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WO2011127118A1 - Procédés de production d'huile dans des organismes non végétaux - Google Patents

Procédés de production d'huile dans des organismes non végétaux Download PDF

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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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Nicholas John Roberts
Kurtis Gale Knapp
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ALGENETIX Inc
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ALGENETIX Inc
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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

La présente invention concerne des procédés de production d'huile dans des cellules microbiennes. L'invention concerne également des procédés de production de ces cellules ainsi que leurs utilisations.
PCT/US2011/031336 2010-04-06 2011-04-06 Procédés de production d'huile dans des organismes non végétaux Ceased WO2011127118A1 (fr)

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US20150322468A1 (en) * 2014-05-07 2015-11-12 Arizona Board Of Regents On Behalf Of Arizona State University Method of producing biofuel using microalgae cultures
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WO2019113463A1 (fr) 2017-12-08 2019-06-13 Synthetic Genomics, Inc. Amélioration de la productivité de lipides d'algues par modification génétique d'une protéine contenant un domaine tpr
US20200024313A1 (en) * 2018-07-18 2020-01-23 Alcantara Research Group Inc. Recombinant polypeptide-enriched chloroplasts or accumulated lipid particles and methods for producing the same in algae
US10563232B2 (en) 2015-07-14 2020-02-18 Synthetic Genomics, Inc. Microorganisms having increased lipid productivity
US10689676B2 (en) 2015-11-02 2020-06-23 Synthetic Genomics, Inc. Algal mutants with increased lipid productivity
WO2021097230A1 (fr) * 2019-11-15 2021-05-20 Xylome Corporation Compositions de corps lipidiques, produits fabriqués à partir de ceux-ci, leurs procédés de fabrication et procédés d'utilisation
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US11859193B2 (en) 2016-09-02 2024-01-02 Nuseed Global Innovation Ltd. Plants with modified traits
US12252513B2 (en) 2018-07-16 2025-03-18 Lumen Bioscience, Inc. Thermostable phycobiliproteins produced from recombinant arthrospira
US12447202B2 (en) 2018-05-17 2025-10-21 Lumen Bioscience, Inc. Arthrospira platensis oral vaccine delivery platform

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030074695A1 (en) * 1998-06-24 2003-04-17 Farese Robert V. Plant diacylglycerol O-acyltransferase and uses thereof
US6878527B1 (en) * 1998-07-10 2005-04-12 Commonwealth Scientific & Industrial Research Organization Modified proteins
US20070122826A1 (en) * 2005-10-12 2007-05-31 J. Craig Venter Institute, Inc. Minimal bacterial genome
US20090133160A1 (en) * 2005-10-19 2009-05-21 Agriculture Victoria Services Pty Ltd Polyoleosins
US20090229007A1 (en) * 2005-07-01 2009-09-10 Tanksley Steven D Oleosin Genes and Promoters From Coffee
US20090269828A1 (en) * 2003-07-02 2009-10-29 E. I. Du Pont De Nemours And Company Acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous yeasts
US20090293151A1 (en) * 2008-05-23 2009-11-26 E. I. Du Pont De Nemours And Company Dgat genes from oleaginous organisms for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030074695A1 (en) * 1998-06-24 2003-04-17 Farese Robert V. Plant diacylglycerol O-acyltransferase and uses thereof
US6878527B1 (en) * 1998-07-10 2005-04-12 Commonwealth Scientific & Industrial Research Organization Modified proteins
US20090269828A1 (en) * 2003-07-02 2009-10-29 E. I. Du Pont De Nemours And Company Acyltransferases for alteration of polyunsaturated fatty acids and oil content in oleaginous yeasts
US20090229007A1 (en) * 2005-07-01 2009-09-10 Tanksley Steven D Oleosin Genes and Promoters From Coffee
US20070122826A1 (en) * 2005-10-12 2007-05-31 J. Craig Venter Institute, Inc. Minimal bacterial genome
US20090133160A1 (en) * 2005-10-19 2009-05-21 Agriculture Victoria Services Pty Ltd Polyoleosins
US20090293151A1 (en) * 2008-05-23 2009-11-26 E. I. Du Pont De Nemours And Company Dgat genes from oleaginous organisms for increased seed storage lipid production and altered fatty acid profiles in oilseed plants

