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.

Putative methyltransferases are thought to be involved in the regulation of secondary metabolites in filamentous fungi. Here, we report the effects of overexpression of a predicted LaeA-like methyltransferase gene llm1 on the synthesis of secondary metabolites in Aspergillus cristatus. Our results revealed that overexpression of the gene llm1 in A. cristatus significantly hindered the production of conidia and enhanced sexual development, and reduced oxidative tolerance to hydrogen peroxide. Compared with the wild-type, the metabolic profile of the overexpression transformant was distinct, and the contents of multiple secondary metabolites were markedly increased, mainly including terpenoids and flavonoids, such as (S)-olEuropeic acid, gibberellin A62, gibberellin A95, ovalitenone, PD 98059, and 1-isomangostin. A total of 600 significantly differentially expressed genes (DEGs) were identified utilizing transcriptome sequencing, and the DEGs were predominantly enriched in transmembrane transport and secondary metabolism-related biological processes. In summary, the strategy of overexpressing global secondary metabolite regulators successfully activated the expression of secondary metabolite gene clusters, and the numerous secondary metabolites were greatly strengthened in A. cristatus. This study provides new insights into the in-depth exploitation and utilization of novel secondary metabolites of A. cristatus.

Microbial fermentation is a useful method for improving the biological activity of Chinese herbal medicine. Herein, we revealed the effects of solid-state fermentation by Lactiplantibacillus plantarum, Bacillus licheniformis, Saccharomyces cerevisiae, Eurotium cristatum and multiple strains on total flavonoid content, total phenol content, as well as antioxidants, α-amylase inhibitory activities and α-glucosidase inhibitory activities in white ginseng (WG). Metabolite differences between non-fermented and fermented WG by different probiotics were comprehensively investigated using ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC-TOF-MS). Results showed that the total flavonoid content, ferric reducing antioxidant power, scavenging activities of DPPH radical and ABTS radical, α-amylase inhibitory activities and α-glucosidase inhibitory activities of WG were considerably enhanced after processing by solid-state fermentation in all strains. The total phenol content was increased by E. cristatum and B. licheniformis fermentation, but decreased by L. plantarum, S. cerevisiae and multi-strain fermentation. Additionally, E. cristatum exhibited stronger biotransformation activity on WG compared to other strains. Significant differential metabolites were mainly annotated as prenol lipids, carboxylic acids and derivatives, flavonoids, polyphenols, coumarins and derivatives. Correlation analysis further showed that changes of these metabolites were closely related to antioxidant and hypoglycemic effects. Our results confirmed that fermentation of WG by different probiotics has distinct effects on biological activities and metabolite composition, and indicating fermentation as an important novel strategy to promote components and bioactivities of WG.

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