Engineered Photosynthetic Organisms

Abstract

The present invention provides unicellular photosynthetic organisms that have been engineered to express fatty acid transporters in their cytoplasmic membranes and are capable of transporting fatty acids into the culture medium in which they are grown. These engineered organisms can be used to produce large amounts of fatty acids on a large scale without the need for an energy-intensive destruction step to harvest the fatty acids. The present invention also relates to nucleic acid constructs for producing such engineered organisms. The present invention further provides a method for culturing these organisms in a medium suitable for the growth of such organisms to produce fatty acids.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to UK Patent Application No. 2205111.4, filed April 7, 2022, the disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This specification refers to a sequence listing submitted electronically on April 6, 2023 with the .xml filename "2023-03-27 P50270WO Sequence Listing." The .xml file was created on March 27, 2023 and is 40KB in size. The entire contents of the sequence listing are incorporated herein by reference.

[0003] The present invention relates to engineered unicellular photosynthetic organisms, such as microalgae, that can be used to produce large amounts of fatty acids on a large scale. These organisms may have applications in the energy-efficient production of microalgae-derived biofuels. [Background technology]

[0004] Microalgae are single-celled photosynthetic organisms that live in water bodies. They are one of the most efficient photosynthesizers, and can be 400 times more efficient than trees. Moreover, they are responsible for 50% of all oxygen production each year. Microalgae cultivation does not require arable land, so a simple dug pond on wasteland is practically sufficient.

[0005] Since the early 2000s, there has been a boom in research into microalgae fuels, especially with the support of a vibrant investment environment. Large-scale farms have been established, infrastructure has been built, and even prototype fuels have been provided to the US Air Force with good results. However, as the wars in the Middle East subsided and crude oil prices stabilized, the high costs associated with microalgae fuels made the technology economically unviable.

[0006] The main factor that has prevented the success of microalgae fuels at large scale is the cost of processing. Microalgae grow on sunlight and CO2 for millions of years. 2 Microalgae have evolved to make and store oil very well from algae, but extracting the oil is difficult and expensive. The cost of the energy required to collect microalgae cells, remove water from the cells, cut them open, and separate the useful material from the cell debris greatly overshadows the revenues derived from the product, especially when the competition is crude oil (Patel et al. (2020) An Overview of Potential Oleaginous Microorganisms and Their Role in Biodiesel and Omega-3 Fatty Acid-Based Industries. Microorganisms, 8(3), 434). Processing can account for up to 70% of the final cost of the fuel. Many major microalgae fuel companies, including Algenol, Sapphire Energy, and Solazyme (later TerraVia, a subsidiary of Dutch food and biochemical company Corbion NV), have gone bankrupt or transitioned to producing products with much higher retail prices. Other types of advanced biofuels involve the cultivation of non-crop grasses and their subsequent enzymatic conversion to fuel or thermal liquefaction, but their development has not progressed beyond the experimental stage and their potential has never become economically feasible.

[0007] Therefore, there is a need to provide a means for producing biofuels that is less energy and resource intensive and easily scalable so as to be economically viable. Summary of the Invention

[0008] The present invention is based on the discovery that unicellular photosynthetic organisms can be engineered to express fatty acid transporters. The inventors have demonstrated that these engineered organisms can secrete fatty acids into the culture medium in which they are grown. This avoids energy-intensive destruction steps to harvest fatty acids, as they can be easily extracted from the medium or the cultured organism. Using established techniques, the extracted fatty acids can be converted into biofuels via transesterification, decarboxylation, or hydrogenolysis.

[0009] In particular, the present invention relates to an engineered unicellular photosynthetic organism comprising an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence, wherein, according to the present invention, the fatty acid transporter is localized to the cytoplasmic membrane of the organism upon expression.

[0010] In some embodiments, the fatty acid transporter is capable of transporting fatty acids having carbon chain lengths of C6 to C22 from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting fatty acids having carbon chain lengths of C14 to C22 from the interior of a cell through the cytoplasmic membrane of an organism.

[0011] In some embodiments, the fatty acid transporter is capable of transporting saturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting polyunsaturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting monounsaturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism.

[0012] In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 to C18 from the inside of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 and / or C18 from the inside of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acids having a carbon chain length of C16 and / or C18 are monounsaturated. In some embodiments, the fatty acid transporter is capable of transporting palmitoleic acid and / or oleic acid from the inside of a cell through the cytoplasmic membrane of an organism.

[0013] In some embodiments, the fatty acid transporter is capable of transporting a fatty acid having a carbon chain length of C18 from the interior of a cell through the cytoplasmic membrane of the organism. In some embodiments, the fatty acid transporter is capable of transporting oleic acid from the interior of a cell through the cytoplasmic membrane of the organism.

[0014] In some embodiments, the coding sequence of the fatty acid transporter is derived from the genome of a plant cell or a mammalian cell. In certain embodiments, the fatty acid transporter is derived from the genome of a plant cell. In a specific embodiment, the fatty acid transporter is an ABC transporter. For example, the ABC transporter can be the Arabidopsis thaliana ABCG11 protein (also known as WBC11) or a functional homolog thereof. Alternatively, the ABC transporter can be the Oryza sativa ABCG15 protein or a functional homolog thereof.

[0015] In further specific embodiments, the fatty acid transporter coding sequence is derived from the genome of a mammalian cell. In specific embodiments, the fatty acid transporter is an ABC transporter or flippase. For example, the flippase can be Homo sapiens FATP1 protein or a functional homolog thereof.

[0016] The promoter sequence may be derived from the genome of a different organism than the genome from which the coding sequence is derived. In some embodiments, the promoter sequence is exogenous to the engineered organism. In other embodiments, the promoter sequence is endogenous to the engineered organism. For example, the promoter may be selected from the group consisting of cauliflower mosaic virus (CaMV) 35S promoter, nitrogen reductase (NR) promoter, photosystem I reaction center subunit II (PSAD) promoter, and HSP70A-RBCS2 (AR) promoter.

[0017] In some embodiments, the coding sequence is codon optimized for expression in an engineered organism, hi some embodiments, the coding sequence comprises one or more introns.

[0018] In some embodiments, the exogenous nucleic acid sequence further comprises a selectable marker. In specific embodiments, the selectable marker confers antibiotic resistance.

[0019] In some embodiments, the exogenous nucleic acid sequence further comprises a terminator sequence operably linked to the coding sequence. In some embodiments, the terminator sequence is derived from a genome of an organism different from the genome from which the coding sequence and / or promoter sequence is derived. In some embodiments, the terminator sequence is exogenous to the engineered organism. In other embodiments, the terminator sequence is endogenous to the engineered organism. In certain embodiments, the terminator sequence encodes the Agrobacterium tumefaciens nopaline synthase (NOS) terminator.

[0020] In some embodiments, the unicellular photosynthetic organism is an algae. In some embodiments, the algae is an oleaginous algae. In some embodiments, the unicellular photosynthetic organism may be grown in freshwater. In some embodiments, the unicellular photosynthetic organism may be grown in seawater. In some embodiments, the unicellular photosynthetic organism is an algae selected from Chlorophyta, Phaeophyta, Rhodophyta, Xanthophyta, Chrysophyta, Bacillariophyta, Cryptophyta, Dinophyta, Euglenophyta, Cyanophyta, and Myxophyta. In some embodiments, the unicellular photosynthetic organism is a member of the genera Chlorella, Chlamydomonas, Dunaliella, Haematococcus, Phaeodactylum, Tetraselmis, Isochrysis, Diacronena, Schizochytrium, Thraustochytrium, Nannochloris, Nannochloropsis, or the like. In some embodiments, the unicellular photosynthetic organism is selected from the group consisting of Chlorella sorokiniana, Chlorella serotinus, Chlorella nigricans ...sorokiniana, Chlorella vulgaris, Chlorella protothecoides, Chlamydomonas reinhardtii, Dunaliella salina, Dunaliella tertiolecta, Dunaliella sp., Haematococcus pluvialis, Phaeodactylum tricornutum, Tetraselmis suecica, Tetraselmis chuii, Isochrysis galbana, Diacronema borchianum volkianum, Schizochytrium sp., Thraustochytrium sp., Nannochloris sp., Nannochloropsis sp., Nannochloropsis gaditana, Porphyridium sp., Nanofrustulum sp., Cryptheiconidium cohnii, Scenedesmus sp., Euglena gracilis, Tetraselmis elliptica, Auxenochlorella protothecoides protothecoides, Botryococcus braunii, Chlorella minutissima, Nannochloropsis salina, Alexandrium sanguineasanguinea, Fistulifera solaris, and Nitzschia laevis.

[0021] The present invention also relates to a nucleic acid comprising a coding sequence of a fatty acid transporter operably linked to a promoter sequence. According to the present invention, the nucleic acid is suitable for inducing the expression of such a fatty acid transporter in a unicellular photosynthetic organism that does not naturally contain a fatty acid transporter in its cytoplasmic membrane (i.e., the coding sequence of the fatty acid transporter is exogenous to the unicellular photosynthetic organism). In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C6 to C22 from the inside of the cell through the cytoplasmic membrane of the organism. In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C14 to C22 from the inside of the cell through the cytoplasmic membrane of the organism.

[0022] In some embodiments, the fatty acid transporter is capable of transporting saturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting polyunsaturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting monounsaturated fatty acids from the interior of a cell through the cytoplasmic membrane of an organism.

[0023] In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 to C18 from the inside of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 and / or C18 from the inside of a cell through the cytoplasmic membrane of an organism. In some embodiments, the fatty acids having a carbon chain length of C16 and / or C18 are monounsaturated. In some embodiments, the fatty acid transporter is capable of transporting palmitoleic acid and / or oleic acid from the inside of a cell through the cytoplasmic membrane of an organism.

[0024] In some embodiments, the fatty acid transporter is capable of transporting a fatty acid having a carbon chain length of C18 from the interior of a cell through the cytoplasmic membrane of the organism. In some embodiments, the fatty acid transporter is capable of transporting oleic acid from the interior of a cell through the cytoplasmic membrane of the organism.

[0025] In some embodiments, the coding sequence of the fatty acid transporter is derived from the genome of a plant cell or a mammalian cell. In certain embodiments, the coding sequence of the fatty acid transporter is derived from the genome of a plant cell. In specific embodiments, the coding sequence of the fatty acid transporter encodes an ATP-binding cassette (ABC) transporter. For example, the ABC transporter can be the Arabidopsis thaliana ABCG11 protein (also known as WBC11) or a functional homolog thereof. Alternatively, the ABC transporter can be the Oryza sativa ABCG15 protein or a functional homolog thereof.

[0026] In some embodiments, the fatty acid transporter coding sequence is derived from the genome of a mammalian cell. In some embodiments, the fatty acid transporter coding sequence encodes an ABC transporter or a flippase. In certain embodiments, the fatty acid transporter coding sequence encodes a Homo sapiens flippase FATP1 protein or a functional homolog thereof.

[0027] In some embodiments, the promoter sequence is derived from a genome of an organism different from the genome from which the coding sequence is derived. In some embodiments, the promoter is selected from the group consisting of cauliflower mosaic virus (CaMV) 35S promoter, nitrogen reductase (NR) promoter, photosystem I reaction center subunit II (PSAD) promoter, and HSP70A-RBCS2 (AR) promoter. In some embodiments, the promoter is derived from an organism different from the fatty acid transporter.

[0028] In some embodiments, the fatty acid transporter coding sequence is codon optimized. In some embodiments, the coding sequence further comprises one or more introns.

[0029] In some embodiments, the nucleic acid further comprises a selectable marker. In some embodiments, the selectable marker confers antibiotic resistance.

[0030] In some embodiments, the nucleic acid further comprises a terminator sequence operably linked to the coding sequence. In some embodiments, the terminator sequence is derived from the genome of a different organism than the genome from which the coding sequence and / or promoter sequence are derived. In some embodiments, the terminator sequence is derived from the genome of a different organism than the genome from which the coding sequence is derived. In some embodiments, the terminator sequence is derived from the genome of a different organism than the genome from which the promoter sequence is derived. In some embodiments, the terminator sequence is derived from the genome of a different organism than the genome from which the coding sequence and promoter sequence are derived. In some embodiments, the terminator is an Agrobacterium tumefaciens nopaline synthase (NOS) terminator.

[0031] The present invention also relates to cultures comprising the engineered unicellular photosynthetic organisms of the present invention.

[0032] The present invention also relates to a method for producing fatty acids, comprising culturing an engineered unicellular photosynthetic organism of the present invention in a medium suitable for the growth of the organism. In some embodiments, the culturing step is continuous at a steady state. In some embodiments, the method further comprises separating the medium from the organism. In some embodiments, the separating step comprises sedimentation or filtration. In some embodiments, the sedimentation involves centrifugation. In some embodiments, the sedimentation involves incubating the medium for a period of time without stirring. In some embodiments, the method further comprises extracting fatty acids from the abiotic medium obtained by sedimentation or filtration using a liquid-liquid extraction process. In some embodiments, the method further comprises scooping fatty acid-containing droplets from the surface of the abiotic culture medium obtained by sedimentation or filtration to extract the fatty acids. In some embodiments, the method further comprises processing the extracted fatty acids by transesterification. In some embodiments, the method further comprises processing the extracted fatty acids by decarboxylation. In some embodiments, the method further comprises processing the extracted fatty acids by hydrogenolysis.

[0033] In some embodiments, the method for producing fatty acids according to the invention produces at least 1 μg of fatty acid per liter of culture medium per day. In some embodiments, the method for producing fatty acids according to the invention produces at least 10 μg of fatty acid per liter of culture medium per day. In some embodiments, the method for producing fatty acids according to the invention produces at least 100 μg of fatty acid per liter of culture medium per day. In some embodiments, the method for producing fatty acids according to the invention produces at least 1 g of fatty acid per liter of culture medium per day.

