Resurrection of efficient Precambrian endoglucanases for lignocellulosic biomass hydrolysis

Abstract

Cellulases catalyze the hydrolysis of cellulose. Improving their catalytic efficiency is a longstanding goal in biotechnology given the interest in lignocellulosic biomass decomposition. Although methods based on sequence alteration exist, improving cellulases is still a challenge. Here we show that Ancestral Sequence Reconstruction can "resurrect" efficient cellulases. This technique reconstructs enzymes from extinct organisms that lived in the harsh environments of ancient Earth. We obtain ancestral bacterial endoglucanases from the late Archean eon that efficiently work in a broad range of temperatures (30-90°C), pH values (4-10). The oldest enzyme (~2800 million years) processes different lignocellulosic substrates, showing processive activity and doubling the activity of modern enzymes in some conditions. We solve its crystal structure to 1.45 Å which, together with molecular dynamics simulations, uncovers key features underlying its activity. This ancestral endoglucanase shows good synergy in combination with other lignocellulosic enzymes as well as when integrated into a bacterial cellulosome.

High performance crystalline nanocellulose using an ancestral endoglucanase

“…Luckily, ASR brings back the ability to generate CNC in one single reaction by one enzyme alone. This may be related to the processive activity reported for the ancestral EG, that demonstrated endo-activity and exo-activity, unlike its modern counterparts 14 .…”

“…EnCNC production and characterization. ANC EG belongs to the last common ancestor of firmicutes (LFCA) and has been fully characterized previously, including its substrate-interaction mechanism 14 , demonstrating high activity over a wide range of conditions and in several substrates. This enzyme can be used in two forms, only the catalytic domain or linked to a carbohydratebinding module (CBM).…”

“…Ancestral sequence reconstruction (ASR) has been proposed for enzyme engineering 14,15 . This technique is commonly used in molecular evolution studies 16,17 , but previous studies reported that ancient enzymes show extraordinary physicochemical properties [18][19][20][21] , making them potentially suitable for biotechnology.…”

“…This technique is commonly used in molecular evolution studies 16,17 , but previous studies reported that ancient enzymes show extraordinary physicochemical properties [18][19][20][21] , making them potentially suitable for biotechnology. We have recently reconstructed a 2.8 billion-year-old ancestral endoglucanase (ANC EG) from bacteria species that showed superior performance in comparison with modern ones 14 . Here, we show that ANC EG alone can produce pure nanocellulose (EnCNC).…”

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

“…The pH stability of cellulase is essential for the efficient degradation of lignocellulosic biomass hydrolysis at wide range of pH (4-10) [153]. Directed evolution employed on endo-β-1,4-glucanase III (EG III) from T. reesei QM9414 enhanced pH stability and specific activity [154].…”