Interrogating the Preferential Catabolism of Dietary Sialic Acids

Lactobacillus delbreuckii subsp. bulgaricus, a homofermentative generally regarded as safe (GRAS) probiotic lactic acid bacterium (LAB), crucial in yogurt production, excels at producing optically pure D-lactic acid (DLA). Despite its role in dairy sector, limited research exists in genetically engineering this organism due to the lack of molecular cloning tools. This study aims to bridge this gap by engaging in the metabolic engineering Lactobacillus delbreuckii subsp. bulgaricus VI104, with the overarching goal of enhancing and fine-tuning DLA production. DLA holds exceptional significance as a crucial organic compound, for synthesis of biodegradable polymer, Poly lactic acid (PLA). The linchpin for PLA production lies in obtaining optically pure DLA, and the principal pathway responsible for DLA biosynthesis is glycolytic pathway. Our approach entails simultaneous overexpression of key glycolytic genes—phosphofructokinase (pfk), phosphoglycerate kinase (pgk), and pyruvate kinase (pyrk)—in conjunction with primary d-lactate dehydrogenase (d-ldh) gene downstream of ldhP promoter from Lb. sakei. This strategic augmentation resulted in a remarkable fourfold increase in DLA production within recombinant Lactobacillus delbreuckii subsp. bulgaricus VI104. Importantly, these improvements were achieved without compromising overall cellular ATP levels, concurrently bolstering the strain's acid tolerance properties. Subsequently, we optimized DLA production through batch fermentation, both under static and shaking conditions. Subsequent investigations will focus on optimizing the growth medium and conducting fermentation studies at the bioreactor level. We introduced an engineered DLA-inducible promoter to VI104, allowing dynamic DLA production control. This research broadens Lactobacillus delbreuckii subsp. bulgaricus applications and sets a genetic manipulation foundation for future studies.