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

## TL;DR

This study uses 13C-labeled glucose to track metabolic pathways in Gluconobacter oxydans 621H during glucose catabolism.

## Contribution

The first use of microtiter plate cultivation with 13C-labeled glucose to study pathway activity in G. oxydans.

## Key findings

- EDP is more active in the first growth phase, while PPP dominates in the second.
- A decarboxylation-carboxylation cycle around the pyruvate node was observed.
- The method is cost-effective and allows for replicable experiments on a small scale.

## Abstract

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.