WO2013056212A2 - Chlorophyte genes for the optimization of productivity - Google Patents

Chlorophyte genes for the optimization of productivity Download PDF

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Publication number
WO2013056212A2
WO2013056212A2 PCT/US2012/060202 US2012060202W WO2013056212A2 WO 2013056212 A2 WO2013056212 A2 WO 2013056212A2 US 2012060202 W US2012060202 W US 2012060202W WO 2013056212 A2 WO2013056212 A2 WO 2013056212A2
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sequence
complementary
nucleic acid
acid molecule
encoding
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PCT/US2012/060202
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French (fr)
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WO2013056212A3 (en
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Mark Scott Abad
Daniel Adam COURY
Zoee Gokhale Perrine
Bradley Lynn Postier
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Phycal Llc
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13122—Chlorophyllide-a oxygenase (1.14.13.122)
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    • C12Y202/01—Transketolases and transaldolases (2.2.1)
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    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
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    • C12Y402/0102—Tryptophan synthase (4.2.1.20)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

  • RFS2 requires 36 million gallons of renewable fuel to be blended into transportation fuel by 2022. Additionally, many states have responded by enacting their own renewable portfolio standards mandating electricity providers to obtain a certain percentage of their power from renewable energy sources. As a result of these concerns and RPS requirements, domestically produced biofuels have become an increasingly attractive alternative to foreign fossil fuels.
  • Microalgae are some of the most productive and therefore desirable sources of biofuels.
  • the Department of Energy (DOE) has determined that biofuel yield per acre from microalgal culture exceeds that of many competing organisms and land crops.
  • DOE's National Renewable Energy Laboratory (NREL) evaluated the economic feasibility of producing biofuels from a variety of aquatic and terrestrial photosynthetic organisms.
  • Biofuel production from microalgae was determined to have the greatest yield per acre potential of any of the organisms screened.
  • Microalgal biofuel production was estimated to be 8 to 24 fold greater than the best terrestrial biofuel production systems. Current estimates of the potential productivity for algal biofuel production range from 2,000 to 10,000 gallons/acre.
  • microalgae yield "30 times more energy per acre than land crops such as soybeans.”
  • Auxenochlorella protothecoides previously known in the literature as Chlorella protothecoides, is a preferred species of algae to use in the production of biofuels because it has a "relatively high specific growth rate" which can help achieve "high productivity and yield of biomass and metabolites.”
  • Auxenochlorella protothecoides is particularly desirable in the biofuel context because it can accumulate large quantities of lipids when grown under certain conditions.
  • Auxenochlorella protothecoides is also widely used in biotechnology, aquaculture feeds, human food supplements, and pharmaceuticals.
  • Green algae have been variously defined through the ages and it is prudent to describe the green algae as belonging to the phylum Chlorophyta to which this invention could apply.
  • the organism currently being described as Axenochlorella protothecoides is considered to be the same organism as previously described as Chorella protothecoides.
  • the class Trebouxiophyceae shall be defined as set forth by Volker A.R. Huss et al., in Biochemical Taxonomy and Molecular Phylogeny of the Genus Chlorella Sensu Lato (Chlorophyta), Journal of Phycolology, volume 35, pages 587- 589 (1999).
  • SEQ ID NO: sequence identifier number
  • compositions, systems, and methods disclosed herein improve the process of producing biofuels from Auxenochlorella protothecoides. This is achieved by identification and manipulation of various gene sequences in Auxenochlorella protothecoides.
  • an embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Chlorophyll a oxygenase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Sedoheptulose-1 ,7-bisphosphatase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding GrpE.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding alternative oxidase 1A.
  • Yet another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carboxyl-terminal processing protease.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Grana-Deficient Chloroplastl .
  • Another embodiment of the present application provides the following promoters and terminators of nuclear encoded genes for green microalgae species: HUP2, RPL40, Rbcs2, GDC1 , PsaD, EF1 a, Actin, and CYC6.
  • Yet another embodiment of the present application provides the following 5' UTR untranslated leader elements of organelle encoded genes in green microalgae: atpB, atpH, atpl, tufA, rps4, rps12, Nad2, Cox1 , and rps2.
  • Another embodiment of the present application provides the following 3' UTR untranslated leader elements of organelle encoded genes in green microalgae: atpl , atpE, atpB, rps12, rps4, rps2, Nad2, and Cox1 .
  • Another embodiment of the present application provides a vector comprising one of the previously mentioned isolated nucleic acid molecules.
  • transgenic Auxenochlorella protothecoides comprising a nucleic acid molecule having an overexpressed sequence of nucleotides encoding, or complementary to, one or more of the following sequences: Sedoheptulose-1 ,7-bisphosphatase, GrpE, alternative oxidase 1A, and carboxyl-terminal processing protease.
  • a transgenic Auxenochlorella protothecoides comprising a nucleic acid molecule having a suppressed sequence of nucleotides encoding, or complementary to, one or more of the following sequences: Chlorophyll a oxygenase and Grana-Deficient Chloroplastl .
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding transketolase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein LHCPII type 1 CAB2B.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein LHCI type III CAB4.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding plastocyanin.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding rubisco activase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carbonic anhydrase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b binding protein CP29.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll antenna size regulatory protein TLA1 .
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP synthase gamma subunit.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding component of cytochrome b6/f complex.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding albino3-like protein.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b-binding protein LCHSR3.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ferredoxin reductase-like protein.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding polyadenylate binding protein (NABI- RRMS).
  • NABI- RRMS polyadenylate binding protein
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ribulose-1 ,5-biphosphate carboxylase/oxygenase small unit.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding protein gradient regulation 5.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding phototropin, blue light receptor.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding photosystem I protein PsaD.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chloroplast photosystem II associated 22 kDa protein PsbS.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase beta subunit.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase alpha subunit.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carotenoid epsilon-ring hydroxylase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding lycopene epsilon cyclase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding cytochrome P450.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding H(+)/hexose cotransporter 1 (HUP1 ).
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol cyclase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol O-methyltransferase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding gamma tocopherol methyltransferase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP-NAD kinase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose pyrophosphorylase large subunit (STA1 ).
  • STA1 ADP-glucose pyrophosphorylase large subunit
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose pyrophosphorylase small subunit (STA6).
  • STA6 ADP-glucose pyrophosphorylase small subunit
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding pyruvate decarboxylase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p-hydroxyphenylpyruvate dioxygenase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding heat shock protein A (HSP70A).
  • HSP70A heat shock protein A
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding zeaxanthin epoxidase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding violaxanthin de-epoxidase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chaperone protein dnaJ-related.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrogen regulatory protein Pll.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding argininosuccinate lyase ARG7.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrate reductase.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding target of rapamycin kinase (TOR).
  • TOR rapamycin kinase
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding 60S ribosomal protein L402.
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p53 induced protein 8 (PIG8).
  • PAG8 p53 induced protein 8
  • Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding dicer.
  • Figure 1 Sample BLAST Output Comparing a Hypothetical Protein of the Indica Group Subspecies of Oryza sativa to the Closest Homologous Gene Hit in Auxenochlorella protothecoides.
  • FIG. 2 BLAST Output Comparing Chlorophyll a oxygenase (CAO) in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
  • Figure 3 BLAST Output Comparing SEBP1 in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
  • Figure 5 BLAST Output Comparing AOX1A in Arabidopsis thaliana and Auxenochlorella protothecoides
  • Figure 6 BLAST Output Comparing CtpA Amino Acid Sequence in Arabidopsis thaliana and Auxenochlorella protothecoides
  • Figure 7 BLAST Output Comparing Grana-Deficient Chloroplastl Amino Acid Sequence in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
  • Figure 8 Vector Comprising HUP2 Promoter
  • Figure 9 Gene Construct to Induce RNA; Silencing of the CAO Gene in Auxenochlorella protothecoides
  • Figure 11 a & b Auxenochlorella protothecoides Nuclear Codon Preference Table
  • Figure 12 Auxenochlorella protothecoides Chloroplast Codon Preference Table
  • Figure 13 Auxenochlorella protothecoides Mitochonarial Codon Preference Table
  • Figure 14 Graphic Example of Gene Arrangement and Suspected Operon including the rps19 and rps3 Genes
  • Figure 16A-H Chloroplast and Mitochondrial genome contig sequences Table of Open Reading Frame Information.
  • the exemplary embodiments of the present application are directed toward enhancing the productivity of Auxenochlorella protothecoides.
  • the first step in obtaining both the Auxenochlorella protothecoides genome and transcriptome was to grow Auxenochlorella protothecoides cultures in 1 -liter flasks. The flasks were exposed to cycles of 12 hours of daylight followed by 12 hours of darkness until they reached a desired density of A750 0.4-0.6 OD (optical density units). All cultures were grown on a stir plate operating at 350 rpm with ambient air pumped into the 1 -liter cultures.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • RNA samples were prepared to obtain the transcriptome of Auxenochlorella protothecoides under different conditions. These six conditions included: heat, cold, high light, low light, salt water, and autotrophic to heterotrophic or HeteroboostTM conditions. (See Sayre & Pereira PCT/US2008/085597).
  • the culture subjected to heat treatment was placed on a shaker platform operating at 225 rpm in a 37°C incubator for 2 hours.
  • the culture exposed to cold treatment was placed on a shaker set at 225 rpm in a 4°C walk-in cold room for 2 hours.
  • the culture exposed to high light treatment was grown with illumination to simulate outdoor sunlight at noon.
  • the culture exposed to low light treatment was grown in the dark.
  • the saltwater culture was exposed to salt treatment by adding 10 ml of a 3M NaCI stock solution to 90 ml of algae culture for a final NaCI concentration of 300 mM NaCI.
  • the culture was then placed on a shaker platform operating at 225 rpm for 2 hours.
  • HeteroboostTM culture 1 ml of a 2M glucose stock solution was added to 99 ml of algae culture. The flask containing this particular culture was then wrapped in aluminum foil and placed on a shaker platform operating at 225 rpm for 24 hours. Samples of the HeteroboostTM culture were taken at 8 and 24 hours.
  • RNA samples were bead beat for 160 seconds with 1 ml_ Trizol (Invitrogen, Carlsbad, CA) and 300 mg RNase free glass beads. The Trizol extraction was completed and the RNA was purified using an RNeasy kit (QIAGEN, Valencia, CA), a RNA purification kit. The sample was then treated twice with deoxyribonuclease or DNAse. Approximately 20-25 g of transcriptomic total RNA samples were submitted to Cofactor.
  • RNA samples Cofactor pooled the RNA samples at equal proportions before using them to make copy DNA or cDNA libraries. Although normalizing the RNA samples sacrificed data on the relative expression of the genes, it increased the ability to obtain information on more genes.
  • the whole transcriptome RNA was extracted from total RNA by removing large and small ribosomal RNA (rRNA) using the RiboMinus Plant Eukaryote Kit (Invitrogen), a Kit for depleting ribosomal RNA. A total of five micrograms of total RNA was hybridized to rRNA-specific biotin labeled probes at 70°C degree for 5 minutes. The rRNA-probe complexes were then removed by streptavid in-coated magnetic beads. The rRNA free transcriptome RNA was concentrated using ethanol precipitation.
  • transcriptome RNA was then fragmented by incubation with fragmentation buffer included in the lllumina RNA-seq kit (lllumina, San Diego, CA) for 5 minutes at 94°C. Fragmented RNA was purified with ethanol precipitation. First stranded cDNA was prepared by priming the fragmented RNA using random hexamer and followed by reverse transcription using Superscript II, a DNA polymerase (Invitrogen).
  • the second stranded cDNA was synthesized by first incubating with a second stranded buffer, RNaseOUT, a solution of chemicals that retard RNAse activity and deoxynucleotide triphosphates (dNTPs) included in the lllumina RNA-seq kit on ice for 5 minutes. The reaction mix was then treated with DNA Polyermase I and RNase H at 16°C for 2.5 hours (Invitrogen).
  • dNTPs deoxynucleotide triphosphates
  • Cluster generation and the following sequencing were performed according to the cluster generation manual and sequencing manual from lllumina (Cluster Station User Guide and Genome Analyzer Operations Guide).
  • the RNA sequences were clustered into sufficiently long contigs to allow for thorough mapping to the genomic sequence and produce an informative annotation for the RNA data set.
  • After pooling the RNA Cofactor reverse transcribed it to make cDNA using random hexamers. This cDNA was then broken into pieces to increase the effectiveness of sequencing.
  • Double-stranded cDNA was treated with a mix of T4 DNA polymerase, Klenow large fragment, and T4 polynucleotide kinase to create blunt-ended DNA. Subsequently, a single adenine (A) base was added at the 3' end using a Klenow fragment (with 3' to 5' exonuclease activity) and dATP. The A-tailed DNA was ligated with paired end adaptors using T4-DNA ligase provided by the lllumina RNA-seq kit. Size selection of adaptor-ligated DNA was performed by cutting the target fragment out of a 4-12% acrylamide gel. The amplified DNA library with ideal fragment size (-250 to 300 base pair) was obtained by in-gel PCR using the Phusion High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA).
