WO2011127118A1 - Methods of producing oil in non-plant organisms - Google Patents

Methods of producing oil in non-plant organisms Download PDF

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Publication number
WO2011127118A1
WO2011127118A1 PCT/US2011/031336 US2011031336W WO2011127118A1 WO 2011127118 A1 WO2011127118 A1 WO 2011127118A1 US 2011031336 W US2011031336 W US 2011031336W WO 2011127118 A1 WO2011127118 A1 WO 2011127118A1
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cell
neutral lipid
microbial
microbial cell
nucleic acid
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PCT/US2011/031336
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French (fr)
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Nicholas John Roberts
Kurtis Gale Knapp
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Algenetix, Inc.
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Publication of WO2011127118A1 publication Critical patent/WO2011127118A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the invention relates generally to oil production in microbial organisms and more specifically to use of constructs to modify organisms to produce and encapsulate oil.
  • Petroleum serves as the feedstock for the global fuel and chemical industries, however supplies are limited and nations are seeking renewable alternatives. While biological oils derived from crop species such as soybeans or palm are renewable, they lack the yield and resource efficiency needed to offset significant portions of the petroleum economy. Therefore, efficient microbial species that produce oil are needed.
  • the hydrocarbon product is not well tolerated by the modified microbial species and it is often secreted into the growth medium.
  • These production processes are limited to batch processes on account of the cell's limited tolerance of the hydrocarbon product. Therefore, there is a need for microbial organisms that simultaneously undergo cellular division, produce oil, and encapsulate that oil.
  • the present invention is based on discovery that certain microbial cells can be modified to produce and/or secrete oil.
  • methods are provided herein to introduce one or more nucleic acid molecules encoding specific enzymes and/or proteins into certain microbial cells.
  • the invention provides a method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle.
  • the method includes introducing into a microbial cell at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein and culturing the microbial cell in order to express the neutral lipid synthesizing enzyme and the neutral lipid encapsulation protein.
  • the nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in a single construct.
  • nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in separate constructs.
  • the constructs are incorporated into the nuclear genome, the chloroplast genome, autonomously replicating plasmid, or artificial chromosome of the microbial cell or the chloroplast genome.
  • two or more neutral lipid synthesizing enzymes are expressed in the cell.
  • two or more neutral lipid encapsulation proteins are expressed in the cell.
  • the invention provides a method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle by means of cross breeding two modified microbial cells.
  • the method includes introducing a first nucleic acid construct into a first microbial cell, wherein the first construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid encapsulation protein, and introducing a second nucleic acid construct into a second microbial cell, wherein the second construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme.
  • the first and second microbial cells are cross-bred to produce a third microbial cell comprising the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein and the nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme.
  • the third microbial cell is then cultured in order to express the at least one neutral lipid encapsulation protein and the at least one neutral lipid synthesizing enzyme.
  • two or more neutral lipid synthesizing enzymes are expressed in the third cell.
  • two or more neutral lipid encapsulation proteins are expressed in the third cell.
  • the invention provides a method for producing an algal cell expressing at least one neutral lipid synthesizing enzyme.
  • the method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and culturing the algal cell in order to express the at least one neutral lipid synthesizing enzyme.
  • two or more neutral lipid synthesizing enzymes are expressed in the cell.
  • the invention provides a method for producing an algal cell expressing at least one neutral lipid encapsulation protein.
  • the method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, and culturing the algal cell in order to express the neutral lipid encapsulation protein.
  • two or more neutral lipid encapsulation proteins are expressed in the cell.
  • the invention provides a microbial cell that has been manipulated to produce at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein.
  • the invention also provides an algal cell which has been manipulated to produce at least one neutral lipid synthesizing enzyme, and/or an algal cell that has been manipulated to produce at least one neutral lipid encapsulation protein.
  • Exemplary neutral lipids include, but are not limited to triacylglycerol (TAG), sterol ester (SE), and wax ester (WE).
  • Exemplary neutral lipid synthesizing enzymes include, but are not limited to, acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol acyltransferase3 (DGAT3),
  • phospholipid:diacylglycerol acyltransferase PDAT
  • diacylglycerol:diacylglycerol transacylase PDAT
  • bifunctional wax ester synthase DGAT WS/DGAT
  • LCAT cholesterol acyltransferase
  • ACAT acyl-CoA: cholesterol acyltransferase
  • neutral lipid encapsulation proteins include, but are not limited to, oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
  • the nucleic acid molecule encoding the at least one neutral lipid synthesizing enzyme encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, any homolog thereof, and any ortholog thereof.
  • the neutral lipid synthesizing enzyme is an acyltransferase with enzyme classification 2.3.1.X, where X is a variable that can be any integer.
  • X is a variable that can be any integer.
  • "2.3.1.X" represents enzyme classifications 2.3.1.20, 2.3.1.75, 2.3.1.158, etc.
  • the at least one neutral lipid encapsulation protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-29, a homolog thereof, and an ortholog thereof.
  • the neutral lipid encapsulation protein is modified. Exemplary modifications include, but are not limited to, one or more gene fusions on a single polypeptide, and at least one cysteine residue introduced into hydrophilic portion of the encapsulation protein.
  • the microbial cell may be a prokaryote or a eukaryote.
  • the phase of the cell cycle includes the GO, Gl, S, G2, or M phase.
  • the microbial cell is an oleaginous species.
  • the microbial cell is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates).
  • the microbial cell is an algal cell of the species Chlamydomonas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridi m, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parietochloris.
  • the algal cell is Chlamydomonas reinhardtii.
  • the cell is from the genus Yarrowia, Candida,
  • Rhodotorula Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, Pythium,
  • the cell is a bacterium of the genus Rhodococcus, Escherichia, or a cyanobacterium.
  • the cell is a yeast cell.
  • the cell is a synthetic cell.
  • the microbial cell may be cultured in a batch culture, fed-batch culture, or continuous culture.
  • the cell produces a neutral lipid and undergoes cellular division simultaneously.
  • the cell produces a neutral lipid, stores a neutral lipid, and/or excretes a neutral lipid.
  • the cell is cultured in a fermentor, photobioreactor, open pond, or any combination thereof.
  • the cell is part of a culture that is in the lag, logarithmic, or stationary growth phase.
  • the microbial cell is manipulated to produce a neutral lipid independent of an external stressor.
  • An exemplary external stressor is an abiotic stress, such as nutrient deprivation.
  • the microbial cell simultaneously produces and accumulates a neutral lipid while continuing to grow.
  • the invention provides a construct including at least one microbial promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, wherein the at least one microbial promoter is operatively linked to the nucleic acid molecules so as to cause expression of the at least one neutral lipid synthesizing enzyme and the at least one neutral lipid encapsulation protein in a microbial cell.
  • the invention also provides uses of the constructs of the invention to induce a microbial cell to express the at least one neutral lipid synthesizing enzyme and the at least one neutral lipid encapsulation protein.
  • the nucleic acid molecule encoding the at least one neutral lipid synthesizing enzyme has been modified or designed to enhance expression of the neutral lipid synthesizing enzyme in the microbial cell. In another embodiment, the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein has been modified or designed to enhance expression of the neutral lipid encapsulation protein in the microbial cell.
  • the modifications to the nucleic acid molecules include matching the approximate proportion of guanine and cytosine to adenine and thymine in the construct to the proportion of guanine and cytosine to adenine and thymine in the genome of the microbial cell, choosing codons that are most highly representative of proteins encoding genes in the genome of the microbial cell, avoiding codons that are used in less than 10% of all possible instances in the genome of the microbial cell, inclusion of an intron, exclusion of unwanted mRNA splice sites, and/or minimization of mRNA degradation.
  • FIG. 1 is a pictorial diagram showing the biochemistry of triacylglycerol (TAG) production in most organisms.
  • TAG triacylglycerol
  • Figure 2 is a pictorial diagram showing fatty acid and triacylglycerol production in plants and algae.
  • Bold numbers represent key enzymes: 1, plastidic pyruvate kinase; 2, acetyl CoA carboxylase; 3, acyl ACP thioesterasees Fat A and FatB; 4, glycerol-3-phosphate acyl transferase; 5, lyso-phophatidic acid acyl -transferase; 6, diacylglycerol acyl transferase; and 7, lyso-phosphatidylcholine acyl transferase.
  • 3PGA 3-phosphoglycerate
  • DAG diacylglycerol
  • ER endoplasmic reticulum
  • FAS fatty acid synthesis
  • G3P glycerol-3- phosphate
  • G6P glucose-6-phosphate
  • LP A lyso-phosphatidic acid
  • LPC lyso- phosphatidylcholine
  • PA phosphatidic acid
  • PC phosphatidylcholine
  • PEP
  • FIG. 3 is a pictorial diagram showing the structure of oil bodies and their mechanism of production. Lipids are irreversibly converted to oil and deposited between the lipid bilayer by the enzyme DGAT in the ER. The oleosin protein targets to this region and eventually dissociates into an oil body. In the cut-away of an oil body on the left, the hydrophobic tails of oleosin (lighter area) anchor into the oil (darker area) and a charged surface is presented to the outside.
  • Figure 4 is a pictorial diagram showing cross sections of an oil body, triglyceride oil is surrounded by a phospholipid layer and oleosin proteins.
  • the left panel shows individual oleosins
  • the next panel is the fusion protein form of polyoleosin
  • the third panel is the disulfide bond form of polyoleosin
  • the fourth panel (far right) shows the engineered oleosins in a reduced form with the cysteine residues facing the cytoplasm.
  • FIG. 5 is a pictorial diagram showing an example of a genetic modification strategy to produce fermentation products photosynthetically in cyanobacteria.
  • TAG is many steps away from pyruvate, but the scheme demonstrates the large fundamental changes to biology being done using modern biotechnology.
  • FIG. 6 is a pictorial diagram showing the biochemistry of a microbial cell modified to produce and excrete oil.
  • DGATl is expressed in the endoplasmic reticulum to convert lipids into the storage form of oil.
  • the increased oil will be stabilized by encapsulating it in neutral lipid storage structures surrounded by oleosin.
  • FIG. 7 is a pictorial diagram showing the biochemistry of the "chloroplast-based" route to improved oil accumulation.
  • acyltransferases such as DGAT are expressed in the chloroplast to convert lipids into the storage form of oil.
  • the increased oil will be stabilized by encapsulating it in oil bodies in either plastid expressed oleosin or plastoglobulin.
  • Figure 8 is a pictorial diagram showing the structural arrangement of cyanobacteria, demonstrating that the methods of the invention can be adapted to both the algal chloroplast and to cyanobacteria.
  • Figure 9 is a pictorial diagram showing various pathways for production of various types of biofuel in microbial species. Note that this diagram does not explicitly show the production of neutral lipids, which must be subsequently processed into a fuel after production in the cell. Note as well that this diagram shows alcohols which are not part of the invention.
  • Figure 10 is a graphical diagram depicting the advantage of a continuous process compared to a batch process in utilization of capital intensive process equipment.
  • Figure 11 is a pictorial diagram showing the process of neutral lipid production in unmodified microbial species. The process is necessarily a batch process where cells are first grown (“biomass”) then starved of nitrogen to produce oil.
  • Figure 12 is a pictorial diagram showing the features of neutral lipid production in microbial species modified using the methods of the invention. The process can now be continuous because the cell produces biomass and oil at the same time.
  • Figure 13 is a pictorial diagram showing a batch process for the production of fuel from algae utilizing the "lipid trigger.”
  • Figure 14 is a pictorial diagram showing a continuous process for making fuel utilizing the methods of the invention.
  • Figure 15 is a pictorial diagram showing a map (MAP 1) of an expression cassette for one gene of interest for transformation into Chlamydomonas reinhardtii.
  • Figure 16 is a pictorial diagram showing a map (MAP 2) of an expression cassette for two genes of interest for transformation into Chlamydomonas reinhardtii.
  • Figure 17 is a pictorial diagram showing a map (MAP 3) of an expression cassette for two genes of interest for transformation into Chlamydomonas reinhardtii.
  • Figure 18 is a pictorial diagram showing a map (MAP 4) of an expression cassette for transformation of 01e_3,3 and AtDGATl into Saccharomyces cerevisiae.
  • Figure 18 discloses "6x His" as SEQ ID NO: 209.
  • Figure 19 is a pictorial diagram showing a map (MAP 5) of an expression cassette for transformation of MLDP and CsDGAT2 into Saccharomyces cerevisiae.
  • Figure 20 is a pictorial diagram showing a map (MAP 6) of an expression cassette for transformation of 01e_3,3 and CsDGAT2 into Saccharomyces cerevisiae.
  • Figure 21 is a graphical diagram showing hydrophobicity plots of MLDP from both Chlamydomonas and Volvox. Arrows indicate regions with relatively high hydrophilic properties that may be exploited to engineer insertion of cysteine residues.
  • Figure 22 is a map showing 5' UTR, position of intron 1 and exons relative to nucleic acid sequence (SEQ ID NO: 39) and peptide sequence (SEQ ID NO: 63) of Chlamydomonas reinhardtii native MLDP.
  • Figure 23 is a microscopy comparison of Nile Red fluorescence from Chlamydomonas reinhardtii cells that were either: from a nitrogen starved culture (left), from a vector only transformed culture at log phase (center), or from a log phase culture that had been transformed with the full length HRp-DGATl-V5-RBCS2At;HRIp-oleo 0,0-RBSCA2t construct (right).
  • Figure 24 is a relative quantitative comparison of neutral lipid accumulation in transgenic Chlamydomonas reinhardtiii expressing NITlp-CrDGAT2-RBCS2At where cells harboring the NITlp-CrDGAT2-RBSC2At cassette were shown to accumulate nearly 2-fold more neutral lipids than those harboring the NITlp-CrDGAT2-CrDGAT2-3'UTR cassette after 8h of induction.
  • Figure 25 is a graphical diagram showing the total lipids extracted per milligram of dry yeast cells expressing DGAT and lipid encapsulating proteins during the lag phase.
  • Figure 26 is a series of confocal microscopy images of Nile Red stained
  • the present invention is based on the discovery that certain microbial cells can be modified to produce and encapsulate oil. Using the techniques provided herein, nucleic acid molecules encoding certain enzymes and/or proteins are introduced into microbial cells, thereby causing the cells to produce oil.
  • oil refers to any hydrocarbon including all alkanes, alkenes, alkynes, and aromatic hydrocarbons.
  • Oils also include biological oils that are largely, but not entirely composed of carbon and hydrogen.
  • Exemplary biological oils include, but are not limited to, lipids, fats, waxes, sterols, fatty acids, fatty alcohols, fatty esters, polyketides, isoprenes, monoglycerides, diglycerides, phospholipids, and neutral lipids. Oils are largely immiscible in water.
  • neutral lipid refers to any lipid having no polar group thereby rendering the lipid unable to integrate into bilayer membranes in substantial amounts.
  • neutral lipids have hydrophobic tails, they do not have a hydrophilic (charged) head.
  • neutral lipids include, but are not limited to, triacylglycerols (TAGs), sterol esters (SEs) and wax esters (WEs).
  • TAGs triacylglycerols
  • SEs sterol esters
  • WEs wax esters
  • the main storage lipids in eukaryotes are triacyl glycerol (TAG) and sterol esters.
  • TAG triacyl glycerol
  • WEs are also used as an energy store
  • neutral lipid fractions may also contain one or more additional lipidic compounds, including, but not limited to,
  • triacylglycerol or “TAG” refers to is a glyceride in which the glycerol is esterified with three fatty acids. Triglycerides are formed from a single molecule of glycerol, combined with three fatty acids on each of the OH groups. Ester bonds form between each fatty acid and the glycerol molecule. Most plants synthesize and store significant amounts of TAG only in developing seeds and pollen cells where it is subsequently utilized to provide catabolizable energy during germination and pollen tube growth. Dicotyledonous plants can accumulate up to approximately 60% of their seed weight as TAG. Ordinarily, this level is considerably lower in the monocotyledonous seeds where the main form of energy storage is carbohydrates ⁇ e.g., starch).
  • TAG is the desired cellular metabolite for a number of products including vegetable oil, omega-3 oils and renewable fuels among others.
  • Acetyl-CoA is a general cellular metabolite that can be synthesized through a number of basic metabolic pathways. There are differences among species, but the enzyme ACC (acetyl-CoA carboxylase) draws on the common pool of Acetyl-Co A. This is the first committed step in the production of TAG in a plant. (See Figures 1 and 2). Once carbon is committed toward lipid or oil production by ACCase, fatty-acid synthase (FAS) produces the individual fatty acid chains in the chloroplast.
  • FAS fatty-acid synthase
  • oil storage structure refers to a microscopic droplet of oil surrounded by a means for encapsulation.
  • the means for encapsulation can include a monolayer of an "oil encapsulation protein".
  • the means for encapsulation can also include a layer of phospholipid.
  • the function of the oil storage structure is to isolate the hydrophobic oil component from the aqueous environment in a controlled manner.
  • the outer layers of the oil storage structure usually present a hydrophobic surface to the interior of the oil storage structure and a hydrophilic surface to the exterior of the structure. 2011/031336
  • neutral lipid storage structures where the droplet of an oil is more specifically a neutral lipid (e.g., triacyl glycerol (TAG)) surrounded by a monolayer of phospholipid where the hydrophobic acyl moieties of the phospholipids interact with the encapsulated lipid and the hydrophilic head groups face the exterior.
  • a neutral lipid e.g., triacyl glycerol (TAG)
  • TAG triacyl glycerol
  • a neutral lipid storage structure is an "oil body”.
  • Oil bodies are typically found in plant seeds. Oil bodies are typically 0.5-2.5 ⁇ in diameter and consist of a TAG core surrounded by a phospholipid monolayer embedded with proteinaceous emulsifiers - predominantly oleosins. The size and number of oil bodies depends on the ratio of oleosin to TAG within the plant cell (Siloto, Findlay et al. 2006). Oil bodies consist of only 0.5-3.5% protein; of this 80-90% is oleosin with the remainder predominantly consisting of the calcium binding (caoleosin) and sterol binding (steroleosin) proteins. It should be understood that the term "neutral lipid storage structure” also includes artificial or synthetic oil bodies that are formed in microbial hosts using the methods disclosed in this invention.
  • plastoglobule refers to a lipoprotein particle inside chloroplasts that contains biosynthetic enzymes and a variety of lipidic compounds, including
  • Plastoglobules contain compounds that confer color to fruits and flowers ⁇ e.g., ripening peppers structurally reorganize their thylakoid membranes and accumulate plastoglobuli).
  • oil encapsulation protein refers to a protein that presents hydrophilic amino acids to the exterior of an oil storage structure and hydrophobic amino acids to the interior. It is known that oil encapsulation proteins can also include a long consecutive stretch of hydrophobic amino acids that extend into the bulk of the encapsulated oil to anchor the oil encapsulation protein to the oil storage structure.
  • neutral lipid encapsulation proteins where the droplet of oil is more specifically a neutral lipid (e.g., triacylglycerol (TAG)).
  • neutral lipid encapsulation protein refers to any protein that surrounds a neutral lipid to produce a neutral lipid storage structure. Nature provides many examples of neutral lipid storage structures and neutral lipid encapsulation proteins.
  • Exemplary natural lipid encapsulation proteins include, but are not limited to, oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
  • neutral lipid encapsulation protein is synthetic versions of any of the proteins having the emulsification and encapsulation properties of the encapsulation proteins as a function of its arrangement and sequence of hydrophilic and hydrophobic amino acid residues.
  • Oleosin Zea mays NM 001 153560.1 76 NP 001147032.1 77
  • Steroleosin Zea mays NM 001 159142.1 88 NP 001 152614.1 89
  • Caoleosin Zea mays NM 001 158434.1 96 NP 001 151906 97
  • Oleosin madephdq tdviksylpekgpstsqvlavvtlfplgavllclagliltgtiiglavatplfVifspilv 15 (S. indicum) paaltialavtgfltsgafgitalssiswllnyvrrmrgslpeqldharrrvqetvgqktreagqrsqdv
  • Caoleosin mssyspppppppprdqsmdteapnapitrerrlnpdlqeqlpkpylaraleavdpshpqgtkgrdpr 20 (Z mays) gmsvlqqhaaffdrngdgviypwetfqglraigcgltvsfafsilinlflsyptqpgwlpspllsirid
  • MDLP maesagkplkhlefVhtyahkfasgaayveggyqkaktyvpavaqpyiakaeetclayaaplat 21 (C. reinhardtii) katdhaekilrstdaqldalyaasaswlsssqkladsniaafrgaadkyydlvkstaqhvtsklptdl
  • oleosin refers to specific plant proteins that are usually found only in seeds and pollen. The properties of the major oleosins are relatively conserved among plants. Oleosins allow oil bodies to become tightly packed discrete organelles without coalescing as the cells desiccate or undergo freezing conditions (Siloto, Findlay et al. 2006). Such oleosins are typically 15-25kDa proteins, which corresponds to approximately 140-230 amino acid residues. Oleosins have three functional domains consisting of an amphipathic N- terminal arm, a highly conserved central hydrophobic core (-72 residues) and a C-terminal amphipathic arm.
  • the accepted topological model is one in which the N- and C-terminal amphipathic arms are located on the outside of the oil body and the central hydrophobic core is located inside the oil body.
  • the negatively charged residues of the N- and C-terminal amphipathic arms are exposed to the aqueous exterior whereas the positively charged residues are exposed to the oil body interior and face the negatively charged lipids.
  • the amphipathic arms with their outward facing negative charge are responsible for maintaining the oil bodies as individual entities via steric hindrance and electrostatic repulsion both in vivo and in isolated preparation.
  • the N-terminal amphipathic arm is highly variable and as such no specific secondary structure can describe all examples.
  • the C-terminal arm contains an oc-helical domain of 30-40 residues (Tzen, Wang et al. 2003).
  • the central core is highly conserved and thought to be the longest hydrophobic region known to occur in nature; at the center is a conserved 12 residue proline knot motif which includes three spaced proline residues.
  • the secondary, tertiary and quaternary structure of the central domain is still unclear.
  • Modeling, Fourier Transformation-Infra Red (FT-IR) and Circular Dichromism (CD) evidence exists for a number of different arrangements (for review, see Roberts et al., 2008; Frandsen et al, 2001; Tzen et al, 2003).
  • oil bodies also include "caoleosin”.
  • Caoleosin has a slightly different proline knot than do the basic oleosins, and contain a calcium-binding motif and several potential phosphorylation sites in the hydrophilic arms (Frandsen, Mundy et al. 2001). Similar to oleosin, caoleosin is proposed to have three structural domains, where the N- and C-terminal arms are hydrophilic while the central domain is hydrophobic and acts as the oil body anchor.
  • the N-terminal hydrophilic domain consists of a helix-turn-helix calcium binding EF-hand motif of 28 residues including an invariable glycine residue as a structural turning point and five conserved oxygen-containing residues as calcium-binding ligands (Frandsen, Mundy et al. 2001).
  • the C-terminal hydrophilic domain contains several phosphorylation sites and near the C- terminus is an invariable cysteine that is not involved in any intra- or inter-disulfide linkages.
  • the hydrophilic N- and C-termini of caoleosin are approximately 3 times larger than those of oleosin.
  • the hydrophobic domain is thought to consist of an amphipathic a-helix and an anchoring region (which includes a proline knot).
  • oil bodies also include "steroleosin” (Tzen, Wang et al. 2003).
  • Steroleosins include an N-terminal anchoring segment that includes two
  • amphipathic a-helices (approximately 912 residues in each helix) connected by a hydrophobic anchoring region of 14 residues.
  • the soluble dehydrogenase domain contains a NADP+- binding subdomain and a sterol-binding subdomain.
  • the apparent distinction between steroleosins-A and -B occurs in their diverse sterol-binding subdomains (Lin and Tzen 2004).
  • Steroleosins have a proline knob in their hydrophobic domain and contains a sterol-binding dehydrogenase in one of their hydrophilic arms.
  • Plastoglobules have a number of associated metabolic enzymes and structural proteins called "plastoglobulins". It has been shown that the availability of plastoglobulins regulates the formation of plastoglobuli in much the same way that oleosin is required for the formation of oil bodies. Plastogobulins surround
  • the coat may contain receptors for attachment to the thylakoid membrane as well as regulatory proteins that may function in the transfer of lipids to and from the thylakoid membranes.
  • MLDP Major Lipid Drop Protein
  • apolipoproteins form low-density lipoproteins (LDLs) when they encapsulate a core of cholesterol and cholesterol esters (sterol ester) to transport dietary fats through the bloodstream.
  • LDLs low-density lipoproteins
  • Apolipoproteins also serve as enzyme co-factors, receptor ligands, and lipid transfer carriers that regulate the metabolism of lipoproteins and their uptake in tissues.
  • Perilipin also known as “lipid droplet-associated protein” or PLIN, is a protein which coats lipid droplets in adipocytes, the fat-storing cells in adipose tissue. Perilipin acts as a protective coating from the body's natural lipases, which break TAG into glycerol and free fatty acids for use in metabolism. In humans, perilipin is expressed in three different isoforms, A, B and C, with perilipin A being the most abundant.
  • Neutral lipid encapsulation proteins such as oleosin, steroleosin, caoleosin, plastoglobulin, MLDP, apolipoprotein and perilipin are well known to those skilled in the art. Further sequences from many different species can be readily identified by methods well-known to those skilled in the art. In various embodiments, the neutral lipid encapsulation protein (e.g., oleosin) may be modified or mutated.
  • polyoleosin was formed from the end to end fusion of two or more oleosin units (Roberts et al. 2008).
  • polyoleosin refers to the fusion of any number of any neutral lipid encapsulation proteins.
  • altering the number of oleosin units enables the properties (thermal stability and degradation rate) of the oil bodies to be tailored.
  • Polyoleosin allows oil bodies to withstand extreme conditions such as heating at 95°C or incubation in rumen fluid for 24 hours.
  • the exposed portions of the protein can be digested with proteinase K and the remaining hydrophobic core still stabilizes the oil body.
  • Expression of polyoleosin in planta leads to incorporation of the polyoleosin units to the oil bodies as per single oleosin units (Scott et al, 2007).
  • oleosins containing a cysteine on the exposed arm can impart properties that prevent it's break down in the cell (PCT NZ2010/000218).
  • PCT NZ2010/0002128 By altering the number and position of the cysteines engineered into the hydrophilic arms of oleosins it is also possible to modulate the degree of stability of the oil bodies.
  • the preferred means for oil encapsulation is to adapt one of nature's neutral lipid encapsulation proteins to form a neutral lipid storage structure.
  • neutral lipid encapsulation proteins could be adapted or improved to encapsulate other oils.
  • an oil encapsulation protein could be designed or selected that bears little sequence homology to proteins found in nature, but is suitable as a means for encapsulation.
  • neutral lipid synthesizing enzyme refers to any enzyme required to convert fatty acids to neutral lipid within a cell.
  • neutral lipid synthesizing enzymes include, but are not limited to, acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl
  • DGAT2 CoA:diacylglycerol acyltransferase2
  • DGAT3 acyl CoA:diacylglycerol acyltransferase3
  • PDAT phospholipid:diacylglycerol acyltransferase
  • WS/DGAT bifunctional wax ester synthase DGAT
  • the preferred embodiment employs neutral lipid as the oil of choice, but it would be recognized by one skilled in the art that the teachings can be applied to other oils.
  • neutral lipid synthesis enzymes are employed as the means for synthesizing neutral lipid, but other means known in the art such as expressing transcription factors or starving an algal cell of nitrogen may similarly be employed.
  • neutral lipid synthesis enzymes are the method to focus on hereafter.
  • DGAT Diacylglycerol acyltransferase
  • Figure 3 Diacylglycerol acyltransferase
  • DGAT deposits TAG between the leaves of the endoplasmic reticulum bilayer.
  • a neutral lipid encapsulation protein is then targeted to these portions of the ER, where eventually an oil body dissociates into the cytoplasm.
  • DGAT is the branch point in the oil pathway where lipids destined for storage are split from those destined to constitute biological membranes.
  • DAG diacylglycerol
  • Over-expression of DGAT deprives the cell of diacylglycerol (DAG) needed for membranes, leading to increased DAG synthesis in the cell to compensate.
  • DAG diacylglycerol
  • increasing the rate of oil synthesis is only half of the challenge in accumulating neutral lipids.
  • the oil must also be encapsulated in the cell to prevent catabolism of the oil or harmful effects on the cell.
  • the coupled interaction between the cell's means for oil synthesis and means for oil encapsulation ( Figure 6) is critical and is the phenomena that inspired the best mode of this disclosure.
  • DGAT1 maildsagvttvtengggefvdldrlrrksrsdssnglllsgsdnnspsddvgapadvrdridsvv 1 (A. thaliana) nddaqgtanlagdnngggdnngggrgggegrgnadatftyrpsvpahrraresplssdaifkqsh
  • DGAT1 mavaessqntttmsghgdsdlnnfrrrkpsssviepsssgftstngvpatghvaenrdqdrvgam 2 (T. majus) enatgsvnligngggvvigneekqvgetdirftyrpsfpahrrvresplssdaifkqshaglfhlciv
  • DGATl generally has a broad substrate specificity, whereby the fatty acid found in the sn-3 position of TAG is proportional to the concentration of that fatty acid in the larger pool.
  • DGAT2 has tighter substrate specificity, and might channel unusual fatty acids into TAG so they do not upset the function of the biological membranes.
  • DGAT3 a new version was recently found to be located in the cytoplasm of peanut.
  • DGATl was first cloned from Arabidopsis in 1999. Over-expression of the gene increased TAG accumulation by 10-70% in Arabidopsis seeds. In tobacco, TAG was increased seven-fold and appeared as lipid droplets in the cells. It has subsequently been discovered that changing a single amino acid in the enzyme abolishes a post-translational phosphorylation regulatory mechanism, such that the enzyme remains active in non-seed tissues. The mutant increased DGATl activity by 38-80%, which led to a 20-50%) increase in oil content on a per seed basis in Arabidopsis.
  • the DGAT1 enzyme from Arabidopsis is functional between kingdoms, as expression of the gene in yeast resulted in a 200-600 fold increase in DGAT enzyme activity, which led to a 3-9 fold increase in TAG observable as a floating layer of oil in the culture. Quite a lot is known about the specificities and modulating factors of the enzyme, but it does appear that the specific activity of the enzyme has been measured.
  • DGAT1 has been shown to be applicable, provided it accepts the fatty acid of choice. Plants generally incorporate long chain PUFAs in the sn-2 position. For the improved specificity for PUFAs, however, a DGAT2 that prefers these fatty acids may be beneficial, or the properties of DGAT 1 could be altered using a directed evolution procedure similar to those previously described.
  • Phospholipid:DAG acyltransferase forms TAG from a molecule of phospholipid and a molecule of diacylglycerol.
  • PDAT is quite active when expressed in yeast but does not appreciably increase TAG yields when expressed in plant seeds.
  • PDAT and a proposed DAG:DAG transacylase are neutral lipid synthesizing enzymes that produce TAG, but are not considered part of the Kennedy Pathway.
  • a combination wax ester synthase and DGAT enzyme has been found in all neutral lipid producing prokaryotes studied so far, and M. tuberculosis has 15 homologues thereof.
  • WS/DGAT has extraordinarily broad activity on a variety of unusual fatty acids, alcohols and even thiols.
  • This enzyme has a putative membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2 families from eukaryotes or the WE synthase from jojoba. (Jojoba is the only eukaryote that has been found to accumulate wax ester.)
  • LCAT Lecithin-cholesterol acyltransferase
  • ACAT cholesterol acyltransferase
  • nucleic acid constructs may be delivered using viral and non-viral methods.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, or algal organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined.
  • the term "construct" refers to an artificially assembled or isolated nucleic acid molecule that includes the gene or nucleic acid molecule of interest. In general, a construct may include the gene or genes of interest and appropriate regulatory sequences.
  • vectors encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources. Thus, an "expression vector” refers to a cloning vector that also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene of interest, so that the gene product of the gene can be expressed.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art ⁇ e.g. , Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current
  • Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
  • DNA plasmid via the vasculature U.S. Pat. No. 6,867,196, incorporated herein by reference
  • liposome mediated transfection Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987;
  • the nucleic acid encoding the enzyme or protein of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the enzyme or protein of interest may be stably integrated into the genome of the cell.
  • the term "genome” refers to the DNA or set of chromosomes or genes that make up an organism, and is passed to the organism's offspring. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene
  • the nucleic acid may be stably integrated in the chloroplast of the microbial cell. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers.
  • the addition of nucleic acid molecules to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. These DNA-lipid complexes are potential non-viral vectors for use in nucleic acid delivery.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a nucleic acid molecule into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993). Where liposomes are employed, other proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. [0089] Another transformation method includes ballistic transformation (U.S. Pat. No.
  • naked DNA is coated onto carrier particles such as gold and forcefully impacted into the cell, for example via compressed gas.
  • carrier particles such as gold
  • This method is useful for transformation of species with a thick cell wall, and also for transformation of the chloroplast (see, e.g.,(Manuell, Beligni et al. 2007), incorporated herein by reference).
