Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA).
The filamentous fungus Paecilomyces variotii NBRC 109023 (teleomorph: Byssochlamys spectabilis NBRC 109023) degrades formaldehyde at concentrations as high as 2.4 % (w/v). In many prokaryotes and in all known eukaryotes, formaldehyde degradation is catalyzed by S-hydroxymethylglutathione (S-HMGSH) dehydrogenase. We report here the isolation and characterization of the gene encoding S-HMGSH dehydrogenase activity in P. variotii. The 1.6-kb fldA gene contained 5 introns and 6 exons, and the corresponding cDNA was 1143 bp, encoding a 40-kDa protein composed of 380 amino acids. FldA was predicted to have 74.3, 73.7, 68.5, and 67.4 % amino acid identity to the S-HMGSH dehydrogenases of Hansenula polymorpha, Candida boidinii, Saccharomyces cerevisiae, and Kluyveromyces lactis, respectively. The predicted protein also showed high amino acid similarity (84∼86 %) to the products of putative fldA genes from other filamentous fungi, including Aspergillus sp. and Penicillium sp. Notably, the P. variotii fldA gene was able to functionally complement a Saccharomyces cerevisiae strain (BY4741 ∆sfa1) lacking the gene for S-HMGSH dehydrogenase. The heterologous expression construct rendered BY4741 ∆sfa1 tolerant to exogenous formaldehyde. Although BY4741 (parental wild-type strain) was unable to degrade even low concentrations of formaldehyde, BY4741 ∆sfa1 harboring Paecilomyces fldA was able to degrade 4 mM formaldehyde within 30 h. The findings from this study confirm the essential role of S-HMGSH dehydrogenase in detoxifying formaldehyde.
Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Kumamoto, 860-0082, Japan.
Takuji
T
Oka
Yuji
Y
Komachi
Kazufumi
K
Ohshima
Yoichi
Y
Kawano
Kohsai
K
Fukuda
Kazuhiro
K
Nagahama
Keisuke
K
Ekino
Yoshiyuki
Y
Nomura
D001711:Biotransformation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005091:Exons; D005557:Formaldehyde / Q000378:metabolism; D015870:Gene Expression; D005816:Genetic Complementation Test; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D007438:Introns; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010143:Paecilomyces / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D004271:DNA, Fungal; D011994:Recombinant Proteins; D005557:Formaldehyde; C029469:S-hydroxymethylglutathione; D010088:Oxidoreductases; D005978:Glutathione