[0034] The present invention also relates to a culture medium inoculated with an engineered unicellular organism of the present invention and incubated for a sufficient period of time to obtain at least 1 μg fatty acid per liter of culture medium. In some embodiments, the culture medium has been incubated for a sufficient period of time to obtain at least 10 μg fatty acid per liter of culture medium. In some embodiments, the culture medium has been incubated for a sufficient period of time to obtain at least 100 μg fatty acid per liter of culture medium. In some embodiments, the culture medium has been incubated for a sufficient period of time to obtain at least 1 g fatty acid per liter of culture medium. In some embodiments, the culture medium does not contain the organism used for inoculation.

[0035] The present invention also relates to a method of extracting fatty acids from a unicellular photosynthetic organism of the present invention, comprising the steps of (i) providing a culture medium inoculated with an engineered unicellular organism of the present invention and incubated for a period of time sufficient to obtain at least 1 μg fatty acids per liter of culture medium, and (ii) extracting the fatty acids. In some embodiments, the extraction step comprises a liquid-liquid extraction process. In some embodiments, the extraction step comprises spooning droplets containing the fatty acids from the surface of the culture medium.

[0036] The present invention also relates to a method for producing a biofuel, comprising the steps of providing fatty acids obtained by one of the methods described in the previous paragraphs, and processing the fatty acids by transesterification, decarboxylation, or hydrocracking. The present invention also relates to a biofuel obtained by such a method.

[0037] Embodiments of the invention will now be described, by way of example, with reference to the following drawings, in which: [Brief description of the drawings]

[0038] [Figure 1-1]Figure 1 shows a phylogenetic tree identifying homologs of representative fatty acid transporters suitable for use with the present invention. Figure 1a shows a representative predicted functional homolog of Arabidopsis thaliana ABC transporter ABCG11. Figure 1b shows a representative functional homolog of Oryza sativa subsp. japonica ABC transporter ABCG15. The homologs shown typically have at least 80% sequence identity to ABCG11 and ABCG15, respectively. [Figure 1-2] (As stated above.) [Diagram 2] FIG. 1 shows a phylogenetic tree identifying representative functional homologs of human flippase FATP1 (NP_940982.1) suitable for use with the present invention. The homologs in the animalia and fungal phyla shown have at least 90% and at least 25% sequence identity to human FATP1, respectively. [Diagram 3] FIG. 1 is a schematic representation of an exemplary nucleic acid comprising a coding sequence for a fatty acid transporter suitable for use with the present invention. The nucleic acid comprises a selection marker (aadA) operably linked to a promoter sequence and a terminator sequence for β-tubulin. Nucleic acid (A) does not have a coding sequence for a fatty acid transporter. It is used as a mock control. Nucleic acids (B)-(G) comprise coding sequences for a fatty acid transporter (B,E: FATP1; C,F: ABCG11; D,G: ABCG15). The coding sequence for the fatty acid transporter is located 3' from the selection marker. It is operably linked to a promoter sequence (CaMV35S) at the 5' end and a terminator sequence (NOS) at the 3' end. Nucleic acids (E)-(G) further comprise a sequence encoding a fluorescent marker protein (Clover) between the coding sequence for the fatty acid transporter and the terminator sequence. The fluorescent marker protein is expressed together with the fatty acid transporter as a fusion protein upon successful transformation of a target organism of interest. The locations of introns within the fatty acid transporter coding sequence and the fluorescent marker protein coding sequence are also indicated. [Figure 4] Figure 4 shows confirmation by PCR of the insertion of an exogenous nucleic acid sequence, including a coding sequence for a fatty acid transporter, into the genome of C. reinhardtii, a unicellular organism suitable for use with the present invention. Figure 4 shows the results of gel electrophoresis of a representative clone. A 567 bp fragment of the selection marker sequence (aadA) was PCR amplified from genomic DNA obtained from transformed C. reinhardtii. Untransformed wild-type cells were used as control. Lanes 1: wild type (untransformed), 2: cells transformed with only the antibiotic cassette, 3-4: FATP1, 5-6: FATP1-clover, 7-8: ABCG11, 9-10: ABCG11-clover, 11-12: ABCG15, 13-14: ABCG15-clover. The detailed gene construct is shown in Figure 3. [Figure 5-1] Immunofluorescence assay images of C. reinhardtii transformed with an exogenous fatty acid transporter fused to the clover fluorescent protein. Figure 5a shows C. reinhardtii transformed only with nucleic acid encoding an antibiotic selection marker (mock control). In Figure 5a, only background fluorescence is visible. Figure 5b shows C. reinhardtii transformed with nucleic acid encoding an ABCG15-clover fusion protein. Clusters of fluorescent cells can be clearly seen in Figure 5b, with the fluorescent staining being dense at the cytoplasmic membrane (scale bar: 20 μm). [Figure 5-2] (As stated above.) [Figure 6]FIG. 1 shows the results of a BODIPY staining assay showing extracellular oil deposits from C. reinhardtii transformed with nucleic acid encoding an exogenous fatty acid transporter according to the invention. BODIPY is a stain for oil and other non-polar lipids. Panels a, c, e, and g are bright field images. Panels b, d, f, and h are fluorescent images of the same fields. Panels a and b show C. reinhardtii transformed with only nucleic acid encoding an antibiotic selection marker (mock control). No extracellular oil deposits are seen in panel b. Panels c and d show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter FATP1. Panels e and f show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter ABCG11. Panels g and h show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter ABCG15. Extracellular oil deposits are clearly visible in panels d, f, and h (see arrows). [Figure 7-1]FIG. 7 shows an exemplary liquid chromatography-mass spectrometry (LC-MS) analysis of fatty acids present in culture media containing C. reinhardtii cells. Sample A corresponds to a C. reinhardtii clone expressing a fatty acid transporter after transformation with an expression cassette containing the human gene SLC27A1 (encoding FATP1) and an antibiotic resistance gene. Sample B corresponds to a clone transformed with only the antibiotic resistance gene (i.e., not expressing SLC27A1, mock control). FIG. 7a shows the detection of palmitoleic acid (C16:1) in culture media recovered from these C. reinhardtii clones. FIG. 7b shows the detection of oleic acid (C18:1) in culture media recovered from these C. reinhardtii clones. Data are visualized in box plots. The SLC27A1-expressing C. reinhardtii clones in sample A secreted significantly greater amounts of the monounsaturated fatty acids C16:1 and C18:1 into the culture medium than the clones not expressing SLC27A1 in sample B. [Figure 7-2] (As stated above.) [Figure 8] Figure 2. Successful expression of fatty acid ABC transporter ABCG11 in the microalga C. sorokiniana (strain SAG211-32). Western blot of C. sorokiniana lysate was probed with anti-His tag antibody. Lane "L" shows the molecular weight ladder. Lane "1" contains lysate of wild type cells not expressing the ABCG11 transporter (negative control). Lane "2" contains lysate of ABCG11 expressing cells. Lane "3" contains His tagged protein used as positive control. Expression of the ABCG11 transgene is shown in lane 2 by a protein band at approximately 118 kDa corresponding to ABCG11+6His tag. [Figure 9-1]Transformation of the microalga Chlorella sorokiniana (strain SAG211-32) with exogenous nucleic acids encoding various fatty acid transporters. Cells were transformed with nucleic acids encoding human flippase (FATP1) or plant ABC transporters from rice (Oryza sativa) (ABCG15) or Arabidopsis thaliana (ABCG11), each fused to a His tag for ease of detection. To select for transformants, cells were plated on agar-TAP plates in the presence of G418. An illustrative set of plates is shown in FIG. 9a. Transformants were picked, grown in culture medium in the presence of G418, lysed, and screened for expression of fatty acid transporters by Western blot. Western blots were probed with anti-His tag antibodies to detect expression of the transgenes. FIG. 9b and FIG. 9c are representative examples of Western blots performed as part of the screening. Figure 9b shows the detection of His-tags in lysates of cells transformed with transgenes encoding FATP1 (lanes 1-8) and ABCG11 (lanes 9-11), respectively. Figure 9c shows the detection of His-tags in lysates of cells transformed with transgenes encoding ABCG11 (lanes 1-4) and ABCG15 (lanes 5-11), respectively. Not all clones successfully expressed the transgenes. Samples in which successful transgene expression was detected are indicated by black rectangles. Lane "L" shows the molecular weight ladder. In both panels, lane 12 contains lysate of wild-type cells (negative control) and lane 13 contains His-tagged protein (positive control). [Figure 9-2] (As stated above.) [Figure 10-1]Secretion of fatty acids into the culture medium by C. sorokiniana cells expressing various fatty acid transporters. Figure 10a shows that cells were successfully transformed with exogenous nucleic acids encoding the human flippase FATP1, the ABC transporter ABCG15 from rice (Oryza sativa), or the ABC transporter ABCG11 from Arabidopsis thaliana (ABCG11), respectively. Anti-His tag antibody was used to detect bands whose molecular weights corresponded to FAPT1 (lanes 1-4), ABCG11 (lanes 5-8), and ABCG15 (lanes 9-12), respectively. Lane 13 contains His-tagged protein (positive control). Lane "L" shows the molecular weight ladder. [Figure 10-2] (As stated above.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Liquid cultures (in TAP medium) were prepared with one of the transformants from each set (ABCG15, FATP1, and ABCG11). Selected transformants are indicated by black rectangles in FIG. 10a. Cultures with corresponding wild-type cells were incubated under identical conditions. Culture medium was sampled to examine for the presence of free fatty acids, normalized by cell density to facilitate comparison. FIG. 10b shows the normalized free fatty acid concentrations in the culture medium after 48 hours of incubation with cells expressing the indicated fatty acid transporters. All fatty acid transporter-expressing cells secreted significantly greater amounts of fatty acids into the culture medium than wild-type (WT) control cells.

[0040] definition In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms, as well as other terms, are set forth throughout the specification.

[0041] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0042] Unless specifically stated or apparent from the context, as used herein, the term "or" is understood to be inclusive and cover both "or" and "and."

[0043] As used herein, the term "unicellular photosynthetic organism" refers to any unicellular organism that can convert light energy into chemical energy. Examples of photosynthetic organisms include cyanobacteria, plants, and algae.

[0044] As used herein, the term "fatty acid transporter" means any protein capable of transporting one or more fatty acids across a lipid bilayer membrane.

[0045] As used herein, the terms "transport" and "transporting" refer to the movement of a molecule from one side of a lipid bilayer membrane to the other.

[0046] The terms "secrete" and "secreting" are used interchangeably herein with the terms "transport" and "transporting."

[0047] As used herein, the term "operably linked" means that a sequence exerts its function on the coding sequence to which it is linked. For example, an operably linked promoter can drive expression of the coding sequence to which it is operably linked. An operably linked terminator can terminate transcription of the coding sequence to which it is operably linked.

[0048] As used herein, the term "target organism" refers to a unicellular photosynthetic organism suitable for use with the present invention that can be transformed with a nucleic acid containing a coding sequence for a fatty acid transporter described herein.

[0049] As used herein, the term "ATP-binding cassette transporter" (ABC transporter) refers to any member of the superfamily of ABC transport systems. In certain embodiments, the term is used herein to describe eukaryotic ABC transporters. Typically, ABC transporters couple the hydrolysis of ATP to the translocation of a substrate across a biological membrane.

[0050] As used herein, the term "flippase" refers to proteins that are part of the subfamily of P-type ATPases. Flippases typically act as transmembrane lipid transporter proteins.

[0051] As used herein, the term "functional homolog" refers to a homologous protein that can have the same function as a reference protein.

[0052] All technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs. Publications and other reference materials referenced herein to describe the background of the invention and to provide further details regarding the practice of the invention are incorporated herein by reference.

[0053] Detailed Description of the Invention The present invention relates to unicellular photosynthetic organisms that have been engineered to express fatty acid transporters in their cytoplasmic membrane and are capable of transporting fatty acids into the culture medium in which they are grown.

[0054] In particular, the present invention relates to an engineered unicellular photosynthetic organism comprising an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence. The engineered organism of the present invention comprising the exogenous nucleic acid is capable of transporting a greater amount of fatty acid across its cytoplasmic membrane than an otherwise identical organism lacking the exogenous nucleic acid cultured under identical conditions. Typically, a culture medium comprising an engineered unicellular photosynthetic organism comprising the exogenous nucleic acid according to the present invention comprises at least a three-fold higher concentration of fatty acid than a corresponding culture medium comprising an otherwise identical organism lacking the exogenous nucleic acid cultured under identical conditions. In some embodiments, the fatty acid concentration in a culture medium comprising an engineered unicellular photosynthetic organism comprising an exogenous nucleic acid in accordance with the invention is at least 5 times higher, at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher, than a corresponding culture medium comprising an identical organism but lacking the exogenous nucleic acid cultured under identical conditions.

[0055] The present invention also relates to a culture medium conditioned by an engineered unicellular photosynthetic organism according to the present invention that is enriched in monounsaturated fatty acids having carbon chain lengths of C16 to C18 compared to a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions. In some embodiments, the culture medium conditioned by an engineered unicellular photosynthetic organism according to the present invention contains at least about 5 times (e.g., at least about 10 times) more palmitoleic acid (C16:1) than a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions. In some embodiments, the culture medium conditioned by an engineered unicellular photosynthetic organism according to the present invention contains at least about 1.5 times (e.g., at least about 3 times or at least about 5 times) more oleic acid (C18:1) than a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0056] The present invention also relates to cell-free fatty acid compositions secreted by engineered unicellular photosynthetic organisms according to the invention that are enriched in monounsaturated fatty acids having carbon chain lengths of C16 to C18 compared to compositions secreted by corresponding wild-type unicellular photosynthetic organisms under identical conditions. In some embodiments, the cell-free fatty acid compositions secreted by engineered unicellular photosynthetic organisms according to the invention contain at least about 5-fold (e.g., at least about 10-fold) more palmitoleic acid (C16:1) than compositions secreted by corresponding wild-type unicellular photosynthetic organisms under identical conditions. In some embodiments, the cell-free fatty acid compositions secreted by engineered unicellular photosynthetic organisms according to the invention contain at least about 1.5-fold (e.g., at least about 3-fold or at least about 5-fold) more oleic acid (C18:1) than compositions secreted by corresponding wild-type unicellular photosynthetic organisms under identical conditions.