  • agrobacterium-mediated transfection is used for transformation in plants. It has been shown that this method is applicable to algal species (see, e.g., (Bellucci, De Marchis et al. 2008), incorporated herein by reference).
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
  • the invention provides a method for producing a microbial cell expressing at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein.
  • the method includes introducing a nucleic acid construct into a microbial cell, wherein the construct includes, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid
  • the construct further includes at least one promoter.
  • nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein may either be contained in a single construct, or more likely in separate constructs, each of which being introduced into the microbial cell. In one embodiment, a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of constructs, each containing a nucleic acid molecule encoding a neutral lipid synthesizing enzyme or a neutral lipid encapsulation protein may be introduced into the cell.
  • microbial cell refers to any cell derived from a microbial organism.
  • microbial organism or “microbe” refers to any non-plant and non-animal single-celled organism to which the methods of the invention may be performed.
  • the microbial organism may be prokaryotic or eukaryotic.
  • Exemplary microbial organisms include, but are not limited to yeasts, bacteria, algae, protists, archaebacteria, and synthetic forms thereof (i.e., synthetic cells).
  • the method involves use of cross breeding two or more cells that have been modified with one or more nucleic acid molecules to express a neutral lipid
  • the method includes introducing a first nucleic acid construct into a first microbial cell, wherein the first construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid encapsulation protein, and introducing a second nucleic acid construct into a second microbial cell, wherein the second construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme.
  • the first and second microbial cells are cross-bred to produce a third microbial cell comprising the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein and the nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme.
  • the third microbial cell is then cultured in order to express the at least one neutral lipid encapsulation protein and the at least one neutral lipid synthesizing enzyme.
  • nucleic acid sequences are embodied in the present invention.
  • nucleic acid sequence or equivalents thereof refer to a DNA or RNA sequence.
  • the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
  • pseudoisocytosine 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- mefhylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5
  • variant refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantially 60% or greater homology with the nucleotide or amino acid sequence of the Figures, preferably 75% homology and most preferably 90-95% homology to the sequences of the present invention. - as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides) or BLAST X (nucleotides).
  • the variant may result from modification of the native nucleotide or amino acid sequence by such modifications as insertion, substitution or deletion of one or more nucleotides or amino acids or it may be a naturally-occurring variant.
  • variant also includes homologous sequences which hybridize to the sequences of the invention under standard hybridization conditions defined as 2 x SCC at 55°C, or preferably under stringent hybridization conditions defined as 2 x SSC at 65°C or very stringent hybridization conditions defined as 0.1 x SSC at 65°C, provided that the variant is capable of substantially performing the equivalent biological function of the neutral lipid/oil encapsulation protein; or the neutral lipid synthesizing enzyme; as would be required to perform the present invention.
  • the nucleotide sequence of the native DNA is altered appropriately. This alteration can be effected by synthesis of the DNA or by modification of the native DNA, for example, by site-specific or cassette mutagenesis.
  • portions of cDNA or genomic DNA require sequence
  • site-specific primer directed mutagenesis is employed, using techniques standard in the art.
  • the term 'manipulated', 'manipulation' or grammatical variations thereof refers to the alteration of genetic information in a microbial cell (e.g., an algal or plant cell), by a number of suitable genetic techniques, including, but not limited to:
  • nucleic acid molecule of interest to a cell; mutagenesis techniques; and/or traditional microbial breeding techniques (unless specifically excluded); or any combination thereof.
  • introducing when used in the context of inserting a nucleic acid molecule into a cell, means “transfection” or “transformation” or
  • transduction and includes reference to the incorporation or transfer of a nucleic acid molecule into a eukaryotic or prokaryotic cell where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
  • nucleic acid sequence is a "promoter” sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters.”
  • the promoter molecule may be an RNA, cRNA, genomic DNA or cDNA molecule, and me be single or double stranded.
  • the promoter molecule may also optionally include one or more synthetic, non-natural or altered nucleotide bases, or any combination thereof.
  • constitutive plant promoters examples include, but are not limited to, the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is incorporated herein by reference. Exemplary promoters and selection genes suitable for transformation and selection of algae are summarized in, e.g., (Walker, Collet et al. 2005) and (Hallmann 2007), both of which are incorporated herein by reference. Methods for transformation, promoters and selectable marker genes suitable for transformation of the chloroplast are known to those skilled in the art (see, e.g., (Bateman and Purton 2000), incorporated herein by reference).
  • transgenes include inclusion of an intron, optimization of G/C content, optimization of codon usage to match that of the host organism, elimination of cryptic splice sites, and elimination of mRNA degradation signals are known.
  • inclusion of an intron optimization of G/C content, optimization of codon usage to match that of the host organism, elimination of cryptic splice sites, and elimination of mRNA degradation signals are known.
  • a strain of Chlamydomonas has been developed that has high predictable levels of transgene expression (see, e.g., (Neupert, Karcher et al. 2008), incorporated herein by reference).
  • Exemplary terminators that are commonly used in plant transformation genetic construct include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
  • CaMV cauliflower mosaic virus
  • Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators the Zea mays zein gene terminator
  • the Oryza sativa ADP-glucose pyrophosphorylase terminator the Solarium tuberosum PI-II terminator.
  • nucleic acids embodied in the present invention are "operably linked" to each other or linked to a protein or peptide.
  • "operatively linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence.
  • the control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between a promoter . sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • polypeptide for example, neutral lipid synthesizing enzymes and/or neutral lipid encapsulation proteins.
  • Polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • ortholog refers to a functionally equivalent yet distinct corresponding nucleotide or amino acid sequence that may be derived from another plant.
  • an ortholog may have a substantially identical nucleotide or amino acid sequence to the sequences of the present invention as set forth in the sequence listing.
  • homolog refers to a related gene from a different but related species.
  • the invention provides a method for producing an algal cell expressing at least one neutral lipid synthesizing enzyme.
  • the method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and culturing the algal cell in order to express the at least one neutral lipid synthesizing enzyme.
  • the invention provides a method for producing an algal cell expressing at least one neutral lipid encapsulation protein.
  • the method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, and culturing the algal cell in order to express the neutral lipid encapsulation protein.
  • algae refers to a family of aquatic, eukaryotic single cell or multicellular organisms without stems, roots and leaves, that are typically autotrophic, photosynthetic, contain chlorophyll, and grow in bodies of water, including fresh water, sea water, and brackish water, with the degree of growth being in relative proportion to the amount of nutrients available.
  • microalgae refers to photosynthetic protists that include a variety of unicellular, coenocytic, colonial, and multicellular organisms, such as the protozoans, slime molds, brown and red algae, algal strains, diatoms, dinoflagellates, etc. It should be understood that while some definitions of "algae” include cyanobacteria because they are unicellular and photosynthetic, cyanobacteria are prokaryotic and are included in the definition of "bacteria” for the purposes of this invention.
  • Exemplary algae include, but are not limited to, organisms of the division of
  • Chlorophyta green algae
  • Rhodophyta red algae
  • Phaeophyceae brown algae
  • the alga is a species of Chlamydomonas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis,
  • the alga is Chlamydomonas reinhardtii.
  • Exemplary yeasts include, but are not limited to, oleaginous species of the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. It is also recognized that yeast species commonly used in biotechnology such as Saccharomyces or Pichia could be engineered to produce neutral lipid, and are therefore suitable host species for the methods of this invention.
  • Exemplary bacteria include, but are not limited to, oleaginous organisms from the genus Rhodococcus. It is also recognized that bacterial species commonly used in biotechnology such as Escherichia coli or Bacillus subtilis could be engineered to produce neutral lipid, and are therefore suitable host species for the methods of this invention. Exemplary bacteria also include cyanobacteria including, but not limited to, organisms from the genus Spirulina, Synechococcus or Synechocystis.
  • omega-3 fatty acids can be made in protists.
  • protists include, but are not limited to, the Heteromonyphyta and Alveolata.
  • the protist is an oleaginous species of the order Thraustochytriales.
  • the protist is from the genus Schizochytrium, Thraustochytrium, Ulkenia, or Pythium
  • synthetic cell refers to a single-celled organism that is created by man and not found in nature. While it is well known that all transgenic organisms are man-made and not found in nature, synthetic cells differ in the methods of modification and extensive degree to which they are modified or holistically designed. Typically, synthetic cells, rather than a simple transgenic organism, are created to provide an organism of minimal genome size and complexity upon which useful products can be made in a precise manner. (For a review of the current state of the field refer to (Carr and Church 2009), incorporated herein by reference).
  • a synthetic cell an entire genome is synthesized from chemical building blocks and transplanted into a naturally occurring organism, replacing the natural genome and therefore changing the species identity of the resulting cell.
  • researchers created Mycoplasma laboratoriwn from Mycoplasma genitalium through genome transplantation (U.S. Pub. No. 20070122826, incorporated herein by reference; and (Gibson, Benders et al.
  • the invention provides a microbial cell that has been manipulated to produce at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein.
  • the invention also provides an algal cell which has been manipulated to produce at least one neutral lipid synthesizing enzyme, and/or an algal cell that has been manipulated to produce at least one neutral lipid encapsulation protein.
  • DGAT and PDAT homologs can be identified in all algal genomes sequenced to date, although they exhibit distinct distribution patterns. Chlamydomonas reinhardtii has three genes encoding for DGATs but is missing a PDAT homolog. The diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum have both DGAT and PDAT. Ostreococcus tauri lacks a recognizable DGAT, but does have a PDAT. Cyanidioschyzon merolae has DGAT but not PDAT. Microscopy reveals that eukaryotic algae synthesize neutral lipids in the ER and deposit them in the cytoplasm. In addition, algae are also known to form plastoglobules. Plastoglobule-like structures constitute the eyespot of
  • neutral lipids do not seem to be a storage end-point in algae like they are in oil seeds.
  • neutral lipids are rapidly remobilized to rebuild the chloroplast when nitrogen is reintroduced after starvation. Oil Production in the Algal Chloroplast
  • the chloroplast is descended from an endosymbiotic cyanobacteria and is the "fat factory" of the cell. As such, it is envisioned that forcing this cell-within-a-cell to resemble a plant seed.
  • the chloroplast contains plastoglobules that share superficial similarities with plant oil bodies, but are dynamic structures and never fully dissociate for long term oil storage.
  • the instant invention provides methods to up-regulate the synthesis of oil into the plastoglobules by expressing acyltransferases such as DGAT.
  • acyltransferases such as DGAT.
  • the concomitant expression of oleosin or the native plastoglobulin may subvert the remobilization of oil and even dissociate a storage structure.
  • the chloroplast is an ideal organelle. Not only is it the cell's "fat factory", but it also accumulates much higher levels of recombinant protein due to a lack of epigenetic phenomena and ability to accommodate multiple copies of the transgene.
  • algae contain a single large chloroplast (compared with high plants which have many small ones) which greatly simplifies their transformation and regulation ( Figure 7).
  • the chloroplast-based strategy would aid extraction.
  • the cell wall and chloroplast outer membranes would still need to be ruptured, but by over expressing plastoglubulin or oleosin, stable storage structures are created. The purification of these structures by density gradient centrifugation has been demonstrated, similar to ER generated oil bodies.
  • DGAT over-expression causes a floating layer of oil to develop in the culture (Bouvier-Nave, Benveniste et al. 2000) (Beaudoin and Napier 2002) (Froissard, D'Andrea et al. 2009) (Beaudoin, Wilkinson et al. 2000). It has been shown that caoleosin can be expressed in yeast (Froissard, D'Andrea et al. 2009). Yeast also have an oil production system of their own consisting of four partially redundant enzymes. Dgalp is homologous to DGAT2 of plants. It is targeted to the ER, but also dissociates with and remains active on the lipid particle.
  • Lrolp has PDAT activity and some sequence homology to the plant PDAT, but the enzymes are evolutionarily distant.
  • Arelp and Are2p are DGATl-type enzymes, but minor in yeast compared with Dgalp and Lrolp.
  • Yeast also produce a number of sterol esters in addition to neutral lipids.
  • the DGAT1 - type enzymes (Arelp and Are2p) are also known as sterol acyltransferases (or sterol ester synthases) because of this activity.
  • Yeast store neutral lipids and sterol esters in a common "lipid particle" that has several ordered shells of sterol esters below the surface phospholipid monolayer, whereas the neutral lipids are randomly packed in the center.
  • the lipid particle typically has a diameter of about 0.4 ⁇ and has approximately 40 associated proteins.
  • yeast lipid particles are able to form neutral lipids autonomously.
  • the lipid particle associated multi-enzyme complex from the oleaginous yeast Rhodotorula glutinis was isolated, the proteins characterized, and shown to incorporate free fatty acids or fatty acyl-coenzyme A into neutral lipids quickly.
  • a pathway for complete de novo synthesis was reviewed.
  • a picture is emerging that the lipid particles of yeast are active organelles with a number of roles potentially including trafficking of lipids and proteins within the cell, chaperone activity and sequestration of small molecules and misfolded proteins. Oil Production in Bacteria
  • Bacteria generally produce polyhydroxyalkanoates rather than neutral lipids as an energy storage molecule, but exceptions include some Mycobacterium and Streptomyces species. For example, Rhodococcus opacus can accumulate neutral lipids to 87% by weight. The two pathways compete for acetyl-CoA in cases where both are present. As with other microbes, neutral lipids are produced during times of stress such as nitrogen starvation.
  • the responsible enzyme is a combination wax ester synthase and DGAT (WS/DGAT) that has extraordinarily broad activity on a variety of unusual fatty acids, alcohols and even thiols.
  • This enzyme has a putative membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2 families from eukaryotes or the WE synthase from jojoba. (Jojoba is the only eukaryote that has been found to accumulate wax ester.)
  • the WS/DGAT enzyme has been found in all neutral lipid producing prokaryotes studied so far, and M. tuberculosis has 15 homologues thereof.
  • the enzyme doesn't synthesize oil between the leaflets of the phospholipid bilayer. Rather, an oily layer develops just inside the plasma membrane and eventually blubs off oil drops of a characteristic size (-200 nm in Acinetobacter calcoaceticus). These droplets have a phospholipid membrane, but don't seem to have any structural proteins.
  • Cyanobacteria could have advantages over eukaryotic algae for the production of renewable oils, most significantly the ability of some species to fix atmospheric nitrogen.
  • GAP glyceraldehyde-3 -phosphate
  • the invention provides methods that are generally performed in a continuous process. Compared with a batch or fed-batch process, continuous processes better utilize large capital intensive pieces of equipment (Figure 10). Additionally, labor costs are generally higher for batch processes than for continuous processes. Furthermore, batch processes can be more difficult and expensive to control and produce a consistent product than a continuous process that operates at a steady state. The advantages of continuous processes over batch processes are magnified for fuel production where scales on the order of millions of metric tons per year are required and the fuel must be produced at a price competitive with petroleum-based products.
  • Category (a) is by definition a batch process because the cells alternate between periods of cell division and periods of oil production. Once sufficient oil is produced the cells must be harvested as a batch because returning to conditions of cellular growth would result in degradation of oil as an energy source for the cell ( Figure 11).
  • Category (b) is not by definition a batch process, but seems to be limited to a batch process in practice.
  • microbial species could be genetically modified to produce oil constitutively while continuing to grow, however nature does not have examples of organisms that make high levels of oil at all times.
  • Oil production is generally either i) a transient process in response to temporary nutrient limitation or stress (as in algae), ii) confined to a reproductive phase of life (as in plants), or iii) a backup of excess energy (as in animals).
  • nature usually stores the hydrocarbon in some kind of protective storage structure (e.g., oil body, plastoglobule, perilipin, apolipoprotien, etc.).
  • the present invention provides methods that enable microbial cells to grow vigorously and produce oil at the same time ( Figure 12).
  • the methods of the invention produce a microbial cell that resembles a plant seed in that it accumulates oil steadily and at the same time packages the oil into stable droplets.
  • Algae and plant seeds both accumulate lipids for energy storage, but do so under different circumstances and using different genetics.
  • plants synthesize oil in the seed to provide energy for germination.
  • lysed Once harvested, the cell typically needs to be broken open (i.e., "lysed") to release its oil.
  • Methods for lysing cells are known to those of skill in the art, and may be dependent upon strain selection, as some cells have thick, rigid cell walls like a vascular plant.
  • Options for lysis include, but are not limited to, osmosis, mechanical crushing, extraction with chemicals, sonication, and genetic modification.
  • the oil may be "partitioned" from the residual biomass for various reasons. For example, the value of the biomass as an animal feed is decreased if it contains too much residual oil.
  • the primary objective for creating oil storage structures in algae or other microbial species is to slow or eliminate oil remobilization and reduce feedback inhibition of oil synthesis, leading to continuous production of higher levels of oil.
  • the tighter physical structure and the negative charge of oleosin, for example will also likely reduce interactions between the oil and residual biomass during extraction. This is advantageous for 'cleanly' removing the oil from the lysed biomass.
  • Oil bodies for example are more than 97% pure oil, so being less dense than water, are easily extractable upon rupture of only the outer cell wall. Oil storage structures will not coalesce and therefore will not trap cell debris as unprotected oils would. Methods for converting storage oils into useful products are well known to those skilled in the art.
  • the methods of the invention will supply a superior feedstock for transesterification to biodiesel.
  • virgin oils have a higher proportion of TAG than waste oils, and therefore yield a better product requiring less refinement.
  • the oil neutral lipid structure formed by the methods of the invention is predominantly TAG for example.
  • accession numbers throughout this description are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A., and are all incorporated herein by reference. The accession numbers are provided in the database as of March 1, 2010.
  • Enzyme Classification Numbers The EC numbers provided throughout this description are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo, and are incorporated herein by reference. The EC numbers are as provided in the database as of March 1, 2010. [0150] All references, including any patents, patent applications, and literature, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art.
  • the genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.
  • the genetic constructs of the invention are intended to include expression constructs as herein defined.
  • the invention provides a host cell which comprises a genetic construct or vector of the invention.
  • Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g., Sambrook et al , Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al , Current
  • Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
  • the expressed recombinant polypeptide which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g., Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
  • Transformation strategies are available ⁇ e.g., electroporation, heat shock, glass beads).
  • Nuclear transformation in Chlamydomonas is achieved through electroporation (Brown et al., 1991, Shimogawara et al., 1998) or through vortexing in the presence of DNA of interest and glass beads (Kindle, 1990). Transformation of Chlamydomonas via electroporation for the strains ccl690 and ccl24 (from the Chlamydomonas Resource Center) which have intact cell walls does not require sucrose and hence it is omitted. Voltage parameters are 2000 V/cm, 25 uF capacitance, infinite ohms, 0.4 cm cuvettes using the Gene Pulser (Bio-Rad).
  • TAP media Harris, 1989
  • selection medium hygromycin 20 ⁇ g/mL, paromomycin 15 ⁇ g/mL
  • Single colonies are inoculated into liquid selection media (TAP + 5 ⁇ g/mL hyg, 10 ⁇ g/mL paro) for phenotypic and genetic analysis.
  • Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.
  • Strategies may be designed to increase expression of a polynucleotide/polypeptide in an algal cell, and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, and/or at a particular developmental stage which/when it is not normally expressed.
  • the expressed polynucleotide/polypeptide may be derived from the algal species to be transformed or may be derived from a different species.
  • Transformation strategies may be designed to reduce expression of a
  • polynucleotide/polypeptide in an algal cell or at a particular developmental stage which/when it is normally expressed.
  • Such strategies are known as gene silencing strategies.
  • Genetic constructs for expression of genes in transgenic algae typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed algae.
  • the promoters suitable for use in the constructs of this invention are functional in an algal cell, inducible promoters, constitutive promoters that are active in most algal cells, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired.
  • the promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other algae, plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention.
  • constitutive plant promoters include the ⁇ -tubulin ( ⁇ -TUBp) promoter, the Rubisco small subunit2A (RBCS2Ap) promoter, the heat shock 70A promoter (HSP70Ap) fused to the RBCS2A promoter (HRp).
  • Chlamydomonas promoter sequences have been shown to drive expression of heterologous reporter and selectable marker genes at low levels (e.g., Heitzer M. & Zschoernig, B., 2007, Fuhrmann et al., 1999).
  • heterologous reporter and selectable marker genes e.g., Heitzer M. & Zschoernig, B., 2007, Fuhrmann et al., 1999.
  • chimeric HSP70A-RBCS2A promoter which has been shown to drive the strongest level of expression of a reporter gene, luciferase.
  • Examples of an inducible promoter in algae include the nitrate reductasel (NITlp) promoter. Examples of promoter sequences are listed in Table 5.
  • Exemplary terminators that are commonly used in algae transformation genetic construct include, e.g., COPt, RBCS2At, MLDPt, DGAT2t, TUB2t (such sequences are listed in Table 5).
  • Selectable markers commonly used in algae transformation include the Streptomyces hygroscopicus aphVII and Streptomyces rimosus aphVIII selectable marker genes, whose gene products render the cells resistant to hygromycin and paromomycin, respectively (Berthold et al., 2002, Sizovaa, et al, 2001). aphVII and aphVIII sequences are listed in Table 6 and Table 7.
  • reporter genes coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in algae are also contemplated.
  • a visible signal e.g., luciferase, GUS, GFP
  • the algae of the invention may be grown and either allowed to divide or crossed with a different algal strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Algae resulting from such standard breeding approaches also form an aspect of the present invention.
  • HSP70Ap :RBCS2Ap chimera (with or without the RBCS2A intron 1 sequence embedded in it) (see sequence Tables 5 and 6 as well as Heitzer M. & Zschoernig, B., 2007, Schroda et al, 2000), was chosen to drive expression of the genes of interest.
  • NITlp The endogenous nitrate inducible promoter (NITlp), which in vivo drives expression of nitrate reductase, was also chosen to potentially overcome silencing.
  • This promoter was shown to drive expression of the arylsulphatase reporter gene (ARS) in the absence of ammonium and presence of nitrate (Ohresser et al, 1997, Loppes et al, 1999). Since this promoter does not drive expression in the presence of ammonium (the nitrogen source in TAP media), downstream genes of interest are not expressed until ammonium is removed from the media and replaced with nitrate.
  • ARS arylsulphatase reporter gene
  • the cell may not recognize such genes as foreign until their expression is turned on by the removal of ammonium (i.e., after they have been transformed, selected and grown enough biomass for phenotypic assays).
  • the GFP reporter is not expressed at high enough levels to visualize either by microscopy or Western blot unless it is fused to a selectable marker (e.g., the phleomycin resistance gene ble from Streptoalloteichus hindustanus) or an endogenous Chlamydomonas gene (chlamyopsin (COP)) (Fuhrmann et al, 1999).
  • a selectable marker e.g., the phleomycin resistance gene ble from Streptoalloteichus hindustanus
  • COP endogenous Chlamydomonas gene
  • Each promoter has been tested by placing the promoter upstream of the aphVII gene and the resulting cassette was used to transform Chlamydomonas with selection on paromomycin containing TAP agar plates.
  • Chlamydomonas genome More specifically using the 5' UTR from an endogenous gene, placing an endogenous Chlamydomonas intron into the coding sequence or 5'UTR, changing the codons to only those that are predominantly used by Chlamydomonas, using a 3' UTR from an endogenous gene, where possible using the 5' UTR as well as the first 8 amino acids and intron from the endogenous MLDP (GenBank: XP_001697668) or the first 8 amino acids and intron from the endogenous gene beta-2 tubulin (GenBank: AAA33102.1).
  • each construct or construct preparation typically minimizes the quantity of DNA used for transformation since it is recognized that silencing is enhanced when excessive DNA is used in the transformation.
  • the backbone of plasmids is typically of prokaryotic origin and therefore very different to the high GC rich gene coding regions of algae. Removal of as much as this as possible is preferable to reduce the degree of silencing. Cutting right at the end of the terminator or promoter is an option although this may have unintended consequences when the construct is inside the nucleus prior to its integration into the genome where exonuclease activity is high.
  • One approach to minimizing the selection of constructs in which the gene of interest has been partially or completely degraded but the selectable marker is still intact is to flank the gene of interest between two separate selectable markers. In this case, both paromomycin and hygromycin were chosen as the two selectable markers.
  • the genes of interest are in a back to back orientation facing the flanking selectable marker cassettes which themselves are both facing outwards (Table 8, Figures 15, 16 and 17).
  • wild-type algal cultures were grown to log phase, harvested by centrifugation (5 min @ 2000xg) and resuspended in nitrogen-free TAP media (i.e., TAP with NH 4 C1 omitted). Resuspended cells were transferred to 6-well plates and incubated with constant shaking under continuous light @ 25°C for 1-8 days.
  • the yeast expression vector pYES2.1 V5-His Topo was purchased from Invitrogen (Carlsbad, CA).
  • the quadruple yeast mutant Saccharomyces cerevisiae strain HI 246 was obtained from the Swedish University of Agricultural Sciences, Sweden. This yeast strain is deficient in all four genes (DGA1, LROl, ARE1 and ARE2) that encode the enzymes for lipid biosynthesis in yeast (Sandager, et al., 2002).
  • Yeast competent cells were prepared using S. c. EasyCompTM Transformation Kit (Invitrogen, Carlsbad, CA).
  • the Yeast synthetic incomplete medium (without uracil, histidine, leucine and tryptophan), yeast nitrogen base (YNB), D(+) Glucose, and D(+) Raffinose pentahydrate were purchased from Sigma (Sigma Aldrich Co., USA.). Bacto agar was procured from Difco (Detroit, MI, USA).
  • yeast cells were grown aerobically overnight in a synthetic medium with 0.67% YNB, without uracil (SC-U), and containing 2% raffinose. Cells from overnight culture were used to inoculate 200 mL of induction medium (SC-U containing 2% galactose and 1% raffinose) to an initial OD600 of 0.6. Cells were allowed to further grow at 30°C, with shaking at 200 rpm for 24 h. Cell pellets were collected by centrifugation at 1500 x g for 5 min then washed with distilled water and either used immediately for subsequent analysis or kept in -80°C until required. Cell pellets for neutral lipid extraction were freeze-dried for 48 h and stored in -20°C freezer until required.
  • nucleotide or translated amino acid sequences of the putative DGAT1 gene or protein were obtained from GenBank or TAIR with the following identification: Arabidopsis DGAT1 or AtDGATl (GenBank Accession no. AJ238008.1). The sequence was optimized for protein expression in yeast and were synthesized by Geneart (Geneart AG, Germany). The Chlamydomonans reinhardtii DGAT2 gene (GenBank Accession no XP_001693189);
  • Chlamydomonas reinhardtii MLDP gene (GenBank Accession no XP_001697668); Sesamum indicum oleosin gene (GenBank Accession no AAD42942) and Sesamum indicum oleosin gene engineered to contain cysteine residues were optimized for protein expression in Saccharomyces cerivisiae and were synthesized by GenScript (GenScript USA Inc.). Nucleic and translated sequences optimized for expression in S. cerivisiae are listed in Tables 9 and 10.
  • Chlamydomonas reinhardtii DGAT2 and Chlamydomonas reinhardtii MLDP were organized into the same plasmid in a back to back orientation each under the control of their own separate GALl promoter (Table 9 and Figure 19).
  • the Chlamydomonas reinhardtii DGAT2 and Sesamum indicum oleosin modified to contain cysteine residues were organized into the same plasmid in a back to back orientation each under the control of their own separate GAL1 promoter (Table 9 and Figure 20).
  • PCR program was executed: initial denaturation of 95°C for 5 min; then five cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 sec, and extension at 72°C for 1 min and 45 s; concluded by a final extension of 7 min.
  • the DGAT1 gene was cloned into the pYES2.1/V5 His topo vector using Topo TA cloning Kit (Invitrogen) following the manufacturer's protocol.
  • Topo TA cloning Kit Invitrogen
  • full length DNAs were sequenced using different sets of primers designed from different regions of the DNA. DNA sequencing was carried out using an ABI3730 DNA Analyser, Applied Biosystems, Inc.
  • Yeast transformation was conducted using the S. c. EasyCompTM Transformation Kit (Invitrogen). Briefly, after thawing competent cells, 50 xL was aliquoted into an Eppendorf tube and one microgram of DNA was added. After addition of Solution III (Invitrogen), the
  • DNA/competent cell mixture was vortexed vigorously and then incubated at 30°C for one hour, with the mixture being vortexed every 15 min.
  • the transformation reaction was added with one milliliter of SC medium and incubated at 30°C with shaking at 200 rpm for lh.
  • cells were pelleted by centrifugation at 3000 x g for 5 min, and resuspended in 100 ih of Solution III (Invitrogen). Finally, cells were plated onto SC- U selection plates and incubated at 30°C for 3 days.
  • the FAMES GC/MS was analyzed using the SGE capillary column BPX70 (50m x 0.22 mm x 0.25 ⁇ ).
  • the condition of GC-MS was as follows: the temperature was programmed from 80 °C to 150°C at 15°C /min and then to 250°C at 8°C /min and held isothermal for 10 min. Samples were injected in a split mode; total flow of 28.4 mL/min; column flow of 0.82 mL/min; and a purge flow of 3.0 mL/min.
  • the pressure was kept at 150 kPa; ion source temperature was 200°C and an interface temperature was kept at 260°C.
  • the target compounds were acquired by mass spectrometry in a scan mode starting at 50 m/z and ending at 350 m/z.
  • Neutral lipid from yeast was extracted using a modified method of that described by Ruiz-Lopez et al, (2003). For each analysis, 30 mg of freeze-dried yeast or algal cells (powdered using the glass beads) were placed in 13-mm screw cap tube, added with 2.4 mL of 0.17 M NaCl in methanol and mixed by vortexing. Following the addition of 4.8 mL heptanes and 10 iL of C14:0 (10 ⁇ g. ⁇ L "1 ) internal standard, the suspension was mixed gently and incubated without shaking in 80°C water bath for 2h. After cooling to room temperature, the upper lipidic phase was transferred to fresh screw-cap tube and evaporated to dryness under the stream of N gas. Finally, the dried powder was resuspended in 100 x heptane, mixed thoroughly then transferred to a flat- bottom glass insert fitted into a brown glass vial for GC MS analysis.
  • TAG analysis was performed on a Hewlett Packard (hp) gas chromatograph/mass spectrometer (QP2010) (Shimadzu Scientific Instruments Inc). All analyses were performed with a RESTEK capillary column, MXT®-65TG (65% diphenyl -35% dimethyl polysiloxane, 30.0 m x 0.10 ⁇ thickness x 0.25 mm diameter) in Electron Impact (EI) ionization mode. Helium was used as the carrier gas. All samples were injected in splitless mode at 1.0 ⁇ , aliquots and a column flow of 1.2 mL.min "1 .
  • EI Electron Impact
  • the gas chromatograph was programmed from 200 to 370°C at 15°C.min "1 then isothermal at 370°C for 15 min.
  • the sample injector port temperature was maintained at 350°C, column oven temperature at 200°C, with a pressure of 131.1 kPa, and a purge flow of 3.0 mL.min "1 .
  • the mass spectrometric conditions were as follows: the ion source temperature was held at 260°C during the GC-MS runs, the mass spectra were obtained at ionization voltage of 70 eV at an emission current of 60 ⁇ and an interface temperature of 350°C. Acquisition mode was by scanning at a speed of 5000, 0.25s per scan. Chromatograph peaks with mass to charge ratio of 150 m/z to 1090m/z were collected starting at 9 min and ending at 25 min.
  • Nile Red fluorescence assays were essentially performed as per James et al, 2011. Briefly, OD750 measurements were determined for transgenic and nitrogen starved algal cultures, the cells were diluted to an OD750 of 0.2-0.4 in appropriate media (selective media for transformants containing constitutive promoters or induction media for transformants containing the NITlp).
  • TAP-Nile Red TAP + 3.37 ⁇ g/mL Nile Red in acetone (or TAP + 4mM KN03 for NITlp constructs+ 3.37 ⁇ g/mL Nile Red in acetone)
  • TAP + 3.37 ⁇ g/mL Nile Red in acetone or TAP + 4mM KN03 for NITlp constructs+ 3.37 ⁇ g/mL Nile Red in acetone
  • fluorescence measurements were taken using the Gemini SpectraMax plate reader ex 485nm, em 600nm. Nitrogen starved algal cultures and vector only transformants were grown and analyzed side-by-side as positive and negative controls, respectively.
  • Nile Red fluorescence microscopy of algal transformants cells were grown as above (see growth, induction and nitrogen starvation section) and ⁇ , cells were aliquoted into new 96-well plates. 0.5 ⁇ of Q.5 xglmh Nile Red in acetone was added to each sample and allowed to incubate at RT in the dark for 10 min. ⁇ , aliquots were used to prepare slides and samples were visualized with the Olympus BX50 fluorescence microscope using filter cube U- MWIB excitation 460-495 nM, emission 515-700 nM. Nitrogen starved algal cultures and vector only transformants were grown and analyzed side-by-side as positive and negative controls, respectively.
  • lipid associated proteins engineered to contain additional cysteine residues in the N- and C- terminal hydrophilic arms (MLDP)
  • MLDP has no cysteine residues.
  • oleosins do not typically possess cysteine residues and when they are present they are only found in the hydrophobic portions and not the hydrophilic regions.
