Abstract
Enzymes capable of catalysing the production of hydrocarbons hold promise for sustainable fuel synthesis. However, the native activities of these enzymes are often insufficient for their exploitation in industrial bioprocesses. Enzyme engineering approaches including directed evolution (DE) can be used to improve the properties of enzymes to meet desirable standards for their industrial application. In this review, we summarise DE methods for engineering hydrocarbon-producing enzymes, including both screening- and selection procedures. The efficacy of DE depends on several factors, including sensitive and accurate detection of enzyme activity, the throughput of screening or selection steps, and the scale of diversity generation. Although DE is a well-established approach, its application in engineering hydrocarbon-producing enzymes has not been widely demonstrated. This can be attributed to the physiochemical properties of the target molecules, such as aliphatic hydrocarbons, which can be insoluble, gaseous, and chemically inert. Detection of these molecules in vivo presents several unique challenges, as does dynamically coupling their abundance to cell fitness. We conclude with a discussion on future directions and potential advancements in this field.