13C Tracers for Glucose Degrading Pathway Discrimination in Gluconobacter oxydans 621H

Metabolites

Steffen Ostermann²  Janine Richhardt²  Stephanie Bringer²  Michael Bott²  Wolfgang Wiechert²  Marco Oldiges¹ 

[1] Institute of Bio- and Geosciences—IBG-1: Biotechnology, Leo-Brandt-Straße, 52428 Jülich, Germany;

关键词: microtiter cultivation;    ¹³C tracer;    pentose phosphate pathway;    Entner–Doudoroff pathway;    glucose;   

DOI  :  10.3390/metabo5030455

来源: mdpi

【 摘 要 】

Gluconobacter oxydans 621H is used as an industrial production organism due to its exceptional ability to incompletely oxidize a great variety of carbohydrates in the periplasm. With glucose as the carbon source, up to 90% of the initial concentration is oxidized periplasmatically to gluconate and ketogluconates. Growth on glucose is biphasic and intracellular sugar catabolism proceeds via the Entner–Doudoroff pathway (EDP) and the pentose phosphate pathway (PPP). Here we studied the in vivo contributions of the two pathways to glucose catabolism on a microtiter scale. In our approach we applied specifically ¹³C labeled glucose, whereby a labeling pattern in alanine was generated intracellularly. This method revealed a dynamic growth phase-dependent pathway activity with increased activity of EDP in the first and PPP in the second growth phase, respectively. Evidence for a growth phase-independent decarboxylation-carboxylation cycle around the pyruvate node was obtained from ¹³C fragmentation patterns of alanine. For the first time, down-scaled microtiter plate cultivation together with ¹³C-labeled substrate was applied for G. oxydans to elucidate pathway operation, exhibiting reasonable labeling costs and allowing for sufficient replicate experiments.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.