Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism.

Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas.

MicroRNAs (miRNAs) play important roles in diverse biological processes in eukaryotes, generally through degradation and/or inhibition of the translation of target mRNAs. MicroRNAs are loaded into Argonaute (AGO) proteins to form the RNA-induced silencing complex (RISC) and used as guides to identify complementary transcripts. The distinct functions and features, such as associated small RNA classes and modes of silencing, of individual AGO paralogs have been well documented in multicellular eukaryotes.

Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains.

Chlamydomonas is a very attractive candidate plant cell factory. However, its main drawback is the difficulty to find the transformants that robustly express heterologous genes randomly inserted in the nuclear genome. We previously showed that domestic squalene synthase (SQS) gene of Chlamydomonas was much more efficiently overexpressed in a mutant strain [UV-mediated mutant (UVM) 4] than in wild type.

Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains.

We attempted to overexpress three types of expression cassettes, each of which contained a different open reading frame (ORF) of domestic Chlamydomonas cDNAs. Each ORF was strongly driven by an artificial hybrid promoter. We used two wild-type Chlamydomonas strains (i.

Release of single cells from the colonial oil-producing alga Botryococcus braunii by chemical treatments.

We tested for chemical reagents that would be useful in preparing a large number of vital single cells from colonial Botryococcus braunii B-race, variety Showa. Among the 18 reagents assayed, glycerol and erythritol showed the highest potency for releasing single cells. Incubation in medium containing these reagents released 40-50 % single cells in 15 min.

THE ROLE OF ZINC FINGER PROTEIN IN RNAi INTERFERENCE IN A UNICELLULAR GREEN ALGA CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE).

In our previous study, we generated a strain of 19-P (1030) in which artificial RNA interference (RNAi) was induced by transcribing a hairpin RNA of ~780-bp stem. We utilized this RNAi-induced strain to uncover RNAi-related genes. Random insertional mutagenesis was performed to generate tag-mutants that show a RNAi deficient phenotype.

Involvement of Elongin C in the spread of repressive histone modifications.

In our previous work, we induced RNA interference (RNAi) against the spectinomycin resistance-conferring aadA transgene by transcribing a long inverted repeat in Chlamydomonas reinhardtii. However, after long-term culture, the level of transcripts of the inverted repeat was markedly decreased. In this study, we performed random insertional mutagenesis of the RNAi strain to identify the genes that contribute to the transcriptional silencing of the silencer construct.

Degenerated recognition property of a mitochondrial homing enzyme in the unicellular green alga Chlamydomonas smithii.

Target sequence cleavage is the essential step for intron invasion into an intronless allele. DNA cleavage at a specific site is performed by an endonuclease, termed a homing enzyme, which is encoded by an open reading frame within the intron. The recognition properties of them have only been analyzed in vitro, using purified, recombinant homing enzyme and various mutated DNA substrates, but it is unclear whether the homing enzyme behaves similarly in vivo.

Shared molecular characteristics of successfully transformed mitochondrial genomes in Chlamydomonas reinhardtii.

Three types of respiratory deficient mitochondrial strains have been reported in Chlamydomonas reinhardtii: a deficiency due to (i) two base substitutions causing an amino acid change in the apocytochrome b (COB) gene (i.e., strain named dum-15), (ii) one base deletion in the COXI gene (dum-19), or (iii) a large deletion extending from the left terminus of the genome to somewhere in the COB gene (dum-1, -14, and -16).

Adaptation of intronic homing endonuclease for successful horizontal transmission.

Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion.