Screening of a soil metatranscriptomic library by functional complementation of Saccharomyces cerevisiae mutants.
Metatranscriptomics applied to environmental transcripts provides unique opportunities to reveal microbial activity in the environment and to discover novel enzymes of potential use in biotechnological applications. Here, by functional complementation of a pho5(-) mutation (affecting a repressible acid phosphatase) and a his3(-) mutation in Saccharomyces cerevisiae, we identified fungal genes encoding an acid phosphatase and an imidazoleglycerol-phosphate dehydratase in a metatranscriptomic library, which was obtained by reverse-transcribed polyA fraction of total RNA extracted from the organic layer of a sugar maple forest soil, constructed in the modified yeast secretion vector pTEF-MF-SfiI A/B. Yeast transformants exhibiting phosphatase activity were identified in a colony-staining assay and transformants with his3(-)-complementing genes were detected by plating on histidine-deficient medium. In each screen one DNA insert was found and sequenced. The sequenced his3(-)-complementing gene showed strong similarity to a basidiomycete imidazoleglycerol-phosphate dehydratase (76% identity to a Phaffia rhodozyma enzyme). The candidate showing phosphatase activity was found to produce phosphatase extracellularly, the enzyme showing highest activity at pH 4 and between 40 and 50°C when 4-nitrophenyl phosphate was used as substrate. The sequenced insert showed strong similarity to a basidiomycete acid phosphatase (60% identity to Postia placenta).
Kellner
Harald
H
Unité de Biologie animale et microbienne, Gembloux Agro-Bio Tech, Université de Liège, B-5030 Gembloux, Belgium. microbio.gembloux@ulg.ac.be
Luis
Patricia
P
Portetelle
Daniel
D
Vandenbol
Micheline
M
D031002:Acer; D000135:Acid Phosphatase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D015723:Gene Library; D005816:Genetic Complementation Test; D006836:Hydro-Lyases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D012988:Soil Microbiology; D014197:Trees
D004271:DNA, Fungal; D000135:Acid Phosphatase; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase