02/01/2008
New Technique for Analysis of Metabolic Flux in Microbial Communities
This information is central to many studies, including physiological characterization of stress responses and impact of metabolic engineering.
The Science
A new approach has been developed by scientists at the Lawrence Berkeley National Laboratory to overcome the significant challenge of studying a microbe in its natural environment. The ability to develop biotechnology-based strategies for environmental remediation or bioenergy applications with microbes depends on understanding microbial metabolism under rapidly changing conditions. Moreover, the metabolism of a single microbe is difficult to selectively monitor in the presence of many other microbial community species The Lawrence Berkeley scientists engineered a reporter gene encoding the green fluorescent protein (GFP) into a microbe, then fed the microbe glucose labeled with the stable radioisotope carbon-13. Subsequent analysis of the metabolism of carbon-13 label from glucose into amino acid building blocks within the GFP reflected the metabolism of all the proteins in that microbe. This proof of concept of the technique lays the foundation for analysis of a range of metabolic activities within a specific microbe, rather than the entire microbial community in which it is found.
The Impact
The isotopomer labeling patterns of amino acids derived from purified GFP and total cell protein are indistinguishable, indicating that amino acids from a purified protein can be used to infer metabolic fluxes of targeted organisms in a mixed culture. This study provides the foundation to extend isotopomer-based flux analysis to study metabolism of individual strains in microbial communities. As it is the isotopomer data from these key amino acids that are used to determine flux through central metabolic pathways, the flux distribution information of a target organism in a mixed culture can be obtained from a single purified protein.
Principal Investigator
Jay Keasling
Joint BioEnergy Institute
[email protected]
Funding
The research was directed by Jay Keasling, with funding from the Genomics:GTL program in the Office of Biological and Environmental Research, and was published in the February 1, 2008, issue of the journal Analytical Chemistry.
References
Shaikh, A. S., Y. J. Tang, A. Mukhopadhyay, and J. Keasling. 2008. “Isotopomer Distributions in Amino Acids from a Highly Expressed Protein as a Proxy for Those from Total Protein,” Analytical Chemistry 80(3), 886–90.