KnowVolution of a GH5 Cellulase from Penicillium verruculosum to Improve Thermal Stability for Biomass Degradation

Abstract

Understanding the thermostability of cellulases is of high importance for their application in ligno­cellulosic biomass degradation, feedstock, and pulp and paper production. Cellulases have to withstand high temperatures and harsh conditions in various application areas, for instance, in bioethanol production. Engineering thermo­stable cellulases increases the cellulase lifetime in processes and contributes to more-sustainable production. Here we report the first Know­Volution campaign toward improving the thermo­stability of the endo-β-1,4-glucanase PvCel5A from Penicillium verrucu­losum. The C-terminal region of PvCel5A (eighth α-helix, amino acid residues 280–314) was identified as a key structural determinant to improve the thermo­stability of PvCel5A without affecting its specific activity. The most beneficial variant, PvCel5A-R17, harbors three substitutions (F16L/​Y293F/​Q289G); its half-life at 75 °C improved 5.5-fold (from 32 to 175 min) and the melting temperature was raised 7.7 °C (from 70.8 °C) when compared to those of wild-type PvCel5A. Exceptionally, the thermally improved PvCel5A-R17 variant retained its specific activity at low temperatures (40 °C). Computational analyses revealed that the stabilization of the C-terminal region of PvCel5A is responsible for the improved thermo­stability. This knowledge will facilitate shorter times in cellulase engineering and thereby enhance the performance and sustainability of processes.

Can constraint network analysis guide the identification phase of KnowVolution? A case study on improved thermostability of an endo-β-glucanase

“…EGLII mutant libraries were cloned into the shuttle vector pBSYA1S1Z (Bisy e.U., Hofstaetten/Raab, Austria) and generated in P. pastoris BSYBG11. Endo-β-glucanase gene eglII from Penicillium verruculosum (EGLII; UniProtKB/Swiss-Prot: A0A1U7Q1U3) was purchased as codon-optimized synthetic gene fragment from ThermoFisher (Germany) and cloned into pBSYA1S1Z as described previously [38] .…”

“…Random mutagenesis was generated in the endoglucanase egl II gene by error-prone PCR (ep-PCR), as described by Contreras et al [38] . Briefly, test libraries (consisting of 180 clones) were generated by ep-PCR with varying concentrations of MnCl 2 ranging from 0.1 to 0.4 mM.…”

“…Site-saturation mutagenesis libraries at positions 76, 77, 92, 93, 114, 129, 130, 134, 189, 190, 222, 240, 244, 255, 256, 273, 299, 308, and 312 were generated by site-saturation mutagenesis (SSM) method [40] . SSM libraries were produced as described previously [38] , and used NNK primers are detailed in Table S1 in SI. The resulting PCR product was digested using DpnI (37 °C, 18 h), purified by using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel), and transformed into P. pastoris BSYBG11 for expression.…”

“…Purification of the endoglucanase EGLII was performed by anion exchange chromatography as described previously [38] . After flask expression, 50 mL of EGLII containing supernatant was concentrated by centrifugal ultrafiltration (10 kDa MWCO PES; VivaSpin turbo 15, Sartorius) to 2 mL, and the buffer was exchanged to Bis-Tris buffer (pH 6.5, 20 mM; buffer A).…”

“…A two-step screening system was employed, as described by Contreras et al [38] . Briefly, an agar-based pre-screening step was performed with Azo-carboxymethyl cellulose (Azo-CMC; Megazyme, Bray, Ireland) supplemented YPD agar plates, colonies presenting clear halos were selected as active for hydrolytic activity.…”