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Introduction    Cupriavidus necator  H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO 2 ) through Calvin–Benson–Bassham (CBB) cycle. The potential to utilize synthesis gas (Syngas) and the prospects of rerouting carbon from polyhydroxybutyrate synthesis to value-added compounds makes  C. necator  an excellent chassis for industrial application.    Objectives  In the context of lack of sufficient quantitative information of the metabolic pathways and to advance in rational metabolic engineering for optimized product synthesis in  C. necator  H16, we carried out a metabolic flux analysis based on steady-state  13 C-labelling.    Methods  In this study, steady-state carbon labelling experiments, using either  d -[1- 13 C]fructose or [1,2- 13 C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in  C. necator  H16 under heterotrophic and mixotrophic growth conditions, respectively.    Results  We found that the CBB cycle is active even under heterotrophic condition, and growth is indeed mixotrophic. While Entner–Doudoroff (ED) pathway is shown to be the major route for sugar degradation, tricarboxylic acid (TCA) cycle is highly active in mixotrophic condition. Enhanced flux is observed in reductive pentose phosphate pathway (redPPP) under the mixotrophic condition to supplement the precursor requirement for CBB cycle. The flux distribution was compared to the mRNA abundance of genes encoding enzymes involved in key enzymatic reactions of the central carbon metabolism.    Conclusion  This study leads the way to establishing  13 C-based quantitative fluxomics for rational pathway engineering in  C. necator  H16.    Electronic supplementary material  The online version of this article (10.1007/s11306-017-1302-z) contains supplementary material, which is available to authorized users.

13C-assisted metabolic flux analysis to investigate heterotrophic and mixotrophic metabolism in Cupriavidus necator H16