Genome and Transcriptome Analysis of the Food-Yeast Candida utilis

The industrially important food-yeast Candida utilis is a Crabtree effect-negative yeast used to produce valuable chemicals and recombinant proteins. In the present study, we conducted whole genome sequencing and phylogenetic analysis of C. utilis, which showed that this yeast diverged long before the formation of the CUG and Saccharomyces/Kluyveromyces clades. In addition, we performed comparative genome and transcriptome analyses using next-generation sequencing, which resulted in the identification of genes important for characteristic phenotypes of C. utilis such as those involved in nitrate assimilation, in addition to the gene encoding the functional hexose transporter. We also found that an antisense transcript of the alcohol dehydrogenase gene, which in silico analysis did not predict to be a functional gene, was transcribed in the stationary-phase, suggesting a novel system of repression of ethanol production. These findings should facilitate the development of more sophisticated systems for the production of useful reagents using C. utilis

Gene Cloning and Molecular Characterization of an Extracellular Poly(l-Lactic Acid) Depolymerase from Amycolatopsis sp. Strain K104-1

We have isolated a polylactide or poly(l-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H(57), D(102), and S(195) (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H(74), D(111), and S(197). The G(193) residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S(195) and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H(74), D(111), and S(197) are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, ɛ-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA

Transcription results of 2 putative hexose transporters of <i>C. utilis</i>.

<p>Transcription results of 2 putative hexose transporters of <i>C. utilis</i>.</p

Phylogenetic tree of nitrate assimilation related genes.

<p>This tree was built on concatenated homologous sequences of all three genes (nitrate/nitrite transporter, nitrate reductases, and nitrite reductase) in Ascomycota and Basidiomycota from the subkingdom Dikarya (Materials and Methods) by the ML method.</p

Antisense transcripts of <i>C. utilis ADH1</i> (cut01g0000110) differentially expressed between log- and stationary-phases.

<p>Expressions at the <i>ADH1</i> locus was visualized using the Genaris integrated Next-Generation Sequencing Data Analysis Platform (GiNeS) (Genaris, Inc., Kanagawa, Japan).</p

Genome sequencing overview.

<p>Genome sequencing overview.</p