAS), and cannabidiolic acid synthase (CBDAS). Amino acid sequences of cannabinoid biosynthetic pathway enzymes as described herein are provided in Table 1.

[0049] Table 1. Sequences _ [0050] The term “cannabinoid” is generally understood to include any chemical compound that acts upon a cannabinoid receptor. Examples of cannabinoids include cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabichromanon (CBCN), cannabielsoin (CBE), cannbifuran (CBF), tetrahydrocannabinol (THC), cannabinodiol (CBDL), cannabicyclol (CBL), cannabitriol (CBT), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabinerolic acid, cannabidiolic acid (CBDA), cannabinodiol (CBND), cannabinol propyl variant (CBNV), cannabitriol (CBO), cannabigerolic acid (CBGA), tetrahydrocannabinolic acid (THCA), cannabichromenic acid (CBCA), tetrahydrocannabivarinic acid (THCVA), cannabigerovarinic acid (CBGVA), cannabidivarinic acid (CBDVA), cannabichromevarinic acid (CBCVA), and derivatives thereof. Further examples of cannabinoids are discussed in PCT Patent Application Pub. No. WO2017/190249 and US Patent Application Pub. No. US2014/0271940. A cannabinoid may be in an acid form or a non-acid form, the latter also being referred to as the decarboxylated form since the non-acid form can be generated by decarboxylating the acid form.

[0051] A cannabinoid biosynthetic pathway product is a cannabinoid or a product associated with the production of a cannabinoid such as precursor or intermediate compound. Examples of cannabinoid biosynthetic pathway products include, but are not limited to, hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, divarinol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol and cannabidiol. In an embodiment, the cannabinoid biosynthetic pathway product is at least one of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, divarinol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, D9- tetrahydrocanannabinol and cannabidiol.

[0052] Figure 1 shows an exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa : Tetraketide synthase (TKS) condenses hexanoyl-CoA and malonyl-CoA to form the intermediate trioxododecanoyl-CoA; Olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation to yield olivetolic acid (OA); aromatic prenyltransferase transfers a geranyldiphosphate (GPP) onto OA to produce cannabigerolic acid (CBGA); tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase catalyze the oxidative cyclization of CBGA into tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), respectively. Decarboxylation of THCA or CBDA to remove the carboxyl group will produce decarboxylated cannabinoids tetrahydrocannabinol (THC) or cannabidiol (CBD), respectively.

[0053] In addition to the exemplary cannabinoid biosynthetic pathway from Cannabis sativa shown in Figure 1 , alternative biosynthetic intermediates can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, olivetol is an intermediate that lacks the carboxyl group of olivetolic acid. Use of olivetol instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly lack a carboxyl group such as cannabigerol (CBG), tetrahydrocannabinol (THC), or cannabidiol (CBD). In another example, tetraketide synthase (TKS) condenses butyryl-CoA and malonyl-CoA to form the intermediate trioxodecanoyl-CoA, and olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation of trioxodecanoyl-CoA to yield divarinolic acid. Divarinolic acid is an intermediate containing an n-propyl group in place of the n-pentyl group found in olivetolic acid. Use of divarinolic acid instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly contain an n-propyl group such as cannabigerovarinic acid (CBGVA), tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), or cannabichromevarinic acid (CBCVA). In another example, divarinol is an intermediate that lacks the carboxyl group of divarinolic acid, and contains an n-propyl group in place of the n-pentyl group found in olivetol. Use of divarinol instead of divarinolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly contain an n-propyl group and lack a carboxyl group such as cannabigerovarin (CBGV), tetrahydrocannabivarin (THCV), cannabidivarinic acid (CBDV), or cannabichromevarinic acid (CBCV).

