Genome Engineering Technologies for Rapid Programming & Evolution of Organisms

Farren Isaacs

Harnessing the potential of species diversity makes biological systems ideal to address global challenges, such as producing new drugs to alleviate human disease and generating biologically derived fuels, chemicals and materials to ensure environmental sustainability. In addition to a thorough understanding of biological systems, achieving these goals requires safe and programmable control of biological systems. In this regard, our ability to measure and modify genetic and biochemical components and understand their interactions in pathways, cells and the environment remain defining challenges.

One of the key cellular engineering challenges is the development of high-throughput and automated methodologies for precise manipulation of genomes from the nucleotide to megabase scales. To address these challenges, I develop methods for versatile genome modification and evolution of cells. Multiplex Automated Genome Engineering (MAGE) simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Hierarchical Conjugative Assembly Genome Engineering (CAGE) facilitates the large-scale assembly of many modified genomes. I applied MAGE to optimize the DXP biosynthesis pathway in E. coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. In three days, variants were isolated with more than fivefold increase in lycopene production, a significant improvement over existing metabolic engineering techniques. I have also applied MAGE and CAGE to engineer strains of E. coli in which the entire genome is recoded, replacing the 314 UAG stop codons with UAA synonymous major stop codons.

These changes to the genetic code allow us to construct safer and multi-virus resistant strains and enhance the incorporation of unnatural amino acids into proteins. Our methods treat the chromosome as both an editable and evolvable template and are capable of fundamentally re-engineering genomes from the nucleotide to the megabase scale. Moreover, this work expands the repertoire of technologies for cellular engineering and could generate useful biological systems for compelling applications, such as the development of new drugs, fuels, chemicals and materials.