Continuous Evolution of Proteins In Vivo

Directed evolution is a powerful approach for engineering new biomolecular and cellular functions [1–3]. In contrast to rational design approaches, directed evolution exploits diversity and evolution to shape the behavior of biological matter by applying the Darwinian cycle of mutation, selection, and amplification of genes and genomes. By doing so, the field of directed evolution has generated important insights into the evolutionary process [4–6] as well as useful RNAs, proteins, and systems with wide-ranging applications across biotechnology and medicine [7–11]. To mimic the evolutionary process, classical directed evolution approaches carry out cycles of ex vivo diversification on genes of interest (GOIs), transformation of the resulting gene libraries into cells, and selection of the desired function (Figure 1.1). Each iteration of this cycle is defined as a round of evolution, and as selection stringency increases over rounds, either automatically through competition or manually through changing conditions (or both), this process can lead GOIs closer and closer to the desired function. This overall process makes practical sense for a number of reasons, especially for the goal of protein engineering (i.e. GOI encodes a protein). First, ex vivo diversification is appropriate, because test tube molecular biology techniques such as DNA shuffling, site-directed saturation mutagenesis, and error-prone (ep) polymerase chain reaction (PCR) [2] are capable of generating exceptionally high and precise levels of sequence diversity for any GOI. Second, transforming diversified libraries of the GOI into cells is appropriate, because each GOI variant needs to be translated into a protein in order to express its function, and cells, especially model microbes, are naturally robust hosts for protein expression. Third, carrying out selection inside cells is appropriate, because (i) cells automatically maintain the genotype–phenotype connection between the GOI and expressed protein that is necessary for amplification of desired variants,