  • a hydrophobicity plot (using the yte and Doolittle hydrophobicity scale below with a window size of 19) of the MLDPs suggests their topology is not as simple as oleosins ( Figure 21).
  • GLU -3 500 GLY: -0 400 HIS: -3.200 ILE: 4 500 LEU: 3 800 ' LYS: -3 900
  • TYR -1 300 VAL: 4 200 : -3 500 : -3.500 -0 490
  • Chlamydomonas MLDP engineered peptide sequence is shown below:
  • transformant 17-4 which contains the full length HRp-DGATl-V5-
  • SEQ ID NOs: 49 and 50 accumulated levels of neutral lipid similar to the nitrogen starved control, as shown by fluorescence microscopy, while in the vector only no detectable neutral lipids were detected (Figure 23). Both transgenic samples (17-4 and vector only) were grown to log phase in selection media (as in growth, induction, and nitrogen starvation section above) and analyzed side-by-side with the nitrogen starved control.
  • RBSC2At cassette accumulate nearly 2-fold more neutral lipids than those harboring the NITlp-
  • Neutral lipids accumulate in Saccharomyces cerivisiae during lag growth phase when expressing AtDGATl and 01e_3,3 or CrDGAT2 and MLDP
  • Saccharomyces cerivisiae cells were transformed with constructs harboring the AtDGATl (SEQ NO: 60) or CrDGAT2 (SEQ NO: 62) and 01e_3,3 (SEQ NO: 61) or MLDP (SEQ NO: 63) arranged in various configurations (such as those seen in expression vectors SEQ NOs: 57, 58 and 59). Cells were induced by the addition galactose and allowed to grow for 8hr (transition of lag to log phase). Samples were taken for FAMES-GC/MS analysis (Figure 25) as well as for Nile Red fluorescence analysis by confocal microscopy ( Figure 26).
  • Table 5 Promoter and terminator sequences used to control the expression of nucleotide sequences encoding Arabidopsis thaliana DGATl, Sesamum inducum oleosin and oleosin engineered to contain cysteines, Chlamydomonas reinhardtii DGATl, MLDP and MLDP engineered to contain cysteines, Streptomyces rimosus aphVIII gene (paramomycin resistance), Streptomyces hygroscopicus aphVII gene (hygromycin resistance) in
  • COP terminator gatccggcaagactggccccgcttggcaacgcaacagtgagcccctcctagtgtgtttggggatgtgactat 35 (COPt) gtattcgtgtgttggccaacgggtcaacccgaacagattgatacccgccttggcatttcctgtcagaatgtaacgt
  • Tubulin2 atgccggcacctccatgcgccactgaacgtgtagcgtgactgtggcggccttggcagtttttgaccgtgactgac 37 3 'UTR and cctggacaaaggatccctgactgaagacaacttgacatgtgattgccatttgacgctttggtgtggaggcggatt
  • RBCS2A gcttcggcgtggcggccctgagcgtgctgtcctggatctaccgctacctgaccggcaagcacccgcccggcg
  • hygroscopicus aphVII gene (hygromycin resistance)]. and one gene of interest
  • ⁇ Chlamydomonas reinhardtii DGAT2, or Arabidopsis thaliana DGAT1_V5) both under the control of a separate promoter and terminator.
  • Nucleic Acid Sequences arranged in expression cassettes for transformation into Chlamydomonas reinhardtii consisting of two selectable markers [Streptomyces rimosus aphVIII gene (paramomycin resistance), and Streptomyces hygroscopicus aphVII gene (hygromycin resistance)] flanking two back to back genes of interest including a neutral lipid synthesising enzyme
  • Chlamydomonas expression vectors and a new dominant selectable marker.” Molecular & general genetics: MGG 263(3): 404.
  • GFP green Fluorescent protein

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Abstract

The present invention provides methods for producing oil in microbial cells. Also provided are methods for producing such cells and uses thereof.

Description

METHODS OF PRODUCING OIL IN NON-PLANT ORGANISMS
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims the benefit of priority under 35 U.S.C. § 1 19(e) of U.S. Serial No. 61/321,454, filed April 6, 2010, the entire content of which is incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 5, 201 1, is named CF127822.txt and is 539,799 bytes in size.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0003] The invention relates generally to oil production in microbial organisms and more specifically to use of constructs to modify organisms to produce and encapsulate oil.
BACKGROUND INFORMATION
[0004] Petroleum serves as the feedstock for the global fuel and chemical industries, however supplies are limited and nations are seeking renewable alternatives. While biological oils derived from crop species such as soybeans or palm are renewable, they lack the yield and resource efficiency needed to offset significant portions of the petroleum economy. Therefore, efficient microbial species that produce oil are needed.
[0005] Algae grow rapidly and produce oil, but not at the same time. Algae must be starved of nitrogen or otherwise stressed to produce and store oil, necessitating an inefficient batch process. Scientists have searched many years for the genetics that control this "lipid trigger" without success. The first genetically modified algae was a failed attempt to produce a strain that simultaneously undergoes cellular division and produces oil (Dunahay 1996).
[0006] Recently, genetic modification has been used to create microbial organisms that produce hydrocarbon compounds ("oil") without the need to starve the cell of nitrogen or otherwise purposely stress the cell. Examples of microbial production species include bacteria (PCT Pub. No. WO2007/136762), yeast (PCT Pub. No. WO2008/045555) and algae (PCT Pub. No. WO2009/11 1513). Examples of hydrocarbon products include isoprenoids (U.S. Pub. No. 2009/0280545) and fatty acids (U.S. Pub. No. 2009/0298143). However, these methods known in the art do not provide a means for encapsulation of the hydrocarbon, as would be found in most biological oil production mechanisms found in nature. As a result of this deficiency, the hydrocarbon product is not well tolerated by the modified microbial species and it is often secreted into the growth medium. These production processes are limited to batch processes on account of the cell's limited tolerance of the hydrocarbon product. Therefore, there is a need for microbial organisms that simultaneously undergo cellular division, produce oil, and encapsulate that oil.
SUMMARY OF THE INVENTION
[0007] The present invention is based on discovery that certain microbial cells can be modified to produce and/or secrete oil. Thus methods are provided herein to introduce one or more nucleic acid molecules encoding specific enzymes and/or proteins into certain microbial cells.
[0008] In one aspect, the invention provides a method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle. The method includes introducing into a microbial cell at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein and culturing the microbial cell in order to express the neutral lipid synthesizing enzyme and the neutral lipid encapsulation protein. In one embodiment, the nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in a single construct. In another embodiment, the nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in separate constructs. In yet another embodiment, the constructs are incorporated into the nuclear genome, the chloroplast genome, autonomously replicating plasmid, or artificial chromosome of the microbial cell or the chloroplast genome. In yet another embodiment, two or more neutral lipid synthesizing enzymes are expressed in the cell. In yet another embodiment, two or more neutral lipid encapsulation proteins are expressed in the cell.
[0009] In another aspect, the invention provides a method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle by means of cross breeding two modified microbial cells. The method includes introducing a first nucleic acid construct into a first microbial cell, wherein the first construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid encapsulation protein, and introducing a second nucleic acid construct into a second microbial cell, wherein the second construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme. Thereafter, the first and second microbial cells are cross-bred to produce a third microbial cell comprising the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein and the nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme. The third microbial cell is then cultured in order to express the at least one neutral lipid encapsulation protein and the at least one neutral lipid synthesizing enzyme. In one embodiment, two or more neutral lipid synthesizing enzymes are expressed in the third cell. In another embodiment, two or more neutral lipid encapsulation proteins are expressed in the third cell.
[0010] In another aspect, the invention provides a method for producing an algal cell expressing at least one neutral lipid synthesizing enzyme. The method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and culturing the algal cell in order to express the at least one neutral lipid synthesizing enzyme. In one embodiment, two or more neutral lipid synthesizing enzymes are expressed in the cell.
[0011] In another aspect, the invention provides a method for producing an algal cell expressing at least one neutral lipid encapsulation protein. The method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, and culturing the algal cell in order to express the neutral lipid encapsulation protein. In one embodiment, two or more neutral lipid encapsulation proteins are expressed in the cell.
[0012] In another aspect, the invention provides a microbial cell that has been manipulated to produce at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein. Likewise, the invention also provides an algal cell which has been manipulated to produce at least one neutral lipid synthesizing enzyme, and/or an algal cell that has been manipulated to produce at least one neutral lipid encapsulation protein.
[0013] Exemplary neutral lipids include, but are not limited to triacylglycerol (TAG), sterol ester (SE), and wax ester (WE). Exemplary neutral lipid synthesizing enzymes include, but are not limited to, acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol acyltransferase3 (DGAT3),
phospholipid:diacylglycerol acyltransferase (PDAT), diacylglycerol:diacylglycerol transacylase, bifunctional wax ester synthase DGAT (WS/DGAT), lecithin holesterol acyltransferase
(LCAT), and an acyl-CoA: cholesterol acyltransferase (ACAT). Exemplary neutral lipid encapsulation proteins include, but are not limited to, oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
[0014] In various embodiments, the nucleic acid molecule encoding the at least one neutral lipid synthesizing enzyme encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, any homolog thereof, and any ortholog thereof. In one embodiment, the neutral lipid synthesizing enzyme is an acyltransferase with enzyme classification 2.3.1.X, where X is a variable that can be any integer. For example, "2.3.1.X" represents enzyme classifications 2.3.1.20, 2.3.1.75, 2.3.1.158, etc. Likewise, in various embodiments, the at least one neutral lipid encapsulation protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-29, a homolog thereof, and an ortholog thereof. In one embodiment, the neutral lipid encapsulation protein is modified. Exemplary modifications include, but are not limited to, one or more gene fusions on a single polypeptide, and at least one cysteine residue introduced into hydrophilic portion of the encapsulation protein.
[0015] In various embodiments, the microbial cell may be a prokaryote or a eukaryote. In one embodiment, the phase of the cell cycle includes the GO, Gl, S, G2, or M phase. In yet another embodiment, the microbial cell is an oleaginous species. In another embodiment, the microbial cell is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates). In another embodiment, the microbial cell is an algal cell of the species Chlamydomonas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridi m, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parietochloris. In another embodiment, the algal cell is Chlamydomonas reinhardtii. In yet another embodiment, the cell is from the genus Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, Pythium,
Schizochytrium, Thraustochytrium, or Ulkenia. In yet another embodiment, the cell is a bacterium of the genus Rhodococcus, Escherichia, or a cyanobacterium. In yet another embodiment, the cell is a yeast cell. In yet another embodiment, the cell is a synthetic cell. [0016] In all aspects of the invention, the microbial cell may be cultured in a batch culture, fed-batch culture, or continuous culture. In one embodiment, the cell produces a neutral lipid and undergoes cellular division simultaneously. In another embodiment, the cell produces a neutral lipid, stores a neutral lipid, and/or excretes a neutral lipid. In yet another embodiment, the cell is cultured in a fermentor, photobioreactor, open pond, or any combination thereof. In yet another embodiment, the cell is part of a culture that is in the lag, logarithmic, or stationary growth phase. In yet another embodiment, the microbial cell is manipulated to produce a neutral lipid independent of an external stressor. An exemplary external stressor is an abiotic stress, such as nutrient deprivation. In yet another embodiment, the microbial cell simultaneously produces and accumulates a neutral lipid while continuing to grow.
[0017] In another aspect, the invention provides a construct including at least one microbial promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, wherein the at least one microbial promoter is operatively linked to the nucleic acid molecules so as to cause expression of the at least one neutral lipid synthesizing enzyme and the at least one neutral lipid encapsulation protein in a microbial cell. As such, the invention also provides uses of the constructs of the invention to induce a microbial cell to express the at least one neutral lipid synthesizing enzyme and the at least one neutral lipid encapsulation protein. In one embodiment, the nucleic acid molecule encoding the at least one neutral lipid synthesizing enzyme has been modified or designed to enhance expression of the neutral lipid synthesizing enzyme in the microbial cell. In another embodiment, the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein has been modified or designed to enhance expression of the neutral lipid encapsulation protein in the microbial cell. In yet another embodiment, the modifications to the nucleic acid molecules include matching the approximate proportion of guanine and cytosine to adenine and thymine in the construct to the proportion of guanine and cytosine to adenine and thymine in the genome of the microbial cell, choosing codons that are most highly representative of proteins encoding genes in the genome of the microbial cell, avoiding codons that are used in less than 10% of all possible instances in the genome of the microbial cell, inclusion of an intron, exclusion of unwanted mRNA splice sites, and/or minimization of mRNA degradation. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is a pictorial diagram showing the biochemistry of triacylglycerol (TAG) production in most organisms. The pathway shows the metabolites, biochemical steps and some of the enzymes responsible for the conversions.
[0019] Figure 2 is a pictorial diagram showing fatty acid and triacylglycerol production in plants and algae. Bold numbers represent key enzymes: 1, plastidic pyruvate kinase; 2, acetyl CoA carboxylase; 3, acyl ACP thioesterasees Fat A and FatB; 4, glycerol-3-phosphate acyl transferase; 5, lyso-phophatidic acid acyl -transferase; 6, diacylglycerol acyl transferase; and 7, lyso-phosphatidylcholine acyl transferase. Abbreviations are: 3PGA, 3-phosphoglycerate; DAG, diacylglycerol; ER, endoplasmic reticulum; FAS, fatty acid synthesis; G3P, glycerol-3- phosphate; G6P, glucose-6-phosphate; LP A, lyso-phosphatidic acid; LPC, lyso- phosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PEP,
phosphoenolpyruvate; PM, plasma membrane; Pyr, pyruvate; TAG, triacylglycerol.
[0020] Figure 3 is a pictorial diagram showing the structure of oil bodies and their mechanism of production. Lipids are irreversibly converted to oil and deposited between the lipid bilayer by the enzyme DGAT in the ER. The oleosin protein targets to this region and eventually dissociates into an oil body. In the cut-away of an oil body on the left, the hydrophobic tails of oleosin (lighter area) anchor into the oil (darker area) and a charged surface is presented to the outside.
[0021] Figure 4 is a pictorial diagram showing cross sections of an oil body, triglyceride oil is surrounded by a phospholipid layer and oleosin proteins. The left panel shows individual oleosins, the next panel is the fusion protein form of polyoleosin, the third panel is the disulfide bond form of polyoleosin, the fourth panel (far right) shows the engineered oleosins in a reduced form with the cysteine residues facing the cytoplasm.
[0022] Figure 5 is a pictorial diagram showing an example of a genetic modification strategy to produce fermentation products photosynthetically in cyanobacteria. In this case, TAG is many steps away from pyruvate, but the scheme demonstrates the large fundamental changes to biology being done using modern biotechnology.
[0023] Figure 6 is a pictorial diagram showing the biochemistry of a microbial cell modified to produce and excrete oil. In this example, DGATl is expressed in the endoplasmic reticulum to convert lipids into the storage form of oil. The increased oil will be stabilized by encapsulating it in neutral lipid storage structures surrounded by oleosin.
[0024] Figure 7 is a pictorial diagram showing the biochemistry of the "chloroplast-based" route to improved oil accumulation. In this example, acyltransferases such as DGAT are expressed in the chloroplast to convert lipids into the storage form of oil. The increased oil will be stabilized by encapsulating it in oil bodies in either plastid expressed oleosin or plastoglobulin.
[0025] Figure 8 is a pictorial diagram showing the structural arrangement of cyanobacteria, demonstrating that the methods of the invention can be adapted to both the algal chloroplast and to cyanobacteria.
[0026] Figure 9 is a pictorial diagram showing various pathways for production of various types of biofuel in microbial species. Note that this diagram does not explicitly show the production of neutral lipids, which must be subsequently processed into a fuel after production in the cell. Note as well that this diagram shows alcohols which are not part of the invention.
[0027] Figure 10 is a graphical diagram depicting the advantage of a continuous process compared to a batch process in utilization of capital intensive process equipment.
[0028] Figure 11 is a pictorial diagram showing the process of neutral lipid production in unmodified microbial species. The process is necessarily a batch process where cells are first grown ("biomass") then starved of nitrogen to produce oil.
[0029] Figure 12 is a pictorial diagram showing the features of neutral lipid production in microbial species modified using the methods of the invention. The process can now be continuous because the cell produces biomass and oil at the same time.
[0030] Figure 13 is a pictorial diagram showing a batch process for the production of fuel from algae utilizing the "lipid trigger."
[0031] Figure 14 is a pictorial diagram showing a continuous process for making fuel utilizing the methods of the invention.
[0032] Figure 15 is a pictorial diagram showing a map (MAP 1) of an expression cassette for one gene of interest for transformation into Chlamydomonas reinhardtii.
[0033] Figure 16 is a pictorial diagram showing a map (MAP 2) of an expression cassette for two genes of interest for transformation into Chlamydomonas reinhardtii. 10034] Figure 17 is a pictorial diagram showing a map (MAP 3) of an expression cassette for two genes of interest for transformation into Chlamydomonas reinhardtii.
[0035] Figure 18 is a pictorial diagram showing a map (MAP 4) of an expression cassette for transformation of 01e_3,3 and AtDGATl into Saccharomyces cerevisiae. Figure 18 discloses "6x His" as SEQ ID NO: 209.
[0036] Figure 19 is a pictorial diagram showing a map (MAP 5) of an expression cassette for transformation of MLDP and CsDGAT2 into Saccharomyces cerevisiae.
[0037] Figure 20 is a pictorial diagram showing a map (MAP 6) of an expression cassette for transformation of 01e_3,3 and CsDGAT2 into Saccharomyces cerevisiae.
[0038] Figure 21 is a graphical diagram showing hydrophobicity plots of MLDP from both Chlamydomonas and Volvox. Arrows indicate regions with relatively high hydrophilic properties that may be exploited to engineer insertion of cysteine residues.
[0039] Figure 22 is a map showing 5' UTR, position of intron 1 and exons relative to nucleic acid sequence (SEQ ID NO: 39) and peptide sequence (SEQ ID NO: 63) of Chlamydomonas reinhardtii native MLDP.
[0040] Figure 23 is a microscopy comparison of Nile Red fluorescence from Chlamydomonas reinhardtii cells that were either: from a nitrogen starved culture (left), from a vector only transformed culture at log phase (center), or from a log phase culture that had been transformed with the full length HRp-DGATl-V5-RBCS2At;HRIp-oleo 0,0-RBSCA2t construct (right).
[0041] Figure 24 is a relative quantitative comparison of neutral lipid accumulation in transgenic Chlamydomonas reinhardtiii expressing NITlp-CrDGAT2-RBCS2At where cells harboring the NITlp-CrDGAT2-RBSC2At cassette were shown to accumulate nearly 2-fold more neutral lipids than those harboring the NITlp-CrDGAT2-CrDGAT2-3'UTR cassette after 8h of induction.
[0042] Figure 25 is a graphical diagram showing the total lipids extracted per milligram of dry yeast cells expressing DGAT and lipid encapsulating proteins during the lag phase.
[0043] Figure 26 is a series of confocal microscopy images of Nile Red stained
Saccharomyces cerevisiae cells. DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention is based on the discovery that certain microbial cells can be modified to produce and encapsulate oil. Using the techniques provided herein, nucleic acid molecules encoding certain enzymes and/or proteins are introduced into microbial cells, thereby causing the cells to produce oil.
[0045] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described.
[0047] As used herein, the term "oil" refers to any hydrocarbon including all alkanes, alkenes, alkynes, and aromatic hydrocarbons. "Oils" also include biological oils that are largely, but not entirely composed of carbon and hydrogen. Exemplary biological oils include, but are not limited to, lipids, fats, waxes, sterols, fatty acids, fatty alcohols, fatty esters, polyketides, isoprenes, monoglycerides, diglycerides, phospholipids, and neutral lipids. Oils are largely immiscible in water.
[0048] As used herein, a "neutral lipid" refers to any lipid having no polar group thereby rendering the lipid unable to integrate into bilayer membranes in substantial amounts. Thus, while such neutral lipids have hydrophobic tails, they do not have a hydrophilic (charged) head.
Exemplary "neutral lipids" include, but are not limited to, triacylglycerols (TAGs), sterol esters (SEs) and wax esters (WEs). The main storage lipids in eukaryotes are triacyl glycerol (TAG) and sterol esters. In algae, plankton and in bacteria, WEs are also used as an energy store
(Athenstaedt and Daum 2006). It should be understood that neutral lipid fractions may also contain one or more additional lipidic compounds, including, but not limited to,
monogalactosyldiacylglycerol, digalactosyldiacylgycerol, free fatty acid, carotenoids, prenylquinones (tocopherols, plastoquinone, and menaquinones), and chlorophylls (Austin, Frost et al. 2006).
[0049] As used herein, "triacylglycerol" or "TAG" refers to is a glyceride in which the glycerol is esterified with three fatty acids. Triglycerides are formed from a single molecule of glycerol, combined with three fatty acids on each of the OH groups. Ester bonds form between each fatty acid and the glycerol molecule. Most plants synthesize and store significant amounts of TAG only in developing seeds and pollen cells where it is subsequently utilized to provide catabolizable energy during germination and pollen tube growth. Dicotyledonous plants can accumulate up to approximately 60% of their seed weight as TAG. Ordinarily, this level is considerably lower in the monocotyledonous seeds where the main form of energy storage is carbohydrates {e.g., starch).
[0050] TAG is the desired cellular metabolite for a number of products including vegetable oil, omega-3 oils and renewable fuels among others. Acetyl-CoA is a general cellular metabolite that can be synthesized through a number of basic metabolic pathways. There are differences among species, but the enzyme ACC (acetyl-CoA carboxylase) draws on the common pool of Acetyl-Co A. This is the first committed step in the production of TAG in a plant. (See Figures 1 and 2). Once carbon is committed toward lipid or oil production by ACCase, fatty-acid synthase (FAS) produces the individual fatty acid chains in the chloroplast. In this intermediate step toward making TAG, considerable diversity is generated with respect to both the length and degree of saturation of lipids between species. The fatty acids are then transported out of the chloroplast and into the ER where their eventual fate is determined. Biological membranes are composed of glycerol backbones with two of the three positions occupied by fatty acids, while TAG has all three positions occupied. The pathway by which fatty acids are added sequentially to the glycerol backbone is called the "Kennedy Pathway".
[0051] As used herein, the term "oil storage structure" refers to a microscopic droplet of oil surrounded by a means for encapsulation. The means for encapsulation can include a monolayer of an "oil encapsulation protein". The means for encapsulation can also include a layer of phospholipid. The function of the oil storage structure is to isolate the hydrophobic oil component from the aqueous environment in a controlled manner. The outer layers of the oil storage structure usually present a hydrophobic surface to the interior of the oil storage structure and a hydrophilic surface to the exterior of the structure. 2011/031336
[0052] Included in the "oil storage structures" are "neutral lipid storage structures" where the droplet of an oil is more specifically a neutral lipid (e.g., triacyl glycerol (TAG)) surrounded by a monolayer of phospholipid where the hydrophobic acyl moieties of the phospholipids interact with the encapsulated lipid and the hydrophilic head groups face the exterior. Surrounding the phospholipid layer is a layer of "neutral lipid encapsulation protein".
[0053] One embodiment of a "neutral lipid storage structure" is an "oil body". Oil bodies are typically found in plant seeds. Oil bodies are typically 0.5-2.5μηι in diameter and consist of a TAG core surrounded by a phospholipid monolayer embedded with proteinaceous emulsifiers - predominantly oleosins. The size and number of oil bodies depends on the ratio of oleosin to TAG within the plant cell (Siloto, Findlay et al. 2006). Oil bodies consist of only 0.5-3.5% protein; of this 80-90% is oleosin with the remainder predominantly consisting of the calcium binding (caoleosin) and sterol binding (steroleosin) proteins. It should be understood that the term "neutral lipid storage structure" also includes artificial or synthetic oil bodies that are formed in microbial hosts using the methods disclosed in this invention.
[0054] Another example of a "neutral lipid storage structure" is a "plastoglobule". As used herein, the term "plastoglobule" refer to a lipoprotein particle inside chloroplasts that contains biosynthetic enzymes and a variety of lipidic compounds, including
monogalactosyldiacyglycerol, digalactosyldiacyglycerol, free fatty acid, triacylglycerols, carotenoids, prenylquinones (tocopherols, plastoquinone, and menaquinones), and chlorophylls (for a review, see Austin et ah, 2006). It has been shown that plastoglobules function as both lipid biosynthesis and storage sub-compartments of thylakoid membranes. Light stress, nitrogen starvation, osmotic stress and oxidative stress are all known to cause the formation of, or increase the size of, oil deposits on the thylakoid membranes in the chloroplast. These structures are called plastoglobules, tubules, fibrils or crystalloids and vary in size from 30 nm to several micrometers. They do not generally dissociate from the thylakoid like an oil body. They contain lipid soluble β-carotene as sunscreen during light stress. Plastoglobules contain compounds that confer color to fruits and flowers {e.g., ripening peppers structurally reorganize their thylakoid membranes and accumulate plastoglobuli).
[0055] As used herein, the term "oil encapsulation protein" refers to a protein that presents hydrophilic amino acids to the exterior of an oil storage structure and hydrophobic amino acids to the interior. It is known that oil encapsulation proteins can also include a long consecutive stretch of hydrophobic amino acids that extend into the bulk of the encapsulated oil to anchor the oil encapsulation protein to the oil storage structure.
[0056] Included in the "oil encapsulation proteins" are "neutral lipid encapsulation proteins" where the droplet of oil is more specifically a neutral lipid (e.g., triacylglycerol (TAG)). As used herein, the term "neutral lipid encapsulation protein" refers to any protein that surrounds a neutral lipid to produce a neutral lipid storage structure. Nature provides many examples of neutral lipid storage structures and neutral lipid encapsulation proteins. Exemplary natural lipid encapsulation proteins include, but are not limited to, oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein. Tables 1 and 2 provide lists of known neutral lipid encapsulation proteins that may be used in wild-type or modified form in the methods of the invention. It should be understood that included in the term "neutral lipid encapsulation protein" are synthetic versions of any of the proteins having the emulsification and encapsulation properties of the encapsulation proteins as a function of its arrangement and sequence of hydrophilic and hydrophobic amino acid residues.
