Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

McCarthy, James K.; Smith, Sarah R.; McCrow, John P.; Tan, Maxine; Zheng, Hong; Beeri, Karen; Roth, Robyn; Lichtlé, Christiane; Goodenough, Ursula; Bowler, Chris; Dupont, Cristopher L; Allen, Andrew E.

doi:10.1105/tpc.16.00910

Cited by 81 publications

(80 citation statements)

References 115 publications

(151 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The sgRNAs were designed to mutate the nitrate reductase (NR) gene in Phaeodactylum. NR was picked as a genomic target because the function of NR has been well studied in Phaeodactylum and NR knockout cell mutants exhibit an easily screenable growth phenotype of cell death when supplemented with nitrate as a sole nitrogen source (McCarthy et al, 2017). The two sgRNAs were designed to target regions 55-bp apart so that the same amplification and sequencing primers could be used for genotyping efforts.…”

Section: Single Gene Mutagenesismentioning

confidence: 99%

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

et al. 2020

Self Cite

View full text Add to dashboard Cite

Marine diatoms are eukaryotic microalgae that play significant ecological and biogeochemical roles in oceans. They also have significant potential as organismal platforms for exploitation to address biotechnological and industrial goals. In order to address both modes of research, sophisticated molecular and genetic tools are required. We presented here new and improved methodologies for introducing CRISPR-Cas9 to the model diatom Phaeodactylum tricornutum cells and a streamlined protocol for genotyping mutant cell lines with previously unknown phenotypes. First, bacterialconjugation was optimized for the delivery of Cas9 by transcriptionally fusing Cas9 to a selectable marker by the 2A peptide. An episome cloning strategy using both negative and positive selection was developed to streamline CRISPR-episome assembly. Next, cell line picking and genotyping strategies, that utilize manual sequencing curation, TIDE sequencing analysis, and a T7 endonuclease assay, were developed to shorten the time required to generate mutants. Following this new experimental pipeline, both singlegene and two-gene knockout cell lines were generated at mutagenesis efficiencies of 48% and 25%, respectively. Lastly, a protocol for precise gene insertions via CRISPR-Cas9 targeting was developed using particle-bombardment transformation methods. Overall, the novel Cas9 episome design and improved genotyping methods presented here allow for quick and easy genotyping and isolation of Phaeodactylum mutant cell lines (less than 3 weeks) without relying on a known phenotype to screen for mutants.

show abstract

Section: Single Gene Mutagenesismentioning

confidence: 99%

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

et al. 2020

Self Cite

View full text Add to dashboard Cite

show abstract

“…The waning of the diurnal cycle and macronutrient deficiency have been shown to cause co-ordinated changes in transcriptional regulation, re-organization of metabolic flux and the reallocation of cellular carbon towards lipid production [83,85,[88][89][90][91]. Similar effects can be achieved by disrupting nutrient assimilation, such as knocking-out or knocking-down nitrate reductase [92,93]. Neutral lipid accumulation can also be induced by exogenous addition of a variety of compounds, including fatty acids [94], sodium bicarbonate [95], nitric oxide [96] or other compounds [97,98].…”

Section: Biogenesismentioning

confidence: 99%

A Review of Diatom Lipid Droplets

Leyland

Boussiba

Khozin‐Goldberg

2020

Biology

View full text Add to dashboard Cite

The dynamic nutrient availability and photon flux density of diatom habitats necessitate buffering capabilities in order to maintain metabolic homeostasis. This is accomplished by the biosynthesis and turnover of storage lipids, which are sequestered in lipid droplets (LDs). LDs are an organelle conserved among eukaryotes, composed of a neutral lipid core surrounded by a polar lipid monolayer. LDs shield the intracellular environment from the accumulation of hydrophobic compounds and function as a carbon and electron sink. These functions are implemented by interconnections with other intracellular systems, including photosynthesis and autophagy. Since diatom lipid production may be a promising objective for biotechnological exploitation, a deeper understanding of LDs may offer targets for metabolic engineering. In this review, we provide an overview of diatom LD biology and biotechnological potential.

show abstract

“…Such reduction of nitrate and assimilation into urea may also occur during high nitrogen growth. Nitrate reductase combined with the ornithine-urea cycle supports urea as nitrogen storage product in diatoms and has been suggested to be key to the evolutionary success of diatoms (Bowler et al, 2008;Allen et al, 2011;McCarthy et al, 2017). This trait may indeed be advantageous in environments with highly dynamic nutrient sources and may also partly explain the large variation of nitrate assimilation rates observed at both clone-specific and cell-specific level.…”

Section: Resultsmentioning

confidence: 99%

High single‐cell diversity in carbon and nitrogen assimilations by a chain‐forming diatom across a century

Olofsson

Kourtchenko

Zetsche

et al. 2018

Environmental Microbiology

View full text Add to dashboard Cite

Summary Almost a century ago Redfield discovered a relatively constant ratio between carbon, nitrogen and phosphorus in particulate organic matter and nitrogen and phosphorus of dissolved nutrients in seawater. Since then, the riverine export of nitrogen to the ocean has increased 20 fold. High abundance of resting stages in sediment layers dated more than a century back indicate that the common planktonic diatom Skeletonema marinoi has endured this eutrophication. We germinated unique genotypes from resting stages originating from isotope‐dated sediment layers (15 and 80 years old) in a eutrophied fjord. Using secondary ion mass spectrometry (SIMS) combined with stable isotopic tracers, we show that the cell‐specific carbon and nitrogen assimilation rates vary by an order of magnitude on a single‐cell level but are significantly correlated during the exponential growth phase, resulting in constant assimilation quota in cells with identical genotypes. The assimilation quota varies largely between different clones independent of age. We hypothesize that the success of S. marinoi in coastal waters may be explained by its high diversity of nutrient demand not only at a clone‐specific level but also at the single‐cell level, whereby the population can sustain and adapt to dynamic nutrient conditions in the environment.

show abstract

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

Cited by 81 publications

References 115 publications

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

Multiplexed Knockouts in the Model Diatom Phaeodactylum by Episomal Delivery of a Selectable Cas9

A Review of Diatom Lipid Droplets

High single‐cell diversity in carbon and nitrogen assimilations by a chain‐forming diatom across a century

Contact Info

Product

Resources

About