(5cs) Meeting in the Middle: Top-Down and Bottom-up Approaches to Metabolic Engineering

Metabolic engineering has been tasked with harnessing the amazingly specific and complex chemistry of biological life for the improvement of human existence. Unlike other fields of engineering, where mathematical models can accurately described the system and are central to design, metabolic engineering has and continues to rely on engineering strategies that must be robust in the face of large biological uncertainty. The understanding of both small sets of interacting molecules (e.g. a short metabolic pathway), and global systems (e.g. regulatory stress responses), present a formidable challenge to further unlock the usefulness of cells. In my research, I have contributed by both top-down and bottom-up approaches for metabolic engineering

Bottom-up approaches have been used in (1) improving genetic stability of recombinant expression and (2) analyzing the biopolymer, polyhydroxybutyrate (PHB), production in E. coli. (1) Recombinant productivity loss, as a result of plasmid instability, was studied by a simple probabilistic model of plasmid propagation. The model identified a new genetic instability pathway, termed ?allele segregation.? A novel high-copy genomic integration strategy, Chemically Induced Chromosomal Evolution (CIChE), was developed that avoided allele segregation and can improve genetic stability 10 fold. CIChE was implemented experimentally and significantly improved genetic stability and specific productivity over plasmids. We expect CIChE to find application in many industrial recombinant processes because of CIChE's superior properties compared to plasmids. (2) Systematic evaluation of the PHB metabolic pathway enzymes identified the acetoacetyl-CoA reductase as the major limitation in the pathway, despite the long standing hypothesis that production was substrate limited. The key insight to measure flux to PHB, rather than batch accumulation, delineated growth rate dilution effects from changes in pathway flux. Through systematic overexpression and chemostat studies, these biological observations could be made, and PHB flux was increased by 3.7 fold.

Top-down approaches were successfully used in (3) PHB improvement in photosynthetic organisms and are currently being applied to (4) yeast protein secretion. (3) Regulatory pathways in the photosynthetic bacterium Synechocystis PCC6803 are largely unknown, but strictly control PHB production. The development of a mutational library and screening strategy resulted in the identification of distal genes that improve PHB production, by as much as 3 fold. These biological results may be useful in the engineering of higher plants for biopolymer production, where such screening would be impossible. (4) I am currently expanding my top-down competence to include high-throughput data analysis (X-omics) in the important eukaryotic organism, S. cerevisiae. Work is focused on improving throughput and trafficking of secreted proteins in the yeast secretory pathway. This will be valuable for the production of protein therapeutics and industrial enzymes, such as cellulases.

In future challenges, the flexibility to approach problems from a ?top-down? or ?bottom-up?, or most importantly, the development of theories and methods for integration of the two approaches will be essential. Experience in both of these approaches should prove useful in metabolic engineering pursuits.