MOLECULAR-CLONING, SEQUENCING, AND HETEROLOGOUS EXPRESSION OF THE VAOA GENE FROM PENICILLIUM-SIMPLICISSIMUM CBS-170.90 ENCODING VANILLYL-ALCOHOL OXIDASE

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected f rom a cDNA library constructed from mRNA isolated from Penicillium sim plicissimum CBS 170.90 grown on veratryl alcohol by immunochemical scr eening. The vaoA-cDNA nucleotide sequence revealed an open reading fra me of 1680 base pairs encoding a 560-amino acid protein with a deduced mass of 62,915 Da excluding the covalently bound FAD. The deduced pri mary structure shares 31% sequence identity with the 8 alpha-(O-tyrosy l)-FAD containing subunit of the bacterial flavocytochrome p-cresol me thyl hydroxylase. The vaoA gene was isolated from a P. simplicissimum genomic library constructed in lambda(EMBL3) using the vaoA-cDNA as a probe. Comparison of the nucleotide sequence of the vaoA gene with the cDNA nucleotide sequence demonstrated that the gene is interrupted by five short introns. Aspergillus niger NW156 prtF pyrA leuA cspA trans formed with the pyrA containing plasmid and a plasmid harboring the co mplete vaoA gene including the promoter and terminator was able to pro duce vaoA mRNA and active vanillyl-alcohol oxidase when grown on verat ryl alcohol and anisyl alcohol. A similar induction of the vaoA gene w as found for P. simplicissimum, indicating that similar regulatory sys tems are involved in the induction of the vaoA gene in these fungi. In troduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA -cDNA resulted in elevated expression levels of active vanillyl-alcoho l oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indis tinguishable from the native enzyme.