Physical and computational analysis of the yeast Kluyveromyces lactis secreted proteome

New England Biolabs, Ipswich, MA 01938, USA.
Proteomics (Impact Factor: 3.81). 07/2008; 8(13):2714-23. DOI: 10.1002/pmic.200700764
Source: PubMed


Secretion of proteins is the most common approach to protein expression in Kluyveromyces lactis. A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by K. lactis cells. Multidimensional separations were conducted and RP online ESI-MS/MS analysis identified 81 secreted proteins. In addition, an in silico analysis predicted 178 K. lactis proteins to be secreted via the general secretory pathway (GSP). These two datasets were compared and approximately 70% of the K. lactis proteins detected in the culture medium possessed a GSP sequence. The detected proteins included those involved with cell wall structure and synthesis, carbohydrate metabolism, and proteolysis, a result that may have significant bearing on heterologous protein expression. Additionally, both the experimental and in silico datasets were compared to similar, previously published datasets for Candida albicans. With the methodology presented here, we provide the deepest penetration into a yeast secretome yet reported.

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Available from: Christopher Taron, May 24, 2015
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    • "In the classical pathway, proteins that contain a specific N-terminal signal sequence are translocated into the ER, transported to the Golgi and then secreted by secretory vesicles [30,32,33]. Accumulating evidence indicates that a large number of signal-less proteins can also be secreted through the non-classical pathway [1,2,4,5,10,21,23,31], [34], including metabolic enzymes, chaperones, translation factors, and transcriptional regulators [5,8,9,11,20-24,31,34-37]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme and has been shown by immuno-transmission electron microscopy (immuno-TEM) to be present on the surface of Candida albicans and Saccharomyces cerevisiae[14]. Using the immuno-TEM technique, another glycolytic enzyme, enolase, was also found on the surface of Listeria monocytogenes[24]. "
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    ABSTRACT: Protein secretion is a fundamental process in all living cells. Proteins can either be secreted via the classical or non-classical pathways. In Saccharomyces cerevisiae, gluconeogenic enzymes are in the extracellular fraction/periplasm when cells are grown in media containing low glucose. Following a transfer of cells to high glucose media, their levels in the extracellular fraction are reduced rapidly. We hypothesized that changes in the secretome were not restricted to gluconeogenic enzymes. The goal of the current study was to use a proteomic approach to identify extracellular proteins whose levels changed when cells were transferred from low to high glucose media. We performed two iTRAQ experiments and identified 347 proteins that were present in the extracellular fraction including metabolic enzymes, proteins involved in oxidative stress, protein folding, and proteins with unknown functions. Most of these proteins did not contain typical ER-Golgi signal sequences. Moreover, levels of many of these proteins decreased upon a transfer of cells from media containing low to glucose media. Using an extraction procedure and Western blotting, we confirmed that the metabolic enzymes (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, glucose-6-phosphate dehydrogenase, pyruvate decarboxylase), proteins involved in oxidative stress (superoxide dismutase and thioredoxin), and heat shock proteins (Ssa1p, Hsc82p, and Hsp104p) were in the extracellular fraction during growth in low glucose and that the levels of these extracellular proteins were reduced when cells were transferred to media containing high glucose. These proteins were associated with membranes in vesicle-enriched fraction. We also showed that small vesicles were present in the extracellular fraction in cells grown in low glucose. Following a transfer from low to high glucose media for 30 minutes, 98% of these vesicles disappeared from the extracellular fraction. Our data indicate that transferring cells from low to high glucose media induces a rapid decline in levels of a large number of extracellular proteins and the disappearance of small vesicles from the extracellular fraction. Therefore, we conclude that the secretome undergoes dynamic changes during transition from glucose-deficient to glucose-rich media. Most of these extracellular proteins do not contain typical ER signal sequences, suggesting that they are secreted via the non-classical pathway.
    Proteome Science 02/2014; 12(1):9. DOI:10.1186/1477-5956-12-9 · 1.73 Impact Factor
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    • "Recently, the secretomes of several yeast species have been analysed (Swaim et al., 2008; Madinger et al., 2009; Mattanovich et al., 2009; Sorgo et al., 2010; Stead et al., 2010). Comparisons of the secretome of C. utilis with those of K. lactis and P. pastoris grown in a glucose-containing medium identified 10 proteins that occur in all three species. "
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    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.
    Microbiology 06/2011; 157(Pt 9):2493-503. DOI:10.1099/mic.0.049320-0 · 2.56 Impact Factor
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    • "Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved propagated at high density in a bioreactor (Swaim et al., 2008; Madinger et al., 2009). Not knowing a priori as to which pfam00026 proteases may be most detrimental to heterologous proteins secreted from the K. lactis GG799 manufacturing strain, we elected to construct a set of five individual pfam00026 protease null mutants and assess each strain for its growth characteristics, protein expression performance and ability to improve recombinant protein quality. "
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    ABSTRACT: Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.
    FEMS Yeast Research 03/2011; 11(2):168-78. DOI:10.1111/j.1567-1364.2010.00703.x · 2.82 Impact Factor
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