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

Andres García,Pau Ferrer,Joan Albiol,Daniel Segura,Tania Castillo,Carlos Peña

doi:10.1186/s12934-018-0860-8

Abstract

BackgroundAzotobacter 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.ResultsThe 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−1 h−1), 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−1 h−1). 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−1 h−1. 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).ConclusionsWe 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.

Highlights

Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown
dissolved oxygen tension (DOT) and OTR under oxygen‐limiting and non‐limiting conditions The main purpose of this study was to understand the effect of the oxygen transfer rate (OTR) on the distribution of metabolic fluxes and the intracellular redox state (i.e. NADH/NAD+ and NADPH/NADP+ ratios) in A. vinelandii growing under oxygen-limiting and non-limiting conditions
During the steady-state there is no oxygen accumulation (dO2/dt ≈ 0); the OTR is equal to the oxygen uptake rate (OUR)

Summary

Introduction

Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. Azotobacter vinelandii is a Gram negative bacterium that produces two polymers of biotechnological importance, poly-3-hydroxybutyrate (P3HB), an intracellular polyester of the polyhydroxyalkanoates (PHAs) family, and alginate, an extracellular polysaccharide [1, 2] This bacterium fixes nitrogen under aerobic conditions, being. Genetic manipulations aiming at perturbing NADH cofactor levels and/or regeneration rates were employed as a tool for the metabolic engineering of Escherichia coli [11,12,13], Lactococcus lactis [14], Bacillus subtilis [15, 16] and Saccharomyces cerevisiae [17] These studies showed that changes in the ratio of NADH/NAD+ determined the metabolic products. Other studies have shown that strategies increasing the NADPH cofactor levels improved, for example, penicillin formation [18], methylenomycin biosynthesis [19] and P3HB biosynthesis [20,21,22,23]

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