Identification of efficient prokaryotic cell-penetrating peptides with applications in bacterial biotechnology

Abstract

In bacterial biotechnology, instead of producing functional proteins from plasmids, it is often necessary to deliver functional proteins directly into live cells for genetic manipulation or physiological modification. We constructed a library of cell-penetrating peptides (CPPs) capable of delivering protein cargo into bacteria and developed an efficient delivery method for CPP-conjugated proteins. We screened the library for highly efficient CPPs with no significant cytotoxicity in Escherichia coli and developed a model for predicting the penetration efficiency of a query peptide, enabling the design of new and efficient CPPs. As a proof-of-concept, we used the CPPs for plasmid curing in E. coli and marker gene excision in Methylomonas sp. DH-1. In summary, we demonstrated the utility of CPPs in bacterial engineering. The use of CPPs would facilitate bacterial biotechnology such as genetic engineering, synthetic biology, metabolic engineering, and physiology studies. Lee et al. construct a cell-penetrating peptides (CPP) library and identify CPPs that can penetrate bacterial cells with minimum or no impact on cell viability. For the identified top CPP candidates, their abilities to deliver macromolecules such as I-SceI and Cre recombinase proteins to bacteria are evaluated as proof-of-concept studies for potential applications.