Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

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-beta-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.