Table 1 - Neutral Lipid Encapsulation Proteins
Neutral Lipid Species cDNA accession SEQ Protein accession no. SEQ
Encapsulation no. ID ID
Protein NO: NO:
Oleosin Sesamum indicum AF302907 64 AAG23840 65
Oleosin Sesamum indicum U97700 66 AAB58402 67
Oleosin Arabidopsis. X62353 68 CAA44225 69
thaliana
Oleosin Arabidopsis. BT023738 70 AAZ23930 71
thaliana
Oleosin Helianthus annuus X62352.1 72 CAA44224.1 73
Oleosin Brassica napus X82020.1 74 CAA57545.1 75
Oleosin Zea mays NM 001 153560.1 76 NP 001147032.1 77
Oleosin Oryza sativa AAL40177.1 78 AAL40177.1 79
Oleosin Brassica oleracea AF1 17126.1 80 AAD24547.1 81
Oleosin Coffea arabica AY928084.1 82 AAY14574.1 83
Steroleosin Sesamum indicum AAL13315 84 AAL13315 85
Steroleosin Brassica napus EU678274 86 ACG69522 87
Steroleosin Zea mays NM 001 159142.1 88 NP 001 152614.1 89
Steroleosin Brassica napus EF143915.1 90 ABM30178.1 91
Caoleosin Sesamum indicum AF 109921 92 AAF13743 93
Caoleosin Glycine max AF004809 94 AAB71227 95
Caoleosin Zea mays NM 001 158434.1 96 NP 001 151906 97
Caoleosin Brassica napus AY966447.1 98 AAY40837 99
Caoleosin Cycas revoluta FJ455154.1 100 ACJ70083 101
Caoleosin Cucumis sativus EU232173.1 102 ABY56103.1 103
Chlamydomonas 104 105
MLDP reinhardtii XM 001697616 XP 001697668
Arabidopsis 106 107
Plastoglobulin thaliana BT001242 Q9M2P7 Arabidopsis 108 109
Plastoglobulin thaliana AY120766 AAM53324
Arabidopsis 1 10 111
Plastoglobulin thaliana BT020480 AAW3898
Arabidopsis 1 12 1 13
Plastoglobulin thaliana AY042905 AAK68845
Arabidopsis 1 14 1 15
Plastoglobulin thaliana NM 11351 1 NP 189236
Arabidopsis 1 16 1 17
Plastoglobulin thaliana NM 1 13512 NP 189237
Arabidopsis 1 18 1 19
Plastoglobulin thaliana BT024911 ABD91502
Arabidopsis 120 121
Plastoglobulin thaliana NM 001036785 NP 001031862
Arabidopsis 122 123
Plastoglobulin thaliana NM 122001 NP 197494
Arabidopsis 124 125
Plastoglobulin thaliana AY954828 AAX55154
Arabidopsis 126 127
Plastoglobulin thaliana AY062597 AAL32675
Arabidopsis 128 129
Plastoglobulin thaliana NM 1 16220 NP 191914
Arabidopsis 130 131
Plastoglobulin thaliana NM 103989 NP 175522
Arabidopsis 132 133
Plastoglobulin thaliana BT008410 AAP37769
Synechocystis PCC 134 135
Plastoglobulin 6803 NC 00091 1 NP 440566
Synechocystis PCC 136 137
Plastoglobulin 6803 NC 00091 1 NP 440481
Perilipin Mus mu cuius NM_007408 138 NP 031434 139
Perilipin Mus musculus NM 175640 140 NP 783571 141
Perilipin Mus musculus NM 025836 142 NP 080112 143
Perilipin Mus musculus NM 020568 144 NP 065593 145
Perilipin Mus musculus - - EDL07050 146
Perilipin Mus musculus - - EDL07051 147
Perilipin Mus musculus - - EDL07052 148
Perilipin Mus musculus - - EDL07053 149
Perilipin Mus musculus - - EDL07054 150
Perilipin Mus musculus - - EDL07055 151
Apolipoprotein Homo sapiens NM 001643.1 152 NP 001634.1 153
Apolipoprotein Homo sapiens NM_000040.1 154 NP 000031.1 155
Apolipoprotein Homo sapiens NM 000042.2 156 NP 000033.2 157
Apolipoprotein Homo sapiens NM 000041.2 158 NP 000032.1 159
Apolipoprotein Homo sapiens NM 052968.4 160 NP 443200.2 161
Apolipoprotein Homo sapiens NM 019101.2 162 NP 061974.2 163
Apolipoprotein Homo sapiens NM 000039.1 164 NP 000030.1 165
Table 2 - Amino Acid Sequences of Neutral Lipid Encapsulation Proteins
Neutral Lipid Sequence SEQ ID Encapuslation NO: Protein
Oleosin madrd^hphqiqvhpqhphryeggvksllpqkgpsttqilaiitllpisgtllclagitlvgtliglav 14 (S. indicum) atpvfvifspvlvpaailiaga\ afltsgafgltglsslswvlnsfrratgqgpleyakrgvqegtlyv
gektkqageaikstakeggregtart Neutral Lipid Sequence SEQ ID Encapuslation NO:
Protein
Oleosin madephdq tdviksylpekgpstsqvlavvtlfplgavllclagliltgtiiglavatplfVifspilv 15 (S. indicum) paaltialavtgfltsgafgitalssiswllnyvrrmrgslpeqldharrrvqetvgqktreagqrsqdv
ί
Oleosin madtargthhdiigrdqypmmgrdrdqyqmsgrgsdysksrqiakaatavtaggsllvlssltlv 16 {A. thaliana) gtvialtvatpllvifspilvpalitvallitgflssggfgiaaitvfswiykyatgehpqgsdkldsarm
klgskaqdlkdraqyygqqhtggehdrdrtrggqhtt
Oleosin madhqqhqqqqqpimrslhesspstrqivrfVtaatiglsllvlsgltltgtviglivatplmvlfspvl 17 A. thaliana) vpavitiglltmgflfsggcgvaaataltwiykyvtgkhpmgadkvdyarmriaekakelghyth
sqpqqthqttttth
Steroleosin mdlihtflnliappftfffllfflppfqifkfflsilgtlfsedvagkvvvitgassgigeslayeyakrga 18 (S. indicum) clvlaarrerslqevaerardlgspdvvvvradvskaedcrkvvdqtmnrfgrldhlvnnagimsv
smleeveditgyretmdinfwgyvymtrfaapylrnsrgrivvlssssswmptprmsfynaska aisqffetlrvefgpdigitlvlpgfieseltqgkrynagervidqdmrdvqvsttpilrvesaarsivrs airgeryvtepawfrvtywwklfcpevmewvfrlmylaspgepeketfgkkvldytgvksllyp etvqvpepknd
Caoleosin mathvlaaaaernaalapdaplapvtmerpvrtdletsipkpymarglvapdmdhpngtpghv 19 (S. indicum) hdnlsvlqqhcaffdqddngiiypwetysglrqigfiiviaslimaivinvalsyptlpgwipspfip
iylynihkaWigsdsgtydtegiylpmnfenlfskhartmpdrltlgelwsmteanreafdifgwi askmewtllyilardqdgflskeaiiTcydgslfeycakmqrgaedkmk
Caoleosin mssyspppppprdqsmdteapnapitrerrlnpdlqeqlpkpylaraleavdpshpqgtkgrdpr 20 (Z mays) gmsvlqqhaaffdrngdgviypwetfqglraigcgltvsfafsilinlflsyptqpgwlpspllsirid
nihkgkhgsdselydtegrfdpskfdaifskygrthpnaitrdelssmlqgnrntydflgwlaaage wlllyslakdkdgllqretvrglfdgslferleddnnkkkss
MDLP maesagkplkhlefVhtyahkfasgaayveggyqkaktyvpavaqpyiakaeetclayaaplat 21 (C. reinhardtii) katdhaekilrstdaqldalyaasaswlsssqkladsniaafrgaadkyydlvkstaqhvtsklptdl
svakarellsasleqakaladpdaavaaaldawtkfaaipavakvlsaaspltgkgvaaftaahdllv hsalyrygvsvgastlgwatsttpyklsaaylyplvqpvadpaldkvskstyvnaaikywapapv aaa
Plastoglobulin maliqhgsvsgtsavrlsfsssvsppssspplsrvslnfqsekkscyrrmicramvqdsvqgipsv 22 {A. thaliana) yaremerlsakeslilaftldaggfealvtgkitdmqkidvneritnlerlnptp ttspylegrwsfe
wfgvntpgslavrvmferQjstlvslsnmeifikdnntkatanikllnsienkitlsskltiegplrmk eeylegllesptvieeavpdqlrgllgqatttlqqlpepikdtlanglriplgglyqrffmisylddeili vrdtagvpevltrvetsspmsssswenleyns
Plastoglobulin mpnsmdanmdfktnlleaiagknrgllasdrdrvailsavekledynphpkplqeknlldgnwrl 23 (Synechocystis lytssqsilglnrlpllqlgqiyqyidvagsrvvnlaeiegipfleslvsvvasfipvsdkrievkfersil PCC 6803) glqkilnyqsplkfiqqistgkrflpadfhlpgrdnaawleityldedlrisrgnegnvfilakv
Plastoglobulin mslerqtlkqklstliqplqtakrgapltnrtlsattcqqieslvtaiealnpnlspllyspqlldgnwwl 24 {Synechocystis nystareirsldklplglkvgriyqiinvpnqsflnqafvyhplglakgyvkvtakfeiakpagtvlp
PCC 6803) dkrinveflermisiqklmgvp^kldpakvvparspegripfleityldddlrigrggegslfVlsk
vsevtp
Perilipin maaavvdpqqsvvmrvanlplvsstydlvssayvstkdqypylrsvceniaekgvktvtsaamt 25 (M musculus) salpiiqklepqiavantyackgldrmeerlpilnqptseivasargavtgakdvvtttmagakdsv
astvsgvvdkikgavtgsvertksvvngsintvlgmvqfninsgvdnaitksellvdqyfpltqeel emeakkvegfdmvqkpsnyerleslstklcsrayhqalsrvkeakqksqetisqlhstvhliefark nmhsanqkiqgaqdklyvswvewkrsigyddtdeshcvehiesrtlaiarnltqqlqttcqtvlvn aqglpqniqdqakhlgvmagdiysvfrnaasfkevsdgvltsskgqlqkmkesldevmdyfvn ntplnwlvgpfypqstevnkaslkvqqsevkaq
Perilipin msmnkgptlldgdlpeqenvlqrvlqlpvvsgtcecfqktynstkeahplvasvcnayekgvqg 26 (M musculus) asnlaawsmepvvrrlstqftaanelacrgldhleekipalqyppekiaselkgtistrlrsarnsisv
piastsdkvlgatlagcelalgmaketaeyaantrvgrlasggadlalgsiekvvefllppdkesaps sgrqrtqkapkakpslvrrvstlantlsrhtmqttawalkqghslamwipgvaplsslaqwgasaa mqvvsrrqsevrvpwlhnlaasqdeshddqtdtegeetddeeeeeeseaeenvlrevtalpnprg llggvvhtvqntlrntisavtwapaavlgtvgril ltpaqavsstkgramslsdalkgvtdnvvdtv vhyvplprlslmepesefrdidnpsaeaerkgsga aspestp gqprgslrsvrglsa scpgl ddkteasaφgflamprekparrvsdsffi svmepilgraqysqlrkks Neutral Lipid Sequence SEQ ID Encapuslation NO:
Protein
Perilipin mssngtdapaeaqaameepvvqpsvvdrvaglplisstygmvsaaytstkenyphvrtvcdva 27
(M. musculus) ekgvktlttaavstaqpilsklepqiataseyahrgldrlqeslpilqqptekvladtkelvsstvsgaq
emvsssvssaketvatrvtgavdvtlgavqnsvdktksamtsgvqsvmgsrvgqmvisgvdrv lvkseawadnrlplteaelaliatppedsdmaslqqqrqeqnyfvrlgslserlmhayehslgklqn
arqkaqetlqqltsvlglmesvkqgvdqrlgegqeklhqmwlswnqktpqdaekdpakpeqv earalsmfrditqqlqsmcvalgasiqglpshvreqaqqarsqvndlqatfsgihsfqdlsagvlaqt
reriararealdntveyvaqntpamwlvgpfapgitektpegk
Apolipoprotein alvrrqakepcveslvsqyfqtvtdygkdlmekvkspelqaeaksyfekskeqltplikkagtelv 28
(H. sapiens) nflsyfvelgtqpatq
Apolipoprotein seaedasllsfmqgymkhatktakdalssvqesqvaqqargwvtdgfsslkdywstvkdkfsef 29
{H. sapiens) wdldpev tsavaa
[0057] As used herein, the term "oleosin" refers to specific plant proteins that are usually found only in seeds and pollen. The properties of the major oleosins are relatively conserved among plants. Oleosins allow oil bodies to become tightly packed discrete organelles without coalescing as the cells desiccate or undergo freezing conditions (Siloto, Findlay et al. 2006). Such oleosins are typically 15-25kDa proteins, which corresponds to approximately 140-230 amino acid residues. Oleosins have three functional domains consisting of an amphipathic N- terminal arm, a highly conserved central hydrophobic core (-72 residues) and a C-terminal amphipathic arm. The accepted topological model is one in which the N- and C-terminal amphipathic arms are located on the outside of the oil body and the central hydrophobic core is located inside the oil body. The negatively charged residues of the N- and C-terminal amphipathic arms are exposed to the aqueous exterior whereas the positively charged residues are exposed to the oil body interior and face the negatively charged lipids. Thus, the amphipathic arms with their outward facing negative charge are responsible for maintaining the oil bodies as individual entities via steric hindrance and electrostatic repulsion both in vivo and in isolated preparation. The N-terminal amphipathic arm is highly variable and as such no specific secondary structure can describe all examples. In comparison the C-terminal arm contains an oc-helical domain of 30-40 residues (Tzen, Wang et al. 2003). The central core is highly conserved and thought to be the longest hydrophobic region known to occur in nature; at the center is a conserved 12 residue proline knot motif which includes three spaced proline residues. The secondary, tertiary and quaternary structure of the central domain is still unclear. Modeling, Fourier Transformation-Infra Red (FT-IR) and Circular Dichromism (CD) evidence exists for a number of different arrangements (for review, see Roberts et al., 2008; Frandsen et al, 2001; Tzen et al, 2003). [0058] In addition to oleosin, oil bodies also include "caoleosin". Caoleosin has a slightly different proline knot than do the basic oleosins, and contain a calcium-binding motif and several potential phosphorylation sites in the hydrophilic arms (Frandsen, Mundy et al. 2001). Similar to oleosin, caoleosin is proposed to have three structural domains, where the N- and C-terminal arms are hydrophilic while the central domain is hydrophobic and acts as the oil body anchor. The N-terminal hydrophilic domain consists of a helix-turn-helix calcium binding EF-hand motif of 28 residues including an invariable glycine residue as a structural turning point and five conserved oxygen-containing residues as calcium-binding ligands (Frandsen, Mundy et al. 2001). The C-terminal hydrophilic domain contains several phosphorylation sites and near the C- terminus is an invariable cysteine that is not involved in any intra- or inter-disulfide linkages. The hydrophilic N- and C-termini of caoleosin are approximately 3 times larger than those of oleosin. The hydrophobic domain is thought to consist of an amphipathic a-helix and an anchoring region (which includes a proline knot).
[0059] In addition to oleosin and caoleosin, oil bodies also include "steroleosin" (Tzen, Wang et al. 2003). Steroleosins include an N-terminal anchoring segment that includes two
amphipathic a-helices (approximately 912 residues in each helix) connected by a hydrophobic anchoring region of 14 residues. The soluble dehydrogenase domain contains a NADP+- binding subdomain and a sterol-binding subdomain. The apparent distinction between steroleosins-A and -B occurs in their diverse sterol-binding subdomains (Lin and Tzen 2004). Steroleosins have a proline knob in their hydrophobic domain and contains a sterol-binding dehydrogenase in one of their hydrophilic arms.
[0060] Additional exemplary neutral lipid encapsulation proteins can be readily identified from neutral lipid storage structures other than oil bodies. Plastoglobules have a number of associated metabolic enzymes and structural proteins called "plastoglobulins". It has been shown that the availability of plastoglobulins regulates the formation of plastoglobuli in much the same way that oleosin is required for the formation of oil bodies. Plastogobulins surround
plastoglobules forming a coating on the surface of the lipoprotein particle in a plant leaf. The coat may contain receptors for attachment to the thylakoid membrane as well as regulatory proteins that may function in the transfer of lipids to and from the thylakoid membranes.
[0061] In algae, "Major Lipid Drop Protein" ("MLDP") was recently identified in the green alga model Chlamydomonas reinhardtii, and was shown to exist within the lipid droplet enriched fraction during nitrogen deprivation. MLDP appears to be specific to the green algal lineage of photosynthetic organisms. Repression of MLDP gene expression was shown to increase lipid droplet size, but not alter triacyl glycerol content or metabolism (Moellering 2009).
[0062] In animals, "apolipoproteins" form low-density lipoproteins (LDLs) when they encapsulate a core of cholesterol and cholesterol esters (sterol ester) to transport dietary fats through the bloodstream. Apolipoproteins also serve as enzyme co-factors, receptor ligands, and lipid transfer carriers that regulate the metabolism of lipoproteins and their uptake in tissues.
[0063] "Perilipin", also known as "lipid droplet-associated protein" or PLIN, is a protein which coats lipid droplets in adipocytes, the fat-storing cells in adipose tissue. Perilipin acts as a protective coating from the body's natural lipases, which break TAG into glycerol and free fatty acids for use in metabolism. In humans, perilipin is expressed in three different isoforms, A, B and C, with perilipin A being the most abundant.
[0064] Neutral lipid encapsulation proteins such as oleosin, steroleosin, caoleosin, plastoglobulin, MLDP, apolipoprotein and perilipin are well known to those skilled in the art. Further sequences from many different species can be readily identified by methods well-known to those skilled in the art. In various embodiments, the neutral lipid encapsulation protein (e.g., oleosin) may be modified or mutated.
[0065] One desirable modification is to express fusion proteins consisting of two or more neutral lipid encapsulation proteins. In one embodiment, polyoleosin was formed from the end to end fusion of two or more oleosin units (Roberts et al. 2008). As used herein, "polyoleosin" refers to the fusion of any number of any neutral lipid encapsulation proteins. In this
embodiment, altering the number of oleosin units enables the properties (thermal stability and degradation rate) of the oil bodies to be tailored. Polyoleosin allows oil bodies to withstand extreme conditions such as heating at 95°C or incubation in rumen fluid for 24 hours.
Remarkably, the exposed portions of the protein can be digested with proteinase K and the remaining hydrophobic core still stabilizes the oil body. Expression of polyoleosin in planta leads to incorporation of the polyoleosin units to the oil bodies as per single oleosin units (Scott et al, 2007).
[0066] It was subsequently suggested that oleosins containing a cysteine on the exposed arm can impart properties that prevent it's break down in the cell (PCT NZ2010/000218). By altering the number and position of the cysteines engineered into the hydrophilic arms of oleosins it is also possible to modulate the degree of stability of the oil bodies. Furthermore, it may be possible to alter the oxidised/reduced status of the cysteines by manipulation of the oxidising/reducing environment thus enabling the tailoring of oil body stability for different purposes during processing or during their application post extraction (Figure 4).
[0067] The preferred means for oil encapsulation is to adapt one of nature's neutral lipid encapsulation proteins to form a neutral lipid storage structure. However, it would be recognized by one skilled in the art that neutral lipid encapsulation proteins could be adapted or improved to encapsulate other oils. Similarly, an oil encapsulation protein could be designed or selected that bears little sequence homology to proteins found in nature, but is suitable as a means for encapsulation.
[0068] The prior art provides several examples of "means for synthesizing oil" utilizing genetic modification of microbial species (PCT Pub. No. WO2007/136762, PCT Pub. No.
WO2008/045555, PCT Pub. No. WO2009/111513, U.S. Pub. No. 2009/0280545, and U.S. Pub. No. 2009/0298143, each of which is incorporated herein by reference) (see Figure 9). In most cases, a cellular metabolite is diverted from the normal uses of the cell and synthesized into oil by one or more oil synthesis enzymes. However, the prior art does not include a "means for encapsulating" oil produced in microbial species. This disclosure teaches that providing a means for encapsulating oil in addition to a means to synthesizing oil leads to increased efficiency and productivity.
[0069] In order to best match the preferred means for oil encapsulation, the preferred means for oil synthesis should be expression of one or more neutral lipid synthesizing enzymes. As used herein, the term "neutral lipid synthesizing enzyme" refers to any enzyme required to convert fatty acids to neutral lipid within a cell. Exemplary neutral lipid synthesizing enzymes include, but are not limited to, acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl
CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol acyltransferase3 (DGAT3), phospholipid:diacylglycerol acyltransferase (PDAT), diacylglycerohdiacylglycerol transacylase, and bifunctional wax ester synthase DGAT (WS/DGAT). Tables 2 and 3 provide lists of known neutral lipid synthesizing enzymes that may be used in wild-type or modified form in the methods of the invention.
[0070] The preferred embodiment employs neutral lipid as the oil of choice, but it would be recognized by one skilled in the art that the teachings can be applied to other oils. Furthermore, neutral lipid synthesis enzymes are employed as the means for synthesizing neutral lipid, but other means known in the art such as expressing transcription factors or starving an algal cell of nitrogen may similarly be employed. However, neutral lipid synthesis enzymes are the method to focus on hereafter.
[0071] Diacylglycerol acyltransferase (DGAT) is the final committed step in the synthesis of TAG, and the most successful target for increasing oil synthesis in plants (Figure 3). In plants, DGAT deposits TAG between the leaves of the endoplasmic reticulum bilayer. A neutral lipid encapsulation protein is then targeted to these portions of the ER, where eventually an oil body dissociates into the cytoplasm.
[0072] Without being bound by theory, the following is the most likely explanation for DGAT over-expression's proven ability to increase TAG levels in plants. DGAT is the branch point in the oil pathway where lipids destined for storage are split from those destined to constitute biological membranes. Over-expression of DGAT deprives the cell of diacylglycerol (DAG) needed for membranes, leading to increased DAG synthesis in the cell to compensate. However, increasing the rate of oil synthesis is only half of the challenge in accumulating neutral lipids. The oil must also be encapsulated in the cell to prevent catabolism of the oil or harmful effects on the cell. In this regard, the coupled interaction between the cell's means for oil synthesis and means for oil encapsulation (Figure 6) is critical and is the phenomena that inspired the best mode of this disclosure.
Table 3 - Neutral Lipid Synthesizing Enzymes
Neutral Species cDNA accession SEQ Protein accession SEQ Enzyme Lipid no. ID no. ID Classific
Synthesizin NO: NO: ation no.
g Enzyme
DGAT1 Arabidopsis NM_127503 166 NP_179535 167 2.3.1.20
thaliana
DGAT1 Tropaeolum AY084052 168 AAM03340 169 2.3.1.20
majus
DGAT1 Zea mays EU039830 170 ABV91586 171 2.3.1.20
DGAT1 Ricinus XM_002514086 172 XP_002514132 173 2.3.1.20
communis
DGAT2 Arabidopsis NM_11501 1 174 NP_566952 175 2.3.1.20
thaliana
DGAT2 Brassica FJ858270 176 AC090187 177 2.3.1.20
napus
DGAT2 Oryza sativa NM 001064065 178 NP 001057530 179 2.3.1.20
DGAT2 Chlamydomon XM_001693137 180 XP_001693189 181 2.3.1.20
as reinhardtii
DGAT2 Ricinus XM_002528485 182 XP_002528531 183 2.3.1.20
communis
DGAT2 Saccharomyce NM_00183664 184 EEU04881 185 2.3.1.20
s cerevisiae
Figure imgf000022_0001
Table 4 - Amino Acid Sequences of Neutral Lipid Synthesizing Enzymes
Neutral Lipid Sequence SEQ ID Synthesizing NO: Enzyme
DGAT1 maildsagvttvtengggefvdldrlrrrksrsdssnglllsgsdnnspsddvgapadvrdridsvv 1 (A. thaliana) nddaqgtanlagdnngggdnngggrgggegrgnadatftyrpsvpahrraresplssdaifkqsh
aglfhlcvvvliavnsrliienlmkygwlirtdfwfssrslrdwplfmccislsifplaaftveklvlqk yisepvviflhiiitmtevlypvyvtlrcdsaflsgvtlmlltcivwlklvsyahtsydirslanaadka npevsyyvslkslayfmvaptlcyqpsyprsacirkgwvarqfaklvifitgfmgfiieqyinpivr nsldiplkgdllyaiervlklsvpnlyvwlcmfycffhlwlnilaellcfgdrefykdwwnaksvg dywrmwnmpvhkwmvrhiy clrskipktlaiiiaflvsavfhelciavpcrlfklwaflgimf qvplvfitnylqerfgstvgnmifwfifcifgqpmcvllyyhdlmnrkgsms
DGAT1 mavaessqntttmsghgdsdlnnfrrrkpsssviepsssgftstngvpatghvaenrdqdrvgam 2 (T. majus) enatgsvnligngggvvigneekqvgetdirftyrpsfpahrrvresplssdaifkqshaglfhlciv
vliavnsrliienlmkygwlidtgfwfssrslgdwsifmccltlpiqDlaafiveklvqmhiselvav llhvivstaavlypviviltcdsvymsgvvlmlfgcimwlklvsyahtssdirtlaksgykgdahp nstivscsydvslkslayfmvaptlcyqpsyprsscirkgwvvrqfvklivfiglmgfiieqyinpi vmskhplkgdflyaiervlklsvpnlywlcmfysffhlwlnilaellrfgdreiykdwwnaktv aeywkmwnmpvhrwmvrhlyfyclmgipkegaiiiaflvsgafhelciavpchvfklwafigi mfqvplvlitnylqekfsnsmvgnmifwfifcilgqpmcvllyyhdlinlkek
DGAT2 mggsrefraeehsnqfhsiiamaiwlgaih&valvlcsliflppslslmvlgllslfifipidhrskyg 3 (A. thaliana) rklaiyickhacnyfpvslyvedyeafqpnrayvfgyephsvlpigvvalcdltgfmpipnikvla
ssaifylpflrhiwtwlgltaasrknftslldsgyscvlvpggvqetfhmqhdaenvflsrrrgfvria meqgsplvpvfcfgqarvykwwkpdcdlylklsrairftpicf gvfgsplpcrqpmhvvvgk pievtktlkptdeeiakfhgqyvealrdlferhksrvgydlelkil
DGAT2 mplaklrnvvleyaaiaiyvsaiytsvvllpsalalfylfgatspsawlllaaflaltftplqlttgalserf 4 (C. reinhardtii) vqfsvaraaay trvvvtdpeafΓtdrgylfgfcphsalpial iafattspllpkelrgrthglassvc
fsapivrqlywwlgv atrqsisgllraΓkvavlvpggvqevlnmehgkevaylssrtgfvrlav qhgaplvpv afgqtrayswfrpgpplvptwlverisraagavpigmfgqygtpmphrepltiv vg ipvpela gqle epevlaallkrftddlqalydkhkaqfgkgeelvim
Figure imgf000023_0001
Neutral Lipid Sequence SEQ ID
Synthesizing NO:
Enzyme
LCAT mkkisshysvviailvvvtmtsmcqavgsnvyplilvpgnggnqlevrldreykpssvwcsswl 1 1
(A. thaliana) ypihkksggwfrlwfdaavllspftrcfsdrmmlyydpdlddyqnapgvqtrvphfgstksllyl
dprlrdatsymehlvkalekkcgyvndqtilgapydfryglaasghpsrvasqflqdlkqlvektss enegkpvillshslgglfVlhflnrttpswrrkyildifvalaapwggtisqmktfasgntlgvplra^ llvrrhqrtsesnqwllpstkvfhdrtkplvvtpqvnytayemdrffadigfsqgvvpyktrvlplte elmtpgvpvtciygrgvdtpevlmygkggfdkqpeikygdgdgtvnlaslaalkvdslntveidg vshtsilkdeialkeimkqisiinyelanvnavne
ACAT mtetkdllqdeeflkirrlnsaeankrhsvtydnvilpqesmevsprssttslvepmesteaervag 12
S. cerevisiae) kqeqeeeypvdahmqkylshlksksrsrfhrkdaskyvsffgdvsfdprptlldsainvpfqttfk
gpvlekqll nlqltktktkatvkttvkttektdkadappgeklesnfsgiyvfawmflgwiaircct dyyasygsawnkleivqymttdlftiamldlamflctffvvfvhwlvkkriinwkwtgfvavsif elaflpvtφiyvyyfdfhwvtriflflhs fvmkshsfaίyngylwdikqeleyssk^^
lspelreilqkscdfclfelnyqtkdnd^imiscsnffmfc^vlvyqinyprtsrirvvryvlekvc aiigtiflmmvtaqffmhpvamrciqflmtptfggwipatqewfhllfdmipgftvlymltfy
wdallncvaeltrfadryfygdwwncvsfeefsri nvpvhkfllrhvyhssmgalhlsksqatlf tfflsavfhemamfaii -vrgylfmfqlsqfvwtalsntkflrarpqlsnvvfsfgvcsgpsiimtly
Itl
ACAT mdkkkdlleneqflriqklnaadagkrqsitvddegelygldtsgnspanehtattitqnhsvvasn 13
(S. cerevisiae) gdvafipgtategnteivteevietddrunfkthvktlsskekaryrqgssnfisyfddmsfehrpsil
dgsvnepfktkfVgptlekeirrrekelmamrknlhhrksspdavdsvgkndgaapttvptaatse tvvtvettiissnfsglyvaiSvmaiafgavkalidyyyqhngsfkdseilkfmttnlftvasvdllmy lstyfVvgiqylckwgvlkwgttgwiftsiyeflfvifymyltenilklhwlskiflflhslvllmkm hsfafyngylwgikeelqfsksalakykdsindpkvigalekscefcsfelssqslsdqtqkfpnnis aksffwftm tliyqieyprtkeirwsyvlekicaifgtiflmmidaqilmypvamralavrnse wtgildrllkwvgllvdivpgfivmyildfyliwdailncvaellrfgdryfygdwwncvswadfs riwnipvhkfllrhvyhssmssMiiksqatlmtfflssvvhelamy
valtntkfmrnrtiignvirwlgicmgpsvmctlyltf
[0073] There are two structurally unrelated versions of the enzyme. DGATl generally has a broad substrate specificity, whereby the fatty acid found in the sn-3 position of TAG is proportional to the concentration of that fatty acid in the larger pool. DGAT2 has tighter substrate specificity, and might channel unusual fatty acids into TAG so they do not upset the function of the biological membranes. In plants, both DGATl and DGAT2 are located in the ER, however a new version (DGAT3) was recently found to be located in the cytoplasm of peanut.
[0074] DGATl was first cloned from Arabidopsis in 1999. Over-expression of the gene increased TAG accumulation by 10-70% in Arabidopsis seeds. In tobacco, TAG was increased seven-fold and appeared as lipid droplets in the cells. It has subsequently been discovered that changing a single amino acid in the enzyme abolishes a post-translational phosphorylation regulatory mechanism, such that the enzyme remains active in non-seed tissues. The mutant increased DGATl activity by 38-80%, which led to a 20-50%) increase in oil content on a per seed basis in Arabidopsis. [0075] The DGAT1 enzyme from Arabidopsis is functional between kingdoms, as expression of the gene in yeast resulted in a 200-600 fold increase in DGAT enzyme activity, which led to a 3-9 fold increase in TAG observable as a floating layer of oil in the culture. Quite a lot is known about the specificities and modulating factors of the enzyme, but it does appear that the specific activity of the enzyme has been measured.
[0076] Where a specific fatty acid is desirable, such as a long-chain PUFA, DGAT1 has been shown to be applicable, provided it accepts the fatty acid of choice. Plants generally incorporate long chain PUFAs in the sn-2 position. For the improved specificity for PUFAs, however, a DGAT2 that prefers these fatty acids may be beneficial, or the properties of DGAT 1 could be altered using a directed evolution procedure similar to those previously described.
[0077] Phospholipid:DAG acyltransferase (PDAT) forms TAG from a molecule of phospholipid and a molecule of diacylglycerol. PDAT is quite active when expressed in yeast but does not appreciably increase TAG yields when expressed in plant seeds. PDAT and a proposed DAG:DAG transacylase are neutral lipid synthesizing enzymes that produce TAG, but are not considered part of the Kennedy Pathway.
[0078] A combination wax ester synthase and DGAT enzyme (WS/DGAT) has been found in all neutral lipid producing prokaryotes studied so far, and M. tuberculosis has 15 homologues thereof. WS/DGAT has extraordinarily broad activity on a variety of unusual fatty acids, alcohols and even thiols. This enzyme has a putative membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2 families from eukaryotes or the WE synthase from jojoba. (Jojoba is the only eukaryote that has been found to accumulate wax ester.)
[0079] Lecithin-cholesterol acyltransferase (LCAT) and Acyl-coenzyme: cholesterol acyltransferase (ACAT) are enzymes that produce sterol esters, a form of neutral lipid.
[0080] Accordingly, it is an object of the present invention to deliver nucleic acid constructs to a microbial cell. As is known in the art, nucleic acid constructs may be delivered using viral and non-viral methods.
[0081] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, or algal organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined. [0082] As used herein, the term "construct" refers to an artificially assembled or isolated nucleic acid molecule that includes the gene or nucleic acid molecule of interest. In general, a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be understood that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. Also included in the term "construct" are vectors. As used herein, the term "vector" encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources. Thus, an "expression vector" refers to a cloning vector that also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene of interest, so that the gene product of the gene can be expressed.
[0083] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987).
[0084] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art {e.g. , Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current
Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention.
[0085] Several non- viral methods for the transfer of expression constructs into microbial cells are contemplated by the present invention. Suitable methods for nucleic acid delivery for use with the current invention include methods as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of "naked"
DNA plasmid via the vasculature (U.S. Pat. No. 6,867,196, incorporated herein by reference); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al, 1979; Nicolau et al, 1987;
Wong et al, 1980; Kaneda et al, 1989; Kato et al., 1991) and receptor-mediated transfection
(Wu and Wu, 1987; Wu and Wu, 1988); by agitation with silicon carbide fibers (Kaeppler et al,
1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference); use of cationic lipids; naked DNA; or by microencapsulated DNA (U.S. Pat. Appl. No. 2005/0037085 incorporated herein by reference). Through the application of techniques such as these, target cells or tissue can be stably or transiently transformed.
[0086] Once the construct has been delivered into the cell, the nucleic acid encoding the enzyme or protein of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the enzyme or protein of interest may be stably integrated into the genome of the cell. As used herein, the term "genome" refers to the DNA or set of chromosomes or genes that make up an organism, and is passed to the organism's offspring. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene
augmentation). In yet further embodiments, the nucleic acid may be stably integrated in the chloroplast of the microbial cell. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
[0087] In certain embodiments of the invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers. The addition of nucleic acid molecules to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules. These DNA-lipid complexes are potential non-viral vectors for use in nucleic acid delivery.
[0088] Other vector delivery systems which can be employed to deliver a nucleic acid encoding a nucleic acid molecule into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993). Where liposomes are employed, other proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. [0089] Another transformation method includes ballistic transformation (U.S. Pat. No.
4,945,050, incorporated herein by reference). Briefly, naked DNA is coated onto carrier particles such as gold and forcefully impacted into the cell, for example via compressed gas. This method is useful for transformation of species with a thick cell wall, and also for transformation of the chloroplast (see, e.g.,(Manuell, Beligni et al. 2007), incorporated herein by reference).
[0090] In one embodiment, agrobacterium-mediated transfection is used for transformation in plants. It has been shown that this method is applicable to algal species (see, e.g., (Bellucci, De Marchis et al. 2008), incorporated herein by reference).
[0091] Other means for transformation include, but are not limited to, electroporation and agitation of cell- wall deficient mutants glass beads. For a comprehensive review of algae transformation methods review, see, e.g., (Coll 2006), incorporated herein by reference.
[0092] In another embodiment of the invention, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane.
[0093] Accordingly, in one aspect, the invention provides a method for producing a microbial cell expressing at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein. The method includes introducing a nucleic acid construct into a microbial cell, wherein the construct includes, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid
encapsulation protein. Thereafter, the cell is cultured so as to cause the cell to express the neutral lipid synthesizing enzyme and the neutral lipid encapsulation protein. In one embodiment, the construct further includes at least one promoter.
[0094] It should be understood that, as discussed above, the nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the nucleic acid molecule encoding a neutral lipid encapsulation protein may either be contained in a single construct, or more likely in separate constructs, each of which being introduced into the microbial cell. In one embodiment, a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of constructs, each containing a nucleic acid molecule encoding a neutral lipid synthesizing enzyme or a neutral lipid encapsulation protein may be introduced into the cell.
[0095] As used herein, the term "microbial cell" refers to any cell derived from a microbial organism. The term "microbial organism" or "microbe" refers to any non-plant and non-animal single-celled organism to which the methods of the invention may be performed. The microbial organism may be prokaryotic or eukaryotic. Exemplary microbial organisms include, but are not limited to yeasts, bacteria, algae, protists, archaebacteria, and synthetic forms thereof (i.e., synthetic cells).
[0096] In one embodiment, the method involves use of cross breeding two or more cells that have been modified with one or more nucleic acid molecules to express a neutral lipid
synthesizing enzyme and/or a neutral lipid encapsulation protein. The method includes introducing a first nucleic acid construct into a first microbial cell, wherein the first construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid encapsulation protein, and introducing a second nucleic acid construct into a second microbial cell, wherein the second construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme. Thereafter, the first and second microbial cells are cross-bred to produce a third microbial cell comprising the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein and the nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme. The third microbial cell is then cultured in order to express the at least one neutral lipid encapsulation protein and the at least one neutral lipid synthesizing enzyme.
[0097] Various nucleic acid sequences are embodied in the present invention. As used herein, "nucleic acid" sequence or equivalents thereof refer to a DNA or RNA sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- mefhylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5 -oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5 -oxyacetic acid methylester, uracil- 5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine. [0098] The term "variant" as used herein refers to nucleotide and polypeptide sequences wherein the nucleotide or amino acid sequence exhibits substantially 60% or greater homology with the nucleotide or amino acid sequence of the Figures, preferably 75% homology and most preferably 90-95% homology to the sequences of the present invention. - as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides) or BLAST X (nucleotides). The variant may result from modification of the native nucleotide or amino acid sequence by such modifications as insertion, substitution or deletion of one or more nucleotides or amino acids or it may be a naturally-occurring variant. The term "variant" also includes homologous sequences which hybridize to the sequences of the invention under standard hybridization conditions defined as 2 x SCC at 55°C, or preferably under stringent hybridization conditions defined as 2 x SSC at 65°C or very stringent hybridization conditions defined as 0.1 x SSC at 65°C, provided that the variant is capable of substantially performing the equivalent biological function of the neutral lipid/oil encapsulation protein; or the neutral lipid synthesizing enzyme; as would be required to perform the present invention. Where such a variant is desired, the nucleotide sequence of the native DNA is altered appropriately. This alteration can be effected by synthesis of the DNA or by modification of the native DNA, for example, by site-specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence
modifications, site-specific primer directed mutagenesis is employed, using techniques standard in the art.
[0099] It will be appreciated that the term 'manipulated', 'manipulation' or grammatical variations thereof, refers to the alteration of genetic information in a microbial cell (e.g., an algal or plant cell), by a number of suitable genetic techniques, including, but not limited to:
introducing a nucleic acid molecule of interest to a cell; mutagenesis techniques; and/or traditional microbial breeding techniques (unless specifically excluded); or any combination thereof.
[0100] The term "introducing" (or grammatical variants thereof) when used in the context of inserting a nucleic acid molecule into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation or transfer of a nucleic acid molecule into a eukaryotic or prokaryotic cell where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). 6
[0101] Other nucleic acid sequences include "control sequences", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.
