Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.

BackgroundThe major cell cycle control acting at the G2 to mitosis transition is triggered in all eukaryotes by cyclin-dependent kinases (CDKs). In the fission yeast Schizosaccharomyces pombe the activation of the G2/M CDK is regulated primarily by dephosphorylation of the conserved residue Tyr15 in response to the stress-nutritional response and cell geometry sensing pathways. To obtain a more complete view of the G2/M control we have screened systematically for gene deletions that advance cells prematurely into mitosis.ResultsA screen of 82% of fission yeast non-essential genes, comprising approximately 3,000 gene deletion mutants, identified 18 genes that act negatively at mitotic entry, 7 of which have not been previously described as cell cycle regulators. Eleven of the 18 genes function through the stress response and cell geometry sensing pathways, both of which act through CDK Tyr15 phosphorylation, and 4 of the remaining genes regulate the G2/M transition by inputs from hitherto unknown pathways. Three genes act independently of CDK Tyr15 phosphorylation and define additional uncharacterized molecular control mechanisms.ConclusionsDespite extensive investigation of the G2/M control, our work has revealed new components of characterized pathways that regulate CDK Tyr15 phosphorylation and new components of novel mechanisms controlling mitotic entry.

In the yeast Hansenula polymorpha, the YNT1 gene encodes the high affinity nitrate transporter, which is repressed by reduced nitrogen sources such as ammonium or glutamine. Ynt1 protein is degraded in response to glutamine in the growth medium. Ynt1 disappears independently of YNT1 glutamine repression as shown in strains where YNT1 repression is abolished. Ynt1-green fluorescent protein chimera and a mutant defective in vacuolar proteinase A (⌬pep4) showed that Ynt1 is degraded in the vacuole in response to glutamine. The central hydrophilic domain of Ynt1 contains PEST-like sequences whose deletion blocked Ynt1 down-regulation. Site-directed mutagenesis showed that Lys-253 and Lys-270, located in this sequence, were involved in internalization and subsequent vacuolar degradation of Ynt1. Ynt1-ubiquitin conjugates were induced by glutamine and not nitrate. We conclude that glutamine triggers Ynt1 down-regulation via ubiquitinylation of lysines in the central hydrophilic domain, and proteolysis in the vacuole.

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3′UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas , and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.

Ynt1 is the sole high affinity nitrate transporter of the yeast Hansenula polymorpha. It is highly regulated by the nitrogen source, by being down-regulated in response to glutamine by repression of the YNT1 gene and Ynt1 ubiquitinylation, endocytosis, and vacuolar degradation. On the contrary, we show that nitrogen limitation stabilizes Ynt1 levels at the plasma membrane, requiring phosphorylation of the transporter. We determined that Ser-246 in the central intracellular loop plays a key role in the phosphorylation of Ynt1 and that the nitrogen permease reactivator 1 kinase (Npr1) is necessary for Ynt1 phosphorylation. Abolition of phosphorylation led Ynt1 to the vacuole by a pep12-dependent end4-independent pathway, which is also dependent on ubiquitinylation, whereas Ynt1 protein lacking ubiquitinylation sites does not follow this pathway. We found that, under nitrogen limitation, Ynt1 phosphorylation is essential for rapid induction of nitrate assimilation genes. Our results suggest that, under nitrogen limitation, phosphorylation prevents Ynt1 delivery from the secretion route to the vacuole, which, aided by reduced ubiquitinylation, accumulates Ynt1 at the plasma membrane. This mechanism could be part of the response that allows nitrate-assimilatory organisms to cope with nitrogen depletion.