A novel D(−)-lactic acid-inducible promoter regulated by the GntR-family protein D-LldR of Pseudomonas fluorescens

Abstract

Lactic acid has two stereoisomers of D(−)- and L(+)-forms, both of which are important monomers of biodegradable plastic, poly-lactic acid. In this study, a novel d-lactate inducible system was identified in Pseudomonas fluorescens A506, partially characterized and tested as biosensor. The d-lactate catabolic operon (lldP-dld-II) was negatively regulated through the inversely transcribed D-lldR (encoding a GntR-type regulator), where the repression is relieved by addition of d-lactate. The derepression was specific to d-lactate and marginally affected by l-lactate. The D-LldR-responsive operator, showing dyad symmetry and separated by one base, was located between +11 and + 27 from the transcription start site of the lldP-dld-II operon. By site-directed mutagenesis, a motif with a dyad symmetry (AATTGGTAtTACCAATT), present in the upstream region of lldP, was identified as essential for the binding of LldR. d-lactate biosensors were developed by connecting the upregulation by d-lactate to a green fluorescent readout. About ~6.0-fold induction by 100 mM d-lactate was observed compared to l-lactate. Keywords: Lactic acid, Enantiomer, Biosensor, Repression