  • Sequence data was generated using the lllumina platform and sequence data files were compiled. The data was in a FASTA format that is compatible with most, if not all nucleic sequence analysis software. Geneious, a bioinformatics software package, (Biomatters Ltd., Auckland, New Zealand) was used to analyze sequence data for comparison with currently annotated sequences.
  • This application consists of numerous gene sequences encoded in the Auxenochlorella protothecoides genome that can be used to improve its productivity by altering gene expression patterns in the alga and creating valuable traits for lowering the cost of producing algal biomass. These sequences can be ectopically expressed in algae to increase the titer of the gene product to result in more gene activity (e.g., heterologous constitutive expression of a transgene which is normally only expressed conditionally). Conversely, the sequences can be used to repress expression of the endogenous genes (e.g., through the use of RNAi gene expression knockdown technology).
  • genes included in this application can be grouped into the following seven functional categories that are involved in these cellular processes: photosynthesis efficiency, small molecule metabolism, biotic and abiotic stress response, nutrient utilization efficiency, cell division/proliferation, programmed cell death, and nucleic acid metabolism.
  • the sequences for all of the gene sequences are found at SEQ ID Nos. 1 -83 after the claims in this application.
  • any gene function can have an impact in more than once cellular process, the following twenty four genes can be primarily grouped into the photosynthetic efficiency category: chlorophyll a oxygenase, sedoheptulose-1 ,7- bisphosphatase, C-terminal processing protease, grana-deficient chloroplast I transketolase, light harvesting complex protein LHCPII type I CAB2B, light harvesting complex protein LHCI type lll(CAB4), plastocyanin, rubisco activase, carbonic anhydrase, chlorophyll ab binding protein CP29, chlorophyll antenna size regulatory protein TLA1 , ATP synthase gamma subunit, component of cytochrome b6/f complex, albino3-like protein, chlorophyll a/b-binding protein LCHSR3, ferredoxin reductase-like protein, polyadenylate binding protein (NAB1 -RRMS), ribu
  • any of these sequences alone or in combination can be used to enhance photosynthetic efficiency to increase biomass production of cultivated genetically modified algae for the production of oil, biodiesel, and any additional algal bioproducts.
  • One example of a mode of action to effect such an outcome is the reduction of photosynthetic antennae capacity per cell to enhance distribution of incident light energy amongst a higher percentage of the cultivated algal cells. Sequences for these genes can be found at SEQ ID Nos. 1 , 2, 5, 6, and 40 through 58, respectively.
  • tryptophan synthase beta subunit tryptophan synthase alpha subunit, carotenoid epsilon-ring hydroxylase, lycopene epsilon cyclase, cytochrome P450, H(+)/hexose cotransporter 1 (HUP1 ), tocopherol cyclase, tocopherol O-methyltransferase, gamma tocopherol methyltransferase, ATP-NAD kinase, ADP-glucose pyrophosphorylase large subunit (STA1 ), ADP-glucose pyrophosphorylase small subunit (STA6), and pyruvate decarboxylase.
  • tryptophan synthase beta subunit tryptophan synthase alpha subunit
  • carotenoid epsilon-ring hydroxylase carotenoid epsilon-ring hydroxylase
  • any of these sequences alone or in combination can be used to drive the biosynthesis of a particular small molecule in genetically modified algae. Conversely, any of these sequences can be used to cause a paucity of a particular small molecule in genetically modified algae.
  • One example of an application of this approach to add value to cultivated algal biomass is the use of genes encoding enzymes of the tocopherol (vitamin E) biosynthetic pathway to drive accumulation of tocopherol in algae.
  • Such a strategy may also utilize conditional gene promoters and terminator elements to delay production of tocopherol (or other target small molecules) during proliferation of biomass and to direct its accumulation when the algal culture is at an optimum cell density. Sequences for these genes can be found at SEQ ID Nos. 59 through 71 , respectively.
  • the following seven genes can be primarily grouped into a category of biotic and abiotic stress response: molecular chaperone of the GrpE family, alternative oxidase, p-hydroxyphenylpyruvate dioxygenase, heat shock protein A (HSP70A), zeaxanthin epoxidase, violaxanthin de-epoxidase, and chaperone protein dnaJ-related. Any of these sequences alone or in combination can be used to confer stress tolerance in genetically modified algae.
  • One example of engineering stress tolerance to create a value-added algal strain is to ectopically overexpress an enzyme or protein with antioxidant properties to absorb or detoxify excess generation of reactive oxygen molecules (such as hydrogen peroxide, hydroxyl radicals or superoxide radicals) that are created in biological systems as a result of biotic or abiotic stressors such as temperature stress, light stress, osmotic/salinity stress or attack by pathogens and predators.
  • Successful strategies will result in modified algal strains that can perform better than parental control strains during exposure to such stressors, especially in outdoor culture conditions. Sequences for these genes can be found at SEQ ID Nos. 3, 4, and 72 through 76, respectively.
  • the following three genes can be primarily grouped into a category of nutrient utilization efficiency: nitrogen regulatory protein Pll, argininosuccinate lyase ARG7, and nitrate reductase. Any of these sequences alone or in combination can be used to create genetically modified algae that can perform better than parental control lines when cultivated with fewer nutrients. This can result in engineering valuable input traits into algal lines, allowing production of algal biomass at considerably reduced costs. It is analogous to creating a genetically modified crop plant that requires considerably less fertilizer.
  • One example of engineering enhanced nutrient utilization efficiency is to ectopically overexpress argininosuccinate lyase in genetically modified algae to enhance its ability to scavenge nitrogen in the form of the amino acid arginine and reduce its dependency on added nitrogen as a nutrient. Sequences for these genes can be found at SEQ ID Nos. 77 through 79, respectively.
  • the following two genes can be primarily grouped into a category of cell division or proliferation enhancement: target of rapamycin kinase (TOR) and 60S ribosomal protein L402.
  • TOR target of rapamycin kinase
  • 60S ribosomal protein L402. These sequences alone or in combination can be used to drive proliferation in genetically modified algae to enhance growth rates leading to higher cell densities and greater yields of algal biomass.
  • One example of engineering enhanced proliferation in modified algae is the ectopic expression of the TOR gene, which has been shown to be necessary for mitogenesis by driving the cell cycle in yeast (Saccharomyces cerevisiae). Sequences for these genes can be found at SEQ ID Nos. 80 and 81 , respectively.
  • the p53 induced protein 8 (PIG8) gene (SEQ ID No. 82) can be grouped into a category of programmed cell death (apoptosis).
  • This gene can be used to drive programmed cell death in genetically modified algae under the control of a tightly regulated, inducible promoter.
  • Such a mechanism can be applied to control viability of modified algae and can be established as a trait for biocontainment of genetically modified algae in outdoor production ponds.
  • modified algal cells can only live in the intentional confines of an algal cultivation facility. Successful biocontainment traits will enable the commercial production of algal biomass in outdoor growth facilities.
  • the dicer gene regulates nucleic acid metabolism.
  • This sequence can be used to either enhance or attenuate RNA metabolism responsible for gene suppression via the mechanism of small inhibitory RNA.
  • a modified version of dicer could be constructed and overexpressed in modified algae to target a particular essential gene product(s) of a predatory organism (such as a rotifer) and generate a high titer of RNAi species specific for that target. Consumption of the modified algae by the predatory organism would be lethal, controlling the levels of the predatory species and adding a valuable trait for the production of algal biomass.
  • a Basic Local Alignment Search Tool (BLAST) analysis was then performed to compare these four reference genomes to Auxenochlorella protothecoides.
  • a BLAST analysis is a bioinformatics algorithm that uses either nucleic or amino acid query sequences in a reference genome to find related sequences in a target genome that has not been annotated.
  • Figure 1 shows a sample BLAST output comparing a hypothetical protein (110), Osl_24195, from the Indica Group subspecies of rice (Oryza sativa) to Auxenochlorella protothecoides (120).
  • the BLAST algorithm attempts to find a sequence from a reference genome in the target genome.
  • the BLAST output includes a comparison of how well the two sequences match (130).
  • the present application consists of numerous sequences in Auxenochlorella protothecoides that have an impact on its productivity. Several of these sequences will be discussed in further detail. The first of these sequences encodes Chlorophyll a oxygenase (CAO).
  • CAO Chlorophyll a oxygenase
  • Chlorophyll b is a photosynthetic pigment that is predominantly bound by the peripheral light-harvesting antenna complexes associated with Photosystem II. Photoacclimation responses that help optimize light harvesting and photosynthetic growth under various environmental conditions involve the modulation of Chlorophyll b levels and light-harvesting antenna size. Photosynthetic productivity under high light conditions can be enhanced by reducing the levels of Chlorophyll b and hence the size of the Photosystem II light harvesting antenna.
  • RNAi inhibitor RNA
  • a complete “gene knockout” can be achieved using a specific gene deletion meganuclease approach.
  • Figure 2 shows the BLAST output of the comparison between Chlorophyll a oxygenase in Chlamydomonas reinhardtii (210) and Auxenochlorella protothecoides (220). The figure also demonstrates the similarity of this sequence in the two different species (230). A sequence for CAO, SEQ ID No:1 , can be found after the claims in this application.
  • SEBP1 Another homolog of interest is SEBP1 , also found in Chlamydomonas reinhardtii.
  • This gene encodes for the enzyme Sedoheptulose-1 , 7-bisphosphatase (SBPase), which functions during the regenerative phase of the Calvin-Benson and catalyzes the dephosphorylation of sedoheptulose-1 ,7-bisphosphate. Reductions in this enzyme decrease CO2 fixation and growth. Conversely, increasing SBPase transcript levels increases light-saturated photosynthesis and biomass. Therefore, the photosynthetic productivity of Auxenochlorella protothecoides may be increased by increasing the levels of SBPase.
  • Figure 3 shows the BLAST output of the comparison between SEBP1 in Chlamydomonas reinhardtii (310) and Auxenochlorella protothecoides (320). The figure also demonstrates the similarity of this sequence in the two different species (330).
  • a sequence for SEBP1 SEQ ID No:2, can be found after the claims in this application.
  • GrpE nucleotide release factor Yet another homolog that can impact the productivity of Auxenochlorella protothecoides is GrpE nucleotide release factor.
  • the homolog of GrpE in Chlamydomonas reinhardtii is induced by heat shock and light. It is also known to have an impact on DNA replication.
  • GrpE type proteins are labeled in the field of cell biology as "chaperonins" as they have the ability to bind to other proteins in a non-covalent manner and help these other proteins to keep a functional three dimensional structure.
  • Biological systems are induced to make heat shock proteins that become much more abundant under heat stress conditions.
  • GrpE is one such heat shock protein.
  • FIG. 4 shows the BLAST output of the comparison between GrpE nucleotide release factor in Chlamydomonas reinhardtii (410) and Auxenochlorella protothecoides (420). The figure also demonstrates the similarity of this sequence in the two different species (430).
  • a sequence for GrpE, SEQ ID No:3, can be found after the claims in this application.
  • Auxenochlorella protothecoides Another homolog that can be manipulated to enhance productivity of Auxenochlorella protothecoides is Alternative Oxidase 1A, or AOX1A.
  • This homolog found in Arabidopsis thaliana, is known to encode an enzyme that can reduce the production of reactive oxygen species. This function can be very useful if engineered into Auxenochlorella protothecoides to enhance cold temperature stress tolerance and high light tolerance. This is true because reactive oxygen species are generated during photosynthesis and respiration to an extent that can be tolerated under normal conditions. However, under cold temperatures and high light conditions, considerably more reactive oxygen species are generated that can have damaging effects to the algal cells, such as peroxidation of membrane lipids and redox damage to nucleic acids and proteins.
  • FIG. 5 shows the BLAST output of the comparison between AOX1A in Arabidopsis thaliana (510) and Auxenochlorella protothecoides (520). The figure also demonstrates the similarity of this sequence in the two different species (530).
  • a sequence for AOX1A, SEQ ID No:4, can be found after the claims in this application.
  • CtpA Carboxyl-terminal processing protease
  • the amino acid sequence of CtpA found in Arabidopsis thaliana, is known to code for a substrate specific proteolytic enzyme responsible for maturation of the D1 protein, a protein necessary for photosynthetic electron transport.
  • the D1 protein turns over very rapidly which requires continuous CtpA proteolytic activity. Therefore, overexpression of CtpA can result in more robust photochemistry as light-driven electron transport through the D1 protein can be supported at a higher rate.
  • Figure 6 shows the BLAST output of the comparison between CtpA in Arabidopsis thaliana (610) and Auxenochlorella protothecoides (620).
  • the discontinuous bar graph in the figure also identifies the patches of sequence that have greatest similarity between the two different CtpA amino acid sequences from these two species (630).
  • a sequence for CtpA, SEQ ID No:5, can be found after the claims in this application.
  • GDC1 Grana- Deficient Chloroplastl
  • FIG. 7 shows the BLAST output of the comparison between GDC1 in Chlamydomonas reinhardtii (710) and Auxenochlorella protothecoides (720).
  • the discontinuous bar graph in the figure also identifies the patches of sequence that have greatest similarity between the two different GDC1 amino acid sequences from these two species (730).
  • a sequence for GDC1 SEQ ID No:6, can be found after the claims in this application.
