School of Chemistry, The University of Manchester, 131 Princess St, Manchester M1 7DN, UK.
Analyst. 2018 Sep 24;143(19):4747-4755. doi: 10.1039/c8an00851e.
Directed evolution enables the improvement and optimisation of enzymes for particular applications and is a valuable tool for biotechnology and synthetic biology. However, studies are often limited in their scope by the inability to screen very large numbers of variants to identify improved enzymes. One class of enzyme for which a universal, operationally simple ultra-high throughput (>106 variants per day) assay is not available is flavin adenine dinucleotide (FAD) dependent oxidases. The current high throughput assay involves a visual, colourimetric, colony-based screen, however this is not suitable for very large libraries and does not enable quantification of the relative fitness of variants. To address this, we describe an optimised method for the sensitive detection of oxidase activity within single Escherichia coli (E. coli) cells, using the monoamine oxidase from Aspergillus niger, MAO-N, as a model system. In contrast to other methods for the screening of oxidase activity in vivo, this method does not require cell surface expression, emulsion formation or the addition of an extracellular peroxidase. Furthermore, we show that fluorescence activated cell sorting (FACS) of large libraries derived from MAO-N under the assay conditions can enrich the library in functional variants at much higher rates than via the colony-based method. We demonstrate its use for directed evolution by identifying a new mutant of MAO-N with improved activity towards a novel secondary amine substrate. This work demonstrates, for the first time, an ultra-high throughput screening methodology widely applicable for the directed evolution of FAD dependent oxidases in E. coli.
定向进化使人们能够改进和优化特定应用的酶，是生物技术和合成生物学的宝贵工具。然而，由于无法筛选大量变体来鉴定改进的酶，许多研究的范围往往受到限制。目前，还没有一种通用的、操作简单的超高通量（每天超过 106 个变体）测定法可用于黄素腺嘌呤二核苷酸 (FAD) 依赖性氧化酶。目前的高通量测定法涉及一种基于菌落的视觉比色筛选，但这种方法不适合非常大的文库，也无法量化变体的相对适应性。为了解决这个问题，我们描述了一种优化的方法，用于在单个大肠杆菌 (E. coli) 细胞内灵敏检测氧化酶活性，使用黑曲霉单胺氧化酶 MAO-N 作为模型系统。与体内筛选氧化酶活性的其他方法相比，这种方法不需要细胞表面表达、乳液形成或添加细胞外过氧化物酶。此外，我们表明，在测定条件下对源自 MAO-N 的大型文库进行荧光激活细胞分选 (FACS) 可以比基于菌落的方法以更高的速率在功能变体中富集文库。我们通过鉴定一种对新型仲胺底物具有更高活性的 MAO-N 新突变体，证明了其在定向进化中的用途。这项工作首次证明了一种广泛适用于大肠杆菌中 FAD 依赖性氧化酶定向进化的超高通量筛选方法。
