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Characterization of Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) Production in Escherichia Coli

Matthew S. Wong1, Thomas B. Causey2, Nikos Mantzaris3, George N. Bennett2, and Ka-Yiu San1. (1) Bioengineering, Rice University, 6100 Main Street MS#142, Houston, TX 77005, (2) Biochemistry & Cell Biology, Rice University, 6100 Main Street MS#140, Houston, TX 77005, (3) Chemical & Biomolecular Engineering, Rice University, 6100 Main Street MS#362, Houston, TX 77005

A recombinant strain of Escherichia Coli was metabolically engineered to vary the composition of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This was accomplished by placing the production of propionyl-CoA substrate for copolymer synthesis was put under the control of an IPTG-inducible plasmid (pMMB-prpE), which contains the propionyl-CoA synethetase prpE. The polyhydroxyalkanoate synthesis operon (phaBCA) from Alcaligenes eutrophus was coexpressed from a plasmid that is compatible with pMMB-prpE. These plasmids were used in conjunction with the E. coli strain GJT001. The addition of varying concentrations of IPTG was used to tune the composition of the PHBV. The quantity and composition of the PHBV produced were measured by GC. The intracellular concentrations of CoA, acetyl-CoA, and propionyl-CoA were monitored by HPLC. The PHBV molecular weight distribution was examined by GPC. The prpE activity was measured by enzymatic assay. The distribution of PHBV production in the cell population was characterized by Nile red staining of PHBV granules and flow cytometry. This study illustrates the impact of varying PHBV composition on E. coli metabolism and on the characteristics of the PHBV produced.