[0102] Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters." The promoter molecule may be an RNA, cRNA, genomic DNA or cDNA molecule, and me be single or double stranded. The promoter molecule may also optionally include one or more synthetic, non-natural or altered nucleotide bases, or any combination thereof. Examples of constitutive plant promoters that may be effective in algae include, but are not limited to, the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is incorporated herein by reference. Exemplary promoters and selection genes suitable for transformation and selection of algae are summarized in, e.g., (Walker, Collet et al. 2005) and (Hallmann 2007), both of which are incorporated herein by reference. Methods for transformation, promoters and selectable marker genes suitable for transformation of the chloroplast are known to those skilled in the art (see, e.g., (Bateman and Purton 2000), incorporated herein by reference).
[0103] Several strategies for increasing the expression of transgenes are known by those skilled in the art. For example, inclusion of an intron, optimization of G/C content, optimization of codon usage to match that of the host organism, elimination of cryptic splice sites, and elimination of mRNA degradation signals are known. Furthermore, a strain of Chlamydomonas has been developed that has high predictable levels of transgene expression (see, e.g., (Neupert, Karcher et al. 2008), incorporated herein by reference).
[0104] Exemplary terminators that are commonly used in plant transformation genetic construct include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solarium tuberosum PI-II terminator.
[0105] The nucleic acids embodied in the present invention are "operably linked" to each other or linked to a protein or peptide. As used herein, "operatively linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter . sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.
[0106] The polynucleotides described herein encode for a "polypeptide", for example, neutral lipid synthesizing enzymes and/or neutral lipid encapsulation proteins. "Polypeptide" is used in its conventional meaning, i.e., as a sequence of amino acids. The polypeptides are not limited to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise. A polypeptide may be an entire protein, or a subsequence thereof.
[0107] The term "ortholog," "orthologous gene," or "orthologous polypeptide" refers to a functionally equivalent yet distinct corresponding nucleotide or amino acid sequence that may be derived from another plant. In general, an ortholog may have a substantially identical nucleotide or amino acid sequence to the sequences of the present invention as set forth in the sequence listing. The term "homolog" refers to a related gene from a different but related species.
[0108] In another aspect, the invention provides a method for producing an algal cell expressing at least one neutral lipid synthesizing enzyme. The method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter, at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and culturing the algal cell in order to express the at least one neutral lipid synthesizing enzyme.
[0109] In yet another aspect, the invention provides a method for producing an algal cell expressing at least one neutral lipid encapsulation protein. The method includes introducing a nucleic acid construct into an algal cell, wherein the construct comprises at least one promoter and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein, and culturing the algal cell in order to express the neutral lipid encapsulation protein.
[0110] It should be understood that "algae" refers to a family of aquatic, eukaryotic single cell or multicellular organisms without stems, roots and leaves, that are typically autotrophic, photosynthetic, contain chlorophyll, and grow in bodies of water, including fresh water, sea water, and brackish water, with the degree of growth being in relative proportion to the amount of nutrients available. As such, algae are distinguishable from and therefore not included in the term "plant." The term "microalgae" refers to photosynthetic protists that include a variety of unicellular, coenocytic, colonial, and multicellular organisms, such as the protozoans, slime molds, brown and red algae, algal strains, diatoms, dinoflagellates, etc. It should be understood that while some definitions of "algae" include cyanobacteria because they are unicellular and photosynthetic, cyanobacteria are prokaryotic and are included in the definition of "bacteria" for the purposes of this invention.
[0111] Exemplary algae include, but are not limited to, organisms of the division of
Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae),
Bacillariophyceae (diatoms), or Dinoflagellata (dinoflagellates). In one embodiment, the alga is a species of Chlamydomonas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis,
Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parietochloris. In another embodiment, the alga is Chlamydomonas reinhardtii.
[0112] Exemplary yeasts include, but are not limited to, oleaginous species of the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces. It is also recognized that yeast species commonly used in biotechnology such as Saccharomyces or Pichia could be engineered to produce neutral lipid, and are therefore suitable host species for the methods of this invention.
[0113] Exemplary bacteria include, but are not limited to, oleaginous organisms from the genus Rhodococcus. It is also recognized that bacterial species commonly used in biotechnology such as Escherichia coli or Bacillus subtilis could be engineered to produce neutral lipid, and are therefore suitable host species for the methods of this invention. Exemplary bacteria also include cyanobacteria including, but not limited to, organisms from the genus Spirulina, Synechococcus or Synechocystis.
[0114] Certain useful products including omega-3 fatty acids can be made in protists.
Exemplary protists include, but are not limited to, the Heterokontophyta and Alveolata. In one embodiment, the protist is an oleaginous species of the order Thraustochytriales. In yet another embodiment, the protist is from the genus Schizochytrium, Thraustochytrium, Ulkenia, or Pythium
[0115] As used herein, the term "synthetic cell" refers to a single-celled organism that is created by man and not found in nature. While it is well known that all transgenic organisms are man-made and not found in nature, synthetic cells differ in the methods of modification and extensive degree to which they are modified or holistically designed. Typically, synthetic cells, rather than a simple transgenic organism, are created to provide an organism of minimal genome size and complexity upon which useful products can be made in a precise manner. (For a review of the current state of the field refer to (Carr and Church 2009), incorporated herein by reference). In one example of a synthetic cell, an entire genome is synthesized from chemical building blocks and transplanted into a naturally occurring organism, replacing the natural genome and therefore changing the species identity of the resulting cell. As an example of this method, researchers created Mycoplasma laboratoriwn from Mycoplasma genitalium through genome transplantation (U.S. Pub. No. 20070122826, incorporated herein by reference; and (Gibson, Benders et al.
2008). In another example of a synthetic cell, researchers demonstrated a technique for making large numbers of targeted changes to a genome (Wang, Isaacs et al. 2009). In yet another example of a synthetic cell, researchers demonstrated the deletion of large numbers of genes to create a minimal genome size (Posfai, Plunkett et al. 2006). Synthetic cells also include cell-like systems as described in (US Pub. No. 2007/0269862, incorporated herein by reference). [0116] Accordingly, in another aspect, the invention provides a microbial cell that has been manipulated to produce at least one neutral lipid synthesizing enzyme and at least one neutral lipid encapsulation protein. Likewise, the invention also provides an algal cell which has been manipulated to produce at least one neutral lipid synthesizing enzyme, and/or an algal cell that has been manipulated to produce at least one neutral lipid encapsulation protein.
[0117] It is recognized that the methods of the invention can be applied to any microbial species for which suitable methods for transformation and cultivation exist. Furthermore, the particular oil synthesis and oil storage mechanisms of a particular microbial species can be altered, however a discussion of the oil synthesis and storage methods of native species informs someone skilled in the art about which particular embodiments of the invention are likely to have a particular effect in the host organism.
Oil Production in Algae
[0118] Comparison of the lipid profiles of many strains of algae show that there is
considerable diversity between classes. This is an advantage for algae relative to agricultural crops as potential feedstocks for the oleochemical industry (Table 5).
[0119] Regarding neutral lipid synthesizing genes, DGAT and PDAT homologs can be identified in all algal genomes sequenced to date, although they exhibit distinct distribution patterns. Chlamydomonas reinhardtii has three genes encoding for DGATs but is missing a PDAT homolog. The diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum have both DGAT and PDAT. Ostreococcus tauri lacks a recognizable DGAT, but does have a PDAT. Cyanidioschyzon merolae has DGAT but not PDAT. Microscopy reveals that eukaryotic algae synthesize neutral lipids in the ER and deposit them in the cytoplasm. In addition, algae are also known to form plastoglobules. Plastoglobule-like structures constitute the eyespot of
Chlamydomonas.
[0120] Without being bound by theory, neutral lipids do not seem to be a storage end-point in algae like they are in oil seeds. For example, in the red algae, Porphyridium cruentum, neutral lipids are rapidly remobilized to rebuild the chloroplast when nitrogen is reintroduced after starvation. Oil Production in the Algal Chloroplast
[0121] The chloroplast is descended from an endosymbiotic cyanobacteria and is the "fat factory" of the cell. As such, it is envisioned that forcing this cell-within-a-cell to resemble a plant seed. The chloroplast contains plastoglobules that share superficial similarities with plant oil bodies, but are dynamic structures and never fully dissociate for long term oil storage.
Accordingly, the instant invention provides methods to up-regulate the synthesis of oil into the plastoglobules by expressing acyltransferases such as DGAT. The concomitant expression of oleosin or the native plastoglobulin may subvert the remobilization of oil and even dissociate a storage structure.
Table 5 - Lipid profiles of broad classes of algae
r
cmo
[0122] For maximizing the formation of oil storage structures, the chloroplast is an ideal organelle. Not only is it the cell's "fat factory", but it also accumulates much higher levels of recombinant protein due to a lack of epigenetic phenomena and ability to accommodate multiple copies of the transgene. In addition, algae contain a single large chloroplast (compared with high plants which have many small ones) which greatly simplifies their transformation and regulation (Figure 7). [0123] It is also expected that the chloroplast-based strategy would aid extraction. The cell wall and chloroplast outer membranes would still need to be ruptured, but by over expressing plastoglubulin or oleosin, stable storage structures are created. The purification of these structures by density gradient centrifugation has been demonstrated, similar to ER generated oil bodies.
Oil Production in Yeast
[0124] With regard to expression of plant genes in yeast, DGAT over-expression causes a floating layer of oil to develop in the culture (Bouvier-Nave, Benveniste et al. 2000) (Beaudoin and Napier 2002) (Froissard, D'Andrea et al. 2009) (Beaudoin, Wilkinson et al. 2000). It has been shown that caoleosin can be expressed in yeast (Froissard, D'Andrea et al. 2009). Yeast also have an oil production system of their own consisting of four partially redundant enzymes. Dgalp is homologous to DGAT2 of plants. It is targeted to the ER, but also dissociates with and remains active on the lipid particle. The other three neutral lipid producing enzymes are only associated with the ER. Lrolp has PDAT activity and some sequence homology to the plant PDAT, but the enzymes are evolutionarily distant. Arelp and Are2p are DGATl-type enzymes, but minor in yeast compared with Dgalp and Lrolp.
[0125] Yeast also produce a number of sterol esters in addition to neutral lipids. The DGAT1 - type enzymes (Arelp and Are2p) are also known as sterol acyltransferases (or sterol ester synthases) because of this activity. Yeast store neutral lipids and sterol esters in a common "lipid particle" that has several ordered shells of sterol esters below the surface phospholipid monolayer, whereas the neutral lipids are randomly packed in the center. The lipid particle typically has a diameter of about 0.4 μιη and has approximately 40 associated proteins.
[0126] Unlike plants, the yeast lipid particles are able to form neutral lipids autonomously. The lipid particle associated multi-enzyme complex from the oleaginous yeast Rhodotorula glutinis was isolated, the proteins characterized, and shown to incorporate free fatty acids or fatty acyl-coenzyme A into neutral lipids quickly. In another example, a pathway for complete de novo synthesis was reviewed. A picture is emerging that the lipid particles of yeast are active organelles with a number of roles potentially including trafficking of lipids and proteins within the cell, chaperone activity and sequestration of small molecules and misfolded proteins. Oil Production in Bacteria
[0127] Bacteria generally produce polyhydroxyalkanoates rather than neutral lipids as an energy storage molecule, but exceptions include some Mycobacterium and Streptomyces species. For example, Rhodococcus opacus can accumulate neutral lipids to 87% by weight. The two pathways compete for acetyl-CoA in cases where both are present. As with other microbes, neutral lipids are produced during times of stress such as nitrogen starvation. The responsible enzyme is a combination wax ester synthase and DGAT (WS/DGAT) that has extraordinarily broad activity on a variety of unusual fatty acids, alcohols and even thiols. This enzyme has a putative membrane-spanning region but shows no sequence homology to the DGAT1 and DGAT2 families from eukaryotes or the WE synthase from jojoba. (Jojoba is the only eukaryote that has been found to accumulate wax ester.) The WS/DGAT enzyme has been found in all neutral lipid producing prokaryotes studied so far, and M. tuberculosis has 15 homologues thereof.
[0128] However, the enzyme doesn't synthesize oil between the leaflets of the phospholipid bilayer. Rather, an oily layer develops just inside the plasma membrane and eventually blubs off oil drops of a characteristic size (-200 nm in Acinetobacter calcoaceticus). These droplets have a phospholipid membrane, but don't seem to have any structural proteins.
[0129] When fed oleate, E. coli over expressing WS/DGAT and the fatty alcohol-producing bifunctional acyl-Coenzyme A reductase from jojoba produces neutral storage lipids. This definitively showed that WS/DGAT makes oil structures without any other structural proteins. WS/DGAT was also shown to restore TAG synthesis in the (Adgal Alrol arel Aare2) yeast quadruple mutant.
Oil Production in Cyanobacteria
[0130] Cyanobacteria could have advantages over eukaryotic algae for the production of renewable oils, most significantly the ability of some species to fix atmospheric nitrogen.
However, the species are not naturally oily. In one suggested cyanobacterial GMO scheme, the position of glyceraldehyde-3 -phosphate (GAP) as a key metabolite in the Calvin cycle
(phototrophy) and as a substrate for biosynthesis (anabolism) would allow diversion of sunlight energy to products such as oil (Figure 5). [0131] While cyanobacteria are technically bacteria, no evidence has proven their use of the WS/DGAT system. Since living cyanobacteria and the chloroplast share a common ancestor, and both have thylakoid membranes, the oil synthesis mechanism is similar (Figure 8). Indeed, "lipid droplets" have been reported among the thylakoids in cyanobacteria. TAG, sterol esters and carotenoids are stored in these structures under stress. Furthermore, Synechocystis has two plastoglobulin homologs.
Oil Production Process
[0132] While it is recognized that the methods of the invention can be performed in batch processes, in certain aspects, the invention provides methods that are generally performed in a continuous process. Compared with a batch or fed-batch process, continuous processes better utilize large capital intensive pieces of equipment (Figure 10). Additionally, labor costs are generally higher for batch processes than for continuous processes. Furthermore, batch processes can be more difficult and expensive to control and produce a consistent product than a continuous process that operates at a steady state. The advantages of continuous processes over batch processes are magnified for fuel production where scales on the order of millions of metric tons per year are required and the fuel must be produced at a price competitive with petroleum-based products.
[0133] Current oil production processes utilizing microbial species can be categorized into two groups a) methods that employ unmodified species and the "lipid trigger"; and b) methods that employ modified species without a means for oil encapsulation. Both of these categories are limited to batch processes.
[0134] Category (a) is by definition a batch process because the cells alternate between periods of cell division and periods of oil production. Once sufficient oil is produced the cells must be harvested as a batch because returning to conditions of cellular growth would result in degradation of oil as an energy source for the cell (Figure 11).
[0135] Category (b) is not by definition a batch process, but seems to be limited to a batch process in practice. In theory microbial species could be genetically modified to produce oil constitutively while continuing to grow, however nature does not have examples of organisms that make high levels of oil at all times. Oil production is generally either i) a transient process in response to temporary nutrient limitation or stress (as in algae), ii) confined to a reproductive phase of life (as in plants), or iii) a backup of excess energy (as in animals). Furthermore, nature usually stores the hydrocarbon in some kind of protective storage structure (e.g., oil body, plastoglobule, perilipin, apolipoprotien, etc.).
[0136] Prior art methods for oil production of Category (b) do not provide a means for oil encapsulation as would be found in nature (Figure 9), and is an important limitation of the prior art. As continuous production of unprotected hydrocarbons do not exist in nature, the cell's health is adversely affected. The cell seems to deal with this disturbance by secreting the hydrocarbon. This solution is inadequate because the process is not perfectly continuous because the cells cannot survive the stress of unprotected oil indefinitely and their ability to grow is compromised by the unprotected and/or secreted hydrocarbon.
[0137] In contrast to methods of both Categories (a) and (b), the present invention provides methods that enable microbial cells to grow vigorously and produce oil at the same time (Figure 12). Without being bound by theory, the methods of the invention produce a microbial cell that resembles a plant seed in that it accumulates oil steadily and at the same time packages the oil into stable droplets. Algae and plant seeds both accumulate lipids for energy storage, but do so under different circumstances and using different genetics. Unlike in microbial species, plants synthesize oil in the seed to provide energy for germination.
[0138] Changing the biochemistry of oil production and storage in algae also leads to simplification of the process and equipment needed to grow the cells. Using the nitrogen starvation trigger forces the process engineer to design a batch process where all of the cells are grown, then all of the cells are fattened and finally all processed. At industrial scale this requires large and expensive storage tanks between all of the steps of the process. The reactor and other equipment also sit idle for significant portions of time as they are being cleaned and prepared for a new batch.
[0139] Furthermore, methods of oil production that result in secretion of the oil from the cell are often cited as advantageous because a) the productive period of a batch is significantly lengthened, but is still not continuous; b) the oil is currently more valuable in the market than the biomass of the cell and a larger proportion of oil to biomass is produced in secretion processes; and c) recovery of the oil is simplified because secreted oil is immiscible in the aqueous growth medium and rises to the surface of the medium where it is easily collected. [0140] However, there are disadvantages to a secretion process that are not generally recognized by those skilled in the art. First of all, the layer of secreted oil would impede light penetration into the medium for use by photosynthetic production cells. Secondly, the advantages of oil recovery are mitigated by the fact that the cellular mass must also be recovered from the medium regardless. At fuel production scales of millions of metric tons, cellular biomass must be put to economic use and cannot be a costly waste stream when producing a fuel on a cost competitive basis with petroleum-based fuels. It could be easier to harvest cells that have stored oil, and subsequently extract the oil from the cell. Furthermore, given that economically profitable outlets for biomass must be found at large scale, the relative economic value of oil versus biomass is likely to be mitigated.
[0141] By growing and fattening simultaneously, a continuous process is enabled whereby the cells flow from step to step without the need for intermediate storage. At the initial production stage, reactors can be utilized at nearly all times and their design simplified without the need to alternate between two growth conditions (Figure 10).
[0142] Methods for growing microbial species are known to those skilled in the art. In addition, methods for removing grown/cultured microbial cells from their liquid growth medium ("harvesting") are known to those skilled in the art.
[0143] Once harvested, the cell typically needs to be broken open (i.e., "lysed") to release its oil. Methods for lysing cells are known to those of skill in the art, and may be dependent upon strain selection, as some cells have thick, rigid cell walls like a vascular plant. Options for lysis include, but are not limited to, osmosis, mechanical crushing, extraction with chemicals, sonication, and genetic modification.
[0144] Following lysis, the oil may be "partitioned" from the residual biomass for various reasons. For example, the value of the biomass as an animal feed is decreased if it contains too much residual oil. The primary objective for creating oil storage structures in algae or other microbial species is to slow or eliminate oil remobilization and reduce feedback inhibition of oil synthesis, leading to continuous production of higher levels of oil. However, the tighter physical structure and the negative charge of oleosin, for example, will also likely reduce interactions between the oil and residual biomass during extraction. This is advantageous for 'cleanly' removing the oil from the lysed biomass. [0145] Without being bound by theory, it is suspected that the majority of the non-extractable oil is located in the chloroplast because this organelle is smaller and greasier and therefore harder to rupture than the cell itself The chloroplast has two concentric outer membranes and is packed with folded thylakoid membranes. These hydrophobic membranes create a chemical
environment that could make isolation of oil difficult. By creating oil storage structures, a sink for oil is created that metabolically pulls lipids from the chloroplast to the ER and then into an oil storage structure. Oil bodies for example are more than 97% pure oil, so being less dense than water, are easily extractable upon rupture of only the outer cell wall. Oil storage structures will not coalesce and therefore will not trap cell debris as unprotected oils would. Methods for converting storage oils into useful products are well known to those skilled in the art.
[0146] Thus, in another aspect, the methods of the invention will supply a superior feedstock for transesterification to biodiesel. For one, virgin oils have a higher proportion of TAG than waste oils, and therefore yield a better product requiring less refinement. Secondly, the oil neutral lipid structure formed by the methods of the invention is predominantly TAG for example. There will be a single layer of phospholipid and a single layer of oleosin for example, but these are only a small percentage of the total mass.
[0147] In summary, the methods of the invention used in conjunction with methods known in the art for microbial growth, harvesting, extraction, and conversion of oil to useful products are transformative. Oil production methods that were previously inefficient batch processes (Figure 13) can now be operated in an efficient continuous manner (Figure 14).
[0148] Accession Numbers: The accession numbers throughout this description are derived from the NCBI database (National Center for Biotechnology Information) maintained by the National Institute of Health, U.S.A., and are all incorporated herein by reference. The accession numbers are provided in the database as of March 1, 2010.
[0149] Enzyme Classification Numbers (EC): The EC numbers provided throughout this description are derived from the KEGG Ligand database, maintained by the Kyoto Encyclopedia of Genes and Genomics, sponsored in part by the University of Tokyo, and are incorporated herein by reference. The EC numbers are as provided in the database as of March 1, 2010. [0150] All references, including any patents, patent applications, and literature, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art.
EXAMPLE 1
Methods
[0151] This invention will now be illustrated with reference to the following non-limiting methods.
Methods for producing constructs and vectors
[0152] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
[0153] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al , Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for producing host cells comprising polynucleotides, constructs or vectors
[0154] The invention provides a host cell which comprises a genetic construct or vector of the invention.
[0155] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g., Sambrook et al , Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al , Current
Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g., Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
Methods for genetic manipulation of algae
[0156] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the algal species a number of algal
transformation strategies are available {e.g., electroporation, heat shock, glass beads). Nuclear transformation in Chlamydomonas is achieved through electroporation (Brown et al., 1991, Shimogawara et al., 1998) or through vortexing in the presence of DNA of interest and glass beads (Kindle, 1990). Transformation of Chlamydomonas via electroporation for the strains ccl690 and ccl24 (from the Chlamydomonas Resource Center) which have intact cell walls does not require sucrose and hence it is omitted. Voltage parameters are 2000 V/cm, 25 uF capacitance, infinite ohms, 0.4 cm cuvettes using the Gene Pulser (Bio-Rad). Cells are electroporated, incubated overnight in TAP media (Harris, 1989) and plated onto selection medium (hygromycin 20μg/mL, paromomycin 15μg/mL) and grown under continuous light for 5-7 days. Single colonies are inoculated into liquid selection media (TAP + 5μg/mL hyg, 10μg/mL paro) for phenotypic and genetic analysis.
[0157] Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.
[0158] Strategies may be designed to increase expression of a polynucleotide/polypeptide in an algal cell, and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the algal species to be transformed or may be derived from a different species.
[0159] Transformation strategies may be designed to reduce expression of a
polynucleotide/polypeptide in an algal cell, or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
[0160] Genetic constructs for expression of genes in transgenic algae typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed algae. [0161] The promoters suitable for use in the constructs of this invention are functional in an algal cell, inducible promoters, constitutive promoters that are active in most algal cells, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other algae, plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the β-tubulin (β-TUBp) promoter, the Rubisco small subunit2A (RBCS2Ap) promoter, the heat shock 70A promoter (HSP70Ap) fused to the RBCS2A promoter (HRp). Several Chlamydomonas promoter sequences have been shown to drive expression of heterologous reporter and selectable marker genes at low levels (e.g., Heitzer M. & Zschoernig, B., 2007, Fuhrmann et al., 1999). Of interest is the chimeric HSP70A-RBCS2A promoter which has been shown to drive the strongest level of expression of a reporter gene, luciferase.
[0162] Examples of an inducible promoter in algae include the nitrate reductasel (NITlp) promoter. Examples of promoter sequences are listed in Table 5.
[0163] Exemplary terminators that are commonly used in algae transformation genetic construct include, e.g., COPt, RBCS2At, MLDPt, DGAT2t, TUB2t (such sequences are listed in Table 5).
[0164] Selectable markers commonly used in algae transformation include the Streptomyces hygroscopicus aphVII and Streptomyces rimosus aphVIII selectable marker genes, whose gene products render the cells resistant to hygromycin and paromomycin, respectively (Berthold et al., 2002, Sizovaa, et al, 2001). aphVII and aphVIII sequences are listed in Table 6 and Table 7.
[0165] Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in algae are also contemplated.
[0166] The algae of the invention may be grown and either allowed to divide or crossed with a different algal strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Algae resulting from such standard breeding approaches also form an aspect of the present invention.
Optimizing construct design and transformation to reduce silencing in C. reinhardtii
[0167] The HSP70Ap: :RBCS2Ap chimera (with or without the RBCS2A intron 1 sequence embedded in it) (see sequence Tables 5 and 6 as well as Heitzer M. & Zschoernig, B., 2007, Schroda et al, 2000), was chosen to drive expression of the genes of interest. Another promoter ICLp which drives expression of isocitrate lyase in vivo was also chosen to drive expression of the genes of interest (Table 8).
[0168] The endogenous nitrate inducible promoter (NITlp), which in vivo drives expression of nitrate reductase, was also chosen to potentially overcome silencing. This promoter was shown to drive expression of the arylsulphatase reporter gene (ARS) in the absence of ammonium and presence of nitrate (Ohresser et al, 1997, Loppes et al, 1999). Since this promoter does not drive expression in the presence of ammonium (the nitrogen source in TAP media), downstream genes of interest are not expressed until ammonium is removed from the media and replaced with nitrate. Therefore the cell may not recognize such genes as foreign until their expression is turned on by the removal of ammonium (i.e., after they have been transformed, selected and grown enough biomass for phenotypic assays). It should be noted that the GFP reporter is not expressed at high enough levels to visualize either by microscopy or Western blot unless it is fused to a selectable marker (e.g., the phleomycin resistance gene ble from Streptoalloteichus hindustanus) or an endogenous Chlamydomonas gene (chlamyopsin (COP)) (Fuhrmann et al, 1999).
[0169] Each promoter has been tested by placing the promoter upstream of the aphVII gene and the resulting cassette was used to transform Chlamydomonas with selection on paromomycin containing TAP agar plates.
[0170] Over expression of foreign genes in Chlamydomonas is difficult due to the numerous efficient silencing mechanisms that algae possess (for reviews see Kim, E-J, Cerutti, H, 2009; Eichler-Stahlberg et al., 2009). A number of modifications can be made to foreign genes in an attempt to reduce the degree of silencing. The majority of these approaches involve modifying the sequences so that they more closely resemble genes found endogenously within the
Chlamydomonas genome. More specifically using the 5' UTR from an endogenous gene, placing an endogenous Chlamydomonas intron into the coding sequence or 5'UTR, changing the codons to only those that are predominantly used by Chlamydomonas, using a 3' UTR from an endogenous gene, where possible using the 5' UTR as well as the first 8 amino acids and intron from the endogenous MLDP (GenBank: XP_001697668) or the first 8 amino acids and intron from the endogenous gene beta-2 tubulin (GenBank: AAA33102.1).
[0171] It is also preferable to reduce the degree of duplication in the construct. As such, different promoter and terminator sequences are typically used for each gene (including selectable markers) and in many cases use different intron sequences.
Optimized DNA levels for transformation of Chlamydomonas reinhardtii
[0172] Typically, the more DNA used for transformation the more DNA will be incorporated into the genome (at the same loci as well as at different loci). It is preferable to titre each construct or construct preparation to minimize the quantity of DNA used for transformation since it is recognized that silencing is enhanced when excessive DNA is used in the transformation.
Designing plasmids so the backbone can be removed prior to transformation
[0173] The backbone of plasmids is typically of prokaryotic origin and therefore very different to the high GC rich gene coding regions of algae. Removal of as much as this as possible is preferable to reduce the degree of silencing. Cutting right at the end of the terminator or promoter is an option although this may have unintended consequences when the construct is inside the nucleus prior to its integration into the genome where exonuclease activity is high. One approach to minimizing the selection of constructs in which the gene of interest has been partially or completely degraded but the selectable marker is still intact is to flank the gene of interest between two separate selectable markers. In this case, both paromomycin and hygromycin were chosen as the two selectable markers. In addition the genes of interest are in a back to back orientation facing the flanking selectable marker cassettes which themselves are both facing outwards (Table 8, Figures 15, 16 and 17).
Working in lines with reduced nuclear gene silencing
[0174] Substantial work has gone into identifying algal lines with reduced levels of nuclear gene silencing (Casas-Mollano et al., 2008; Bock and Karcher 2009 and references therein). The majority of these have been found through the release of silencing of a marker gene. Some of these have been generated through natural mutation while others have been selected after mutagenesis (e.g., chemical or UV induced). Expression of a foreign gene or genes in algal lines with reduced silencing is anticipated to result in a higher expression of the genes of interest compared to lines in which silencing is fully functional.
Methods for Growth, Induction and Nitrogen Starvation of algal cells
[0175] Independent Chlamydomonas transformants were inoculated into 200μΙ liquid selection media (TAP + l(^g/mL paro, 5μg/mL hyg (where appropriate)) in 96-well plates, allowed to grow for 3-4 days under continuous moderate light at 25C, diluted 1 : 100 into 6-well plates (60μΙ, inoculum into 6mL fresh selection media), and allowed to grow with shaking under continuous light at 25 C for 3-4 days. Cultures were then analyzed for gene insertion and gene expression by standard molecular biology techniques (Sambrook, et al) including PCR, Southern analysis, and RT-PCR. Cultures were also analyzed phenotypically via fluorescence microscopy and Nile Red fluorescence assays.
[0176] For transformants containing the NITlp, cells were grown as above, harvested by centrifugation (5 min @ 2,000xg) and resuspended in induction media (TAP + 4mM KN03 (no NH4C1) l(^g/mL paro, 5μg/mL hyg (where appropriate) and transferred to new 6-well plates. Plates were incubated under continuous light with constant shaking at 25°C. Samples were removed at 4h, 8h and 24h time points and analyzed for gene expression and phenotype via RT- PCR and Nile Red fluorescence assays.
[0177] For nitrogen starved controls, wild-type algal cultures were grown to log phase, harvested by centrifugation (5 min @ 2000xg) and resuspended in nitrogen-free TAP media (i.e., TAP with NH4C1 omitted). Resuspended cells were transferred to 6-well plates and incubated with constant shaking under continuous light @ 25°C for 1-8 days.
Methods for Expression in Saccharomyces cerivisiae
[0178] The yeast expression vector pYES2.1 V5-His Topo was purchased from Invitrogen (Carlsbad, CA). The quadruple yeast mutant Saccharomyces cerevisiae strain HI 246 was obtained from the Swedish University of Agricultural Sciences, Sweden. This yeast strain is deficient in all four genes (DGA1, LROl, ARE1 and ARE2) that encode the enzymes for lipid biosynthesis in yeast (Sandager, et al., 2002). Yeast competent cells were prepared using S. c. EasyComp™ Transformation Kit (Invitrogen, Carlsbad, CA). The Yeast synthetic incomplete medium (without uracil, histidine, leucine and tryptophan), yeast nitrogen base (YNB), D(+) Glucose, and D(+) Raffinose pentahydrate were purchased from Sigma (Sigma Aldrich Co., USA.). Bacto agar was procured from Difco (Detroit, MI, USA).
[0179] Routinely, yeast cells were grown aerobically overnight in a synthetic medium with 0.67% YNB, without uracil (SC-U), and containing 2% raffinose. Cells from overnight culture were used to inoculate 200 mL of induction medium (SC-U containing 2% galactose and 1% raffinose) to an initial OD600 of 0.6. Cells were allowed to further grow at 30°C, with shaking at 200 rpm for 24 h. Cell pellets were collected by centrifugation at 1500 x g for 5 min then washed with distilled water and either used immediately for subsequent analysis or kept in -80°C until required. Cell pellets for neutral lipid extraction were freeze-dried for 48 h and stored in -20°C freezer until required.
Methods for Construct Design for Expression in Saccharomyces cerivisiae
[0180] The nucleotide or translated amino acid sequences of the putative DGAT1 gene or protein were obtained from GenBank or TAIR with the following identification: Arabidopsis DGAT1 or AtDGATl (GenBank Accession no. AJ238008.1). The sequence was optimized for protein expression in yeast and were synthesized by Geneart (Geneart AG, Germany). The Chlamydomonans reinhardtii DGAT2 gene (GenBank Accession no XP_001693189);
Chlamydomonas reinhardtii MLDP gene (GenBank Accession no XP_001697668); Sesamum indicum oleosin gene (GenBank Accession no AAD42942) and Sesamum indicum oleosin gene engineered to contain cysteine residues were optimized for protein expression in Saccharomyces cerivisiae and were synthesized by GenScript (GenScript USA Inc.). Nucleic and translated sequences optimized for expression in S. cerivisiae are listed in Tables 9 and 10.
[0181] The Arabidopsis thaliana DGAT1 and Sesamum indicum oleosin were organized into the same plasmid in a back to back orientation each under the control of their own separate GALl promoter (Table 9).
[0182] The Arabidopsis thaliana DGAT1 and Sesamum indicum oleosin modified to contain cysteine residues were organized into the same plasmid in a back to back orientation each under the control of their own separate GALl promoter (Table 9 and Figure 18).
[0183] The Chlamydomonas reinhardtii DGAT2 and Chlamydomonas reinhardtii MLDP were organized into the same plasmid in a back to back orientation each under the control of their own separate GALl promoter (Table 9 and Figure 19). [0184] The Chlamydomonas reinhardtii DGAT2 and Sesamum indicum oleosin modified to contain cysteine residues were organized into the same plasmid in a back to back orientation each under the control of their own separate GAL1 promoter (Table 9 and Figure 20).
