Directed evolution 2.0: improving and deciphering enzyme properties.

Directed evolution has matured to a routinely applied algorithm to tailor enzyme properties to meet the demands in various applications. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, many different strategies have been used in directed evolution campaigns. Analysis of directed evolution campaigns reveals that traditional approaches, in which several iterative rounds of diversity generation and screening are performed, are gradually replaced by strategies which require less time, less screening efforts, and generate a molecular understanding of the targeted properties. In this review, conceptual advances in knowledge generating directed evolution strategies are summarized, compared to each other and to traditional directed evolution strategies. Finally, a 'KnowVolution' (knowledge gaining directed evolution) termed strategy is proposed.