  • the present invention also includes isolated nucleic acid molecules comprising a sequence of nucleotides encoding, or complementary which hybridize under low stringency conditions to the nucleotide sequences set forth in SEQ ID No:1 through SEQ ID No:6 or to complementary strains thereof.
  • An example of low stringency conditions is exposure to wash buffer containing 0.5X SSC at 65°C.
  • the present application also includes sequences of nucleotides from the Auxenochlorella protothecoides genomes that encode gene expression elements, such as promoters of nuclear genes, terminator sequences of nuclear genes, untranslated leader sequences of organellar genes and untranslated terminators of organellar genes are indispensible tools used to construct gene expression vectors that are able to drive the expression of homologous or heterologous genes in modified strains of Auxenochlorella protothecoides and in some other green microalgae species as well. Promoters of nuclear encoded genes and 5' untranslated leader elements of organelle encoded genes are cis acting elements that can direct the docking of RNA polymerase to initiate transcription.
  • gene expression elements such as promoters of nuclear genes, terminator sequences of nuclear genes, untranslated leader sequences of organellar genes and untranslated terminators of organellar genes are indispensible tools used to construct gene expression vectors that are able to drive the expression of homologous or heterologous genes in modified strains
  • Examples include: the polymerization of ribonucleotide triphosphates into a biologically active RNA molecule such as a messenger RNA that is used to program protein synthesis on ribosomes, a ribosomal RNA that plays a structural role, and RNA cofactors required for correct splicing of message, and other related processes. Promoters may also contribute to gene expression elements, such as constitutive expression, particularly EF1 a and Actin, and to nickel inducibility of heterologous gene expression, particularly CYC6.
  • sequences of the following nuclear encoded promoters, in FASTA format: HUP2, RbcS2, RPL40, GDC1 , PsaD, EF1 a, Actin, and CYC6 can be found in SEQ ID Nos.:7 through 10, 57, and 34 through 36, respectively.
  • sequences of the following 5' UTR untranslated leader elements of chloroplast encoded genes: atpB, atpH, atpl, tufA, rps4, and rps12, can be found in SEQ ID Nos.:1 1 through 16, respectively.
  • Terminators of nuclear encoded genes and 3' untranslated leader elements of organelle encoded genes are cis acting elements that can curtail gene transcription and result in release of RNA polymerase from the genomic DNA, and in copper repressibility. In other words, they define the end of a unit of gene function.
  • Sequences for the following nuclear encoded terminators: HUP2, RbcS2, RPL40, GDC1 , PsaD , EF1 a, Actin, and CYC6, can be found at SEQ ID Nos.:21 through 24, 54, and 37 through 39, respectively.
  • the sequences of the following 3' UTR untranslated leader elements of chloroplast encoded genes: atpl , atpE, atpB, rps12, and rps4 can be found in SEQ ID Nos.:25 through 29 respectively.
  • sequences of the following 3' UTR untranslated leader elements of mitochondrial encoded genes: Atpl, rps2, Nad2, and Cox1 can be found in SEQ ID Nos.:30 through 33, respectively.
  • Selected chloroplast and mitochondrial genome contig sequences may have additional open reading frames inserted in order to "hi-jack" the function of the expression elements, the 5' UTRs and the 3' UTRs, in these organellar genomes.
  • the sequences of the mitrochondrial genome contigs can be found in SEQ ID Nos. 84, 87, 91 , 95 through 100, 102, and 104 through 108, respectively.
  • the sequences of the chloroplast genome contigs can be found in SEQ ID Nos.
  • Figures 16A through 16H provide information as to the insertion of additional open reading frames to "hi-jack" the function of the expressional elements in these organellar genomes.
  • the steps involved in isolating a promoter element and a terminator element and using them to create an expression vector are described below for the example of the HUP2 gene.
  • the cDNA coding sequence for the HUP2 transporter protein can be found in the set of sequences obtained through RNA sequencing. This nucleic acid sequence can then be used to BLAST search the genomic DNA contigs and scaffolds to find nucleotide sequence both upstream and downstream of the RNA coding sequence of the gene.
  • Upstream contigs may be composed of a long enough stretch of sequence to contain the necessary cis-acting sequence elements that make up a functional promoter.
  • a 900 base pair (bp) long genomic DNA fragment (810) was identified upstream of the HUP2 RNA coding sequence.
  • This 900 base pair sequence is then amplified using the polymerase chain reaction with an upstream primer designed from the DNA sequence at the 5' end of the contig and with a downstream primer from the region of the contig sequence at or near the initiator methionine codon.
  • the fragment is sequence verified to ensure that it is the same as expected based on the genomic sequence, or if any changes have been introduced, they are subtle enough to most likely not impact the promoter function.
  • the downstream primer has a Pstl site incorporated such that the 900 base pair promoter fragment can be cloned into a vector to allow the 3' end to be joined to the 5' end of the 700 base pair HUP2 terminator fragment (820) which also contains a Pstl site.
  • This HUP2 terminator fragment is also amplified from Auxenochlorella protothecoides genomic DNA via the polymerase chain reaction, sequence confirmed and cloned into the vector, which can now be opened at the Pstl restriction site and a DNA coding sequence for a protein or functional RNA can be inserted at the Pstl restriction site (830), in the correct sense orientation in relation to the direction of transcription as determined by the directionality of both the promoter and terminator elements, to create a transgene cassette that consists of a promoter immediately upstream of a coding sequence which is immediately upstream of a transcriptional terminator.
  • transgene cassette (promoter plus RNA coding sequence plus terminator) (840) can now be inserted into an algal DNA transformation vector that already contains another transgene cassette containing a selectable marker gene between promoter and terminator elements which enables selection of modified Auxenochlorella protothecoides on media containing the selection agent, such as an antibiotic or an herbicide.
  • Auxenochlorella protothecoides can be improved via molecular engineering to enhance photosynthetic efficiency.
  • a transformation vector for modification of Auxenochlorella protothecoides can be constructed.
  • RNAi inhibitory RNA
  • CAO Chlorophyll a oxygenase
  • Two gene cassettes are created: one for the RNAi gene suppression function (910) and one for providing a selectable marker (selection cassette) to facilitate recovery of correctly modified isolates of Auxenochlorella protothecoides (920).
  • molecular cloning is performed to make a gene cassette using the constitutive Auxenochlorella protothecoides RPL40 promoter sequence element (930) upstream of an inverted repeat sequence composed of portions of the Auxenochlorella protothecoides CAO gene, including portions of the genes naturally occurring intron and portions of the CAO coding gene (940), all followed by the Auxenochlorella protothecoides RPL40 terminator sequence element to control the end of transcription (950).
  • the selection cassette is made using the Auxenochlorella protothecoides light activated psaD promoter (960) driving expression of a fluridone herbicide resistance selectable marker (970) which was created by site-directed mutagenesis of the phytoene desaturase gene PDS1 from Chlamydomonas reinhardtii.
  • the psaD terminator sequence element (980) is positioned downstream of the selectable marker to control the end of transcription.
  • These two gene cassettes can be cloned into a high copy number plasmid DNA molecule to facilitate maintenance of the sequence integrity, confirmation of structure by nucleotide sequencing and vector propagation to make DNA for transformation.
  • the pUC19 vector backbone is used to construct the transformation vector.
  • the vector backbone is removed via restriction enzyme digestion and the resulting DNA fragments are introduced into algal cells via biolistic bombardment, glass bead agitation or other methods.
  • Algal colonies representing the progeny of transformed cells acquiring the expression vector are selected on plates of solid agar media supplemented with the selective agent, in this case fluridone herbicide.
  • Transformed algae cells are isolated and cultured. Upon confirmation that the cells have incorporated the transgene DNA, cultures are tested for phenotypic attributes imparted by the transgenes. In this case, the transgenics will be assessed for decreased Photosystem II light-harvesting antenna size using biophysical measurements such as flash-induced Ch1 fluorescence, increased photosynthetic oxygen evolution rates and increased growth rates under high light intensities.
  • the expression of the antisense constructs targeting CAO and GDC1 would be driven by light-inducible promoters such that gene silencing and the RNAi phenotype would be maximal under high light and minimal under low light. This would result in metabolically engineered algae strains with the ability to self-regulate antenna size depending on the prevailing light intensity, being large at limiting light and smaller under higher light conditions.
  • the Auxenochlorella protothecoides sequence identified could then be used to design oligonucleotide primers that could drive a polymerase chain reaction to generate a complementary DNA (cDNA) molecule from Auxenochlorella protothecoides RNA.
  • cDNA complementary DNA
  • the DNA fragment containing the RbcS2 promoter element is amplified using the polymerase chain reaction to also include a short portion of the sequence at the 5' end of the ctpA coding sequence (1010).
  • fusion PCR a technique termed "fusion PCR” is used to create a DNA segment consisting of both the promoter element and the coding sequence in the correct juxtaposition and including a unique restriction enzyme site at the 3' end of the coding (CtpA) sequence (1020).
  • This fragment can then be inserted into a vector via standard molecular cloning technique and subsequently a DNA fragment containing the RbcS2 terminator element, previously produced via polymerase chain reaction from Auxenochlorella protothecoides genomic DNA and incorporating the same restriction enzyme site at the 5' end, is inserted downstream of the CtpA coding sequence via standard molecular cloning technique (1030).
  • the DNA transformation vector also contains a gene cassette encoding a selectable marker that allows transformed algal cells to be selected when grown on media containing a selection agent, such as an antibiotic or an herbicide.
  • a selection agent such as an antibiotic or an herbicide.
  • the resulting CtpA expression vector could be introduced into Auxenochlorella protothecoides using DNA coated microparticle bombardment to generate engineered versions of this algal strain that express significantly higher levels of the CtpA proteolytic enzyme. This is accomplished by insertion of the transgene cassette (promoter: CtpA coding sequence::terminator) into the nuclear genome.
  • the nucleotide sequence encoding the Gaussia princeps luciferase enzyme is taken from The National Center for Biotechnology Information (NCBI) and the deduced amino acid sequence is used to design a synthetic gene using the Auxenochlorella protothecoides nuclear codon preference table ( Figure 11 a & b.). Figure 11 a & b were made using 10,779 codons.
  • Table 1 the 185 residue deduced amino acid sequence is listed from top to bottom in the left most column using the one letter amino acid code with a translational stop signal following the last amino acid residue ("STOP").
  • STOP last amino acid residue
  • a synthetic gene can then be made via a vendor such as Integrated DNA Technologies (http://www.idtdna.com/Home/Home.aspx) or GeneWiz (http://www.genewiz.com/), by providing them the nucleotide sequence data and paying a fee.
  • the synthetic gene can be made to include unique restriction enzyme sites, such as GAATTC for EcoRI and AGATCT for Bglll (http://rebase.neb.com/rebase/rebase.html) , at either end to facilitate restriction digestion and ligation into a nuclear expression vector.
  • the coding sequence with restriction enzyme sites at either side can be ligated downstream of the PsaD promoter and upstream of the PsaD terminator, both from the Auxenochlorella protothecoides genome to create a transgene cassette capable of expression.
  • This nuclear expression vector can then be introduced into Auxenochlorella protothecoides cells, along with a herbicide resistance gene cassette or a antibiotic resistance gene cassette, in a co- transformation experiment, using a particle bombardment (gene gun) procedure.
  • fluridone can be used as the selection agent to propagate cells that have acquired the synthetic Gaussia princeps luciferase transgene. Presence of the transgene can be verified by polymerase chain reaction using oligonucleotide primers specific for the Gaussia princeps luciferase transgene to detect the gene sequence directly. Alternatively, the function of the luciferase transgene can be verified using a luminescence assay with a specific substrate for the Gaussia princeps luciferase and a 96 well luminescence plate reader. The coding sequence for the A. protothecoides optimized Gaussia princeps luciferase nucleotide sequence is shown in SEQ ID No. 110.
  • Table 1 Coding Sequence Design Table Gaussia princeps luciferase amino acid sequence for G. princeps luciferase
  • Efficient protein expression of a transgene within the mitochondria or chloroplast of any given organism is dependent on the bias in utilization of specific codons and availability of specific tRNAs to translate any given gene.
  • the most efficient systems utilize a codon preference that matches that of the naturally occurring genes of the target genome.
  • Figures 12 and 13 are derived from the identified coding regions of the near complete genomic sequences of Auxenochlorella protothecoides chloroplast and mitochondria, respectively. Column one on the left identifies all specific codons grouped by their corresponding amino acid residue in column 2 as identified by their conventional single letter designation. Column three identifies the uses of a specific codon among all uses for its given amino acid.
  • Column 4 identifies the frequency of use of any given codon within the pool of selected protein encoding sequences. Codons with higher frequency in use or higher percent use for any given amino acid are considered preferred codons.
  • alanine (A) usage in the chloroplast suggests a codon preference in the following order: GCT > GCA >> GCC > GCG.
  • cysteine (C) usage suggests no preference between TGC and TGT. Alteration of a target genes nucleotide sequence to accommodate for this given codon usage preference will then permit efficient translation of expressed mRNAs.
  • nucleotide modification process is exemplified by efforts to express a selectable marker gene conferring resistance to the protein synthesis inhibitor G418.
  • the KanMX gene encodes an enzyme with a phosphotransferase activity that neutralizes several antibiotics including the protein synthesis inhibitor G418.
