Pinpointing metabolite cross feeding interactions in microbial communities using 13C metabolic flux analysis

Maciek R. Antoniewicz is a Full Professor of Chemical Engineering at the University of Michigan. Dr. Antoniewicz earned his B.S. and M.S. degrees in Chemical Engineering from Delft University of Technology (2000), and his Ph.D. in Chemical Engineering from the Massachusetts Institute of Technology (2006). After graduating he performed post-doctoral research at the DuPont Company. Dr. Antoniewicz started as an Assistant Professor in 2007 at the University of Delaware and was promoted to Associate Professor in 2013 and to Full Professor in 2017. In 2019, Dr. Antoniewicz moved to the University of Michigan.

Dr. Antoniewicz is an expert and a pioneer in the field of 13C-metabolic flux analysis (13C-MFA). Dr. Antoniewicz has received many awards for his research accomplishments, including the DuPont Young Professor Award (2008), the James E. Bailey Young Investigator Award in Metabolic Engineering (2008), the NSF CAREER Award (2011), and the Biotechnology and Bioengineering Daniel I.C. Wang Award (2015). In 2018, Dr. Antoniewicz was elected as a Fellow of the American Institute for Medical and Biological Engineering (AIMBE). His current interests include elucidating syntrophic interactions in microbial communities, adaptive laboratory evolution, optimizing CHO cell cultures for therapeutic protein production, and metabolic engineering of microbes for enhanced utilization of renewable substrates for production of value-added chemicals.

Abstract

One of the most important and difficult to quantify interactions in microbial communities is the cross-feeding of nutrients between community members. Experimental approaches that are based on metabolic measurements cannot precisely identify which metabolites are exchanged between community members. To address this challenge, we have developed a 13C-metabolic flux analysis technique that can precisely pinpoint exact metabolite that are being cross-fed. In this presentation, I will demonstrate applications of our new 13C-flux analysis approach to elucidate metabolite exchanges in several microbial co-cultures. To determine which metabolites are cross-fed (and at what rates), cells are cultured in parallel in the presence of carefully selected isotopic tracers. Isotopic labeling is then measured by mass spectrometry and the data is then analyzed using our new 13C-flux analysis approach. Using statistical methods, we can then precisely identify what metabolites are exchanged by the different strains, in which direction the cross-feeding occurs, and what the rates are for these cross-feeding fluxes. As an example, in E. coli co-cultures we identified nine metabolites that are consistently cross-fed. These include organic acids, intermediates of the glycolysis pathway, intermediates of the citric acid cycle, and some (but not all) amino acids. To verify these predictions, additional 13C-tracer experiments were conducted and complemented with 13C exo-metabolomic measurements. Taken together, these examples demonstrate that our new analysis techniques can significantly improve our understanding of metabolic interactions in microbial communities (which cannot be resolved with current approaches), and in the future will enable rational engineering of microbial interactions and assembly of microbiomes for practical applications in medicine and biotechnology.