The effect of carbon source on the secretome of Kluyveromyces lactis
New England Biolabs, Ipswich, MA 01938, USA. Proteomics
(Impact Factor: 3.81).
10/2009; 9(20):4744-54. DOI: 10.1002/pmic.200800915
A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by Kluyveromyces lactis cells. Here, we report changes detected in the K. lactis secretome as a result of growth in three different carbon sources: glucose, galactose and glycerol. A total of 151 secreted proteins were detected by multi-dimensional separations and reversed-phase online nanoESI-MS/MS analysis. From these, we were able to identify 63 proteins (termed the "base secretome") that were common to all three fermentation conditions. The majority of base secretome proteins, 79%, possessed general secretory pathway (GSP) sequences and were involved with cell wall structure, glycosylation, carbohydrate metabolism and proteolysis. There was little variation in the functional groupings of base secretome GSP proteins and GSP proteins that were not part of the base secretome. In contrast, the majority of non-GSP proteins detected were not part of the base secretome and the functions of these proteins varied significantly. Finally, through further identification of non-GSP proteins in carbon sources not originally tested, we have gained further evidence of a protein export mechanism separate from the GSP in K. lactis.
Available from: Bruce A Stanley
- "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
, 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. "
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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.
Available from: Farah Diba Abu Bakar
- "Conversely, feeding glucose into the designed medium of bioreactors may further increase recombinant Xyn2 production levels using K. lactis GG799. The reduction in recombinant Xyn2 activity could also be due to proteolysis by proteases because K. lactis secretes proteases in the presence of glucose, galactose and glycerol (Madinger et al. 2009). Proteolytic degradation has been an ongoing concern for the production of recombinant proteins in other hosts (Zhou and Zhang 2002), such as P. pastoris (Sinha et al. 2005) and S. cerevisiae (Rao et al. 1999). "
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ABSTRACT: Kluyveromyces lactis is an excellent host for a high cell density culture, which allows high expression levels of recombinant enzymes. Nutrient
composition and culture conditions affect the secretion, production level and stability of the recombinant host. Therefore,
it is technologically important to formulate a medium that stimulates high cell density and enhances the desired enzyme production
using K. lactis GG799. In this study, six media were initially compared, and a Plackett-Burman experimental design was employed to screen
for important components and trace elements. Nitrogen sources such as ammonium sulfate and free amino acid (casamino acid)
as well as compounds like MgSO4∙7H2O, Na2SO4, ZnSO4∙6H2O, MnSO4∙4H2O and KH2PO4 affected biomass concentrations (5.67 g/l) and recombinant endo-β-1,4-xylanase (Xyn2) production (49.73 U/ml). Optimum productivity
was obtained at shorter incubation times (i.e., 6 h), making the medium suitable for use when seeking efficient production.
Expression of recombinant Xyn2 by K. lactis GG799 in the designed medium resulted in satisfactory recombinant Xyn2 volumetric productivity (vp) at 8.29 U/ml/h. When compared to the rich, non-selective YPD medium, the designed medium improved biomass output and recombinant
Xyn2 production in K. lactis GG799 by approximately 9 and 22%, respectively.
Available from: Mehul Ganatra
- "These methods involved testing the stability of fluorescently labeled casein and measuring the hydrolysis of a chromogenic peptide substrate containing dibasic amino acids. Additionally, growth of yeast cells on different carbon sources may either increase or repress the expression of individual proteases and influence the proteins that cells secrete (Sinha et al., 2004; Madinger et al., 2009 "
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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.
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