  • Auxenochlorella protothecoides is naturally susceptible to G418 at very low concentrations (>10 ug/ml).
  • Optimization of codon usage within the KanMX gene will permit efficient translation of its transcript upon expression in one of the Auxenochlorella protothecoides organellar genomes and confer resistance to G418.
  • For chloroplast expression this is achieved using the codon usage table in Figure 12 and the KanMX amino acid sequence derived from pUG30 (Genbank accession number AF298781 ).
  • the nucleotide sequence for KanMX was previously optimized for expression in yeast and is shown in SEQ ID No. 111 . This sequence encodes the amino acid sequence shown in SEQ ID No. 112.
  • nucleotide sequence was manually edited to a nucleotide sequence which upon transcription will be translated into the same amino acid sequence as expressed from pUG30.
  • the nucleotide and amino acid sequences can be found at SEQ ID Nos. 113 and 114, respectively.
  • transgenes in the chloroplast or mitochondria of green algae can enhance any one of various attributes related to biofuels production. This includes resistance to abiotic or biotic stress, enhanced carbon fixation, improved photosynthetic rates, improved efficiency in electron transfer events, or others. To do this generally requires a target gene or genes of interest, a selectable marker gene, promoters and terminators for both of these genes to drive their expression and two regions of sequence homologous to the host genome which permits homologous recombination.
  • An operon is a region of genomic DNA that encodes more than one gene expressed as a single mRNA transcript.
  • the ribosomal complex binds to the 5'UTR and initiates translation of the first coding region. Processing continues toward the 3' end of the transcript terminating translation at stop codons and reinitiating expression at the next methionine codon until all encoded proteins are expressed. When the complex reaches the end of the mRNA it detaches from the mRNA. Secondary structures and post transcriptional modification, regulate the processivity of the operon as well as the relative abundance of proteins derived from each gene. The utilization of operons permits higher gene density in small genomes and coordinated expression of genes with related function. The use of operons is common to many bacterial species and some organelles specifically including the mitochondria and chloroplasts of green algae.
  • the target gene KanAp has been codon optimized as described in Example 4 for efficient translation in the chloroplast of Auxenochlorella protothecoides.
  • the KanAp gene also serves as a selective marker as it confers resistance to the antibiotic G418.
  • the rps19-rps3 (rps193) operon was modified to include a coding region for KanAp between the two genes. This reduces the need for promoter and terminator elements for expression of the target gene.
  • the codon optimized KanAp gene was inserted between the rps19 termination codon and the rps3 initiation codon.
  • the intergenic region was modified to include addition restriction sites (EcoRI and EcoRV) flanking the KanAp gene to permit downstream modification if necessary.
  • the final construct has the sequence as shown in SEQ ID No. 42. This sequence is represented graphically as shown in Figure 15. Biolistic transformation with this linearized product yielded transformant colonies of Auxenochlorella protothecoides selected on 10ug/ml G418 grown under photoautotrophic conditions. The presence of the KanAp gene was confirnned by colony PCR.

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Abstract

The present application is comprised of various genes in Auxenochlorella (Chlorella) protothecoides that can be used to enhance productivity of the algae species. For example, these genes have an impact on photosynthetic efficiency, tolerance to environmental stress, and biomass yield. The present application also includes promoters and terminators which can be used to control gene expression in Auxenochlorella protothecoides.

Description

CHLOROPHYTE GENES FOR THE OPTIMIZATION OF PRODUCTIVITY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 61/547,416, filed October 14, 2011 .
TECHNICAL FIELD
[0002] The disclosed embodiments of the present application are in the field of algal biomass and biofuel production.
BACKGROUND
[0003] Biofuels will play an increasing role in the United States energy market as energy prices increase, political emphasis on establishing national energy independence intensifies and apprehension about climate change continues to grow. The price of petroleum has fluctuated dramatically, reaching record highs of more than US$140 per barrel in 2008. In part, those price increases reflected economic, political, and supply chain uncertainties. Political concerns about the availability of petroleum supplies have led to the realization that the United States' energy independence is of critical strategic importance, both economically and militarily. The release of CO2 from fossil fuel combustion may also substantially contribute to global warming, intensifying efforts to develop biofuels. The United States has responded by issuing a renewable fuel standard update (RFS2) that encourages a shift to more advanced biofuels in the market. For example, RFS2 requires 36 million gallons of renewable fuel to be blended into transportation fuel by 2022. Additionally, many states have responded by enacting their own renewable portfolio standards mandating electricity providers to obtain a certain percentage of their power from renewable energy sources. As a result of these concerns and RPS requirements, domestically produced biofuels have become an increasingly attractive alternative to foreign fossil fuels.
[0004] Microalgae are some of the most productive and therefore desirable sources of biofuels. The Department of Energy (DOE) has determined that biofuel yield per acre from microalgal culture exceeds that of many competing organisms and land crops. Between the late 1970s and 1990s, the DOE's National Renewable Energy Laboratory (NREL) evaluated the economic feasibility of producing biofuels from a variety of aquatic and terrestrial photosynthetic organisms. Biofuel production from microalgae was determined to have the greatest yield per acre potential of any of the organisms screened. Microalgal biofuel production was estimated to be 8 to 24 fold greater than the best terrestrial biofuel production systems. Current estimates of the potential productivity for algal biofuel production range from 2,000 to 10,000 gallons/acre. According to the DOE, microalgae yield "30 times more energy per acre than land crops such as soybeans." Although existing technologies are promising, there is still a need for systems and methods that create even greater efficiencies in biofuel production from microalgae to meet economic targets needed for successful commercialization.
[0005] Auxenochlorella protothecoides, previously known in the literature as Chlorella protothecoides, is a preferred species of algae to use in the production of biofuels because it has a "relatively high specific growth rate" which can help achieve "high productivity and yield of biomass and metabolites." Auxenochlorella protothecoides is particularly desirable in the biofuel context because it can accumulate large quantities of lipids when grown under certain conditions. Auxenochlorella protothecoides is also widely used in biotechnology, aquaculture feeds, human food supplements, and pharmaceuticals.
[0006] Further optimization of biofuel production from Auxenochlorella protothecoides can be achieved through genetic modification. Numerous genes have been identified in other species which convey various desirable characteristics. For example, some genes may control traits for enhanced abiotic stress tolerance, broad spectrum utilization of reduced carbon feedstocks, enhanced photosynthetic efficiency, reduced dependence upon nutrients, and facilitated harvesting and concentrating algal cells in culture. The sequences of these genes in Auxenochlorella protothecoides, however, have been unknown until now. Identifying these genes allows one to target them and modify their expression in order to enhance biofuel production. DEFINITIONS
[0007] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary embodiments, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0008] Green algae have been variously defined through the ages and it is prudent to describe the green algae as belonging to the phylum Chlorophyta to which this invention could apply. In the current application, the organism currently being described as Axenochlorella protothecoides is considered to be the same organism as previously described as Chorella protothecoides. As referenced in this application, the class Trebouxiophyceae shall be defined as set forth by Volker A.R. Huss et al., in Biochemical Taxonomy and Molecular Phylogeny of the Genus Chlorella Sensu Lato (Chlorophyta), Journal of Phycolology, volume 35, pages 587- 589 (1999).
[0009] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the application as a whole. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably. Furthermore, as used in the description of the application and the appended claims, the singular forms "a", "an", and "the" are inclusive of their plural forms, unless contraindicated by the context surrounding such. The singular "alga" is likewise intended to be inclusive of the plural "algae."
[0010] Nucleotide and amino acid sequences are referred to by sequence identifier number (SEQ ID NO:). The SEQ ID NOs: are listed after the claims in this application.
SUMMARY OF THE APPLICATION [0011] Exemplary embodiments of the compositions, systems, and methods disclosed herein improve the process of producing biofuels from Auxenochlorella protothecoides. This is achieved by identification and manipulation of various gene sequences in Auxenochlorella protothecoides.
[0012] In one aspect, an embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Chlorophyll a oxygenase.
[0013] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Sedoheptulose-1 ,7-bisphosphatase.
[0014] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding GrpE.
[0015] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding alternative oxidase 1A.
[0016] Yet another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carboxyl-terminal processing protease.
[0017] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Grana-Deficient Chloroplastl .
[0018] Another embodiment of the present application provides the following promoters and terminators of nuclear encoded genes for green microalgae species: HUP2, RPL40, Rbcs2, GDC1 , PsaD, EF1 a, Actin, and CYC6.
[0019] Yet another embodiment of the present application provides the following 5' UTR untranslated leader elements of organelle encoded genes in green microalgae: atpB, atpH, atpl, tufA, rps4, rps12, Nad2, Cox1 , and rps2.
[0020] Another embodiment of the present application provides the following 3' UTR untranslated leader elements of organelle encoded genes in green microalgae: atpl , atpE, atpB, rps12, rps4, rps2, Nad2, and Cox1 . [0021] Another embodiment of the present application provides a vector comprising one of the previously mentioned isolated nucleic acid molecules.
[0022] Another embodiment of the present application is a transgenic Auxenochlorella protothecoides comprising a nucleic acid molecule having an overexpressed sequence of nucleotides encoding, or complementary to, one or more of the following sequences: Sedoheptulose-1 ,7-bisphosphatase, GrpE, alternative oxidase 1A, and carboxyl-terminal processing protease.
[0023] Another embodiment of the present application A transgenic Auxenochlorella protothecoides comprising a nucleic acid molecule having a suppressed sequence of nucleotides encoding, or complementary to, one or more of the following sequences: Chlorophyll a oxygenase and Grana-Deficient Chloroplastl .
[0024] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding transketolase.
[0025] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein LHCPII type 1 CAB2B.
[0026] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein LHCI type III CAB4.
[0027] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding plastocyanin.
[0028] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding rubisco activase.
[0029] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carbonic anhydrase.
[0030] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b binding protein CP29.
[0031] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll antenna size regulatory protein TLA1 .
[0032] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP synthase gamma subunit.
[0033] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding component of cytochrome b6/f complex.
[0034] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding albino3-like protein.
[0035] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b-binding protein LCHSR3.
[0036] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ferredoxin reductase-like protein.
[0037] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding polyadenylate binding protein (NABI- RRMS).
[0038] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ribulose-1 ,5-biphosphate carboxylase/oxygenase small unit. [0039] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding protein gradient regulation 5.
[0040] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding phototropin, blue light receptor.
[0041] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding photosystem I protein PsaD.
[0042] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chloroplast photosystem II associated 22 kDa protein PsbS.
[0043] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase beta subunit.
[0044] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase alpha subunit.
[0045] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carotenoid epsilon-ring hydroxylase.
[0046] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding lycopene epsilon cyclase.
[0047] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding cytochrome P450.
[0048] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding H(+)/hexose cotransporter 1 (HUP1 ). [0049] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol cyclase.
[0050] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol O-methyltransferase.
[0051] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding gamma tocopherol methyltransferase.
[0052] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP-NAD kinase.
[0053] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose pyrophosphorylase large subunit (STA1 ).
[0054] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose pyrophosphorylase small subunit (STA6).
[0055] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding pyruvate decarboxylase.
[0056] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p-hydroxyphenylpyruvate dioxygenase.
[0057] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding heat shock protein A (HSP70A).
[0058] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding zeaxanthin epoxidase.
[0059] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding violaxanthin de-epoxidase.
[0060] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chaperone protein dnaJ-related.
[0061] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrogen regulatory protein Pll.
[0062] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding argininosuccinate lyase ARG7.
[0063] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrate reductase.
[0064] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding target of rapamycin kinase (TOR).
[0065] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding 60S ribosomal protein L402.
[0066] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p53 induced protein 8 (PIG8).
[0067] Another embodiment of the present application provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding dicer. BRIEF DESCRIPTION OF THE DRAWINGS
[0068] A better understanding of the exemplary embodiments of the present application will be had when reference is made to the accompanying drawings, and wherein:
[0069] Figure 1 : Sample BLAST Output Comparing a Hypothetical Protein of the Indica Group Subspecies of Oryza sativa to the Closest Homologous Gene Hit in Auxenochlorella protothecoides.
[0070] Figure 2: BLAST Output Comparing Chlorophyll a oxygenase (CAO) in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
[0071] Figure 3: BLAST Output Comparing SEBP1 in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
[0072] Figure 4: BLAST Output Comparing GrpE Nucleotide Release Factor in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
[0073] Figure 5: BLAST Output Comparing AOX1A in Arabidopsis thaliana and Auxenochlorella protothecoides
[0074] Figure 6: BLAST Output Comparing CtpA Amino Acid Sequence in Arabidopsis thaliana and Auxenochlorella protothecoides
[0075] Figure 7: BLAST Output Comparing Grana-Deficient Chloroplastl Amino Acid Sequence in Chlamydomonas reinhardtii and Auxenochlorella protothecoides
[0076] Figure 8: Vector Comprising HUP2 Promoter
[0077] Figure 9: Gene Construct to Induce RNA; Silencing of the CAO Gene in Auxenochlorella protothecoides
[0078] Figure 10: Gene Construct to Overexpress Endogenous CtpA in Auxenochlorella protothecoides
[0079] Figure 11 a & b: Auxenochlorella protothecoides Nuclear Codon Preference Table
[0080] Figure 12: Auxenochlorella protothecoides Chloroplast Codon Preference Table [0081] Figure 13: Auxenochlorella protothecoides Mitochonarial Codon Preference Table
[0082] Figure 14: Graphic Example of Gene Arrangement and Suspected Operon including the rps19 and rps3 Genes
[0083] Figure 15: Graphic Example of KanAp Gene
[0084] Figure 16A-H: Chloroplast and Mitochondrial genome contig sequences Table of Open Reading Frame Information.