[0054] In addition to the exemplary cannabinoid biosynthetic pathway from Cannabis sativa shown in Figure 1 , alternative enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, in addition to the enzymes found in Cannabis sativa , alternative enzymes of a cannabinoid biosynthetic pathway may be found in other plants (e.g., Humulus lupulus), in bacteria (e.g., Streptomyces), or in protists (e.g., Dictyostelium discoideum). Enzymes that differ in structure, but perform the same function, may be used interchangeably in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. For example, the aromatic prenyltransferases CsPT1 (SEQ ID NO: 3) and CsPT4 (SEQ ID NO: 10) from Cannabis sativa , HIPT1 from Humulus lupulus (SEQ ID NO: 11), and Orf2 (SEQ ID NO: 9) from Streptomyces Sp. Strain CI190 are all aromatic prenyltransferases that catalyze the synthesis of CBGA from GPP and OA. In a further example, the Steelyl (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 into olivetol, and may be used in place of TKS to produce olivetol in the absence of OAC.

[0055] In addition to the wild-type enzymes found in organisms discussed herein, modified variants of these enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism. Variants of enzymes for use in a cannabinoid biosynthetic pathway can be generated by altering the nucleic acid sequence encoding said enzyme to, for example, increase/decrease the activity of a domain, add/remove a domain, add/remove a signaling sequences, or to otherwise alter the activity or specificity of the enzyme. For example, the sequence of Steelyl can be modified to reduce the activity of a methyltransferase domain in order to produce non-methylated cannabinoids. By way of 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. In a further example, the sequences of tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase can be modified to remove an N-terminal secretion peptide. By way of example, this can be done by removing amino acids 1-28 of SEQ ID NO: 5 or 6 to produce a truncated enzyme as disclosed in WO/2018/200888.

[0056] A acyl-CoA synthetase is an acyl-activating enzyme that ligates CoA and a straight-chain alkanoic acid or alkanoate containing 2 to 6 carbon atoms to produce alkanoyl-CoA, wherein the alkanoyl-CoA is a thioester of coenzyme A containing an alkanoyl group of 2 to 6 carbon atoms. In one embodiment, the acyl-CoA synthetase is hexanoyl-CoA synthetase, which ligates CoA and hexanoic acid or hexanoate to produce hexanoyl-CoA. A hexanoyl-CoA synthetase may have the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence with at least 90% identity to SEQ ID NO: 4. In another embodiment, an acyl-CoA synthetase ligates CoA and butyric acid or butyrate to produce butyryl-CoA.

[0057] A type III polyketide synthase is an enzyme that produces polyketides by catalyzing the condensation reaction of acetyl units to thioester-linked starter molecules.

A type III polyketide synthase may have the amino acid sequence of SEQ ID NO: 1 , 7, or

8 or an amino acid sequence with at least 90% identity to SEQ ID NO: 1 , 7, or 8. In an embodiment, a type III polyketide synthase condenses an alkanoyl-CoA with three malonyl-CoA in a multi-step reaction to form a 3,5,7-trioxoalkanoyl-CoA, wherein the 3,5,7-trioxoalkanoyl-CoA contains 8 to 12 carbon atoms. In another embodiment, the type III polyketide synthase is tetraketide synthase from Cannabis sativa which is also known in the art as olivetol synthase and 3,5,7-trioxododecanoyl-CoA synthase. In one embodiment, tetraketide synthase condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxododecanoyl-CoA. In another embodiment, tetraketide synthase condenses butyryl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxodecanoyl-CoA. In another embodiment, the type III polyketide synthase is Steelyl or Steely 2 from Dictyostelium discoideum, comprising a domain with type III polyketide synthase activity, ora variant thereof (e.g., Steelyl (G1516D+G1518A) or Steelyl (G1516R) disclosed in WO/2018/148849). Steelyl is also known in the art as DiPKS or DiPKSI , and Steely2 is also known in the art as DiPKS37.

[0058] An olivetolic acid cyclase, as used herein, refers to an enzyme that catalyzes an intramolecular aldol condensation of a 3,5,7-trioxoalkanoyl-CoA to form a 2,4-dihydroxy-6-alkylbenzoic acid, wherein the alkyl group of the benzoic acid contains 1 to 5 carbons. In an embodiment, an olivetolic acid cyclase catalyzes the formation of olivetolic acid from 3,5,7-trioxododecanoyl-CoA. In another embodiment, an olivetolic acid cyclase catalyzes the formation of divarinolic acid from 3,5,7-trioxodecanoyl-CoA. An olivetolic acid cyclase may have the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with at least 90% identity to SEQ ID NO: 2. Olivetolic acid cyclase from Cannabis sativa is also known in the art as olivetolic acid synthase and 3,5,7- trioxododecanoyl-CoA CoA-lyase.

