In vivo Characterization of the Inducible Promoter System of 3-hydroxypropionic Dehydrogenase in Pseudomonas denitrificans

Abstract

Here, we characterized a gene expression system induced by 3-hydroxypropionic acid (3-HP) in Pseudomonas denitrificans. The system consists of a putative LysR-type transcriptional regulator (LTTR) encoded by hpdR and a LTTR-responsive promoter that positively controls the expression of hpdH, encoding 3-HP dehydrogenase in the presence of 3-HP. In the hpdH-responsive promoter region, two operators exhibiting dyad symmetry and designated O-1 and O-2 and centered at the -73 and -30 positions, respectively, were located upstream of the hpdH transcription start site. When either O-1, O-2, or both regions were mutated, the inducibility by the HpdR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcriptional activation. The HpdR protein and its operator sites on hpdH were highly specific for each other, and did not engage in cross-talk with another similar 3-HP-inducible system that is also present in P. denitrificans. This 3-HP-inducible promoter system should be useful in developing biosensors for 3-HP, and/or dynamic gene expression systems for metabolic engineering purposes.