A new butenolide with antifungal activity from solid co-cultivation of Irpex lacteus and Nigrospora oryzae

Abstract A new antifungal butenolide irperide (1) along with five known compounds were isolated from the co-culture of endophyte Irpex lacteus and pathogenic Nigrospora oryzae. The structure of 1, including the absolute configuration, was elucidated by analysis of NMR, HR-ESI-MS data and ECD spectra. Compounds 1, 4 and 6 exhibited significant antifungal activity against Aspergillus fumigatus, with MIC values of 1, 2 and 1 μg/mL, respectively. Graphical Abstract


Introduction
Many microbial gene clusters may be silent under standard laboratory growth conditions, which led to repeated separation of a large number of known compounds and finally hindered the speed of the discovery of new active drugs (Jones and Wang 2018;Peng et al. 2021). Therefore, in order to overcome these limitations, several methods have been developed to help activating silent microbial gene clusters in recent years, one of which is microbial co-cultivation Arora et al. 2020). By inducing metabolic pathways in coculture, the chemical diversity of isolated compounds has been increased and new active molecules can be discovered easier (Boruta et al. 2021). Aspergillus fumigatus is a saprophytic fungus and the most prevalent airborne fungal pathogen, capable of adapting and multiplying in harsh environments. It can cause severe and usually fatal invasive aspergillosis in immunocompromised patients, resulting in extremely high morbidity and mortality of lung infections (Park and Yu 2016; Latg e and Chamilos 2019). Our previous work afforded new azaphilones and tremulane sesquiterpene from the liquid coculture fermentation of Nigrospora oryzae and Irpex lacteus . In order to further explore the metabolic potential of these two strains, the antifungal metabolites (1-6) against A. fumigatus ( Figure 1) from solid co-culture of endophyte Irpex lacteus, phytopathogen Nigrospora oryzae were studied.

General experimental procedures
The chromatographic silica gel was bought from Qingdao Haiyang Chemical Company, Lichroprep RP-18 and Sephadex LH-20 were bought from Merck, GE Healthcare Company. The nuclear magnetic resonance (NMR) spectra and mass spectroscopy (MS) were acquired respectively by a Bruker AVANCE, Agilent G3250AA, and Waters AutoSpec Premier P776 spectrometers. Optical rotations and circular dichroism were acquired respectively by JASCO AP-1020 polarimeter, and Chirascan spectrometer.

Fungal material, fermentation and isolation
The fungi TPZ 10-1 isolated from the seeds of Dendrobium officinale and ZPY 45-2 isolated from the leaves of Dendrobium devoninum were collected from Wenshan of Yunnan Province in People's Republic of China. The species were determined to be Nigrospora oryzae (TPZ 10-1) and Irpex lacteus (ZPY 45-2) by the morphological and genetic characteristics. The voucher specimens of fungi (TPZ 10-1) and (ZPY 45-2) were conserved at the functional molecules analysis and biotransformation key laboratory of Yunnan University.
The fermentation products of N. oryzae, and I. lacteus, were co-cultured in 0.5 L Erlenmeyer flasks containing 200 mL of potato dextrose broth (PDB) prepared by infusion of potato (200 g), dextrose (20 g), and 1.0 L of water at 128 rpm and 28 C for 3 days for the seed culture. The seed culture of N. oryzae, and I. lacteus was cultured in rice-spinach medium (10 kg of rice, 5 kg of fresh spinach) for 40 days.
The fermentation products of rice-spinach solid medium were extracted with MeOH, then the extract was concentrated under reduced pressure to give a crude extract. The resulting aqueous concentrate was partitioned into EtOAc layer and then concentrated under reduced pressure to give a crude extract of EtOAc (64.9 g).

ECD calculation
The absolute configurations of compound 1 was determined by calculated ECD data using Gaussian 09 program with the DFT (Density Functional Theory) . The conformers were optimized by Gaussian 09 program at the B3LYP/6-31(d,p) level. ECD calculation data were acquired with the PCM in MeOH.

Antifungal assay
In the in vitro antifungal test, the minimal inhibitory concentrations (MICs) of the metabolites against Aspergillus fumigatus were measured with the broth dilution method. Test compounds were dissolved in dimethyl sulfoxide (DMSO). The final concentrations were 512,256,128,64,32,16,8,4, 2 and 1 mg/mL in medium. The 96-well microplates containing nystatin (Taicheng Pharmaceutical Company, purity >99%) was used as positive control. The wells containing samples were cultured for 18 hours at 28 C. The tests were repeated three times.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was subsidized by the National Natural Science Foundation of China (81960640)