DETAILED DESCRIPTION
[0085] The exemplary embodiments of the present application are directed toward enhancing the productivity of Auxenochlorella protothecoides. The first step in obtaining both the Auxenochlorella protothecoides genome and transcriptome was to grow Auxenochlorella protothecoides cultures in 1 -liter flasks. The flasks were exposed to cycles of 12 hours of daylight followed by 12 hours of darkness until they reached a desired density of A750 0.4-0.6 OD (optical density units). All cultures were grown on a stir plate operating at 350 rpm with ambient air pumped into the 1 -liter cultures.
[0086] Once the culture reached this desired density, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) samples were prepared. To prepare the DNA samples, a sufficient volume of culture usually 50 ml of culture was centrifuged at 5000 rpm for 10 minutes to obtain a pellet of cells weighing approximately 100 mg. The pellet was flash frozen and processed by breaking open the algal cells with glass beads and purifying the DNA using the Nucleospin II Plant Kit. The genomic DNA samples were then submitted to Cofactor Genomics ("Cofactor") at a concentration of 300 ng/μΙ.
[0087] Six different RNA samples were prepared to obtain the transcriptome of Auxenochlorella protothecoides under different conditions. These six conditions included: heat, cold, high light, low light, salt water, and autotrophic to heterotrophic or Heteroboost™ conditions. (See Sayre & Pereira PCT/US2008/085597).
[0088] The culture subjected to heat treatment was placed on a shaker platform operating at 225 rpm in a 37°C incubator for 2 hours. The culture exposed to cold treatment was placed on a shaker set at 225 rpm in a 4°C walk-in cold room for 2 hours. The culture exposed to high light treatment was grown with illumination to simulate outdoor sunlight at noon. The culture exposed to low light treatment was grown in the dark. The saltwater culture was exposed to salt treatment by adding 10 ml of a 3M NaCI stock solution to 90 ml of algae culture for a final NaCI concentration of 300 mM NaCI. The culture was then placed on a shaker platform operating at 225 rpm for 2 hours. For the Heteroboost™ culture, 1 ml of a 2M glucose stock solution was added to 99 ml of algae culture. The flask containing this particular culture was then wrapped in aluminum foil and placed on a shaker platform operating at 225 rpm for 24 hours. Samples of the Heteroboost™ culture were taken at 8 and 24 hours.
[0089] Three 50 ml volumes of each of the six cultures were centrifuged at 5000 rpm. Supernatant was then removed from each aliquot and the cells, in the form of a pellet at the bottom of the flask, were flash frozen and stored at -80°C.
[0090] All of the RNA samples were bead beat for 160 seconds with 1 ml_ Trizol (Invitrogen, Carlsbad, CA) and 300 mg RNase free glass beads. The Trizol extraction was completed and the RNA was purified using an RNeasy kit (QIAGEN, Valencia, CA), a RNA purification kit. The sample was then treated twice with deoxyribonuclease or DNAse. Approximately 20-25 g of transcriptomic total RNA samples were submitted to Cofactor.
[0091] Cofactor pooled the RNA samples at equal proportions before using them to make copy DNA or cDNA libraries. Although normalizing the RNA samples sacrificed data on the relative expression of the genes, it increased the ability to obtain information on more genes.
[0092] The whole transcriptome RNA was extracted from total RNA by removing large and small ribosomal RNA (rRNA) using the RiboMinus Plant Eukaryote Kit (Invitrogen), a Kit for depleting ribosomal RNA. A total of five micrograms of total RNA was hybridized to rRNA-specific biotin labeled probes at 70°C degree for 5 minutes. The rRNA-probe complexes were then removed by streptavid in-coated magnetic beads. The rRNA free transcriptome RNA was concentrated using ethanol precipitation. [0093] One microgram of transcriptome RNA was then fragmented by incubation with fragmentation buffer included in the lllumina RNA-seq kit (lllumina, San Diego, CA) for 5 minutes at 94°C. Fragmented RNA was purified with ethanol precipitation. First stranded cDNA was prepared by priming the fragmented RNA using random hexamer and followed by reverse transcription using Superscript II, a DNA polymerase (Invitrogen). The second stranded cDNA was synthesized by first incubating with a second stranded buffer, RNaseOUT, a solution of chemicals that retard RNAse activity and deoxynucleotide triphosphates (dNTPs) included in the lllumina RNA-seq kit on ice for 5 minutes. The reaction mix was then treated with DNA Polyermase I and RNase H at 16°C for 2.5 hours (Invitrogen).
[0094] Cluster generation and the following sequencing were performed according to the cluster generation manual and sequencing manual from lllumina (Cluster Station User Guide and Genome Analyzer Operations Guide). The RNA sequences were clustered into sufficiently long contigs to allow for thorough mapping to the genomic sequence and produce an informative annotation for the RNA data set. After pooling the RNA, Cofactor reverse transcribed it to make cDNA using random hexamers. This cDNA was then broken into pieces to increase the effectiveness of sequencing.
[0095] Double-stranded cDNA was treated with a mix of T4 DNA polymerase, Klenow large fragment, and T4 polynucleotide kinase to create blunt-ended DNA. Subsequently, a single adenine (A) base was added at the 3' end using a Klenow fragment (with 3' to 5' exonuclease activity) and dATP. The A-tailed DNA was ligated with paired end adaptors using T4-DNA ligase provided by the lllumina RNA-seq kit. Size selection of adaptor-ligated DNA was performed by cutting the target fragment out of a 4-12% acrylamide gel. The amplified DNA library with ideal fragment size (-250 to 300 base pair) was obtained by in-gel PCR using the Phusion High-Fidelity PCR Kit (New England Biolabs, Ipswich, MA).
[0096] Sequence data was generated using the lllumina platform and sequence data files were compiled. The data was in a FASTA format that is compatible with most, if not all nucleic sequence analysis software. Geneious, a bioinformatics software package, (Biomatters Ltd., Auckland, New Zealand) was used to analyze sequence data for comparison with currently annotated sequences.
[0097] To identify sequences of interest, homologs were identified in four reference genomes. These four genomes were selected because they are both well annotated and had the potential to be similar to Auxenochlorella protothecoides. These four reference genomes are: (1 ) Arabidopsis thaliana (a model system flowering plant), (2) Chlorella variabilis NC64A, (the model system Chlorella strain used for modeling Chlorella/Chlorella virus interactions), (3) Chlamydomonas reinhardtii (a model system green microalga), and (4) Saccharomyces cerevisiae (Baker's Yeast, the classical model system for eukaryotic genetics).
[0098] This application consists of numerous gene sequences encoded in the Auxenochlorella protothecoides genome that can be used to improve its productivity by altering gene expression patterns in the alga and creating valuable traits for lowering the cost of producing algal biomass. These sequences can be ectopically expressed in algae to increase the titer of the gene product to result in more gene activity (e.g., heterologous constitutive expression of a transgene which is normally only expressed conditionally). Conversely, the sequences can be used to repress expression of the endogenous genes (e.g., through the use of RNAi gene expression knockdown technology). The genes included in this application can be grouped into the following seven functional categories that are involved in these cellular processes: photosynthesis efficiency, small molecule metabolism, biotic and abiotic stress response, nutrient utilization efficiency, cell division/proliferation, programmed cell death, and nucleic acid metabolism. The sequences for all of the gene sequences are found at SEQ ID Nos. 1 -83 after the claims in this application.
[0099] Although any gene function can have an impact in more than once cellular process, the following twenty four genes can be primarily grouped into the photosynthetic efficiency category: chlorophyll a oxygenase, sedoheptulose-1 ,7- bisphosphatase, C-terminal processing protease, grana-deficient chloroplast I transketolase, light harvesting complex protein LHCPII type I CAB2B, light harvesting complex protein LHCI type lll(CAB4), plastocyanin, rubisco activase, carbonic anhydrase, chlorophyll ab binding protein CP29, chlorophyll antenna size regulatory protein TLA1 , ATP synthase gamma subunit, component of cytochrome b6/f complex, albino3-like protein, chlorophyll a/b-binding protein LCHSR3, ferredoxin reductase-like protein, polyadenylate binding protein (NAB1 -RRMS), ribulose-1 ,5-bisphosphate carboxyase/oxygenase small subunit, proton gradient regulation 5, phototropin, blue light receptor, photosystem I protein PsaD, and chloroplast photosystem II associated 22 kDa protein PsbS. Any of these sequences alone or in combination can be used to enhance photosynthetic efficiency to increase biomass production of cultivated genetically modified algae for the production of oil, biodiesel, and any additional algal bioproducts. One example of a mode of action to effect such an outcome is the reduction of photosynthetic antennae capacity per cell to enhance distribution of incident light energy amongst a higher percentage of the cultivated algal cells. Sequences for these genes can be found at SEQ ID Nos. 1 , 2, 5, 6, and 40 through 58, respectively.
[0100] Similarly, the following twelve genes can be primarily grouped into a category of small molecule metabolism processes: tryptophan synthase beta subunit, tryptophan synthase alpha subunit, carotenoid epsilon-ring hydroxylase, lycopene epsilon cyclase, cytochrome P450, H(+)/hexose cotransporter 1 (HUP1 ), tocopherol cyclase, tocopherol O-methyltransferase, gamma tocopherol methyltransferase, ATP-NAD kinase, ADP-glucose pyrophosphorylase large subunit (STA1 ), ADP-glucose pyrophosphorylase small subunit (STA6), and pyruvate decarboxylase. Any of these sequences alone or in combination can be used to drive the biosynthesis of a particular small molecule in genetically modified algae. Conversely, any of these sequences can be used to cause a paucity of a particular small molecule in genetically modified algae. One example of an application of this approach to add value to cultivated algal biomass is the use of genes encoding enzymes of the tocopherol (vitamin E) biosynthetic pathway to drive accumulation of tocopherol in algae. Such a strategy may also utilize conditional gene promoters and terminator elements to delay production of tocopherol (or other target small molecules) during proliferation of biomass and to direct its accumulation when the algal culture is at an optimum cell density. Sequences for these genes can be found at SEQ ID Nos. 59 through 71 , respectively.
[0101] In a similar manner, the following seven genes can be primarily grouped into a category of biotic and abiotic stress response: molecular chaperone of the GrpE family, alternative oxidase, p-hydroxyphenylpyruvate dioxygenase, heat shock protein A (HSP70A), zeaxanthin epoxidase, violaxanthin de-epoxidase, and chaperone protein dnaJ-related. Any of these sequences alone or in combination can be used to confer stress tolerance in genetically modified algae. One example of engineering stress tolerance to create a value-added algal strain is to ectopically overexpress an enzyme or protein with antioxidant properties to absorb or detoxify excess generation of reactive oxygen molecules (such as hydrogen peroxide, hydroxyl radicals or superoxide radicals) that are created in biological systems as a result of biotic or abiotic stressors such as temperature stress, light stress, osmotic/salinity stress or attack by pathogens and predators. Successful strategies will result in modified algal strains that can perform better than parental control strains during exposure to such stressors, especially in outdoor culture conditions. Sequences for these genes can be found at SEQ ID Nos. 3, 4, and 72 through 76, respectively.
[0102] In a similar manner, the following three genes can be primarily grouped into a category of nutrient utilization efficiency: nitrogen regulatory protein Pll, argininosuccinate lyase ARG7, and nitrate reductase. Any of these sequences alone or in combination can be used to create genetically modified algae that can perform better than parental control lines when cultivated with fewer nutrients. This can result in engineering valuable input traits into algal lines, allowing production of algal biomass at considerably reduced costs. It is analogous to creating a genetically modified crop plant that requires considerably less fertilizer. One example of engineering enhanced nutrient utilization efficiency is to ectopically overexpress argininosuccinate lyase in genetically modified algae to enhance its ability to scavenge nitrogen in the form of the amino acid arginine and reduce its dependency on added nitrogen as a nutrient. Sequences for these genes can be found at SEQ ID Nos. 77 through 79, respectively.
[0103] In a similar manner, the following two genes can be primarily grouped into a category of cell division or proliferation enhancement: target of rapamycin kinase (TOR) and 60S ribosomal protein L402. These sequences alone or in combination can be used to drive proliferation in genetically modified algae to enhance growth rates leading to higher cell densities and greater yields of algal biomass. One example of engineering enhanced proliferation in modified algae is the ectopic expression of the TOR gene, which has been shown to be necessary for mitogenesis by driving the cell cycle in yeast (Saccharomyces cerevisiae). Sequences for these genes can be found at SEQ ID Nos. 80 and 81 , respectively.