[0057] The engineered unicellular photosynthetic organisms of the invention can be used for large-scale production of large amounts of fatty acids without the need for an energy-intensive destruction step to harvest the fatty acids. The invention also relates to nucleic acid constructs for producing such engineered organisms. The invention further provides methods for culturing these organisms in media suitable for the growth of such organisms to produce fatty acids.

[0058] fatty acid Fatty acids are composed of a carboxylic acid and a saturated or unsaturated aliphatic chain. They are primary metabolic products used by cells for energy storage. Natural fatty acids typically have an unbranched aliphatic chain consisting of an even number of carbon atoms from 4 to 28 (C4 to C28). Fatty acids can be the major component of lipids (up to 70% by weight) in some microalgae species. Such microalgae species may be particularly suitable for use with the present invention.

[0059] Fatty acids with carbon chain lengths from C6 to C22 are of particular interest for industrial applications such as the production of biofuels.

[0060] Fatty acid transporters Biological membranes are composed of lipid bilayers with hydrophobic sides facing each other but polar sides facing each other. Lipid bilayer membranes are semi-permeable and small molecules can diffuse across the membrane, but as the size of the molecule increases, the diffusion rate across the lipid bilayer membrane decreases inversely by several orders of magnitude. Lipid bilayer membranes are virtually impermeable to macromolecules. Cells have dedicated mechanisms for the transport of macromolecules, such as fatty acids, across the lipid bilayer membrane.

[0061] Certain transporters can transport fatty acids of various lengths through lipid bilayer membranes. Fatty acid transporters can transport fatty acids of any length from inside the cell through the cytoplasmic membrane of an organism. The fatty acids can include fatty acids with carbon chain lengths of C6 to C22. Certain fatty acid transporters can preferentially transport fatty acids of certain lengths. For example, typical substrates of natural fatty acid transporters are fatty acids with carbon chain lengths of C14 to C22. Fatty acid transporters are particularly effective in transporting fatty acids with carbon chain lengths of about C18 through cytoplasmic membranes and other lipid bilayer membranes. For example, the fatty acid transporters disclosed herein are particularly effective in transporting fatty acids with carbon chain lengths of C16 to C18 through cytoplasmic membranes and other lipid bilayer membranes. These fatty acid transporters are also particularly effective in transporting monounsaturated fatty acids (e.g., C16:1 and C18:1) through cytoplasmic membranes and other lipid bilayer membranes.

[0062] Thus, in some embodiments, fatty acid transporters suitable for use with the present invention are capable of transporting fatty acids having a carbon chain length of C6 to C22. In some embodiments, fatty acid transporters suitable for use with the present invention are capable of transporting fatty acids having a carbon chain length of C14 to C22 (e.g., C16 to C18) from the interior of a cell through the cytoplasmic membrane of an organism. In certain embodiments, fatty acid transporters suitable for use with the present invention are capable of transporting fatty acids having a carbon chain length of about C16 and / or C18 from the interior of a cell through the cytoplasmic membrane of an organism. In some embodiments, fatty acids having a carbon chain length of C16 and / or C18 are monounsaturated. In some embodiments, fatty acid transporters for use with the present invention are capable of transporting palmitoleic acid and / or oleic acid.

[0063] Fatty acid transporters suitable for use with the present invention are typically derived from eukaryotic cells. In particular, the inventors have identified plant and mammalian fatty acid transporters and demonstrated successful secretion of fatty acids from unicellular photosynthetic organisms engineered to express these fatty acid transporters.

[0064] Homologs have been identified for many fatty acid transporters.Without wishing to be bound by theory, it is expected that functional homologs of fatty acid transporters transport fatty acids through lipid bilayer membranes with similar specificity for certain fatty acids of length (e.g., fatty acids with carbon chain length of C6 to C22).Functional homologs of fatty acid transporters described herein can be used in place of the exemplary fatty acid transporters described herein.

[0065] Although the invention is described with reference to representative fatty acid transporters, the invention should not be construed as limited to the particular fatty acid transporters described herein.

[0066] ATP-binding cassette (ABC) transporters ATP-binding cassette (ABC) transporters constitute a ubiquitous integral membrane protein superfamily responsible for the ATP-driven transport of many substrates across membranes. This superfamily is conserved from prokaryotes to eukaryotes only in structure, rather than in sequence. The proteins form channels in the lipid bilayer and regulate the influx or efflux of specific molecules. One ATP molecule is hydrolyzed per transported cargo (e.g., a single substrate molecule, such as a fatty acid).

[0067] Substrate specificity varies among members of the superfamily. ABC transporters have been identified to transport a variety of substrates, including lipids, retinoic acid derivatives, bile acids, iron, nucleosides, and peptides. Some ABC transporters are believed to play a role in the transport of lipids and lipid-related compounds. For example, about half of the 48 identified human ABC transporter proteins are believed to facilitate the transport of lipids or lipid-related compounds. In some embodiments, any ABC transporter that facilitates the transport of lipids or lipid-related compounds can be used in place of the fatty acid transporters described herein. More typically, the ABC transporters for use with the present invention are capable of transporting fatty acids through the cytoplasmic membrane of the target organism, particularly fatty acids that are primary metabolites used by the target organism for energy conservation (e.g., fatty acids with carbon chain lengths of C14 to C22).

[0068] Flippase Flippases are a subfamily of P-type ATPases characterized as P4-ATPases. Flippases typically flip phospholipids across cell membranes and have roles in a myriad of processes, including vesicle budding and trafficking, cell signaling, blood clotting, apoptosis, bile, cholesterol homeostasis, and neuronal survival. Different flippases have different targets and specificities, e.g., they may preferentially transport fatty acids with certain chain lengths and certain degrees of saturation across lipid bilayer membranes. For example, some flippases transport phosphatidylethanolamine across lipid bilayer membranes, while others are specific for phosphatidylcholine.

[0069] In typical embodiments, the flippase for use with the present invention is a eukaryotic flippase. For example, a suitable flippase may be derived from a plant cell or a mammalian cell. In certain embodiments, the flippase derived from a mammalian cell may be a Homo sapiens flippase.

[0070] Substrate specificity The inventors have found that fatty acid transporters for use with the present invention can preferentially transport fatty acids having a particular chain length and a particular degree of saturation from the interior of a cell through the cytoplasmic membrane of an organism. For example, the inventors have found that fatty acid transporters (e.g., flippases) preferentially transport monounsaturated fatty acids having carbon chain lengths of C16 and C18 (C16:1 and C18:1) through the cytoplasmic membrane.

[0071] The term "preferentially transport" as used in this context refers to the substrate specificity of a fatty acid transporter. For example, a fatty acid transporter may transport at least 1.5-2 times more (e.g., at least 3 times more, at least 5 times more, or at least 10 times more) of one fatty acid relative to one or more other fatty acids. In some embodiments, a fatty acid transporter (e.g., Homo sapiens flippase) for use with the invention transports monounsaturated fatty acids having carbon chain lengths of C16 and C18 (C16:1 and C18:1) across the cytoplasmic membrane at least 1.5-5 times more than saturated or polyunsaturated fatty acids (e.g., C14:0, C16:0, C18:0, C18:2).

[0072] In some embodiments, the substrate specificity of the fatty acid transporters of the present invention can be exploited by further engineering the organism to overproduce fatty acids that are preferentially transported by the fatty acid transporter. For example, metabolic engineering can be used to provide engineered unicellular photosynthetic organisms that overproduce fatty acids having a particular chain length and a particular degree of saturation (as determined relative to a corresponding unicellular photosynthetic organism that has not been metabolically engineered).

[0073] In certain embodiments, unicellular photosynthetic organisms are metabolically engineered to overproduce monounsaturated fatty acids having carbon chain lengths of C16 and / or C18 (e.g., palmitoleic acid and / or oleic acid). As shown herein, flippases (e.g., Homo sapiens flippases) can preferentially transport monounsaturated fatty acids having carbon chain lengths of C16 and / or C18 across the cytoplasmic membrane of unicellular photosynthetic organisms such as microalgae.

[0074] In some embodiments, the fatty acid transporter for use with the present invention has minimal transport activity for fatty acids with carbon chain lengths less than C14. In some embodiments, the fatty acid transporter for use with the present invention has minimal transport activity for fatty acids with carbon chain lengths greater than C22. In certain embodiments, the fatty acid transporter for use with the present invention has minimal transport activity for fatty acids with carbon chain lengths less than C14 and greater than C22. The term "minimal" in this context means that less than 1% of the fatty acids transported from the interior of the cell through the plasma membrane have carbon chain lengths less than C14 and / or greater than C22.

[0075] In some embodiments, transport activity is assessed by determining the transport of fatty acids through the lipid bilayer of artificial phospholipid vesicles that contain the fatty acid transporter of interest. Such vesicles can be incubated with fatty acids, and the appearance of fatty acids in the internal aqueous phase of the vesicles is monitored (see, for example, Glatz et al., Physiol Rev. 2010; 90(1): 367-417, which is incorporated herein by reference).

[0076] Exemplary Amino Acid Sequences Encoding Fatty Acid Transporter Proteins Exemplary amino acid sequences of fatty acid transporter proteins suitable for use with the present invention are shown in Table 1.

[0077] [Table 1-1]

[0078] [Table 1-2]

[0079] In some embodiments, the invention relates to an engineered unicellular photosynthetic organism that has been recombinantly engineered to be capable of expressing a fatty acid transporter having the amino acid sequence of SEQ ID NO: 1, or a functional homolog thereof. In some embodiments, the invention relates to an engineered unicellular photosynthetic organism that has been recombinantly engineered to be capable of expressing a fatty acid transporter having the amino acid sequence of SEQ ID NO: 2, or a functional homolog thereof. In some embodiments, the invention relates to an engineered unicellular photosynthetic organism that has been recombinantly engineered to be capable of expressing a fatty acid transporter having the amino acid sequence of SEQ ID NO: 3, or a functional homolog thereof.

[0080] In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 25% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 90% identical to the amino acid sequence of SEQ ID NO: 1.

[0081] In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 90% identical to the amino acid sequence of SEQ ID NO: 2.

[0082] In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 80% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence of a functional homolog of a fatty acid transporter of the present invention is at least 90% identical to the amino acid sequence of SEQ ID NO: 3.

[0083] In some embodiments, the amino acid sequence of a fatty acid transporter according to the invention is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of a fatty acid transporter according to the invention is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of a fatty acid transporter according to the invention is at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0084] In some embodiments, a fatty acid transporter according to the invention can preferentially transport monounsaturated fatty acids having carbon chain lengths of C16 and C18 (e.g., palmitoleic acid and / or oleic acid) across a lipid bilayer membrane and has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a fatty acid transporter according to the invention can preferentially transport monounsaturated fatty acids having carbon chain lengths of C16 and C18 (e.g., palmitoleic acid and / or oleic acid) across a lipid bilayer membrane and has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2. In some embodiments, a fatty acid transporter according to the invention can preferentially transport monounsaturated fatty acids having carbon chain lengths of C16 and C18 (e.g., palmitoleic acid and / or oleic acid) across a lipid bilayer membrane and has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:3.

[0085] In some embodiments, the amino acid sequence of a fatty acid transporter in accordance with the invention is the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of a fatty acid transporter in accordance with the invention is the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of a fatty acid transporter in accordance with the invention is the amino acid sequence of SEQ ID NO: 3.

[0086] Nucleic acid encoding a fatty acid transporter Nucleic acids comprising coding sequences for the fatty acid transporters of the invention can be used to engineer unicellular photosynthetic organisms capable of transporting fatty acids from the interior of the cell across the cytoplasmic membrane. Exemplary nucleic acids are shown diagrammatically in FIG.

[0087] A suitable nucleic acid comprises a coding sequence of a fatty acid transporter operably linked to a promoter sequence. The coding sequence may code for one of the amino acid sequences of SEQ ID NOs: 1 to 3. Typically, the promoter sequence codes for a strong promoter, such as a viral promoter (e.g., the cauliflower mosaic virus 35S promoter coded for by SEQ ID NOs: 7 or 8). In some embodiments, a suitable promoter native to the target organism may be used. More typically, the suitable promoter is exogenous to the target organism.

[0088] Typically, the nucleic acid also contains a terminator sequence (e.g., one of SEQ ID NO: 22 or 23) operably linked to the coding sequence. The presence of the terminator sequence prevents the transcription of the sequence located downstream of the insertion site of the exogenous nucleic acid in the engineered organism from being controlled by the promoter sequence that controls the expression of the sequence encoding the fatty acid transporter. A suitable terminator sequence is, for example, the nopaline synthase (NOS) terminator encoded by SEQ ID NO: 22.

[0089] In some embodiments, the nucleic acid also comprises a selection marker sequence. The selection marker can be used to select organisms that contain the exogenous nucleic acid sequence in their genome. A suitable selection marker sequence for use with the present invention encodes an aminoglycoside-3'-adenylate transferase. The selection marker is operably linked to a suitable promoter sequence. Suitable promoters include constitutively active promoters (e.g., promoters that control the expression of tubulin).

