GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania, 19426, USA.
GE Global Research, One Research Circle, Niskayuna, NY, 12309, USA.
Sci Rep. 2020 Jun 24;10(1):10279. doi: 10.1038/s41598-020-67307-9.
The ability to quickly and easily assess the activity of large collections of enzymes for a desired substrate holds great promise in the field of biocatalysis. Cell-free synthesis, although not practically amenable for large-scale enzyme production, provides a way to accelerate the timeline for screening enzyme candidates using small-scale reactions. However, because cell-free enzyme synthesis requires a considerable amount of template DNA, the preparation of high-quality DNA "parts" in large quantities represents a costly and rate-limiting prerequisite for high throughput screening. Based on time-cost analysis and comparative activity data, a cell-free workflow using synthetic DNA minicircles and rolling circle amplification enables comparable biocatalytic activity to cell-based workflows in almost half the time. We demonstrate this capability using a panel of sequences from the carbon-nitrogen hydrolase superfamily that represent possible green catalysts for synthesizing small molecules with less waste compared to traditional industrial chemistry. This method provides a new alternative to more cumbersome plasmid- or PCR-based protein expression workflows and should be amenable to automation for accelerating enzyme screening in industrial applications.
快速、轻松地评估大量酶对目标底物的活性，这在生物催化领域具有巨大的应用前景。无细胞合成虽然不太适用于大规模的酶生产，但为使用小规模反应筛选酶候选物提供了一种加速时间表的方法。然而，由于无细胞酶合成需要相当数量的模板 DNA，因此大量制备高质量的 DNA“部件”是高通量筛选的一个昂贵且具有限速性的前提条件。基于时间成本分析和比较活性数据，使用合成 DNA 迷你环和滚环扩增的无细胞工作流程可以在几乎一半的时间内实现与基于细胞的工作流程相当的生物催化活性。我们使用来自碳氮水解酶超家族的一系列序列证明了这一能力，这些序列代表了与传统工业化学相比，用于合成小分子的可能绿色催化剂，产生的废物更少。这种方法为更繁琐的基于质粒或 PCR 的蛋白质表达工作流程提供了一种新的替代方法，并且应该适合自动化，以加速工业应用中的酶筛选。
