A Cell-Free Microtiter Plate Screen for Improved [FeFe] Hydrogenases

Background[FeFe] hydrogenase enzymes catalyze the production and dissociation of H2, a potential renewable fuel. Attempts to exploit these catalysts in engineered systems have been hindered by the biotechnologically inconvenient properties of the natural enzymes, including their extreme oxygen sensitivity. Directed evolution has been used to improve the characteristics of a range of natural catalysts, but has been largely unsuccessful for [FeFe] hydrogenases because of a lack of convenient screening platforms.
Methodology/Principal FindingsHere we describe an in vitro screening technology for oxygen-tolerant and highly active [FeFe] hydrogenases. Despite the complexity of the protocol, we demonstrate a level of reproducibility that allows moderately improved mutants to be isolated. We have used the platform to identify a mutant of the Chlamydomonas reinhardtii [FeFe] hydrogenase HydA1 with a specific activity ∼4 times that of the wild-type enzyme.
Conclusions/SignificanceOur results demonstrate the feasibility of using the screen presented here for large-scale efforts to identify improved biocatalysts for energy applications. The system is based on our ability to activate these complex enzymes in E. coli cell extracts, which allows unhindered access to the protein maturation and assay environment.

背景
[FeFe]氢化酶（[FeFe] hydrogenase）可催化氢气（H₂）的合成与解离，氢气是一种极具应用潜力的可再生燃料。此前尝试在工程化系统中应用这类催化剂的研究，均因天然酶存在诸多生物技术层面的不便特性而受阻，其中尤以其极强的氧敏感性最为突出。定向进化（Directed evolution）技术已被广泛用于优化多种天然催化剂的性能，但针对[FeFe]氢化酶的相关研究基本未能取得成功，核心原因在于缺乏便捷的筛选平台。

研究方法与主要发现
本文报道了一种可用于筛选耐氧且高活性[FeFe]氢化酶的体外筛选技术。尽管该实验流程较为复杂，但我们证实其具备足够的可重复性，能够分离得到性能适度优化的突变体。我们依托该平台筛选得到了莱茵衣藻（Chlamydomonas reinhardtii）[FeFe]氢化酶HydA1的一个突变体，其比活约为野生型酶的4倍。

结论与意义
本研究结果证实，采用本文所提出的筛选方法开展大规模筛选工作，以获取适用于能源应用的优化生物催化剂具备切实可行性。该系统的核心优势在于，我们能够在大肠杆菌（E. coli）细胞提取物中激活这些结构复杂的酶，从而不受限制地获取蛋白质成熟与检测所需的完整环境条件。