Inducing Secondary Metabolite Production of Aspergillus Sydowii Through Microbial Co-culture With Bacillus Subtilis

Abstract

Background: The co-culture strategy which mimics natural ecology by constructing an artificial microbial community is a useful tool to activate the biosynthetic gene clusters to generate new metabolites. However, the conventional method to study the co-culture is to isolate and purify compounds separated by HPLC, which was inefficient and time-consuming. Furthermore, the overall changes in the metabolite profile cannot be well characterized. Results: A new approach which integrates computational programs, MS-DIAL, MS-FINDER and web-based tools including GNPS and MetaboAnalyst, was developed to analyze and identify the metabolites of the co-culture of Aspergillus sydowii and Bacillus subtilis. A total of 25 new bio-synthesized metabolites were detected only in co-culture. The structures of the new synthesized metabolites were elucidated, four of which were identified as novel compounds by the new approach. The accuracy of the new approach was confirmed by purification and NMR data analysis of 7 new-bio-synthesized metabolites. The bioassay of new synthesized metabolites showed that four of the compounds had different degrees of PTP1b inhibitory activity, and compound N2 had the strongest inhibition activity with an IC50 value of 7.967 μM. Conclusions: Co-culture led to global changes of the metabolite profile and is an effective way to induce the biosynthesis of novel natural products. The new approach in this study is one of the effective and relatively accurate methods to characterize the change of metabolite profiles and to identify novel compounds in co-culture systems.

Interspecific interaction mediated crosstalk in the co-culture of Aspergillus sydowii and Bacillus subtilis produces novel benzoic acid derivates

“…Instead, the microbes formed a relatively neat boundary at the confrontation area, indicating that after the co-culture, the microbes produced chemical substances that could inhibit the growth of the opposite microorganism and prevented its further invasion. This result was also demonstrated in our previous article showing that the 7 new compounds induced by co-culture had antibacterial activity, and that these compounds were not produced in pure cultures [10]. Furthermore, A. sydowii presented smooth and complete conidiophores, and the spores were proli c on BDA plate (Fig.…”

“…The thalli of A. sydowii and B. subtilis were obtained respectively for pure cultures. The thalli of the confrontation zone was obtained for co-culture, and transferred into 100 mL glass asks using the same method as introduced in our previous article [10]. The thalli were extract for 3 times with 70 mL EtOAc to obtain the extract solutions, and the extract was then ltered through lter paper.…”

“…The mass spectrometry analysis was performed with an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scienti c, Hemel Hempstead, UK) at a ow rate of 0.6 mL/min with an electrospray interface (ESI), using the same method described in our previous article [10]. In separate runs, the m/z range was set as 50-1200 Da in centroid mode with a scan rate of 1.5 spectra/s.…”

“…For the co-culture and pure cultures supplemented with benzoic acid or 3-OH-benzoic acid, 100 µL of 20 mg/mL benzoic acid or 20 mg/mL 3-OH-benzoic acid dissolved in sterile ethanol was supplemented to the 20 mL BDA medium before the inoculation of the microbes. The identi cation of compounds by MS-DIAL and MS-FINDER was based on the approach provided in our previous article [10].…”

“…Moreover, Bacillus subtilis is a well-known producer of bacterial agents and has been successfully used in agricultural biological control, which can produce a large number of metabolites with potential antibacterial and anti-insect activities [9]. In our previous study, the co-culture of A. sydowii and B. subtilis induced 25 newly biosynthesized metabolites [10]. Four of the newly biosynthesized metabolites were novel compounds and 5 compounds including one novel compound belong to benzoic acid derivates.…”