Creation of a functional hyperthermostable designer cellulosome

Amaranta Kahn,Dimitris G Hatzinikolaou,Anastasia P Galanopoulou,Neal Hengge,Nicholas S Sarai,Sarah Moraïs,Yannick J Bomble,Michael E Himmel,Edward A Bayer,Daehwan Chung

doi:10.1186/s13068-019-1386-y

Abstract

BackgroundRenewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria.ResultsHere, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum.ConclusionThe functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

Highlights

Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a high potential as an alternative source of energy
The catalytic modules were selected from bifunctional cellulase Cel9/48A and endoglucanase Cel5D of known properties from the hyperthermophilic bacterium, Ca. bescii
The cohesins and dockerins were selected from components of thermophilic microbes, since mesophilic cohesin–dockerin interactions were shown to be unstable at high temperatures [33]

Summary

Introduction

Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a high potential as an alternative source of energy. The use and production of the various cellulases remain costly, due to problematic production steps and demanding process parameters, such as optimizing concentrations, pH, and maintenance of ambient temperatures throughout an exothermic process [11]. In this context, thermostable cellulolytic enzymes are attractive candidates for biomass deconstruction. Owing to the elevated reaction temperatures, pre-treatment conditions may be relieved or even eliminated in biomass-to-biofuels conversion processes [13]

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Discussion

Conclusion