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

Microbial adaptation to fluctuating nutrient and oxygen conditions requires coordinated regulation of the metabolic network 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^T using label-free quantitative proteomics integrated with physiological and metabolite analyses. Distinct carbon sources defined discrete metabolic states shaped by redox constraints, carbon routing, and biosynthetic prioritization. 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 µg 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 consistent with an alternative electron sink that could facilitate NADH reoxidation under redox-constrained conditions. Together, these findings establish that carbon source shapes physiological state through carbon routing, redox homeostasis, and differential resource allocation, with cofactor biosynthesis emerging as a system-level property rather than a simple consequence of biosynthetic enzyme abundance.

Propionibacterium freudenreichii is used in food fermentations and is one of the few microorganisms able to synthesize biologically active vitamin B12, making it valuable for industry and biotechnology. Yet the metabolic principles governing its performance across 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 identify an adaptation in sugar-grown cells in which aspartate metabolism provides a potential 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. This work provides mechanistic insight into redox-constrained metabolism and a framework for improving vitamin B12 production and other microbial bioprocesses.