Metabolic engineering of the Escherichia coli polyphosphate pathways

Many organisms store energy and phosphate in polymers of three to more than one thousand residues called polyphosphate. In Escherichia coli, these compounds are synthesized by polyphosphate kinase (PPK), which reversibly transfers the terminal phosphate group from ATP to the polyphosphate chain. The gene encoding polyphosphatase (ppx), which irreversibly and processively hydrolyzes phosphate residues from polyphosphate, is located on the same operon as ppk. The goal of this project is to investigate how polyphosphate metabolism is regulated, and how it influences other cellular processes.;Studies using promoter-reporter gene fusions demonstrated that the native polyphosphate operon is constitutively transcribed at low levels; therefore, use of the E. coli native promoter is unsuitable for both overproduction of polyphosphate and control over polyphosphate metabolism. An engineered E. coli strain was developed by placing the ppk and ppx genes under control of separate, inducible promoters on different plasmids, enabling polyphosphate to be selectively synthesized and degraded by the addition of different inducers. When polyphosphate was degraded by PPX, excess phosphate was released from the cell. When polyphosphate degradation coincided with a shift to phosphate-free medium, the cell's natural response to phosphate starvation (the Pho regulon) was repressed. By varying the level of ppx induction in the engineered strain, precise control over expression from Pho promoters can be achieved.;The ability to overproduce PPK was also used to explore the relationship between polyphosphate and the energy status of the cell. Specifically, experiments were performed to see if polyphosphate could supply energy to the cell during periods of energy limitation. Although the synthesis of polyphosphate by PPK is reversible in vitro, no degradation of polyphosphate was observed in E. coli upon a shift to energy-starved conditions.;In parallel with the experimental work described above, a structured mathematical model of the phosphate starvation response consisting of 21 ordinary differential equations was also developed. With only two adjustable parameters, experimental results were matched for the production of alkaline phosphatase, an enzyme produced under control of the Pho regulon during periods of low external phosphate concentration. The model was then applied to cells engineered for the controlled synthesis and degradation of polyphosphate. In agreement with the experimental observations, the model predicted release of phosphate from the cell and subsequent repression of the Pho regulon when polyphosphate was degraded by PPX.