Characterizing Cell-Free Metabolism through Physiochemical Optimization for Biosynthetic Pathway Engineering

Traditional in vivo methods for building genetic constructs, studying enzymes, and probing metabolism is slow and difficult. A platform is needed in order to rapidly and efficiently study metabolism, observe enzyme kinetics, and prototype pathways for industrially relevant chemical production. Crude lysate cell-free systems have been utilized to quickly produce chemicals through cell-free metabolic engineering (CFME), such as n-butanol via a 17-step pathway. By combining cell-free protein synthesis with metabolic engineering (CFPS-ME), we are able to rapidly monitor and manipulate physiochemical conditions in order to test metabolism and prototype enzymes. We describe a series of experiments outlining the differences in metabolic profiles of the two systems, the effects of alternative energy sources, and the ability of CFPS-ME for enzyme titrations in order to optimize pathways. We found that the components of CFPS reactions inhibit the production of n-butanol, and that phosphate-minimal and pH controlled setups are necessary. This is the first time that metabolite profiles have been extensively characterized in CFME, and this gives insight into how to expand these cell-free frameworks to accelerate the metabolic engineering design-build-test cycle.