Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution

Abstract

BackgroundEnzymatic hydrolysis of cellulose involves the spatiotemporally correlated action of distinct polysaccharide chain cleaving activities confined to the surface of an insoluble substrate. Because cellulases differ in preference for attacking crystalline compared to amorphous cellulose, the spatial distribution of structural order across the cellulose surface imposes additional constraints on the dynamic interplay between the enzymes. Reconstruction of total system behavior from single-molecule activity parameters is a longstanding key goal in the field.ResultsWe have developed a stochastic, cellular automata-based modeling approach to describe degradation of cellulosic material by a cellulase system at single-molecule resolution. Substrate morphology was modeled to represent the amorphous and crystalline phases as well as the different spatial orientations of the polysaccharide chains. The enzyme system model consisted of an internally chain-cleaving endoglucanase (EG) as well as two processively acting, reducing and non-reducing chain end-cleaving cellobiohydrolases (CBHs). Substrate preference (amorphous: EG, CBH II; crystalline: CBH I) and characteristic frequencies for chain cleavage, processive movement, and dissociation were assigned from biochemical data. Once adsorbed, enzymes were allowed to reach surface-exposed substrate sites through “random-walk” lateral diffusion or processive motion. Simulations revealed that slow dissociation of processive enzymes at obstacles obstructing further movement resulted in local jamming of the cellulases, with consequent delay in the degradation of the surface area affected. Exploiting validation against evidence from atomic force microscopy imaging as a unique opportunity opened up by the modeling approach, we show that spatiotemporal characteristics of cellulose surface degradation by the system of synergizing cellulases were reproduced quantitatively at the nanometer resolution of the experimental data. This in turn gave useful prediction of the soluble sugar release rate.ConclusionsSalient dynamic features of cellulose surface degradation by different cellulases acting in synergy were reproduced in simulations in good agreement with evidence from high-resolution visualization experiments. Due to the single-molecule resolution of the modeling approach, the utility of the presented model lies not only in predicting system behavior but also in elucidating inherently complex (e.g., stochastic) phenomena involved in enzymatic cellulose degradation. Thus, it creates synergy with experiment to advance the mechanistic understanding for improved application.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0463-8) contains supplementary material, which is available to authorized users.

Improvement of ethanol production from crystalline cellulose via optimizing cellulase ratios in cellulolytic Saccharomyces cerevisiae

“…This observation is consistent with the known function of EG in preferentially cleaving the amorphous regions of cellulose; this activity reduces the chance of CBH being unable to pass through this region, which would result in stalling of CBH upon encountering an amorphous region (Jalak et al, 2012). In a free-form cellulase system, CBH can escape from being stuck by dissociating from the cellulose surface (Eibinger et al, 2016); however, in a cellsurface display system, the immobilized CBH will be unable to "get off" the cellulose. As a result, more EG will be required to clear the obstacles.…”

“…In this study, the optimized strain (A26) indicated the high transcript levels of EG and CBH1 (Fig. In a free-form cellulase system, CBH can escape from being stuck by dissociating from the cellulose surface (Eibinger et al, 2016); however, in a cellsurface display system, the immobilized CBH will be unable to "get off" the cellulose. This observation is consistent with the known function of EG in preferentially cleaving the amorphous regions of cellulose; this activity reduces the chance of CBH being unable to pass through this region, which would result in stalling of CBH upon encountering an amorphous region (Jalak et al, 2012).…”

“…Surprisingly, approximately 57% of the ethanol theoretical yield was obtained from Avicel using the ratio-optimized cellulolytic S. cerevisiae in the present study. Nonetheless, natural lignocellulose consists of crosslinked cellulose, hemicellulose, and lignin (Eibinger et al, 2016), making natural lignocellulose more resistant to digestion than Avicel. Nonetheless, natural lignocellulose consists of crosslinked cellulose, hemicellulose, and lignin (Eibinger et al, 2016), making natural lignocellulose more resistant to digestion than Avicel.…”

“…Among current reports, our results represents the highest reported ethanol yield from Avicel obtained using cellulolytic yeast (Table II), suggesting that our technique greatly promotes the feasibility of the efficient utilization of crystalline cellulose. Nonetheless, natural lignocellulose consists of crosslinked cellulose, hemicellulose, and lignin (Eibinger et al, 2016), making natural lignocellulose more resistant to digestion than Avicel. Although, we have successfully obtained a 60% increase of ethanol yield from rice straw through optimizing the cellulase ratio in cellulolytic yeast, the titer is still low.…”

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

“…An important role in deciphering the mechanism of cellulose hydrolysis is played by mathematical modeling (Bansal, Hall, Realff, Lee, & Bommarius, ; Jeoh, Cardona, Karuna, Mudinoor, & Nill, ), including both the models based on ordinary differential equations (Bommarius et al, ; Griggs, Stickel, & Lischeske, ; Praestgaard et al, ) and on particle‐based simulations (Eibinger, Zahel, Ganner, Plank, & Nidetzky, ; Shang, Chang, & Chu, ; Shang & Chu, ; Warden, Little, & Haritos, ).…”