[0185] Standard DNA procedures were performed as described by Sambrook, et al. (1989). If high purity DNA was required, plasmid DNA was extracted using High Pure Plasmid Isolation Kit (Roche Diagnostics) following manufacturer's protocol. For cloning of the DGAT1 construct into the yeast expression vector, Topo TA cloning was carried out. To do this, an A' overhang was created into the gene using a five-cycle PCR. This was performed using forward primer 5'- gccgcatttaattaagaattc-3 ' (SEQ ID NO: 207). This primer was paired with a reverse primer 5'- ggcagacatagaaccctttcta-3 ' (SEQ ID NO: 208). The following PCR program was executed: initial denaturation of 95°C for 5 min; then five cycles of denaturation at 95°C for 30 s, annealing at 60°C for 45 sec, and extension at 72°C for 1 min and 45 s; concluded by a final extension of 7 min. Immediately after PCR, the DGAT1 gene was cloned into the pYES2.1/V5 His topo vector using Topo TA cloning Kit (Invitrogen) following the manufacturer's protocol. For confirmation of right DNA sequence of each construct and chimeras, full length DNAs were sequenced using different sets of primers designed from different regions of the DNA. DNA sequencing was carried out using an ABI3730 DNA Analyser, Applied Biosystems, Inc.
[0186] Yeast transformation was conducted using the S. c. EasyComp™ Transformation Kit (Invitrogen). Briefly, after thawing competent cells, 50 xL was aliquoted into an Eppendorf tube and one microgram of DNA was added. After addition of Solution III (Invitrogen), the
DNA/competent cell mixture was vortexed vigorously and then incubated at 30°C for one hour, with the mixture being vortexed every 15 min. Shortly after this incubation, the transformation reaction was added with one milliliter of SC medium and incubated at 30°C with shaking at 200 rpm for lh. At the conclusion of incubation, cells were pelleted by centrifugation at 3000 x g for 5 min, and resuspended in 100 ih of Solution III (Invitrogen). Finally, cells were plated onto SC- U selection plates and incubated at 30°C for 3 days.
Lipid extraction and analysis
Preparation of material for FAMES-GC/MS analysis
[0187] 10 mg of freeze-dried powdered algal cells was placed in a 13 x 100 mm screw-cap tube, 10 \iL of non methylated internal standard (CI 5:0 FA, 4 mg/mL dissolved in heptane) was added, To this mixture, 1 mL of the methanolic HCl reagent (1 mL of 3 M solution diluted to 1 M using dry methanol which had been treated with 5% 2,2-dimeethoxypropane as a water scavenger. The tube was then flushed with N2 gas then sealed immediately with Teflon-lined cap, and heated at 80°C for 1 h. After the tubes had cooled to room temperature, 10 μΐ, pre-methylated standard (4 mg/mL of CI 7:0 dissolved in heptane) was added. To this mixture, 0.6 mL heptane and 1.0 mL of 0.9% (w/v) NaCl was added, and mixed thoroughly by vortexing. Following centrifugation at 500 rpm for 1 min at room temperature, 100 μΐ, of the top layer (containing heptanes) was collected and transferred to a flat-bottom glass insert fitted into a brown vial for GC/MS analysis.
FAMES GC/MS Analysis
[0188] The FAMES GC/MS was analyzed using the SGE capillary column BPX70 (50m x 0.22 mm x 0.25 μηι). The condition of GC-MS was as follows: the temperature was programmed from 80 °C to 150°C at 15°C /min and then to 250°C at 8°C /min and held isothermal for 10 min. Samples were injected in a split mode; total flow of 28.4 mL/min; column flow of 0.82 mL/min; and a purge flow of 3.0 mL/min. The pressure was kept at 150 kPa; ion source temperature was 200°C and an interface temperature was kept at 260°C. The target compounds were acquired by mass spectrometry in a scan mode starting at 50 m/z and ending at 350 m/z.
TAG extraction
[0189] Neutral lipid from yeast was extracted using a modified method of that described by Ruiz-Lopez et al, (2003). For each analysis, 30 mg of freeze-dried yeast or algal cells (powdered using the glass beads) were placed in 13-mm screw cap tube, added with 2.4 mL of 0.17 M NaCl in methanol and mixed by vortexing. Following the addition of 4.8 mL heptanes and 10 iL of C14:0 (10 μg.μL"1) internal standard, the suspension was mixed gently and incubated without shaking in 80°C water bath for 2h. After cooling to room temperature, the upper lipidic phase was transferred to fresh screw-cap tube and evaporated to dryness under the stream of N gas. Finally, the dried powder was resuspended in 100 x heptane, mixed thoroughly then transferred to a flat- bottom glass insert fitted into a brown glass vial for GC MS analysis.
TAG GC-MS analysis
[0190] TAG analysis was performed on a Hewlett Packard (hp) gas chromatograph/mass spectrometer (QP2010) (Shimadzu Scientific Instruments Inc). All analyses were performed with a RESTEK capillary column, MXT®-65TG (65% diphenyl -35% dimethyl polysiloxane, 30.0 m x 0.10 μηι thickness x 0.25 mm diameter) in Electron Impact (EI) ionization mode. Helium was used as the carrier gas. All samples were injected in splitless mode at 1.0 μΐ, aliquots and a column flow of 1.2 mL.min"1. The gas chromatograph was programmed from 200 to 370°C at 15°C.min"1 then isothermal at 370°C for 15 min. The sample injector port temperature was maintained at 350°C, column oven temperature at 200°C, with a pressure of 131.1 kPa, and a purge flow of 3.0 mL.min"1. The mass spectrometric conditions were as follows: the ion source temperature was held at 260°C during the GC-MS runs, the mass spectra were obtained at ionization voltage of 70 eV at an emission current of 60 μΑ and an interface temperature of 350°C. Acquisition mode was by scanning at a speed of 5000, 0.25s per scan. Chromatograph peaks with mass to charge ratio of 150 m/z to 1090m/z were collected starting at 9 min and ending at 25 min.
[0191] Determination of relative amounts of each TAG species was made using Glyceryl trinonadecanoate (CI 9:0) as an internal standard. Qualitative quantification was carried out by peak integration based on Total ionization chromatogram (TIC) of the sample peak area relative to the internal standard.
Methods for Nile Red Fluorescence Assays of algal cells
[0192] Nile Red fluorescence assays were essentially performed as per James et al, 2011. Briefly, OD750 measurements were determined for transgenic and nitrogen starved algal cultures, the cells were diluted to an OD750 of 0.2-0.4 in appropriate media (selective media for transformants containing constitutive promoters or induction media for transformants containing the NITlp). ΙΟΟμΙ, of these dilutions were aliquoted in triplicate into black 96- well plates and ΙΟΟμΙ, TAP-Nile Red (TAP + 3.37μg/mL Nile Red in acetone (or TAP + 4mM KN03 for NITlp constructs+ 3.37μg/mL Nile Red in acetone)) was added to the samples using a multichannel pipette. After a 15-25 min incubation at RT in the dark, fluorescence measurements were taken using the Gemini SpectraMax plate reader ex 485nm, em 600nm. Nitrogen starved algal cultures and vector only transformants were grown and analyzed side-by-side as positive and negative controls, respectively.
Methods for Nile Red microscopy of algal and yeast cells
[0193] For Nile Red fluorescence microscopy of algal transformants, cells were grown as above (see growth, induction and nitrogen starvation section) and ΙΟΟμΙ, cells were aliquoted into new 96-well plates. 0.5μΙν of Q.5 xglmh Nile Red in acetone was added to each sample and allowed to incubate at RT in the dark for 10 min. ΙΟμΙ, aliquots were used to prepare slides and samples were visualized with the Olympus BX50 fluorescence microscope using filter cube U- MWIB excitation 460-495 nM, emission 515-700 nM. Nitrogen starved algal cultures and vector only transformants were grown and analyzed side-by-side as positive and negative controls, respectively.
EXAMPLE 2
Further lipid associated proteins engineered to contain additional cysteine residues in the N- and C- terminal hydrophilic arms (MLDP)
[0194] The applicants have used the same strategy as described in PCT NZ2010/000218 for sesame seed oleosin, accession number AAD42942, (i.e., substituting charged residues predicted to be on the surface of OBs with cysteines) to engineer cysteines into the hydrophilic regions of the Chlamydomonas reindhartii Major Lipid Droplet Protein (MLDP, accession number
XP_001697668, SEQ ID NO: 47).
[0195] The MLDP sequences from Chlamydomonas and Volvox carteri f. nagariensis (accession number EFJ40256) were aligned:
Alignment of Chlamydomonas and Volvox MLDPs
(XP_001697668 (SEQ ID NO: 105) ( EFJ40256 (SEQ ID NO: 206)
(XP_001697668 (SEQ ID NO: : 105) ( EFJ40256 (SEQ ID NO: :206)
(XP_001697668 (SEQ ID NO: : 105) ( EFJ40256 (SEQ ID NO: :206)
(XP_001697668 (SEQ ID NO: : 105) ( EFJ4 0256 (SEQ ID NO: :206)
(XP_001697668 (SEQ ID NO: : 105) (EFJ4 0256 (SEQ ID NO: :206)
(XP_001697668 TYVNAAI KYWAP AP VAAA (SEQ ID NO: : 105) ( EFJ40256 TYVNATLKY APVAAA- - (SEQ ID NO: :206)
[0196] Alignment of the Chlamy and Volvox MLDPs showed a high degree of identity along the full length of the proteins. The Chlamydomonas MLDP has only one cysteine residue (grey boxed) which is predicted to be located in the centre of a hydrophobic portion while the Volvox
MLDP has no cysteine residues. Similarly, oleosins do not typically possess cysteine residues and when they are present they are only found in the hydrophobic portions and not the hydrophilic regions. However, a hydrophobicity plot (using the yte and Doolittle hydrophobicity scale below with a window size of 19) of the MLDPs suggests their topology is not as simple as oleosins (Figure 21).
KYTE & DOOLITTLE, THE INDIVIDUAL VALUES FOR THE 20 AMINO ACIDS ARE:
ALA: 1 800 ARG: -4 500 ASN : -3.500 ASP: -3 500 CYS: 2 500 GLN: -3 500
GLU: -3 500 GLY: -0 400 HIS: -3.200 ILE: 4 500 LEU: 3 800 ' LYS: -3 900
MET: 1 900 PHE: 2 800 PRO: -1.600 SER: -0 800 THR: -0 700 TRP: -0 900
TYR: -1 300 VAL: 4 200 : -3 500 : -3.500 -0 490
[0197] The same strategy as used for sesame oleosin (SEQ ID NO: 50)
(PCT/NZ2010/000218) was used herein for the identification of potential residues for substitution with cysteine residues. Where glutamate (E) and aspartate (D) residues were preferentially substituted for cysteine since in oleosin they are predicted to be on the outside of the oil body providing a negative charge. When these residues were unavailable, the neutral charged glutamine (Q) was selected. Hence, not conserved E, D or Q residues in predicted hydrophilic regions were identified. Conserved residues were not used since they may indicate structural function. Using these criteria the potential residues for substitution are highlighted by grey boxes below. It should be noted that D100 was not chosen for use since it was considered too be close to Q96, which was selected.
MA!SAG PLK HLEFVHTYAH KFASGAAYVE GGYQKAKTYV PAVAQPYIAK 50
AEETCLAYAA PLATKATDHA EKILRSTDAQ LDALYAASAS WLSSSQXLAD 100
SNIAAFRGAA DKYYgLVKST AQHVTSKLPT DLSVAKAREL LSASLEQAKA 150
LADPDAAVAA ALDAWTKFAA IPAVAKVLSA ASPLTGKGVA AFTAAHDLLV 200
HSALYRYGVS VGASTLGWAT STTPYKLSAA YLYPLVQPVA DPALDKVSKS 250
TYVNAAIKYW APAPVAAA* (SEQ ID NO: 105)
[0198] After substituting these residues for cysteine residues the Chlamydomonas MLDP engineered peptide sequence is shown below:
MACSAGKPLK HLCFVH YAH KFASGAAYVE GGYCKAKTYV PAVAQPYIAK 50
AEETCLAYAA PLATKATDHA EKILRSTDAQ LDALYAASAS WLSSSCKLAD 100
SNIAAFRGAA DKYYGLVKST AQHVTSKLPT DLSVAKARCL LSASLECAKA 150
LADPDAAVAA ALDAWTKFAA IPAVAKVLSA ASPLTGKGVA AFTAAHDLLV 200
HSALYRYGVS VGASTLGWAT STTPYKLSAA YLYPLVQPVA DPALCKVSXS 250
TYVNAAIKYW APAPVAAA* (SEQ ID NO: 48)
[0199] The original 5'UTR and intron 1 were left, while a single G to C mutation was introduced to delete an internal NotI site (without changing the translated peptide sequence), and Xbal and EcoRI sites were added to the 5' and 3' flanking regions respectively. This sequence (SEQ ID NO: 39) was used to generate an expression construct encoding the native MLDP. Using TGC as the optimized codon for cysteine in Chlamydomonas, this was substituted into SEQ ID NO: 39 (which encodes for SEQ ID NO: 47) at the positions indicated above to create SEQ ID NO: 40 (which encodes for SEQ ID NO: 48. The final nucleic acid sequence and translated peptide sequences of the modified MLDP are listed as SEQ ID NOs: 39 and 40, and SEQ ID NOs: 47 and 48, respectively.
EXAMPLE 3
Neutral lipids accumulate during log phase in transgenic Chlamydomonas reinhardtii harboring the AtDGATl-V5 and oleo 0,0 genes
[0200] In this example, transformant 17-4 which contains the full length HRp-DGATl-V5-
RBCS2At;HRIp-oleo 0,0-RBSCA2t construct (as confirmed via PCR), enabling it to express both
SEQ ID NOs: 49 and 50, accumulated levels of neutral lipid similar to the nitrogen starved control, as shown by fluorescence microscopy, while in the vector only no detectable neutral lipids were detected (Figure 23). Both transgenic samples (17-4 and vector only) were grown to log phase in selection media (as in growth, induction, and nitrogen starvation section above) and analyzed side-by-side with the nitrogen starved control.
EXAMPLE 4
Neutral lipids accumulate in transgenic
Chlamydomonas reinhardtiii expressing NITlp-CrDGAT2-RBCS2At
[0201] In this example, transgenic Chlamydomonas harboring the NITlp-CrDGAT2-
RBSC2At cassette accumulate nearly 2-fold more neutral lipids than those harboring the NITlp-
CrDGAT2-CrDGAT2-3'UTR cassette after 8h of induction as shown by the Nile Red fluorescence assay (Figure 24). Transgenic algal cultures were grown side-by-side to log phase, harvested and resuspended in induction media and grown for 8h (as in grown, induction and nitrogen starvation section above) and Nile Red fluorescence assays were performed in triplicate
(as per paragraph [0186], above).
EXAMPLE 5
Neutral lipids accumulate in Saccharomyces cerivisiae during lag growth phase when expressing AtDGATl and 01e_3,3 or CrDGAT2 and MLDP
[0202] In this example, Saccharomyces cerivisiae cells were transformed with constructs harboring the AtDGATl (SEQ NO: 60) or CrDGAT2 (SEQ NO: 62) and 01e_3,3 (SEQ NO: 61) or MLDP (SEQ NO: 63) arranged in various configurations (such as those seen in expression vectors SEQ NOs: 57, 58 and 59). Cells were induced by the addition galactose and allowed to grow for 8hr (transition of lag to log phase). Samples were taken for FAMES-GC/MS analysis (Figure 25) as well as for Nile Red fluorescence analysis by confocal microscopy (Figure 26).
Table 5 - Promoter and terminator sequences used to control the expression of nucleotide sequences encoding Arabidopsis thaliana DGATl, Sesamum inducum oleosin and oleosin engineered to contain cysteines, Chlamydomonas reinhardtii DGATl, MLDP and MLDP engineered to contain cysteines, Streptomyces rimosus aphVIII gene (paramomycin resistance), Streptomyces hygroscopicus aphVII gene (hygromycin resistance) in
Chlamydomonas reinhardtii.
Function Sequence SEQ ID
NO:
Nitrate tcgaggcgagacgtcgagggcgtgggctctgtatggctgggtaacggtacgtataattccaggtacaagctag 30 reductase 1 agcagacggtggtgagaagcattagaagcattgtcccgagtgtggtggctagaatcccggcccacgaatcac
promoter + agtgaatgggtacatgtacaggtgccccgccagcccccgctcctctgctgcctctgatgcctcatgccaaaagt
5'UTR (Nitlp) cctgacgcggcgccctcacatccccgtccgggtaatctatgagtttcccttatcgagcatgtacgcgatagtgg
acggggctcagggtggggggtgggtgggtgggaggggcgttccttcagacaccctggaggggtggctaga
aaaacggccgcgcgccagaaatgtctcgctgccctgtgcaataagcaccggctatattgctcagcgctgttcg
gcgcaacggggggtcagcccttgggaagcgttggactatatggtagggtgcgagtgaccccgcgcgacttg
gagctcgatggccccgggttgtttggggcgtccgcctctcgcgctattctgagctggagaccgaggcgcatga aaatgcattcgcttccataggacgctgcattgtggcttgaaggttcaagggaagggttcaaacgaccccgccgt acgaacttttgtcggggggcgctcccggccccgggctcttgtgcgcgcattagggcttcgggtcgcaagcaa
gacgatacaggaaccgaccaatcgatagtcttgtgcgaccgtgcacgtgtgcagcaatagttaggtcgataac
cacgttgaacttgcgtctctcttcgtggcgcctcctgcttggtgctccacttcacttgtcgctatatagcacagcgtt gaaagcaaaggccacactaatacagccgggctcgagagtccgtctgcgtttgcattgttggccaagggctgct ttgtagccaaagccatacacgaagcttcacttgattagctttacgaccctcagccgaatcctgccag
Isocitrate lyase attctgcccgtttcctccaagcctttatagacccgccaggcacatgctgcttatttcccagcgttccaataaagaa 31 promoter + agcttggtctaagcttctcttgccacagagagtcgacacacgttcacaggatcagctgcacgttcaaaacgtgc
5'UTR (ICLp) gtcaccatttcagttctgtagtgagctgagtacgtagtgagcatgctacacaggaacagtagtacacccatgtgc
agctcgatgtcgactttgaatttggatcgagctcgcccccaggccccccaggcccccaacctcgcccccctctt ctctgcgtgcccatcctgcgtatcccagcgcaattccattccacagcaaccatggcatgcgcataccatgccag
agtgccgtcgcagcacccctaccgtacgccctgcgcacccctggtcctgcaacgctatgatttgaaacagctg
catgctgcgaagctgttggccaagcgcccgtggtgtaaaccgtgcaccgccaagcagcaatacatactaggc
tcccacgacagccaactgcagcccggtaagtgcacccgggcaaacccgggcaaatactccccgccgggcc
agtcagggcacggtggcacgccctccaaacgccttcactgctccctcaatactctgatcaggaagctatatacg
ttcatagaaaaggccttcggcaatgcacacgcgacgtgcggaacatgtcatgcgtaagcttgtcatcgagtcag ctttggctgtgcaccggtttgtgcatttgcagcggacatgaatatcacgatgagcgcattgcgtagaatgaaaga
aagcttcagactggagggactgggctgcgttcagcagtaggtcgacttgacaccgcttgacgtggaaaactgc attggatccaaaccgacccaccgcccataaagcccttccgggctcttcttgcgtttgtagcatacttaagtgtgag cgctccgaacccggcactcgctcgtgtgcctactcgttcttgtaagccctcgcttcgctcctcgtctcgctcgtcg
tcgtcgactctagattcaaggagtgctgtatgagtctataaaccagtcaggagcttgcctctttcttagggccgaa
cgaga
Heatshock 70A gagctcgctgaggcttgacatgattggtgcgtatgtttgtatgaagctacaggactgatttggcgggctatgagg 32 linked with gcgggggaagctctggaagggccgcgatggggcgcgcggcgtccagaaggcgccatacggcccgctgg
Rubisco2A cggcacccatccggtataaaagcccgcgaccccgaacggtgacctccactttcagcgacaaacgagcactta
promoter + tacatacgcgactattctgccgctatacataaccactcagctagcgatcccgggcgcgccagaaggagcgca
5'UTR fusion gccaaaccaggatgatgtttgatggggtatttgagcacttgcaacccttatccggaagccccctggcccacaaa
(HSP:RBCS2A ggctaggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcctgataaaccggccagggg
P) gcctatgttctttacttttttacaagagaagtcactcaacatcttaaa
Heatshock 70A gagctcgctgaggcttgacatgattggtgcgtatgtttgtatgaagctacaggactgatttggcgggctatgagg 33 and Rubisco2A gcgggggaagctctggaagggccgcgatggggcgcgcggcgtccagaaggcgccatacggcccgctgg +5' UTR + cggcacccatccggtataaaagcccgcgaccccgaacggtgacctccactttcagcgacaaacgagcactta intron promoter tacatacgcgactattctgccgctatacataaccactcagctagcttaagatcccatcaagcttgcatgccgggc
fusion (HRIp) gcgccagaaggagcgcagccaaaccaggatgatgtttgatggggtatttgagcacttgcaacccttatccgga
agccccctggcccacaaaggctaggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcc
tgataaaccggccagggggcctatgttctttacttttttacaagagaagtcactcaacatcttaaaatggccaggt
gagtcgacgagcaagcccggcggatcaggcagcgtgcttgcagatttgacttgcaacgcccgcattgtgtcg
acgaaggcttttggctcctctgtcgctgtctcaagcagcatctaaccctgcgtcgccgtttccatttgcag p-Tubulin2 tctttcttgcgctatgacacttccagcaaaaggtagggcgggctgcgagacggcttcccggcgctgcatgcaa 34 promter and 5' caccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgag
UTR (βΤρ) cgctgtttaaatagccaggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctag
atcactaccacttctacacaggccactcgagcttgtgatcgcactccgctaagggggcgcctcttcctcttcgttt
cagtcacaacccgcaaac
COP terminator gatccggcaagactggccccgcttggcaacgcaacagtgagcccctccctagtgtgtttggggatgtgactat 35 (COPt) gtattcgtgtgttggccaacgggtcaacccgaacagattgatacccgccttggcatttcctgtcagaatgtaacgt
cagttgatggtac
Rubisco2A ggatccccgctccgtgtaaatggaggcgctcgttgatctgagccttgccccctgacgaacggcggtggatgga 36 3 'UTR and agatactgctctcaagtgctgaagcggtagcttagctccccgtttcgtgctgatcagtctttttcaacacgtaaaaa
terminator gcggaggagttttgcaattttgttggttgtaacgatcctccgttgattttggcctctttctccatgggcgggctggg
(RBCS2At) cgtatttgaagcg
Tubulin2 atgccggcacctccatgcgccactgaacgtgtagcgtgactgtggcggccttggcagttttgaccgtgactgac 37 3 'UTR and cctggacaaaggatccctgactgaagacaacttgacatgtgattgccatttgacgctttggtgtggaggcggatt
terminator gtgagatgggaggggggcccattgccttcgtgaccataacgacatcgaatttcatacatgtgaacagttcagca
(Tub2t) tggacattcatctcgtcggattagctcttgtgtgataggccatagcagctggactgttgtgagctctcgatctgcgt
agctactggctgtgattgtgcttcaggcggcaggggcaggtaactgccctgaacgtaaaggtgcagcagcag
acagcggatgtgcagaacgaatagcgcagtggataaggttgatgggtggccacacactcgtgcacgtgtaat
aagatacacgaatcgtcctccttttctttgtcgtaaccatgggagggaagagagcgtgaccagatgacagcctg
ggacatctaagaacttggtaggcgggtaagtccctcgatggcgtggtgtattcccaccagtcgcaagcaaagc
caaggggcgtagcagtttgtggggctgagaggtggatgggaggttgcattggttacacaagggacctg
Table 6 - Nucleic Acid Sequences adapted for expression in Chlamydomonas reinhardtii encoding: Chlamydomonas reinhardtii DGAT2, MLDP and engineered MLDP_4,4, Arabidopsis thaliana DGAT1 V5, Sesamum indicum oleosin and engineered oleosin_3,3, Streptomyces rimosus aphVIII gene (paramomycin resistance), Streptomyces hygroscopicus aphVII gene (hygromycin resistance).
Description of Sequence SEQ ID nucleic acid NO: sequence
C. reinhardtii ttcaaggagtgctgtatgagtctataaaccagtcaggagcttgcctctttcttagggccgaacgagaatgccgct 38 DGAT2, cgcaaagctgcgaaacgtggtgctggagtacgcggccatagccatctacgtcaggtaattttgcttaagacgc
includes gactgttctgtgaaactgacgagctcaggaaatcggctgggccgaaccaccatggcgtctcccgtccaaagc
5 'UTR, exons gttcttgcgcaccccctcccccgcccaagctctcgccccgctgccacacgccctgcaaccccaagcctccaa
and introns 1, 2 cccccaaacccccatcctctccacagcgccatctacacctcggtggtgctgctgccctcggcgctcgcgctgtt
and 3 then ctacctgtttggggccaccagcccctcggcctggctgctgctagccgccttcctggccctcaccttcacgccgc
fused exons 4,5 tgcagctgaccaccggtgcgctgtcggagcggttcgtgcagttcagtgtggcgcgggcggcggcctacttcc
and 6 ccacccgcgtggtggtcacggacccggaggtgagggcgctgtgggggcgctgtacggggggctgctggg
ggtggggggcgaaggttgtgggggccggacctgtggggaaagggggaggagtagtctggggtcacgagg
gaggagacagggggcggggctgcacggttaaggcagtggaaactgggtaagagcatcgggaacagggaa
cggtgggcagtgcatcaggcgtaggtgagtggttgcgtgccgatgaccggagcaggtggggaaggcgggg
tttattgcactcccaaaagaaaccaagacccggaaccagcccacaaagggtatcgtagtggcatcgggttgaa
cggcgacaccaccgccctgcattggctttggcattgactgcggtcctgtgccctccccccccccaggccttcc
gcactgaccgcggctacttgttcggattctgcccgcactcggctctgcccatcgcactgcccatcgccttcgcc accacctcgccgctgctgcccaaggagctgcgcggccggacacacggcttggcgtcgtccgtgtgcttcagc gcgcccataggtgtgtgtggcggggggggcggcgcggggggaggatgggccgggagagcacccggtga caggttggtggtgtggcgggttgttctccgggtaggtggcgaagccgtcctgtgctctccaccgctaccgcga ctgctaccgtggctgctccccagaacatcacccacatgtgtgttccctctcctgctcctcctacgcctccctcctc ctcctccctccccacgtgctgcagtgcggcagctgtactggtggctgggcgtgcggcccgccacgcggcag agcatcagcggcctgttgcgggcgcgcaaggtggcggtgctggtgccggggggcgtgcaggaggtgctca acatggagcacggcaaggaggtggcctacctctccagccgcaccggcttcgtgcgactggccgtgcagcac ggcgcgccgctggtgccagtgtgggcgttcggccagacgcgcgcgtacagctggttccggccggggccgc cgctcgtgcccacgtggctcgtggagcgcatctcacgtgccgccggcgccgtacccatcggcatgtttgggc agtacggcacgcccatgccgcaccgcgagcccctcaccattgtggtgggtcgccccatcccggtgccggag ctggcgccgggccagctcgagcccgagcccgaggtgctggcggcgctcctcaagcgcttcacggacgacc tgcaggcgctgtacgacaagcacaaggcgcagttcggcaagggcgaggagctggtcataatgtag
C. reinhardtii tctagaaatcgatactgtattcgcaacaaagtcaagctcaatttacaacattatccaaagctacggtatccacagc 39 MLDP + 5' atttagaaatggccgagtctgctggaaagcctctgaagcaccttgagtttgtgcacacctacgcgcacaagtttg UTR, intron 1 cgagtggtgccgcttacgttgagggcggctaccagaaggccaagacctatgttcccgcggtcgctcagccct and point acatcgccaaagccgaggagacctgcctagcatacgccgctcctcttgcgacaaaggcgacggaccacgta mutation agttaattcatccggggtctctgccaccgggtggccttggaagccgactcgtcgactccgcaacaccccctcc removing ggttctgcccgagatgctgacttcctggtgctttgaatgcttttacaggcagagaagattctccggagcaccgac internal Notl gcacagctggacgcgctgtacgcggcctccgccagctggctgagcagctcccagaagctggccgactccaa site catcgcggccttcaggggcgccgccgacaagtactacgacctggtcaagtccactgcgcagcacgtgacgtc caagctgcccaccgacctgtcggtggccaaggcccgcgagctgctgtcggcctcgctggagcaggccaag gctctggctgacccggacgctgcggtggcggcggcgctggatgcctggaccaagttcgcagccatcccggc ggttgccaaggtgctgtccgccgcctccccgctgacgggcaagggcgtggcggccttcacggcggcacac gacctcctggtgcactcggcgctgtaccgctacggcgtgtcggtgggcgcctccaccctgggctgggccacc agcaccaccccctacaagttgagcgcggcttacctgtacccgctggtgcagcccgtggcggaccccgcgctg gacaaggtgtccaagagcacctacgtcaacgcagccatcaagtactgggcgcccgcacccgtggccgccgc gtaagaattc
C. reinhardtii tctagaaatcgatactgtattcgcaacaaagtcaagctcaatttacaacattatccaaagctacggtatccacagc 40 MLDP 4,4 + 5' atttagaaatggcctgctctgctggaaagcctctgaagcacctttgctttgtgcacacctacgcgcacaagtttgc UTR, intron 1 gagtggtgccgcttacgttgagggcggctactgcaaggccaagacctatgttcccgcggtcgctcagccctac and point atcgccaaagccgaggagacctgcctagcatacgccgctcctcttgcgacaaaggcgacggaccacgtaag mutation ttaattcatccggggtctctgccaccgggtggccttggaagccgactcgtcgactccgcaacaccccctccggt removing tctgcccgagatgctgacttcctggtgctttgaatgcttttacaggcagagaagattctccggagcaccgacgca internal Notl cagctggacgcgctgtacgcggcctccgccagctggctgagcagctcctgcaagctggccgactccaacatc site gcggccttcaggggcgccgccgacaagtactactgcctggtcaagtccactgcgcagcacgtgacgtccaa gctgcccaccgacctgtcggtggccaaggcccgctgcctgctgtcggcctcgctggagtgcgccaaggctct ggctgacccggacgctgcggtggcggcggcgctggatgcctggaccaagttcgcagccatcccggcggttg ccaaggtgctgtccgccgcctccccgctgacgggcaagggcgtggcggccttcacggcggcacacgacct cctggtgcactcggcgctgtaccgctacggcgtgtcggtgggcgcctccaccctgggctgggccaccagca ccaccccctacaagttgagcgcggcttacctgtacccgctggtgcagcccgtggcggaccccgcgctgtgca aggtgtccaagagcacctacgtcaacgcagccatcaagtactgggcgcccgcacccgtggccgccgcgtaa gaattc
A. thaliana atggccatcctggacagcgcgggcgtgaccaccgtgaccgagaacggcggcggcgagttcgtggacctgg 41 DGAT1 accgcctgcgccgccgcaagtccgtgagtcgacgagcaagcccggcggatcaggcagcgtgcttgcagatt includes tgacttgcaacgcccgcattgtgtcgacgaaggcttttggctcctctgtcgctgtctcaagcagcatctaaccctg RBCS2A intron cgtcgccgtttccatttgcagcgctcggacagctccaacggcctgctgctgtcgggcagcgacaacaactccc 1 and Cterminal cgtcggacgacgtgggcgcccccgcggacgtgcgcgaccgcatcgacagcgtggtgaacgacgacgccc V5 and His tag agggcaccgcgaacctggccggcgacaacaacggcggcggcgacaacaacggcggcggccggggcgg cggcgagggccgcggcaacgcggacgccaccttcacctaccgcccgtccgtgcccgcgcaccgccgcgc ccgcgagtcgccgctgagctcggacgcgatcttcaagcagtcccacgccggcctgttcaacctgtgcgtggt ggtgctgatcgcggtgaacagccgcctgatcatcgagaacctgatgaagtacggctggctgatccgcaccga cttctggttctcgtcgcgcagcctgcgcgactggcccctgttcatgtgctgcatctccctgtcgatcttcccgctg gccgcgttcaccgtggagaagctggtgctgcagaagtacatcgccgagcccgtggtgatcttcctgcacatca tcatcaccatgaccgaggtgctgtacccggtgtacgtgaccctgcgctgcgacagcgcgttcctgtcgggcgt gaccctgatgctgctgacctgcatcgtgtggctgaagctggtgtcctacgcccacaccagctacgacatccgct cgctggcgaacgccgcggacaaggccaaccccgaggtgtcgtactacgtgagcctgaagtccctggcgtac ttcatggtggccccgaccctgtgctaccagccctcgtacccgcgcagcgcgtgcatccgcaagggctgggtg gcccgccagttcgcgaagctggtgatcttcaccggcttcatgggcttcatcatcgagcagtacatcaaccccat cgtgcgcaactcgaagcacccgctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtccgt gcccaacctgtacgtgtggctgtgcatgttctactgcttcttccacctgtggctgaacatcctggcggagctgctg tgcttcggcgaccgcgagttctacaaggactggtggaacgccaagagcgtgggcgactactggcgcatgtgg aacatgccggtgcacaagtggatggtgcgccacatctacttcccctgcctgcgctcgaagatcccgaagaccc tggcgatcatcatcgccttcctggtgtcggcggtgttccacgagctgtgcatcgccgtgccctgccgcctgttca agctgtgggcgttcctgggcatcatgttccaggtgccgctggtgttcatcaccaactacctgcaggagcgcttc ggcagcaccgtgggcaacatgatcttctggttcatcttctgcatcttcggccagcccatgtgcgtgctgctgtact accacgacctgatgaaccgcaagggctccatgtccaagggcgagctgcgcggccaccccttcgagggcaa gccgatccccaacccgctgctgggcctggacagcacccgcaccggc
S. indicum atggccgagcactacggccagcagcagcagacccgcgcgcccgtgagtcgacgagcaagcccggcggat 42 oleosin includes caggcagcgtgcttgcagatttgacttgcaacgcccgcattgtgtcgacgaaggcttttggctcctctgtcgctgt RBCS2A intron ctcaagcagcatctaaccctgcgtcgccgtttccatttgcagcacctgcagctgcagccgcgcgcccagcgcg 1 tggtgaaggcggccaccgcggtgaccgccggcggcagcctgctggtgctgtccggcctgaccctggcggg caccgtgatcgccctgaccatcgcgacccccctgctggtgatcttctcgccggtgctggtgcccgccgtgatca ccatcttcctgctgggcgcgggcttcctggccagcggcggcttcggcgtggcggccctgtccgtgctgtcgtg gatctaccgctacctgaccggcaagcacccgcccggcgcggaccagctggagagcgccaagaccaagctg gcgtccaaggcccgcgagatgaaggaccgcgcggagcagttctcgcagcagccggtggccggcagccag acctcctaa
5. indicum gagaagtcactcaacatcttaaaattctagacaaaatggcctgccactacggccagcagcagcagacctgcgc 43 oleosin gccgcacgtgagtcgacgagcaagcccggcggatcaggcagcgtgcttgcagatttgacttgcaacgcccg engineered to cattgtgtcgacgaaggcttttggctcctctgtcgctgtctcaagcagcatctaaccctgcgtcgccgtttccattt contain gcagctgcagctgcagccgcgcgcctgccgcgtggtgaaggcggccaccgcggtgaccgccggcggcag cysteines, cctgctggtgctgtcgggcctgaccctggcgggcaccgtgatcgccctgaccatcgcgacccccctgctggt includes gatcttctccccggtgctggtgcccgccgtgatcaccatcttcctgctgggcgcgggcttcctggcctcgggcg
RBCS2A gcttcggcgtggcggccctgagcgtgctgtcctggatctaccgctacctgaccggcaagcacccgcccggcg
5'UTR and cggactgcctggagtcggccaagaccaagctggcgtcctgcgcccgcgagatgaaggaccgcgcggagc
RBCS2A intron agttcagctgccagccggtggccggctcgcagacctcctaa
1
S. rimosus atggacgatgcgttgcgtgcactgcggggtcggtatcccggttgtgagtgggttgttgtggaggatggggcct 44 aphVIII ORF cgggggctggtgtttatcggcttcggggtggtgggcgggagttgtttgtcaaggtggcagctctgggggccgg and 3 'UTR ggtgggcttgttgggtgaggctgaacggctggtgtggttggcggaggtggggattcccgtacctcgtgttgtg gagggtggtggggacgagagggtcgcctggttggtcaccgaagcggttccggggcgtccggccagtgcgc ggtggccgcgggagcagcggctggacgtggcggtggcgctcgcggggctcgctcgttcgctgcacgcgct ggactgggagcggtgtccgttcgatcgcagtctcgcggtgacggtgccgcaggcggcccgtgctgtcgctga agggagcgtcgacttggaggatctggacgaggagcggaaggggtggtcgggggagcggcttctcgccga gctggagcggactcggcctgcggacgaggatctggcggtttgccacggtgacctgtgcccggacaacgtgc tgctcgaccctcgtacctgcgaggtgaccgggctgatcgacgtggggcgggtcggccgtgcggaccggcac tccgatctcgcgctggtgctgcgcgagctggcccacgaggaggacccgtggttcgggccggagtgttccgc ggcgttcctgcgggagtacgggcgcgggtgggatggggcggtatcggaggaaaagctggcgttttaccggc tgttggacgagttcttctgagggacctgatggtgttggtggctgggtagggttgcgtcgcgtgggtgacagcac agtgtggacgttgg
5. atgacacaagaatccctgttacttctcgaccgtattgattcggatgattcctacgcgagcctgcggaacgaccag 45 hygroscopicus gagttctgggagccgctggcccgccgagccctggaggagctcgggctgccggtgccgccggtgctgcggg aphVII ORF tgcccggcgagagcaccaaccccgtactggtcggcgagcccggcccggtgatcaagctgttcggcgagca ctggtgcggtccggagagcctcgcgtcggagtcggaggcgtacgcggtcctggcggacgccccggtgccg gtgccccgcctcctcggccgcggcgagctgcggcccggcaccggagcctggccgtggccctacctggtgat gagccggatgaccggcaccacctggcggtccgcgatggacggcacgaccgaccggaacgcgctgctcgc cctggcccgcgaactcggccgggtgctcggccggctgcacagggtgccgctgaccgggaacaccgtgctc accccccattccgaggtcttcccggaactgctgcgggaacgccgcgcggcgaccgtcgaggaccaccgcg ggtggggctacctctcgccccggctgctggaccgcctggaggactggctgccggacgtggacacgctgctg gccggccgcgaaccccggttcgtccacggcgacctgcacgggaccaacatcttcgtggacctggccgcgac cgaggtcaccgggatcgtcgacttcaccgacgtctatgcgggagactcccgctacagcctggtgcaactgcat ctcaacgccttccggggcgaccgcgagatcctggccgcgctgctcgacggggcgcagtggaagcggaccg aggacttcgcccgcgaactgctcgccttcaccttcctgcacgacttcgaggtgttcgaggagaccccgctggat ctctccggcttcaccgatccggaggaactggcgcagttcctctgggggccgccggacaccgcccccggcgc ctga Table 7 - Translated amino acid sequences of constructs expressed in Chlamydomonas reinhardtii, including: Chlamydomonas reinhardtii DGAT2, MLDP and engineered MLDP_4,4, Arabidopsis thaliana DGAT1_V5, Sesamum indicum oleosin and engineered oleosin_3,3, Streptomyces rimosus aphVIII gene (paramomycin resistance), Streptomyces hygroscopicus aphVII gene (hygromycin resistance).