[0090] In some embodiments, additional elements are included to facilitate detection of the fatty acid transporter (e.g., to ensure its correct localization), for example, when expressed inside a cell. For example, the coding sequence of the fatty acid transporter may be fused to a coding sequence that encodes a marker polypeptide (e.g., a fluorescent marker protein such as clover protein). The resulting fusion protein can be easily detected by fluorescence microscopy.

[0091] Code Sequence Coding sequences for fatty acid transporters suitable for use with the present invention may be codon-optimized for expression in a target organism, such as a suitable unicellular photosynthetic organism. Codon optimization can be performed using any suitable algorithm known to those skilled in the art.

[0092] Furthermore, the presence of introns in genes can aid in expression after transformation of eukaryotic cells. Thus, in some embodiments, the coding sequence comprises one or more introns. For example, the Intronserter tool can be used to create codon-optimized, intron-containing coding sequences for optimal expression in unicellular photosynthetic organisms (Jaeger et al. Intronserter, an advanced online tool for design of intron containing transgenes, Algal Research, Volume 42, 2019). Thus, in some embodiments, the nucleic acid for use with the present invention comprises a codon-optimized, intron-containing fatty acid transporter coding sequence.

[0093] Exemplary Optimized Coding Sequences Encoding Fatty Acid Transporters Exemplary coding sequences for fatty acid transporters optimized for expression in the target organisms of the present invention are shown in Table 2.

[0094] [Table 2-1]

[0095] [Table 2-2]

[0096] [Table 2-3]

[0097] [Table 2-4]

[0098] [Table 2-5]

[0099] In some embodiments, an optimized coding sequence of a fatty acid transporter according to the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, comprises, or consists of the nucleic acid sequence of SEQ ID NO: 4 and encodes the amino acid sequence of SEQ ID NO: 1. In some embodiments, an optimized coding sequence of a fatty acid transporter according to the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, comprises, or consists of the nucleic acid sequence of SEQ ID NO: 5 and encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, an optimized coding sequence of a fatty acid transporter according to the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, comprises, or consists of the nucleic acid sequence of SEQ ID NO:6 and encodes the amino acid sequence of SEQ ID NO:3.

[0100] Promoter sequence Nucleic acids for use with the present invention include promoter sequences for expression of sequences encoding fatty acid transporters after transformation into a target organism. The promoter may be a native (endogenous) promoter of the target organism. The coding sequence may be inserted into the genome of the organism so that it is operably linked to the native promoter. Suitable endogenous promoter sequences may include promoter sequences that control the expression of constitutively active genes in the target organism. More typically, the promoter sequence is exogenous to the target organism. Suitable exogenous promoter sequences may include promoter sequences from viruses (e.g., promoter sequences from one or more plant viruses known to infect the target organism). Exemplary promoters suitable for use with the present invention include the cauliflower mosaic virus (CaMV) 35s promoter (including enhanced forms).

[0101] Thus, in some embodiments, the promoter sequence may be derived from the genome of a different organism than the genome from which the coding sequence is derived. For example, the promoter sequence may be derived from the genome of the same organism as the genome from which the coding sequence is derived. More typically, the coding sequence and the promoter sequence are derived from different genomes.

[0102] In some embodiments, promoter can be constitutively active.For example, promoter sequence can be derived from gene known to be constitutively active in target organism.Examples include promoter sequence derived from gene coding for photosystem I reaction center subunit II (PSAD), ribulose bisphosphate carboxylase small subunit (RBCS), tubulin (e.g., α-tubulin or β-tubulin), and ubiquitin extension protein. Exemplary constitutively active promoters suitable for use with the present invention include the Nannochloropsis gaditana β-tubulin 2 promoter, the Nannochloropsis gaditana ubiquitin extension protein promoter, the C. reinhardtii HSP70A / RBCS2 hybrid promoter, the C. reinhardtii photosystem I reaction center subunit II (PSAD) promoter, and the C. reinhardtii ribulose bisphosphate carboxylase small subunit (RBCS) promoter.

[0103] In some embodiments, the promoter sequence may be a non-natural hybrid promoter sequence of two different promoter sequences. An exemplary hybrid promoter is the Chlamydomonas reinhardtii HSP70A-RBCS2 promoter.

[0104] In other embodiments, the promoter sequence may be an inducible promoter. An inducible promoter may induce expression of a coding sequence only under certain conditions (e.g., under certain growth or environmental conditions, including certain chemical or physical conditions). Inducible promoters may allow growth of the target organism without expression of the coding sequence, and may be used to induce expression of the coding sequence when the organism is cultured under certain growth conditions, such as nitrogen depletion, vitamin depletion, or in a particularly light-rich environment. Exemplary inducible promoters suitable for use with the present invention are the Nannochloropsis gaditana and Phaeodactylum tricornutum nitrogen reductase (NR) promoters. Other suitable inducible promoter sequences are derived from genes encoding methionine synthase (METE) and vesicle-inducing protein of plastid 2 (VIPP2), respectively.

[0105] Exemplary Promoter Sequences Exemplary promoter sequences for use with the present invention are shown in Table 3.

[0106] [Table 3-1]

[0107] [Table 3-2]

[0108] [Table 3-3]

[0109] [Table 3-4]

[0110] [Table 3-5]

[0111] Terminator sequence The nucleic acid for use with the present invention may include a terminator sequence. The terminator sequence may be located at the 3' end of the coding sequence of the fatty acid transporter. A suitable terminator sequence may terminate the transcription of the coding sequence in the target organism. In some embodiments, a suitable promoter sequence and a suitable terminator sequence are derived from the genome of different organisms. In some embodiments, the promoter sequence, the coding sequence and the terminator sequence are derived from different organisms. The terminator sequence may be a native (endogenous) terminator of the target organism. More typically, the terminator sequence is exogenous to the target organism.

[0112] An exemplary terminator sequence suitable for use with the present invention is the Agrobacterium tumefaciens nopaline synthase (NOS) terminator. Another exemplary terminator sequence suitable for use with the present invention is the PSAD terminator native to Chlamydomonas. A further exemplary terminator suitable for use with the present invention is the Phaeodactylum tricornutum nitrate reductase terminator.

[0113] Exemplary Terminator Sequences Exemplary terminator sequences for use with the present invention are shown in Table 4.

[0114] [Table 4]

[0115] Selection marker Nucleic acids for use with the present invention may include a selection marker. The selection marker allows for the selection of successfully transformed organisms. In one embodiment, the selection marker confers antibiotic resistance. Suitable genes encoding selection markers (e.g., antibiotic resistance) include aadA (aminoglycoside-3'-adenylate transferase, conferring resistance to spectinomycin), AphVII (confers resistance to hygromycin), APhVIII (confers resistance to paromomycin), NptII (confers resistance to neomycin phosphotransferase II, conferring resistance to kanamycin), Ble (derived from Streptoalloteichus hindustanus, conferring resistance to zeocin or bleomycin), CAT (chloramphenicol acetyltransferase, conferring resistance to chloramphenicol), and BSR (derived from Streptomyces Griseochromogenes, conferring resistance to blasticidin). In some embodiments, the selection marker is aminoglycoside-3'-adenylate transferase. Aminoglycoside-3'-adenylate transferase confers resistance to spectinomycin. Nucleic acids for use with the invention can include (in 5' to 3' order) a promoter sequence, a coding sequence for a fatty acid transporter, and a terminator sequence, including a selectable marker sequence with another promoter sequence and optionally another terminator sequence.

[0116] Thus, in some embodiments, a suitable selectable marker is operably linked to a promoter and terminator sequence, hi some embodiments, the promoter and terminator sequences operably linked to the selectable marker are different from the promoter and terminator sequences operably linked to the coding sequence.

[0117] Additional Elements The nucleic acid for use with the present invention may include additional sequence elements. The nucleic acid may include a sequence that codes for a marker polypeptide. The marker polypeptide includes various tags, including His tag, c-myc tag. In some embodiments, the marker polypeptide is a protein, such as glutathione-S-transferase (GST). In certain embodiments, the marker polypeptide is a fluorescent protein.

[0118] In typical embodiments, the coding sequence for the marker polypeptide is fused to the coding sequence for the fatty acid transporter to encode a fusion protein. The coding sequence encoding the fusion protein may be operably linked to a promoter sequence and a terminator sequence located at the 5' and 3' ends, respectively, of the coding sequence for the fusion protein.

[0119] The inclusion of a marker polypeptide or protein can be used to detect the expression of the fatty acid transporter protein after transformation into an organism. In some embodiments, the marker polypeptide or protein is used to localize the fatty acid transporter in the cell. For example, a fusion protein containing a fatty acid transporter fused to a fluorescent marker protein can be used to detect and localize the fatty acid transporter by fluorescence microscopy.

[0120] In certain embodiments, the nucleic acid comprises a first coding sequence for fatty acid transporter and a second coding sequence for fluorescent marker protein, such that the first and second coding sequences are operably linked to be co-expressed.The co-expression of fatty acid transporter and fluorescent marker protein can be used to identify organisms that express fatty acid transporter.

[0121] Suitable fluorescent marker sequences are known in the art. In some embodiments, the fluorescent marker is a clover fluorescent protein.

[0122] Exemplary Fluorescent Marker Sequences Exemplary fluorescent marker sequences for use with the present invention are shown in Table 5.

[0123] [Table 5]

[0124] Single-celled photosynthetic organisms Oleaginous organisms that can be used to produce biofuels include algae, yeasts, filamentous fungi, bacteria (including cyanobacteria), and thraustochytrids. The present invention is particularly directed to engineered unicellular photosynthetic organisms such as algae and cyanobacteria.

[0125] Typically, the organism is a unicellular eukaryote, such as a microalgae. Unicellular photosynthetic organisms (such as microalgae) are found in both freshwater and seawater. Thus, in some embodiments, the unicellular photosynthetic organisms for use with the present invention can be grown in freshwater. In other embodiments, the unicellular photosynthetic organisms for use with the present invention can be grown in seawater.

[0126] algae Many algae species are single-celled photosynthetic organisms that live in water. These organisms (commonly called microalgae) are among the most efficient photosynthesizers. They have been consuming sunlight and CO for millions of years. 2 Our bodies have evolved to be extremely good at making fatty acids and lipids from these foods and storing them inside cells.

[0127] Thus, in some embodiments, the unicellular photosynthetic organism for use with the present invention is an algae. In some embodiments, the unicellular photosynthetic organism for use with the present invention is a green algae. In some embodiments, the unicellular photosynthetic organism for use with the present invention is a brown algae. In some embodiments, the unicellular photosynthetic organism for use with the present invention is a red algae. The algae may be selected from any of the following phyla: Chlorophyta, Phaeophyta, Rhodophyta, Xanthophyta, Chrysophyta, Bacillariophyta, Cryptophyta, Dinophyta, Chlorophyta, Euglenophyta, Cyanophyta, and Myxophyta.

[0128] Particularly suitable for use with the present invention are oleaginous algae. Examples of oleaginous algae include Tetraselmis elliptica, Auxenochlorella protothecoides, Botryococcus braunii, Chlorella minutissima, Nannochloropsis salina, Alexandrium sanguinea, Fistulifera solaris, Nitzschia laevis, Chlorella sorokiniana, and Chlamydomonas reinhardtii.

[0129] In some embodiments, oleaginous algae can be identified by culturing the organism and determining the percentage of fatty material per dry weight of the resulting culture. For example, to determine the percentage of fatty material per dry weight of the culture, a sample of the culture can be collected and centrifuged. The resulting cell pellet may be dried (e.g., using an oven). The weight of the dried pellet is then determined. The cells are then cut open (e.g., using a pestle and mortar, or a cell homogenizer). The cell debris is removed (e.g., by sedimentation and centrifugation). The fatty material in the resulting cell-free solution (supernatant) can then be quantified using gas chromatography-flame ionization detection (GC-FID) or gas chromatography-mass spectrometry (GC-MS). The results can be used to calculate the percentage of fatty material per dry weight of the culture.

[0130] In some embodiments, oleaginous algae suitable for use with the present invention comprise at least 15% fatty acid material by dry weight. In some embodiments, oleaginous algae suitable for use with the present invention comprise at least 20% fatty acid material by dry weight. In some embodiments, oleaginous algae suitable for use with the present invention comprise at least 25% fatty acid material by dry weight. In some embodiments, oleaginous algae suitable for use with the present invention comprise at least 30% fatty acid material by dry weight.

[0131] In some embodiments, algae for use with the present invention have been used in industrial applications, such as the production of biofuels, including algae from the genera Chlorella, Dunaliella, Haematococcus, Phaeodactylum, Tetraselmis, Isochrysis, Diacronena, Schizochytrium, Thraustochytrium, Nannochloris, Nannochloropsis, and the like. s), Microchloropsis, Porphyridium, Nanofrustulum, Cryptheiconidium, Schenedesmus, Euglena, Auxenochlorella, Botryococcus, Alexandrium, Fistulifera, and Nitzschia. Exemplary preferred algae include Chlorella sorokiniana, Chlorella vulgaris, Chlorella protothecoides, Dunaliella salina, Dunaliella tertiolecta, Dunaliella sp.), Haematococcus pluvialis, Phaeodactylum tricornutum, Tetraselmis suecica, Tetraselmis chuii, Isochrysis galbana, Diacronena volkianum, Schizochytrium sp., Thraustochytrium sp., Nannochloris sp., Nannochloropsis sp., Nannochloropsis gaditana, Porphyridium sp. sp., Nanofrustulum sp., Cryptheiconidium cohnii, Scenedesmus sp., Euglena gracilis, Tetraselmis elliptica, Auxenochlorella protothecoides, Botryococcus braunii, Chlorella minutissima, Nannochloropsis salina, Alexandrium sanguinea, Fistulifera solaris, and Nitzschia laevis. .

