Identification, Biochemical Characterization, and Evolution of the Rhizopus oryzae 99-880 Polygalacturonase Gene Family

Authors: Mertens, Jeffrey; Burdick, Ryan; Rooney, Alejandro - Alex

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2008
Publication Date: 11/1/2008
Citation: Mertens, J.A., Burdick, R.C., Rooney, A.P. 2008. Identification, Biochemical Characterization, and Evolution of the Rhizopus oryzae 99-880 Polygalacturonase Gene Family. Fungal Genetics and Biology. 45(12):1616-1624.

Interpretive Summary: The use of agricultural biomass will be required to meet the future needs and requirements of fuel ethanol production. Biomass typically needs to be broken down by chemical pretreatments and enzymes into simple sugars in order to be utilized. The fungus Rhizopus oryzae produces enzymes called polygalacturonases that are very effective in the conversion of pectin, a major component of plant cell walls, into simple sugars. Industrial interest in this enzyme is strong due to the use of this enzyme in the clarification of fruit juices, retting of flax as well as its potential in the conversion of biomass to simple sugars. In this work, we have identified a family of genes that are responsible for production of previously uncharacterized polygalacturonase enzymes from Rhizopus. This discovery further improves the ability to control the efficiency of polygalacturonase production and will ultimately decrease the cost of using agricultural crops for the production of high value products.

Technical Abstract: A search of the recently sequenced Rhizopus oryzae strain 99-880 genome database uncovered 18 putative polygalacturonase genes with 2 genes being identical and only 1 with similarity to a previously reported R. oryzae polygalacturonase gene. The 17 different genes share 50% to greater than 90% identity at the nucleotide level as well as the deduced protein sequence level. The cDNA of the different genes was isolated directly or recombinantly and used to express the encoded proteins in Pichia pastoris. Recombinant protein expression demonstrated that 15 of the 17 genes encode active enzymes with 12 genes encoding for endo-polygalacturonase enzymes and 3 genes encoding for exo-polygalacturonase enzymes. Phylogenetic analysis indicates that the genes form a distinct monophyletic group among fungal polygalacturonase enzymes. Finally, our results also suggest that the ancestral form of polygacturonase in fungi is endolytic and exolytic function evolved later, at least two independent times.