Abstract
The first demonstration that macromolecules could be evolved in a test tube was reported twenty-five years ago. That breakthrough meant that billions of years of chance discovery and refinement could be compressed into a few weeks, and provided a powerful tool that now dominates all aspects of protein engineering. A challenge has been to extend this scientific advance into synthetic chemical space: to enable the directed evolution of abiotic molecules. The problem has been tackled in many ways. These include expanding the natural genetic code to include unnatural amino acids, engineering polyketide and polypeptide synthases to produce novel products, and tagging combinatorial chemistry libraries with DNA. Importantly, there is still no small-molecule analog of directed protein evolution, i.e. a substantiated approach for optimizing complex (≥ 10^9 diversity) populations of synthetic small molecules over successive generations. We present a key advance towards this goal: a tool for genetically-programmed synthesis of small-molecule libraries from large chemical alphabets. The approach accommodates alphabets that are one to two orders of magnitude larger than any in Nature, and facilitates evolution within the chemical spaces they create. This is critical for small molecules, which are built up from numerous and highly varied chemical fragments. We report a proof-of-concept chemical evolution experiment utilizing an outsized genetic code, and demonstrate that fitness traits can be passed from an initial small-molecule population through to the great-grandchildren of that population. The results establish the practical feasibility of engineering synthetic small molecules through accelerated evolution.
Highlights
Evolution, the change in the inherited characteristics of biological populations over successive generations, accounts for the diversity of life on earth
We designed a peptide library that was chemically assembled in four coupling steps, incorporating seventeen different amino acids in the first three steps, and eighteen in the fourth step (Fig 2)
2.17Ã1010 different DNA genes programmed the synthesis of a peptide, but only 88,434 unique tetramer and pentamer amino-acid sequences were produced because of redundancy in the genetic code
Summary
The change in the inherited characteristics of biological populations over successive generations, accounts for the diversity of life on earth. Thirty thousand years ago humans began to harness it as a tool for the selective breeding of animals and crops. Evolution is routinely used to engineer macromolecules [1,2,3,4]. The enabling technology involves the creation of a microscopic ecosystem populated by autonomous DNA, RNA or protein units. PLOS ONE | DOI:10.1371/journal.pone.0154765 August 10, 2016
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