[0132] Algae species that are particularly suitable for biofuel production include those of the genera Chlorella, Tetraselmis, Nannochloropsis, Phaeodactylum, and Porphyridium.

[0133] Genetic manipulation of unicellular photosynthetic organisms The unicellular photosynthetic organism is transformed with exogenous nucleic acid to produce the engineered organism according to the present invention. Suitable methods of transformation are known in the art. A representative method for producing the engineered unicellular photosynthetic organism according to the present invention is described in Example 4.

[0134] After transformation, organisms containing the exogenous nucleic acid sequence can be selected. In some embodiments, a selectable marker contained in the nucleic acid is used to select transformants. In other embodiments, a fluorescent marker contained in the nucleic acid is used to select transformants.

[0135] The engineered organism of the present invention may comprise one or more exogenous nucleic acids comprising a fatty acid transporter coding sequence operably linked to a promoter sequence. In some embodiments, the organism may comprise two or more exogenous nucleic acids comprising the same fatty acid transporter coding sequence. In other embodiments, the organism may comprise two or more exogenous nucleic acids comprising two or more different fatty acid transporter coding sequences.

[0136] Optimization of biosynthetic pathways The engineered unicellular photosynthetic organism for use with the present invention can include one or more additional genetic modifications to increase the amount of free fatty acid in the cell.Suitable genetic modifications are described, for example, in US Patent Application Publication No. 2013 / 245339, US Patent Application Publication No. 2013 / 005003, and US Patent Application Publication No. 2014 / 213826, each of which is incorporated herein by reference.

[0137] In some embodiments, engineered unicellular photosynthetic organisms for use with the invention comprise one or more endogenous genes that have been modified to overproduce fatty acids having carbon chain lengths from C6 to C22. In some embodiments, the one or more endogenous genes are selected from the group consisting of acyl-acyl carrier protein (acyl-ACP) thioesterase, acetyl-CoA carboxylase, pyruvate dehydrogenase, acyl-CoA synthase, acyl carrier protein, acyl carrier protein synthase, acyl-CoA reductase, decarboxylase, aldehyde decarbonylase triglyceride lipase, alkane deformylating monooxygenase, and carboxylic acid reductase.

[0138] In some embodiments, an engineered unicellular photosynthetic organism for use with the invention comprises one or more exogenous genes for overproducing one or more fatty acids having carbon chain lengths from C6 to C22. In some embodiments, the one or more exogenous genes are selected from the group consisting of acyl-acyl carrier protein (acyl-ACP) thioesterase, acetyl-CoA carboxylase, pyruvate dehydrogenase, acyl-CoA synthase, acyl carrier protein, acyl carrier protein synthase, acyl-CoA reductase, decarboxylase, aldehyde decarbonylase triglyceride lipase, alkane deformylating monooxygenase, and carboxylic acid reductase.

[0139] In some embodiments, the one or more fatty acids overproduced by the engineered unicellular photosynthetic organisms for use with the present invention have a carbon chain length of C14 to C22. In some embodiments, the one or more fatty acids have a carbon chain length of C16 to C18 and are optionally monounsaturated. In some embodiments, the one or more fatty acids are palmitoleic acid and / or oleic acid.

[0140] Accumulation of free fatty acids can depend on acyl-ACP thioesterases expressed by unicellular photosynthetic organisms (see, e.g., Zhang et al., Metab Eng. 2011; 13(6):713-2, incorporated herein by reference). For example, it has been shown that Eschericha coli strains carrying the Diploknema butyracea acyl-ACP thioesterase gene can produce approximately 0.2 g / L of free fatty acids in 48 hours. The same strains carrying the Ricinus communis or Jatropha curcas acyl-ACP thioesterase genes can produce more than 2.0 g / L in 48 hours. Eschericha coli strains carrying the acyl-ACP thioesterase genes from Ricinus communis or Jatropha curcas can accumulate the three major linear free fatty acids C14, C16:1, and C16.

[0141] Thus, in some embodiments, the engineered unicellular photosynthetic organism of the invention further comprises an exogenous nucleic acid sequence comprising a coding sequence for an acyl-acyl carrier protein (ACP) thioesterase. In some embodiments, the acyl-ACP thioesterase preferentially releases fatty acids having carbon chain lengths of C6 to C22. In some embodiments, the acyl-ACP thioesterase preferentially releases fatty acids having carbon chain lengths of C14 to C22. In some embodiments, the acyl-ACP thioesterase preferentially releases fatty acids having carbon chain lengths of C16 to C18.

[0142] In some embodiments, the acyl-ACP thioesterase is an oleoyl-ACP thioesterase. In some embodiments, the oleoyl-ACP thioesterase is a fatA thioesterase. In some embodiments, the fatA thioesterase is derived from a bacterium or a plant. In some embodiments, the fatA thioesterase is derived from Eschericha coli, Diploknema butyracea, Gossypium hirsutum, Ricinus communis, Jatropha curcas, Helianthus annuus, Umbellularia californica, Arabidopsis thaliana, Brassica juncea, or Cuphea hookeriana. In some embodiments, the fatA thioesterase is derived from microalgae.

[0143] In some embodiments, the acyl-ACP thioesterase is a palmitoyl-ACP thioesterase. In some embodiments, the palmitoyl-ACP thioesterase is a fatB thioesterase. In some embodiments, the fatB thioesterase is derived from a bacterium or a plant. In some embodiments, the fatB thioesterase is derived from Eschericha coli, Diploknema butyracea, Gossypium hirsutum, Ricinus communis, Jatropha curcas, Helianthus annuus, Umbellularia californica, Arabidopsis thaliana, Brassica juncea, or Cuphea hookeriana. In some embodiments, the fatB thioesterase is derived from microalgae.

[0144] In some embodiments, the engineered unicellular photosynthetic organisms of the invention further comprise a first exogenous nucleic acid sequence that comprises a coding sequence for an oleoyl-ACP thioesterase (e.g., a bacterial or plant fatA thioesterase), and a second exogenous nucleic acid sequence that comprises a coding sequence for a palmitoyl-ACP thioesterase (e.g., a bacterial or plant fatB thioesterase).

[0145] culture In some embodiments, the invention relates to a culture comprising the engineered organisms described herein. Typically, the culture comprises multiple organisms of identical genetic makeup suspended in a suitable medium, e.g., a culture medium suitable for supporting the growth or survival of the engineered organisms. In some embodiments, the culture medium comprises an agent (e.g., an antibiotic) for which the exogenous nucleic acid contained in the engineered organism comprises a selection marker (e.g., a nucleic acid sequence that can confer resistance to an antibiotic).

[0146] The engineered unicellular photosynthetic organisms described herein can be grown in a suitable liquid medium. For example, to support the growth of the engineered unicellular photosynthetic organisms described herein, an inorganic carbon source (typically CO 2 ) and exposed to sunlight (e.g., rainwater) may be sufficient.

[0147] In some embodiments, the medium is supplemented with an inorganic carbon source (typically CO 2 ), for example, the medium may be enriched with ambient CO 2 CO concentrations higher than the level (approximately 0.04% by volume) 2 For example, the medium may contain CO 2 CO by bubbling gas into the medium 2 In some embodiments, the medium can be enriched with about 1% CO by volume. 2 In other embodiments, the medium contains at least 1% by volume CO 2 In some embodiments, the medium comprises about 2% CO by volume. 2 In other embodiments, the medium contains at least 2% by volume CO 2 Includes.

[0148] Method for Producing Fatty Acids The present invention is also directed to a method of producing fatty acids utilizing the engineered unicellular photosynthetic organisms described herein, the method comprising culturing the engineered organism of the invention in a medium suitable for growth of the organism.

[0149] In some embodiments, the culture medium inoculated with the engineered organism of the invention is incubated (e.g., in the culture medium) for a period of time sufficient to obtain fatty acids. In some embodiments, the incubation period is 1-7 days. In some embodiments, the incubation period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 1 month, 2 months, or 3 months. Methods for culturing unicellular photosynthetic organisms are known in the art.

[0150] Methods for producing fatty acids typically involve culturing an engineered organism of the invention in the presence of light and an inorganic carbon source (typically CO 2 ), where the exposure to light results in the conversion of the inorganic carbon source to fatty acids by the engineered organism. 2 is easily soluble in water and CO 2 is carbonic acid (H 2 CO 3 ), bicarbonate (HCO 3 - ), and carbonate (CO 3 - )

[0151] In some embodiments, the culture medium contains CO2-containing media, such as acetate (which can be obtained directly from primary photosynthesis). 2 A low emission liquid carbon source is supplemented.

[0152] As shown in Example 6, fatty acids produced by engineered organisms of the invention are readily secreted into the culture medium by fatty acid transporters encoded by exogenous nucleic acids of the invention. The culture medium containing the fatty acids can then be separated from the engineered organism. Typically, the organism is not destroyed or otherwise damaged to obtain the fatty acids.

[0153] The method for producing fatty acids according to the invention can be carried out in laboratory scale culture volumes (less than 2.5 L), which can be scaled up to pilot plant scale (2.5-25 L) and commercial scale (greater than 25 L) culture volumes for industrial production of fatty acids, typically for use in biofuels.

[0154] The method for producing fatty acids according to the present invention is capable of producing at least 1 μg of fatty acid per liter (L) of culture medium per day (d). In some embodiments, methods according to the invention produce fatty acids at a rate of at least 10 μg / L / d, at least 20 μg / L / d, at least 30 μg / L / d, at least 40 μg / L / d, at least 50 μg / L / d, at least 60 μg / L / d, at least 70 μg / L / d, at least 80 μg / L / d, at least 90 μg / L / d, at least 100 μg / L / d, at least 200 μg / L / d, at least 300 μg / L / d, at least 400 μg / L / d, at least 500 μg / L / d, at least 600 μg / L / d, at least 700 μg / L / d, at least 800 μg / L / d, at least 900 μg / L / d, or at least 1 g / L / d.

[0155] The culture medium inoculated with an engineered organism of the invention and secreting fatty acids may be cultured for at least one week. For example, the inoculated culture medium secreting fatty acids may be cultured for at least two weeks, at least three weeks, at least one month, at least two months, at least three months, at least four months, or at least five months.

[0156] In some embodiments, the culture medium comprising an engineered unicellular photosynthetic organism according to the invention is enriched in monounsaturated fatty acids having carbon chain lengths of C16 to C18, as compared to the culture medium of a corresponding wild-type unicellular photosynthetic organism incubated under identical conditions. In some embodiments, the culture medium comprises at least about 5-fold (e.g., at least about 10-fold) more palmitoleic acid (C16:1) than the culture medium of a corresponding wild-type unicellular photosynthetic organism incubated under identical conditions after incubation with an engineered unicellular photosynthetic organism of the invention. In some embodiments, the culture medium comprises at least about 1.5-fold (e.g., at least about 3-fold or at least about 5-fold) more oleic acid (C18:1) than the culture medium of a corresponding wild-type unicellular photosynthetic organism incubated under identical conditions after incubation with an engineered unicellular photosynthetic organism of the invention.

[0157] In some embodiments, culturing the engineered organisms of the invention is continuous at steady state, in these embodiments, the culture medium containing the secreted fatty acids is separated from the organism and replaced with fresh culture medium without disturbing the cultivation of the organism.

[0158] Separation of fatty acids The method of producing fatty acids according to the present invention may further include a step of separating the culture medium from the organism. In some embodiments, the step of separating the medium from the organism includes sedimentation. Sedimentation may include centrifugation of the medium. In some embodiments, sedimentation occurs naturally (e.g., by incubating the medium without stirring). In other embodiments, the step of separating the medium from the organism may include filtration. Typically, separation of the medium from the organism does not damage (e.g., destroy) the organism.

[0159] Both sedimentation (e.g., by centrifugation or by incubating the medium without stirring) and filtration can be used to obtain the abiotic culture medium. In some embodiments, the abiotic culture medium may be subjected to liquid-liquid extraction to isolate fatty acids from the abiotic culture medium. In other embodiments, fatty acids are recovered from the abiotic culture medium, for example, by scooping fatty acid-containing droplets from the surface of the abiotic culture medium. The scooping can be performed by an automated mechanical device.

[0160] For example, a culture medium inoculated with an engineered organism of the invention and then incubated with the organism for a sufficient period of time may comprise at least 1 μg fatty acid per liter of culture medium. In some embodiments, the culture medium comprises at least 10 μg fatty acid per liter of culture medium. In some embodiments, the culture medium comprises at least 100 μg fatty acid per liter of culture medium. In some embodiments, the culture medium comprises at least 1 g fatty acid per liter of culture medium.

[0161] The extracted fatty acids may be subjected to further processing steps, for example to obtain a product suitable for use in biofuels. Further processing, for example for the production of biofuels, may include transesterification, decarboxylation, or hydrocracking (also called hydrotreating).

[0162] Downstream applications The fatty acids obtainable from the engineered unicellular photosynthetic organisms described herein can be used (directly or after further purification) in cosmetics, pharmaceuticals, chemicals, and nutraceuticals, or may be further processed for use in biofuels.

[0163] Biofuel Production In some embodiments, fatty acids obtainable from the engineered unicellular photosynthetic organisms described herein may be subjected to transesterification with methanol to produce biofuel. For example, the resulting methyl-transesterified fatty acids can be used directly as biofuel. In some embodiments, the resulting methyl-transesterified fatty acids are blended with fossil fuels (e.g. diesel).