[0059] An aromatic prenyltransferase, as used herein, refers to an enzyme capable of transferring a geranyl diphosphate onto a 5-alkylbenzene-1 ,3-diol to synthesize a 2- geranyl-5-alkylbenzene-1 ,3-diol, wherein the alkyl group of the product contains 1 to 5 carbons. In one embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetol to synthesize cannabigerol (CBG). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetolic acid (OA) to synthesize cannabigerolic acid (CBGA). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarin (CBGV). In another embodiment, an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarinic acid (CBGVA). An example of an aromatic prenyltransferase is aromatic prenyltransferase from Cannabis sativa which is also known in the art as CsPT1 , prenyltransferase 1 , geranylpyrophosphate-olivetolic acid geranyltransferase, and geranyl-diphosphate: olivetolate geranytransferase. Further examples of aromatic prenyltransferase include HIPT1 from Humulus lupulus , CsPT4 from Cannabis sativa, and Orf2 (NphB). Orf2 enzyme can be wild type Orf2 (e.g. from Streptomyces Sp. Strain CI190) or a mutant Orf2 enzyme engineered to produce a higher amount of enzymatic product and/or to to have higher activity in relation to a specific substrate. Examples of mutant Orf2 enzymes are disclosed in WO2019/183152A1 , the contents of which are herein incorporated by reference. An aromatic prenyltransferase may have the amino acid sequence of SEQ ID NO: 3, 9, 10 or 11 , or an amino acid sequence with at least 90% identity to SEQ ID NO: 3, 9, 10 or 11 .

[0060] A tetrahydrocannabinolic acid synthase is also known in the art as D9- tetrahydrocannabinolic acid synthase, and synthesizes A9-tetrahydrocannabinolic acid by catalyzing the cyclization of the monoterpene moiety in cannabigerolic acid. A tetrahydrocannabinolic acid synthase may have the amino acid sequence of SEQ ID NO:

5 or an amino acid sequence with at least 90% identity to SEQ ID NO: 5.

[0061] A cannabidiolic acid synthase synthesizes cannabidiolic acid by catalyzing the stereoselective oxidative cyclization of the monoterpene moiety in cannabigerolic acid. A cannabidiolic acid synthase may have the amino acid sequence of SEQ ID NO:

6 or an amino acid sequence with at least 90% identity to SEQ ID NO: 6.

[0062] In an embodiment, genetically modified microorganisms provided herein comprise at least one nucleic acid molecule that encodes at least one, two, three, four, five, or six of hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase.

[0063] In an embodiment, the at least nucleic acid molecule encodes at least one of a hexanoyl-CoA synthetase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 4; a type III polyketide synthase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 1 , 7 or 8; an olivetolic acid cyclase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 2 or 17; an aromatic prenyltransferase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 3, 9, 10, or 11 ; a tetrahydrocannabinolic acid synthase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 5; and a cannabidiolic acid synthase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 6.

[0064] In some embodiments, microorganisms for use in the present invention can be microorganisms capable of producing at least one cannabinoid biosynthetic pathway product. In some embodiments, the microorganism capable of producing at least one cannabinoid biosynthetic pathway product is cultured in a culture medium comprising a precursor or intermediate compound of the cannabinoid biosynthetic pathway. In such embodiments, the microorganism may convert the precursor of intermediate compound into another cannabinoid biosynthetic pathway product. It has been discovered that a microorganism transformed with an exogenous nucleic acid molecule that encodes tetraketide synthase and olivetolic acid cyclase produced cannabinoids in the absence of any other exogenous cannabinoid biosynthetic pathway enzymes (US 62/927,321). In an embodiment, the microorganism capable of producing at least one cannabinoid biosynthetic pathway product is cultured in a culture medium comprising a 2,4-dihydroxy- 6-alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.

