(127b) Genetic and Metabolic Perturbation Analysis of the Polyhydroxybutyrate Pathway

Continuous biocatalyst reactors can have economic advantages over batch reactors. For this, the desired product must be produced at high levels during growth phase. However, in growth-associated production, growth rates and product formation rates are delicately balanced. In the case of poly-3-hydroxybutyrate (PHB) production in Escherichia coli, the carbon supply must be adequate for biomass production, but simultaneously, the flux of carbon to PHB must be high for attractive yields to be achieved. In this study, growth-phase PHB productivity was studied by (1), manipulation of gene expression in the product-forming pathway and (2) varying growth rates in a nitrogen-limited chemostat. Systematic overexpression of genes in the product forming pathway revealed the acetoacetyl-CoA reductase (phaB) limits flux to PHB, but the highest diversion of carbon to PHB was found by overexpressing the entire pathway. As PHB pathway expression levels increased, the maximal growth rate decreased. The observed trade-off between PHB productivity and growth rate was largely consistent with the stoichiometrically maximal trade-off, as calculated from a stoichiometric model of E. coli metabolism, implying changes in growth could be accounted for by the diversion of resources to PHB. In a nitrogen-limited chemostat, PHB productivity was insensitive to growth rate. These results imply that specific PHB productivity is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors and should allow the design of tradeoff between product and biomass for optimal process conditions.