[0164] In some embodiments, fatty acids obtainable from the engineered unicellular photosynthetic organisms described herein may be subjected to a decarboxylation reaction. In some embodiments, the decarboxylation is carried out by decarbonating the fatty acids with hydrogen (H 2 ) and the addition of a metal catalyst (typically at high pressure). In other embodiments, the decarboxylation is carried out enzymatically. The decarboxylation reaction produces an alkane or alkene with an unbranched aliphatic chain that is one carbon atom shorter than the aliphatic chain of the fatty acid from which it is derived.

[0165] In some embodiments, fatty acids obtainable from the engineered unicellular photosynthetic organisms described herein are treated at high temperature and pressure by adding hydrogen and water ("hydrocracking") to produce a variety of alkanes and alkenes with different aliphatic chain lengths. The resulting hydrogenated vegetable oils (HVO) or hydrotreated esters and fatty acids (HEFA) can be used as renewable drop-in fuels. They do not require blending with fossil fuels.

[0166] Numbered embodiments 1. An engineered unicellular photosynthetic organism comprising an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence.

[0167] 2. The engineered organism of embodiment 1, wherein the fatty acid transporter is localized to the cytoplasmic membrane of the organism upon expression.

[0168] 3. The engineered organism of embodiment 1 or embodiment 2, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of from C6 to C22 from the interior of the cell through the cytoplasmic membrane of the organism.

[0169] 4. The engineered organism of any one of embodiments 1 to 3, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C14 to C22 from the interior of the cell through the cytoplasmic membrane of the organism.

[0170] 5. The engineered organism of any one of embodiments 1 to 4, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 to C18 from the interior of the cell through the cytoplasmic membrane of the organism.

[0171] 6. The engineered organism of any one of embodiments 1 to 5, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 and / or C18 from the interior of the cell through the cytoplasmic membrane of the organism.

[0172] 7. The engineered organism of embodiment 6, wherein the fatty acid is monounsaturated.

[0173] 8. The engineered organism of any one of embodiments 1 to 7, wherein the fatty acid transporter is capable of transporting palmitoleic acid and / or oleic acid from the interior of the cell through the cytoplasmic membrane of the organism.

[0174] 9. The engineered organism of any one of embodiments 1 to 8, wherein the coding sequence for the fatty acid transporter is derived from the genome of a plant cell or a mammalian cell.

[0175] 10. The engineered organism of embodiment 9, wherein the coding sequence for the fatty acid transporter is derived from the genome of a plant cell.

[0176] 11. The engineered organism of embodiment 10, wherein the fatty acid transporter is an ABC transporter.

[0177] 12. The engineered organism of embodiment 11, wherein the ABC transporter is the Arabidopsis thaliana ABCG11 protein or a functional homolog thereof.

[0178] 13. The engineered organism of embodiment 11, wherein the ABC transporter is the rice (Oryza sativa) ABCG15 protein or a functional homolog thereof.

[0179] 14. The engineered organism of embodiment 9, wherein the coding sequence for the fatty acid transporter is derived from the genome of a mammalian cell.

[0180] 15. The engineered organism of embodiment 14, wherein the fatty acid transporter is an ABC transporter or flippase.

[0181] 16. The engineered organism of embodiment 15, wherein the flippase is a Homo sapiens FATP1 protein or a functional homolog thereof.

[0182] 17. The engineered organism of any one of embodiments 1 to 16, wherein the promoter sequence is derived from the genome of a different organism than the genome from which the coding sequence is derived.

[0183] 18. The engineered organism of embodiment 17, wherein the promoter sequence is exogenous to the engineered organism.

[0184] 19. The engineered organism of embodiment 17, wherein the promoter sequence is endogenous to the engineered organism.

[0185] 20. The engineered organism of embodiment 17 or 18, wherein the promoter is selected from the group consisting of the cauliflower mosaic virus (CaMV) 35S promoter, the nitrogen reductase (NR) promoter, the photosystem I reaction center subunit II (PSAD) promoter, and the HSP70A-RBCS2 (AR) promoter.

[0186] 21. The engineered organism of any one of embodiments 1 to 20, wherein the coding sequence is codon-optimized for expression in the engineered organism.

[0187] 22. The engineered organism of any one of embodiments 1 to 21, wherein the coding sequence comprises one or more introns.

[0188] 23. The engineered organism of any one of embodiments 1 to 22, wherein the exogenous nucleic acid sequence further comprises a selectable marker.

[0189] 24. The engineered organism of embodiment 23, wherein the selection marker confers antibiotic resistance.

[0190] 25. The engineered organism of any one of embodiments 1 to 24, wherein the exogenous nucleic acid sequence further comprises a terminator sequence operably linked to the coding sequence.

[0191] 26. The engineered organism of embodiment 25, wherein the terminator sequence is derived from the genome of a different organism than the genome from which the coding sequence and / or promoter sequence is derived.

[0192] 27. The engineered organism of embodiment 26, wherein the terminator sequence is exogenous to the engineered organism.

[0193] 28. The engineered organism of embodiment 26, wherein the terminator sequence is endogenous to the engineered organism.

[0194] 29. The engineered organism of any one of embodiments 25 to 27, wherein the terminator sequence encodes the Agrobacterium tumefaciens nopaline synthase (NOS) terminator.

[0195] 30. The engineered organism of any one of embodiments 1 to 29, which is an algae.

[0196] 31. The engineered organism of embodiment 30, wherein the algae is an oleaginous algae.

[0197] 32. The engineered organism of any one of embodiments 1 to 31, which is capable of growing in freshwater.

[0198] 33. An engineered organism according to any one of embodiments 1 to 31, capable of growing in seawater.

[0199] 34. The engineered organism of embodiment 30, which is an alga selected from Chlorophyta, Phaeophyta, Rhodophyta, Xanthophyta, Chrysophyta, Bacillariophyta, Cryptophyta, Dinophyta, Euglenophyta, Cyanophyta, and Myxophyta.

[0200] 35. Chlorella, Chlamydomonas, Dunaliella, Haematococcus, Phaeodactylum, Tetraselmis, Isochrysis, Diacronena, Schizochytrium, Thraustochytrium, Nannochloris, Nannochloropsis, Microchloropsis 35. The engineered organism of embodiment 34, wherein the engineered organism is selected from the group consisting of Microchloropsis, Porphyridium, Nanofrustulum, Cryptheiconidium, Schenedesmus, Euglena, Auxenochlorella, Botryococcus, Alexandrium, Fistulifera, and Nitzschia.

[0201] 36. Chlorella sorokiniana, Chlorella vulgaris, Chlorella protothecoides, Chlamydomonas reinhardtii, Dunaliella salina, Dunaliella tertiolecta, Dunaliella sp., Haematococcus pluvialis, Phaeodactylum tricornutum, Tetraselmis suecica, Tetraselmis chuii, Isochrysis galbana galbana, Diacronena volkianum, Schizochytrium sp., Thraustochytrium sp., Nannochloris sp., Nannochloropsis sp., Nannochloropsis gaditana, Porphyridium sp., Nanofrustulum sp., Cryptheiconidium cohnii, Scenedesmus sp.36. The engineered organism of embodiment 35, wherein the engineered organism is selected from the group consisting of Chlorella protothecoides, Euglena gracilis, Tetraselmis elliptica, Auxenochlorella protothecoides, Botryococcus braunii, Chlorella minutissima, Nannochloropsis salina, Alexandrium sanguinea, Fistulifera solaris, and Nitzschia laevis.

[0202] 37. The engineered organism of any one of embodiments 1-36, comprising one or more endogenous genes that have been modified to overproduce fatty acids having a carbon chain length of C6 to C22.

[0203] 38. The engineered organism of embodiment 37, wherein the one or more endogenous genes are selected from the group consisting of acyl-acyl carrier protein (acyl-ACP) thioesterase, acetyl-CoA carboxylase, pyruvate dehydrogenase, acyl-CoA synthase, acyl carrier protein, acyl carrier protein synthase, acyl-CoA reductase, decarboxylase, aldehyde decarbonylase triglyceride lipase, alkane deformylating monooxygenase, and carboxylic acid reductase.

[0204] 39. The engineered organism of any one of embodiments 1 to 38, comprising one or more exogenous genes for overproducing one or more fatty acids having a carbon chain length of C6 to C22.

[0205] 40. The engineered organism of embodiment 39, wherein the one or more exogenous genes are selected from the group consisting of acyl-acyl carrier protein (acyl-ACP) thioesterase, acetyl-CoA carboxylase, pyruvate dehydrogenase, acyl-CoA synthase, acyl carrier protein, acyl carrier protein synthase, acyl-CoA reductase, decarboxylase, aldehyde decarbonylase triglyceride lipase, alkane deformylating monooxygenase, and carboxylic acid reductase.

[0206] 41. The engineered organism of any one of embodiments 37-40, wherein the one or more fatty acids have a carbon chain length of from C14 to C22.

[0207] 42. The engineered organism of any one of embodiments 37-40, wherein the one or more fatty acids have a carbon chain length of from C16 to C18 and are optionally monounsaturated.

[0208] 43. The engineered organism of any one of embodiments 37-40, wherein the one or more fatty acids are palmitoleic acid and / or oleic acid.

[0209] 44. The engineered organism of any one of embodiments 37 to 40, further comprising an exogenous nucleic acid sequence comprising a coding sequence for an acyl-acyl carrier protein (ACP) thioesterase.

[0210] 45. An acyl-ACP thioesterase according to embodiment 44, which preferentially liberates fatty acids having carbon chain lengths from C6 to C22.

[0211] 46. ​​An acyl-ACP thioesterase according to embodiment 44, which preferentially liberates fatty acids having a carbon chain length from C14 to C22.

[0212] 47. An acyl-ACP thioesterase according to embodiment 44, which preferentially liberates fatty acids having a carbon chain length of from C16 to C18.

[0213] 48. The acyl-ACP of any one of embodiments 44-47, wherein the acyl-ACP is an oleoyl-ACP thioesterase, and optionally, the oleoyl-ACP is a fatA thioesterase.

[0214] 49. The fatA thioesterase according to embodiment 48, which is derived from a bacterium or a plant, optionally from (a) Eschericha coli, Diploknema butyracea, Gossypium hirsutum, Ricinus communis, Jatropha curcas, Helianthus annuus, Umbellularia californica, Arabidopsis thaliana, Brassica juncea, or Cuphea hookeriana, or (b) microalgae.

[0215] 50. The acyl-ACP of any one of embodiments 44-47, wherein the acyl-ACP is a palmitoyl-ACP thioesterase, and optionally, the palmitoyl-ACP is a fatB thioesterase.

[0216] 51. The fatB thioesterase according to embodiment 50, which is derived from a bacterium or a plant, optionally from (a) Eschericha coli, Diploknema butyracea, Gossypium hirsutum, Ricinus communis, Jatropha curcas, Helianthus annuus, Umbellularia californica, Arabidopsis thaliana, Brassica juncea, or Cuphea hookeriana, or (b) microalgae.

[0217] 52. The engineered organism of any one of embodiments 37 to 40, further comprising a first exogenous nucleic acid sequence comprising a coding sequence for an oleoyl-ACP thioesterase, and a second exogenous nucleic acid sequence comprising a coding sequence for a palmitoyl-ACP thioesterase.

[0218] 53. The exogenous nucleic acid sequence of embodiment 52, wherein (a) the oleoyl-ACP thioesterase is a bacterial or plant fatA thioesterase and / or (b) the palmitoyl-ACP thioesterase is a bacterial or plant fatB thioesterase.

[0219] 54. A nucleic acid comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence.

[0220] 55. The nucleic acid of embodiment 54, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of from C6 to C22 from the interior of the cell through the cytoplasmic membrane of the organism.

[0221] 56. The nucleic acid of embodiment 54 or 55, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of from C14 to C22 from the interior of the cell through the cytoplasmic membrane of the organism.

[0222] 57. The nucleic acid according to any one of embodiments 54 to 56, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 to C18 from the interior of the cell through the cytoplasmic membrane of the organism.

[0223] 58. The nucleic acid according to any one of embodiments 54 to 57, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of C16 and / or C18 from the interior of the cell through the cytoplasmic membrane of the organism.

[0224] 59. The nucleic acid of embodiment 58, wherein the fatty acid is monounsaturated.

[0225] 60. The nucleic acid according to any one of embodiments 54 to 59, wherein the fatty acid transporter is capable of transporting palmitoleic acid and / or oleic acid from the interior of the cell through the cytoplasmic membrane of the organism.

[0226] 61. The nucleic acid according to any one of embodiments 54 to 60, wherein the coding sequence of the fatty acid transporter is derived from the genome of a plant cell or a mammalian cell.

[0227] 62. The nucleic acid of embodiment 61, wherein the coding sequence of the fatty acid transporter is derived from the genome of a plant cell.

[0228] 63. The nucleic acid of embodiment 62, wherein the fatty acid transporter coding sequence encodes an ATP-binding cassette (ABC) transporter.

[0229] 64. The nucleic acid of embodiment 63, wherein the ABC transporter is the Arabidopsis thaliana ABCG11 protein or a functional homolog thereof.

[0230] 65. The nucleic acid of embodiment 63, wherein the ABC transporter is the rice (Oryza sativa) ABCG15 protein or a functional homologue thereof.

[0231] 66. The nucleic acid of embodiment 61, wherein the coding sequence of the fatty acid transporter is derived from the genome of a mammalian cell.

[0232] 67. The nucleic acid of embodiment 66, wherein the fatty acid transporter coding sequence encodes an ABC transporter or flippase.

[0233] 68. The nucleic acid of embodiment 67, wherein the coding sequence of the fatty acid transporter encodes a Homo sapiens flippase FATP1 protein or a functional homolog thereof.

[0234] 69. The nucleic acid according to any one of embodiments 54 to 68, wherein the promoter sequence is derived from the genome of a different organism than the genome from which the coding sequence is derived.