[0065] In an embodiment, the microalga is from the genera Ankistrodesmus, Asteromonas, Auxenochlorella, Basichlamys, Botryococcus, Botryokoryne, Borodinella, Brachiomonas, Catena, Carteria, Chaetophora, Characiochloris, Characiosiphon, Chlainomonas, Chlamydomonas, Chlorella, Chlorochytrium, Chlorococcum, Chlorogonium, Chloromonas, Closteriopsis, Dictyochloropsis, Dunaliella, Ellipsoidon, Eremosphaera, Eudorina, Floydiella, Friedmania, Haematococcus, Hafniomonas, Heterochlorella, Gonium, Halosarcinochlamys, Koliella, Lobocharacium, Lobochlamys, Lobomonas, Lobosphaera, Lobosphaeropsis, Marvania, Monoraphidium, Myrmecia, Nannochloris, Oocystis, Oogamochlamys, Pabia, Pandorina, Parietochloris, Phacotus, Platydorina, Platymonas, Pleodorina, Polulichloris, Polytoma, Polytomella, Prasiola, Prasiolopsis, Prasiococcus, Prototheca, Pseudochlorella, Pseudocarteria, Pseudotrebouxia, Pteromonas, Pyrobotrys, Rosenvingiella, Scenedesmus, Spirogyra, Stephanosphaera, Tetrabaena, Tetraedron, Tetraselmis, Trebouxia, Trochisciopsis, Viridiella, Vitreochlamys, Volvox, Volvulina, Vulcanochloris, Watanabea, Yamagishiella, Euglena, Isochrysis, Nannochloropsis. In an embodiment, the microalga is Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta , or Heamatococus plucialis. In another embodiment, the microalga is a diatom, optionally Phaeodactylum tricornutum or Thalassiosira pseudonana. In another embodiment, the cyanobacterium is from Spirulinaceae, Phormidiaceae, Synechococcaceae, or Nostocaceae. In an embodiment, the cyanobacterium is Arthrospira plantesis , Arthrospira maxima, Synechococcus elongatus, or Aphanizomenon flos-aquae.

[0066] Microorganisms can be grown in organic conditions without the use of chemicals or additives that contravene the standards for organically-produced products. Microalgae can be grown organically, for example, by growing them in conditions that comply with jurisdictional standards such as the standards set 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 permits the production of organic plant natural products in microalgae.

[0067] Particular embodiments of the disclosure include, without limitation, the following:

1. A method of culturing a photosynthetic microorganism, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.

2. The method according to embodiment 1 , wherein the photosynthetic microorganism produces at least one biosynthetic product.

3. The method according to embodiment 2, wherein the at least one biosynthetic product is a cannabinoid biosynthetic pathway product.

4. The method according to embodiment 3, wherein the photosynthetic microorganism comprises at least one nucleic acid molecule encoding at least one cannabinoid biosynthetic pathway enzyme. 5. The method according to embodiment 4, wherein the at least one nucleic acid molecule encodes at least one of a hexanoyl-CoA synthetase, a type III polyketide synthase (e.g., tetraketide synthase, Steely 1 and Steely 2), an olivetolic acid cyclase, an aromatic prenyltransferase (e.g. CsPT1 , Orf2, CsPT4, and HIPT1), a tetrahydrocannabinolic acid synthase, or a cannabidiolic acid synthase.

6. The method according to embodiment 5, wherein the hexanoyl-CoA synthetase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 4.

7. The method according to any one of embodiments 5 to 6, wherein the type III polyketide synthase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 1 , 7, or 8.

8. The method according to any one of embodiments 5 to 7, wherein the olivetolic acid cyclase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 2.

9. The method according to any one of embodiments 5 to 8, wherein the aromatic prenyltransferase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 3, 9, 10, or 11.

10. The method according to any one of embodiments 5 to 9, wherein the tetrahydrocannabinolic acid synthase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 5.

11. The method according to any one of embodiments 5 to 10, wherein the cannabidiolic acid synthase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 6.

