Cloning and characterization of a NAD+-dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer.
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.
Chen
Xianzhong
X
Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wuxi, China.
Fang
Huiying
H
Rao
Zhiming
Z
Shen
Wei
W
Zhuge
Bin
B
Wang
Zhengxiang
Z
Zhuge
Jian
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005990:Glycerol / Q000378:metabolism*; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D005990:Glycerol