Unité de Biologie animale et microbienne, Gembloux Agro-Bio Tech, Université de Liège, B-5030 Gembloux, Belgium.
Microbiol Res. 2011 Jul 20;166(5):360-8. doi: 10.1016/j.micres.2010.07.006. Epub 2010 Sep 23.
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).
宏转录组学应用于环境转录本提供了独特的机会来揭示环境中的微生物活性，并发现具有潜在生物技术应用价值的新酶。在这里，通过在酿酒酵母中功能性互补 pho5(-)突变（影响可诱导的酸性磷酸酶）和 his3(-)突变，我们在一个宏转录组文库中鉴定了编码酸性磷酸酶和咪唑甘油磷酸脱水酶的真菌基因，该文库是通过从糖枫森林土壤的有机层中提取的总 RNA 的 polyA 反转录部分构建的，构建在改良的酵母分泌载体 pTEF-MF-SfiI A/B 中。在菌落染色测定中鉴定出具有磷酸酶活性的酵母转化体，并且在缺乏组氨酸的培养基上通过平板筛选检测到具有 his3(-)互补基因的转化体。在每个筛选中都发现并测序了一个 DNA 插入片段。测序的 his3(-)互补基因与担子菌咪唑甘油磷酸脱水酶显示出很强的相似性（与 Phaffia rhodozyma 酶的相似度为 76%）。被发现具有磷酸酶活性的候选物被发现能够在细胞外产生磷酸酶，当使用 4-硝基苯磷酸作为底物时，该酶在 pH 4 和 40 到 50°C 之间具有最高的活性。测序的插入片段与担子菌酸性磷酸酶显示出很强的相似性（与 Postia placenta 的相似度为 60%）。
