Proteomic analysis of Candida magnoliae strains by two‐dimensional gel electrophoresis and mass spectrometry

Abstract

Candida magnoliae which has been newly isolated from honey comb is an osmotolerant yeast to produce erythritol as a major product. Erythritol is a noncariogenic, low calorie sweetener and safe for diabetics. Strain development by chemical mutation to obtain the improved erythritol yield and productivity relative to the parental strain made it necessary to elucidate the physiological differences between the wild and mutant strains. Proteomic analyses of C. magnoliae wild and mutant strains with two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry were carried out to identify intracellular proteins and to estimate the effects of newly characterized metabolic enzymes on the yeast cell growth and erythritol production. Most of the molecular mass of intracellular proteins were distributed in the range of pI 4-8 and molecular mass of approximately 130 kDa. Six out of nine protein spots expressed at different levels between the wild and mutant strains were analyzed with nanoelectrospray tandem mass spectrometry and identified by comparing amino acid sequences with the National Center for Biotechnology Information and Saccharomyces Genome Databases. Except for Ygr086cp, these proteins were believed to be the metabolic enzymes involved in the citric acid cycle (citrate synthase, succinyl-CoA ligase and fumarase) and the glycolysis pathway (pyruvate decarboxylase and enolase). Up-regulated enzymes in the citric acid cycle could explain high growth of the C. magnoliae mutant strain owing to the increased NADH and ATP formation. Down-regulated enolase and up-regulated fumarase in the mutant strain seemed to play a role in the improved bioconversion of erythrose-4-phosphate to erythritol compared with the wild strain.

Yeast Proteome Analysis

“…The osmotolerant Candida magnoliae has been isolated from honey comb and has the potential for production of erythritol, a noncarcinogenic, low calorie sweetener (Lee et al, 2003 ). Chemical mutagenesis has been performed to obtain strains with improved erythritol yield.…”

“…A proteomics approach was now applied to monitor the physiological basis for the improved capacity of the mutant strain relative to the parental isolate. Six proteins out of nine differentially expressed proteins could be identified, among them citrate synthase, succinyl-CoA ligase, fumarase, pyruvate decarboxylase and enolase as an initial result to establish a metabolic network database (Lee et al, 2003 ).…”

Proteomics and physiology of erythritol-producing strains

“…After staining and destaining of proteins on 2-DE, in-gel digestion in which protease, especially trypsin, cuts the peptide bonds of a protein inside SDS-polyacrylamide gel facilitates sample preparation for further mass spectrometric analysis. As an example for C. magnoliae, procedures of in-gel digestion and peptide extraction from 2-DE are described below [37,38]. A protein spot in silver-stained gel without glutaraldehyde treatment was sliced and destained by removing the silver bound with proteins [42].…”

“…The cells were resuspended in disruption buffer containing 10 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 mM Tris-HCl (pH 7.8) for T. coralline or 50 mM potassium phosphate buffer (pH 7.0) for C. magnoliae. For proteomic analysis of cellular proteins in C. magnoliae by the isoelectric focusing and two-dimensional gel electrophoresis, proteins were extracted by the following procedure [37,38]. Resuspension of C. magnoliae cells with hot SDS sample buffer (1% sodium dodecyl sulfate (SDS) and 100 mM Tris-HCl at pH 7.0 and 95 • C) prevented protein modification and degradation and loss of high molecular mass proteins.…”

“…After the addition of thiourea/urea lysis buffer (2 M thiourea, 7 M urea, 4% CHAPS, 1% dithiothreitol (DTT), and 2% carrier ampholytes; pH 3-10), the suspension was shaken moderately for 1 h and centrifuged at 16,000 rpm for 20 min. The clear supernatant was collected and used for isoelectric focusing on an immobilized pH dry strip [37].…”