Computational protocol: The evolution of Jen3 proteins and their role in dicarboxylic acid transport in Yarrowia

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Protocol publication

[…] Twenty-three species were chosen based on their positions in the phylogenetic tree of hemiascomycetes (see Kurtzman ; for reference tree). For each species, genomes were retrieved from the indicated database: S. cerevisiae – SGD (; C. albicans – CGD (; Schizosaccharomyces pombe – Pombase (; Komagataella (Pichia) pastoris – Gent University (; Wickerhamomyces ciferrii, Kazachstania naganishii, and Naumovozyma castellii – the NCBI database (; Candida tropicalis – the Broad Institute (; Candida glabrata, Zygosaccharomyces rouxii, Eremothecium (Ashbya) gossypii, K. lactis, Lachancea kluyveri, Lachancea thermotolerans, and Debaryomyces hansenii – Génolevures (; Blastobotrys (Arxula) adeninivorans, Cyberlindnera fabianii, and Y. lipolytica – GRYC ( Species closely related to Y. lipolytica, that is, Y. yakushimensis CBS10253, Yarrowia galli CBS9722, Y. phangngensis CBS10407, Y. alimentaria CBS10151, and C. hispaniensis CBS9996, were sequenced and annotated in our laboratory at INRA Thiverval-Grignon (data not shown). Homologues of YLJEN genes were found via a homology search based on a two-step reciprocal approach using BLASTp (Altschul et al. ). In the first step, each YLJen protein was used as a query for a BLASTp search of the other species, with a cutoff E-value of 1e-10. Then, we used the top hit of each search to perform a reciprocal BLASTp search; if the top hit returned a Y. lipolytica gene different from a JEN gene, it was discarded. Amino acid sequences of all Jen proteins are given in . Pairwise comparisons of both amino acid identity and similarity were calculated using aligncopypair (EMBOSS; Rice et al. ). [...] The JEN gene tree was constructed based on an alignment of 61 Jen proteins, representing 18 yeast species. The alignment was performed with MultAlin (Corpet ) and manually corrected with GeneDoc v2.7.0 (Nicholas et al. ). The final 405-aa alignment was used to reconstruct trees with both Neighbor-Joining and Maximum-Likelihood algorithms, with three different amino acid substitution matrices (WAG, Dayhoff, and JTT). The models were corrected by a Γ-law distribution with four categories of evolution rates; both invariable sites and the α-parameter of the Γ-law distribution were optimized according to the data. Seaview v4.3.3 was used for these analyses (Gouy et al. ). [...] To reconstruct the evolutionary scenario of the Jen protein family in the Yarrowia clade, a species tree based on the concatenation of 912 proteins (data not shown) and a Jenp tree built as described above were reconciled with synteny data. A parsimonious approach was used to minimize the number of duplications and losses of JEN genes. For the phylogeny of JEN genes within the Yarrowia clade, KLJen2p was used as an outgroup. Protein sequences were aligned with MAFFT (Katoh et al. ) and edited with Gblocks (Castresana ). The phylogenetic tree constructed from the Jen protein alignment was constructed with Seaview v4.3.3 using the BioNJ distance method (Gouy et al. ). Bootstrap values were calculated with 100 replicates. […]

Pipeline specifications

Software tools BLASTP, EMBOSS, MultAlin, SeaView, MAFFT, Gblocks
Databases PomBase Genolevures
Application Phylogenetics
Organisms Saccharomyces cerevisiae, Yarrowia lipolytica
Chemicals Dicarboxylic Acids, Pyruvic Acid, Lactic Acid, Succinic Acid