(643c) Investigation of Metabolite Channeling in Central Bacterial Pathways

Bacterial cytoplasm is a distinctive in vivo environment, crowed with macromolecules which creates glass-like properties (1). Intracellular enzymes of central pathways can be organized into a variety of assemblies, forming channels that pass intermediates from one enzyme to the next without release into the cellular medium. Such reaction mechanisms overcome intracellular diffusion limitations, improve reaction thermodynamics, aid low copy number enzymes, stabilize metabolic fluxes, and reduce side products from promiscuous enzymes. Comparing to transcriptional, translational, or allosteric regulations, channeling is an overlooked regulatory mechanism that leads to stability of a cell’s fluxome under environmental/genetic perturbations, allowing cells to remember the topology for distributing substrate influxes throughout its metabolic network (2). Understanding of the scope and mechanisms of metabolite channeling will lead to better design and engineering of heterologous pathways for synthetic biology applications. The channeling can be inferred from in vitro analysis, reaction thermodynamics, transport/reaction modeling, measurements of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling approaches. In this study, ¹³C-pathway tracing and kinetic models demonstrate several evidences for channeling in native pathways: 1. In ¹³C-labeling experiments, both cyanobacteria and Escherichia coli wild type strains demonstrate unsequential labeling order for cascade metabolites through glycolysis, pentose phosphate and TCA pathways; 2. An overexpressed pathway (Entner-Doudoroff)’s flux can be doubled by knocking out the cell’s phosphotransferase system (PTS), the proposed first channeled step; 3. Diffusion/reaction models highlight the relationship between diffusion coefficients, enzyme copy numbers, and enzyme distances and support the necessity of channeling for optimal cell metabolism. This work, together with numerous other studies, shows that native enzymes of bacterial pathways are likely more organized than we thought, and thus impede fluxes towards engineered pathways. If the hypothesis is true, metabolic modeling may have to re-considered, which assumes homogenous intracellular environments.

References:

1. Parry, B.R., Surovtsev, I.V., Cabeen, M.T., O’Hern, C.S., Dufresne, E.R., Jacobs-Wagner, C., 2014. The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity. Cell 156, 183–194. doi:10.1016/j.cell.2013.11.028

2. Hollinshead, W.D., Rodriguez, S., Martin, H.G., Wang, G., Baidoo, E.E.K., Sale, K.L., Keasling, J.D., Mukhopadhyay, A., Tang, Y.J., 2016. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Δpfk mutants. Biotechnol. Biofuels 9, 212. doi:10.1186/s13068-016-0630-y