Cloning of the gene encoding for monapinone dimerizing enzyme involved in atropisomers, dinapinone A1 and A2 produced by Talaromyces pinophilus FKI-3864

Dinapinone A (DPA), produced by Talaromyces pinophilus FKI-3864 is a potent inhibitor of triacylglycerol (TG) accumulation in mammalian cells. The compound is the dimer of monapinone A (MPA) with a biaryl dihydronaphthopyranone skeleton. Furthermore, DPA was found to be a mixture of atropisomers, DPA1 and A2. Interestingly, MPA was exclusively produced by the fungus in the seawater-containing medium. DPA1 and A2 were found to be converted from MPA with the cell free extract of T. pinophilus FKI-3864. MPA dimerizing enzyme (MDE) was purified by three steps, acetone precipitation, TOYOPEARL Phenyl-650M column chromatography and Sephacryl S-200 column chromatography. The molecular mass of MDE was estimated about 60 kDa in native state. The optimum temperature and pH were 50°C and 4.0, respectively, and the enzyme was stable in a pH range of 3.0 to 5.0. The enzyme had no apparent requirement of cofactors, and its activity was strongly inhibited by chloride ion. The sequences of the genomic DNA and cDNA encoding MDE predicted that the MDE polypeptide consists of 603 amino acids. The MDE gene was introduced in Aspergillus oryzae to express under the starch-inducible Taka-amylase A promoter. The MDE activity was confirmed in the cytoplasmic fraction. The recombinant MDE produced a mixture of atropisomers DPA1 and DPA2 from MPA.