Farren J. Isaacs
Programming Genomes to Re-engineer Life's Functional Repertoir
Molecular, Cellular & Developmental Biology
The conservation of the genetic code, with minor exceptions, enables exchange of gene function among species, viruses and across ecosystems. Fundamental changes to the genetic code could significantly enhance our understanding of the origins of the canonical code and reveal new subtleties of how genetic information is encoded and exchanged. Modifying the canonical genetic code could also lead to orthogonal biological systems with new properties. I will describe the construction of a genomically recoded organism (GRO) using high-throughput and automated methodologies for precise manipulation of genomes: multiplex automated genome engineering (MAGE) and conjugative assembly genome engineering (CAGE). In the GRO, all known UAG stop codons in Escherichia coli MG1655 were replaced with synonymous UAA codons, which permitted the deletion of release factor 1 and reassignment of UAG translation function. This GRO exhibited improved properties for incorporation of nonstandard amino acids (NSAAs) that expand the chemical diversity of proteins in vivo. To improve NSAA incorporation into proteins, we used MAGE in the GRO to mutagenize >12 residues in the amino acid binding pocket and tRNA binding interface of a Methanocaldococcus jannaschii tyrosyl-tRNA synthetase resulting in enzyme variants with 17- to 25-fold increased activities. The GRO also exhibited increased resistance to T7 bacteriophage, demonstrating that new genetic codes could enable increased viral resistance. This work increases the toolbox for genomic and cellular engineering with the goal of expanding the functional repertoire of organisms.