Engineering All-Round Cellulase for Bioethanol Production

Abstract

One strategy to decrease both the consumption of crude oil and environmental damage is through the production of bioethanol from biomass. Cellulolytic enzyme stability and enzymatic hydrolysis play important roles in the bioethanol process. However, the gradually increased ethanol concentration often reduces enzyme activity and leads to inactivation, thereby limiting the final ethanol yield. Herein, we employed an optimized Two-Gene Recombination Process (2GenReP) approach to evolve the exemplary cellulase CBHI for practical bioethanol fermentation. Two all-round CBHI variants (named as R2 and R4) were obtained with simultaneously improved ethanol resistance, organic solvent inhibitor tolerance, and enzymolysis stability in simultaneous saccharification and fermentation (SSF). Notably, CBHI R4 had a 7.0-to 34.5-fold enhanced catalytic efficiency (k cat /K M ) in the presence/absence of ethanol. Employing the evolved CBHI R2 and R4 in the 1G bioethanol process resulted in up to 10.27% (6.7 g/L) improved ethanol yield (ethanol concentration) than non-cellulase, which was far more beyond than other optimization strategies. Besides bioenergy fields, this transferable protein engineering routine holds the potential to generate all-round enzymes that meet the requirement in biotransformation and bioenergy fields.

Corner Engineering: Tailoring Enzymes for Enhanced Resistance and Thermostability in Deep Eutectic Solvents

“…Three DESs (30 % (v/v) ChCl: acetamide, 95 % (v/v) ChCl: ethylene glycol, 30 % (v/v) TBPB: ethylene glycol) were selected with screening concentrations to maintain 30–40 % residual activity of WT for the standard screening process (Figure S1) [49] . The latter screening condition was suitable to observe and distinguish the resistance change (e.g., beneficial, non‐beneficial) between variants and BSLA WT [31,54] …”

“…[49] The latter screening condition was suitable to observe and distinguish the resistance change (e.g., beneficial, non-beneficial) between variants and BSLA WT. [31,54] After screening 2200 variants, 216 beneficial variants located at eight corners were identified towards three DES cosolvents (Figure 2b-c). An up to 32 % beneficial rate at the single corner (e.g., corner 161.162.163) can be obtained (Figure 2c-d and S2), suggesting this approach leads to the efficient acquisition of beneficial variants exhibiting enhanced DESs resistance.…”

“…And the temperature sensitive DES‐thermomorphic multiphasic system (DES‐TMS) was developed, which the product and the biocatalyst alcohol dehydrogenase can be separated in a single unit operation step [29] . Besides, directed evolution and (semi−)rational design were often applied for engineering enzyme properties in non‐conventional media, such as cellulases in ethanol, [30,31] lipases in 1,4‐dioxane, [32] and laccases in [C 2 C 1 Im][OAc] [33] . Leveraging experimental techniques and computational analysis, researchers developed various protein engineering strategies to accelerate the evolving process and reduce the laborious work [34–38] .…”

Improving plastic degrading enzymes via directed evolution

“…It has been used extensively for enhancing the activity, thermostability, solubility, solvent tolerance, and pH adaptation of biocatalysts with interest for industrial applications ( Sanchez and Ting 2020 , Wang et al. 2021 , Wang et al. 2023 ).…”

Corner Engineering: Tailoring Enzymes for Enhanced Resistance and Thermostability in Deep Eutectic Solvents

“…[28] And the temperature sensitive DES-thermomorphic multiphasic system (DES-TMS) was developed, which the product and the biocatalyst alcohol dehydrogenase can be separated in a single unit operation step. [29] Besides, directed evolution and (semiÀ )rational design were often applied for engineering enzyme properties in nonconventional media, such as cellulases in ethanol, [30,31] lipases in 1,4-dioxane, [32] and laccases in [C 2 C 1 Im][OAc]. [33] Leveraging experimental techniques and computational analysis, researchers developed various protein engineering strategies to accelerate the evolving process and reduce the laborious work.…”