Alleviating product inhibition in cellulase enzyme Cel7A

Abstract

Enzymes that degrade cellulose into glucose are one of the most expensive components of processes for converting cellulosic biomass to fuels and chemicals. Cellulase enzyme Cel7A is the most abundant enzyme naturally employed by fungi to depolymerize cellulose, and like other cellulases is inhibited by its product, cellobiose. There is thus great economic incentive for minimizing the detrimental effects of product inhibition on Cel7A. In this work, we experimentally generated 10 previously proposed site‐directed mutant Cel7A enzymes expected to have reduced cellobiose binding energies (the majority of mutations were to alanine). We then tested their resilience to cellobiose as well as their hydrolytic activities on microcrystalline cellulose. Although every mutation tested conferred reduced product inhibition (and abolished it for some), our results confirm a trade‐off between Cel7A tolerance to cellobiose and enzymatic activity: Reduced product inhibition was accompanied by lower overall enzymatic activity on crystalline cellulose for the mutants tested. The tempering effect of mutations on inhibition was nearly constant despite relatively large differences in activities of the mutants. Our work identifies an amino acid in the Cel7A product binding site of interest for further mutational studies, and highlights both the challenge and the opportunity of enzyme engineering toward improving product tolerance in Cel7A. Biotechnol. Bioeng. 2016;113: 330–338. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Crystal structures of wild‐type Trichoderma reesei Cel7A catalytic domain in open and closed states

“…The extensive hydrogen bond network between loop A4 R394 and the product +2 moiety supports its role in product inhibition. Mutation of R394 to alanine suppresses this network alleviating product inhibition . In contrast to previous observations, the product +2 glucosyl moiety is not hydrogen bonded to D259 .…”

“…The main chain D259 carbonyl group, however, forms a hydrogen bond with the O2 of the +1 glucosyl moiety (3.0 A) as previously reported [38]. D259A has been shown to mitigate product inhibition to a lesser extent than R394A or R251K [16]. The variability observed across structures in the hydrogen bond interactions between D259 and the product provides structural insight as to why this is the case.…”

“…Overall, the cellobiose-protein interactions in our structure are similar to those observed in the truncated E212Q inactive TrCel7A cellobiose complex (PDB ID 3CEL) [38]. We focus here on the interactions known to be key to product inhibition [16]. O5 and O6 from the +2 glucosyl moiety form hydrogen bonds with R394 NH1 (3.3 and 3.2 A, respectively), while O1 on the same moiety is hydrogen bonded to R394 NH2.…”

Bioethanol and lipid production from the enzymatic hydrolysate of wheat straw after furfural extraction

“…In experiments of integrated storage and microbial pretreatment, 25–50% of the cellulose was converted to glucose (Passoth et al 2013). Probably, even higher values of cellulose degradation might be possible when using simultaneous saccharification and fermentation, to avoid feedback inhibition of cellulase, and in general, by using improved enzyme cocktails (Atreya et al 2016). …”

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose

“…On the mechanistic level, molecular origins of Cel7A's product inhibition remains controversial (Andric et al, ), and this uncertainty impedes engineering of variants that are less prone to inhibition. Such variants have been suggested on the basis of computational work (Bu et al, ; Silveira and Skaf, 2015), and most recently also produced by site directed mutagenesis (Atreya et al, ). This latter work confirmed that the predicted mutations alleviated product inhibition, but also found that this improvement was generally associated with reduced catalytic efficiency.…”