Evolving Robust and Interpretable Enzymes for the Bioethanol Industry

Abstract

Obtaining a robust and applicable enzyme for bioethanol production is a dream for biorefinery engineers. Herein, we describe a general method to evolve an all-round and interpretable enzyme that can be directly employed in the bioethanol industry. By integrating the transferable protein evolution strategy In-SiReP 2.0 (In Silico guided Recombination Process), enzymatic characterization for actual production, and computational molecular understanding, the model cellulase PvCel5A (endoglucanase II Cel5A from Penicillium verruculosum) was successfully evolved to overcome the remaining challenges of low ethanol and temperature tolerance, which primarily limited biomass transformation and bioethanol yield. Remarkably, application of the PvCel5A variants in both first-and secondgeneration bioethanol production processes (i. Conventional corn ethanol fermentation combined with the in situ pretreatment process; ii. cellulosic ethanol fermentation process) resulted in a 5.7-10.1 % increase in the ethanol yield, which was unlikely to be achieved by other optimization techniques.

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

“…These results demonstrated that BSLA variants evolved for DES cosolvents resistance by corner engineering also have enhanced thermostability, especially for K88E/N89K and M137D/N138D. Similar phenomena were observed for protein engineering champaigns towards OSs [51,54] and ILs resistances [30,32,41] …”

“…Similar phenomena were observed for protein engineering champaigns towards OSs [51,54] and ILs resistances. [30,32,41] The transferability of corner engineering was confirmed by investigating two more enzymes: BHETase and cellulase Among 13 beneficial BSLA variants obtained (> 1.5-fold), 61.5 % (8/13) variants were identified from coil-helix transition regions, signifying exploring coil-helix corner holds the potential to obtain the most enhanced variants than turn-helix, coil-sheet, and turn-sheet. Two industrial enzymes, Bs2Est and PvCel5A, were examined to validate this finding further (Figure 4a and 4b).…”

“…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] .…”

Enhanced Thermostability of Candida Ketoreductase by Computation-Based Cross-Regional Combinatorial Mutagenesis

“…In the last few decades, biocatalysts have been widely employed for the catalytic synthesis of pharmaceuticals and bioactive molecules in various research and industrial sectors. − Industrial biocatalysts need to tolerate harsh reaction conditions, such as elevated temperatures and high substrate/product concentrations . Excellent robustness is necessary for the enzymes, which includes not only high thermal stability but also exceptional resistance to organic substances (e.g., solvents, the substrate, and the product), drastic pH variation, especially in the cases requiring acid/base neutralization, and even high shear stress, which becomes increasingly important in large-scale stirred tank reactors. − Moreover, considering that new functions of enzymes generated through a series of mutations often encounter more destabilized structures, robust enzymes are also highly desirable because of their evolvability that can accommodate a wider variety of unstable mutations. − Therefore, improving enzyme stability is extremely important for both industrial applications and fundamental studies.…”

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

“…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] .…”