USOO8709766B2 (12) United States Patent (10) Patent No.: US 8,709,766 B2 Radakovits et al. (45) Date of Patent: Apr. 29, 2014 (54) USE OF ENDOGENOUS PROMOTERS IN Cocket al. “The Ectocarpus genome and the independent evolution GENETIC ENGINEERING OF of multicellularity in brown algae', Nature, pp. 617-621 (2010). Conesa et al., “Blast2GO: a universal tool for annotation, visualiza NANNOCHLOROPSIS GADITANA tion and analysis in functional genomics research'. Bioinformatics 21, pp. 3674-3676 (2005). (71) Applicant: Colorado School of Mines, Golden, CO Gobler et al. “Niche of harmful alga Aureococcus anophageferens (US) revealed through ecogenomics'. Proceedings of the National Acad emy of Sciences, pp. 4352-4357 (2011). (72) Inventors: Randor Radakovits, Denver, CO (US); Götz et al., “B2G-FAR, a species centered GO annotation reposi tory'. Bioinformatics (2011). 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Primary Examiner — David T Fox Assistant Examiner — Matthew Keogh (56) References Cited (74) Attorney, Agent, or Firm — Dorsey & Whitney LLP PUBLICATIONS (57) ABSTRACT Armbrust et al., “The genome of the diatom Thalassiosira The present disclosure is directed to novel polynucleotide pseudonana: Ecology, evolution, and metabolism'. Science, 79-86 sequences for use in Nannochloropsis gaditana. The novel (2004). polynucleotide sequences include control sequences and cod Atsumi et al., “Direct photosynthetic recycling of carbon dioxide to ing sequences. Also disclosed are novel gene expression con isobutyraldehyde'. Nature Biotechnology, pp. 1177-1180 (2009). structs wherein N. gaditana promoters/control regions are Blanc et al. “The Chlorella variabilis NC64A genome reveals adap operatively linked to N. gaditana or non-N.gaditana coding tation to photosymbiosis, coevolution with viruses, and cryptic sex’, sequences. These novel polynucleotide sequences and The Plant Cell (2010). expression constructs can be introduced into N. gaditana and Blithgen et al. “Biological Profiling of Gene groups utilizing Gene can recombine into the N.gaditana genome. Expression from ontology—A statistical and software framework'. (2004), Gossip software, http://www.microdiscovery.de/. these polynucleotide sequences and expression constructs Blithgen et al. “Biological Profiling of Gene Groups utilizing Gene can enhance N. gaditana biomass and/or lipid biosynthesis. Ontology”. Genome Informatics, 16 (1): 106-115, 2005. Also disclosed are methods for modifying N. gaditana, for Bowler et al., “The Phaeodactylum genome reveals the evolutionary example by stably transforming N.gaditana with nucleic acid history of diatom genomes”. Nature, pp. 239-244 (2008). sequences, growing the modified N. gaditana, and obtaining Chen et al., “Conditional production of a functional fish growth biomass and biofuels from the modified N. gaditana. hormone in the transgenic line of Nannochloropsis oculata (Eustigmatophyceae)”. Journal of Phycology, pp. 768-776 (2008). 13 Claims, 198 Drawing Sheets US 8,709,766 B2 Page 2 (56) References Cited Wang et al. Algal Lipid Bodies: Stress induction, purification, and biochemical characterization in wild-type and starchless PUBLICATIONS Chlamydomonas reinhardtii. Eukaryotic Cell , pp. 1856-1868 (2009). Pal et al., “The effect of light, salinity, and nitrogen availability on Work et al. “Increased lipid accumulation in the Chlamydomonas lipid production by Nannochloropsis sp.”