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Molecular cloning, sequencing, and heterologous expression of the vaoA gene from Penicillium simplicissimum CBS 170.90 encoding vanillyl-alcohol oxidase. 1998

J A Benen, and P Sánchez-Torres, and M J Wagemaker, and M W Fraaije, and W J van Berkel, and J Visser
Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.

The cDNA encoding vanillyl-alcohol oxidase (EC 1.1.3.7) was selected from a cDNA library constructed from mRNA isolated from Penicillium simplicissimum CBS 170.90 grown on veratryl alcohol by immunochemical screening. The vaoA-cDNA nucleotide sequence revealed an open reading frame 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 primary structure shares 31% sequence identity with the 8alpha-(O-tyrosyl)-FAD containing subunit of the bacterial flavocytochrome p-cresol methyl hydroxylase. The vaoA gene was isolated from a P. simplicissimum genomic library constructed in lambdaEMBL3 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 transformed with the pyrA containing plasmid and a plasmid harboring the complete vaoA gene including the promoter and terminator was able to produce vaoA mRNA and active vanillyl-alcohol oxidase when grown on veratryl alcohol and anisyl alcohol. A similar induction of the vaoA gene was found for P. simplicissimum, indicating that similar regulatory systems are involved in the induction of the vaoA gene in these fungi. Introduction of a consensus ribosome binding site, AGAAGGAG, in the vaoA-cDNA resulted in elevated expression levels of active vanillyl-alcohol oxidase from the lac promoter in Escherichia coli TG2. The catalytic and spectral properties of the purified recombinant enzyme were indistinguishable from the native enzyme.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010407 Penicillium A mitosporic Trichocomaceae fungal genus that develops fruiting organs resembling a broom. When identified, teleomorphs include EUPENICILLIUM and TALAROMYCES. Several species (but especially PENICILLIUM CHRYSOGENUM) are sources of the antibiotic penicillin. Penicilliums
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D005800 Genes, Fungal The functional hereditary units of FUNGI. Fungal Genes,Fungal Gene,Gene, Fungal
D000429 Alcohol Oxidoreductases A subclass of enzymes which includes all dehydrogenases acting on primary and secondary alcohols as well as hemiacetals. They are further classified according to the acceptor which can be NAD+ or NADP+ (subclass 1.1.1), cytochrome (1.1.2), oxygen (1.1.3), quinone (1.1.5), or another acceptor (1.1.99). Carbonyl Reductase,Ketone Reductase,Carbonyl Reductases,Ketone Reductases,Oxidoreductases, Alcohol,Reductase, Carbonyl,Reductase, Ketone,Reductases, Carbonyl,Reductases, Ketone
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D016415 Sequence Alignment The arrangement of two or more amino acid or base sequences from an organism or organisms in such a way as to align areas of the sequences sharing common properties. The degree of relatedness or homology between the sequences is predicted computationally or statistically based on weights assigned to the elements aligned between the sequences. This in turn can serve as a potential indicator of the genetic relatedness between the organisms. Sequence Homology Determination,Determination, Sequence Homology,Alignment, Sequence,Alignments, Sequence,Determinations, Sequence Homology,Sequence Alignments,Sequence Homology Determinations
D017421 Sequence Analysis A multistage process that includes the determination of a sequence (protein, carbohydrate, etc.), its fragmentation and analysis, and the interpretation of the resulting sequence information. Sequence Determination,Analysis, Sequence,Determination, Sequence,Determinations, Sequence,Sequence Determinations,Analyses, Sequence,Sequence Analyses

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