Self-sufficient reductive dehalogenases for bioremediation

卤化有机化合物在化工和制药工业、农业以及作为溶剂和材料得到了广泛的使用，每年的产量可达数吨。它们的持久性和(不)受控释放导致了这类化合物在环境中的全球积累。此外，生物积累和毒性导致许多有机卤化物对人类和环境健康构成重大挑战。事实上，大多数持久性有机污染物(POP)都含有卤素，其中许多现在属于2004年《斯德哥尔摩公约》的管辖范围，该公约旨在消除它们的使用。虽然监管减少了一些污染水平，但沉积物和废物中剩余的水库仍然令人担忧。此外，使用有机卤化物的替代品很少，在管理有机卤化物的生产和使用时，社会正在平衡对人类的好处和对环境的损害。然而，在理解人类/环境暴露的(长期)影响方面，仍然存在重大的知识差距。与流行的看法相反，有机卤化物并不完全是人为来源，越来越多的天然化合物被发现，其中大部分是生物来源。事实上，在过去的几十年里，人们已经对有机卤化物生物化学中的一系列卤酶和脱卤酶进行了鉴定和表征。虽然脱卤酶可以用于持久性有机污染物的生物修复或生物传感，但这些酶的固有特性与人工合成的外源生物谱系不匹配。我们试图研究一种特殊类型的脱卤酶，即还原脱卤酶或有机卤化物还原酶，它们在底物范围、生物修复和生物传感方面提供了独特的机会。近年来，我们自己的团队和其他团队对这些依赖维生素B12进行活动的神秘蛋白质提供了详细的见解。然而，确切的操作模式仍然有些神秘，而旨在基于rdh模板产生强大生物催化剂的蛋白质工程努力尚未成功。我们试图使用我们实验室中选择和研究的特定还原脱卤酶来提供对其作用机制的完整的基础见解。我们寻求这些信息来指导定向进化实验，目的是产生一个可以作用于持久性有机污染物和相关感兴趣化合物的脱卤酶工具箱。

Halogenated organic compounds have found widespread use in the chemical and pharmaceutical industries, in agriculture, and as solvents and materials, with production on the multi-ton scale per annum. Their persistent nature and (un)controlled release has led to worldwide accumulation of such compounds in the environment. Furthermore, bioaccumulation and toxicity has led to many organohalides presenting significant challenges to human and environmental health. Indeed, the majority of persistent organic pollutants (POPs) contain halogens, with many of them now falling under the 2004 Stockholm convention that serves to eliminate their use. While regulation has reduced some levels of contamination, remaining reservoirs in sediments and waste remain of concern. Furthermore, few alternatives are available for the use of organohalides, and society is balancing human benefit with environmental detriment when regulating organohalide production and use. However, significant knowledge gaps remain in understanding the (long-term) effects of human/environmental exposure. Counter to popular perception, organohalides are not exclusively of anthropogenic origin, with an ever-increasing amount of natural compounds identified, the majority of biological origin. Indeed, the last decades have seen identification and characterisation of a range of halogenase and dehalogenase enzymes that feature in organohalide biochemistry. While dehalogenases can serve in bioremediation or biosensing of POPs, the inherent properties of these enzymes do not match the human-made xenobiotic repertoire. We seek to study a particular type of dehalogenases, the reductive dehalogenases (Rdh) or organohalide reductases, which present unique opportunities in terms of substrate scope, bioremediation and biosensing. In recent years, our own group and that of others have provided detailed insights into these enigmatic proteins that depend on vitamin B12 for activity. However, the exact mode of operation remains somewhat enigmatic, while protein engineering efforts aimed at generating robust biocatalyst based on the Rdh-template have yet to meet with success. We seek to use specific reductive dehalogenases selected and studied in our lab to provide a complete fundamental insight into the mechanism of action. We seek this information to guide directed evolution experiments aimed at generating a toolbox of dehalogenases that can act on POPs and related compounds of interest.