Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.
PLoS One. 2010 Dec 6;5(12):e15275. doi: 10.1371/journal.pone.0015275.
[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.
METHODOLOGY/PRINCIPAL FINDINGS: Here we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.
CONCLUSIONS/SIGNIFICANCE: [FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.
[FeFe]氢化酶在由常见元素组成的复杂辅基的帮助下催化分子氢的形成和离解。基于这些可再生催化剂的能量转换技术的发展受到其对氧气极端敏感的阻碍。由于缺乏能够达到足够高通量以充分筛选大量突变 DNA 文库的筛选平台，通过定向进化来改进这些酶的尝试失败了。体外分隔（IVC）是一种能够在非常高的高通量下筛选多次转化酶活性的强大方法。最近的进展使得[FeFe]氢化酶能够在基于 IVC 的无细胞蛋白合成反应中表达和激活；然而，IVC 是一种要求很高的技术，许多酶已经被证明与之不兼容。
方法/主要发现：在这里，我们描述了一种用于筛选耐氧[FeFe]氢化酶的超高通量 IVC 筛选方法。我们证明了[FeFe]氢化酶 CpI 可以在乳液滴中表达和激活，并鉴定出一种荧光底物，它将氧暴露后的活性与荧光信号的产生联系起来。我们提出了一种筛选方案，其中突变基因及其编码的蛋白质通过与微珠表面的连接，然后通过三个单独的乳液步骤进行放大、表达和评估氢化酶突变体。我们表明，通过荧光激活细胞分选可以分离显示活性氢化酶的珠粒，并且我们使用该方法从模拟文库中富集此类珠粒。
结论/意义：[FeFe]氢化酶是无细胞蛋白合成产生的最复杂的酶，也是 IVC 迄今为止应用的最具挑战性的目标。这里描述的技术是开发用于生物制氢经济的生物催化剂的一个推动步骤。