[0235] 70. The nucleic acid of any one of embodiments 54 to 69, wherein the promoter is selected from the group consisting of the cauliflower mosaic virus (CaMV) 35S promoter, the nitrogen reductase (NR) promoter, the photosystem I reaction center subunit II (PSAD) promoter, and the HSP70A-RBCS2 (AR) promoter.

[0236] 71. The nucleic acid according to any one of embodiments 54 to 70, wherein the promoter is derived from a different organism than the fatty acid transporter.

[0237] 72. The nucleic acid of any one of embodiments 54 to 71, wherein the nucleic acid sequence of the fatty acid transporter is codon-optimized.

[0238] 73. The nucleic acid according to any one of embodiments 54 to 72, further comprising one or more introns.

[0239] 74. The nucleic acid according to any one of embodiments 54 to 73, further comprising a selection marker.

[0240] 75. The nucleic acid of embodiment 74, wherein the selection marker confers antibiotic resistance.

[0241] 76. The nucleic acid of any one of embodiments 54 to 75, further comprising a terminator sequence operably linked to the coding sequence.

[0242] 77. The nucleic acid of embodiment 76, wherein the terminator sequence is derived from the genome of a different organism than the genome from which the coding sequence and / or promoter sequence is derived.

[0243] 78. The nucleic acid of embodiment 76 or embodiment 67, wherein the terminator is the Agrobacterium tumefaciens nopaline synthase (NOS) terminator.

[0244] 79. A culture comprising an organism according to any one of embodiments 1 to 53.

[0245] 80. A method for producing fatty acids, comprising culturing an organism according to any one of embodiments 1 to 53 in a medium suitable for the growth of the organism.

[0246] 81. The method of embodiment 80, wherein the culturing step is continuous at steady state.

[0247] 82. The method of embodiment 80 or 81, further comprising a step of separating the medium from the organism.

[0248] 83. The method of embodiment 82, wherein the separation step comprises sedimentation or filtration.

[0249] 84. The method of embodiment 83, wherein the sedimentation involves centrifugation.

[0250] 85. The method of embodiment 83, wherein the sedimentation involves incubating the medium for a period of time without stirring.

[0251] 86. The method of any one of embodiments 83 to 85, further comprising extracting fatty acids from the obtained abiotic medium by sedimentation or filtration using a liquid-liquid extraction process.

[0252] 87. The method of any one of embodiments 83 to 85, further comprising the step of extracting fatty acids by spooning droplets containing the fatty acids from the surface of the abiotic culture medium obtained by sedimentation or filtration.

[0253] 88. The method of embodiment 86 or 87, further comprising processing the extracted fatty acids by transesterification.

[0254] 89. The method of embodiment 86 or 87, further comprising processing the extracted fatty acids by decarboxylation.

[0255] 90. The method of embodiment 86 or 87, further comprising processing the extracted fatty acids by hydrogenolysis.

[0256] 91. The method of any one of embodiments 80-90, wherein the method produces at least 1 μg of fatty acid per liter of culture medium per day.

[0257] 92. The method of embodiment 91, wherein the method produces at least 10 μg of fatty acid per liter of culture medium per day.

[0258] 93. The method of embodiment 92, wherein the method produces at least 100 μg of fatty acid per liter of culture medium per day.

[0259] 94. The method of embodiment 93, wherein at least 1 g of fatty acid is produced per liter of culture medium per day.

[0260] 95. A culture medium inoculated with an engineered organism according to any one of embodiments 1 to 53 and incubated for a period of time sufficient to obtain at least 1 μg fatty acid per liter of culture medium.

[0261] 96. The culture medium of embodiment 95, incubated for a period of time sufficient to obtain at least 10 μg of fatty acid per liter of culture medium.

[0262] 97. The culture medium of embodiment 96, incubated for a period of time sufficient to obtain at least 100 μg of fatty acid per liter of culture medium.

[0263] 98. The culture medium of embodiment 97, incubated for a period of time sufficient to obtain at least 1 g of fatty acids per liter of culture medium.

[0264] 99. A culture medium according to any one of embodiments 95 to 98, which does not contain the organism used for inoculation.

[0265] 100. A method for extracting fatty acids from a unicellular photosynthetic organism, comprising: a. Providing a culture medium according to any one of embodiments 95 to 99; b. Extracting fatty acids A method comprising:

[0266] 101. The method of embodiment 100, wherein the extraction step comprises a liquid-liquid extraction process.

[0267] 102. The method of embodiment 100, wherein the extraction step comprises spooning droplets containing the fatty acids from the surface of the culture medium.

[0268] 103. A method for producing biofuel, comprising: a. providing a fatty acid obtained by the method according to any one of embodiments 80 to 87 and 90 to 92; and b. Processing the fatty acids by transesterification, decarboxylation, or hydrogenolysis A method comprising:

[0269] 104. A biofuel obtained by the method according to embodiment 103.

[0270] 105. A culture medium conditioned by an engineered unicellular photosynthetic organism according to any one of embodiments 1 to 53, which is enriched in monounsaturated fatty acids having carbon chain lengths of C16 to C18, as compared to a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0271] 106. The culture medium of embodiment 105, comprising at least about 5 times more palmitoleic acid (C16:1) than a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0272] 107. The culture medium of embodiment 105, comprising at least about 1.5 times more oleic acid (C18:1) than a culture medium conditioned by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0273] 108. A cell-free fatty acid composition secreted by an engineered unicellular photosynthetic organism according to any one of embodiments 1 to 53, which is enriched in monounsaturated fatty acids having carbon chain lengths of C16 to C18, as compared to a composition secreted by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0274] 109. The composition of embodiment 108, comprising at least about 5 times more palmitoleic acid (C16:1) than the composition secreted by a corresponding wild-type unicellular photosynthetic organism under identical conditions.

[0275] 110. The composition of embodiment 108, comprising at least about 1.5 times more oleic acid (C18:1) than the composition secreted by a corresponding wild-type unicellular photosynthetic organism under identical conditions. EXAMPLES

[0276] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0277] [Example 1] Identification of ABC transporters from plants suitable for the movement of fatty acids across the plasma membrane This example describes the identification of ABC transporters from plants suitable for the movement of fatty acids across the cytoplasmic membrane.

[0278] The present inventors have identified two exemplary ABC transporters, ABCG11 and ABCG15, that are particularly effective in transporting fatty acids across the cytoplasmic membrane of plant cells. Both ABCG11 and ABCG15 are evolutionarily conserved ABC transporters with many known homologs in the plant kingdom (Figures 1a and 1b, respectively).

[0279] The Arabidopsis thaliana ABCG11 protein is essential for the formation of the cuticle. The cuticle is a protective waxy layer on the surface of the leaf. It is also essential for vascular growth. The physiological and in vitro activity of the protein itself has not been tested so far. Without wishing to be bound by a particular theory, the inventors have linked its role in cuticle formation to the transport of long-chain fatty acids to the surface of the leaves of A. thaliana plants to form the cuticle.

[0280] Rice (Oryza sativa) ABCG15 is essential for the development of exine and sporopollenin in rice pollen. Exine and sporopollenin have been suggested to be formed by complex polymerization of long-chain wax and aliphatic cutin polymers (Li et al. The molecular structure of plant sporopollenin, Nature Plants 5, 41-46 (2019)). Without wishing to be bound by a particular theory, the inventors believe that this may be a substrate for ABCG15 and therefore have identified ABCG15 as a suitable fatty acid transporter for use with the present invention.

[0281] Functional homologs to ABCG11 and ABCG15, such as those identified in FIG. 1, are believed to transport fatty acids across the plasma membrane in a similar manner and with similar specificity to ABCG11 and ABCG15, and are therefore contemplated as suitable alternatives for use with the present invention.

[0282] [Example 2] Identification of mammalian flippases suitable for the transfer of fatty acids across the plasma membrane. This example describes the identification of flippases from mammals suitable for the transfer of fatty acids across the cytoplasmic membrane.

[0283] Homo sapiens FATP1 is an exemplary mammalian flippase. It is an evolutionarily conserved membrane protein with functional homologs in many mammalian species (Figure 2). Its function is to maintain fatty acid homeostasis, which is known to act in concert with the insulin signaling pathway. It only has two transmembrane helices, and its function is thought to occur more often in the interior of the cell rather than in the cytoplasmic membrane. However, the subcellular localization of the protein is under debate and may not be constant in different cell types. For example, in 293 cell lines, FATP1 appears to be localized to the cytoplasmic membrane, whereas in 3T3-L1 and muscle cells, it was localized to the endoplasmic reticulum and mitochondria, respectively.

[0284] In yeast, FATP1 homologs are associated with the import of long-chain fatty acids (Obermeyer et al. Topology of the yeast fatty acid transport protein Fat1p: mechanistic implications for functional domains on the cytoplasmic surface of the plasma membrane, J. Lipid Res, 2007; 48(11)). In mammalian blood-brain barrier, FATP1 appears to be involved in the passage of fatty acids through the blood-brain barrier itself by regulating both fatty acid import and export at the cellular level (Ochiai et al. The blood-brain barrier fatty acid transport protein 1 (FATP1 / SLC27A1) supplies docosahexaenoic acid to the brain, and insulin facilitates transport. J Neurochem. 2017 May;141(3):400-412).

[0285] FATP1 and its functional homologues are believed to act by a mechanism different from the plant-derived ABC transporters described in Example 1. In particular, they are believed to act as flippases, facilitating the transfer of phospholipid molecules between the two leaflets that compose the bilayer membrane via lateral diffusion. Without wishing to be bound by a particular theory, the inventors hypothesize that FATP1 and its homologues may be utilized for the transport of fatty acids from unicellular photosynthetic organisms to the surrounding culture medium.

[0286] [Example 3] Creation of expressible and selectable nucleic acids containing coding sequences for fatty acid transporters This example describes the generation of an exemplary nucleic acid comprising a fatty acid transporter coding sequence operably linked to a promoter sequence that can be used for expression of the fatty acid transporter in an engineered unicellular photosynthetic organism.

[0287] The ABC transporter genes ABCG11 and ABCG15 and the flippase FATP1 were codon-optimized with the Intronserter open tool to ensure optimal expression in Chlamydomonas (Jaeger et al.). Introns were also inserted into the genes to facilitate expression. Expression of the genes was driven by one copy of the Cauliflower Mosaic Virus (CaMV) 35s promoter, and the nucleic acid also encoded the Agrobacterium tumefaciens nopaline synthase (NOS) terminator to generate expression cassettes for each of ABCG11, ABCG15, and FATP1. The expression cassettes were linked to a selection marker cassette for aminoglycoside-3'-adenyltransferase driven and terminated by the Chlamydomonas native β-tubulin 2 promoter and PSAD terminator. The selectable marker confers antibiotic resistance to spectinomycin.

[0288] A separate nucleic acid was created in which the expression cassette contained the fatty acid transporter followed by a clover tag. The cassette is shown in FIG.

[0289] [Example 4] Transformation of nucleic acids encoding fatty acid transporters into unicellular photosynthetic organisms This example demonstrates the successful transformation of an exemplary unicellular photosynthetic organism with the exemplary nucleic acids produced in Example 3.

[0290] Chlamydomonas reinhardtii cells were transformed with the nucleic acid prepared in Example 3. C. reinhardtii strain CC-5416 was obtained from the Chlamydomonas Resource Center at the University of Minnesota (Kurniasih et al. (2016) UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems. Plant Mol Biol. 92:629-641).

[0291] The transformation method was adapted from Crozet et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii, ACS Synth. Biol., 2018 7 (9), 2074-2086. Cells were inoculated at low density in TAP medium and grown in wide-bottom Fernbach flasks with large magnetic stirrers at 25 °C with a 16 / 8 illumination light cycle. Cells were inoculated at 5 × 10 8The cells were resuspended in fresh TAP to a cell density of 100 μL. 250 μL of cells were mixed with 250 ng of linear DNA in a 0.4 cm gap electroporation cuvette and placed on ice for 10 min. The cuvette was then electroporated at 800 V (2 kV / cm), 25 μF, and infinite shunt resistance. The cells were removed, placed in 10 mL of TAP + 60 mM sucrose, incubated at 25 °C in the dark with gentle shaking for 16 h, and harvested. The cells were centrifuged at 1500 g for 10 min at 4 °C, resuspended in 500 μL of TAP + 60 mM sucrose, and plated on TAP agar with 100 μM spectinomycin and incubated with a 16 / 8 illumination light cycle until colonies formed. The colonies were passaged two more rounds on fresh antibiotic plates.

[0292] After three successive passages, the genomic insert was confirmed by PCR on genomic DNA extracted from the growing culture. Genomic DNA was extracted and purified (Barbier et al. A phenol / chloroform-free method to extract nucleic acids from recalcitrant, woody tropical species for gene expression and sequencing. Plant Methods 15, 62 (2019)). The PCR target was the aadA gene, which codes for spectinomycin resistance. Genomic insertion was confirmed using VeriFi polymerase or Repliqa Toughmix 2x master mix. Successful transformation is shown in Figure 4.

[0293] [Example 5] Fatty acid transporters are localized in the plasma membrane This example demonstrates that a nucleic acid sequence of the present invention comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence is expressed by an exemplary unicellular photosynthetic organism and localizes to the cytoplasmic membrane.

