title: Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16.
abstract: Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P(lac), P(tac), or P(BAD) derived from Escherichia coli, or promoter regions of phaC1 (P(phaC)) or phaP1 (P(phaP)) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P(lac), P(tac), P(phaC), and P(phaP ) mediated constitutive gene expression, among which P(tac) was the strongest promoter. lacI-P(tac) was not thoroughly functional even after addition of isopropyl-β-D-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P(BAD) could be regulated by varying L-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P(phaP) exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P(phaP ) for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.
authors.affiliation: Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-37 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. tfukui@bio.titech.ac.jp
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Fukui
authors.forename: Kei
authors.initials: K
authors.lastname: Ohsawa
authors.forename: Jun
authors.initials: J
authors.lastname: Mifune
authors.forename: Izumi
authors.initials: I
authors.lastname: Orita
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nakamura
mesh_terms: D003470:Culture Media / Q000737:chemistry; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism*; D005632:Fructose / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D054813:Polyhydroxyalkanoates / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D013025:Glycine max / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D003470:Culture Media; D054813:Polyhydroxyalkanoates; D049452:Green Fluorescent Proteins; D005632:Fructose; D007544:Isopropyl Thiogalactoside