Growth-dependent secretome of Candida utilis

Abstract

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.

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

“…To eliminate acetonitrile, all samples were evaporated using a Speedvac (Genevac, Ipswich, England), and the peptides were resolubilized in 20 l of 0.1% trifluoroacetic acid (TFA). The amount of peptides in all samples was determined using a NanoDrop ND-1000 (Isogen Life Science, IJsselstein, The Netherlands) at 205 nm as described before (14,16,41,42).…”

Candida utilis and Cyberlindnera (Pichia) jadinii: yeast relatives with expanding applications

“…Alignment of multiple sequence reads to an arbitrarily assigned C. jadinii genomic consensus sequence revealed 50 % of matches, consistent with a diploid genome for this yeast. On the other hand, mapping of C. utilis sequence reads (Buerth et al 2011) revealed broad SNP occurrence peaks at 33 and 66 % on the same C. jadinii consensus, which suggests for C. utilis a ploidy of 3; triploidy was additionally confirmed by FACS of nuclear stained cells (Rupp et al 2015). Triploidy and not tetraploidy of C. utilis is consistent with its inability to sporulate (Kurtzman and BasehoarPowers 2011).…”

“…Automated gene prediction indicated 6147 and 5689 ORFs in C. utilis and C. jadinii, respectively; 16 unique ORFs for C. utilis and 64 unique ORFs for C. jadinii may not only represent genuine genetic differences but may also reflect larger gap sizes in the reported C. utilis genome sequence. Phylogenetic tree analyses of orthologous genes revealed that both yeast species are not members of the CUG-clade comprising the species Candida albicans, Debaromyces hansenii, and Pichia stipitis (Tomita et al 2012), which was confirmed by verifying a CUG-encoded conserved leucine residue in the Inv1 protein (Buerth et al 2011). Until today, functional gene analyses to verify phenotypic traits experimentally have not been reported for either yeast.…”

“…Progress was greatly aided by the finding that C. utilis is not a member of the CUG-clade (see above), allowing adaptation of vector components and transformation procedures from other yeasts, especially from S. cerevisiae. As transformation host for C. utilis strain ATCC 9950 (alternative designations DSM2361, CBS5609, NRRL Y-900, NBRC0988) has been used with few exceptions; this strain is safely used in the food industry for at least seven decades (Kurtzman et al 1979;Lawford et al 1979) and its genomic sequence has recently been established (Buerth et al 2011;Tomita et al 2012). The first transformation system for C. utilis was described by Kondo et al (1995), who used an integrative transformation vector expressing a gene encoding a mutated ribosomal protein L41 conferring cycloheximide resistance as dominant selection marker.…”

“…Sporulation of C. jadinii but not C. utilis was observed; therefore, it was suggested that C. utilis represents the anamorphic (asexual) growth form of C. jadinii as the corresponding parental teleomorph (Kurtzman et al 1979). The close relation of both species was definitely clarified by comparing full genomic sequences of C. utilis strain ATCC9950 (Buerth et al 2011;Tomita et al 2012) and C. jadinii strain CBS1600 (Rupp et al 2015). This analysis revealed similar genome sizes (12.8 and 12.7 Mbp, respectively) and an overall sequence identity of 98 %; furthermore, the alignment of both genomes suggested that genomes of both species are located on 7-8 chromosomes within the size range of 1.265-2.451 Mbp (Rupp et al 2015).…”