HIGH-LEVEL POLY(BETA-HYDROXYBUTYRATE) PRODUCTION IN RECOMBINANT ESCHERICHIA-COLI IN SUGAR FREE, COMPLEX MEDIUM

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Alcaligenes eutrophus that are organized in a single operon (phbCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type phb operon from plasmid p4A leads to the formation of 10 or 50-80% PHB/cell dry mass when the cells are grown in Luria-Bertani medium alone or supplemented with 1% glucose (w/v), respectively. To further stimulate PHB formation independent of additional carbon source in Luria-Bertani medium, molecular methods have been applied to provide efficient E, coli transcription and translation signals for the PHB synthase gene (phbC). The lac promoter present upstream of the phbC sequence allows its expression to be controlled depending on the LacI status of the chosen host strain. The T7 gene 10 ribosome binding site is utilized for translational initiation. PHB production in E, coli was compared in strains either harboring plasmid p4A containing the intact phbCAB operon or harboring two compatible plasmids carrying the beta-ketothiolase (phbA) and acetoacetyl-CoA-reductase (phbB) genes under transcriptional control of the lac promoter-operator region and also carrying separately the phbC gene with its natural promoter sequence. In addition, plasmid pSYN allowing the phbC gene to be expressed under new transcription and translation conditions combined with plasmid pUMS gave rise to the same amount of PHB formation (70% PHB cell dry mass) in E, coli when grown in Luria-Bertani medium without glucose supplement.