[Cloning of the gene encoding a key enzyme involved in production of glycerol in Candida glycerinogenes].

Candida glycerinogenes WL2002-5, an excellent glycerol producer, has been used for industrial scale fermentation of glycerol by an aerobic process. However, our knowledge about glycerol biosynthesis at the molecular level and genetic background of this yeast species lags far behind those of model yeasts such as Saccharomyces cerevisiae et al. In this report, inverse primers, in conjunction with degenerated primers, were used to amplify the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) encoding gene from C. glycerinogenes. The completed nucleotide sequence of the coding, as well as flanking genomic regions was determined (GenBank accession No. EU186536). DNA sequence analysis revealed the present of the open reading frame (ORF) of 1,167 bp, encoding a polypeptide with 388-amino-acid with a molecular mass of 42,695 Da. The CgGPD did not exhibit significant sequence similarity with others described in other eukaryotic systems by comparative analysis. However, it consisted of two typical functional domains which belong to almost all eukaryotic GPDs: a co-enzyme binding domain in the N-terminal, and a catalytic domain. Moreover, some relevant features involved in initiation, regulation and stress response element of gene transcription were observed in the nucleotide sequence of the 5'-non-coding regions. Heterologous expression of CgGPD gene in S. cerevisiae improved its glycerol production significantly. In conclusion, the functional CgGPD has been cloned and identified successfully from C. glycerinogenes genome.