Abstract
Regulated promoters are an important basic genetic tool allowing, for example, gene-dosage and gene depletion studies. We have previously described a cumate-inducible promoter (P(Q5)) that is functional in diverse Alphaproteobacteria. This promoter has been engineered by combining a synthetic minimal promoter, P(syn2), and operator sites and the repressor of the Pseudomonas putida F1 cym/cmt system. In the present study, we engineered a vanillate-regulated promoter using P(syn2) and the regulatory elements of the Caulobacter crescentus vanR-vanAB system. We show that the resulting promoter, which we called P(V10), responds rapidly to the inducer vanillate with an induction ratio of about two orders of magnitude in Sphingomonas melonis Fr1. In contrast to the switch-like behavior of P(Q5), P(V10) shows a linear dose-response curve at intermediate vanillate concentrations, allowing graded gene expression. P(V10) is functionally compatible with and independent of P(Q5) and cumate, and vice versa, suggesting that both systems can be used simultaneously.