Proteomic analysis of Candida magnoliae strains by two-dimensional gel electrophoresis and mass spectrometry.
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.
Lee
Do-Yup
DY
Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Park
Yong-Cheol
YC
Kim
Hyo-Jin
HJ
Ryu
Yeon-Woo
YW
Seo
Jin-Ho
JH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D002175:Candida / Q000737:chemistry / Q000201:enzymology*; D002950:Citrate (si)-Synthase / Q000378:metabolism; D019343:Citric Acid / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004896:Erythritol / Q000096:biosynthesis*; D005649:Fumarate Hydratase / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D015966:Gene Expression Regulation, Fungal; D006019:Glycolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009243:NAD / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005656:Fungal Proteins; D009243:NAD; D019343:Citric Acid; D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D005649:Fumarate Hydratase; D004896:Erythritol