Dynamic Changes in the Transcriptome and Methylome of Chlamydomonas reinhardtii throughout Its Life Cycle

The green alga Chlamydomonas reinhardtii undergoes gametogenesis and mating upon nitrogen starvation. While the steps involved in its sexual reproductive cycle have been extensively characterized, the genome-wide transcriptional and epigenetic changes underlying different life cycle stages have yet to be fully described. Here, we performed transcriptome and methylome sequencing to quantify expression and DNA methylation from vegetative and gametic cells of each mating type and from zygotes. We identified 361 gametic genes with mating type-specific expression patterns and 627 genes that are specifically induced in zygotes; furthermore, these sex-related gene sets were enriched for secretory pathway and alga-specific genes. We also examined the C. reinhardtii nuclear methylation map with base-level resolution at different life cycle stages. Despite having low global levels of nuclear methylation, we detected 23 hypermethylated loci in gene-poor, repeat-rich regions. We observed mating type-specific differences in chloroplast DNA methylation levels in plus versus minus mating type gametes followed by chloroplast DNA hypermethylation in zygotes. Lastly, we examined the expression of candidate DNA methyltransferases and found three, DMT1a, DMT1b, and DMT4, that are differentially expressed during the life cycle and are candidate DNA methylases. The expression and methylation data we present provide insight into cell type-specific transcriptional and epigenetic programs during key stages of the C. reinhardtii life cycle

Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype

BACKGROUND: Improvement 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. RESULTS: We 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. CONCLUSIONS: Functional 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 system

A Toolkit for the Characterization of the Photoprotective Capacity of Green Algae

International audienceWhile light is a crucial energy source in photosynthetic organisms, if its intensity exceeds their photosynthetic capacity it may cause light-induced damage. A dominant photoprotective mechanism in plants and algae is the qE (quenching of energy), the major component of nonphotochemical quenching (NPQ). qE is a process that dissipates absorbed excitation energy as heat, ensuring cell survival even under adverse conditions. The present protocol gathers together a set of experimental approaches (in vivo chlorophyll fluorescence, western blotting, growth and cellular chlorophyll content at very strong light) that collectively allow for the characterization of the qE capacity of the model green algae Chlamydomonas reinhardtii

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis.

The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k cat/K m) of ~10 4 M −1 s −1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R P isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities. The redeployment of catalytic machinery in naturally occurring enzyme active sites for noncognate reactions holds considerable promise as a method for obtaining new biocatalysts 1,2. Alterations of substrate specificity and stereospecificity and the enhancement of preexisting promiscuous activities of enzymes have been accomplished by library-based directed evolution approaches 3. However, introducing completely new catalytic activities remains