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Heterologous protein secretion by Candida utilis

  • Applied genetics and molecular biotechnology
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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.

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References

  • Bekatorou A, Psaríanos C, Koutinas AA (2006) Production of food grade yeasts. Food Technol Biotechnol 44:407–415

    Google Scholar 

  • Boze H, Moulin G, Galzy P (1992) Production of food and fodder yeasts. Crit Rev Biotechnol 12:65–86

    Google Scholar 

  • Buerth C, Heilmann CJ, Klis FM, de Koster CG, Ernst JF, Tielker D (2011) Growth-dependent secretome of Candida utilis. Microbiology 157:2493–2503

    Article  PubMed  CAS  Google Scholar 

  • Cantero PD, Lengsfeld C, Prill SK, Subanović M, Román E, Pla J, Ernst JF (2007) Transcriptional and physiological adaptation to defective protein-O-mannosylation in Candida albicans. Mol Microbiol 64:1115–1128

    Article  PubMed  CAS  Google Scholar 

  • Chávez FP, Pons T, Delgado JM, Rodriguez L (1998) Cloning and sequence analysis of the gene encoding invertase (INV1) from the yeast Candida utilis. Yeast 14:1223–1232

    Article  PubMed  Google Scholar 

  • Eisenhaber B, Schneider G, Wildpaner M, Eisenhaber F (2004) A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe. J Mol Biol 337:243–253

    Article  PubMed  CAS  Google Scholar 

  • Fonzi WA, Irwin MY (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134:717–728

    PubMed  CAS  Google Scholar 

  • Frieman MB, Cormack BP (2004) Multiple sequence signals determine the distribution of glycosylphophosphatidylinositol proteins between the plasma membrane and cell wall in Saccharomyces cerevisiae. Microbiology 150:3105–3114

    Article  PubMed  CAS  Google Scholar 

  • Gerami-Nejad M, Berman J, Gale CA (2001) Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans. Yeast 18:859–864

    Article  PubMed  CAS  Google Scholar 

  • Grinna LS, Tschopp JF (1989) Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast 5:107–115

    Article  PubMed  CAS  Google Scholar 

  • Hong YR, Chen YL, Farh L, Yang WJ, Liao CH, Shiuan D (2006) Recombinant Candida utilis for the production of biotin. Appl Microbiol Biotechnol 71:211–221

    Article  PubMed  CAS  Google Scholar 

  • Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86:403–417

    Article  PubMed  CAS  Google Scholar 

  • Ikushima S, Fujii T, Kobayashi O (2009a) Efficient gene disruption in the high-ploidy yeast Candida utilis using the Cre-loxP system. Biosci Biotechnol Biochem 73:879–884

    Article  PubMed  CAS  Google Scholar 

  • Ikushima S, Fujii T, Kobayashi O, Yoshida S, Yoshida A (2009b) Genetic engineering of Candida utilis yeast for efficient production of L-lactic acid. Biosci Biotechnol Biochem 73:1818–1824

    Article  PubMed  CAS  Google Scholar 

  • Ikushima S, Minato T, Kondo K (2009c) Identification and application of novel autonomously replicating sequences (ARSs) for promoter-cloning and co-transformation in Candida utilis. Biosci Biotechnol Biochem 73:152–159

    Article  PubMed  CAS  Google Scholar 

  • Iwakiri R, Noda Y, Adachi H, Yoda K (2005) Isolation of the YAP1 homologue of Candida utilis and its use as an efficient selection marker. Yeast 22:1079–1087

    Article  PubMed  CAS  Google Scholar 

  • Kok RG, Christoffels VM, Vosman B, Hellingwerf KJ (1993) Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases. J Gen Microbiol 139:2329–2342

    Article  PubMed  CAS  Google Scholar 

  • Kondo K, Saito T, Kajiwara S, Takagi M, Misawa N (1995) A transformation system for the yeast Candida utilis: use of a modified endogenous ribosomal protein gene as a drug-resistance marker and ribosomal DNA as an integration target for vector DNA. J Bacteriol 177:7171–7177

    PubMed  CAS  Google Scholar 

  • Kondo K, Miura Y, Sone H, Kobayashi K, Iijima H (1997) High-level expression of a sweet protein, monellin, in the food yeast Candida utilis. Nat Biotechnol 15:453–457

    Article  PubMed  CAS  Google Scholar 

  • Kurtzman CP, Johnson CJ, Smiley MJ (1979) Determination of conspecificity of Candida utilis and Hansenula jadinii through DNA reassociation. Mycologia 71:844–847

    Article  Google Scholar 

  • Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D (2012) Recombinant protein production in yeasts. Methods Mol Biol 824:329–358

    Google Scholar 

  • Miura Y, Kondo K, Saito S, Shimada H, Fraser PD, Misawa N (1998) Production of the carotenoids lycopene, β-carotene, and astaxanthin in the food yeast Candida utilis. Appl Environ Microbiol 64:1226–1229

