An In-Cell Localized Mutagenesis Method for Engineering Bacterial Phenotypes

Mutagenesis is an essential component of strain improvement. Traditional methods of mutagenesis include site-specific methods, aimed at making defined changes to a DNA sequence, untargeted mutagenesis, aimed at introducing changes nearly at random into a cell's DNA, and localized mutagenesis methods. Localized mutagenesis offers an attractive compromise as it does not require an extensive knowledge of protein and gene structure as does site specific mutagenesis and it limits the amount of DNA where mutations must be analyzed and characterized compared with untargeted mutagenesis. Some disadvantages of traditional localized mutagenesis methods are the necessity for library construction with error-prone PCR and the requirement for sequence homology with the transducing phage approach. We have developed an in-cell method of localized mutagenesis aimed at overcoming some of the disadvantages of traditional localized mutagenesis methods. Our system, called "Rapidaptor," which is short for rapid-adaptor, makes use of error-prone double strand break repair by Escherichia coli DNA polymerase IV to introduce mutations into a specified region of plasmid DNA in-cell. It does this while leaving the Rapidaptor strain's chromosomal DNA relatively untouched. Furthermore, the rate of this localized mutagenesis is tightly controlled by a non-toxic inducer in our system. We have shown that our system can produce localized mutation rates between 10 and 200 fold higher than chromosomal mutation rates, depending on inducer concentration, for a specific G:C to T:A transversion mutation. The applications of this system to directed evolution and strain improvement will be discussed.