Identification of additive formulations for enhanced shelf‐life and productivity of E. coli cell‐free protein synthesis extracts

Abstract

Cell‐Free Protein Synthesis (CFPS) is a platform biotechnology that enables a breadth of applications, from expressing traditionally intractable proteins to metabolic engineering and point‐of‐care diagnostics. However, field applications have remained limited due to the poor shelf stability of aqueous E. coli cell‐free extracts, which contain a complex mixture of cellular machinery. Lyophilization of the E. coli extract improves shelf‐life, but remains insufficient for maintaining extract productivity over extended storage periods at room temperature. For many pure protein‐based biologics, a variety of additives have been identified that help to stabilize the protein for lyophilization, transportation, and storage. However, the advances made for pure proteins cannot be directly applied to the complex cell extract, and current investigations into improving cell extract stability via additives has been limited. Thus, we have addressed the cold chain limitation in cell‐free by mapping the chemical space of ten low‐cost additives with four distinct mechanisms of action in a combinatorial manner to elucidate their capacity to further stabilize lyophilized cell extract. We report three key findings: 1) unique additive formulations that maintain full productivity of the cell extract at 4°C and 23°C; 2) additive formulations that enhance extract productivity up to 195%; 3) a machine learning algorithm that provides predictive capacity for the stabilizing effects of additive formulations that were not tested experimentally. The identified additive formulations provide a simple and low‐cost advance toward making CFPS field‐ready.Support or Funding InformationBill and Linda Frost Fund, Center for Applications in Biotechnology’s Chevron Biotechnology Applied Research Endowment Grant, Cal Poly Research, Scholarly, and Creative Activities Grant Program (RSCA 2017), and the National Science Foundation (NSF‐1708919)Comparison of traditional cellfree extract production and reaction setup with our modified workflow for improved stability and productivity to enable field applications.Figure 1