MOLECULAR-CLONING AND HETEROLOGOUS EXPRESSION OF THE GENE ENCODING DIHYDROGEODIN OXIDASE, A MULTICOPPER BLUE ENZYME FROM ASPERGILLUS-TERREUS

Aspergillus terreus dihydrogeodin oxidase (DHGO) is an enzyme catalyzi ng the stereospecific phenol oxidative coupling reaction converting di hydrogeodin to (+)-geodin, We previously reported the purification of DHGO from A. terreus and raised polyclonal antibody against DHGO. From the first cDNA library constructed in lambda gt11 using mRNA from 3-d ay-old mycelium of A. terreus, four clones were identified using anti- DHGO antibody, but all contained partial cDNA inserts around 280 base pairs. This cDNA fragment was used as a probe to clone the genomic DNA and cDNA for dihydrogeodin oxidase from A. terreus. The sequence of t he cloned DHGO genomic DNA and cDNA predicted that the DHGO polypeptid e consists of 605 amino acids showing significant homology with multic opper blue proteins such as laccase and ascorbate oxidase. Four potent ial copper binding domains exist in DHGO polypeptide, The DHGO gene co nsists of seven exons separated by six short introns. Expression of th e DHGO gene in Aspergillus nidulans under the starch or maltose-induci ble Taka-amylase A promoter as an active enzyme established the functi onal identity of the gene. Also, introduction of the genomic DNA for D HGO into Penicillium frequentans led to the production of DHGO polypep tide as judged by Western blot analysis.