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

Hyang-Mi Lee,Dae-Sung Kim,Byeong-Min Jeon,Myungback Choi,Kyung Eun Lee,Dokyun Na,Yuna Oh,Hyunjoo Kim,Jun Ren,Kha Mong Tran,Won-Ung Park

doi:10.1038/s42003-021-01726-w

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.

Highlights

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
cell-penetrating peptides (CPPs) have been used without additional treatments, but recently, the chemical treatment of cells has improved the delivery efficiency of CPP conjugates[46]
We constructed a library of CPPs and evaluated their cytotoxicity and efficiency in penetrating bacteria (E. coli), to deliver biologically functional proteins

Summary

Introduction

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. The prediction models have been based on simple chemico–physical properties of the CPP sequences and designed for binary classification, which means that they predict whether or not, the peptide in query can penetrate cellular membranes in mammalian cells. CPP-conjugated transcription activator-like effector nucleases (TALENs) and CPP-mediated Cas9/sgRNA delivery systems have been reported to improve the efficiency of genome modification with reduced off-target effects in various human cell lines[6,40]

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