Characterization of the unique pectinolytic system of Paenibacillus amylolyticus 27C64

P. amylolyticus 27C64, a Gram positive bacterium originally isolated from the hindgut of Tipula abdominalis (aquatic cranefly) larvae, is capable of degrading major plant cell wall polysaccharides, including pectin. Two pectate lyases capable of degrading recalcitrant, highly methylated pectins have been previously identified suggesting that this organism may be a useful source of pectinases. Whole-genome sequencing and analysis for carbohydrate active enzymes identified a system of 17 putative pectinases including esterases, lyases, and hydrolases responsible for deconstructing both polygalacturonic acid and rhamnogalacturonan. Notably the sole pectin methylesterase lacks a signal peptide, suggesting that P. amylolyticus demethylates pectin intracellularly unlike Erwinia and Aspergillus. Also unique to this system is one protein which contains both rhamnogalacturonan lyase and acetylesterase domains. A protein BLAST of this pectinase against the CAZy database identified no other enzymes with both of these conserved domains. Each gene will be overexpressed in E. coli and activities on various pectic substances will be characterized with thin layer chromatography. To our knowledge, this is the first attempt to characterize a complete pectinolytic system within the genus Paenibacillus.