BackgroundImprovement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.ResultsWe sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches.ConclusionsFunctional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0670-3) contains supplementary material, which is available to authorized users.

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10-20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined. First, WRPW and related tetrapeptide motifs have been well characterized in several repressors. Second, a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. Here we describe a yeast two-hybrid screen that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. We show that one of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation. Comparison between diverse eh1 motifs reveals a bias for the phosphoacceptor amino acids serine and threonine at a fixed position, and a mutational analysis of Oddskipped indicates that these residues are critical for efficient interactions with Groucho and for repression in vivo. Our data suggest that phosphorylation of these phosphomeric residues, if it occurs, will down-regulate Groucho binding and therefore repression, providing a mechanism for posttranslational control of Grouchomediated repression.Negative transcriptional regulation is a strategy that has been commonly selected in evolution for setting up and maintaining gene expression patterns. A striking case in point is the process of segmentation in the early Drosophila embryo. This developmental system is regulated almost exclusively by transcription factors, many of which are repressors that silence the expression of their targets (35; reviewed in reference 57). Mutations in genes encoding these transcriptional repressors lead to the loss of repressor activity that normally restricts the expression domains of downstream genes, causing disruptions in the metameric subdivision of the fly embryo.One key principle to emerge from studies on Drosophila segmentation and on developmental processes in other model organisms is the fact that DNA-binding repressors in general do not operate on their own. Instead, they complex with nuclear coregulators, called corepressors, tethering them to promoters whose expression is subsequently blocked (43). Groucho (Gro), one such ubiquitously expressed corepressor that is highly conserved throughout evolution from worms to humans, has been shown to interact with and to potentiate the repressor function of a vast number of transcription factors, including many of those acting in segmentation (8). It is not fully understood how Gro and its Transducin-like Enhancer-of-split (TLE) mammalian homologues elicit transcriptional repression, although given that these corepressors associate with histones and bind histone deacetylases, they are likely to ...

Fly stocks w 1118 , ab 1 , omb 1, w [1118] P{Ubi-GFP(S65T)nls}X P{neoFRT}18A and MKRS, P{hsFLP}86E/TM6B, Tb[1] stocks were obtained from the Bloomington stock center. The y w brk m68 f 36a FRT18a/FM7a stock was kindly provided by C. Rushlow (Jazwinska et al., 1999a), the yw hsFLP f 36a ; ab>f + >GAL4-lacZ/CyO stock was kindly provided by K. Basler (Moreno et al., 2002), and the omb D4 w/FM6 stock was kindly provided by G. Pflugfelder. Flies used for expression pattern markers included: X47 (Campbell and Tomlinson, 1999) for brk-lacZ expression; bi x35 for omb-lacZ expression (Sun et al., 1995); and P{ry +t7.2 =PZ}salm 03602 cn 1 /CyO; ry 506 (Drosophila genome project) for sal-lacZ expression. Lines for ectopic expression using the GAL4/UAS system (Brand and Perrimon, 1993) included: MS1096-GAL4, C765-GAL4 (kindly provided by Gomez-Skarmeta) and Vg B GAL4 (kindly provided by S. Carroll), UAS-brk (C. Rushlow and E. Moreno) and UAS-omb (Grimm and Pflugfelder, 1996). Clonal analysisHomozygous loss-of-function clones were generated by hsFLP-FRT recombination (Xu and Rubin, 1993 MKRS, P{hsFLP}86E/TM6B, Tb[1] and larvae were heat shocked 24-72 hours after egg-laying at 37°C for 1-2 hours. Wing discs were dissected and analyzed after 24-72 hours, or vials were kept at 25°C until flies hatched and wings were analyzed. Mutant clones in the wing disc were detected by lack of GFP expression, and in the adult wing by f 36a phenotype.Flip-out clones ectopically expressing ab were generated in larvae of the genotype yw hsFLP f 36a ; ab>f + >GAL4-lacZ/UAS-ab following recombination between FRT elements (>), initiated by heat induction of the HS-FLP recombinase transgene for 30 minutes at 34°C. These clones were marked by gain of lacZ expression in the disc, and by the cell-autonomous f 36a trichome phenotype in adult wings. A similar set of crosses was used to generate flip-out clones misexpressing high levels of omb.