[0294] Cells transformed in Example 4 with nucleic acid encoding ABCG15 containing a fatty acid clover tag resulted in expression of exogenous fatty acid transporter fused to the clover tag. The cells were imaged using fluorescence microscopy with immunofluorescence assay. All fluorescence microscopy was performed on a Leica DMIL equipped with a 100w Osram HBO light source and a 100x Leica Fluotar 1.30 oil immersion objective. Microscopic images were taken with an Altair hypercam 183M camera. Images were annotated with a scale bar using ImageJ. A liquid blocker marker was used to mark a poly-L-lysine glass slide. 100 μl of late log phase cell culture was placed inside the marked area and left to fix for 1 hour. Excess liquid was pipetted out and replaced with 150 μL of freshly thawed 4% paraformaldehyde dissolved in PBS. After fixation, cells were washed twice with PBS for 5 minutes per wash. The cell membrane was then permeabilized with 150 μL of 0.5% saponin dissolved in PBS. The cells were washed twice with PBS and once with 1 mM Tris for 5 min each. The cells were then blocked with 150 μL of 2% BSA dissolved in PBS for 30 min. A primary antibody incubation was then performed without a washing step for 60 min using anti-GFP (66002-1-Ig, Proteintech) diluted 1:100 in 2% BSA / PBS. The cells were then washed three times with PBS for 5 min per wash. A secondary antibody incubation was performed for 60 min using DL488-conjugated secondary antibody (GtxRb-003-D488NHSX, Immunoreagents) diluted 1:100 in 2% BSA / PBS. The cells were then washed twice with PBS and twice with milli-Q water for 5 min per wash. The slides were then allowed to dry in the dark, overlaid with 5 μL of Prolong Diamond (Thermofisher), and sealed with a #1.5 coverslip and nail polish. The slides were then stored overnight at 4° C. and observed under a fluorescent microscope.

[0295] Fluorescence microscopy demonstrated that the exogenous fatty acid transporter is localized to the cytoplasmic membrane of engineered unicellular photosynthetic organisms containing the exogenous nucleic acid sequences of the invention. Figure 5a shows C. reinhardtii transformed with only the nucleic acid encoding an antibiotic selection marker (mock control). In Figure 5a, only background fluorescence is visible. Figure 5b shows C. reinhardtii transformed with the nucleic acid encoding the ABCG15-clover fusion protein. Clusters of fluorescent cells can be clearly seen in Figure 5b, with the fluorescent staining being intense at the cytoplasmic membrane.

[0296] The experimental results described in this Example demonstrate that unicellular photosynthetic organisms engineered to contain an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence express the fatty acid transporter, and that the fatty acid transporter is localized to the cytoplasmic membrane of the organism upon expression.

[0297] [Example 6] Fatty acid transporters efficiently transport fatty acids from the interior of the cell across the plasma membrane. This example illustrates that an engineered unicellular photosynthetic organism containing an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence can transport fatty acid from the interior of the cell through the cytoplasmic membrane into the surrounding culture medium.

[0298] To demonstrate that the fatty acid transporter functions by secreting fatty acids from the cell, C. reinhardtii cells were transformed with one of the nucleic acids described in Example 3 that contains a coding sequence for a fatty acid transporter operably linked to a promoter sequence. No antibiotics were used during this assay.

[0299] The cells were artificially fattened to increase the intracellular fatty acid concentration (Bai et al. Long-chain acyl-CoA synthetases activate fatty acids for lipid synthesis, remodeling and energy production in Chlamydomonas, New Phytologist (2022) 233: 823-837). Briefly, the transformed cells were cultured at 2 × 10 7 Cells were grown to a cell density of 1000 and then transferred to nitrogen-depleted TAP medium with regular acetate or triacetate (TAP-N / T5AP-N). Cells were stained with the lipophilic fluorescent dye BODIPY: 30 μL of late logarithmic phase cells were gently mixed with 0.3 μL of BODIPY 495 / 505 (1:100 dilution) at a concentration of 1 mg / mL in DMSO. Cells were protected from light and incubated for 5 min, after which 5 μL was placed on a regular slide and sealed with a #1.5 coverslip and nail polish.

[0300] BODIPY is a dye for oil and other non-polar lipids. After staining with BODIPY, the cells were directly subjected to fluorescence microscopy. Representative images are shown in Figure 6. Panels a, c, e, and g of Figure 6 are bright field images. Panels b, d, f, and h of Figure 6 are fluorescent images of the same fields. Panels a and b show C. reinhardtii transformed with only nucleic acid encoding an antibiotic selection marker (mock control). Panels c and d show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter FATP1. Panels e and f show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter ABCG11. Panels g and h show C. reinhardtii transformed with nucleic acid encoding the fatty acid transporter ABCG15.

[0301] After 72 hours of nitrogen starvation, mock-transformed cells were much fatter, as indicated by the distinct lipid bodies that occupied a large fraction of the intracellular volume (see panels b, d, f, and h in Figure 6). No extracellular oil deposits were visible in the control cells. Cells transformed with fatty acid transporters showed a major difference, as shown in the representative images in Figure 6. Extracellular oil globules were seen in almost perfect spheres or were deposited on the surface of the cells (see arrows in panels d, f, and h in Figure 6).

[0302] The experimental results described in this Example demonstrate that a unicellular photosynthetic organism engineered to contain an exogenous nucleic acid sequence comprising a fatty acid transporter coding sequence operably linked to a promoter sequence is capable of transporting fatty acids from the interior of the cell across the cytoplasmic membrane as a result of expression of the fatty acid transporter.

[0303] [Example 7] Fatty acid transporters preferentially transport monounsaturated fatty acids This example illustrates that an engineered unicellular photosynthetic organism comprising an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence can preferentially transport monounsaturated long-chain free fatty acids (C16:1 and C18:1) from the interior of the cell across the cytoplasmic membrane to the surrounding culture medium.

[0304] C. reinhardtii cells were transformed with SLC27A1 (encoding the Homo sapiens flippase FATP1) as described in Example 3 or with the antibiotic cassette alone. Clones expressing SLC27A1 (sample A in Figures 7a and 7b) were selected to assess fatty acids secreted into the cell medium and compared with clones not expressing SLC27A1 (sample B in Figures 7a and 7b). Clones were cultured at 2 × 10 5The cells were grown to a cell density of 0.01 mg / ml and resuspended in high carbon, low nitrogen medium in the absence of antibiotics. Samples of cell-free medium were collected 5 days after treatment, diluted with isopropanol, and analyzed by liquid chromatography-mass spectrometry (LC-MS).

[0305] The cell-free medium samples were evaluated for the presence of saturated free fatty acids (C14:0, C16:0, and C18:0), monounsaturated free fatty acids (C16:1 and C18:1), and polyunsaturated free fatty acids (C18:2). Compared to the medium obtained from the clones that do not express SLC27A1, the medium obtained from the clones that express SLC27A1 contained significantly more monounsaturated long-chain free fatty acids, specifically about 5 times more palmitoleic acid (C16:1) and about 1.5 times more oleic acid (C18:1), respectively (see Figures 7a and 7b). No statistical differences were found for the saturated or polyunsaturated free fatty acids tested.

[0306] The results described in this Example demonstrate that unicellular photosynthetic organisms can be engineered to preferentially secrete monounsaturated fatty acids with chain lengths of C16 to C18 from the interior of the cell.

[0307] [Example 8] Expression of exogenous fatty acid ABC transporters in unicellular photosynthetic organisms suitable for biofuel production This example demonstrates successful transformation of the unicellular photosynthetic organism Chlorella sorokiniana with exogenous nucleic acid containing a coding sequence for an exemplary fatty acid ABC transporter (ABCG11).

[0308] Chlorella sorokiniana is one of the most promising microalgae strains provided by the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium project. C. sorokiniana cells (strain number: SAG211-32) were transformed using a PEG-based protoplast preparation method. Cells were grown to mid-log phase, concentrated, and resuspended in pectinase and cellulase to enzymatically remove the cell wall. Cells were washed and then incubated with PEG8000 and linearized plasmids carrying expression cassettes containing the fatty acid transporter gene ABCG11 and / or antibiotic resistance genes prepared in Example 3.

[0309] Cells were cultured in TAP medium, followed by cell lysis in 2xLDS buffer at 95°C. Gel electrophoresis was performed using Thermo Bis-Tris polyacrylamide gels, followed by protein transfer to PVDF membranes using Invitrogen iBlot2. Membranes were blocked with 7.5% skim milk for 20 min, then incubated with anti-His tag antibody (66005-1-Ig). Protein expression was detected using a goat anti-mouse secondary antibody that can be visualized with Li-Cor C-Digit. Expression of the ABCG11 transgene was visualized by a protein band of approximately 118 kDa corresponding to the ABCG11+6His tag (see Figure 8).

[0310] This example demonstrates successful transformation of the unicellular photosynthetic organism Chlorella sorokiniana (C. sorokiniana) cells with exogenous nucleic acid containing a coding sequence for a fatty acid ABC transporter.

[0311] [Example 9] Expression of various exogenous fatty acid transporters This example demonstrates successful transformation of the unicellular photosynthetic organism Chlorella sorokiniana with exogenous nucleic acids encoding a plant-derived fatty acid ABC transporter (ABCG11 or ABCG15) or a mammalian-derived flippase (FATP1).

[0312] Chlorella sorokiniana cells (strain number: SAG211-32) were transformed via a biolistic transformation process. Expression plasmids were precipitated onto solid-phase gold particles, which were fired into the microalgae using a PDS-1000 Biolistic Particle Delivery System (BioRad) according to the manufacturer's instructions. After particle bombardment, cells were resuspended and plated on agar-TAP plates in the presence of G418 (see Figure 9a) to select for transformants.

[0313] Transformed cells were identified after 5 days of selection. Clones were isolated and grown in liquid TAP medium. Expression of FATP1, ABCG15, and ABCG11 transgenes was determined using Western blot as described in Example 8 (Figure 9b and Figure 9c). Not all clones successfully expressed the respective transgenes. Samples in which successful transgene expression was detected are indicated by black rectangles in Figure 9b and Figure 9c.

[0314] This example demonstrates successful transformation of the unicellular photosynthetic organism Chlorella sorokiniana (C. sorokiniana) cells with exogenous nucleic acids encoding either a plant-derived fatty acid ABC transporter (ABCG11 or ABCG15) or a mammalian-derived flippase (FATP1).

[0315] [Example 10] Engineered unicellular photosynthetic cells expressing exogenous fatty acid transporters secrete fatty acids This example demonstrates that engineered unicellular photosynthetic organisms expressing exogenous fatty acid transporters secrete fatty acids into the culture medium.

[0316] Chlorella sorokiniana cells were transformed with one of the three fatty acid transporter-encoding nucleic acids described in Example 9. For each transgene, a set of four representative transformants that successfully expressed the transgene was selected to evaluate the secretion of fatty acids into the liquid culture medium. Prior to the secretion assay, the expression of the transgene was confirmed by Western blot as described in Example 9. The results are shown in Figure 10a. Bands whose molecular weights corresponded to FATP1, ABCG11, and ABCG15, respectively, were detected using an anti-His tag antibody.

[0317] Liquid cultures (in TAP medium) were prepared with one of the transformants from each set. The corresponding cultures with wild-type cells were incubated under identical conditions. Each culture was nitrogen starved for 2 days as described in Example 7. Cell-free medium samples were harvested and their free fatty acid content was determined using a free fatty acid quantification kit (Abcam). The free fatty acid content was normalized by cell density.

[0318] Unlike the wild-type controls, all transformants expressing the fatty acid transporters secreted readily detectable amounts of fatty acids into the growth medium (see FIG. 10b).

[0319] This example demonstrates that engineered unicellular photosynthetic organisms containing exogenous nucleic acids to express either a plant-derived fatty acid ABC transporter (ABCG11 or ABCG15) or a mammalian-derived flippase (FATP1) readily secrete fatty acids into the culture medium.

Claims

1. An engineered unicellular photosynthetic organism comprising an exogenous nucleic acid sequence comprising a coding sequence for a fatty acid transporter operably linked to a promoter sequence.

2. The engineered organism of claim 1 , wherein the fatty acid transporter is localized to the plasma membrane of the organism upon expression.

3. 3. The engineered organism of claim 1 or claim 2, wherein the fatty acid transporter is capable of transporting fatty acids having a carbon chain length of from C14 to C22 from the interior of a cell through the plasma membrane of the organism, and optionally the fatty acid is monounsaturated.

4. 4. The engineered organism of any one of claims 1 to 3, wherein the coding sequence for the fatty acid transporter is derived from the genome of a plant cell or a mammalian cell.

5. The engineered organism of claim 4 , wherein the coding sequence for the fatty acid transporter is derived from the genome of a plant cell.

6. The engineered organism of claim 5 , wherein the fatty acid transporter is an ABC transporter.

7. 7. The engineered organism of claim 6, wherein the ABC transporter is (a) the Arabidopsis thaliana ABCG11 protein or a functional homolog thereof, or (b) the Oryza sativa ABCG15 protein or a functional homolog thereof.

8. The engineered organism of claim 4 , wherein the coding sequence for the fatty acid transporter is derived from the genome of a mammalian cell.

9. The engineered organism of claim 8 , wherein the fatty acid transporter is an ABC transporter or a flippase.

10. 10. The engineered organism of claim 9, wherein the flippase is the Homo sapiens FATP1 protein or a functional homolog thereof.

11. 11. The engineered organism of any one of claims 1 to 10, wherein the organism is an algae, optionally wherein the algae is an oil-producing algae.

12. 12. The engineered organism of claim 11, which is an alga selected from Chlorophyta, Phaeophyta, Rhodophyta, Xanthophyta, Chrysophyta, Bacillariophyta, Cryptophyta, Dinophyta, Euglenophyta, Cyanophyta, and Myxophyta.

13. A culture comprising an organism according to any one of claims 1 to 12.

14. 13. A method for producing fatty acids, comprising culturing an organism according to any one of claims 1 to 12 in a medium suitable for the growth of said organism.

15. 1. A method for producing biofuel, comprising: a. providing a fatty acid obtained by the method of claim 14; and b. processing the fatty acids by transesterification, decarboxylation, or hydrogenolysis. A method comprising:

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