By following the reduction of 3,4-Dimethoxybenzaldehyde (Veratraldehyde) in Nitrogen-limited cultures of P. chrysosporium, Muheim et al.[19] purified an intracellular aryl-alcohol dehydrogenase (EC 1.1.1.91) from this lignin-degrading fungus. A cDNA coding for this protein was later isolated
and characterized [20]. Opaganib in vivo However, the biochemical properties of the Aadp enzyme were not extensively studied. Due to its high efficiency in lignin degradation, and to its potential applications in the textile, fuel and paper industries, the 35-Mb haploid genome of P. chrysosporium strain RP78 has been sequenced [2]. The current draft release, version 2.0, includes a total of 10,048 gene models [21] and reveals that the secreted oxidases, peroxidases and hydrolytic enzymes that cooperate in wood decay exist as large multi-gene families. Taking advantage of this genome sequence, this work describes the cloning Selleckchem CHIR-99021 of an AAD cDNA and the comprehensive biochemical characterization
of the encoded enzyme in order to get deeper insight into its biological relevance and biotechnological applications potential such as the degradation of aromatic inhibitors in lignocellulosic hydrolysates that strongly impair ethanol fermentation by yeast [22], as well as for the microbial production of natural flavour and fragrance molecules like 2-Phenylethanol. Results and discussion Cloning of a cDNA from Phanerochaete chrysosporium encoding an aryl-alcohol dehydrogenase Using the amino acid sequence coded by a previously cloned Quisqualic acid AAD ORF from Phanerochaete chrysosporium (Pc) strain OGC101 [20] as query, a BLAST alignment was performed against the translated predicted ORFs of the genome sequence of P. chrysosporium strain RP78 [2, 21]. The results showed the existence of 8 AAD homologues that consist of six to nine exons and encode proteins from 240 to 398 amino acids. The presence of multiple AAD genes in the Pc genome is in accordance with strong multiple bands observed in a Southern blot by Reiser et al.[20]. Interestingly, in
scaffold_1, two tandem AAD homologues (scaffold_1:1025231 to 1023962, and scaffold_1:1027063 to 1025827) were found adjacent to each other. The distance between these two adjacent ORFs is only 596 base-pairs. This extensive genetic diversity was also observed for other lignin-biodegradation related genes encoding peroxidases, oxidases, glycosydases and cytochrome P450s [2]. The existence of multiple AAD genes might suggest multiple specificities required to reduce various aryl-aldehydes arising from the catabolism of complex wood polymers. Among the 8 predicted homologous ORFs in the genome of Pc strain RP78, the one in scaffold_3:2235704–2237287 (JGI Transcript Id: 11055) has only 37 base pairs differences with the cDNA previously cloned by Reiser et al.
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