[0104] In a similar manner, the p53 induced protein 8 (PIG8) gene (SEQ ID No. 82) can be grouped into a category of programmed cell death (apoptosis). This gene can be used to drive programmed cell death in genetically modified algae under the control of a tightly regulated, inducible promoter. Such a mechanism can be applied to control viability of modified algae and can be established as a trait for biocontainment of genetically modified algae in outdoor production ponds. In this scenario, modified algal cells can only live in the intentional confines of an algal cultivation facility. Successful biocontainment traits will enable the commercial production of algal biomass in outdoor growth facilities.
[0105] In a similar manner, the dicer gene (SEQ ID No. 83) regulates nucleic acid metabolism. This sequence can be used to either enhance or attenuate RNA metabolism responsible for gene suppression via the mechanism of small inhibitory RNA. For example, a modified version of dicer could be constructed and overexpressed in modified algae to target a particular essential gene product(s) of a predatory organism (such as a rotifer) and generate a high titer of RNAi species specific for that target. Consumption of the modified algae by the predatory organism would be lethal, controlling the levels of the predatory species and adding a valuable trait for the production of algal biomass.
[0106] A Basic Local Alignment Search Tool (BLAST) analysis was then performed to compare these four reference genomes to Auxenochlorella protothecoides. A BLAST analysis is a bioinformatics algorithm that uses either nucleic or amino acid query sequences in a reference genome to find related sequences in a target genome that has not been annotated.
[0107] Figure 1 shows a sample BLAST output comparing a hypothetical protein (110), Osl_24195, from the Indica Group subspecies of rice (Oryza sativa) to Auxenochlorella protothecoides (120). The BLAST algorithm attempts to find a sequence from a reference genome in the target genome. The BLAST output includes a comparison of how well the two sequences match (130). [0108] As discussed above, the present application consists of numerous sequences in Auxenochlorella protothecoides that have an impact on its productivity. Several of these sequences will be discussed in further detail. The first of these sequences encodes Chlorophyll a oxygenase (CAO). The homolog of this gene in Chlamydomonas reinhardtii, is known to be involved in chlorophyll b formation. Chlorophyll b is a photosynthetic pigment that is predominantly bound by the peripheral light-harvesting antenna complexes associated with Photosystem II. Photoacclimation responses that help optimize light harvesting and photosynthetic growth under various environmental conditions involve the modulation of Chlorophyll b levels and light-harvesting antenna size. Photosynthetic productivity under high light conditions can be enhanced by reducing the levels of Chlorophyll b and hence the size of the Photosystem II light harvesting antenna. This can be achieved using an RNAi (inhibitory RNA)- mediated "gene knockdown" approach to suppress levels of the endogenous CAO transcript using an antisense or inverted repeat-containing RNA specific to the CAO gene sequence. In addition, a complete "gene knockout" can be achieved using a specific gene deletion meganuclease approach.
[0109] Figure 2 shows the BLAST output of the comparison between Chlorophyll a oxygenase in Chlamydomonas reinhardtii (210) and Auxenochlorella protothecoides (220). The figure also demonstrates the similarity of this sequence in the two different species (230). A sequence for CAO, SEQ ID No:1 , can be found after the claims in this application.
[0110] Another homolog of interest is SEBP1 , also found in Chlamydomonas reinhardtii. This gene encodes for the enzyme Sedoheptulose-1 , 7-bisphosphatase (SBPase), which functions during the regenerative phase of the Calvin-Benson and catalyzes the dephosphorylation of sedoheptulose-1 ,7-bisphosphate. Reductions in this enzyme decrease CO2 fixation and growth. Conversely, increasing SBPase transcript levels increases light-saturated photosynthesis and biomass. Therefore, the photosynthetic productivity of Auxenochlorella protothecoides may be increased by increasing the levels of SBPase. Figure 3 shows the BLAST output of the comparison between SEBP1 in Chlamydomonas reinhardtii (310) and Auxenochlorella protothecoides (320). The figure also demonstrates the similarity of this sequence in the two different species (330). A sequence for SEBP1 , SEQ ID No:2, can be found after the claims in this application.
[0111] Yet another homolog that can impact the productivity of Auxenochlorella protothecoides is GrpE nucleotide release factor. The homolog of GrpE in Chlamydomonas reinhardtii, is induced by heat shock and light. It is also known to have an impact on DNA replication. GrpE type proteins are labeled in the field of cell biology as "chaperonins" as they have the ability to bind to other proteins in a non-covalent manner and help these other proteins to keep a functional three dimensional structure. Biological systems are induced to make heat shock proteins that become much more abundant under heat stress conditions. GrpE is one such heat shock protein. Engineering Auxenochlorella protothecoides to ectopically express GrpE can render the algal cell more heat stress tolerant by way of the additional GrpE protein bestowing this kind of chaperonin-like activity. Some of the cellular processes that are important to protect from heat stress are protein translation, RNA transcription, RNA processing and DNA replication. Having extra chaperonin-like activity present during heat stress can result in much greater survival under extreme heat conditions and can result in much more production of biomass at intermediate heat stress temperatures. Figure 4 shows the BLAST output of the comparison between GrpE nucleotide release factor in Chlamydomonas reinhardtii (410) and Auxenochlorella protothecoides (420). The figure also demonstrates the similarity of this sequence in the two different species (430). A sequence for GrpE, SEQ ID No:3, can be found after the claims in this application.
[0112] Another homolog that can be manipulated to enhance productivity of Auxenochlorella protothecoides is Alternative Oxidase 1A, or AOX1A. This homolog, found in Arabidopsis thaliana, is known to encode an enzyme that can reduce the production of reactive oxygen species. This function can be very useful if engineered into Auxenochlorella protothecoides to enhance cold temperature stress tolerance and high light tolerance. This is true because reactive oxygen species are generated during photosynthesis and respiration to an extent that can be tolerated under normal conditions. However, under cold temperatures and high light conditions, considerably more reactive oxygen species are generated that can have damaging effects to the algal cells, such as peroxidation of membrane lipids and redox damage to nucleic acids and proteins. By ectopically expressing a transgene encoding additional AOX1A and increasing the level of this enzyme activity, engineered algal cells will be protected under these types of stressful conditions and give rise to algal cultures that can produce significantly more biomass in cold temperatures and in high light conditions. Figure 5 shows the BLAST output of the comparison between AOX1A in Arabidopsis thaliana (510) and Auxenochlorella protothecoides (520). The figure also demonstrates the similarity of this sequence in the two different species (530). A sequence for AOX1A, SEQ ID No:4, can be found after the claims in this application.
[0113] Yet another homolog that has characteristics that can affect productivity in Auxenochlorella protothecoides is Carboxyl-terminal processing protease, or CtpA. The amino acid sequence of CtpA found in Arabidopsis thaliana, is known to code for a substrate specific proteolytic enzyme responsible for maturation of the D1 protein, a protein necessary for photosynthetic electron transport. The D1 protein turns over very rapidly which requires continuous CtpA proteolytic activity. Therefore, overexpression of CtpA can result in more robust photochemistry as light-driven electron transport through the D1 protein can be supported at a higher rate. Figure 6 shows the BLAST output of the comparison between CtpA in Arabidopsis thaliana (610) and Auxenochlorella protothecoides (620). The discontinuous bar graph in the figure also identifies the patches of sequence that have greatest similarity between the two different CtpA amino acid sequences from these two species (630). A sequence for CtpA, SEQ ID No:5, can be found after the claims in this application.
[0114] Another Auxenochlorella protothecoides gene homolog that may be targeted for enhancing light utilization and photosynthetic productivity is Grana- Deficient Chloroplastl (GDC1 ). The gene knockout of GDC1 in Arabidopsis thaliana results in plants that are deficient in grana and assemble only stromal thylakoids. These transgenics also have low levels of light-harvesting complex II (LHCII) and a high ratio of chlorophyll a to b, both of which are characteristic high light responses. Further, in Arabidopsis, the GDC1 gene transcript is induced upon illumination. Hence the DGC1 gene homolog in Auxenochlorella protothecoides can be targeted by RNAi or knockout approach as elucidated above for the CAO gene, as a strategy for increasing light utilization, photosynthetic efficiency and biomass yield in Auxenochlorella protothecoides under high light conditions. In addition, the Auxenochlorella protothecoides GDC1 promoter can be employed for the light-dependent expression of heterologious genes. Figure 7 shows the BLAST output of the comparison between GDC1 in Chlamydomonas reinhardtii (710) and Auxenochlorella protothecoides (720). The discontinuous bar graph in the figure also identifies the patches of sequence that have greatest similarity between the two different GDC1 amino acid sequences from these two species (730). A sequence for GDC1 , SEQ ID No:6, can be found after the claims in this application.
[0115] The present invention also includes isolated nucleic acid molecules comprising a sequence of nucleotides encoding, or complementary which hybridize under low stringency conditions to the nucleotide sequences set forth in SEQ ID No:1 through SEQ ID No:6 or to complementary strains thereof. An example of low stringency conditions is exposure to wash buffer containing 0.5X SSC at 65°C.
[0116] The present application also includes sequences of nucleotides from the Auxenochlorella protothecoides genomes that encode gene expression elements, such as promoters of nuclear genes, terminator sequences of nuclear genes, untranslated leader sequences of organellar genes and untranslated terminators of organellar genes are indispensible tools used to construct gene expression vectors that are able to drive the expression of homologous or heterologous genes in modified strains of Auxenochlorella protothecoides and in some other green microalgae species as well. Promoters of nuclear encoded genes and 5' untranslated leader elements of organelle encoded genes are cis acting elements that can direct the docking of RNA polymerase to initiate transcription. Examples include: the polymerization of ribonucleotide triphosphates into a biologically active RNA molecule such as a messenger RNA that is used to program protein synthesis on ribosomes, a ribosomal RNA that plays a structural role, and RNA cofactors required for correct splicing of message, and other related processes. Promoters may also contribute to gene expression elements, such as constitutive expression, particularly EF1 a and Actin, and to nickel inducibility of heterologous gene expression, particularly CYC6. The sequences of the following nuclear encoded promoters, in FASTA format: HUP2, RbcS2, RPL40, GDC1 , PsaD, EF1 a, Actin, and CYC6 can be found in SEQ ID Nos.:7 through 10, 57, and 34 through 36, respectively. The sequences of the following 5' UTR untranslated leader elements of chloroplast encoded genes: atpB, atpH, atpl, tufA, rps4, and rps12, can be found in SEQ ID Nos.:1 1 through 16, respectively. The sequences of the following 5' UTR untranslated leader elements of mitochondrial encoded genes: Nad2, Cox1 , rps2, and Atp1 can be found in SEQ ID Nos.:17 through 20, respectively. Terminators of nuclear encoded genes and 3' untranslated leader elements of organelle encoded genes are cis acting elements that can curtail gene transcription and result in release of RNA polymerase from the genomic DNA, and in copper repressibility. In other words, they define the end of a unit of gene function. Sequences for the following nuclear encoded terminators: HUP2, RbcS2, RPL40, GDC1 , PsaD , EF1 a, Actin, and CYC6, can be found at SEQ ID Nos.:21 through 24, 54, and 37 through 39, respectively. The sequences of the following 3' UTR untranslated leader elements of chloroplast encoded genes: atpl , atpE, atpB, rps12, and rps4 can be found in SEQ ID Nos.:25 through 29 respectively. The sequences of the following 3' UTR untranslated leader elements of mitochondrial encoded genes: Atpl, rps2, Nad2, and Cox1 , can be found in SEQ ID Nos.:30 through 33, respectively. Selected chloroplast and mitochondrial genome contig sequences may have additional open reading frames inserted in order to "hi-jack" the function of the expression elements, the 5' UTRs and the 3' UTRs, in these organellar genomes. The sequences of the mitrochondrial genome contigs can be found in SEQ ID Nos. 84, 87, 91 , 95 through 100, 102, and 104 through 108, respectively. The sequences of the chloroplast genome contigs can be found in SEQ ID Nos. 85, 86, 88, 89, 90, 92, 93, 94, 101 , 103, and 109, respectively. Figures 16A through 16H provide information as to the insertion of additional open reading frames to "hi-jack" the function of the expressional elements in these organellar genomes.
[0117] The steps involved in isolating a promoter element and a terminator element and using them to create an expression vector are described below for the example of the HUP2 gene. Beginning with the HUP2 gene homolog of Auxenochlorella protothecoides, the cDNA coding sequence for the HUP2 transporter protein can be found in the set of sequences obtained through RNA sequencing. This nucleic acid sequence can then be used to BLAST search the genomic DNA contigs and scaffolds to find nucleotide sequence both upstream and downstream of the RNA coding sequence of the gene. Upstream contigs may be composed of a long enough stretch of sequence to contain the necessary cis-acting sequence elements that make up a functional promoter. As shown in Figure 8, a 900 base pair (bp) long genomic DNA fragment (810) was identified upstream of the HUP2 RNA coding sequence. This 900 base pair sequence is then amplified using the polymerase chain reaction with an upstream primer designed from the DNA sequence at the 5' end of the contig and with a downstream primer from the region of the contig sequence at or near the initiator methionine codon. The fragment is sequence verified to ensure that it is the same as expected based on the genomic sequence, or if any changes have been introduced, they are subtle enough to most likely not impact the promoter function. The downstream primer has a Pstl site incorporated such that the 900 base pair promoter fragment can be cloned into a vector to allow the 3' end to be joined to the 5' end of the 700 base pair HUP2 terminator fragment (820) which also contains a Pstl site. This HUP2 terminator fragment is also amplified from Auxenochlorella protothecoides genomic DNA via the polymerase chain reaction, sequence confirmed and cloned into the vector, which can now be opened at the Pstl restriction site and a DNA coding sequence for a protein or functional RNA can be inserted at the Pstl restriction site (830), in the correct sense orientation in relation to the direction of transcription as determined by the directionality of both the promoter and terminator elements, to create a transgene cassette that consists of a promoter immediately upstream of a coding sequence which is immediately upstream of a transcriptional terminator. The whole transgene cassette (promoter plus RNA coding sequence plus terminator) (840) can now be inserted into an algal DNA transformation vector that already contains another transgene cassette containing a selectable marker gene between promoter and terminator elements which enables selection of modified Auxenochlorella protothecoides on media containing the selection agent, such as an antibiotic or an herbicide.
