The metabolic pathways of any particular cell form a single interconnected network. The Investigator's goal is to understand how this network evolves in response to the changing needs of the organism. He and his team have developed a model system that streamlines laboratory investigations of adaptive genome evolution. They expressed the lux operon of Photorhabdus luminescens, which encodes an anabolic pathway producing visible light, in Acinetobacter baylyi ADP1, which naturally exchanges and recombines its chromosomal DNA as it grows. The model system has an active natural recombination system that enables an efficient microbial breeding strategy and at the same time, permits the selection of gene-based metabolic traits that contribute to the production of visible light. The Investigator proposes to direct the evolution of strains that produce even more light, and to investigate the most hypermorphic strain by genome sequencing, microarray analysis and biochemical studies of the evolved proteins. This work will show how evolutionary tradeoffs can optimize the flux through a model anabolic pathway. These lessons could be applied to increase the biosynthetic yields of biofuels, materials, and other economically valuable compounds.
Broader Impact:
Many prospective molecular biologists lack the financial resources to conduct research. The cost of the basic equipment necessary to transform Escherichia coli (centrifuge, 80° C freezer) imposes a formidable entry barrier. A. baylyi is naturally competent, so it can be transformed without expensive hardware. The Investigator's team has created a novel expression vector for this organism, and has written software that shows users how to combine DNA fragments in overlap PCR reactions. A determined high school student or hobbyist could use these tools to create and express novel genes in minimally equipped home laboratories. This study will inspire novice scientists to design and conduct their own experiments, and eventually to seek formal training.
Intellectual Merit Metabolic engineers can insert foreign genes in microorganisms, thereby causing them to produce valuable compounds, including pharmaceuticals and fuels. Production yields, however, tend to be low because microorganisms normally produce energy and materials for themselves rather than for human engineers. The original goal of this project was to use classical breeding strategies to "domesticate" an unusual bacterium, Acinetobacter baylyi ADP1, to maximize the production yields of a foreign substance (in this case, visible light). Light production, however, was not stably inherited, so a better understood bacterium, Escherichia coli, was studied. The effects of every possible gene knock-out upon light production and growth rate in E. coli was measured. Mutations that impart improvements in one trait were almost always coupled to a diminishment of the other. These results suggest that there is no such thing as a free lunch, even for evolving bacteria. Broader Impacts This project produced tangible returns to taxpayers. The study of the E. coli genome, which was viewed by over 500 scientists during its first month after publication, will motivate more efficient metabolic engineering strategies. The genetic tools that were developed for Acinetobacter baylyi ADP1, which have been distributed to over 100 researchers worldwide, will help others to use this organism in new ways. Synthetic biologists could exploit its natural ability to import and recombine DNA to construct whole chromosomes. Students at high schools and community colleges could use it to clone genes without the high capital costs of refrigerated centrifuges and ultra-cold freezers. Moreover, the post-doctoral fellow and technicians who were trained during this project will continue to enhance the productivity and competitiveness of the national economy for many years to come.
