Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations

Process engineering of biotechnological productions can benefit greatly from comprehensive analysis of microbial physiology and metabolism.Ralstonia eutropha (syn. Cupriavidus necator) is one of the best studied organisms for the synthesis of biodegradable polyhydroxyalkanoate (PHA). A comprehensive metabolomic study during bioreactor cultivations with the wild-type (H16) and an engineered (Re2058/pCB113)R. eutropha strain for short– and or medium-chain-length PHA synthesis has been carried out. PHA production from plant oil was triggered through nitrogen limitation. Sample quenching allowed to conserve the metabolic states of the cells for subsequent untargeted metabolomic analysis, which consisted of GC–MS and LC–MS analysis.


Fig: Bioreactor cultivations of R. eutropha (a) and exploratory analysis of annotated compounds from untargeted metabolite profiling (b–i). (a) Strains R. eutrophaH16 (upper graph) and Re2058/pCB113 (lower graph) were grown in batch cultivations in MSM using 3% (w v−1) PO as the main carbon source and 0.4% (w v−1) NH4Cl as the sole nitrogen source. Vertical dashed lines indicate the time points of ammonia depletion. Cell dry weight (CDW; g L−1, black circles), PHB content (wt%, green diamonds) and HHx content (mol%, red triangles) are shown. Data points represent values of biological triplicate cultivations and the solid lines represent the best fit through the data points. Multivariate analysis results of samples from cultivations of R. eutropha H16 (b–c), Re2058/pCB113 (d–e) or both strains (f–i) are shown: PCA (b,d) grouped by time points (ascending order: brown circles, yellow triangles, blue diamonds, cyan stars) and ANOVA (c,e) with significantly changing metabolite concentrations (FDR-adjusted p value ≤ 0.05) colored in orange. PCA (f) and volcano plot (g) of samples classified by nitrogen availability from both strains (before depletion: dark yellow stars, after depletion: dark cyan circles). PCA (h) and volcano plot (i) of samples classified by the strain (H16: green circles, Re2058/pCB113: red stars). PC principal component, FC fold-change.

Multivariate data analysis resulted in identification of significant changes in concentrations of oxidative stress-related metabolites and a subsequent accumulation of antioxidative compounds. Moreover, metabolites involved in the de novo synthesis of GDP-L-fucose as well as the fucose salvage pathway were identified. The related formation of fucose-containing exopolysaccharides potentially supports the emulsion-based growth of R. eutropha on plant oils.

Gutschmann, B., Bock, M.C.E., Jahns, S. et al. Untargeted metabolomics analysis of Ralstonia eutropha during plant oil cultivations reveals the presence of a fucose salvage pathway. Sci Rep 11, 14267 (2021). https://doi.org/10.1038/s41598-021-93720-9

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