12. The method according any one of embodiments 3 to 11 , wherein the at least one cannabinoid biosynthetic pathway product is at least one of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, D9- tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol and cannabidiol.

13. The method according to any one of embodiments 1 to 12, wherein the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm in wavelength.

14. The method according to any one of embodiments 1 to 12, wherein the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 625 nm to about 740 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm in wavelength.

15. The method according to any one of embodiments 1 to 12, wherein the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 600 nm to about 700 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm in wavelength.

16. The method according to any one of embodiments 1 to 12, wherein the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 600 nm to about 640 nm; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm in wavelength.

17. The method according to any one of embodiments 1 to 12, wherein the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 680 nm to about 700 nm; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about 80%, about 75% to about 80%, about 65% to about 75%, about 70% to about 75% of light within about 590 nm to about 740 nm in wavelength.

18. The method according to any one of embodiments 1 to 17, wherein the second light spectrum consists essentially of light within about 400 nm to about 700 nm in wavelength. 19. The method according to any one of embodiments 1 to 17, wherein the second light spectrum consists essentially of solar light.

20. The method according to any one of embodiments 1 to 17, wherein the second light spectrum consists essentially of light within about 450 nm to about 485 nm in wavelength.

21. The method according to any one of embodiments 1 to 20, wherein the first period of time comprises a part of the exponential phase.

22. The method according to embodiment 21 , wherein the first period of time starts at the beginning of the exponential phase.

23. The method according to embodiment 21 , wherein the first period of time starts during or at the beginning of the lag phase.

24. The method according to embodiment 21 , wherein the first period of time consists of the exponential phase and the lag phase.

25. The method according to any one of embodiments 1 to 23, wherein the second period of time starts during the exponential phase.

26. The method according to any one of embodiments 1 to 23, wherein the second period of time starts at the beginning of the stationary phase.

27. The method according to any one of embodiments 1 to 26, wherein the photosynthetic microorganism undergoes autotrophic or mixotrophic growth.

28. The method according to any one of embodiments 1 to 27, which results in an increase in the biomass of the culture and/or an increase in the lipid content of the photosynthetic microorganism compared to a method comprising culturing the photosynthetic microorganism only under the first light spectrum.

29. The method according to any one of embodiments 1 to 28, which results in an increase in the production of medium-chain fatty acids and/or omega-3 fatty acids in the photosynthetic microorganism compared to the method comprising culturing the photosynthetic microorganism only under the first light spectrum.

30. The method according to any one of embodiments 1 to 29, which results in an increased yield of at least one biosynthetic product compared to the method comprising culturing the photosynthetic microorganism only under the first light spectrum. 31. The method according to any one of embodiments 1 to 30, wherein the photosynthetic microorganism is a photosynthetic microalga or cyanobacterium.

32. The method according to embodiment 31 , wherein the photosynthetic microorganism is a diatom.

33. The method according to embodiment 32, wherein the diatom is Phaeodactylum tricorn utum.

34. A method of culturing a photosynthetic microorganism capable of producing at least one cannabinoid biosynthetic pathway product in a culture medium, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6-alkylbenzoic acid or a 2,4-dihydroxy-6- alkylbenzoate.

35. The method according to embodiment 34, wherein the at least one cannabinoid biosynthetic pathway product is at least one of cannabigerolic acid, cannabigerol, D9- tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol, and cannabidiol.

36. The method according to any one of embodiments 34 to 35, wherein the first light spectrum comprises at least 65% of light within about 590 nm to about 740 nm in wavelength.

37. The method according to any one of embodiments 34 to 35, wherein the first light spectrum comprises at least 65% of light within about 625 nm to about 740 nm in wavelength.

38. The method according to any one of embodiments 34 to 35, wherein the first light spectrum comprises at least 65% of light within about 600 nm to about 700 nm in wavelength.

39. The method according to any one of embodiments 34 to 35, wherein the first light spectrum comprises at least 65% of light within about 600 nm to about 640 nm in wavelength. 40. The method according to any one of embodiments 34 to 35, wherein the first light spectrum comprises at least 65% of light within about 680 nm to about 700 nm in wavelength.

