(83a) Evolving Robust and Interpretable Enzymes for the Bioethanol Industry

The large-scale production of bioethanol, one of the most promising sustainable renewable resources, holds the potential to address the world’s current environmental and energy challenges.¹ Since the widespread production of 1^st generation ethanol from sugar- and starch-containing agricultural resources would result in a food supply crisis,² integrating lignocellulosic biomass into 1G ethanol processes has become a transition strategy.³ Lignocellulosic biomass can replace fossil resources if it can be converted into ethanol economically and efficiently.⁴ The main components of lignocellulosic biomass are cellulose, hemicellulose, and lignin, which can be chemically or enzymatically hydrolyzed to produce fermentable monomers after appropriate pretreatment.⁵ Enzymatic hydrolysis is a practical method for biomass hydrolysis using less energy under mild conditions.⁶ Stable cellulases play a critical role in bioethanol fermentation, benefitting from reduced enzyme addition and fewer inhibitor effects.⁷ However, a remaining challenge in bioethanol production is that gradually increased ethanol concentrations significantly reduce cellulase activity or even lead to enzyme inactivation.⁸

In our study, we employed endo-β-1,4-glucanase Cel5A from Penicillium verruculosum (PvCel5A) as a model enzyme to investigate the feasibility of applying an evolved robust cellulase in industrial bioethanol production. PvCel5A is promising endoglucanase for lignocellulose hydrolysis due to its high activity.⁹ The optimized InSiReP (named InSiReP 2.0) was developed to generate an all-round enzyme, including three steps: (i) eleven potential amino acid positions were obtained by cepPCR; (ii) seven beneficial positions and12 beneficial substitutions were identified by SSM with the 20c-Tangmethod. (iii) InSiReP 2.0 analysis and recombination. The obtained beneficial substitutions were grouped based on the CompassR rule: Subset 1: ΔΔGfold ≤+0.36kcal/mol, predictable beneficial recombination; Subset 2:0.36 < ΔΔGfold ≤+7.52kcalmol 1, unpredictable recombination. Then, the single substitutions of Subset 1 were recombined in silico and ranked according to their thermodynamic stability (ΔΔGfold). The top 10% of ranked recombinants were subjected to gene construction, expression, and experimental validation. The improved recombinants were further recombined with single substitutions in Subset 2. Finally，five all-round PvCel5A variants (V1:S3T/S12K/T77M/E85Y/G264H/D290G; V2:S3T/S12C/T77M/E85Y/G264R; V3:S3T/S12K/T77M/E85Y/G264H; R6:S12K/T77M/E85Y/G264H/D290G; R2:S12K/ T77M/E85Y/G264R/D290G) were yielded with minimal experimental effort. Therefore, the InSiReP 2.0 strategy was proved to successfully engineer PvCel5A with significant ethanol tolerance (up to 7.5-fold improvement in 60 % (v/v) ethanol), thermostability (t1/2 up to 1.42-fold at 75 °C, Tm improved by 3.1 and 5.4 °C in buffer and 40 % (v/v) ethanol, respectively), and tolerance to six representative OSs. The detailed molecular understanding based on MD simulation and the inherent rationality of the InSiReP 2.0 approach (based on the internal stability of enzymes during evolution) enabled us to confidently apply these interpretable cellulases in industrial production processes. Notably, purified PvCel5A R6 (S12K/T77M/E85Y/G264H/D290G) increased the ethanol yield by 8.47 % compared to no cellulase addition in the ADISET process. At the same time, up to 10.08 % improvement of the ethanol yield was obtained when applying PvCel5A R6 in the bioethanol fermentation process of corn stover compared to the addition of only commercial cellulase. The successful application of these robust cellulases, especially PvCel5A R6, in both 1st and 2nd generation ethanol production processes demonstrated the great prospects of using engineered cellulases in industrial production, even beyond the bioethanol field.

Reference

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(4) Liu, Y.-J.; Li, B.; Feng, Y.; Cui, Q. Consolidated Bio-Saccharification: Leading Lignocellulose Bioconversion into the Real World. Biotechnology Advances 2020, 40, 107535. https://doi.org/10.1016/j.biotechadv.2020.107535.

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Figure. (a) The production performance of corn ethanol fermentation combined with in-situ cellulase-based pretreatment. No cellulase was added to 1; Only crude PvCel5A R6 was added to 2; Only commercial cellulase was added to 3. Equal amounts of commercial cellulase (U/mL) and PvCel5A (U/mL) were added to cellulase cocktails 4-9, which contain different with PvCel5A variants (4: crude PvCel5A WT; 5: crude PvCel5A V1; 6: crude PvCel5A R6; 7: purified PvCel5A WT; 8: purified PvCel5A V1; 9: purified PvCel5A R6). The crude enzyme was concentrated, and all experiments were based on the fermentation of 100 g of liquefied mixture. (b) The ethanol production performance in the fermentation of corn stover with the addition of different cellulases. Equal amounts of commercial cellulase (U/mL) were added to cellulase cocktails 2-5 (2: purified PvCel5A WT; 3: purified PvCel5A V1; 4: purified PvCel5A V2; 5: purified PvCel5A R6). All experiments were based on 10 g of dilute alkali pretreated corn stover. (c) ADISET process. Conventional corn fermentation with SSF (in black) was combined with in-situ pretreatment (in green). (d) The fermentation process of corn stover to yield ethanol through pretreatment, enzyme hydrolysis, and fermentation