A systems approach using OSMAC, Log P and NMR fingerprinting: An approach to novelty

Abstract

The growing number of sequenced microbial genomes has revealed a remarkably large number of secondary metabolite biosynthetic clusters for which the compounds are still unknown. The aim of the present work was to apply a strategy to detect newly induced natural products by cultivating microorganisms in different fermentation conditions. The metabolomic analysis of 4160 fractions generated from 13 actinomycetes under 32 different culture conditions was carried out by ¹H NMR spectroscopy and multivariate analysis. The principal component analysis (PCA) of the ¹H NMR spectra showed a clear discrimination between those samples within PC1 and PC2. The fractions with induced metabolites that are only produced under specific growth conditions was identified by PCA analysis. This method allows an efficient differentiation within a large dataset with only one fractionation step. This work demonstrates the potential of NMR spectroscopy in combination with metabolomic data analysis for the screening of large sets of fractions.

Keywords: Metabolites; NMR fingerprints; OSMAC.

Graphical abstract

Fig. 1

Flowchart for OSMAC-NMR-PCA-based secondary metabolomics.

Fig. 2

Distribution of total NMR intensities among 32 OSMAC conditions as an indicator of metabolite production.

Fig. 3

Pie chart of anti-BCG activities of 1271 active LLE fractions (A. 646 active fractions from media variation, B. 625 active fractions from stress conditions).

Fig. 4

Scores plots and loadings plots of PC1 and PC2 of 4160 fractions, fractions from endophytes, land strains and marine strains in scores plots were shown in red, orange and green, respectively. Bucket values in loadings plots were shown as numbers below each circle. A. Scores plot of region 2 (3.5–6 ppm). B. Loadings plot of region 2 (3.5–6 ppm).

Fig. 5

¹H NMR spectra of 3 outliers indicated by PCA results of region 2.

Fig. 6

PCA results of 320 fractions from MS110104. Anti-BCG activity was shown in different colors coded according to the legend (blue lowest and red highest activity). A. Scores plot of region 1 (0–2.4 ppm). Fraction numbers were shown below B. Loadings plot of region 1 (0–2.4 ppm). Bucket values in loadings plots were shown as numbers below each circle.

Fig. 7

¹H NMR spectra of the outlier MS110104_Medium6_7d_Biomass_fr.5 indicated by PCA result of region 1 and the corresponding standard fraction.

Fig. 8

PCA results of 320 fractions from ES120127. Anti-BCG activity was shown in different colors coded according to the legend (blue lowest and red highest activity). A. Scores plot of region 3 (6–10 ppm). Fraction numbers were shown below each symbol. B. Loadings plot of region 3 (6–10 ppm). Bucket values in loadings plots were shown as numbers below each circle.

Fig. 9

¹H NMR spectra of the outlier ES120127_Medium9_14d_Supernatant_fr.3 indicated by PCA result of region 3 and the corresponding standard fraction and ES120127_Medium9_7d_Supernatant_fr.3.

Fig. 10

PCA results of 320 fractions from MS110149. Anti-BCG activity was shown in different colors coded according to the legend (blue lowest and red highest activity). A. Scores plot of region 1 (0–2.4 ppm). Fraction numbers were shown below each symbol. B. Loadings plot of region 1 (0–2.4 ppm). Bucket values in loadings plots were shown as numbers below each circle.

Fig. 11

¹H NMR spectra of the outliers MS110149_1mM_Supernatant_fr.3, MS110149_10mM_Supernatant_fr.3 and MS110149_100mM_Supernatant_fr.3 indicated by PCA result of region 1 and the corresponding standard fraction.

Fig. 12

Scores plots of 320 fractions from MS110109. Fraction numbers were shown below each symbol. Anti-BCG activity was shown in different colors coded according to the legend (blue lowest and red highest activity). A. Scores plot of region 1 (0–2.4 ppm). B. Scores plot of region 2 (3.5–6 ppm). C. Scores plot of region 3 (6–10 ppm). D. Scores plot of region 4 (10–15 ppm).

Fig. 13

¹H NMR spectra of the outliers of MS110109 and the corresponding standard fraction.

Fig. 14

The distribution of 37 prioritized outliers.

Fig. 15

Scores plots of 37 outliers fractions. Anti-BCG activity was shown in different colors coded according to the legend (blue lowest and red highest activity). A. Scores plot of region 1 (0–2.4 ppm). B. Scores plot of region 2 (3.5–6 ppm). C. Scores plot of region 3 (6–10 ppm). D. Scores plot of region 4 (10–15 ppm).