Heterologous protein secretion by Candida utilis

Abstract

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.

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

“…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. In the following years, other dominant markers including heterologous genes aph, hph, nat, and ble genes (conferring resistance to G418, hygromycin B, nourseothricin, or zeocin, respectively [Shimada et al 1998;Kunigo et al 2013;Boňková et al 20 14]) and end oge nou s ge ne YA P 1 (conferr ing cycloheximide-resistance [Iwakiri et al 2005b]) were added for selection of transformants. Antibiotic-resistance markers were used to generate specific mutations in the C. utilis genome by homologous recombination with a disruption cassette encompassing the dominant marker gene.…”

“…Correct genomic integration in the desired locus has not been verified extensively in all cases, but the availability of the whole genome sequence should ameliorate this situation in the future. Multiple plasmid integrations have been reported for the rDNA and URA3 loci (Kondo et al 1997), while single-plasmid integrations were reported for the TDH3 and HIS3 loci (Kunigo et al 2013. For unknown reasons, high plasmid copy numbers were found to occur by integration at the URA3 locus (reported up to 90 copies); this property was exploited in a Bcocktail multicopy integration method^to randomly integrate different expression units at this site (Tamakawa et al 2013b).…”

“…With regard to the stability of chromosomally integrated plasmids, in the absence of a selective agent, variable results were obtained. High plasmid stability was observed for integrations in URA3 (Kondo et al 1997) and HIS3 (Kunigo et al 2013), while instability was shown for integrations in the rDNA (Kondo et al 1997) and both stability and instability were reported for integrations at the TDH3 locus (Kunigo et al 2013. Conceivably, mitotic recombination between homologous chromosomes may evict sectors of chromosomes carrying expression vectors in some cells, which may outcompete the parental cells in time, especially if the respective product is a Bbiosynthetic burden.T wo chromosomal autonomously replicating sequences (ARS) sequences have been described for C. utilis (Ikushima et al 2009c), although they provide only low copy numbers and their instability is undesirable for high-level production.…”

“…These promoters effected strong but unregulated expression of various heterologous genes. Kunigo et al (2013) recently identified highly xylose-inducible promoters of XDH1 and GXS1 genes encoding NAD-xylitol dehydrogenase and a glucose/xylose symporter, which on glucose media were completely inactive to express genes for the GFP reporter or the CalB lipase. In media containing both glucose and xylose, cells containing a XDH1/GXS1-promoter driven expression unit first grow without and-after glucose depletion-with strong production of heterologous proteins, which is a desired feature, especially for production of toxic proteins.…”

“…C. utilis secretes a limited set of proteins, lacking protease activity, into the medium (Buerth et al 2011). Homologous and heterologous signal sequences were successfully used to secrete heterologous proteins into the Wei et al 2010 growth medium of C. utilis (Table 2) including lipase B (CalB) of Pseudozyma aphidis (previously named Candida antarctica) (Kunigo et al 2013). The signal sequence of invertase, one of the most abundant proteins in the C. utilis secretome (Buerth et al 2011), directed higher secretion levels than that of the native CalB but surprisingly, the highest secretion levels were achieved using the heterologous pre-pro region of the α-factor precursor protein (Mfα1) of S. cerevisiae .…”