Carbon source-dependent metabolic states govern redox homeostasis and cofactor biosynthesis in Propionibacterium freudenreichii

Microbial adaptation to fluctuating nutrient and oxygen conditions requires coordinated regulation of metabolic networks to maintain redox homeostasis within physicochemical and energetic constraints. While oxygen-dependent responses in Propionibacterium freudenreichii (PFR) have been characterized at the transcriptomic level, the role of carbon source in defining system-level metabolic states remains unclear. Here, we investigated carbon source-dependent metabolic reprogramming and cofactor biosynthesis in PFR strain DSM 20271 using label-free quantitative proteomics integrated with physiological and metabolite analyses. Distinct carbon sources defined discrete metabolic states shaped by redox balance and flux distribution. Lactate supported a comparatively balanced physiological state characterized by enhanced respiratory metabolism, amino acid biosynthesis, and riboflavin metabolism, enabling high specific vitamin B12 yields ([~]100 {micro}g g-1 wet biomass). In contrast, hexose metabolism (glucose and fructose) imposed a redox-constrained state marked by upregulation of transport systems, glycolysis, and the pentose phosphate pathway, resulting in increased biomass but reduced biosynthetic efficiency. A defining feature of the hexose-driven state was activation of aspartate metabolism. Proteomic and metabolite data, together with functional assays, support a model in which aspartate is converted to fumarate and subsequently reduced to succinate, providing an alternative electron sink that facilitates NADH reoxidation under redox-constrained conditions. Together, these findings establish that carbon source shapes physiological state through flux distribution, redox homeostasis, and resource allocation, with cofactor biosynthesis emerging as a system-level property rather than a simple consequence of biosynthetic enzyme abundance. IMPORTANCEPropionibacterium freudenreichii is a central bacterium used in food fermentations and one of the few microorganisms able to synthesize biologically active vitamin B12, making it valuable for industry and biotechnology. Yet the metabolic principles that govern its performance under different growth conditions remain poorly understood. Here, we show that carbon source is a key determinant of metabolic state, dictating how cells resolve redox constraints and allocate biosynthetic resources. We uncover a previously unrecognized adaptation in sugar-grown cells, where aspartate functions as an alternative electron sink to sustain redox balance under constrained conditions. By contrast, lactate supports a physiological state that promotes efficient vitamin B12 biosynthesis. These findings reveal a central role for carbon source in shaping metabolic configuration and identify redox balancing as a critical lever linking environmental inputs to biosynthetic output. More broadly, this work provides mechanistic insight into redox-constrained metabolism and a framework for improving vitamin B12 production and other microbial bioprocesses.