Shotgun Metagenomics Reveals Systemic Metabolic Pathways Promoting Organic Acid Production in Traditional Sunki...

Salt-free lacto-fermentation is gaining attention owing to the global demand for sodium reduction. Sunki, a traditional unsalted Japanese turnip pickle, provides a unique ecological model. In this study, we investigated the microbial balance and systemic metabolic pathways that promote organic acid accumulation using shotgun metagenomics. We revealed that a high-quality sunki cluster exhibiting an abundant accumulation of organic acids (lactic acid 8.58 ± 2.42 g/L, acetic acid 2.70 ± 0.76 g/L, succinic acid 1.50 ± 0.52 g/L) and low pH (3.91 ± 0.15) was characterized by the co-dominance of specific lactic acid bacteria along with the foundational species Limosilactobacillus fermentum. This co-dominance enables the synergistic operation of both the homofermentative and heterofermentative pathways, whose functional potentials positively correlated with lactic acid (r = 0.50) and acetic acid (r = 0.48) concentrations, respectively, thereby achieving both effective preservation and flavor complexity. Furthermore, we propose a dual-pathway model for succinic acid production involving the reductive tricarboxylic acid cycle from malate and the γ-aminobutyric acid (GABA) shunt. Succinic acid positively correlated with the functional potential of the GABA shunt enzyme (puuE, r = 0.57) and negatively correlated with that of glutamate dehydrogenase (gdhA, r = -0.51), suggesting a metabolic switch redirecting glutamate flux toward the GABA shunt. This mechanism highlights an advanced ecological adaptation for pH homeostasis, emphasizing that a balanced co-dominance structure determines the quality of salt-free fermented foods.