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

Growth-dependent secretome of Candida utilis

Recently, the food yeast Candida utilis has emerged as an excellent host for production of heterologous proteins. Since secretion of the recombinant product is advantageous for its purification, we characterized the secreted proteome of C. utilis. Cells were cultivated to the exponential or stationary growth phase, and the proteins in the medium were identified by MS. In parallel, a draft genome sequence of C. utilis strain DSM 2361 was determined by massively parallel sequencing. Comparisons of protein and coding sequences established that C. utilis is not a member of the CUG clade of Candida species. In total, we identified 37 proteins in the culture solution, 17 of which were exclusively present in the stationary phase, whereas three proteins were specific to the exponential growth phase. Identified proteins represented mostly carbohydrate-active enzymes associated with cell wall organization, while no proteolytic enzymes and only a few cytoplasmic proteins were detected. Remarkably, cultivation in xylose-based medium generated a protein pattern that diverged significantly from glucose-grown cells, containing the invertase Inv1 as the major extracellular protein, particularly in its highly glycosylated S-form (slow-migrating). Furthermore, cultivation without ammonium sulfate induced the secretion of the asparaginase Asp3. Comparisons of the secretome of C. utilis with those of Kluyveromyces lactis and Pichia pastoris, as well as with those of the human fungal pathogens Candida albicans and Candida glabrata, revealed a conserved set of 10 and six secretory proteins, respectively

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nudleotide polymorphisms

The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7 Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C. jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C. utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid

Uml2 is a novel CalB-type lipase of Ustilago maydis with phospholipase A activity

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities

T cell proliferation is reduced in MOG_35-55 treated animals.

<p><b>(A)</b> T cell proliferation in response to rechallenge with MOG_35-55 at the indicated concentrations. Spleen cell proliferation at day 15 of EAE of animals of the indicated groups was measured by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155082#pone.0155082.ref003" target="_blank">3</a>]H-thymidine incorporation in the presence of increasing concentrations of MOG_35-55 (0–50 μg/ml) in quadruplicates of four independent experiments. Depicted is the mean ± SEM of six mice per group. <b>(B)</b> Number of FoxP3 positive cells gated on CD4 T cells of lymphoid organs. Cells from the indicated organs were prepared at day 15 of EAE from the four groups. Intranuclear FoxP3 staining of was used to determine frequencies of regulatory T cell within the CD4 population in three independent experiments with 4 mice per group. dLN: immunization site draining lymph node; mLN: mesenteric (gut-draining) lymph node; PP: Peyer’s patches. EAE = immunized mice, untreated; CU ctrl = <i>C</i>. <i>utilis</i> wild-type; CU MOG = CBCu17. Asterisks indicate significance (* p < 0.05; *** p < 0.01 *** p < 0.001) student’s t test.</p

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms

Rupp O, Brinkrolf K, Buerth C, et al. The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms. Journal of Biotechnology. 2015;211:20-30.The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid. (C) 2015 Elsevier B.V. All rights reserved

Clinical manifestation of EAE in mice after oral gavage of <i>C</i>. <i>utilis</i> expressing MOG_35-55.

<p><b>(A)</b> Mean clinical score of four independent experiments testing the preventive potential of <i>C</i>. <i>utilis</i> expressing MOG_35-55. C57BL/6 mice (n = 6–8) were immunized and 1.5 x 10⁸ <i>C</i>. <i>utilis</i> were applied daily by oral gavage for 7 days prior immunization for 10 days. <i>C</i>. <i>utilis</i> expressing MOG_35-55 (CBCu17) was tested either alive or heat killed. Control groups received 1.5 x 10⁸ wild-type <i>Candida utilis</i> (ctrl.) or no cells (EAE). <b>(B)</b> Incidence of EAE and <b>(C)</b> maximal score at the peak of the disease after oral gavage of <i>C</i>. <i>utilis</i> to C57BL/6 mice in the indicated groups. Incidence was calculated as percentage of mice that displayed clinical symptoms of a score of one for more than two days deviated to number of immunized mice in the given groups for each experiment. Clinical scoring was assessed daily using the following system: 0 = no clinical signs; 1 = tail paralysis; 2 = hind limb paresis; 3 = hind limb paralysis; 4 = fore limp paresis; 5 = death. Depicted is the mean ± SEM of four independent experiments. Asterisks indicate significance (* p < 0.05; *** p < 0.01 *** p < 0.001) student’s t test.</p

MOG production and localization in <i>C</i>. <i>utilis</i>.

<p><b>(A)</b> Transformants of CBCu7 and CBCu8 were incubated for 72 h in YPD medium supplemented with 10 μg/ml Nourseothricin. Cells were harvested and washed two times with PBS. 20 μl of cells were loaded on a SDS gel (4–20% acrylamide) and the MOG peptide was detected in an immunoblot with a mouse anti-MOG_35-55 antibody and a secondary horseradish peroxidase coupled anti-mouse antibody. Protein masses are indicated in kDa. Strain MKCu1 (vc) was used as control. Lane 1–3: three independent transformants. <b>(B)</b> Strain CBCu8 was incubated for 72 h in YPD medium and MOG was detected on the surface of non permeabilized cells with a mouse anti-MOG_35-55 antibody and a secondary FITC-coupled anti-mouse antibody. Nuclear DNA was stained with 4′,6-Diamidin-2-phenylindol (DAPI). Strain MKCu1 (<i>TDH3</i>p) was used as negative control. <b>(C)</b> Feeding protocol. On day 0 (d0) fecal pellets were collected and one group (n = 6) was administered with 1x10⁷ CBCu17 cells, another group with 1x10⁸ cells. Fecal pellets were collected every 24 hours and on day 2 (d2) mice were fed with CBCu17 cells again. On day 11 the experiment was stopped. The collected fecal pellets were plated out on YPD agar plates containing ampicillin (100 μg/ml) and Nourseothricin (10 μg/ml) to prevent bacterial growth and colony forming units were counted. Mean values of three measurements and standard deviations are shown.</p

MOG expression vector.

<p><b>(A)</b> Scheme of the expression unit of the single MOG plasmid pCB13 and the corresponding encoded amino acid sequence. <b>(B)</b> Scheme of the tandem MOG expression plasmid pCB10 and amino acid sequence. The coding sequence for the MOG epitope was inserted between the secretion signal sequence (red) and GPI anchor sequence (blue) of the <i>C</i>. <i>utilis GAS1</i> sequence. The immunogenic MOG epitope (green) is linked to the <i>GAS1</i> GPI sequence with a non immunogenic MOG spacer sequence (black). The potential GPI attachment site (ω) is highlighted in orange. <i>Nhe</i>I restriction sites are in <i>italics</i>.</p