Metabolic flux analysis of the central carbon metabolism of the industrial vitamin B ₁₂ producing strain Pseudomonas denitrificans using ¹³C-labeled glucose

The network topology and metabolic fluxes of central carbon metabolism in the industrial vitamin B ₁₂ producing strain Pseudomonas denitrificans were characterized under oxygen limiting levels. Cultivations were carried out with 100% [1- ¹³C] or 20% [U- ¹³C] glucose as substrates under different oxygen supply conditions. The labeling patterns of the proteinogenic amino acids of exponentially growing cells were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. Metabolic flux analysis showed that glucose was mostly catabolized by the Entner-Doudoroff and pentose phosphate pathways. Up to 33% of glucose was consumed via the PP pathway under high specific oxygen uptake rate (SOUR) conditions. This amount was 77.9% higher than that under low oxygen uptake conditions. Quantitative evidence was also found for reversible serine hydroxymethyl transferase and threonine aldolase activities. Metabolic flux and cofactor analyses further showed that higher SOUR accelerated the supply of precursors and methyl groups. SOUR also provided more NADPH for higher vitamin B ₁₂ production under the same glucose consumption.