Abstract
IntroductionCupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) 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.ObjectivesIn 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 13C-labelling.MethodsIn this study, steady-state carbon labelling experiments, using either d-[1-13C]fructose or [1,2-13C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively.ResultsWe 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.ConclusionThis study leads the way to establishing 13C-based quantitative fluxomics for rational pathway engineering in C. necator H16.
Highlights
Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) through Calvin–Benson–Bassham (CBB) cycle
Analysis of culture growth profile showed that the growth rate was highest under heterotrophic condition with fructose, while it was slowest for heterotrophic growth with glycerol (Table 1), complementing previous studies (Friedrich et al 1981; Lopar et al 2014)
The amino acid fractional labelling was corrected for natural isotope abundance to generate the mass isotopomer distribution (MID) values (Supplementary Tables S4, S5, S6)
Summary
Cupriavidus necator H16 is a gram-negative bacterium, capable of lithoautotrophic growth by utilizing hydrogen as an energy source and fixing carbon dioxide (CO2) through Calvin–Benson–Bassham (CBB) cycle. Methods In this study, steady-state carbon labelling experiments, using either d-[1-13C]fructose or [1,2-13C]glycerol, were undertaken to investigate the carbon flux through the central carbon metabolism in C. necator H16 under heterotrophic and mixotrophic growth conditions, respectively. Cupriavidus necator H16 is a facultative anaerobe, which can switch to anaerobic respiration by using N O3 and N O2 as electron acceptors It grows utilizing a variety of organic substrates ranging from sugars, fatty acids, amino acids and citric acid cycle intermediates. It is capable to fix directly a carbon dioxide (CO2) through the CBB cycle using hydrogen as the energy source (Bowien and Kusian 2002), making it a useful organism for studying mixotrophic and autotrophic metabolism. The ability to utilize a range of organic and inorganic substrates, and possibility of re-routing the stored carbon to valuable products
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
