Open AccessCloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway
Author(s) -
Steven Slater,
William H. Voige,
Douglas Dennis
Publication year - 1988
Publication title -
journal of bacteriology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.652
H-Index - 246
eISSN - 1067-8832
pISSN - 0021-9193
DOI - 10.1128/jb.170.10.4431-4436.1988
Subject(s) - cosmid , biology , subcloning , escherichia coli , alcaligenes , ecori , biochemistry , molecular cloning , biosynthesis , microbiology and biotechnology , enzyme , genomic library , plasmid , dna , bacteria , gene , genetics , peptide sequence , pseudomonas
The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.