To address this issue, we applied metabolic flux analysis using 13C labelled isotopes to gain a first insight into the central catabolic OSI-906 cost pathways of Dinoroseobacter shibae DFL12 [1] and Phaeobacter gallaeciensis DSM 17395 [14]. These species represent find more two prominent members of the Roseobacter clade. P. gallaeciensis has received strong interest due to its ability to produce the antibiotic tropodithietic acid. D. shibae was isolated as a novel species from marine dinoflagellates and lives in a symbiotic

relationship with eukaryotic algae [15]. Metabolic flux analysis using 13C labelled isotopes has proven a key technology in the unravelling of metabolic pathways and has recently been used to study different microorganisms mainly linked to biotechnological production processes [16–19]. No such

study has yet been performed for members of the Roseobacter clade. Results and Discussion Cultivation profile The cultivation E7080 price profile of D. shibae on defined medium with glucose as the sole carbon source is displayed in Figure 2. After an initial adaptation phase, cells grew exponentially with a constant specific growth rate of 0.11 h-1. After 50 hours of cultivation the carbon source was depleted and cells entered a stationary phase. The biomass yield was 0.45 g cell dry mass per g glucose consumed, indicating efficient utilisation of the carbon source for growth. A similar growth profile was determined for P. gallaeciensis. Figure 2 Time courses of glucose concentration and optical density during a batch cultivation of D. shibae in shake flasks under constant light. Pathways for glucose catabolism The carbon core metabolism of D. shibae and P. gallaeciensis consists of three potential routes for glucose catabolism. Glucose can be alternatively catabolised via glycolysis (EMP), the pentose phosphate pathway (PPP) and the Entner-Doudoroff ID-8 pathway (EDP). The use of [1-13C] glucose by each

individual pathway leads to a different labelling pattern in specific fragments of alanine and serine, which can be taken as a clear differentiation of flux (Figure 3). For D. shibae the corresponding [M-57] fragment of serine did not show any enrichment of 13C but rather reflected the pattern resulting from the natural abundance of 13C only (Table 1). Any contribution of glycolysis to formation of this metabolite and its precursor 3-phosphoglycerate can therefore be excluded as this would lead to enrichment of 13C at the C3 position, yielding a higher fraction of M+1 labelled molecules of Ser. Thus glycolytic flux obviously was not present. The two remaining possibilities, the PPP and the ED pathway, can be differentiated by the labelling pattern of alanine, which represents the pyruvate pool in the cell.