REGULATED EXPRESSION OF THE ALCALIGENES-EUTROPHUS PHA BIOSYNTHESIS GENES IN ESCHERICHIA-COLI

A novel poly-beta-hydroxybutyrate (PHB) production system in which the expression and gene dosage of the Alcaligenes eutrophus pha biosynthe tic operon were effectively regulated by cultivation temperature was c onstructed in Escherichia coil. The pha operon was fused to the negati vely, regulated tac promoter and cloned into a vector in which the cop y number is temperature dependent. A two-phase process was employed to produce PHB during fed-batch growth. In the growth phase, the culture was maintained at a low temperature. Under this condition, the plasmi d copy number was depressed and the number of LacI proteins was suffic ient to repress tac::pha transcription. The production phase was initi ated by temperature upshift. At the elevated temperature, the number o f plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tac::pha transcription, synthesis of poly-beta-hydr oxyalkanoate-specific proteins, and polymer synthesis. During the prod uction phase, the PHB production rate was 1.07 g of PHB liter(-1) h(-1 ) under optimized conditions. This rate is comparable to that of bacte ria which naturally produce this polymer.