Metabolic flux analysis and the NAD(P)H/NAD(P) ratios in chemostat cultures of Azotobacter vinelandii.

BACKGROUND

Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P) ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions.

RESULTS

The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP ratio, showing that at the lowest OTR (2.4 mmol L h), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L h). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L h. At the highest OTR condition, a decrease in the NADPH/NADP ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH).

CONCLUSIONS

We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.