Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer

FEMS Yeast Res. 2008 Aug;8(5):725-34. doi: 10.1111/j.1567-1364.2008.00382.x. Epub 2008 Apr 29.

Abstract

The osmotolerant yeast Candida glycerinogenes produces glycerol as a major metabolite on an industrial scale, but the underlying molecular mechanisms are poorly understood. We cloned and characterized a 4900-bp genomic fragment containing the CgGPD gene encoding a glycerol-3-phosphate dehydrogenase homologous to GPD genes in other yeasts using degenerate primers in conjunction with inverse PCR. Sequence analysis revealed a 1167-bp open reading frame encoding a putative peptide of 388 deduced amino acids with a molecular mass of 42 695 Da. The CgGPD gene consisted of an N-terminal NAD(+)-binding domain and a central catalytic domain, whereas seven stress response elements were found in the upstream region. Functional analysis revealed that Saccharomyces cerevisiae gpd1Delta and gpd1Delta/gpd2Delta osmosensitive mutants transformed with CgGPD were restored to the wild-type phenotype when cultured in high osmolarity media, suggesting that it is a functional GPD protein. Transformants also accumulated glycerol intracellularly and GPD-specific activity increased significantly when stressed with NaCl, whereas the S. cerevisiae mutants transformed with the empty plasmid showed only slight increases. The full-length CgGPD gene sequence including upstream and downstream regions has been deposited in GenBank under accession no. EU186536.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Candida / enzymology*
  • Candida / genetics
  • Catalytic Domain
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Fungal / chemistry
  • DNA, Fungal / genetics
  • Gene Deletion
  • Genetic Complementation Test
  • Glycerol / metabolism*
  • Glycerol-3-Phosphate Dehydrogenase (NAD+) / chemistry
  • Glycerol-3-Phosphate Dehydrogenase (NAD+) / genetics
  • Glycerol-3-Phosphate Dehydrogenase (NAD+) / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Open Reading Frames
  • Phylogeny
  • Polymerase Chain Reaction / methods
  • Promoter Regions, Genetic
  • Protein Structure, Tertiary
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment
  • Sequence Analysis, DNA

Substances

  • DNA Primers
  • DNA, Fungal
  • Saccharomyces cerevisiae Proteins
  • Glycerol-3-Phosphate Dehydrogenase (NAD+)
  • Glycerol

Associated data

  • GENBANK/EU186536