41. The method according to any one of embodiments 34 to 40, wherein the second light spectrum consists essentially of light within about 400 nm to about 700 nm in wavelength.

42. The method according to any one of embodiments 34 to 40, wherein the second light spectrum consists essentially of solar light.

43. The method according to any one of embodiments 34 to 40, wherein the second light spectrum consists essentially of light within about 450 nm to about 485 nm in wavelength.

44. The method according to any one of embodiments 34 to 43, wherein the first period of time comprises a part of the exponential phase.

45. The method according to embodiment 44, wherein the first period of time starts at the beginning of the exponential phase.

46. The method according to embodiment 44, wherein the first period of time starts during or at the beginning of the lag phase.

47. The method according to embodiment 44, wherein the first period of time consists of the exponential phase and the lag phase.

48. The method according to any one of embodiments 34 to 46, wherein the second period of time starts during the exponential phase.

49. The method according to any one of embodiments 34 to 46, wherein the second period of time starts at the beginning of the stationary phase.

50. The method according to any one of embodiments 34 to 49, wherein the photosynthetic microorganism undergoes autotrophic or mixotrophic growth.

51. The method according to any one of embodiments 34 to 50, which results in an increase in the biomass of the culture and/or an increase in the lipid content of the photosynthetic microorganism compared to a method comprising culturing the photosynthetic microorganism only under the first light spectrum.

52. The method according to any one of embodiments 34 to 51 , which results in an increase in the production of medium-chain fatty acids and/or omega-3 fatty acids in the photosynthetic microorganism compared to the method comprising culturing the photosynthetic microorganism only under the first light spectrum.

53. The method according to any one of embodiments 34 to 52, which results in an increased yield of the at least one cannabinoid biosynthetic pathway product compared to the method comprising culturing the photosynthetic microorganism only under the first light spectrum.

54. The method according to any one of embodiment 34 to 53, wherein the photosynthetic microorganism is a photosynthetic microalga or cyanobacterium.

55. The method according to embodiment 54, wherein the photosynthetic microorganism is a diatom.

56. The method according to embodiment 55, wherein the diatom is Phaeodactylum tricorn utum.

[0068] The invention will now be described by way of non-limiting examples having regard to the appended drawings.

[0069] EXAMPLE 1

[0070] METHODS

[0071] Microalgae strain and growth conditions

[0072] Axenic cultures of Phaetodactylum Tricornutum (Culture Collection of Algae and Protozoa CCAP 1055/1) were grown in L1 Media without silica (Artificial Sea Water) in 250 mL Erlenmeyer flasks (50 mL culture volume) for analysis of biomass and lipids or in flat-bottom 6 well culture plates (5 mL culture colume) for analysis of cell growth. P.tricornutum cultures were grown at 18±1 °C and maintained under a continuous light- dark cycle of 16/8 hours using white LED light (F54T5/841) kept 60cm above the bottom of the culture flask. Coloured light (red light and yellow light) was provided by covering the flasks with coloured cellophane. Experiments were conducted in a CMP6050 chamber with a light intensity of 75 pMOL nr ² s ^_1 , humidity of 50%, and shaker speed of 130 rpm.

[0073] Experimental Setup

[0074] The study was divided into two series of experiments. The first set of experiments examined the impact of red light (RL), yellow light (YL) and white light (WL) in both mixotrophic and autotrophic conditions under growth conditions explained above. P.tricornutum was cultivated in mixotrophic conditions and autotrophic conditions in the three different light conditions (Table 2).

[0075] Table 2

[0076] Autotrophic conditions with white light (WL) or with coloured cellophane sheets to provide coloured light (YL or RL): the P.tricornutum culture was grown in 50 mL cultures in triplicates under one of three different light conditions in standard L1 media.

[0077] Mixotrophic conditions with or without coloured cellophane sheets (WL, YL, or RL): The P.tricornutum culture was grown in 50 mL cultures in triplicates under one of three different light conditions in L1 media supplemented with 1% Glucose and 1% Glycine.

