Engineering the D-lactic acid responsive promoter/repressor system as dynamic metabolic engineering tool in Lactobacillus delbrueckii subsp. bulgaricus for controlled D-lactic acid biosynthesis

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2025-02-07 DOI:10.1016/j.enzmictec.2025.110606

Payal Mukherjee , Senthilkumar Sivaprakasam

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引用次数: 0

Abstract

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.

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.