Elucidating the Extent of Endogenous Metabolic Activity in Vibrio Natriegens crude Extracts for Cell-Free Metabolic Engineering Applications

Leveraging active endogenous metabolic pathways in crude cell extracts towards targeted metabolite production addresses limitations inherent to engineering live cells while providing a cheaper alternative to purified enzyme systems. For these reasons, crude lysate engineering is thought to be a promising CFME platform for the bioproduction of high-value metabolites that are challenging to synthesize at high titers in vivo. While bioproduction has been successfully demonstrated in E. coli lysates, product yields could theoretically be increased in lysates with higher metabolic activity. Vibrio natriegens is poised to succeed E. coli as a workhorse for biotechnology applications. Due to this microbe’s shorter doubling time and higher central carbon metabolic rates, its utility in rapidly preparing cell-free systems with high native metabolic activity is intriguing. Here, we characterized the metabolome in V. natriegens lysates to determine the extent of endogenous glucose metabolism in this cell-free system. By feeding V. natriegens lysates fully-labeled ¹³C glucose, metabolite labeling patterns were elucidated over time. We demonstrate that intermediates and end-products of the central carbon metabolic pathways as well as the shikimate pathway for aromatic amino acid biosynthesis are actively produced in our lysates and that the productivities of these pathways have temporal differences over 24 hours. Interestingly, significant glucose consumption and accumulation of labeled metabolites occur within minutes of lysate incubation on ice. We aim to further leverage ¹³C labeling metabolite patterns to interrogate optimal V. natriegens lysate preparation and reaction conditions for CFME purposes. Further, by implementing available genetic tools for this organism, proteome engineering approaches can establish V. natriegens lysates as a useful CFME platform.