Figure imgf000060_0001
aphVII relgrvlgrlhrvplt ntvl^hsev^ellreiraalA'edhrgwgylsprlldrledwlpdvdtllagreprfv (accession # hgdlhgtaifvdlaatevtgivdftdvyagdsryslvqlhlnafrgdreilaalldgaqwkitedfarellaftfl
P09979) hdfevfeetpldlsgftdpeelaqflwgppdtapga
Table 8 - Examples of Nucleic Acid Sequences arranged in expression cassettes for transformation into Chlamydomonas reinhardtii, consisting of at least one selectable marker [Streptomyces rimosus aphVIII gene (paramomycin resistance), or Streptomyces
hygroscopicus aphVII gene (hygromycin resistance)]. and one gene of interest
{Chlamydomonas reinhardtii DGAT2, or Arabidopsis thaliana DGAT1_V5), both under the control of a separate promoter and terminator. Examples of Nucleic Acid Sequences arranged in expression cassettes for transformation into Chlamydomonas reinhardtii, consisting of two selectable markers [Streptomyces rimosus aphVIII gene (paramomycin resistance), and Streptomyces hygroscopicus aphVII gene (hygromycin resistance)] flanking two back to back genes of interest including a neutral lipid synthesising enzyme
{Chlamydomonas reinhardtii DGAT2, or Arabidopsis thaliana DGAT1_V5) and a lipid encapsulating protein {Chlamydomonas reinhardtii MLDP/MLDP_4,4, or Sesamum indicum Oleosin_0,0/Oleosin_3,3).
Cassette Sequence SEQ ID description NO:
Nitlp, tcgaggcgagacgtcgagggcgtgggctctgtatggctgggtaacggtacgtataattccaggtacaagctag 54 AtDGATl, agcagacggtggtgagaagcattagaagcattgtcccgagtgtggtggctagaatcccggcccacgaatcac
RBCS2At; βΤρ, agtgaatgggtacatgtacaggtgccccgccagcccccgctcctctgctgcctctgatgcctcatgccaaaagt
aphVIII gene, cctgacgcggcgccctcacatccccgtccgggtaatctatgagtttcccttatcgagcatgtacgcgatagtgg
COPt acggggctcagggtggggggtgggtgggtgggaggggcgttccttcagacaccctggaggggtggctaga
aaaacggccgcgcgccagaaatgtctcgctgccctgtgcaataagcaccggctatattgctcagcgctgttcg
[SEE MAP 1] gcgcaacggggggtcagcccttgggaagcgttggactatatggtagggtgcgagtgaccccgcgcgacttg
gagctcgatggccccgggttgtttggggcgtccgcctctcgcgctattctgagctggagaccgaggcgcatga
aaatgcattcgcttccataggacgctgcattgtggcttgaaggttcaagggaagggttcaaacgaccccgccgt
acgaacttttgtcggggggcgctcccggccccgggctcttgtgcgcgcattagggcttcgggtcgcaagcaa
gacgatacaggaaccgaccaatcgatagtcttgtgcgaccgtgcacgtgtgcagcaatagttaggtcgataac
cacgttgaacttgcgtctctcttcgtggcgcctcctgcttggtgctccacttcacttgtcgctatatagcacagcgtt
gaaagcaaaggccacactaatacagccgggctcgagagtccgtctgcgtttgcattgttggccaagggctgct
ttgtagccaaagccatacacgaagcttcacttgattagctttacgaccctcagccgaatcctgccagtctagaca
aaatggccatcctggacagcgcgggcgtgaccaccgtgaccgagaacggcggcggcgagttcgtggacct
ggaccgcctgcgccgccgcaagtccgtgagtcgacgagcaagcccggcggatcaggcagcgtgcttgcag
atttgacttgcaacgcccgcattgtgtcgacgaaggcttttggctcctctgtcgctgtctcaagcagcatctaacc
ctgcgtcgccgtttccatttgcagcgctcggacagctccaacggcctgctgctgtcgggcagcgacaacaact
ccccgtcggacgacgtgggcgcccccgcggacgtgcgcgaccgcatcgacagcgtggtgaacgacgacg
cccagggcaccgcgaacctggccggcgacaacaacggcggcggcgacaacaacggcggcggccgggg
cggcggcgagggccgcggcaacgcggacgccaccttcacctaccgcccgtccgtgcccgcgcaccgccg
cgcccgcgagtcgccgctgagctcggacgcgatcttcaagcagtcccacgccggcctgttcaacctgtgcgt
ggtggtgctgatcgcggtgaacagccgcctgatcatcgagaacctgatgaagtacggctggctgatccgcac
cgacttctggttctcgtcgcgcagcctgcgcgactggcccctgttcatgtgctgcatctccctgtcgatcttcccg
ctggccgcgttcaccgtggagaagctggtgctgcagaagtacatcgccgagcccgtggtgatcttcctgcaca tcatcatcaccatgaccgaggtgctgtacccggtgtacgtgaccctgcgctgcgacagcgcgttcctgtcggg cgtgaccctgatgctgctgacctgcatcgtgtggctgaagctggtgtcctacgcccacaccagctacgacatcc gctcgctggcgaacgccgcggacaaggccaaccccgaggtgtcgtactacgtgagcctgaagtccctggcg tacttcatggtggccccgaccctgtgctaccagccctcgtacccgcgcagcgcgtgcatccgcaagggctgg gtggcccgccagttcgcgaagctggtgatcttcaccggcttcatgggcttcatcatcgagcagtacatcaaccc catcgtgcgcaactcgaagcacccgctgaagggcgacctgctgtacgccatcgagcgcgtgctgaagctgtc cgtgcccaacctgtacgtgtggctgtgcatgttctactgcttcttccacctgtggctgaacatcctggcggagctg ctgtgcttcggcgaccgcgagttctacaaggactggtggaacgccaagagcgtgggcgactactggcgcatg tggaacatgccggtgcacaagtggatggtgcgccacatctacttcccctgcctgcgctcgaagatcccgaaga ccctggcgatcatcatcgccttcctggtgtcggcggtgttccacgagctgtgcatcgccgtgccctgccgcctg ttcaagctgtgggcgttcctgggcatcatgttccaggtgccgctggtgttcatcaccaactacctgcaggagcg cttcggcagcaccgtgggcaacatgatcttctggttcatcttctgcatcttcggccagcccatgtgcgtgctgctg tactaccacgacctgatgaaccgcaagggctccatgtccaagggcgagctgcgcggccaccccttcgaggg caagccgatccccaacccgctgctgggcctggacagcacccgcaccggccaccaccaccaccaccactaa ggaattcggatccccgctccgtgtaaatggaggcgctcgttgatctgagccttgccccctgacgaacggcggt ggatggaagatactgctctcaagtgctgaagcggtagcttagctccccgtttcgtgctgatcagtctttttcaaca cgtaaaaagcggaggagttttgcaattttgttggttgtaacgatcctccgttgattttggcctctttctccatgggcg ggctgggcgtatttgaagcgaccggtcatatgtctttcttgcgctatgacacttccagcaaaaggtagggcggg ctgcgagacggcttcccggcgctgcatgcaacaccgatgatgcttcgaccccccgaagctccttcggggctg catgggcgctccgatgccgctccagggcgagcgctgtttaaatagccaggcccccgattgcaaagacattata gcgagctaccaaagccatattcaaacacctagatcactaccacttctacacaggccactcgagcttgtgatcgc actccgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaacatggtcgagattcgaagcatgg acgatgcgttgcgtgcactgcggggtcggtatcccggttgtgagtgggttgttgtggaggatggggcctcggg ggctggtgtttatcggcttcggggtggtgggcgggagttgtttgtcaaggtggcagctctgggggccggggtg ggcttgttgggtgaggctgaacggctggtgtggttggcggaggtggggattcccgtacctcgtgttgtggagg gtggtggggacgagagggtcgcctggttggtcaccgaagcggttccggggcgtccggccagtgcgcggtg gccgcgggagcagcggctggacgtggcggtggcgctcgcggggctcgctcgttcgctgcacgcgctggac tgggagcggtgtccgttcgatcgcagtctcgcggtgacggtgccgcaggcggcccgtgctgtcgctgaagg gagcgtcgacttggaggatctggacgaggagcggaaggggtggtcgggggagcggcttctcgccgagctg gagcggactcggcctgcggacgaggatctggcggtttgccacggtgacctgtgcccggacaacgtgctgctc gaccctcgtacctgcgaggtgaccgggctgatcgacgtggggcgggtcggccgtgcggaccggcactccg atctcgcgctggtgctgcgcgagctggcccacgaggaggacccgtggttcgggccggagtgttccgcggcg ttcctgcgggagtacgggcgcgggtgggatggggcggtatcggaggaaaagctggcgttttaccggctgttg gacgagttcttctgagggacctgatggtgttggtggctgggtagggttgcgtcgcgtgggtgacagcacagtgt ggacgttgggatccggcaagactggccccgcttggcaacgcaacagtgagcccctccctagtgtgtttgggg atgtgactatgtattcgtgtgttggccaacgggtcaacccgaacagattgatacccgccttggcatttcctgtcag aatgtaa
COPt, ttacattctgacaggaaatgccaaggcgggtatcaatctgttcgggttgacccgttggccaacacacgaatacat 55 aphVIII,PTUBp agtcacatccccaaacacactagggaggggctcactgttgcgttgccaagcggggccagtcttgccggatcc , RBCS2At, caacgtccacactgtgctgtcacccacgcgacgcaaccctacccagccaccaacaccatcaggtccctcaga CrDGAT2, agaactcgtccaacagccggtaaaacgccagcttttcctccgataccgccccatcccacccgcgcccgtactc ICLp;HRp, ccgcaggaacgccgcggaacactccggcccgaaccacgggtcctcctcgtgggccagctcgcgcagcacc MLDP 4,4, agcgcgagatcggagtgccggtccgcacggccgacccgccccacgtcgatcagcccggtcacctcgcagg RBCSTAt; tacgagggtcgagcagcacgttgtccgggcacaggtcaccgtggcaaaccgccagatcctcgtccgcaggc TUB2p, cgagtccgctccagctcggcgagaagccgctcccccgaccaccccttccgctcctcgtccagatcctccaagt aphVII, TUB2t cgacgctcccttcagcgacagcacgggccgcctgcggcaccgtcaccgcgagactgcgatcgaacggaca (note aphVIII ccgctcccagtccagcgcgtgcagcgaacgagcgagccccgcgagcgccaccgccacgtccagccgctg and CrDGAT2 ctcccgcggccaccgcgcactggccggacgccccggaaccgcttcggtgaccaaccaggcgaccctctcgt are reversed) ccccaccaccctccacaacacgaggtacgggaatccccacctccgccaaccacaccagccgttcagcctca
cccaacaagcccaccccggcccccagagctgccaccttgacaaacaactcccgcccaccaccccgaagcc
[SEE MAP 2] gataaacaccagcccccgaggccccatcctccacaacaacccactcacaaccgggataccgaccccgcagt
gcacgcaacgcatcgtccatgcttcgaatctcgaccatgtttgcgggttgtgactgaaacgaagaggaagagg cgcccccttagcggagtgcgatcacaagctcgagtggcctgtgtagaagtggtagtgatctaggtgtttgaatat ggctttggtagctcgctataatgtctttgcaatcgggggcctggctatttaaacagcgctcgccctggagcggca tcggagcgcccatgcagccccgaaggagcttcggggggtcgaagcatcatcggtgttgcatgcagcgccgg gaagccgtctcgcagcccgccctaccttttgctggaagtgtcatagcgcaagaaagacatatgccccgagctc cgcttcaaatacgcccagcccgcccatggagaaagaggccaaaatcaacggaggatcgttacaaccaacaa aattgcaaaactcctccgctttttacgtgttgaaaaagactgatcagcacgaaacggggagctaagctaccgctt 11 031336 cagcacttgagagcagtatcttccatccaccgccgttcgtcagggggcaaggctcagatcaacgagcgcctcc atttacacggagcggggatccgaattcctacattatgaccagctcctcgcccttgccgaactgcgccttgtgctt gtcgtacagcgcctgcaggtcgtccgtgaagcgcttgaggagcgccgccagcacctcgggctcgggctcga gctggcccggcgccagctccggcaccgggatggggcgacccaccacaatggtgaggggctcgcggtgcg gcatgggcgtgccgtactgcccaaacatgccgatgggtacggcgccggcggcacgtgagatgcgctccacg agccacgtgggcacgagcggcggccccggccggaaccagctgtacgcgcgcgtctggccgaacgcccac actggcaccagcggcgcgccgtgctgcacggccagtcgcacgaagccggtgcggctggagaggtaggcc acctccttgccgtgctccatgttgagcacctcctgcacgccccccggcaccagcaccgccaccttgcgcgccc gcaacaggccgctgatgctctgccgcgtggcgggccgcacgcccagccaccagtacagctgccgcactgc agcacgtggggagggaggaggaggagggaggcgtaggaggagcaggagagggaacacacatgtgggt gatgttctggggagcagccacggtagcagtcgcggtagcggtggagagcacaggacggcttcgccacctac ccggagaacaacccgccacaccaccaacctgtcaccgggtgctctcccggcccatcctccccccgcgccgc cccccccgccacacacacctatgggcgcgctgaagcacacggacgacgccaagccgtgtgtccggccgcg cagctccttgggcagcagcggcgaggtggtggcgaaggcgatgggcagtgcgatgggcagagccgagtg cgggcagaatccgaacaagtagccgcggtcagtgcggaaggcctggggggggggagggcacaggaccg cagtcaatgccaaagccaatgcagggcggtggtgtcgccgttcaacccgatgccactacgataccctttgtgg gctggttccgggtcttggtttcttttgggagtgcaataaaccccgccttccccacctgctccggtcatcggcacg caaccactcacctacgcctgatgcactgcccaccgttccctgttcccgatgctcttacccagtttccactgcctta accgtgcagccccgccccctgtctcctccctcgtgaccccagactactcctccccctttccccacaggtccggc ccccacaaccttcgccccccacccccagcagccccccgtacagcgcccccacagcgccctcacctccgggt ccgtgaccaccacgcgggtggggaagtaggccgccgcccgcgccacactgaactgcacgaaccgctccga cagcgcaccggtggtcagctgcagcggcgtgaaggtgagggccaggaaggcggctagcagcagccaggc cgaggggctggtggccccaaacaggtagaacagcgcgagcgccgagggcagcagcaccaccgaggtgta gatggcgctgtggagaggatgggggtttgggggttggaggcttggggttgcagggcgtgtggcagcggggc gagagcttgggcgggggagggggtgcgcaagaacgctttggacgggagacgccatggtggttcggcccag ccgatttcctgagctcgtcagtttcacagaacagtcgcgtcttaagcaaaattacctgacgtagatggctatggc cgcgtactccagcaccacgtttcgcagctttgcgagcggcattctcgttcggccctaagaaagaggcaagctc ctgactggtttatagactcatacagcactccttgaatctagagtcgacgacgacgagcgagacgaggagcgaa gcgagggcttacaagaacgagtaggcacacgagcgagtgccgggttcggagcgctcacacttaagtatgcta caaacgcaagaagagcccggaagggctttatgggcggtgggtcggtttggatccaatgcagttttccacgtca agcggtgtcaagtcgacctactgctgaacgcagcccagtccctccagtctgaagctttctttcattctacgcaatg cgctcatcgtgatattcatgtccgctgcaaatgcacaaaccggtgcacagccaaagctgactcgatgacaagct tacgcatgacatgttccgcacgtcgcgtgtgcattgccgaaggccttttctatgaacgtatatagcttcctgatca gagtattgagggagcagtgaaggcgtttggagggcgtgccaccgtgccctgactggcccggcggggagtat ttgcccgggtttgcccgggtgcacttaccgggctgcagttggctgtcgtgggagcctagtatgtattgctgcttg gcggtgcacggtttacaccacgggcgcttggccaacagcttcgcagcatgcagctgtttcaaatcatagcgttg caggaccaggggtgcgcagggcgtacggtaggggtgctgcgacggcactctggcatggtatgcgcatgcc atggttgctgtggaatggaattgcgctgggatacgcaggatgggcacgcagagaagaggggggcgaggttg ggggcctggggggcctgggggcgagctcgatccaaattcaaagtcgacatcgagctgcacatgggtgtacta ctgttcctgtgtagcatgctcactacgtactcagctcactacagaactgaaatggtgacgcacgttttgaacgtgc agctgatcctgtgaacgtgtgtcgactctctgtggcaagagaagcttagaccaagctttctttattggaacgctgg gaaataagcagcatgtgcctggcgggtctataaaggcttggaggaaacgggcagaatagatctcccccccga gctcgctgaggcttgacatgattggtgcgtatgtttgtatgaagctacaggactgatttggcgggctatgagggc gggggaagctctggaagggccgcgatggggcgcgcggcgtccagaaggcgccatacggcccgctggcg gcacccatccggtataaaagcccgcgaccccgaacggtgacctccactttcagcgacaaacgagcacttata catacgcgactattctgccgctatacataaccactcagctagcgatcccgggcgcgccagaaggagcgcagc caaaccaggatgatgtttgatggggtatttgagcacttgcaacccttatccggaagccccctggcccacaaagg ctaggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcctgataaaccggccagggggc ctatgttctttacttttttacaagagaagtcactcaacatcttaaaattctagaaatcgatactgtattcgcaacaaag tcaagctcaatttacaacattatccaaagctacggtatccacagcatttagaaatggcctgctctgctggaaagcc tctgaagcacctttgctttgtgcacacctacgcgcacaagtttgcgagtggtgccgcttacgttgagggcggcta ctgcaaggccaagacctatgttcccgcggtcgctcagccctacatcgccaaagccgaggagacctgcctagc atacgccgctcctcttgcgacaaaggcgacggaccacgtaagttaattcatccggggtctctgccaccgggtg gccttggaagccgactcgtcgactccgcaacaccccctccggttctgcccgagatgctgacttcctggtgcttt gaatgcttttacaggcagagaagattctccggagcaccgacgcacagctggacgcgctgtacgcggcctccg ccagctggctgagcagctcctgcaagctggccgactccaacatcgcggccttcaggggcgccgccgacaag tactactgcctggtcaagtccactgcgcagcacgtgacgtccaagctgcccaccgacctgtcggtggccaag
gcccgctgcctgctgtcggcctcgctggagtgcgccaaggctctggctgacccggacgctgcggtggcggc ggcgctggatgcctggaccaagttcgcagccatcccggcggttgccaaggtgctgtccgccgcctccccgct gacgggcaagggcgtggcggccttcacggcggcacacgacctcctggtgcactcggcgctgtaccgctacg gcgtgtcggtgggcgcctccaccctgggctgggccaccagcaccaccccctacaagttgagcgcggcttac ctgtacccgctggtgcagcccgtggcggaccccgcgctgtgcaaggtgtccaagagcacctacgtcaacgc agccatcaagtactgggcgcccgcacccgtggccgccgcgtaagaattcggatccccgctccgtgtaaatgg aggcgctcgttgatctgagccttgccccctgacgaacggcggtggatggaagatactgctctcaagtgctgaa gcggtagcttagctccccgtttcgtgctgatcagtctttttcaacacgtaaaaagcggaggagttttgcaattttgtt ggttgtaacgatcctccgttgattttggcctctttctccatgggcgggctgggcgtatttgaagcgaccggtcatat gctttcttgcgctatgacacttccagcaaaaggtagggcgggctgcgagacggcttcccggcgctgcatgcaa caccgatgatgcttcgaccccccgaagctccttcggggctgcatgggcgctccgatgccgctccagggcgag cgctgtttaaatagccaggcccccgattgcaaagacattatagcgagctaccaaagccatattcaaacacctag atcactaccacttctacacaggccactcgagcttgtgatcgcactccgctaagggggcgcctcttcctcttcgttt cagtcacaacccgcaaacatgacacaagaatccctgttacttctcgaccgtattgattcggatgattcctacgcg agcctgcggaacgaccaggagttctgggagccgctggcccgccgagccctggaggagctcgggctgccg gtgccgccggtgctgcgggtgcccggcgagagcaccaaccccgtactggtcggcgagcccggcccggtga tcaagctgttcggcgagcactggtgcggtccggagagcctcgcgtcggagtcggaggcgtacgcggtcctg gcggacgccccggtgccggtgccccgcctcctcggccgcggcgagctgcggcccggcaccggagcctgg ccgtggccctacctggtgatgagccggatgaccggcaccacctggcggtccgcgatggacggcacgaccg accggaacgcgctgctcgccctggcccgcgaactcggccgggtgctcggccggctgcacagggtgccgct gaccgggaacaccgtgctcaccccccattccgaggtcttcccggaactgctgcgggaacgccgcgcggcga ccgtcgaggaccaccgcgggtggggctacctctcgccccggctgctggaccgcctggaggactggctgcc ggacgtggacacgctgctggccggccgcgaaccccggttcgtccacggcgacctgcacgggaccaacatct tcgtggacctggccgcgaccgaggtcaccgggatcgtcgacttcaccgacgtctatgcgggagactcccgct acagcctggtgcaactgcatctcaacgccttccggggcgaccgcgagatcctggccgcgctgctcgacggg gcgcagtggaagcggaccgaggacttcgcccgcgaactgctcgccttcaccttcctgcacgacttcgaggtgt tcgaggagaccccgctggatctctccggcttcaccgatccggaggaactggcgcagttcctctgggggccgc cggacaccgcccccggcgcctgataaggatctatgccggcacctccatgcgccactgaacgtgtagcgtgac tgtggcggccttggcagttttgaccgtgactgaccctggacaaaggatccctgactgaagacaacttgacatgt gattgccatttgacgctttggtgtggaggcggattgtgagatgggaggggggcccattgccttcgtgaccataa cgacatcgaatttcatacatgtgaacagttcagcatggacattcatctcgtcggattagctcttgtgtgataggcca tagcagctggactgttgtgagctctcgatctgcgtagctactggctgtgattgtgcttcaggcggcaggggcag gtaactgccctgaacgtaaaggtgcagcagcagacagcggatgtgcagaacgaatagcgcagtggataagg ttgatgggtggccacacactcgtgcacgtgtaataagatacacgaatcgtcctccttttctttgtcgtaaccatgg gagggaagagagcgtgaccagatgacagcctgggacatctaagaacttggtaggcgggtaagtccctcgat ggcgtggtgtattcccaccagtcgcaagcaaagccaaggggcgtagcagtttgtggggctgagaggtggatg ggaggttgcattggttacacaagggacctg
COPt, ttacattctgacaggaaatgccaaggcgggtatcaatctgttcgggttgacccgttggccaacacacgaatacat 56 aphVIII.pTUBp agtcacatccccaaacacactagggaggggctcactgttgcgttgccaagcggggccagtcttgccggatcc , RBCS2At, caacgtccacactgtgctgtcacccacgcgacgcaaccctacccagccaccaacaccatcaggtccctcaga AtDGATl , agaactcgtccaacagccggtaaaacgccagcttttcctccgataccgccccatcccacccgcgcccgtactc ICLp;HRp, ccgcaggaacgccgcggaacactccggcccgaaccacgggtcctcctcgtgggccagctcgcgcagcacc Ole 3,3, agcgcgagatcggagtgccggtccgcacggccgacccgccccacgtcgatcagcccggtcacctcgcagg RBCSTAt; tacgagggtcgagcagcacgttgtccgggcacaggtcaccgtggcaaaccgccagatcctcgtccgcaggc TUB2p, cgagtccgctccagctcggcgagaagccgctcccccgaccaccccttccgctcctcgtccagatcctccaagt aphVII, TUB2t cgacgctcccttcagcgacagcacgggccgcctgcggcaccgtcaccgcgagactgcgatcgaacggaca (note aphVIII ccgctcccagtccagcgcgtgcagcgaacgagcgagccccgcgagcgccaccgccacgtccagccgctg and AtDGATl ctcccgcggccaccgcgcactggccggacgccccggaaccgcttcggtgaccaaccaggcgaccctctcgt are reversed) ccccaccaccctccacaacacgaggtacgggaatccccacctccgccaaccacaccagccgttcagcctca
cccaacaagcccaccccggcccccagagctgccaccttgacaaacaactcccgcccaccaccccgaagcc
[SEE MAP 3] gataaacaccagcccccgaggccccatcctccacaacaacccactcacaaccgggataccgaccccgcagt
gcacgcaacgcatcgtccatgcttcgaatctcgaccatgtttgcgggttgtgactgaaacgaagaggaagagg cgcccccttagcggagtgcgatcacaagctcgagtggcctgtgtagaagtggtagtgatctaggtgtttgaatat ggctttggtagctcgctataatgtctttgcaatcgggggcctggctatttaaacagcgctcgccctggagcggca tcggagcgcccatgcagccccgaaggagcttcggggggtcgaagcatcatcggtgttgcatgcagcgccgg gaagccgtctcgcagcccgccctaccttttgctggaagtgtcatagcgcaagaaagacatatgccccgagctc cgcttcaaatacgcccagcccgcccatggagaaagaggccaaaatcaacggaggatcgttacaaccaacaa aattgcaaaactcctccgctttttacgtgttgaaaaagactgatcagcacgaaacggggagctaagctaccgctt cagcacttgagagcagtatcttccatccaccgccgttcgtcagggggcaaggctcagatcaacgagcgcctcc atttacacggagcggggatccgaattccttagtggtggtggtggtggtggccggtgcgggtgctgtccaggcc cagcagcgggttggggatcggcttgccctcgaaggggtggccgcgcagctcgcccttggacatggagccct tgcggttcatcaggtcgtggtagtacagcagcacgcacatgggctggccgaagatgcagaagatgaaccaga agatcatgttgcccacggtgctgccgaagcgctcctgcaggtagttggtgatgaacaccagcggcacctgga acatgatgcccaggaacgcccacagcttgaacaggcggcagggcacggcgatgcacagctcgtggaacac cgccgacaccaggaaggcgatgatgatcgccagggtcttcgggatcttcgagcgcaggcaggggaagtag atgtggcgcaccatccacttgtgcaccggcatgttccacatgcgccagtagtcgcccacgctcttggcgttcca ccagtccttgtagaactcgcggtcgccgaagcacagcagctccgccaggatgttcagccacaggtggaagaa gcagtagaacatgcacagccacacgtacaggttgggcacggacagcttcagcacgcgctcgatggcgtaca gcaggtcgcccttcagcgggtgcttcgagttgcgcacgatggggttgatgtactgctcgatgatgaagcccatg aagccggtgaagatcaccagcttcgcgaactggcgggccacccagcccttgcggatgcacgcgctgcgcg ggtacgagggctggtagcacagggtcggggccaccatgaagtacgccagggacttcaggctcacgtagtac gacacctcggggttggccttgtccgcggcgttcgccagcgagcggatgtcgtagctggtgtgggcgtaggac accagcttcagccacacgatgcaggtcagcagcatcagggtcacgcccgacaggaacgcgctgtcgcagc gcagggtcacgtacaccgggtacagcacctcggtcatggtgatgatgatgtgcaggaagatcaccacgggct cggcgatgtacttctgcagcaccagcttctccacggtgaacgcggccagcgggaagatcgacagggagatg cagcacatgaacaggggccagtcgcgcaggctgcgcgacgagaaccagaagtcggtgcggatcagccag ccgtacttcatcaggttctcgatgatcaggcggctgttcaccgcgatcagcaccaccacgcacaggttgaaca ggccggcgtgggactgcttgaagatcgcgtccgagctcagcggcgactcgcgggcgcggcggtgcgcgg gcacggacgggcggtaggtgaaggtggcgtccgcgttgccgcggccctcgccgccgccccggccgccgc cgttgttgtcgccgccgccgttgttgtcgccggccaggttcgcggtgccctgggcgtcgtcgttcaccacgctg tcgatgcggtcgcgcacgtccgcgggggcgcccacgtcgtccgacggggagttgttgtcgctgcccgacag cagcaggccgttggagctgtccgagcgctgcaaatggaaacggcgacgcagggttagatgctgcttgagac agcgacagaggagccaaaagccttcgtcgacacaatgcgggcgttgcaagtcaaatctgcaagcacgctgc ctgatccgccgggcttgctcgtcgactcacggacttgcggcggcgcaggcggtccaggtccacgaactcgcc gccgccgttctcggtcacggtggtcacgcccgcgctgtccaggatggccattttgtctagagtcgacgacgac gagcgagacgaggagcgaagcgagggcttacaagaacgagtaggcacacgagcgagtgccgggttcgga gcgctcacacttaagtatgctacaaacgcaagaagagcccggaagggctttatgggcggtgggtcggtttgga tccaatgcagttttccacgtcaagcggtgtcaagtcgacctactgctgaacgcagcccagtccctccagtctga agctttctttcattctacgcaatgcgctcatcgtgatattcatgtccgctgcaaatgcacaaaccggtgcacagcc aaagctgactcgatgacaagcttacgcatgacatgttccgcacgtcgcgtgtgcattgccgaaggccttttctat gaacgtatatagcttcctgatcagagtattgagggagcagtgaaggcgtttggagggcgtgccaccgtgccct gactggcccggcggggagtatttgcccgggtttgcccgggtgcacttaccgggctgcagttggctgtcgtggg agcctagtatgtattgctgcttggcggtgcacggtttacaccacgggcgcttggccaacagcttcgcagcatgc agctgtttcaaatcatagcgttgcaggaccaggggtgcgcagggcgtacggtaggggtgctgcgacggcact ctggcatggtatgcgcatgccatggttgctgtggaatggaattgcgctgggatacgcaggatgggcacgcaga gaagaggggggcgaggttgggggcctggggggcctgggggcgagctcgatccaaattcaaagtcgacatc gagctgcacatgggtgtactactgttcctgtgtagcatgctcactacgtactcagctcactacagaactgaaatg gtgacgcacgttttgaacgtgcagctgatcctgtgaacgtgtgtcgactctctgtggcaagagaagcttagacc aagctttctttattggaacgctgggaaataagcagcatgtgcctggcgggtctataaaggcttggaggaaacgg gcagaatagatctcccccccgagctcgctgaggcttgacatgattggtgcgtatgtttgtatgaagctacaggac tgatttggcgggctatgagggcgggggaagctctggaagggccgcgatggggcgcgcggcgtccagaag gcgccatacggcccgctggcggcacccatccggtataaaagcccgcgaccccgaacggtgacctccactttc agcgacaaacgagcacttatacatacgcgactattctgccgctatacataaccactcagctagcgatcccgggc gcgccagaaggagcgcagccaaaccaggatgatgtttgatggggtatttgagcacttgcaacccttatccgga agccccctggcccacaaaggctaggcgccaatgcaagcagttcgcatgcagcccctggagcggtgccctcc tgataaaccggccagggggcctatgttctttacttttttacaagagaagtcactcaacatcttaaaattctagacaa aatggcctgccactacggccagcagcagcagacctgcgcgccgcacgtgagtcgacgagcaagcccggc ggatcaggcagcgtgcttgcagatttgacttgcaacgcccgcattgtgtcgacgaaggcttttggctcctctgtc gctgtctcaagcagcatctaaccctgcgtcgccgtttccatttgcagctgcagctgcagccgcgcgcctgccgc gtggtgaaggcggccaccgcggtgaccgccggcggcagcctgctggtgctgtcgggcctgaccctggcgg gcaccgtgatcgccctgaccatcgcgacccccctgctggtgatcttctccccggtgctggtgcccgccgtgatc accatcttcctgctgggcgcgggcttcctggcctcgggcggcttcggcgtggcggccctgagcgtgctgtcct ggatctaccgctacctgaccggcaagcacccgcccggcgcggactgcctggagtcggccaagaccaagct ggcgtcctgcgcccgcgagatgaaggaccgcgcggagcagttcagctgccagccggtggccggctcgcag acctcctaaggaattcggatccccgctccgtgtaaatggaggcgctcgttgatctgagccttgccccctgacga acggcggtggatggaagatactgctctcaagtgctgaagcggtagcttagctccccgtttcgtgctgatcagtct ttttcaacacgtaaaaagcggaggagttttgcaattttgttggttgtaacgatcctccgttgattttggcctctttctcc 2011/031336 atgggcgggctgggcgtatttgaagcgaccggtcatatgctttcttgcgctatgacacttccagcaaaaggtag ggcgggctgcgagacggcttcccggcgctgcatgcaacaccgatgatgcttcgaccccccgaagctccttcg gggctgcatgggcgctccgatgccgctccagggcgagcgctgtttaaatagccaggcccccgattgcaaaga cattatagcgagctaccaaagccatattcaaacacctagatcactaccacttctacacaggccactcgagcttgt gatcgcactccgctaagggggcgcctcttcctcttcgtttcagtcacaacccgcaaacatgacacaagaatccc tgttacttctcgaccgtattgattcggatgattcctacgcgagcctgcggaacgaccaggagttctgggagccg ctggcccgccgagccctggaggagctcgggctgccggtgccgccggtgctgcgggtgcccggcgagagc accaaccccgtactggtcggcgagcccggcccggtgatcaagctgttcggcgagcactggtgcggtccgga gagcctcgcgtcggagtcggaggcgtacgcggtcctggcggacgccccggtgccggtgccccgcctcctc ggccgcggcgagctgcggcccggcaccggagcctggccgtggccctacctggtgatgagccggatgacc ggcaccacctggcggtccgcgatggacggcacgaccgaccggaacgcgctgctcgccctggcccgcgaa ctcggccgggtgctcggccggctgcacagggtgccgctgaccgggaacaccgtgctcaccccccattccga ggtcttcccggaactgctgcgggaacgccgcgcggcgaccgtcgaggaccaccgcgggtggggctacctc tcgccccggctgctggaccgcctggaggactggctgccggacgtggacacgctgctggccggccgcgaac cccggttcgtccacggcgacctgcacgggaccaacatcttcgtggacctggccgcgaccgaggtcaccggg atcgtcgacttcaccgacgtctatgcgggagactcccgctacagcctggtgcaactgcatctcaacgccttccg gggcgaccgcgagatcctggccgcgctgctcgacggggcgcagtggaagcggaccgaggacttcgcccg cgaactgctcgccttcaccttcctgcacgacttcgaggtgttcgaggagaccccgctggatctctccggcttcac cgatccggaggaactggcgcagttcctctgggggccgccggacaccgcccccggcgcctgataaggatcta tgccggcacctccatgcgccactgaacgtgtagcgtgactgtggcggccttggcagttttgaccgtgactgacc ctggacaaaggatccctgactgaagacaacttgacatgtgattgccatttgacgctttggtgtggaggcggattg tgagatgggaggggggcccattgccttcgtgaccataacgacatcgaatttcatacatgtgaacagttcagcat ggacattcatctcgtcggattagctcttgtgtgataggccatagcagctggactgttgtgagctctcgatctgcgt agctactggctgtgattgtgcttcaggcggcaggggcaggtaactgccctgaacgtaaaggtgcagcagcag acagcggatgtgcagaacgaatagcgcagtggataaggttgatgggtggccacacactcgtgcacgtgtaat aagatacacgaatcgtcctccttttctttgtcgtaaccatgggagggaagagagcgtgaccagatgacagcctg ggacatctaagaacttggtaggcgggtaagtccctcgatggcgtggtgtattcccaccagtcgcaagcaaagc
caaggggcgtagcagtttgtggggctgagaggtggatgggaggttgcattggttacacaagggacctg
Table 9 - Nucleic Acid Sequences of Yeast constructs expressing DGAT and lipid encapsulation proteins
Construct Sequence SEQ ID design NO:
Gal l Op- ggccggtagaggtgtggtcaataagagcgacctcatgctatacctgagaaagcaacctgacctacaggaaa 57 gagttactcaagaataagaattttcgttttaaaacctaagagtcactttaaaatttgtatacacttattttttttataactt oleo_3,3- atttaataataaaaatcataaatcataagaaattcgcttatttagaagtgtcaacaacgtatctaccaacgatttga
ADHl :Gall - cccttttccatcttttcgtaaatttctggcaaggtagacaagccgacaaccttgattggagacttgaccaaacctc
