The efficiency for recombineering is dependent on the source of the phage recombinase function unit

Phage recombinase function units (PRFUs) such as lambda-Red or Rac RecET have been proven to be powerful genetic tools in the recombineering of Escherichia coli . Studies have focused on developing such systems in other bacteria as it is believed that these PRFUs have limited efficiency in distant species. However, how the species evolution distance relates to the efficiency of recombineering remains unclear. Here, we present a thorough study of PRFUs to find features that might be related to the efficiency of PRFUs for recombineering. We first identified 59 unique sets of PRFUs in the genus Corynebacterium and classified them based on their sequence as well as secondary structure similarities. Then both PRFUs from this genus and other bacteria were chosen for experiment based on sequential and secondary structure similarity as well as species distance. These PRFUs were compared for their ability in mediating recombineering with oligo or double-stranded DNA substrates in Corynebacterium glutamicum . We demonstrate that the source of the PRFU is more critical than species distance for the efficiency of recombineering. Our work will provide new ideas for efficient recombineering using PRFUs. Importance Recombineering using phage recombinase function units (PRFUs) such as lambda-Red or Rac RecET has gained success in Escherichia coli , while efforts applying these systems in other bacteria were limited by the efficiency. It is believed that the species distance may be a major reason for the low efficiency. In this study, however, we showed that it is the source of PRFU rather than the species distance that matters for the recombineering in Corynebacterium glutamicum . Besides, we also showed that the lower transformation efficiency in other bacteria compared to that of E. coli could be a major reason for the low performance of heterogeneously expressed RecET. These findings will be helpful for the recombineering using PRFUs.