Metabolic Flux Analysis

Our knowledge of the regulation and compartmentation of metabolism in plants is far from complete. This contributes dramatically to the failure rate of metabolic engineering efforts, which are usually unsuccessful in their practical aims for unforeseen (and often unexamined) reasons. To learn from failures and to increase the chances of future success one needs to analyze the functioning of the metabolism involved and the effects upon it of genetic and environmental manipulations. The flow of material and energy through the metabolic network is at the heart of metabolic function and we work to measure and understand the fluxes of central metabolism that underlie food and biofuel production and the growth of cells.

 As an example, the genetic engineering of altered oil, protein and starch levels in crop seeds has met with limited success due to our limited understanding of metabolic fluxes and their regulation. We have used steady state isotopic labeling and metabolic flux analysis (see figure 1) to map the metabolic flows through central metabolism during seed development in oilseed rape (Brassica napus, embryos shown in the banner image of the website), soybean, maize (endosperm as well as embryo), and sunflower (see figure 2). The results have yielded insights into the routes of carbon flow, the sources of biosynthetic reductant, the carbon efficiency of seed metabolism, and the role of light in seeds that are green during development and have pointed to engineering targets.

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Figure 1. An overview of the approach used to obtain metabolic flux maps like the one shown above. Cultured tissues, such as developing seeds, or cells are grown under controlled conditions of metabolic steady state and measurements are made of growth rates, biochemical composition, rates of substrate uptake and product secretion, and the isotopic patterns in end products and intermediates of metabolism after supplying labeled substrates. These data are supplied to a computer model of metabolism. Figure by Doug Allen.

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Figure 2. Map of fluxes through sunflower embryos showing the forward, reverse and net fluxes through central metabolism that make and use the material, energy (ATP), and NAD(P)H (reducing power) for the growth of this important seed crop. The analysis revealed new patterns of carbon flow, energy dissipation (futile cycling), and highlights potential engineering targets for improved productivity. This study was the work of Ana Alonso et al.

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We are currently investigating the influence of environmental conditions and transgenic alterations on the fluxes through central metabolism in developing seeds and growing cells (see figure 3).

Figure 3. Flux maps of E Coli growing under aerobic (red) or anaerobic (blue) conditions. In this study the changes in central metabolism were quantified (correcting previous ideas about growth and fermentation in this model organism), and the basis for sub-optimal growth was identified. A target for improving biofuel production was identified and predictions were made about the evolution of metabolism under defined conditions. This study was the work of Wilson Chen et al (2011)

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To increase our ability to define accurate metabolic flux maps and models, we have also tested and improved the tools of metabolic flux analysis.  We have emphasized the value of obtaining more and better measurements of fluxes and labeling, and testing our findings with independent functional data.