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Abstract
Paenibacillus amylolyticus 27C64 is a Gram-positive bacterium that was isolated from the hindgut of an aquatic crane fly larva, an insect which relies on a microbial community in its gut to break down the plant material it eats. This bacterium in particular showed promise as a source of novel enzymes that could be used to improve deconstruction of plant cell wall polysaccharides. Whole genome sequencing revealed a diverse set of more than 300 CAZymes including pectinases, xylanases, and cellulases. This organism was able to use pectins and xylan as sole carbon sources for growth and appears to specialize in the deconstruction of non-cellulosic polysaccharides. Dynamic regulation of putative pectinases in response to the specific substrate supplied for growth revealed a complex system with potentially novel elements. Characterizing key enzymes within the homogalacturonan deconstruction system revealed that pectin methylesterase activity is not required for efficient deconstruction. This stands in stark contrast to systems that have already been described. Three of the homogalacturonan degrading enzymes also have significant potential to improve numerous industrial applications. Thegenomic analysis also revealed a novel bifunctional enzyme involved in rhamnogalacturonan I deconstruction. This enzyme, RglA, has a complex multidomain structure that includes both rhamnogalacturonan acetylesterase and rhamnogalacturonan lyase domains along with eight non-catalytic fibronectin type III like domains. Five of these non-catalytic domains are likely carbohydrate binding domains. When characterized in vitro, RglA displayed both lyase and esterase activities as predicted, as well as a preference for acetylated pectins. This ability to function on acetylated substrates has not been previously described; acetylation is typically inhibitory to rhamnogalacturonan lyases. Collectively, this work to understand the pectinolytic system of P. amylolyticus 27C64 has contributed to overall understanding of microbial pectin deconstruction and identified novel enzymes that may have industrial uses.