Cotton, Alexander Ross; (2022) Nature inspired engineering: bioprospecting for novel psychrophilic enzymes from the Antarctic tunicate, Salpa thompsoni. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nature-inspired engineering offers an alternative approach to solving key sustainability challenges in bioprocessing by looking to fundamental mechanisms that occur in nature for inspiration on solutions to key bioprocessing obstacles. The degradation of cellulosic materials at low temperatures poses an interesting challenge of how to degrade a recalcitrant starting material in a context that is thermodynamically challenging. A case study of a set of marine invertebrates known as salps was explored for two key reasons: their ability to rapidly filter feed upon microalgae and their gelatinous composition formed of tunicin, a variety of cellulose. Taking this as a departure point, this research targeted a unique pelagic organism surviving in the cold waters surrounding Antarctica, Salpa thompsoni, from which novel psychrophilic carbohydrate active enzymes (CAZymes) were identified using a bioprospecting approach. A transcriptomic dataset was prepared from S. thompsoni which was interrogated alongside a publicly available draft genome. This resulted in the identification of several novel hydrolytic enzymes. Two of the identified cellulases, a putative β-glucosidase (BglS) and putative endoglucanase (EgS), were recombinantly expressed and assayed. BglS was found to display β-glucosidase-like activity when hydrolysing pNPG as a substrate, with a Topt of 20°C. To the authors knowledge, this is the lowest observed Topt of any previously described β-glucosidase. EgS displayed low levels of endoglucanase-like activity when hydrolysing CMC as a substrate however, further characterisation of this putative endoglucanase was inhibited due to low expression levels. Initial characterisation of BglS and EgS provided some evidence for their ability to hydrolyse cellulosic materials. As such, these results indicate potential future use of these enzymes in cellulosic biomass pretreatment. In addition, bioprospecting for novel biocatalysts was carried out in the field on a British Antarctic Survey (BAS) research cruise. Salpa fusiformis samples, an evolutionarily similar species of salp to S. thompsoni, were captured and interrogated for novel biocatalysts using a functional screening approach. Initial results from 16S analysis of the gut microbiome indicate the presence of a Psychrobacter species which may aid in cellulytic digestion within salp species. Further interrogation of hydrolytic enzymes expressed by this Psychrobacter species may yield novel psychrophilic cellulases involved in the digestive processes of salp species with putative industrial applications. This research provides an initial foundation for future works to elucidate the substrate specificity and role of BglS and EgS in global carbon cycles. The work presented here demonstrates the potential synergies between bioprospecting and Antarctic marine ecology when examining cold adapted versus cold acclimated enzymes.

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