Discovery of secondary metabolites in an extractive liquid-surface immobilization system and its application to high-throughput interfacial screening of antibiotic-producing fungi.

An extractive liquid-surface immobilization (Ext-LSI) system, which consists of a hydrophobic organic solvent (an upper phase), a fungal cell-ballooned microsphere layer (a middle phase) and a liquid medium (a lower phase), is a unique interfacial cultivation system for fungi. The fungal cells growing at the interface between the organic and aqueous phases efficiently produce hydrophobic metabolites, which are continuously extracted into the organic phase, and/or hydrophilic metabolites that migrate into the aqueous phase without carbon catabolite repression and product and/or feed-back inhibitions. Application of the system to fermentation of Penicillium multicolor IAM 7153 and Trichoderma atroviride AG2755-5NM398 afforded remarkably different profiles of secondary metabolites in the organic phase compared with those in an aqueous phase in traditional submerged cultivation (SmC). Various hydrophobic metabolites exhibiting unique UV-visible spectra were accumulated into the organic phase. The system was applied to a novel interfacial screening system of antibiotic-producing fungi. Compared with the SmC, the interfacial cultivation system exhibited some interesting and important advantages, such as the higher accumulation of hydrophobic secondary metabolites, the lack of requirement for shaking and troublesome solvent extraction, and the small scale of the vessels (medium, 5 ml; dimethylsilicone oil, 1 ml), as well as the significantly different metabolite profiles. The interfacial screening system yielded a high incidence of antimicrobial activity, with 21.9% of the fungi tested exhibiting antifungal activity against Pichia anomala NBRC 10213. This novel interfacial high-throughput screening approach has the potential to discover new biologically active secondary metabolites even from strains previously found to be unproductive.