Carbon Flow through the Etylmalonyl-CoA Pathway

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Recently, there has been a paradigm shift in microbial metabolism: the focus is no longer solely on the elucidation of how substrates are converted to products (inputs and outputs), but rather also includes a concerted effort to understand the connectivity of pathways in the context of the whole organism's metabolic activities. This project provides an example of what necessitates this new thinking. The well-known glyoxylate cycle and the recently described ethylmalonyl-CoA pathway are apparently equivalent ways to convert acetate to precursor metabolites used for cell carbon biosynthesis. However, the two pathways differ drastically. The glyoxylate cycle provides a simple strategy for converting acetyl-CoA to malate relying on enzymes of the citric acid cycle in addition to two specialized enzymes, isocitrate lyase and malate synthase. The ethylmalonyl-CoA pathway of isocitrate lyase-negative bacteria is much more complex. It requires at least five unique enzymes and novel metabolic intermediates are involved. The question then arises why one pathway is used over the other by a given organism or even by the same organism under different growth conditions. The hypothesis is that the distinct intermediates for the two pathways and/or differential regulatory events necessitate carbon flux through either pathway. The ultimate goal of this research is to place the ethylmalonyl-CoA pathway in the context of central carbon metabolism of R. sphaeroides, a photosynthetic purple non-sulfur bacterium used as a model organism.Specifically, several steps of the ethylmalonyl-CoA are not yet understood and will be elucidated. Comparative genomic studies, mutant analysis, 14C-tracer studies, in vitro detection of enzymatic activity, heterologous gene expression and characterization of recombinant proteins will be used to identify further intermediates and genes/proteins of the ethylmalonyl-CoA pathway. It is possible that the ethylmalonyl-CoA pathway replaces the glyoxylate cycle in certain organisms because various substrates may enter the pathway at different points through peripheral routes. Wild type R. sphaeroides and mutants of the ethylmalonyl-CoA pathway will be examined for photoheterotrophic and chemoheterotrophic growth on a variety of organic compounds. The mechanism by which the key enzyme of the ethylmalonyl-CoA pathway, crotonyl-CoA carboxylase/reductase, is regulated in R. sphaeroides will be elucidated using a genetic approach. Furthermore, other genes that may be part of the same regulatory circuit will be identified.Broader impacts: These studies will provide further insights into the control of central carbon metabolism in bacteria and should enhance our understanding of transformations underlying the carbon cycles in a given habitat. Graduate and undergraduate students involved in the project will be sensitized to this emerging aspect of microbial metabolism and will be able to contribute towards solutions in their own research. A student with a strong background in modern microbial metabolism will realize the seemingly unlimited potential of biological transformations and will subsequently be able to apply this knowledge in an educational, academic, or industrial setting.