Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production.

[1]  W. J. Dyer,et al.  A rapid method of total lipid extraction and purification. , 1959, Canadian journal of biochemistry and physiology.

[2]  R. Guillard,et al.  Culture of Phytoplankton for Feeding Marine Invertebrates , 1975 .

[3]  F. Falkner Implications for Growth in Human Twins , 1978 .

[4]  R. Guillard,et al.  Carotenoid Distribution Patterns in Bacillariophyceae (Diatoms) , 1988 .

[5]  P. Gőcze,et al.  Factors underlying the variability of lipid droplet fluorescence in MA-10 Leydig tumor cells. , 1994, Cytometry.

[6]  A. Grossman,et al.  Stable nuclear transformation of the diatom , 1996 .

[7]  J. Randerson,et al.  Primary production of the biosphere: integrating terrestrial and oceanic components , 1998, Science.

[8]  M. Jasin,et al.  Analysis of Gene Targeting and Intrachromosomal Homologous Recombination Stimulated by Genomic Double-Strand Breaks in Mouse Embryonic Stem Cells , 1998, Molecular and Cellular Biology.

[9]  P. Falkowski,et al.  Biogeochemical Controls and Feedbacks on Ocean Primary Production , 1998, Science.

[10]  F. Azam,et al.  Accelerated dissolution of diatom silica by marine bacterial assemblages , 1999, Nature.

[11]  R. Gordon,et al.  Beyond micromachining: the potential of diatoms. , 1999, Trends in biotechnology.

[12]  Mark Hildebrand,et al.  SILICON METABOLISM IN DIATOMS: IMPLICATIONS FOR GROWTH  , 2000 .

[13]  J. M. Fernández-Sevilla,et al.  Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum. , 2000, Phytochemistry.

[14]  Samuel T. Edwards,et al.  Mutations altering the cleavage specificity of a homing endonuclease. , 2002, Nucleic acids research.

[15]  P. Kroth Molecular biology and the biotechnological potential of diatoms. , 2007, Advances in experimental medicine and biology.

[16]  Y. Chisti Biodiesel from microalgae. , 2007, Biotechnology advances.

[17]  A. Falciatore,et al.  Molecular toolbox for studying diatom biology in Phaeodactylum tricornutum. , 2007, Gene.

[18]  Q. Hu,et al.  Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. , 2008, The Plant journal : for cell and molecular biology.

[19]  A. Kaplan,et al.  A Model for Carbohydrate Metabolism in the Diatom Phaeodactylum tricornutum Deduced from Comparative Whole Genome Analysis , 2008, PloS one.

[20]  A. Falciatore,et al.  Gene silencing in the marine diatom Phaeodactylum tricornutum , 2009, Nucleic acids research.

[21]  U. Maier,et al.  Diatoms in biotechnology: modern tools and applications , 2009, Applied Microbiology and Biotechnology.

[22]  Maider Villate,et al.  Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease , 2009, Nucleic acids research.

[23]  G. Charles Dismukes,et al.  Increased Lipid Accumulation in the Chlamydomonas reinhardtiista7-10 Starchless Isoamylase Mutant and Increased Carbohydrate Synthesis in Complemented Strains , 2010, Eukaryotic Cell.

[24]  Erin L. Doyle,et al.  Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting , 2011, Nucleic acids research.

[25]  U. Linne,et al.  Microalgae as bioreactors for bioplastic production , 2011, Microbial cell factories.

[26]  P. Duchateau,et al.  Meganucleases and Other Tools for Targeted Genome Engineering: Perspectives and Challenges for Gene Therapy , 2011, Current gene therapy.

[27]  C. Bowler,et al.  Decoding algal genomes: tracing back the history of photosynthetic life on Earth. , 2011, The Plant journal : for cell and molecular biology.

[28]  K. Niyogi,et al.  High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. , 2011, Proceedings of the National Academy of Sciences.

[29]  S. Sym,et al.  Microalgal fatty acid composition: implications for biodiesel quality , 2012, Journal of Applied Phycology.

[30]  S. Grizot,et al.  The I-CreI meganuclease and its engineered derivatives: applications from cell modification to gene therapy. , 2011, Protein engineering, design & selection : PEDS.

[31]  A. Falciatore,et al.  Exploring the molecular basis of responses to light in marine diatoms. , 2012, Journal of experimental botany.

[32]  R. Carlson,et al.  Potential role of multiple carbon fixation pathways during lipid accumulation in Phaeodactylum tricornutum , 2012, Biotechnology for Biofuels.

[33]  U. Maier,et al.  An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency , 2012, Microbial Cell Factories.

[34]  Sarah K. Baxter,et al.  Coupling endonucleases with DNA end–processing enzymes to drive gene disruption , 2012, Nature Methods.

[35]  P. Schenk,et al.  Microalgal biofactories: a promising approach towards sustainable omega-3 fatty acid production , 2012, Microbial Cell Factories.

[36]  P. Winge,et al.  Pathways of Lipid Metabolism in Marine Algae, Co-Expression Network, Bottlenecks and Candidate Genes for Enhanced Production of EPA and DHA in Species of Chromista , 2013, Marine drugs.

[37]  Sarah R. Smith,et al.  Metabolic engineering of lipid catabolism increases microalgal lipid accumulation without compromising growth , 2013, Proceedings of the National Academy of Sciences.

[38]  C. Barbas,et al.  ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. , 2013, Trends in biotechnology.

[39]  S. Schorr-Galindo,et al.  Analysis of neutral lipids from microalgae by HPLC-ELSD and APCI-MS/MS. , 2013, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[40]  P. Hegemann,et al.  Nuclear gene targeting in Chlamydomonas using engineered zinc-finger nucleases. , 2013, The Plant journal : for cell and molecular biology.

[41]  George H. Silva,et al.  High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes , 2013, PloS one.

[42]  Aifen Li,et al.  Production, Characterization, and Antioxidant Activity of Fucoxanthin from the Marine Diatom Odontella aurita , 2013, Marine drugs.

[43]  Wei-dong Yang,et al.  Biochemical and Genetic Engineering of Diatoms for Polyunsaturated Fatty Acid Biosynthesis , 2014, Marine drugs.

[44]  Jeffry D Sander,et al.  FLAsH assembly of TALeNs for high-throughput genome editing , 2022 .