Metabolic engineering of Lactobacillus delbrueckii subsp. bulgaricus VI104 as a D-lactic acid cell factory through...

Lactobacillus delbrueckii subsp. bulgaricus is a homofermentative lactic acid bacterium with recognized utility in dairy fermentation and unique capability to produce optically pure D-lactic acid (DLA), a critical monomer for polylactic acid-based bioplastics. Despite this potential, its industrial deployment has been constrained by poor galactose metabolism, incomplete lactose assimilation, and lack of efficient genetic tools. Here, we report the first comprehensive metabolic engineering of L. bulgaricus VI104 to establish a high-performance DLA cell factory. A tailored molecular toolbox was developed, including high-efficiency electroporation (~ 10⁴ CFU µg⁻¹ DNA), the pLEM415 shuttle vector, and strong constitutive promoters (P_ldhL), enabling stable heterologous gene expression. Galactose utilization was restored through heterologous expression of galK and galT, while ATP homeostasis was rebalanced via pyk overexpression. Further enhancement of glycolytic flux was achieved by co-expressing pfk, pgk, and the major dldh isoform. The engineered strain LdbVI104_07 exhibited a 3.4-fold improvement in acid tolerance and produced 9.39 g L⁻¹ DLA with 99.09% optical purity and a yield of 0.188 g g⁻¹ under optimized bioreactor conditions (3% lactose, 0.15% w/v supplemented glutamine and phenylalanine, 40 °C, pH 6.5). Multi-layer validation via transcriptomics revealed > 2.5-fold upregulation of core metabolic genes and downregulation of the global repressor ccpA, confirming metabolic rewiring. This work not only establishes L. bulgaricus as a genetically tractable and industrially relevant chassis but also provides a versatile platform for precision strain engineering in lactic acid bacteria, with direct applications in sustainable biomanufacturing, therapeutics, and synthetic biology.