Experimental resolution of metabolic fluxes is of major significance in basic and applied biological sciences. Fluxes represent the integrated and functional state of cellular metabolism, and as such provide a critical visibility into biology and inform metabolic engineering efforts. To this end, stable-isotope assisted methods, particularly 13C metabolic flux analysis (13C-MFA), have emerged as the predominant approach. Thus far, 13C-MFA has only been applied to study metabolism of cells grown on small molecule substrates, such as glucose, xylose, and acetate, which has been enabled by the availability of large number of 13C-tracers of these compounds, e.g. [1,2-13C]glucose, [5-13C]xylose, etc. In this contribution, we extend the 13C-MFA approach to analysis of cellulose degrading microbes and communities. Since there are no commercially available 13C-cellulose tracers, first, we had to produce specifically 13C-labeled cellulose ourselves. For this purpose, we cultured the cellulose producing microbe Komagataeibacter xylinus on various 13C-labeled glucose tracers. We demonstrated that K. xylinus converted the majority of 13C-labeled glucose tracers into 13C-labeled cellulose, which could be purified easily and was characterized in detail. Using these 13C-labeled cellulose tracers, we then investigated the metabolism of the cellulose-degrading thermophilic bacterium Thermobifida fusca. Cells were cultured on the various 13C-labeled cellulose tracers in parallel cultures, followed by GC-MS analysis of amino acid labeling and 13C-MFA flux estimation. Using this approach, we successfully elucidated the metabolism of T. fusca grown on cellulose as the only carbon source. By comparing the flux results to cultures grown on simple sugars (e.g. glucose and xylose), we identified significant intracellular flux rewiring when cells were grown on cellulose compared to grown on glucose alone. The flux results also suggested new metabolic engineering targets to improve cellulose degradation. Taken together, this study opens the door for a wide array of other investigations of cellulose-degrading microbes (or microbial communities) using the powerful 13C metabolic flux analysis approach.

Bacterial production of 13C-labeled cellulose for 13C metabolic flux analysis of cellulose degradation

2019 Spring National Meeting

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