A genome-scale metabolic reconstruction provides insight into the metabolism of the thermophilic bacterium Rhodothermus marinus

Abstract

The thermophilic bacterium Rhodothermus marinus has mainly been studied for its thermostable enzymes. More recently, the potential of using the species as a cell factory and in biorefinery platforms has been explored, due to the elevated growth temperature, native production of compounds such as carotenoids and exopolysaccharides, the ability to grow on a wide range of carbon sources including polysaccharides, and available genetic tools. A comprehensive understanding of the metabolism of cell factories is important. Here, we report a genome-scale metabolic model of R. marinus DSM 4252T. Moreover, the genome of the genetically amenable R. marinus ISCaR-493 was sequenced and the analysis of the core genome indicated that the model could be used for both strains. Bioreactor growth data were obtained, used for constraining the model and the predicted and experimental growth rates were compared. The model correctly predicted the growth rates of both strains. During the reconstruction process, different aspects of the R. marinus metabolism were reviewed and subsequently, both cell densities and carotenoid production were investigated for strain ISCaR-493 under different growth conditions. Additionally, the dxs gene, which was not found in the R. marinus genomes, from Thermus thermophilus was cloned on a shuttle vector into strain ISCaR-493 resulting in a higher yield of carotenoids.

Keywords: Rhodothermus marinus; carotenoids; genome-scale metabolic model.

Figure 1.

The alginate degradation pathway of R. marinus and its connections to the EMP pathway, the partial ED pathway and the MEP terpenoid pathway. Names of substrates, products and energy molecules (ATP, NADH, NADPH) are shown, some abbreviated: 4-deoxy-l-erythro 5-hexoseulose uronic acid (DEH), 2-keto 3-deoxygluconate (KDG) and 2-keto 3-deoxygluconate 6-phosphate (KDPG). The molecular structure of a partial alginate molecule is shown: β-d-mannuronate (M) and α-^L-guluronate (G). The missing reaction of the ED pathway in R. marinus is represented by a grey dotted arrow.

Figure 2.

Experimental growth data for strains DSM 4252^T and ISCaR-493 was used to validate model growth predictions. The model was constrained with uptake (glucose and pyruvate) and secretion (lactate and acetate) rates observed in vivo (Supplementary File 1) and optimized for growth. Predicted growth rates were compared to growth rates observed in vivo (a). Experimental data from the exponential growth phase of two replicates (▲ and ■), obtained early in the growth phase (time points 3–6 for strain DSM 4252 and 1–5 for strain ISCaR-493), were used (b).

Figure 3.

Cell density (OD620nm) and carotenoid (OD480nm) production following growth of R. marinus strain ISCaR-493 for 24 h on glucose; mixture of glucose and pyruvate under light and dark conditions; alginate; pyruvate and without any carbon sources. Additionally, the modified strain TK-4 (ΔtrpBΔpurA::trpBdxs_{T.thermophilus}) was grown on a mixture of glucose and pyruvate. The carotenoids were always extracted from a fixed number of cells (1 ml of OD620nm = 1). The total amount of carotenoids was calculated by multiplying the cell density by the measured carotenoids. The average cell density of each culture condition is represented by a blue bar (top graph), the average measured carotenoid value as a red bar (middle graph) and the average total carotenoids by an orange bar (bottom graph). Dots represent individual replicates.