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. 2015 Sep:31:132-9.
doi: 10.1016/j.ymben.2015.07.005. Epub 2015 Jul 26.

(13)C-metabolic flux analysis of co-cultures: A novel approach

Nikodimos A Gebreselassie  1 , Maciek R Antoniewicz  2
Affiliations

(13)C-metabolic flux analysis of co-cultures: A novel approach

Nikodimos A Gebreselassie et al. Metab Eng. 2015 Sep.

Abstract

In this work, we present a novel approach for performing (13)C metabolic flux analysis ((13)C-MFA) 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 of species-specific products. Instead, metabolic fluxes for each species in a co-culture are estimated directly from isotopic labeling of total biomass obtained using conventional mass spectrometry approaches such as GC-MS. In addition to determining metabolic fluxes, this approach estimates the relative population size of each species in a mixed culture and inter-species metabolite exchange. As such, it enables detailed studies of microbial communities including species dynamics and interactions between community members. The methodology is experimentally validated here using a co-culture of two E. coli knockout strains. Taken together, this work greatly extends the scope of (13)C-MFA to a large number of multi-cellular systems that are of significant importance in biotechnology and medicine.

Keywords: Isotopic tracers; Metabolic flux analysis; Microbial communities; Microbial consortia; Multi-organism systems.

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Figures

Figure 1
Figure 1
Simple example metabolic network model. Open circles denote 12C-atoms and filled circles denote 13C-atoms.
Figure 2
Figure 2
Three metabolic network models with increasing level of complexity used to illustrate the general concepts of 13C-metabolic flux analysis of co-cultures.
Figure 3
Figure 3
Results of 13C-MFA obtained using a mono-culture model (results shown with black lines) and co-culture model (results shown with colored lines; blue = fluxes estimated for species #1; green = fluxes estimated for species #2). The input for 13C-MFA were simulated MIDs of Etotal, Ftotal, and Gtotal, assuming the fluxes shown in Figure 2B, and assuming metabolite A was labeled at the first carbon atom. The co-culture model produced statistically acceptable fits of all data sets. The estimated fluxes matched perfectly with the true flux values for both species.
Figure 4
Figure 4
Results of 13C-MFA obtained using a mono-culture model (results shown with black lines) and co-culture model (results shown with colored lines; blue = fluxes estimated for species #1; green = fluxes estimated for species #2). The input for 13C-MFA was the simulated MID of Etotal (left column), Ftotal (middle column), or Gtotal (right column), assuming the fluxes shown in Figure 2B, and assuming metabolite A was labeled at the first carbon atom.
Figure 5
Figure 5
Results of 13C-MFA obtained using a mono-culture model (results shown with black lines), a co-culture model without metabolite exchange, and a co-culture model with metabolite exchange flux (results shown with colored lines; blue = fluxes estimated for species #1; green = fluxes estimated for species #2). The input for 13C-MFA were the simulated MIDs of Etotal, Ftotal, and Gtotal, assuming the fluxes shown in Figure 2C, and assuming metabolite A was labeled at the first carbon atom. Only the co-culture model with inter-species exchange of metabolite F produced statistically acceptable fits. The estimated metabolic fluxes matched perfectly with the true flux values.
Figure 6
Figure 6
Results of 13C-MFA for a simulated E. coli/E. coli co-culture, obtained using a mono-culture model (results shown with black lines) and a co-culture model (results shown with colored lines; blue = fluxes estimated for species #1; green = fluxes estimated for species #2). The input for 13C-MFA consisted of simulated MIDs of total biomass amino acids. Four different choices of glucose labeling were evaluated. Overall, [1,2-13C]glucose was the best tracer for 13C-flux analysis of the E. coli/E. coli co-culture.
Figure 7
Figure 7
Results of 13C-MFA for the co-culture system of two E. coli knockouts (Δpgi and Δzwf) cultured on [1,2-13C]glucose. (A) Minimized SSR values of best fits obtained using a mono-culture model and co-culture model, along with threshold SSR values at 95% confidence level. (B) Estimated species-specific metabolic fluxes for E. coli species #1 (Δpgi) and species #2 (Δzwf) obtained using the co-culture model.
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