Department of Chemical & Biomolecular Engineering, Molecular Biotechnology Laboratory, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
Nucleic Acids Res. 2011 Dec;39(22):e152. doi: 10.1093/nar/gkr817. Epub 2011 Oct 5.
In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA.
在工程新型微生物菌株用于生物技术应用中，除了可以预先确定要工程化的途径之外，开发复杂、定义不明确的细胞表型变得越来越重要。一种方法是筛选基因组或宏基因组文库，以鉴定赋予理想表型的基因，例如耐受应激或新型分解代谢程序。这些文库受到无法识别远距离遗传基因座之间相互作用的限制。为了解决这个问题，我们构建了基于质粒和 fosmid 的大肠杆菌共存/共表达基因组文库 (CoGeL)。作为原理的证明，我们敲除了大肠杆菌染色体上两个相距较远的 l-赖氨酸生物合成途径的四个基因对。在这些营养缺陷型细胞转化为 CoGeL 后，发现无需补充就能生长的细胞中含有敲除基因的文库插入物，证明了这两个文库之间的相互作用。还筛选了 CoGeL 以鉴定协同作用以创建更复杂的耐酸表型的遗传基因座。CoGeL 筛选鉴定了已知增强耐酸能力的基因组合（gadBC 操纵子和 adiC），但还鉴定了 arcZ 和 recA 的新组合，使耐酸能力提高了 9000 倍。arcZ 是一种小 RNA，我们发现它可以单独提高 pH 耐受性，并且与 recA 一起提高。