    PubMed  CAS  Google Scholar 

  • Miura Y, Kettoku M, Kato M, Kobayashi K, Kondo K (1999) High level production of thermostable α-amylase from Sulfolobus solfataricus in high-cell density culture of the food yeast Candida utilis. J Mol Microbiol Biotechnol 1:129–134

    PubMed  CAS  Google Scholar 

  • Müller G (2011) Novel applications for glycosylphosphatidylinositol-anchored proteins in pharmaceutical and industrial biotechnology. Mol Membr Biol 28:187–205

    Article  PubMed  Google Scholar 

  • Prill SK, Klinkert B, Timpel C, Gale CA, Schröppel K, Ernst JF (2005) PMT family of Candida albicans: five protein mannosyltransferase isoforms affect growth, morphogenesis and antifungal resistance. Mol Microbiol 55:546–560

    Article  PubMed  CAS  Google Scholar 

  • Setiadi ER, Doedt T, Cottier F, Noffz C, Ernst JF (2006) Transcriptional response of Candida albicans to hypoxia: linkage of oxygen sensing and Efg1p-regulatory networks. J Mol Biol 361:399–411

    Article  PubMed  CAS  Google Scholar 

  • Sharma R, Chisti Y, Banerjee C (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662

    Article  PubMed  CAS  Google Scholar 

  • Sherman F, Fink G, Hicks J (1986) Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sonneborn A, Tebarth B, Ernst JF (1999) Control of white-opaque phenotypic switching in Candida albicans by the Efg1p morphogenetic regulator. Infect Immun 67:4655–4660

    PubMed  CAS  Google Scholar 

  • Su GD, Huang DF, Han SY, Zheng SP, Lin Y (2010) Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis. Appl Microbiol Biotechnol 86:1493–1501

    Article  PubMed  CAS  Google Scholar 

  • Tamakawa H, Ikushima S, Yoshida S (2010a) Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain. J Biosci Bioeng 113:73–75

    Article  Google Scholar 

  • Tamakawa H, Ikushima S, Yoshida S (2010b) Ethanol production from xylose by a recombinant Candida utilis strain expressing protein-engineered xylose reductase and xylitol dehydrogenase. Biosci Biotechnol Biochem 75:1994–2000

    Article  Google Scholar 

  • Tanaka T, Yamada R, Ogino C, Kondo A (2012) Recent developments in yeast cell surface display toward extended applications in biotechnology. Appl Microbiol Biotechnol 95:577–591

    Article  PubMed  CAS  Google Scholar 

  • Tomita Y, Ikeo K, Tamakawa H, Gojobori T, Ikushima S (2012) Genome and transcriptome analysis of the food-yeast Candida utilis. PLoS One 7:e37226

    Article  PubMed  CAS  Google Scholar 

  • Uhl MA, Johnson AD (2001) Development of Streptococcus thermophilus lacZ as a reporter gene for Candida albicans. Microbiology 147:1189–1195

    PubMed  CAS  Google Scholar 

  • Wei W, Hong-Lan Y, Huifang B, Daoyuan Z, Qi-Mu-Ge S, Wood AJ (2010) The effective expression of xylanase gene in Candida utilis by 18S rDNA targeted homologous recombination in pGLR9K. Mol Biol Rep 37:2615–2620

    Article  Google Scholar 

  • Wilson RB, Davis D, Mitchell AP (1999) Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions. J Bacteriol 181:1868–1874

    PubMed  CAS  Google Scholar 

  • Yamada Y, Matsuda M, Mikata K (1995) The phylogenetic relationships of Pichia jadinii, formerly classified in the genus Hansenula, and related species based on the partial sequence of 18S and 26 S ribosomal RNAs (Saccharomycetaceae). Biosci Biotechnol Biochem 59:518–520

    Article  PubMed  CAS  Google Scholar 

  • Yin QY, de Groot PW, Dekker HL, de Jong L, Klis FM, de Koster CG (2005) Comprehensive proteomic analysis of Saccharomyces cerevisiae cell walls: identification of proteins covalently attached via glycosylphosphatidylinositol remnants or mild alkali-sensitive linkages. J Biol Chem 280:20894–20901

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was funded by the Cluster of Industrial Biotechnology NRW CLIB2021.

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Authors and Affiliations

  1. Department Biologie, Molekulare Mykologie, Heinrich-Heine-Universität, Universitätsstrasse 1/26.12.01, 40225, Düsseldorf, Germany

    Maya Kunigo, Christoph Buerth, Denis Tielker & Joachim F. Ernst

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  1. Maya Kunigo
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  2. Christoph Buerth
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  3. Denis Tielker
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  4. Joachim F. Ernst
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Corresponding author

Correspondence to Joachim F. Ernst.

Additional information

M. Kunigo and C. Buerth contributed equally to this study.

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Kunigo, M., Buerth, C., Tielker, D. et al. Heterologous protein secretion by Candida utilis . Appl Microbiol Biotechnol 97, 7357–7368 (2013). https://doi.org/10.1007/s00253-013-4890-1

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  • DOI: https://doi.org/10.1007/s00253-013-4890-1

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