Intellectual Merit of the Research Project: Nothing in biology makes sense except in the light of evolution, wrote Theodore Dobzhansky. Accordingly, scientists "make sense" of life by understanding biological traits from metabolism at the molecular level to behavior at the societal level in response to eons of selective pressure for enhanced survival. The diversity of life on earth was enabled by a myriad evolutionary trajectories emanating from starting points defined by ancestral sequences that existed billions of years ago. Might different trajectories have evolved if different ancestral sequences had been available? Until now this question could not be answered; collections of primordial sequences were not available for laboratory experimentation. Now however, novel genes and proteins are available. Sequences unbiased by billions of years of evolutionary history can be designed and constructed de novo. Such sequences provide a novel feedstock to restart evolution in the laboratory. Genes that never existed in nature, but which nonetheless encode biological functions that sustain life will enable experiments to assess whether (i) alternative progenitor sequences might lead to different evolutionary trajectories, and (ii) different molecular ancestors might produce fundamentally different progeny. The current project addresses these issues at the level of genes, proteins, genomes, and proteomes. The aims of this work include the following: (1) Discovery of novel sequences that do not resemble natural genes, but nonetheless enable cell growth. Life-sustaining functions will be passaged in both prokaryotic and eukaryotic cells. (2) Probing the functional promiscuity of novel proteins and delineate the extent to which unevolved sequences are multifunctional "generalists." (3) Determination of the biochemical and biophysical properties of novel proteins; and correlation of structural order with catalytic proficiency in sequences that have not benefited from selection. (4) Restart evolution by selecting novel sequences to further enhance microbial growth.
Broader Impacts Resulting from the Research Activity: EDUCATION: Surveys indicate that nearly 50% of Americans do not accept evolution as the overarching theory that "makes sense" of life on earth. Evolution's detractors argue that because it is not possible to go back in time and actually observe evolutionary trajectories from ancestral progenitors to modern organisms, evolution must remain "only" a theory. Although evolution in vitro is now a standard technique for protein engineering in academic labs and biotechnology companies, skeptics argue that such studies rely on feedstocks of sequences provided by nature, and therefore provide only limited support for Darwinian theories about the origin and diversification of life on earth. The current project will provide the first example of laboratory studies demonstrating explicitly that (i) novel genes bearing no sequences resemblance to those from natural organisms can evolve to produce progeny that enhance the fitness of a host organism, and (ii) de novo rather than derived macromolecules can provide essential biological functions. Dissemination of these results will have a broad impact on science education and literacy. This study will provide also multiple research training opportunities for students at all levels and postdoctoral researchers.
IMPACTS ON DIVERSITY AND OUTREACH: Woman and minorities play significant roles in this research, and numerous outreach activities are planned.
BIOTECHNOLOGY: Current biotechnology relies on genes and proteins isolated (or modified) from natural sequences. Yet the number of sequences in natural genomes and proteomes is dwarfed by the possibilities in all of sequence space. This research project explores the functional potential of novel sequences, thereby laying foundations for new technologies in which novel proteins will be developed for specific applications.
