Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens.

Abstract

A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries ( e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 ± 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E.coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V.natriegens-derived CFPS platform is versatile and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.