| Current Projects |
Post-doctoral fellow Wayne Patrick and Research Specialist Erik Quandt have identified promiscuous catalytic activities within the proteome of the model bacterium, Escherichia coli . This work was the first systematic analysis of the evolutionary potential of an extant proteome. It suggests that most proteins have secondary activities, and that these activities provide the seeds of evolutionary innovation. The weak promiscuous activities are being evolved in the laboratory; these experiments should provide further insights into the process of adaptive molecular evolution.
Metabolic network evolution:
The metabolic pathways of any particular cell form a single interconnected network. Our goal is to understand how this network evolves in response to the changing needs of the organism. We have developed a model system that streamlines laboratory studies of adaptive genome evolution. The lux operon of Photorhabdus luminescens, which encodes an anabolic (energy-requiring) pathway that produces visible light, was expressed in Acinetobacter baylyi ADP1, a bacterium that naturally exchanges and recombines its chromosomal DNA as it grows. Classical selective breeding strategies will be applied to evolve bacterial strains that produce even more light. The genome of the brightest strain will be sequenced, the genes that were turned up or down will be identified, and the catalytic properties of the evolved enzymes will be measured. This work will show how evolutionary trade-offs can optimize the flux (flow of matter and energy) through a model anabolic pathway. These lessons could be applied to increase the production yields of biofuels, biomaterials and other environmentally friendly compounds.
All metabolic pathways within a cell interconnect to form a single network, which evolves in response to the chemical needs of the organism. (image from http://www.lce.hut.fi/publications/annual2004/fig43.jpg)
Carbon Dioxide Fixation:
Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the rate-limiting step of the Calvin Cycle, the major conduit of carbon dioxide fixation. Previous attempts to improve the catalytic efficiency and/or CO2 specificity by structure-based site-directed mutagenesis have at best led to modest (5-13%) improvements in specificity. We have developed a novel ultra-high throughput genetic selection for RuBisCO function by engineering the pentose phosphate pathway of wild-type Escherichia coli . Our selection system enables the evaluation of millions of RuBisCO mutants, and is significantly more sensitive and rapid than previously reported systems. We are directing the evolution techniques of Synechococcus PCC6301 RuBisCO variants with improved the catalytic efficiency and specificity.
Consurf image of the large subunit of RuBisCO (1RBL).
We are also collaborating with others to improve the overall efficiency of photosynthesis, and to engineer the process so that it produces higher value fuels. Our strategy is to shunt otherwise unused electron equivalents produced under high light conditions away from RuBisCO in photosynthetic source cells, through extracellular nanowires, and to independently engineered fuel production cells. This trans-cellular, plug-and-play platform will enable the engineering of the light and dark parts of photosynthesis, as well as the conductive (bio)wire between them, in isolation. The project will provide proof of principle that energy can be transferred directly between cells as bio-electricity. Furthermore, the biowire to be developed will serve as a future generic connector for electrically interfacing distinct cell types to create novel, functional biofilms. More generally, modularity and technical standardization promote parallel technology development, and the exchange of components. Components to be used in future systems need not even be biological so long as they interface with the wires developed in this project. The photosynthetic components described in this proposal will thus serve as prototypes, and this study will establish a new design paradigm.
Plug and Play project strategy (cartoon produced by NSF).