. Applied Microbiology and reinhardtii Sta7-10 starchless isoamylase mutant and increased car Biotechnology, pp. 1429-1441 (2011). bohydrate synthesis in complemented Strains’. Eukaryotic Cell, pp. Radakovits et al., “Genetic engineering of fatty acid chain length in 1251-1261 (2010). Phaeodactylum tricornutum', Metabolic Engineering , pp. 1-7 Zaslavskaia et al., “Transformation of the diatom Phaeodactylum (2010). tricornutum (Bacillariophyceae) with a variety of selectable marker Radakovits et al., “Genetic engineering of algae for enhanced biofuel and reporter genes”,Journal of Phycology, pp. 379-386 (2000). production”. Eukaryotic Cell 9, pp. 486-501 (2010). Zou et al. “Production of cell mass and eicosapentaenoic acid (EPA) Samstag, Antisense Nucleic Acid Drug Dev 6: pp. 153-156 (1996). in ultrahigh cell density cultures of Nannochloropsis sp. Steen et al., “Microbial production of fatty-acid-derived fuels and (Eustigmatophyceae). European Journal of Phycology, pp. 127 chemicals from plant biomass', Nature 463, pp. 559-562 (2010). 133 (2000). U.S. Patent Apr. 29, 2014 Sheet 1 of 198 US 8,709,766 B2 Supplementary Table 3. Chlorophyll (tetrapyrrole), carotenoid and sterol biosynthesis genes N. gadiana Transcript Gene Enzyme Description EC number node Support Location Tetrapyrrole Synthesis GS glutamyl-tRNA synthetase 6.1.1.17 Nga04989 GTS glutamyl-tRNA synthetase 6.1.24 Nga02834 GTR glutamyl-tRNA reductase 1.2.70 Nga02604 glutamate-1-semialdehyde GSA aminotransferase f glutamate-1- 5438 Nga30045 semialdehyde 21-aminomutase AAD 5-aminolevulinic acid dehydratase f (HenB) porphobilinogen synthase 4.2.1.24 NgaO0585 PBGO porphobilinogen deaminase (HenC) hydroxymethylbilane synthase 2.5.1.6. NgaO3248 UROS uroporphyrinogen synthase 4.2.1.75 NgaO0807 (HenD) UROD uroporphyrinogen I decarboxylase 4.1.1.37 Nga04120 UROD uroporphyrinogen decarboxylase 4.1.1.37 Nga05706 Gr Coproporphyrinogen Oxidase 1.3.3.3 Nga05151 CPX1 t Nga04278 (HenF) Coproporphyrinogen oxidase 33.3 (partial) PPX protoporphyrinogen X oxidase 1.3.3.4. NgaO3873 Chi protoporphyrin XMg-chelatase subunit D 68. Nga30773 Ch protoporphyrin X MG-chelatase subuint 6.6.1.1 Nga40092 Chi-1 protoporphyrin X Mg-chelatase subunit H 6.6.1.1 Nga30995 Chi-2 protoporphyrin XMg-chelatase subunit H 66.11 Nga06242 PPMT Mg-protoporphyri IX methyltransferase 2.1.1.1 : Nga04808 (ChM) ACSF Mg-protoporphyrin IX monomethyester 14, 138 (ycf59) (Oxidative) cyclase Nga40091 DWR divinyl protochlorophytide a 8-vinyl- 1.3.1.75 Nga05945 reductase POR ight-dependent NADPH-protochlorophyllide oxidoreductase 1.3.1.33 Nga04959 POR light-dependent NADPH protochlorophyllide Oxidoreductase 1.3.1.33 Nga00683 ight-independent protochlorophyllide ChB Oxidoreductase subutit B 1.18.-. Nga40089 tight-independent:protochlorophyllide C Oxidoreductase subunit 1.18.-- Nga40044 tight-independent:protochlorophyllide CN Oxidoreductase Subtit N 1.18.-- Nga40045 (Ch;G)C-S chlorophyli synthase 2.5.1.62 Nga31097 F.G.R. A U.S. Patent Apr. 29, 2014 Sheet 2 of 198 US 8,709,766 B2 W. gadiana Transcript eene Description ECner mode Support location GGR (ChiP) geranylgerary reductase 3. Nga04895 UM uroporphyrinogen C-methyltransferase 2.1.1.107 NgaO5160 Sir B sirohydrochlorin ferrochelatase 4.99.14 NgaO0339 FC ferrochelatase 4.99.1.1 NgaO0748 Caroteroid Biosynthesis 1-deoxy-D-xylulose-5-phosphate DXS synthase 22.7 NgaO2203 Y 1-deoxy-D-xylulose-5-phosphate DXR (IspC) reductoisonerase 1.1.1.267