Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.

A 2-His-I-carboxylate facial triad is a common feature of the active sites in a number of mononuclear non-heme iron(I1) enzymes. This structural motif was established crystallographically for five different classes of enzymes and inferred from sequence similarity for two other classes. The 2-His-1-carboxylate facial triad anchors the iron in the active site and at the same time maintains three additional cis-oriented sites. These sites can be used to bind other endogenous ligands or exogenous ligands such as substrate and/or 02, giving the metal center great flexibility to use different mechanistic strategies to perform a variety of chemical transformations.Keywords: 2-His-I -carboxylate facial triad; non-heme; oxygenase; iron ; mononuclear.Mononuclear non-heme iron(I1) enzymes catalyze a diverse range of important reactions in the metabolism of amino acids, nucleic acids, and fatty acids, the biodegradation of aromatic compounds, and the biosynthesis of antibiotics such as penicillin and cephalosporin (Fig. 1) [ I , 21. This emerging class of enzymes now rivals heme enzymes in the diversity of oxidative transformations catalyzed. While many of the mechanistic strategies employed by these two classes of enzymes are similar, there are some significant differences. Heme enzymes feature an iron porphyrin cofactor that is ligated to an amino acid residue on the polypeptide chain; such a ligand environment leaves only one coordination site available for the binding of exogenous ligands, typically 0, for dioxygen-activating enzymes. As a consequence, NADH is the usual source of the two electrons needed for the reaction and a reductase is required to mediate electron transfer into the heme active site. In contrast, mononuclear nonheme iron enzymes employ a variety of electron sources, often metal-bound substrates or cofactors, to reduce 0, to the peroxide oxidation state. This mechanistic feature requires a more flexible metal-coordination environment to allow for the binding of a number of exogenous ligands. Understanding how the ligand environment around the metal center in mononuclear non-heme iron enzymes affects their chemistry and allows them to perform reactions typically associated with their heme counterparts is an area of intense current interest. Emerging from recent crystallographic studies on mononuclear non-heme enzymes is a com-

No abstract

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 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 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.