Microbes rarely exist in nature alone; from human and soil microbiomes to synthetic consortia for biotechnological applications, bacteria survive in complex communities that foster many different types of metabolic interactions. Dissecting intra- and intercellular metabolism in communities is challenging, but essential to understanding and harnessing microbial consortia for applications in health and sustainability. 13C Metabolic flux analysis (13C-MFA) is the gold standard for quantitative analysis of metabolism in pure cultures, using isotopically labeled substrates to track the flux of carbon through intermediates in central metabolism. The primary challenge with extending 13C-MFA to co-cultures lies in attributing the contribution of each microbe’s metabolism to the isotopic labeling pattern of a particular intermediate, given that most central metabolites are not unique. To overcome this challenge, we engineered short orthogonal affinity tags onto highly abundant E. coli proteins, allowing purification of a unique protein from each community member. In this process, we tested a variety of tags, tag locations, and linkers, all with different levels of success – we found the 6xHis and the Twin-Strep-tags to be the best in terms of balancing protein yield with minimal impact on growth. The purified proteins are then hydrolyzed to the constituent amino acids, which are then analyzed for isotopic enrichment to determine fluxes for each microbe. Targeting highly abundant native proteins ensures the metabolic information is representative of the entire cell and avoids the metabolic burden in previous work resulting from high-level expression of heterologous proteins. This approach was rigorously benchmarked and the most promising proteins identified using a defined co-culture of E. coli strains with knockouts of glycolytic enzymes that result in well characterized flux phenotypes. Our approach is readily extendable to defined consortia of genetically tractable microbes and will facilitate a better understanding of metabolic crosstalk in co-cultures, as well as how individual microbes adapt their internal metabolism to such environments.