EXAMPLES
[0118] The following examples are provided for exemplification purposes only to further demonstrate the instant application. They are in no way meant to limit or restrict the application but provided as examples of how the instant application can be applied. EXAMPLE 1
[0119] Construction of an algae transformation vector to drive suppression of the Auxenochlorella protothecoides endogenous chlorophyll a oxygenase gene resulting in improved photosynthetic efficiency in metabolically engineered algal cultures.
[0120] An example of how Auxenochlorella protothecoides can be improved via molecular engineering to enhance photosynthetic efficiency is described below. With the necessary promoter elements, terminator elements, genes of interest and selectable marker genes in hand, a transformation vector for modification of Auxenochlorella protothecoides can be constructed. Below is an example of a vector constructed specifically for the inhibitory RNA (RNAi) mediated gene expression knockdown of the Chlorophyll a oxygenase (CAO) gene in Auxenochlorella protothecoides. Two gene cassettes are created: one for the RNAi gene suppression function (910) and one for providing a selectable marker (selection cassette) to facilitate recovery of correctly modified isolates of Auxenochlorella protothecoides (920). As illustrated in Figure 9 molecular cloning is performed to make a gene cassette using the constitutive Auxenochlorella protothecoides RPL40 promoter sequence element (930) upstream of an inverted repeat sequence composed of portions of the Auxenochlorella protothecoides CAO gene, including portions of the genes naturally occurring intron and portions of the CAO coding gene (940), all followed by the Auxenochlorella protothecoides RPL40 terminator sequence element to control the end of transcription (950). The selection cassette is made using the Auxenochlorella protothecoides light activated psaD promoter (960) driving expression of a fluridone herbicide resistance selectable marker (970) which was created by site-directed mutagenesis of the phytoene desaturase gene PDS1 from Chlamydomonas reinhardtii. The psaD terminator sequence element (980) is positioned downstream of the selectable marker to control the end of transcription.
[0121] These two gene cassettes can be cloned into a high copy number plasmid DNA molecule to facilitate maintenance of the sequence integrity, confirmation of structure by nucleotide sequencing and vector propagation to make DNA for transformation. In this example, the pUC19 vector backbone is used to construct the transformation vector. In a typical algal transformation experiment, the vector backbone is removed via restriction enzyme digestion and the resulting DNA fragments are introduced into algal cells via biolistic bombardment, glass bead agitation or other methods. Algal colonies representing the progeny of transformed cells acquiring the expression vector are selected on plates of solid agar media supplemented with the selective agent, in this case fluridone herbicide.
[0122] Transformed algae cells are isolated and cultured. Upon confirmation that the cells have incorporated the transgene DNA, cultures are tested for phenotypic attributes imparted by the transgenes. In this case, the transgenics will be assessed for decreased Photosystem II light-harvesting antenna size using biophysical measurements such as flash-induced Ch1 fluorescence, increased photosynthetic oxygen evolution rates and increased growth rates under high light intensities.
[0123] A similar genetic engineering approach as described above for the CAO gene, could be used to suppress the expression of the Grana-Deficient Chloroplastl gene to potentially increase light utilization and photosynthetic productivity under high light.
[0124] For the optimization of light capture and utilization and photosynthetic productivity under all light conditions, the expression of the antisense constructs targeting CAO and GDC1 would be driven by light-inducible promoters such that gene silencing and the RNAi phenotype would be maximal under high light and minimal under low light. This would result in metabolically engineered algae strains with the ability to self-regulate antenna size depending on the prevailing light intensity, being large at limiting light and smaller under higher light conditions.
EXAMPLE 2
[0125] Construction of an algae transformation vector to drive overexpression of a transgene encoding the endogenous carboxyl-terminal processing protease to confer abiotic stress tolerance in metabolically engineered algal cultures of Auxenochlorella protothecoides. [0126] An example of how Auxenochlorella protothecoides can be improved via molecular engineering using a gene homolog is described below. The Auxenochlorella protothecoides gene homolog for the ctpA gene could be identified using bioinformatics software by doing a BLAST comparison as illustrated in Figure 6. The Auxenochlorella protothecoides sequence identified could then be used to design oligonucleotide primers that could drive a polymerase chain reaction to generate a complementary DNA (cDNA) molecule from Auxenochlorella protothecoides RNA. In this example illustrated in Figure 10, the DNA fragment containing the RbcS2 promoter element is amplified using the polymerase chain reaction to also include a short portion of the sequence at the 5' end of the ctpA coding sequence (1010). Subsequently, a technique termed "fusion PCR" is used to create a DNA segment consisting of both the promoter element and the coding sequence in the correct juxtaposition and including a unique restriction enzyme site at the 3' end of the coding (CtpA) sequence (1020). This fragment can then be inserted into a vector via standard molecular cloning technique and subsequently a DNA fragment containing the RbcS2 terminator element, previously produced via polymerase chain reaction from Auxenochlorella protothecoides genomic DNA and incorporating the same restriction enzyme site at the 5' end, is inserted downstream of the CtpA coding sequence via standard molecular cloning technique (1030). The DNA transformation vector also contains a gene cassette encoding a selectable marker that allows transformed algal cells to be selected when grown on media containing a selection agent, such as an antibiotic or an herbicide. The resulting CtpA expression vector could be introduced into Auxenochlorella protothecoides using DNA coated microparticle bombardment to generate engineered versions of this algal strain that express significantly higher levels of the CtpA proteolytic enzyme. This is accomplished by insertion of the transgene cassette (promoter: CtpA coding sequence::terminator) into the nuclear genome. Several independent isolates of engineered Auxenochlorella protothecoides using this vector could be advanced on the selection agent and screened for the presence of the CtpA transgene cassette using polymerase chain reaction with oligonucleotides specific for the transgene cassette. Those isolates that contain the CtpA transgene cassette can be screened for improved phenotypes of photosynthetic efficiency or abiotic stress tolerance using fluorescence detectors and oxygen electrode detection equipment specific for characterizing photosynthesis parameters. Superior performing isolates will have acquired photosynthesis enhanced traits that allow it to perform better in high light stress, temperature stress or herbicide stress. Such superior isolates will demonstrate enhanced productivity with improved biomass yield in outdoor and indoor growth facilities. Similar approaches could be used for Sedoheptulose-1 ,7-bisphosphatase, GrpE, and alternative oxidase 1A.
EXAMPLE 3
[0127] Use of Auxenochlorella protothecoides nuclear codon preference table to design a transgene for expression in microalgae.
[0128] The nucleotide sequence encoding the Gaussia princeps luciferase enzyme is taken from The National Center for Biotechnology Information (NCBI) and the deduced amino acid sequence is used to design a synthetic gene using the Auxenochlorella protothecoides nuclear codon preference table (Figure 11 a & b.). Figure 11 a & b were made using 10,779 codons. In Table 1 , the 185 residue deduced amino acid sequence is listed from top to bottom in the left most column using the one letter amino acid code with a translational stop signal following the last amino acid residue ("STOP"). In the adjacent column, we have listed the most commonly used codons corresponding to each amino acid residue by referring to the nuclear codon preference table. Below the columns, we recreated the new, codon optimized, nucleotide sequence for Gaussia princeps luciferase. A synthetic gene can then be made via a vendor such as Integrated DNA Technologies (http://www.idtdna.com/Home/Home.aspx) or GeneWiz (http://www.genewiz.com/), by providing them the nucleotide sequence data and paying a fee. The synthetic gene can be made to include unique restriction enzyme sites, such as GAATTC for EcoRI and AGATCT for Bglll (http://rebase.neb.com/rebase/rebase.html) , at either end to facilitate restriction digestion and ligation into a nuclear expression vector. Alternatively, a fusion polymerase chain reaction procedure (http://www.youtube.com/watch?v=zSgnKWZLmVM) can be used to incorporate the synthetic gene coding region into a nuclear expression vector seamlessly. For the traditional cloning approach, the coding sequence with restriction enzyme sites at either side can be ligated downstream of the PsaD promoter and upstream of the PsaD terminator, both from the Auxenochlorella protothecoides genome to create a transgene cassette capable of expression. This nuclear expression vector can then be introduced into Auxenochlorella protothecoides cells, along with a herbicide resistance gene cassette or a antibiotic resistance gene cassette, in a co- transformation experiment, using a particle bombardment (gene gun) procedure. If using the fluridone resistance gene cassette as a selectable marker, fluridone can be used as the selection agent to propagate cells that have acquired the synthetic Gaussia princeps luciferase transgene. Presence of the transgene can be verified by polymerase chain reaction using oligonucleotide primers specific for the Gaussia princeps luciferase transgene to detect the gene sequence directly. Alternatively, the function of the luciferase transgene can be verified using a luminescence assay with a specific substrate for the Gaussia princeps luciferase and a 96 well luminescence plate reader. The coding sequence for the A. protothecoides optimized Gaussia princeps luciferase nucleotide sequence is shown in SEQ ID No. 110.
[0129] Table 1 : Coding Sequence Design Table Gaussia princeps luciferase amino acid sequence for G. princeps luciferase
Figure imgf000029_0001
amino acid sequence for
Codon 6. princeps
luciferase
A GCC
K AAG
P CCC
T ACC
E GAG
N AAC
N AAC
E GAG
D GAC
F TTC
N AAC
1 ATC
V GTG
A GCC
V GTG
A GCC
S TCC
N AAC
F TTC
A GCC
T ACC
T ACC
D GAC
L CTG
D GAC
A GCC
D GAC
CGC
G GGC
K AAG
L CTG
P CCC
G GGC
K AAG
K AAG
L CTG
P CCC
L CTG
E GAG
V GTG amino acid sequence for
Codon 6. princeps
luciferase
L CTG
K AAG
E GAG
M ATG
E GAG
A GCC
N AAC
A GCC
CGC
K AAG
A GCC
G GGC
C TGC
T ACC
R CGC
G GGC
C TGC
L CTG
1 ATC
C TGC
L CTG
S TCC
H CAC
1 ATC
K AAG
C TGC
T ACC
P CCC
K AAG
M ATG
K AAG
K AAG
F TTC
1 ATC
P CCC
G GGC
R CGC
C TGC
H CAC
T ACC amino acid sequence for
Codon 6. princeps
luciferase
Y TAC
E GAG
G GGC
D GAC
K AAG
E GAG
S TCC
A GCC
Q CAG
G GGC
G GGC
1 ATC
G GGC
E GAG
A GCC
1 ATC
V GTG
D GAC
1 ATC
P CCC
E GAG
1 ATC
P CCC
G GGC
F TTC
K AAG
D GAC
L CTG
E GAG
P CCC
M ATG
E GAG
Q CAG
F TTC
1 ATC
A GCC
Q CAG
V GTG
D GAC
L CTG amino acid sequence for
Codon 6. princeps luciferase
C TGC
V GTG
D GAC
C TGC
T ACC
T ACC
G GGC
C TGC
L CTG
K AAG
G GGC
L CTG
A GCC
N AAC
V GTG
Q CAG
C TGC
S TCC
D GAC
L CTG
L CTG
K AAG
K AAG
W TGG
L CTG
P CCC
Q CAG
CGC
C TGC
A GCC
T ACC
F TTC
A GCC
S TCC
K AAG
1 ATC
Q CAG
G GGC
Q CAG
V GTG amino acid
sequence for
Codon
6. princeps
luciferase
D GAC
K AAG
1 ATC
K AAG
G GGC
A GCC
G GGC
G GGC
D GAC
STOP TGA
EXAMPLE 4
[0130] Use of Auxenochlorella protothecoides chloroplast and mitochondrial codon preference table to design a transgene for expression in microalgae.
[0131] Efficient protein expression of a transgene within the mitochondria or chloroplast of any given organism is dependent on the bias in utilization of specific codons and availability of specific tRNAs to translate any given gene. The most efficient systems utilize a codon preference that matches that of the naturally occurring genes of the target genome. Figures 12 and 13 are derived from the identified coding regions of the near complete genomic sequences of Auxenochlorella protothecoides chloroplast and mitochondria, respectively. Column one on the left identifies all specific codons grouped by their corresponding amino acid residue in column 2 as identified by their conventional single letter designation. Column three identifies the uses of a specific codon among all uses for its given amino acid. Column 4 identifies the frequency of use of any given codon within the pool of selected protein encoding sequences. Codons with higher frequency in use or higher percent use for any given amino acid are considered preferred codons. For example alanine (A) usage in the chloroplast suggests a codon preference in the following order: GCT > GCA >> GCC > GCG. However cysteine (C) usage suggests no preference between TGC and TGT. Alteration of a target genes nucleotide sequence to accommodate for this given codon usage preference will then permit efficient translation of expressed mRNAs.
