Continuous directed evolution is a high-throughput protein engineering process that seamlessly integrates all fundamental processes in Darwinian evolution (replication, mutation, translation and selection) into an uninterrupted cycle. Traditional directed evolution methods handle each of these four processes separately, while continuous evolution method supports all four stages and allows the surviving genes from one generation to spontaneously enter the subsequent generation.

Continuous directed evolution was traditionally carried out in vitro, where the comparative ease of manipulation and selection stringency adjustment can be exploited compared to in vivo methods. For example, a self-sustaining RNA replication cycle for RNA molecules capable of catalyzing their own ligation to an RNA–DNA substrate was established. Recent advances in technologies for site-specific in vivo mutagenesis and automation have also provided opportunities for the virtually non-stop evolution of strains and proteins. For example, Multiplexed Automated Genome Engineering (MAGE) is used for strain selection, and Phage Assisted Continuous Evolution (PACE) is applied for continuous protein engineering based on error-prone replication and propagation of a gene. Those emerging technologies for continuous evolution are increasingly open-source, inexpensive, and built upon accessible designs.

Creative Enzymes has been in the forefront of these newly developed technologies since a decade ago, providing custom biocatalysts through advanced high-throughput directed evolution strategies. With our world-class capacity and years of professional experience in protein engineering, we offer professional one-stop services:

• Mutagenesis plasmid design and construction.
• Large library construction.
• In vitro and in vivo strategies design.
• Strain selection and verification.
• Strain stability test.

Related Sections

• Enzyme Engineering by Directed Evolution

Figure 1 Examples of methods for in vivo targeted mutagenesis
(Current Opinion in Biotechnology 2018)