Non-targeted metabolomics unravels a media-dependent prodiginines production pathway in Streptomyces coelicolor A3(2)

Abstract

The genus Streptomyces is the best-known source of therapeutic secondary metabolites, especially antibiotics with pharmaceutical applications. Here, we performed a comparative study based on the time-resolved metabolic disparity in S. coelicolor A3(2) subjected to fermentative cultivation in two different types of media (R2YE and RSM3) in order to investigate secondary metabolite production pathways. The relative abundance of secondary metabolites, such as prodiginines, indoles, germicidins, and selected diketopiperazines, was increased in S. coelicolor A3(2) cultivated in R2YE medium compared to that in RSM3 medium, variably at the late-log and stationary phases of fermentative growth. Correlation analysis indicated that "antibiotic prodiginines" contributed maximally to the absorption maxima (A530) of culture supernatants, indicating their optimal production at 96 hours in R2YE medium. A higher abundance of L-proline (48-72 hours) followed by prodiginines (96 hours) was evident, substantiating the intertwined links between precursor and activated prodiginines pathway. Similarly, the higher abundance of indoles was concurrent with tryptophan levels in the shikimate pathway, whereas diketopiperazines were synchronously abundant along with the levels of phenylalanine, leucine, and proline. Additionally, acetyl-CoA induced the acetate pathway, resulting in the production of germicidins. Thus, our results demonstrate that S. coelicolor A3(2) produces specific secondary metabolites by enhancing the dedicated metabolic pathway responsible for their production. In conclusion, our results from this study provide insight into the metabolic pathways of S. coelicolor A3(2), and can be applied to further optimize the production of prodiginines.

Fig 1. Growth curve of S. coelicolor A3(2) in R2YE and RSM3 media.

The absorbance of culture supernatants was measured at 600 nm. (Red square, S. coelicolor A3(2) in R2YE medium; green triangle, S. coelicolor A3(2) in RSM3 medium). ** Significant differences calculated by student t-test (P < 0.001).

Fig 2

PCA score plot (A), OPLS-DA score plot (B), and heat map representations (C) of secondary metabolites produced by S. coelicolor A3(2) grown in R2YE and RSM3 media based on the UPLC-Q-TOF-MS data set. Data were normalized to the growth rates (Fig 1) of S. coelicolor A3(2) in R2YE and RSM3 media and then were scaled to an internal standard using adenosine 5′-monophosphate monohydrate. Significantly different metabolites in the heat map were selected by VIP values (> 0.7). Each column represents fold change normalized to the average of all values and shows blue (0) to red (2). (Square, S. coelicolor A3(2) in R2YE medium; triangle, S. coelicolor A3(2) in RSM3 medium; yellow, 48 h; orange, 72 h; red, 96 h; purple, 120 h; blue, 144 h; green, 168 h).^a It was selected by p-value (< 0.05). TDD: Tryptophan-dehydrobutyrine diketopiperazine.

Fig 3

Absorbance variation in culture supernatants of S. coelicolor A3(2) in R2YE (A) and RSM3 (B) media at different time points, a correlation map between the detected secondary metabolites and absorbance measurements for A₆₄₀, A₅₃₀, and A₄₅₀ (C), and the absorbance chromatogram for prodiginines derived from UHPLC-LTQ-ESI-IT-MS/MS coupled with diode array detector (D). Each square in the correlation map represents Pearson's correlation coefficient values (r), blue color indicates negative correlation (−1 < r < 0), and red color indicates positive correlation (0 < r < 1). (yellow, 48 h; orange, 72 h; red, 96 h; purple, 120 h; blue, 144 h; green, 168 h).

Fig 4

PCA score plots of primary (A) and secondary metabolites (B) and heat map representations of changes in the relative content of significantly discriminant primary metabolites (C) and secondary metabolites (D) in S. coelicolor A3(2) in R2YE medium. Primary and secondary metabolite data derived from GC-TOF-MS and UPLC-Q-TOF-MS, respectively. Data were normalized to the growth rates (Fig 1) of S. coelicolor A3(2) in R2YE medium and were scaled to internal standards using methyl nonadecanoate (for GC-TOF-MS analysis) and adenosine 5′-monophosphate monohydrate (for UPLC-Q-TOF-MS). Significantly discriminant metabolites in the heat map were selected by VIP values (> 0.7) and each column represents the fold change normalized to an average of all values and shows blue (0) to red (1.5). (yellow, 48 h; orange, 72 h; red, 96 h; purple, 120 h; blue, 144 h; green, 168 h). ^a It was selected by p-value (P < 0.05)..TDD: Tryptophandehydrobutyrine diketopiperazine.

Fig 5. The metabolic pathway and relative contents of S. coelicolor A3(2) metabolites produced in R2YE medium at different time points.

Primary and secondary metabolite data derived from GC-TOF-MS and UPLC-Q-TOF-MS, respectively. Data were normalized based on the growth rates (Fig 1) of S. coelicolor A3(2) in R2YE medium and were scaled to internal standards using methyl nonadecanoate (for GC-TOF-MS analysis) and adenosine 5′-monophosphate monohydrate (for UPLC-Q-TOF-MS). Metabolite data was selected by VIP values (> 0.7). The pathway map was modified from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/). Fold change was normalized to an average of all values and is shown as blue (0) to red (2).