Our previous results have shown that some genes such as PDIDSM_85260 and PDIDSM_08010 may be partly responsible for limonene biotransformation to α-terpineol as determined by analysis of genomic and transcriptomic data, protein separation and purification, and mass spectrometry. In order to explore the biological functions of the genes, RNA interference (RNAi) vectors for PDIDSM_85260 and PDIDSM_08010 were constructed and transformed into Penicillium digitatum DSM62840 by Agrobacterium tumefaciens-mediated transformation. RANi strains were obtained by a series of experiments, and the functions of the two genes were analyzed in P. digitatum DSM62840. It was found that the amount of α-terpineol produced by the silenced transformants obviously decreased compared to the wild-type strain, suggesting that PDIDSM_85260 and PDIDSM_08010 may participate in limonene biotransformation to α-terpineol.

The key enzyme involved in the biotransformation of limonene into α-terpineol by Penicillium digitatum DSM62840 was extracted and purified by sequential ultracentrifugation, ammonium sulfate precipitation, hydroxyapatite chromatography and gel filtration chromatography after high pressure disruption of cells. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed an obvious protein band with a molecular mass of approximatelty 50 kDa. This protein was tentatively identified as dihydrolipoyl dehydrogenase (PDIDSM_08010) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results of this study may provide a basis and reference for α-terpineol production.