Engineering the D-lactic acid responsive promoter/repressor system as dynamic metabolic engineering tool in...

Dynamic metabolic engineering integrates synthetic logic circuits into cellular systems, optimizing metabolic fluxes and augmenting biosynthesis of target metabolites. This study evaluated a D-lactic acid (DLA)-responsive promoter-repressor system from Pseudomonas fluorescens A506, re-engineered for heightened sensitivity and functional efficacy in Lactobacillus delbrueckii subsp. bulgaricus VI104. The codon-optimized regulatory architecture exhibited peak performance at DLA inducer concentration range of 60-100 mM, validated by fluorometry and microscopy. As an application, overexpression of D-lactate dehydrogenase (dldh) downstream of the engineered promoter repressor system enabled finely tuned modulation of DLA biosynthesis, autonomously regulating the transition between growth and production phases, thereby attenuating overall metabolic load. Cross-species compatibility was confirmed by excising regulatory elements from the promoter-repressor system and functionally assessing them in recombinant L. bulgaricus. Molecular docking elucidated critical noncovalent interactions between D-LldR repressor and operator nucleotide sequence in absence of inducer DLA. The engineered promoter construct with high efficiency effectively elevated DLA biosynthesis by 2.15-folds and expanded the overall fermentation time relative to constitutive systems, attaining maximum DLA titre of 9.02 g L⁻¹ in bioreactor setup. These results substantially broaden the molecular cloning toolkit available for L. bulgaricus, fostering potential future applications in biotherapeutics and probiotics.