[0078] In the second set of experiments P.tricornutum was grown under RL in autotrophic conditions during a specific growth phase (i.e. lag phase, exponential phase, and/or stationary phase) and cultures were exposed to white light when not growing under RL (Table 3).

[0079] Table 3

[0080] Cell Growth and Biomass Analysis

[0081] The cultures were subjected to consistent shaking in orbital shaker at 130 rpm. Cell growth was monitored by measuring the optical density at 680 nm by Synergy Microplate Reader, BioteK on every second day. All the conditions were studied in triplicates. [0082] The biomass studied for the initial conditions was collected after centrifugation of algal cultures at 7000 g for 10 min on the 10th day. The supernatant was discarded and pellet was dried at 60 °C for biomass analysis by measurement on a scale.. The selected conditions were studied for further experimentation.

[0083] Lipid Analysis

[0084] Intracellular lipid bodies were visualized via modified Nile Red (9- diethylamino-5H-benzo[a]-phenoxazine-5-one) staining. Briefly, 1 mL of the algal culture was centrifuged at 12,000 rpm for 10 minutes. The pellet was resuspended in 500 uL of 20% Dimethyl sulfoxide (DMSO) and vortexed for 1 minute at room temperature. Cells were centrifuged at 12,000 rpm for 5 minutes. The pellet was suspended in 500 uL of water and vortexed before adding Nile Red (250 uL of 0.5 mg/mL dissolved in acetone) and incubated for 5 minutes in the dark at room temperature. Stained cells were visualized under a fluorescent microscope using UV light with excitation and emission at 485 nm and 552 nm, respectively.

[0085] Total lipids were extracted using Bligh and Dyer method (Bligh and Dyer 1959) with some modifications. To a 5 mL glass vial containing a known amount of algal biomass, mixtures of methanol and chloroform was added in 2:1 ratio. The mixture was vortexed for 2 minutes and incubated at room temperature for 24 hours. 1 mL of chloroform and 0.9 mL of water was added. The mixture was vortexed for 2 minutes and the different layers were separated by centrifugation for 10 minutes at 2000 rpm. Evaporation was carried out in safe chamber, and the residue was dried at 80 °C for 30 minutes. The weight of the vial was recorded (W2). The lipid content was calculated by subtracting W1 from W2.

[0086] Nile red assay was performed on 96 well plates containing 250 uL of sample with 15 uL of Nile red dissolved in acetone from the stock solution of 0.5 mg/mL. The fluorescence intensity was measured at excitation of 530 nm and emission of 590 nm using Synergy Microplate Reader, BioteK.

[0087] Lipid profiling

[0088] Lipids were extracted for the preparation of FA methyl esters (FAMEs) following standard protocols as previously described (Budge et al 2006). Each sample was homogenized, and lipids were extracted using a mixture of chloroform/methanol (2:1). FAMEs were prepared through acidic transesterification using sulfuric acid in methanol and quantified using temperature-programmed gas liquid chromatography on a Perkin Elmer Autosystem II Capillary FID gas chromatograph fitted with a 30 m x 0.25 mm FID column coated with 50 % cyanopropyl-methylpolysiloxane (DB-23) and linked to a computerized integration system (Varian Star software).

[0089] RESULTS

[0090] The first series of experiments analyzed the impact of light on cell growth, biomass, and lipids in P.tricornutum in mixotrophic and autotrophic conditions under different light colours (red light, yellow light, white light): Biomass (Figure 2) and lipid production (Figure 3). The maximum biomass productivity of 72 mg/50mL was observed for culture under red light with 1% glycine, which was 1.16 and 1.05-fold higher as compared to white light mixotrophic culture and yellow light mixotrophic culture. Red light proved to be an effective light colour for increasing biomass under both mixotrophic and autotrophic conditions.

[0091] The second series of experiments analyzed the impact of variable light conditions on cell growth, total biomass, and lipids. The impact of shifting from red light to white light during different growth phases (Table 2) was compared with white light, yellow light and red light. Cell growth in these conditions were analyzed at days 1-10 of culture (Figure 4).