[0132] An example of the nucleotide modification process is exemplified by efforts to express a selectable marker gene conferring resistance to the protein synthesis inhibitor G418.
[0133] The KanMX gene encodes an enzyme with a phosphotransferase activity that neutralizes several antibiotics including the protein synthesis inhibitor G418. Auxenochlorella protothecoides is naturally susceptible to G418 at very low concentrations (>10 ug/ml). Optimization of codon usage within the KanMX gene will permit efficient translation of its transcript upon expression in one of the Auxenochlorella protothecoides organellar genomes and confer resistance to G418. For chloroplast expression, this is achieved using the codon usage table in Figure 12 and the KanMX amino acid sequence derived from pUG30 (Genbank accession number AF298781 ). The nucleotide sequence for KanMX was previously optimized for expression in yeast and is shown in SEQ ID No. 111 . This sequence encodes the amino acid sequence shown in SEQ ID No. 112.
[0134] Using the chloroplast codon usage preference table in Figure 12, the nucleotide sequence was manually edited to a nucleotide sequence which upon transcription will be translated into the same amino acid sequence as expressed from pUG30. The nucleotide and amino acid sequences can be found at SEQ ID Nos. 113 and 114, respectively.
EXAMPLE 5
[0135] Utilization of an operonic region for expression of transgenes in the chloroplast or mitochondria of Auxenochlorella protothecoides.
[0136] The expression of transgenes in the chloroplast or mitochondria of green algae can enhance any one of various attributes related to biofuels production. This includes resistance to abiotic or biotic stress, enhanced carbon fixation, improved photosynthetic rates, improved efficiency in electron transfer events, or others. To do this generally requires a target gene or genes of interest, a selectable marker gene, promoters and terminators for both of these genes to drive their expression and two regions of sequence homologous to the host genome which permits homologous recombination.
[0137] In some cases it is possible to reduce these requirements by inserting the target gene(s) and selectable marker into an operonic sequence. An operon is a region of genomic DNA that encodes more than one gene expressed as a single mRNA transcript. The ribosomal complex binds to the 5'UTR and initiates translation of the first coding region. Processing continues toward the 3' end of the transcript terminating translation at stop codons and reinitiating expression at the next methionine codon until all encoded proteins are expressed. When the complex reaches the end of the mRNA it detaches from the mRNA. Secondary structures and post transcriptional modification, regulate the processivity of the operon as well as the relative abundance of proteins derived from each gene. The utilization of operons permits higher gene density in small genomes and coordinated expression of genes with related function. The use of operons is common to many bacterial species and some organelles specifically including the mitochondria and chloroplasts of green algae.
[0138] In this example the target gene KanAp has been codon optimized as described in Example 4 for efficient translation in the chloroplast of Auxenochlorella protothecoides. The KanAp gene also serves as a selective marker as it confers resistance to the antibiotic G418. In this example the rps19-rps3 (rps193) operon was modified to include a coding region for KanAp between the two genes. This reduces the need for promoter and terminator elements for expression of the target gene.
[0139] The native sequence for the PCR amplified region including the rps193 operon is shown in SEQID No. 115. Figure 14 is a graphic example of the gene arrangement and suspected operon including the rps19 and rps3 genes.
[0140] The codon optimized KanAp gene was inserted between the rps19 termination codon and the rps3 initiation codon. The intergenic region was modified to include addition restriction sites (EcoRI and EcoRV) flanking the KanAp gene to permit downstream modification if necessary.
[0141] The final construct has the sequence as shown in SEQ ID No. 42. This sequence is represented graphically as shown in Figure 15. Biolistic transformation with this linearized product yielded transformant colonies of Auxenochlorella protothecoides selected on 10ug/ml G418 grown under photoautotrophic conditions. The presence of the KanAp gene was confirnned by colony PCR.

Claims

CLAIMS What is clamed is:
1 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Chlorophyll a oxygenase in Auxenochlorella protothecoides.
2. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary which: encodes Chlorophyll a oxygenase in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:1 or to a complementary strain thereof.
3. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Sedoheptulose-1 ,7- bisphosphatase in Auxenochlorella protothecoides.
4. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary which: encodes Sedoheptulose-1 , 7-bisphosphatase in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:2 or to a complementary strain thereof.
5. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous, or complementary to, a sequence encoding GrpE in Auxenochlorella protothecoides.
6. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous, or complementary which: encodes GrpE in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:3 or to a complementary strain thereof.
7. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complementary to, a sequence encoding alternative oxidase 1A in Auxenochlorella protothecoides.
8. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary which: encodes alternative oxidase 1A in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:4 or to a complementary strain thereof.
9. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complementary to, a sequence encoding carboxyl- terminal processing protease CtpA in Auxenochlorella protothecoides.
10. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous, or complementary which: encodes carboxyl-terminal processing protease CtpA in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:5 or to a complementary strain thereof.
1 1 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous, or complementary to, a sequence encoding carboxyl- terminal processing protease Grana-Deficient Chloroplasti in Auxenochlorella protothecoides.
12. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous, or complementary which: encodes carboxyl-terminal processing protease Grana-Deficient Chloroplastl in an organism belonging to the class Trebouxiophyceae; hybridizes under low stringency conditions to the nucleotide sequence set forth in SEQ ID No:6 or to a complementary strain thereof.
13. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complimentary to, a sequence encoding a promoter or terminator of nuclear encoded genes selected from the group consisting of: HUP2, RPL40, Rbcs2, GDC1 , PsaD, EF1 a, Actin, and CYC6.
14. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complementary to genes from Auxenochlorella
protothecoides and hybridizes under low stringency conditions to a nucleotide sequence set forth in the group consisting of SEQ ID No:7 through SEQ ID No:10, SEQ ID No:21 through SEQ ID No:24, and SEQ ID No: 54, or to a complementary strain thereof.
15. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complimentary to, a sequence encoding a 5' UTR untranslated leader element of organelle encoded gene from Auxenochlorella protothecoides selected from the group consisting of: atpB, atpH, atpl, tufA, rps4, rpsl 2, Nad2, Cox1 , rps2, and Atp1 .
16. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complementary to genes encoded in Auxenochlorella protothecoides and hybridizes under low stringency conditions to a nucleotide sequence set forth in the group consisting of SEQ ID No:1 1 through SEQ ID No:20 or to a complementary strain thereof.
17. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complimentary to, a sequence encoding 3' UTR untranslated leader elements of organelle encoded genes from Auxenochlorella protothecoides selected from the group consisting of: atpl , atpE, atpB, rps12, rps4, rps2, Nad2, and Cox1 .
18. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, heterologous or complementary to genes from Auxenochlorella protothecoides and hybridizes under low stringency conditions to a nucleotide sequence set forth in the group consisting of SEQ ID No:25 through SEQ ID No:33 or to a complementary strain thereof.
19. A transformation vector comprising the nucleic acid molecule according to any one of claims 1 through 15.
20. A transgenic organism belonging to the phylum Chlorophyta (green algae) comprising a nucleic acid molecule having an overexpressed a sequence of nucleotides encoding, or complementary to, a sequence selected from the group consisting of: Sedoheptulose-1 ,7-bisphosphatase, GrpE, alternative oxidase 1A, and carboxyl-terminal processing protease.
21 . A transgenic organism belonging to the phylum Chlorophyta (green algae) comprising a nucleic acid molecule having a suppressed sequence of nucleotides encoding, or complementary to, a sequence selected from the group consisting of: Chlorophyll a oxygenase and Grana-Deficient Chloroplastl .
22. A transgenic organism belonging to the phylum Chlorophyta (green algae) comprising a nucleic acid molecule having a sequence of nucleotides encoding, or complementary to, a sequence selected from the group consisting of: HUP2, RPL40, Rbcs2, GDC1 , EF1 a, Actin, and CYC6.
23. A transgenic organism belonging to the phylum Chlorophyta (green algae) comprising a nucleic acid molecule having a sequence of nucleotides encoding, or complementary to, a 5' UTR untranslated leader element of organelle encoded gene selected from the group consisting of: atpB, atpH, atpl, tufA, rps4, rps12, Nad2, Cox1 , rps2, and Atp1 .
24. A transgenic organism belonging to the phylum Chlorophyta (green algae) comprising a nucleic acid molecule having a sequence of nucleotides encoding, heterologous or complementary to a sequence encoding 3' UTR untranslated leader elements of organelle encoded genes from Auxenochlorella protothecoides selected from the group: atpl , atpE, atpB, rps12, rps4, rps2, Nad2, and Cox1 .
25. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding transketolase in
Auxenochlorella protothecoides.
26. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein LHCPII type I CAB2B in Auxenochlorella protothecoides.
27. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding light harvesting complex protein type III (CAB4) in Auxenochlorella protothecoides.
28. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding plastocyanin in
Auxenochlorella protothecoides.
29. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding rubisco activase in
Auxenochlorella protothecoides.
30. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carbonic anhydrase in Auxenochlorella protothecoides.
31 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b binding protein CP29 in Auxenochlorella protothecoides.
32. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll antenna size regulatory protein TLA1 in Auxenochlorella protothecoides.
33. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP synthase gamma subunit in Auxenochlorella protothecoides.
34. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding component of cytochrome b6/f complex in Auxenochlorella protothecoides.
35. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding albino 3-like protein in Auxenochlorella protothecoides.
36. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chlorophyll a/b-binding protein LCHSR3 in Auxenochlorella protothecoides.
37. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ferredoxin reductase-like protein in Auxenochlorella protothecoides.
38. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding polyadenylate binding protein (NAB1 -RRMS) in Auxenochlorella protothecoides.
39. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ribulose-1 ,5-bisphosphate carboxylase/oxygenase small subunit in Auxenochlorella protothecoides.
40. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding protein gradient regulation 5 in Auxenochlorella protothecoides.
41 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding phototropin, blue light receptor in Auxenochlorella protothecoides.
42. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding photosystem I protein PsaD in Auxenochlorella protothecoides.
43. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chloroplast photosystem II associated 22 kDa protein PsbS in Auxenochlorella protothecoides.
44. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase beta subunit in Auxenochlorella protothecoides.
45. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tryptophan synthase alpha subunit in Auxenochlorella protothecoides.
46. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding carotenoid epsilon cyclase in Auxenochlorella protothecoides.
47. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding cytochrome P450 in
Auxenochlorella protothecoides.
48. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding H(+)/hexose cotransporter 1 (HUP1 ) in Auxenochlorella protothecoides.
49. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol cyclase in Auxenochlorella protothecoides.
50. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding tocopherol O- methyltransferase in Auxenochlorella protothecoides.
51 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding gamma tocopherol methyltransferase in Auxenochlorella protothecoides.
52. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ATP-NAD kinase in
Auxenochlorella protothecoides.
53. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose
pyrophosphorylase large subunit (STA1 ) in Auxenochlorella protothecoides.
54. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding ADP-glucose pyrophosphorylase small subunit (STA6) in Auxenochlorella protothecoides.
55. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding pyruvate decarboxylase in Auxenochlorella protothecoides.
56. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p-hydroxyphenylpyruvate dioxygenase in Auxenochlorella protothecoides.
57. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding heat shock protein A
(HSP70A) in Auxenochlorella protothecoides.
58. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding zeaxanthin epoxidase in Auxenochlorella protothecoides.
59. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding violaxanthin de-epoxidase in Auxenochlorella protothecoides.
60. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding chaperone protein dnaJ- related in Auxenochlorella protothecoides.
61 . An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrogen regulatory protein PI I in Auxenochlorella protothecoides.
62. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding argininosuccinate lyase ARG7 in Auxenochlorella protothecoides.
63. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding nitrate reductase in
Auxenochlorella protothecoides.
64. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding target of rapamycin (TOR) in Auxenochlorella protothecoides.
65. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding 60S ribosomoal protein L402 in Auxenochlorella protothecoides.
66. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding p53 induced protein 8 (PIG8) in Auxenochlorella protothecoides.
67. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding dicer in Auxenochlorella protothecoides.
25. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding Chlorophyll a oxygenase in Auxenochlorella protothecoides.
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US9518277B2 (en) 2012-12-07 2016-12-13 Terravia Holdings, Inc. Genetically engineered microbial strains including Chlorella protothecoides lipid pathway genes
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US9649368B2 (en) 2011-11-28 2017-05-16 Terravia Holdings, Inc. Genetically engineered microbial strains including prototheca lipid pathway genes
US9518277B2 (en) 2012-12-07 2016-12-13 Terravia Holdings, Inc. Genetically engineered microbial strains including Chlorella protothecoides lipid pathway genes
US11268106B2 (en) 2014-09-11 2022-03-08 Jnc Corporation Method for synthetic genes
US10590398B2 (en) 2015-06-04 2020-03-17 Nmc, Inc. Productivity and bioproduct formation in phototropin knock/out mutants in microalgae
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