Engineered Agarase Systems to Enhance Agarose Degradation

Abstract

Environmental polysaccharides represent a vast and renewable energy source. A subset of terrestrial bacteria have evolved multi-enzyme complexes called cellulosomes that degrade cellulosic biomass. The cellulosome comprises a modular scaffold containing a carbohydrate-binding module and repeating cohesin modules onto which complementary carbohydrate-active enzymes (CAZymes) assemble via their resident dockerin modules. The proximity and targeting effects associated with these structural features contribute to the efficient digestion of cellulose. The high-affinity cohesin-dockerin interaction displays species-specific properties that have been used to produce purpose-built multi-CAZyme complexes, termed designer cellulosomes, that take advantage of the proximity and targeting effects to degrade terrestrial polysaccharides. Marine bacteria also produce CAZymes to digest marine polysaccharides, but a cellulosome-like complex has yet to be identified. The designer cellulosome system, and its associated proximity and targeting effects, affords an opportunity to engineer highly efficient agarose-degrading enzyme complexes.

The purpose of this thesis was to: 1) assemble cellulosome-like multi-enzyme complexes using endolytic and exolytic agarases from Bacteroides uniformis and assess the impact of the proximity effect on agarose degradation; and 2) quantify the increase in agarose degradation resulting from the targeting effect by incorporation of a B. uniformis agarose-binding protein into chimeric scaffolds. Using molecular biology and biochemical approaches we produced B. uniformis agarase (BuGH2C, BuGH16B, BuGH86, and BuGH117B)-dockerin fusion constructs and complementary cohesin-based chimeric scaffolds and showed that while a mixture of the BuGH86 and BuGH117B constructs function synergistically to degrade agarose, complexation did not lead to further enhancement in activity via the proximity effect. We generated an AlphaFold-based structural model of the B. uniformis SusE-like agarose-binding protein, which revealed four distinct modules, the fourth of which was structurally similar to a characterized carbohydrate-binding module. Lastly, incorporating the agarose-binding protein into chimeric scaffolds onto which the endolytic BuGH86 and exolytic BuGH117B agarase-dockerin fusion proteins were attached allowed us to show that the hydrolytic properties of both enzymes were augmented due the targeting effect, and that this effect was further enhanced when these constructs were applied as a mixture. This study provides novel insights into agarose degradation by engineered multi-enzyme complexes and acts as a foundation for future biotechnological applications.