[0092] The growth curve under RS treatment (red light during lag and exponential phases) compared to white light (C), complete red light (R), red light during lag phase (RT1), red light during exponential phase (RT2), and red light during stationary phase (RT3) conditions are shown in Figure 5. P.tricornutum cultures quickly adapt to both white light and red light but the growth slightly slowed down in exponential phase in red light as compared to white light. The RS treatment increased growth and slowed death phase compared to all other conditions. The growth trend under pure red light and white light is quite similar until the exponential phase but the death rate or depletion of the stationary phase is faster under white light as compared to red light.

[0093] The biomass under red light (R) increased 1.3-1.4 fold as compared to the biomass under white light (C) (Figure 6). An increase of 2.5 fold in biomass was noted in the RS culture (red light during lag and exponential phases and white light in stationary phase) as compared to the culture grown under white light (C). The maximum biomass of 100 mg/50mL in the culture RS was 1-2.5 fold higher as compared to other tested conditions. [0094] The total lipids were analysed using dry weight estimation of neutral lipids, Nile red assay for screening, and confocal microscopy for visual analysis. Dry weight estimation revealed that total lipid increased 2.3-fold and 1.8-fold in RS as compared to white light (C) and red light (R)(Figure 7). These results were confirmed by Nile Red Assay for quick screening (Table 4). [0095] Table 4

[0096] No significant change in the size or morphology of lipid droplets under white light or red light conditions were observed (Figure 8).

[0097] The fatty acid methyl ester analysis revealed that: saturated fatty acids (SFA) as a percent of total lipids was 24% in red light (R) and red light shifting to white light (red light during lag and exponential phases and white light in stationary phase, RS) and 26% in white light (C); monounsaturated fatty acids (MUFA) as a percent of total lipids was comparable in C and R conditions at 31% but was lower in RS conditions at 28%; and polyunsaturated fatty acids as a percent of total lipids was 27%, 26%, and 28% in C, R, and RS conditions, respectively. The remaining fraction of total lipids was composed of non-neutral lipids. Individual fatty acid species and their quantity as a percent of total lipids in each condition is shown in Table 5:

[0098] Table 5

[0099] The percent quantities of all three omega-3 fatty acids, eicosapentaenoic acid (C20:5), docosahexaenoic acid (C22:6), and linolenic acid (C18:3), were increased in the RS condition compared to the R condition. For both eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22:6), although the R condition decreased the percent quantity compared to the C condition, the percent quantity of each fatty acid was recovered in the RS condition.

[00100] The R condition also decreased the percent quantity of myristic acid (C14:0), a medium-chain fatty acid, compared to the C condition, but the percent quantity of this fatty acid is recovered in the RS condition. Medium-chain fatty acids (i.e. , fatty acids containing between 6 and 14 carbons (C:6 - C: 14)) may improve the solubility of cannabinoids in the lipid bodies of P.tricornutum.

[00101] The properties and structural features (chain length, unsaturation and branching) of fatty acids determine the properties of the total fatty acid fraction. The overall fatty acid composition was analyzed using Biodiesel Analyzer (Talebi et al 2014) and parameters of the fatty acid fraction from the different conditions were compared to those of Biodiesel standards EN 14214:2008 and ASTM D6751 (Table 6).

[00102] Table 6

[00103] EXAMPLE 2

[00104] Autotrophic cultures of P.tricornutum were prepared as described above. The cultures were grown under white light (C), blue light (B), yellow light (Y), or red light (r), and compared to conditions in which the blue light (Bs), yellow light (Ys), or red light (rs) was switched to white light at the stationary phase.

[00105] Figure 9 shows the transmittance spectrum of the red light filter used to provide the red light for conditions (r) and (rs). As shown in Figure 10, switching to white light at the stationary phase after culturing cells under blue light (Bs) or yellow light (Ys) during the lag and exponential phases did not improve biomass yield, whereas an improvement was seen in cells cultured under red light (rs) before switching to white light at the stationary phase.

[00106] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[00107] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00108] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude 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 the stated features, elements, components, groups, integers, and/or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and/or steps.

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