| Polyhydroxybutyrate (PHB) is a plastic material which is of value because it is produced from renewable resources and is completely biodegradable. Cost barriers to widespread use of PHB may be overcome by application of biotechnology; one approach is the introduction of PHB pathways into new host cells. The yeast Saccharomyces cerevisiae, an organism which does not produce PHB, was used as a model eukaryotic host for introduction of a PHB pathway.; Genetic material from the bacterium Alcaligenes eutrophus, encoding its three PHB biosynthetic enzymes, was used to prepare constructs for expression of these enzyme activities in yeast. The first heterologous gene introduced into the yeast host was PHB synthase. This resulted in accumulation of PHB in the cells at a level of 0.3-0.5% of dry cell mass, and indicated that the native metabolism of yeast is able to synthesize the polymer precursor, D-3-hydroxybutyryl-CoA. The second heterologous gene introduced into the yeast host was reductase. This gene was co-expressed with synthase, resulting in high levels of reductase activity and an accumulation of PHB greater than 1% of dry cell mass. PHB was always observed to accumulate in the stationary phase of batch growth and was synthesized concomitantly with ethanol.; A mathematical analysis of the PHB biosynthetic pathway was undertaken to determine important factors affecting the rate of PHB synthesis inside a cell. Results suggested the importance of all three PHB biosynthetic enzymes in determining overall pathway flux, and demonstrated a need for using reversible enzyme kinetic expressions in such models. The model also supported the theory that thiolase and reductase enzymes play a regulatory role in determining the rate of PHB synthesis under different physiological conditions. Stoichiometric analysis of central metabolism and PHB synthesis was performed with attention to utilization and regeneration of ATP and reducing equivalents. It was shown that PHB synthesis represents a net source of reducing equivalents, and is therefore expected to be problematic in anaerobic culture and to change the distribution of fermentation products. This redox imbalance was suggested as a potentially limiting factor in the achievable carbon flux from carbohydrates to PHB in yeast. |
