Microbial communities play an important role in biofuel production, biomedical research, food production, and waste water treatment. The capabilities of multi-microorganism systems are often enhanced by synergistic interactions at different levels. Co-culture systems particularly have unique advantages over mono-culture systems in optimizing product yield as a result of synergistic interactions, communication and division of labor. Here, we present a novel framework for performing 13C metabolic flux analysis (13C-MFA) studies of co-culture systems. 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 and detection of species-specific products. Instead, metabolic fluxes of each species in a co-culture are estimated directly from isotopic labeling of total biomass obtained using conventional mass spectrometry approaches (GC-MS). In addition to determining metabolic fluxes, this approach estimates relative population size of each species in the mixed culture, and inter-species metabolite exchange. As such, this approach enables detailed studies of microbial communities including species dynamics and interactions between community members. We have applied this methodology to study two co-culture systems: 1) a co-culture of two E. coli knockout strains, ∆zwf (knockout of the first step in the pentose phosphate pathway) and ∆pgi (knockout of the first step in glycolysis pathway); and 2) a co-culture of wild-type E. coli and yeast. For the first time, we have elucidated how these two model microbes grow and interact when grown in co-culture. In summary, the new fluxomic methodology that we have developed for analyzing co-culture systems adds a new dimension to metabolic engineering and significantly extends the scope of 13C-MFA studies to a large number of multi-cellular systems that are of significant importance in biotechnology and medicine.

13C metabolic flux analysis of E. coli/E. coli and E. coli/yeast co-culture

249th National Meeting (2015)

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