Regulatory effects on central carbon metabolism from poly-3-hydroxybutryate synthesis.

Poly-3-hydroxybutyrate (PHB) synthesis in Escherichia coli elicits regulatory responses that affect product yield and productivity. We used controlled, steady-state cultures (chemostats) of a genetically stable strain to determine growth-independent metabolic flux regulation. We measured flux and steady-state intracellular metabolite concentrations across different dilution rates (0.05, 0.15, 0.3h(-1)), limitations (glucose, gluconate and nitrogen), and operon copy counts of the PHB pathway (0, 6, 17, and 29). As PHB flux increases, specific substrate consumption and lactate secretion increase while formate and acetate secretion decreases in N-limited, glucose-fed conditions. To understand the regulatory mechanisms that resulted in these macroscopic changes, we used a flux balance analysis model to analyze intracellular redox conditions. Our model shows that under N-limited conditions, synthesis of PHB creates excess reducing equivalents. Cells, under these conditions, secrete more reduced metabolites in order to recycle reducing equivalents. By switching to a more oxidized substrate (gluconate) that decreased excess reducing equivalents, PHB flux yield increased 1.6 fold compared to glucose-fed fermentations. High flux of PHB (~1.2 mmol/g DCWh) was maintained under these steady-state, oxidized conditions. These results imply redox imbalance is a driving force in industrial production of PHB, and substrates that are more oxidized than glucose can increase productivity.