Effects of recombinant precursor pathway variations on poly[(R)-3-hydroxybutyrate] synthesis in Saccharomyces...

Different recombinant R-3-hydroxybutyryl-CoA (3-HB) synthesis pathways strongly influenced the rate and accumulation of the biopolymer poly[(R)-3-hydroxybutyrate] (PHB) in Saccharomyces cerevisiae. It has been previously shown that expression of the Cupriavidus necator PHB synthase gene leads to PHB accumulation in S. cerevisiae [Leaf, T., Peterson, M., Stoup, S., Somers, D., Srienc, F., 1996. Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiology 142, 1169-1180]. This finding indicates that native S. cerevisiae expresses genes capable of synthesizing the correct stereochemical substrate for the synthase enzyme. The effects of variations of 3-HB precursor pathways on PHB accumulation were investigated by expressing combinations of C. necator PHB pathway genes. When only the PHB synthase gene was expressed, the cells accumulated biopolymer to approximately 0.2% of their cell dry weight. When the PHB synthase and reductase gene were co-expressed, the PHB levels increased approximately 18 fold to about 3.5% of the cell dry weight. When the beta-ketothiolase, reductase and synthase genes were all expressed, the strain accumulated PHB to approximately 9% of the cell dry weight which is 45 fold higher than in the strain with only the synthase gene. Fluorescent microscopic analysis revealed significant cell-to-cell heterogeneity in biopolymer accumulation. While the population average for the strain expressing three PHB genes was approximately 9% of the cell dry weight, some cells accumulated PHB in excess of 50% of their cell volume. Other cells accumulated no biopolymer. In addition, the recombinant strain was shown to co-produce ethanol and PHB under anaerobic conditions. These results demonstrate that the technologically important organism S. cerevisiae is capable of accumulating PHB aerobically and anaerobically at levels similar to some bacterial systems. The easily assayed PHB system also creates a convenient means of probing in vivo the presence of intracellular metabolites which could be useful for studying the intermediary metabolism of S. cerevisiae.