Synthetic vanillate-regulated promoter for graded gene expression in Sphingomonas

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.