Open AccessAnaerobic Conditioning of E. coli Cell Lysate for Enhanced In Vitro Protein Synthesis
Author(s) -
Denis Tamiev,
Jared L. Dopp,
Nigel F. Reuel
Publication year - 2021
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.0c00501
Subject(s) - lysis , escherichia coli , cell free protein synthesis , anaerobic exercise , anaerobic respiration , biochemistry , chemistry , green fluorescent protein , bacteria , mutant , cell , biology , protein biosynthesis , gene , physiology , genetics
Cell-free protein expression (CFPS) from E. coli cell lysate is an established chemical biology technique. Common efforts to improve synthesis capacity, such as strain engineering and process improvements, have overlooked the opportunity to increase productivity by reducing the dependence on limited, dissolved oxygen. Here we demonstrate conditioning E. coli cells for anaerobic respiration which increases the initial protein expression rate up to 4-fold and increases titer by 50% as compared to traditional aerobic cell lysate when using sfGFP as a reporter protein in CFPS reactions run at atmospheric conditions. This enhancement is even more significant when run in an oxygen-depleted environment, where anaerobic respiration preconditioned cells increase yield when supplemented with nitrite as a terminal electron acceptor (TEA). Furthermore, we test knockout mutants to determine key proteins responsible for enhancing the anaerobically prepared CFPS lysate. Further improvements could be made in preconditioning cells by increasing expression levels of critical pathway enzymes or by screening other TEA.