Microbial Screening Based on the Mizoroki–Heck Reaction Permits Exploration of Hydroxyhexylitaconic-Acid-Producing Fungi in Soils

Abstract

Recently, we developed a unique microbial screening method based on the Mizoroki–Heck reaction for itaconic acid (IA)-producing fungi. This method revealed that 37 out of 240 fungal strains isolated from soils produce vinyl compounds, including IA. In this study, we further characterized these compounds in order to verify that the screening method permits the isolation of fungi that produce other vinyl compounds, excluding IA. HPLC analysis showed that 11 out of 37 isolated strains produced IA, similar to Aspergillus terreus S12-1. Surprisingly, the other 8 isolated strains produced two vinyl compounds with HPLC retention times different from that of IA. From these strains, the vinyl compounds of Aspergillus niger S17-5 were characterized. Mass spectrometric and NMR analyses showed that they were identical to 8-hydroxyhexylitaconic acid (8-HHIA) and 9-HHIA. This finding showed that 8-HHIA- and 9-HHIA-producing fungi, as well as IA-producing fungi, are ubiquitously found in soils. Neither 8-HHIA nor 9-HHIA showed antibacterial or anti-inflammatory activities. Interestingly, 8-HHIA and 9-HHIA showed cytotoxicity against the human cervical cancer cell line (HeLa) and human diploid cell line (MRC-5), and MRC-5 only, respectively, compared to IA at the same concentration. This study indicates that the screening method could easily discover fungi producing 8-HHIA and 9-HHIA in soils.

Keywords

• itaconic acid
• hydroxyhexyl itaconic acid
• screening
• Mizoroki–Heck reaction