Solution Properties and Molecular Size of Polyhydroxybutyrate from Recombinant Escherichia coli

Polyhydroxybutyrate molecules produced by recombinant Escherichia coli were evaluated in terms of their size in solution to develop and test a hypothesis concerning the regulation of the molecular weight of PHB during in vivo polymer production. Two recombinant strains of E. coli were used to produce material for this study: DH5a/pAeT41, containing the native operon from Alcaligenes eutrophus encoding the three enzymes necessary for the biosynthesis of PHB and DH5a/pSP2, a strain genetically engineered to optimize synthase production in E. coli. In the latter strain, the induction agent isopropyl-p-D-thiogalactopyranoside is used to induce synthase production. Initial flask cultures of these two recombinant strains produced pure PHB showing greatly different intrinsic viscosities in chloroform. Strain pAeT41 produced PHB molecules nearly an order of magnitude larger in intrinsic viscosity than those produced by strain pSP2. This led to the hypothesis that high synthase concentration led to the production of shorter PHB chains. The same fermentation conditions in a larger scale 2 liter fermentation led to similar results with PHB chains from pSP2 being one half the length of those from pAeT41. In an attempt to regain the drastic difference in molecular weight previously discovered, the molar concentration of the induction agent (IPTG) added to the pSP2 culture was increased, and a lower PHB molecular weight resulted, presumably due to an increase in synthase production. The molecular weight as a function of fermentation time was determined using ten liter fermentations. PHB produced by pAeT41 maintained a relatively constant molecular mass, while PHB from pSP2 distinctly decreased in molecular weight during the fermentation. This led to the hypothesis that initial PHB molecular weight was large due to very low enzyme production prior to IPTG induction, at which time high enzyme concentration caused the production of new shorter PHB chains. SEC and light-scattering showed that as enzyme production reached maximum levels, average PHB molecular weight dropped and the polydispersity index sharply increased due to an immediate increase in the number of short chains produced. Thesis Supervisor: ChoKyun Rha Title: Professor of Biomaterials Science and Engineering