tggcgaagaagtccaaagctccgcggttaggatgtttgggaacctgctactggttggcaactgaattgttcggc
AtDGATl - tctgtccttcatttctctggcgcatgatgctaactttgttttggcagattccaaacaatctgcaccaggtggatgttt
CYC1 TT accagtcaagtatctatagatccatgacaaaacagacaatgcagcgacaccaaaaccacctgaagctaagaa
accggcacctaacaaaaatatggtaattactgcaggtaccaaaactggggagaatataactaacaatggagtt cassette from
gcaatggttaaagcgataacggtaccagctaatgtcaaaccactcaagactaacaaggaaccaccagctgtta pYES2.1 cggcagtagctgccttaacgactctgcatgctctaggttgtaattgcaagtgaggagcacaggtttgttgttgttg
accgtagtgacaggccattttttgccggccctaggttatattgaattttcaaaaattcttactttttttttggatggac gcaaagaagtttaataatcatattacatggcattaccaccatatacatatccatatacatatccatatctaatcttact
[SEE MAP 4] tatatgttgtggaaatgtaaagagccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgct
taactgctcattgctatatcggtccgtctagtacggattagaagccgccgagcgggtgacagccctccgaagg aagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgc tccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccac aaaccttcaaatgaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggt aattaatcagcgaagcgatgatttttgatctattaacagatatataaatgcaaaaactgcataaccactttaactaa tactttcaacattttcggtttgtattacttcttattcaaatgtaataaaagtatcaacaaaaaattgttaatatacctcta tactttaacgtcaaggagaaaaaaccccggatcggactactagcagctgtaatacgactcactatagggaatat taagctcgcccttgccgcatttaattaagaattcgccggcaaaaaatggctattttggattctgctggtgttactac tgttactgaaaacggtggaggtgaatttgttgatttggatagattgagaagaagaaagtcaagatctgattcttct aatggtttgttgttgtctggttctgataacaattctccatctgatgatgttggtgctccagctgatgttagagataga attgattctgttgttaacgatgatgctcaaggtactgctaatttggctggtgataacaacggtggtggagacaac aatggtggtggaagaggaggtggtgaaggtagaggtaatgctgatgctacttttacttatagaccatctgttcca gctcatagaagagctagagaatctccactctcgagtgatgctattttcaaacaatctcatgctggtttgtttaatttg tgtgttgttgttttgattgctgttaactcaagattgattattgaaaacttgatgaaatacggttggttgattagaactg atttttggttctcttcaagatctttgagagattggccattgtttatgtgttgtatttctttgtctatttttccattggctgctt ttactgttgaaaagcttgttttgcaaaagtatatttctgaaccagttgttattttcttgcatattatcattactatgactg aagttttgtatccagtttacgttactttgagatgtgattctgcttttttgtctggtgttactttgatgttgttgacttgtatt gtttggttgaagttggtttcttatgctcatacttcttacgatattagatctttggctaatgctgctgataaagctaatcc agaagtttcttactacgtttctttgaaatctttggcttactttatggttgctccaactttgtgttatcaaccatcttatcca agatctgcttgtattagaaaaggttgggttgctagacaatttgctaagttggttattttcactggttttatgggtttcat tattgaacaatacattaacccaattgttagaaattctaagcatccattgaaaggtgatttgttgtatgctattgaaag agttttgaagctttctgttccaaacttgtacgtttggttgtgcatgttttactgttttttccatttgtggttgaatattttgg ctgaattgttgtgttttggtgatagagaattttacaaggattggtggaatgctaaatctgttggtgattattggagaa tgtggaatatgccagttcataaatggatggttagacacatatactttccatgtttgagatctaaaattccaaagact ttggctatcattattgcttttttggtttctgctgtttttcatgaattgtgtattgctgttccatgtagattgtttaaattgtgg gcttttttgggtataatgttccaagttccattggttttcattacaaactacttgcaagaaagatttggttctactgttgg taatatgattttctggttcattttctgtatttttggtcaaccaatgtgtgttttgttgtactatcatgatttgatgaatagaa agggttctatgtctgccaagggcgagcttcgaggtcacccattcgaaggtaagcctatccctaaccctctcctc ggtctcgattctacgcgtaccggtcatcatcaccatcaccattgagtttctagagggccgcatcatgtaattagtt atgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaag tctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaga cgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgca agctgc
GallOp-MLDP- ggccggtagaggtgtggtcaataagagcgacctcatgctatacctgagaaagcaacctgacctacaggaaag 58 agttactcaagaataagaattttcgttttaaaacctaagagtcactttaaaatttgtatacacttaltttttttataacttat ADHl :Gall - ttaataataaaaatcataaatcataagaaattcgcttatttagaagtgtcaacaacgtatctaccaacgatttgaccc CrDGAT2- ttttccatcttttcgtaaatttctggcaaggtagacaagccgacaaccttgattggagacttgaccaaacctctggc
gaagaagtccaaagctccgcggttatgctgctgcaactggggctggtgcccaatacttaatggctgcgtttacat CYC1 TT
atgtggattttgaaaccttgtctaatgctggatcagctacaggttgaactaatgggtacaaatatgcagcacttaat cassette from ttgtatggtgtagtggaggttgcccaacccaatgtggaagcacctactgaaacaccgtatctatacaaagcgga
gtgaactaacaaatcatgggctgctgtgaaagcggctacacctttaccagttaatggagatgcagcactcaaga pYES2.1
ccttagctacggcaggaatggctgcaaatttagtccaagcatccaaagcggctgcaacagcggcgtctggatc tgctaaagctttggcttgttccaaggaagctgataacaattctcttgccttagcaactgataagtcagtaggcaatt tagaggtaacgtgttgagctgtactcttgactaagtcgtaatatttatctgcagcacctctaaaggctgcgatgttt
[SEE MAP 5]
gaatctgccaacttttgggatgaagacaaccaagaagcactagcggcatataatgcgtccaattgggcatctgt agatctcaatatcttttctgcatgatcagtggctttggttgctaaaggtgcagcgtaagccaaacaagtttcttcgg cttttgcaatatatggttgagcaacggcagggacgtaggtcttagctttttgataaccaccttcaacgtaggcggc accagaggcgaacttgtgtgcgtaggtgtggacaaattccaaatgcttcaaaggtttaccagcggattcagcca ttttttgccggccctaggttatattgaattttcaaaaattcttactttttttttggatggacgcaaagaagtttaataatca tattacatggcattaccaccatatacatatccatatacatatccatatctaatcttacttatatgttgtggaaatgtaaa gagccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatatcg gtccgtctagtacggattagaagccgccgagcgggtgacagccctccgaaggaagactctcctccgtgcgtc ctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaacaataaagattcta caatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaatgaacgaat caaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatg atttttgatctattaacagatatataaatgcaaaaactgcataaccactttaactaatactttcaacattttcggtttgta ttacttcttattcaaatgtaataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaa aaccccggatcggactactagcagctgtaatacgactcactatagggaatattaagctcgcccttgccgcattta attaagaattcgccggcaaaaaatgccattagcaaagttgagaaacgtcgtcttggaatacgcagccatcgcca tctatgtcagtgcaatctatacctctgttgtcttgttaccttccgctttagcattgttttatttgttcggtgccacctctcc atcagcttggttgttattggctgcatttttagctttgacattcaccccattacaattgactacaggtgcattgtctgaa agattcgttcaattctcagtagccagagccgctgcatattttcctactagagttgtagtcacagatccagaagcttt cagaacagacagaggttacttgtttggtttctgtccacattccgcattacctattgccttgccaatagcctttgctac cacttctcctttattgccaaaagaattaagaggtagaactcacggtttggcctcttcagtttgcttctctgctcctatt gtaagacaattatattggtggttgggtgttagaccagcaacaagacaatccataagtggtttattgagagctaga aaagtcgcagttttagtaccaggtggtgtacaagaagtcttgaatatggaacatggtaaagaagttgcttatttgtc 6
Figure imgf000068_0001
Table 10 - Translated amino acid sequences of Yeast constructs expressing DGAT and lipid encapsulation proteins
Protein Sequence SEQ ID description NO:
A. thaliana maildsagvttvtengggefvdldrlrrrksrsdssnglllsgsdnnspsddvgapadvrdridsvvnddaq 60 DGAT1 with gtanlagdnngggdnngggrgggegrgnadatttyrpsvpahrraresplssdaifkqshaglfrilcvvvli C-terminal V5 avnsrliienlmkygwlirtdfwfssrslrdwplfmc^^
epitope and His vlyp\^vtlrcdsaflsgvtlmntcivwlklvsyahtsydirslanaadkanpevsyyvslkslayfmvapt
tag lcyqpsyprsacirkgwvarqfaklviftgfmgfiieqyinpivmskhplkgdllyaiervlklsvpnlyvw
lcmfycffhlwlnilaellcfgdrefykdwwnaksvgdywrmwnmpvhkwmwhiyfpclrskipkt
laiiiaflvsavfhelciavpcrlfklwaflgimfqvplvfitnylqerfgstvgnmifwfifcifgqpmcvlty
yhdlmnrkgsmsakgelrghpfegkpipnpllgldstrtgh hhhh
S. indicum machygqqqqtcaphlqlqpracrvvkaatavtaggsllvlsgltlagtvialtiatpllvifspvlvpavitifll 61 oleosin with gagflasggfgvaalsvlswiyryltgkhppgadclesaktklascaremkdraeqfscqpvagsqts
engineered
cysteine
residues
C. reinhardtii mplaklmvvleyaaiaiyvsaiytsvvllpsalalfylfgatspsawlllaaflaltftplqlttgalserfvqfsv 62 DGAT2 araaay 1 vvvtdpeafrtdrgylfgfcphsalpialpiafatts llpkelrgrthglassvcfsapivrqly
wwlgvrpatrqsisgllrarkvavlvpggvqevlnmehgkevaylssrtgfvrlavqhgaplvpvwafgq
trayswfrpgpplvptwlverisraagav igmfgqygtpmphrepltivvg ipvpelapgqlepepe
vlaallkrftddlqalydkhkaqfgkgeelvim
C. reinhardtii maesagkplkhlefVhtyahkfasgaayveggyqkaktyvpavaqpyiakaeetclayaaplatkatdha 63 MLDP ekilrstdaqldalyaasaswlsssqkladsniaafrgaadkyydlvkstaqhvtsklptdlsvakarellsasl
eqakaladpdaavaaaldawtkfaaipavakvlsaaspltgkgvaaftaahdllvhsalyrygvsvgastlg
watsttpyklsaaylyplvqpvadpaldkvskstyvnaaikywapapvaaa
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[0242] Although the invention has been described with reference to the above embodiments, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:
1. A method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle, the method comprising:
a) introducing into a microbial cell at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme, and at least one nucleic acid molecule encoding a neutral lipid encapsulation protein; and
b) culturing the microbial cell in order to express the neutral lipid synthesizing
enzyme and the neutral lipid encapsulation protein.
2. The method of claim 1, wherein the at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the at least one nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in a single construct.
3. The method of claim 1, wherein the at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the at least one nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in separate constructs.
4. The method of claim 1, wherein the neutral lipid is selected from the group consisting of triacylglycerol (TAG), sterol ester (SE), and wax ester (WE).
5. The method of any one of claims 2 or 3, wherein the constructs are incorporated into the nuclear genome, the chloroplast genome, autonomously replicating plasmid, or artificial chromosome.
6. The method of claim 1, wherein the neutral lipid synthesizing enzyme is selected from the group consisting of acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl
CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol acyltransferase3 (DGAT3), phospholipid:diacylglycerol acyltransferase (PDAT),
diacyl glycerol :diacyl glycerol transacylase, Afunctional wax ester synthase DGAT (WS/DGAT), lecithinxholesterol acyltransferase (LCAT), and an acyl-CoAxholesterol acyltransferase (AC AT).
7. The method of claim 1, wherein the nucleic acid molecule encoding the at least one
neutral lipid synthesizing enzyme encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, any homolog thereof, and any ortholog thereof.
8. The method of claim 1, wherein two or more neutral lipid synthesizing enzymes are expressed in the cell.
9. The method of claim 1 , wherein the neutral lipid synthesizing enzyme is an
acyltransferase with enzyme classification 2.3.I .X.
10. The method of claim 1 , wherein the neutral lipid encapsulation protein is selected from the group consisting of oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
11. The method of claim 1 , wherein the at least one neutral lipid encapsulation protein
consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-29, 50, a homolog thereof, and an ortholog thereof.
12. The method of claim 10, wherein the neutral lipid encapsulation protein is modified by creating a head to tail fusion of a single polypeptide.
13. The method of claim 10, wherein the neutral lipid encapsulation protein is modified by introducing at least one artificial cysteine.
14. The method of claim 1, wherein two or more neutral lipid encapsulation proteins are expressed in the cell.
15. The method of claims 1-14, wherein the microbial cell is a prokaryote or a eukaryote.
16. The method of claim 15, wherein the microbial cell is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates).
17. The method of claim 16, wherein the algal cell is a species of Chlamydomonas,
Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum,
Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parietochloris.
18. The method of claim 17, wherein the algal cell is Chlamydomonas reinhardtii.
19. The method of claim 1, wherein the cell is an oleaginous species.
20. The method of claim 21, wherein the cell is from the genus Yarrowia, Candida,
Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, Pythium, Schizochytrium, Thraustochytrium, or Ulkenia.
21. The method of claim 1 , wherein the cell is a bacterium of the genus Rhodococcus,
Escherichia, or a cyanobacterium.
22. The method of claim 1, wherein the cell is a yeast cell of the class Ascomycota or
Basidiomycota.
23. The method of claim 1, wherein the cell is a synthetic cell.
24. The method of claim 1, wherein the cell is cultured in a batch culture, fed-batch culture, or continuous culture.
25. The method of claims 24, wherein the cell is cultured in a fermentor, photobioreactor, open pond, or any combination thereof.
26. The method of claim 15, wherein the phase of the cell cycle includes the GO, Gl, S, G2, or M phase.
27. The method of claim 15, wherein the cell is part of a culture that is in the lag, logarithmic, or stationary growth phase.
28. The method of claim 15, wherein the microbial cell is manipulated to produce a neutral lipid independent of an external stressor.
29. The method of claim 28, wherein the stressor is an abiotic stress.
30. The method of claim 29, wherein the abiotic stress is nutrient deprivation.
31. The method of claim 15, wherein the microbial cell simultaneously produces and
accumulates a neutral lipid while continuing to grow.
32. A method for producing neutral lipids in a microbial cell independent of the stage in the cell cycle the method comprising:
a) introducing a first nucleic acid construct into a first microbial cell, wherein the first construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid encapsulation protein;
b) introducing a second nucleic acid construct into a second microbial cell, wherein the second construct comprises at least one promoter, at least one nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme; c) cross-breeding the first and second microbial cells to produce a third microbial cell comprising the nucleic acid molecule encoding the at least one neutral lipid encapsulation protein and the nucleic acid molecule encoding at least one neutral lipid synthesizing enzyme; and
d) culturing the third microbial cell in order to express the at least one neutral lipid encapsulation protein and the at least one neutral lipid synthesizing enzyme.
33. The method of claim 32, wherein the neutral lipid is selected from the group consisting of triacylglycerol (TAG), sterol ester (SE), and wax ester (WE).
34. The method of claim 32, wherein the at least one neutral lipid synthesizing enzyme is selected from the group consisting of acyl CoA:diacylglycerol acyltransferasel (DGATl), acyl CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol
acyltransferase3 (DGAT3), phospholipid: diacyl glycerol acyltransferase (PDAT), diacylglycerohdiacylglycerol transacylase, bifunctional wax ester synthase DGAT (WS/DGAT), lecithi cholesterol acyltransferase (LCAT), and an acyl-CoA:cholesterol acyltransferase (AC AT).
35. The method of claim 32, wherein the nucleic acid molecule encoding the at least one
neutral lipid synthesizing enzyme encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, any homolog thereof, and any ortholog thereof.
36. The method of claims 32-35, wherein two or more neutral lipid synthesizing enzymes are expressed in the third microbial cell.
37. The method of claim 32, wherein the neutral lipid synthesizing enzyme is an acyltransferase with enzyme classification 2.3.I .X.
38. The method of claims 32-37, wherein the at least one neutral lipid encapsulation protein is selected from the group consisting of oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
39. The method of claim 32, wherein the at least one neutral lipid encapsulation protein
consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-29, 50, a homolog thereof, and an ortholog thereof.
40. The method of claim 38, wherein the neutral lipid encapsulation protein is modified by creating a head to tail fusion of a single polypeptide.
41. The method of claim 38, wherein the neutral lipid encapsulation protein is modified by introducing at least one artificial cysteine.
42. The method of claim 32-41 , wherein two or more neutral lipid encapsulation proteins are expressed in the third microbial cell.
43. The method of claims 32-42, wherein any of the first, second, or third microbial cells is a prokaryote or a eukaryote.
44. The method of claim 42, wherein any of the first, second, or third microbial cells is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae),
Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata
(dinoflagellates).
45. The method of claim 44, wherein any of the first, second, or third microbial cells is a species of Chlamydomonas, Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum, Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parieto Moris.
46. The method of claim 45, wherein any of the first, second, or third microbial cells is
Chlamydomonas reinhardtii.
47. The method of claim 43, wherein any of the first, second, or third microbial cells is an oleaginous species.
48. The method of claim 47, wherein any of the first, second, or third microbial cells is from the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,
Trichosporon, Lipomyces, Pythium, Schizochytrium, Thraustochytrium, or Ulkenia.
49. The method of claim 43, wherein any of the first, second, or third microbial cells is a bacterium of the genus Rhodococcus, Escherichia, or a cyanobacterium.
50. The method of claim 43, wherein any of the first, second, or third microbial cells is a yeast cell of the class Ascomycota or Basidiomycota.
51. The method of claim 43, wherein any of the first, second, or third microbial cells is a synthetic cell.
52. The method of claim 43, wherein any of the first, second, or third microbial cells is cultured in a batch culture, fed-batch culture, or continuous culture.
53. The method of claims 43, wherein any of the first, second, or third microbial cells is cultured in a fermentor, photobioreactor, open pond, or any combination thereof.
54. The method of claim 43, wherein the phase of the cell cycle includes the GO, Gl, S, G2, or M phase.
55. The method of claim 43, wherein the third cell is part of a culture that is in the lag, logarithmic, or stationary growth phase.
56. The method of claim 43, wherein the third microbial cell is manipulated to produce a neutral lipid independent of an external stressor.
57. The method of claim 56, wherein the stressor is an abiotic stress.
58. The method of claim 57, wherein the abiotic stress is nutrient deprivation.
59. The method of claim 43, wherein the third microbial cell simultaneously produces and accumulates a neutral lipid while continuing to grow.
60. A microbial cell which has been manipulated to produce neutral lipids independent of the stage in the cell cycle comprising:
a) a nucleic acid molecule encoding a neutral lipid synthesizing enzyme; and b) a nucleic acid molecule encoding a neutral lipid encapsulation protein.
61. The microbial cell of claim 60, wherein the at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the at least one nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in a single construct.
62. The microbial cell of claim 60, wherein the at least one nucleic acid molecule encoding a neutral lipid synthesizing enzyme and the at least one nucleic acid molecule encoding a neutral lipid encapsulation protein are contained in separate constructs.
63. The microbial cell of claim 60, wherein the neutral lipid is selected from the group
consisting of triacyl glycerol (TAG), sterol ester (SE), and wax ester (WE).
64. The microbial cell of claim 61 or 62, wherein the constructs are incorporated into the nuclear genome, the chloroplast genome, autonomously replicating plasmid, or artificial chromosome.
65. The microbial cell of claim 60, wherein the neutral lipid synthesizing enzyme is selected from the group consisting of acyl CoA:diacylglycerol acyltransferasel (DGAT1), acyl CoA:diacylglycerol acyltransferase2 (DGAT2), acyl CoA:diacylglycerol acyltransferase3 (DGAT3), phospholipid:diacylglycerol acyltransferase (PDAT),
diacylglycerohdiacylglycerol transacylase, bifunctional wax ester synthase DGAT (WS/DGAT), lecithin: cholesterol acyltransferase (LCAT), and an acyl-CoA:cholesterol acyltransferase (AC AT).
66. The microbial cell of claim 60, wherein the nucleic acid molecule encoding the at least one neutral lipid synthesizing enzyme encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-13, any homolog thereof, and any ortholog thereof.
67. The microbial cell of claim 60, wherein two or more neutral lipid synthesizing enzymes are expressed in the cell.
68. The microbial cell of claim 60, wherein the neutral lipid synthesizing enzyme is an acyltransferase with enzyme classification 2.3.I .X.
69. The microbial cell of claim 60, wherein the neutral lipid encapsulation protein is selected from the group consisting of oleosin, steroleosin, caoleosin, major lipid drop protein (MLDP), plastoglobulin, perilipin, and apolipoprotein.
70. The microbial cell of claim 60, wherein the at least one neutral lipid encapsulation protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-29, 50 a homolog thereof, and an ortholog thereof.
71. The microbial cell of claim 60, wherein the neutral lipid encapsulation protein is modified by creating a head to tail fusion of a single polypeptide
72. The microbial cell of claim 69, wherein the neutral lipid encapsulation protein is modified by introducing at least one artificial cysteine
73. The microbial cell of claim 60, wherein two or more neutral lipid encapsulation proteins are expressed in the cell.
74. The microbial cell of claim 60-73, wherein the microbial cell is a prokaryote or a
eukaryote.
75. The method of claim 74, wherein the microbial cell is an algal cell of the division of Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), or Dinoflagellata (dinoflagellates).
76. The algal cell of claim 75, wherein the algal cell is a species of Chlamydomonas,
Dunaliella, Botrycoccus, Chlorella, Crypthecodinium, Gracilaria, Sargassum,
Pleurochrysis, Porphyridium, Phaeodactylum, Haematococcus, Isochrysis, Scenedesmus, Monodus, Cyclotella, Nitzschia, or Parietochloris.
77. The algal cell of claim 76, wherein the algal cell is Chlamydomonas reinhardtii.
78. The microbial cell of claim 74, wherein the cell is an oleaginous species.
79. The oleaginous cell of claim 78, wherein the cell is from the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, Lipomyces, Pythium, Schizochytrium, Thraustochytrium, or Ulkenia.
80. The microbial cell of claim 74, wherein the cell is a bacterium of the genus Rhodococcus, Escherichia, or a cyanobacterium.
81. The microbial cell of claim 74, wherein the cell is a yeast cell of the class Ascomycota or Basidiomycota.
82. The microbial cell of claim 74, wherein the cell is a synthetic cell.
83. The microbial cell of claim 74, wherein the cell is cultured in a batch culture, fed-batch culture, or continuous culture.
84. The microbial cell of claim 83, wherein the cell is cultured in a fermentor,
photobioreactor, open pond, or any combination thereof.
85. The microbial cell of claim 74, wherein the phase of the cell cycle includes the GO, Gl, S, G2, or M phase.
86. The microbial cell of claim 74, wherein the cell is part of a culture that is in the lag,
logarithmic, or stationary growth phase.
87. The microbial cell of claim 74, wherein the microbial cell is manipulated to produce a neutral lipid independent of an external stressor.
88. The microbial cell of claim 87, wherein the stressor is an abiotic stress.
89. The microbial cell of claim 88, wherein the abiotic stress is nutrient deprivation.
90. The microbial cell of claim 74, wherein the microbial cell produces a neutral lipid and undergoes cellular division simultaneously.
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