Abstract
Synthetic multifunctional enzymes exhibit superior performance to their non-multifunctional enzyme components and exhibit novel cellulose deconstruction mechanisms.
Highlights
CelA is comprised of GH 9 and GH 48 catalytic domains connected by three type III cellulose-binding modules (CBMs)
Examination of the mechanism of the CBM3b-containing construct revealed a novel biomass deconstruction behavior similar to, but yet distinct from that of native CelA. Multifunctional cellulolytic enzymes, such as CelA from Caldicellulosiruptor bescii,[1,2,3] represent a novel intermediate class of biomass deconstructing enzymes – falling between the well-known free enzyme systems found in most fungi and bacteria and the highly complexed, bacterial cellulosome systems produced by microbes such as Clostridium thermocellum.[4,5,6]
Such synergy has been well-established in free fungal and bacterial cellulase systems and this architecture is clearly used in multifunctional enzymes, such as the hyperthermophilic CelA from C. bescii, which utilizes a GH9 endo-cellulase domain, a GH48 exo-cellulase domain, and three GH Family 3 cellulose binding modules (CBMs) all connected by a series of linker domains
Summary
Multifunctional cellulolytic enzymes, such as CelA from Caldicellulosiruptor bescii,[1,2,3] represent a novel intermediate class of biomass deconstructing enzymes – falling between the well-known free enzyme systems found in most fungi and bacteria and the highly complexed, bacterial cellulosome systems produced by microbes such as Clostridium thermocellum.[4,5,6] The extremely high cellulolytic activity, unique pit-forming mechanism, and recently reported cellulose crystallinity-agnostic behavior of CelA makes it a very important enzyme to investigate further.[1,2,3] fully native CelA has not been successfully expressed in fungal systems.We have designed and built a set of synthetic multifunctional enzymes based on the architecture of CelA. We chose the catalytic domain of cellobiohydrolase I (Cel7A) from Penicillium funiculosum (PfCel7Acat)[7,8] to serve as the N-terminal exocellulase domain. The PfCel7A enzyme has higher activity than the native Cel7A in T. reesei and the E1Y245G mutation has been shown to have increased resistance to end-product inhibition by cellobiose.[7,10] Both enzymes are known to express well in T. reesei. They possessed the correct N to C orientation to be combined into one larger construct. To parse out the functionality of critical substructures, we designed three different constructs, keeping the exo and endo catalytic domains constant while varying the linker and CBM arrangement connecting them (see Fig. 1): 478 | Green Chem., 2020, 22, 478–489
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