Boundaries in metagenomic screenings using lacZα-based vectors

Abstract Metagenomics approaches have been of high relevance for providing enzymes used in diverse industrial applications. In the current study, we have focused on the prospection of protease and glycosyl hydrolase activities from a soil sample by using the lacZα -based plasmid pSEVA232. For this, we used a functional screen based on skimmed milk agar and a pH indicator dye for detection of both enzymes, as previously reported in literature. Although we effectively identified positive clones in the screenings, subsequent experiments revealed that this phenotype was not because of the hydrolytic activity encoded in the metagenomic fragments, but rather due to the insertion of small metagenomic DNA fragments in frame within the coding region of the lacZ gene present in the original vector. Analyses of the thermodynamic stability of mRNA secondary structures indicated that recovering of positive clones was probably due to higher expression levels of the chimeric lacZα-genes in respect to the original from empty vector. We concluded that this method has a higher tendency for recovery false positive clones, when used in combination with a lacZα-based vector. As these vectors are massively used in functional metagenomic screenings, we highlight the importance of reporting boundaries in established metagenomic screenings methodologies.

摘要 宏基因组学（Metagenomics）方法在获取各类工业应用所需酶类方面具有极高的应用价值。本研究聚焦于依托基于lacZα的质粒pSEVA232，从土壤样本中筛选蛋白酶（protease）与糖基水解酶（glycosyl hydrolase）活性。为此，我们采用了基于脱脂牛奶琼脂与pH指示剂的功能筛选体系，以同时检测这两类酶的活性，该方法已在既往学术文献中有所报道。尽管我们在筛选过程中成功获得了阳性克隆，但后续实验表明，该阳性表型并非源自宏基因组片段所编码的水解活性，而是由于小型宏基因组DNA片段以可读框的形式插入了原始载体自带的lacZ基因编码区。对mRNA二级结构热力学稳定性的分析显示，阳性克隆的获得可能源于嵌合lacZα基因的表达水平相较于空载体携带的原始lacZα基因更高。我们由此得出结论：当与基于lacZα的载体联用时，该方法更容易产生假阳性克隆。鉴于此类载体在功能宏基因组学筛选中被广泛应用，我们强调在已建立的宏基因组筛选方法学中明确报告其局限性的重要性。