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 biosynthetic operon were effectively regulated by cultivation temperature was constructed in Escherichia coli. The pha operon was fused to the negatively, regulated tac promoter and cloned into a vector in which the copy 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 plasmid copy number was depressed and the number of LacI proteins was sufficient to repress tac::pha transcription. The production phase was initiated by temperature upshift. At the elevated temperature, the number of plasmids surpassed the number of LacI repressors, which resulted in rapid induction of tac::pha transcription, synthesis of poly-beta-hydroxyalkanoate-specific proteins, and polymer synthesis. During the production 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 bacteria which naturally produce this polymer.