Comprehensive Analysis of a Yeast Lipase Family in the Yarrowia Clade.

Lipases are currently the subject of intensive studies due to their large range of industrial applications. The Lip2p lipase from the yeast Yarrowia lipolytica (YlLIP2) was recently shown to be a good candidate for different biotechnological applications. Using a combination of comparative genomics approaches based on sequence similarity, synteny conservation, and phylogeny, we constructed the evolutionary scenario of the lipase family for six species of the Yarrowia clade. RNA-seq based transcriptome analysis revealed the primary role of LIP2 homologues in the assimilation of different substrates. Once identified, these YlLIP2 homologues were expressed in Y. lipolytica. The lipase Lip2a from Candida phangngensis was shown to naturally present better activity and enantioselectivity than YlLip2. Enantioselectivity was further improved by site-directed mutagenesis targeted to the substrate binding site. The mono-substituted variant V232S displayed enantioselectivity greater than 200 and a 2.5 fold increase in velocity. A double-substituted variant 97A-V232F presented reversed enantioselectivity, with a total preference for the R-enantiomer.

The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia.

Jen proteins in yeast are involved in the uptake of mono/dicarboxylic acids. The Jen1 subfamily transports lactate and pyruvate, while the Jen2 subfamily transports fumarate, malate, and succinate. Yarrowia lipolytica has six JEN genes: YALI0B19470g, YALI0C15488g, YALI0C21406g, YALI0D20108g, YALI0D24607g, and YALI0E32901g. Through phylogenetic analyses, we found that these genes represent a new subfamily, Jen3 and that these three Jen subfamilies derivate from three putative ancestral genes. Reverse transcription-PCR. revealed that only four YLJEN genes are expressed and they are upregulated in the presence of lactate, pyruvate, fumarate, malate, and/or succinate, suggesting that they are able to transport these substrates. Analysis of deletion mutant strains revealed that Jen3 subfamily proteins transport fumarate, malate, and succinate. We found evidence that YALI0C15488 encodes the main transporter because its deletion was sufficient to strongly reduce or suppress growth in media containing fumarate, malate, or succinate. It appears that the other YLJEN genes play a minor role, with the exception of YALI0E32901g, which is important for malate uptake. However, the overexpression of each YLJEN gene in the sextuple-deletion mutant strain ΔYLjen1-6 revealed that all six genes are functional and have evolved to transport different substrates with varying degrees of efficacy. In addition, we found that YALI0E32901p transported succinate more efficiently in the presence of lactate or fumarate.

Morphology, genome plasticity, and phylogeny in the genus ostreococcus reveal a cryptic species, O. mediterraneus sp. nov. (Mamiellales, Mamiellophyceae).

Coastal marine waters in many regions worldwide support abundant populations of extremely small (1-3 µm diameter) unicellular eukaryotic green algae, dominant taxa including several species in the class Mamiellophyceae. Their diminutive size conceals surprising levels of genetic diversity and defies classical species' descriptions. We present a detailed analysis within the genus Ostreococcus and show that morphological characteristics cannot be used to describe diversity within this group. Karyotypic analyses of the best-characterized species O. tauri show it to carry two chromosomes that vary in size between individual clonal lines, probably an evolutionarily ancient feature that emerged before species' divergences within the Mamiellales. By using a culturing technique specifically adapted to members of the genus Ostreococcus, we purified >30 clonal lines of a new species, Ostreococcus mediterraneus sp. nov., previously known as Ostreococcus clade D, that has been overlooked in several studies based on PCR-amplification of genetic markers from environment-extracted DNA. Phylogenetic analyses of the S-adenosylmethionine synthetase gene, and of the complete small subunit ribosomal RNA gene, including detailed comparisons of predicted ITS2 (internal transcribed spacer 2) secondary structures, clearly support that this is a separate species. In addition, karyotypic analyses reveal that the chromosomal location of its ribosomal RNA gene cluster differs from other Ostreococcus clades.

Comparative physiology of oleaginous species from the Yarrowia clade.

Yarrowia lipolytica is a genetically tractable yeast species that has become an attractive model for analyses of lipid metabolism, due to its oleaginous nature. We investigated the regulation and evolution of lipid metabolism in non-Saccharomycetaceae yeasts, by carrying out a comparative physiological analysis of eight species recently assigned to the Yarrowia clade: Candida alimentaria, Y. deformans, C. galli, C. hispaniensis, C. hollandica, C. oslonensis, C. phangngensis and Y. yakushimensis. We compared the abilities of type strains of these species to grow on 31 non hydrophobic (sugars and other carbohydrate compounds) and 13 hydrophobic (triglycerides, alkanes and free fatty acids) carbon sources. Limited phenotypic diversity was observed in terms of the range of substrates used and, in the case of short-chain fatty acids, their toxicity. We assessed the oleaginous nature of these species, by evaluating their ability to store and to synthesize lipids. The mean lipid content of cells grown on oleic acid differed considerably between species, ranging from 30% of cell dry weight in C. oslonensis to 67% in C. hispaniensis. Lipid synthesis in cells grown on glucose resulted in the accumulation of C18:1 (n-9) as the major compound in most species, except for C. alimentaria and Y. yakushimensis, which accumulated principally C18:2(n-6), and C. hispaniensis, which accumulated both C16:0 and C18:1(n-9). Thus, all species of the clade were oleaginous, but they presented specific patterns of growth, lipid synthesis and storage, and therefore constitute good models for the comparative analysis of lipid metabolism in this basal yeast clade.

Evolution of codon usage in the smallest photosynthetic eukaryotes and their giant viruses.

Prasinoviruses are among the largest viruses (>200 kb) and encode several hundreds of protein coding genes, including most genes of the DNA replication machinery and several genes involved in transcription and translation, as well as transfer RNAs (tRNAs). They can infect and lyse small eukaryotic planktonic marine green algae, thereby affecting global algal population dynamics. Here, we investigate the causes of codon usage bias (CUB) in one prasinovirus, OtV5, and its host Ostreococcus tauri, during a viral infection using microarray expression data. We show that 1) CUB in the host and in the viral genes increases with expression levels and 2) optimal codons use those tRNAs encoded by the most abundant host tRNA genes, supporting the notion of translational optimization by natural selection. We find evidence that viral tRNA genes complement the host tRNA pool for those viral amino acids whose host tRNAs are in short supply. We further discuss the coevolution of CUB in hosts and prasinoviruses by comparing optimal codons in three evolutionary diverged host-virus-specific pairs whose complete genome sequences are known.