MOLECULAR-CLONING, SEQUENCING, AND HETEROLOGOUS EXPRESSION OF THE VAOA GENE FROM PENICILLIUM-SIMPLICISSIMUM CBS-170.90 ENCODING VANILLYL-ALCOHOL OXIDASE
Jae. Benen et al., MOLECULAR-CLONING, SEQUENCING, AND HETEROLOGOUS EXPRESSION OF THE VAOA GENE FROM PENICILLIUM-SIMPLICISSIMUM CBS-170.90 ENCODING VANILLYL-ALCOHOL OXIDASE, The Journal of biological chemistry, 273(14), 1998, pp. 7865-7872
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.