Isolation, sequencing, and heterologous expression of the Paecilomyces variotii gene encoding S-hydroxymethylglutathione dehydrogenase (fldA)

Abstract

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.

Fig. 1

HPLC separation profiles of peptide fragments produced by protease digestion of S-HMGSH dehydrogenase from Paecilomyces variotii NBRC 109023. a Digestion by Staphylococcus aureus protease and b digestion by Achromobacter protease. HPLC separation of the resulting peptide fragments was performed at a rate of 1.0 ml/min with solvent A (0.1 % (v/v) TFA in water) and solvent B (0.1 % (v/v) TFA in 60 % (v/v) acetonitrile). A TSK gel ODS-120T column was used for the separation. The column was equilibrated with solvent A, and peptide fragments were eluted by a linear gradient of solvent B (from 20 % (v/v) to 100 % (v/v)) over 55 min

Fig. 2

Phylogenetic tree of S-HMGSH dehydrogenases from A. fumigatus Af293 (Afu2g01040), A. niger CBS513.88 (An10g00510), A. nidulans FGSC4 (AN7632), A. oryzae RIB40 (AO090308000002), Arabidopsis thaliana (ADH2), Zea mays (NP_001105485), Oryza sativa Japonica group (Os02g0815500), Homo sapiens (CAG38730), Mus musculus (AAH90978), Cricetulus griseus (XM_007630816), Danio rerio (NP_571924), Kluyveromyces lactis (KLLA0D12342g), and Saccharomyces cerevisiae (YDL168W). Clustal W was used for multiple sequence alignments

Fig. 3

Western blot analysis of S-HMGSH dehydrogenase in S. cerevisiae (∆sfa1) transformed with YEp352GAPII (SU2 strain) and S. cerevisiae (∆sfa1) transformed with YEp352GAPII-fldA (SU3 strain). Cells were cultured in SD (-Ura), collected, and then disrupted. After cell disruption, cell debris was removed by centrifugation, and the supernatant was used as crude enzyme solution. The crude enzyme solutions were subjected to SDS-PAGE and Western blot analysis with Anti-Penta-His antibody and goat anti-mouse IgG antibody. Lane 1: S. cerevisiae BY4741 (∆sfa1) transformed with empty vector YEp352GAPII (SU2 strain). Lane 2: S. cerevisiae BY4741 (∆sfa1) transformant, fldA expression strain (SU3 strain). Lane 3: protein marker

Fig. 4

Growth of S. cerevisiae strains SU1, SU2, and SU3 on SD (-Ura) agar plates containing 0 to 8.0 mM formaldehyde. Cell suspensions were serially diluted at the indicated concentrations and then spotted on SD (-Ura) agar plates. The plates were incubated at 30 °C for 2 days, and growth was then evaluated. SU1: S. cerevisiae BY4741 transformed with empty vector (YEp352GAPII). SU2: S. cerevisiae BY4741 (∆sfa1) transformed with empty vector (YEp352GAPII). SU3: S. cerevisiae BY4741 (∆sfa1) transformed with YEp352GAPII-fldA

Fig. 5

Time courses of formaldehyde degradation by S. cerevisiae expressing the Paecilomyces S-HMGSH gene. a Cell growth and b degradation of formaldehyde. S. cerevisiae strains were cultured aerobically in SD (-Ura) liquid medium containing 4 mM formaldehyde at 30 °C for 60 h. The S. cerevisiae strains used in this experiment were the same as those used in Fig. 4. Symbols: open circle, SU3; triangle, SU2; closed circle, SU1. The error bars represent standard deviation (n = 3)