Mutagenesis techniques for evolutionary engineering of microbes - exploiting CRISPR-Cas, oligonucleotides, recombinases, and polymerases.

The natural process of evolutionary adaptation is often exploited as a powerful tool to obtain microbes with desirable traits. For industrial microbes, evolutionary engineering is often used to generate variants that show increased yields or resistance to stressful industrial environments, thus obtaining superior microbial cell factories. However, even in large populations, the natural supply of beneficial mutations is typically low, which implies that obtaining improved microbes is often time-consuming and inefficient. To overcome this limitation, different techniques have been developed that boost mutation rates. While some of these methods simply increase the overall mutation rate across a genome, others use recent developments in DNA synthesis, synthetic biology, and CRISPR-Cas techniques to control the type and location of mutations. This review summarizes the most important recent developments and methods in the field of evolutionary engineering in model microorganisms. It discusses how both in vitro and in vivo approaches can increase the genetic diversity of the host, with a special emphasis on in vivo techniques for the optimization of metabolic pathways for precision fermentation.