Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

TAG controls chronological lifespan independently of conserved pathways.

<p><i>TOR1</i>, <i>RAS2</i>, and <i>SOD2</i> were deleted from the three core strains with a normal, higher, and lower TAG content. The resultant strains were grown for outgrowth assays to compare their CLS with the corresponding parental strains. (A) Representative plot of one of three biological duplicate outgrowth experiments. (B) Quantitative analysis of lifespan from the outgrowth data seen in panel A. The time (in days) it took for each strain to drop to 10%, 1%, and 0.1% viability was calculated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005878#sec007" target="_blank">Materials and Methods</a>) from two or three independent assays, whenever data available. Some thus did not yield a standard deviation. The lifespan changes of the <i>tor1</i>Δ, <i>ras2</i>Δ, and <i>sod2</i>Δ strains were differences (days to reach 10%, 1%, and 0.1% viability) from the corresponding parental strains. Asterisks (*) indicate that at least one of the two strains being compared maintained the viability higher than 1% or 0.1% throughout the duration of the experiments.</p

TAG-medicated lifespan control is independent of several yeast-specific and common lifespan regulatory regimes.

<p>(A) Semi-quantitative comparison of lifespan of the three “core” strains used in this study: wildtype (WT), TAG lipase knockout (<i>tgl3</i>Δ), and TAG synthesis deficient mutant (<i>dga1</i>Δ <i>lro1</i>Δ) under different growth conditions. Glc, glucose; the medium neutralization experiment (fourth column from left) was done with SC medium supplemented with 64.2 mM Na₂HPO₄ and citric acid to stabilize the pH at 6.0. Shown are representative results of 2 or 3 biological repeats. (B) Quantitation of the spot assay results. All data were from three biological duplicates.</p

Wild yeast strains from different origins accumulate higher levels of triacylglycerol (TAG) and exhibit longer chronological lifespan.

<p>(A) Growth curves of wild strains isolated from oak exudates, vineyards, and clinical samples vs. three laboratory strains. Cells were grown in YPD at 30° in a 96-well plate. Each curve represents the average of growth of 2 to 4 strains in the category. Shown are representative results from two biological replicates. (B) Chronological lifespan in SC medium was measured by the “outgrowth” approach. (C) Chronological lifespan examined by spot assays. Viability of cultures was assayed every 3 to 5 days for two months. Only day 1 (all strains showed close to 100% viability) and day 61 images are shown. Cultures were 10-fold serially diluted in water before inoculation. (D) Quantification of triacylglycerol of cells harvested from day 1 and day 8 post-saturation YPD cultures.</p

Overproducing Dga1p increases TAG abundance and extends chronological lifespan.

<p>(A) Thin-layer chromatography of neutral lipids from eight-day old stationary phase cultures bearing either the empty vector (EV) or one that expresses Dga1p. DAG, diacylglycerol; FA, free fatty acids; ori., origins for chromatography. The two EV lanes in panel A are biological duplicates; each of the two <i>DGA1</i> lanes represents the two different constructs with <i>DGA1</i> or <i>ADH1</i> promoter driving the recombinant gene expression. (B) Survival curves. These are averages of four <i>DGA1</i> overexpression isolates (two of each plasmid transformants), and two vector control duplicates. To accommodate the use of episomal plamids, yeast cells were grown in SC-uracil medium, which, compared with the use of synthetic complete medium seen in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005878#pgen.1005878.g001" target="_blank">1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005878#pgen.1005878.g002" target="_blank">2</a>, likely caused slightly faster viability loss of all strains analyzed.</p

Elevated intracellular TAG level promotes longevity during chronological aging, whereas TAG depletion shortens lifespan.

<p>(A) Abbreviated view of TAG metabolism in yeast. (B) A laboratory strain yMK839 and its single and double lipase knockout derivatives were grown deeply into stationary phase in SC medium. At different time points, cells were spread to fresh YPD plates to quantify the colony forming units, expressed as percent survival. The plot was from two biological replicates of each strain. (C) Deleting <i>DGA1</i> and <i>LRO1</i> causes early death in stationary phase. Shown are averages of five biological repeats by the outgrowth approach. ** p < 0.01. Note the difference in time scale between panel B and panel C. Also, the two different assays for chronological lifespan quantification, i.e., colony forming units and outgrowth method, might give rise to differences in the absolute numbers of percent survival. The former did not differentiate colony size variations, whereas the latter outgrowth assay, which relied on the population growth rate, would be impacted by differences in doubling time and in the time when a cell exited the lag phase. (D) Quantification of intracellular TAG from day-8 post-saturation cultures in SC medium. (E) Growth comparison of yMK839 and its TAG-rich and -depleted derivatives. Growth curves were obtained as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005878#pgen.1005878.g001" target="_blank">Fig 1A</a>.</p

Culture medium pH changes are comparable among strains with different chronological lifespan.

<p>The pH changes of YPD cultures were monitored for 312 hours after inoculating overnight cultures to fresh medium (Panel A). In a second set of experiments (Panel B) the medium pH was measured from 4-day old YPD cultures of the indicated lab and wild strains. n ≥ 3.</p

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga <em>Nannochloropsis oceanica</em> CCMP1779

<div><p>Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes <em>Nannochloropsis oceanica</em> an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of <em>N. oceanica</em> CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the <em>N. oceanica</em> CCMP1779 gene repertoire with the recently published <em>N. gaditana</em> genome identified 2,649 genes likely specific to <em>N. oceanica</em> CCMP1779. Many of these <em>N. oceanica</em>–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of <em>N. oceanica</em> CCMP1779 are provided. The availability of genomic and transcriptomic data for <em>Nannochloropsis oceanica</em> CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.</p> </div