Metabolomic profiles of the liquid state fermentation in co-culture of Eurotium amstelodami and Bacillus licheniformis

Abstract

As an important source of new drug molecules, secondary metabolites (SMs) produced by microorganisms possess important biological activities, such as antibacterial, anti-inflammatory, and hypoglycemic effects. However, the true potential of microbial synthesis of SMs has not been fully elucidated as the SM gene clusters remain silent under laboratory culture conditions. Herein, we evaluated the inhibitory effect of Staphylococcus aureus by co-culture of Eurotium amstelodami and three Bacillus species, including Bacillus licheniformis, Bacillus subtilis, and Bacillus amyloliquefaciens. In addition, a non-target approach based on ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS) was used to detect differences in extracellular and intracellular metabolites. Notably, the co-culture of E. amstelodami and Bacillus spices significantly improved the inhibitory effect against S. aureus, with the combination of E. amstelodami and B. licheniformis showing best performance. Metabolomics data further revealed that the abundant SMs, such as Nummularine B, Lucidenic acid E2, Elatoside G, Aspergillic acid, 4-Hydroxycyclohexylcarboxylic acid, Copaene, and Pipecolic acid were significantly enhanced in co-culture. Intracellularly, the differential metabolites were involved in the metabolism of amino acids, nucleic acids, and glycerophospholipid. Overall, this work demonstrates that the co-culture strategy is beneficial for inducing biosynthesis of active metabolites in E. amstelodami and B. licheniformis.

Keywords: Bacillus licheniformis; Eurotium amstelodami; co-culture; metabolomic profiles; secondary metabolism.

Figure 1

Scanning electron microscopy of Eurotium amstelodami mycelia in pure culture (A), co-cultures (B) and Bacillus licheniformis pure culture (C). Dried mycelial and cells weight of E. amstelodami (D) and B. licheniformis (E). Data are reported as the mean value ± standard deviation of three replicates, Statistical significance (Student’s t-test) is indicated as follows: *p < 0.05.

Figure 2

Principal components analysis (PCA) score plots of extracellular metabolites (A). Partial least squares discriminant analysis (PLS-DA) score plots of extracellular metabolites (B). Hierarchical clustering analysis (HCA) of the 59 extracellular metabolites only overexpressed in co-culture group and represented on a heatmap (C). (LA, extracellular metabolites of E. amstelodami pure culture; LB, extracellular metabolites of B. licheniformis pure culture; LAB, extracellular metabolites of co-culture).

Figure 3

PCA score plots of intracellular metabolites (A). PLS-DA score plots of intracellular metabolites between E. amstelodami pure culture and co-culture(B). PLS-DA score plots of intracellular metabolites between B. licheniformis pure culture and co-culture (C). (SA, intracellular metabolites of E. amstelodami pure culture; SCA, intracellular metabolites of E. amstelodami co-culture; SB, intracellular metabolites of B. licheniformis pure culture; SCB, intracellular metabolites of B. licheniformis co-culture).

Figure 4

Volcano plot displaying the differences in intracellular metabolic profiles between pure culture and co-culture of E. amstelodami (A) and B. licheniformis (B). Pathway analysis for intracellular metabolites of E. amstelodami (C), B. licheniformis (D) pure culture and co-cultures.

Figure 5

Molecular network of the MS/MS spectra for extracellular extracts of E. amstelodami, B. licheniformis pure culture and co-cultures in positive ion mode (A). Chemical classification was achieved by MolNetEnhancer at the subclass level, (B–E) shows clusters of glycerophosphoethanolamines, styrenes, amino acids, peptides, and analogs and flavonoid glycosides, respectively. (F) Shows a putative flavoglaucin. Nodes represent parent ions and edge thickness corresponds to the cosine score, which represents the degree of similarity between the connected nodes. Pie ratio was determined according to scan number of spectra.

Figure 6

Integration map of some metabolic pathways for intracellular and extracellular metabolites of E. amstelodami and B. licheniformis co-cultures.