Microbial communities carry out efficient biological transformations that are the result of multiple complementary metabolic systems working together. Co-culture systems are now increasingly applied in metabolic engineering to optimize utilization of mixed substrates, for production of mixed products, and distribution of complex metabolic pathways among species. Among the many omics tools used to gain insight into the physiology of these systems, fluxomics is the most direct and relevant method to study the in vivo metabolic state of co-cultures. In the past, 13C-metabolic flux analysis (13C-MFA) of co-culture systems required physical separation of proteins or cells to resolve individual population fluxes. In this work, we demonstrate, for the first time, that it is possible to determine metabolic flux distributions in multiple species simultaneously without the need for physical separation of cells or proteins, or overexpression of species-specific products. Instead, metabolic fluxes for each species in a co-culture are estimated directly from isotopic labeling of total biomass. In addition to determining metabolic fluxes, the approach quantifies the relative population size of each species in a mixed culture and interspecies metabolic exchange, thus enabling detailed studies of species dynamics and interactions in co-cultures. We will demonstrate application of our novel co-culture 13C-MFA approach on the following three diverse co-culture systems: A co-culture system in which each species consumes the same substrate but metabolizes the substrate through different pathways, specifically a co-culture of E. coli knockouts Δpgi and Δzwf. A co-culture system in which each species consumes the same substrate but produces different a product, specifically a co-culture of wild-type E. coli and S. cerevisiae. A co-culture system in which one species consumes a substrate and produces a by-product that serves as substrate for the second species, specifically a glucose consuming E. coli and a glucose non-consuming E. coli strain that uses acetate produced by the first strain as carbon source. Through co-culture 13C-MFA, we are able to elucidate the metabolic state of the various co-culture systems as well as investigate species dynamics and interspecies metabolite exchange. In summary, our work provides a powerful novel approach to study co-culture systems that will greatly facilitate rational design of co-culture systems in metabolic engineering.

Elucidating physiology of microbial communities through co-culture 13C-metabolic flux analysis

253rd National Meeting (2017)

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