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2823042A1 (fr) * 2012-03-09 2015-01-14 Matrix Genetics, LLC Protéines modifiées de diacylglycérol acyltransférase et procédés d'utilisation de ces protéines
CN102943048A (zh) * 2012-11-30 2013-02-27 北京联合大学生物化学工程学院 高含油量小环藻突变株及其筛选和培养方法
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EP2935594A4 (fr) * 2012-12-19 2016-09-07 Boston Medical Ct Corp Procédés d'augmentation de la teneur en matières grasses/huile de plantes
US10253325B2 (en) 2012-12-19 2019-04-09 Boston Medical Center Corporation Methods for elevating fat/oil content in plants
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CN103965305A (zh) * 2013-01-31 2014-08-06 中国科学院大连化学物理研究所 一种产油酵母脂滴蛋白及其编码基因与应用
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CN103965304B (zh) * 2013-01-31 2016-06-29 中国科学院大连化学物理研究所 一种产油酵母脂滴蛋白及其编码基因与应用
WO2014164232A1 (fr) * 2013-03-13 2014-10-09 Matrix Genetics, Llc Cyanobactéries qui produisent des protéines d'empaquetage des lipides
CN112961868A (zh) * 2013-12-31 2021-06-15 合成基因组股份有限公司 生物质生产率调节剂
US20150322468A1 (en) * 2014-05-07 2015-11-12 Arizona Board Of Regents On Behalf Of Arizona State University Method of producing biofuel using microalgae cultures
EP4174186A1 (fr) * 2014-05-29 2023-05-03 Ginkgo Bioworks, Inc. Augmentation de la production de lipides dans des levures oléagineuses
US11365369B2 (en) 2014-07-07 2022-06-21 Commonwealth Scientific And Industrial Research Organisation Processes for producing industrial products from plant lipids
EP4303288A2 (fr) 2014-07-07 2024-01-10 Nuseed Global Innovation Ltd Procédés de production de produits industriels à partir de lipides végétaux
US10472587B2 (en) 2014-07-07 2019-11-12 Commonwealth Scientific And Industrial Research Organisation Processes for producing industrial products from plant lipids
US11814600B2 (en) 2014-07-07 2023-11-14 Nuseed Global Innnovation Ltd. Process for producing industrial products from plant lipids
EP2966157A1 (fr) 2014-07-07 2016-01-13 Commonwealth Scientific and Industrial Research Organisation Procédés de production de produits industriels à partir de lipides végétaux
CN105567715A (zh) * 2014-10-17 2016-05-11 丰益(上海)生物技术研发中心有限公司 裂殖壶菌α-微管蛋白相关序列及其应用
CN104593362A (zh) * 2015-01-08 2015-05-06 上海海洋大学 一种编码缺刻缘绿藻油体钙蛋白的dna序列及其应用
US11332764B2 (en) 2015-07-14 2022-05-17 Viridos, Inc. Microorganisms having increased lipid productivity
US10563232B2 (en) 2015-07-14 2020-02-18 Synthetic Genomics, Inc. Microorganisms having increased lipid productivity
US10689676B2 (en) 2015-11-02 2020-06-23 Synthetic Genomics, Inc. Algal mutants with increased lipid productivity
US12421523B2 (en) 2016-09-02 2025-09-23 Nuseed Global Innovation Ltd. Plants with modified traits
US11859193B2 (en) 2016-09-02 2024-01-02 Nuseed Global Innovation Ltd. Plants with modified traits
WO2019113463A1 (fr) 2017-12-08 2019-06-13 Synthetic Genomics, Inc. Amélioration de la productivité de lipides d'algues par modification génétique d'une protéine contenant un domaine tpr
US12447202B2 (en) 2018-05-17 2025-10-21 Lumen Bioscience, Inc. Arthrospira platensis oral vaccine delivery platform
US12252513B2 (en) 2018-07-16 2025-03-18 Lumen Bioscience, Inc. Thermostable phycobiliproteins produced from recombinant arthrospira
US20200024313A1 (en) * 2018-07-18 2020-01-23 Alcantara Research Group Inc. Recombinant polypeptide-enriched chloroplasts or accumulated lipid particles and methods for producing the same in algae
US12350364B2 (en) 2019-11-15 2025-07-08 Xylome Corporation Lipid body compositions, products made therefrom, methods of making same, and methods of use
WO2021097230A1 (fr) * 2019-11-15 2021-05-20 Xylome Corporation Compositions de corps lipidiques, produits fabriqués à partir de ceux-ci, leurs procédés de fabrication et procédés d'utilisation

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