Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library

Abstract

We aimed at constructing thermostable cellulase variants of cellobiohydrolase II, derived from the mesophilic fungus Phanerochaete chrysosporium, by using an advanced evolutionary molecular engineering method. By aligning the amino acid sequences of the catalytic domains of five thermophilic fungal CBH2 and PcCBH2 proteins, we identified 45 positions where the PcCBH2 genes differ from the consensus sequence of two to five thermophilic fungal CBH2s. PcCBH2 variants with the consensus mutations were obtained by a cell-free translation system that was chosen for easy evaluation of thermostability. From the small library of consensus mutations, advantageous mutations for improving thermostability were found to occur with much higher frequency relative to a random library. To further improve thermostability, advantageous mutations were accumulated within the wild-type gene. Finally, we obtained the most thermostable variant Mall4, which contained all 15 advantageous mutations found in this study. This variant had the same specific cellulase activity as the wild type and retained sufficient activity at 50°C for >72 h, whereas wild-type PcCBH2 retained much less activity under the same conditions. The history of the accumulation process indicated that evolution of PcCBH2 toward improved thermostability was ideally and rapidly accomplished through the evolutionary process employed in this study.

Crystal structure of a family 6 cellobiohydrolase from the basidiomycetePhanerochaete chrysosporium

“…Mutagenesis work performed on PcCel6A revealed several properties of noncatalytic residues of this enzyme (see Heinzelman et al, 2009;Ito et al, 2013;Tachioka et al, 2016). In our recent work, the substitution mutation W267C was found to be a critical mutation for the degradation of crystalline cellulose III I but not of amorphous phosphoric acid-swollen cellulose (Tachioka et al, 2016), which was a similar finding to the work on TrCel6A (Koivula et al, 1998).…”

“…In a previous study, we cloned and recombinantly expressed PcCel6A and found that the hydrolysis rate of PcCel6A was greatly accelerated by a polymorphic change of crystalline cellulose (Igarashi et al, 2012). PcCel6A has been the target of protein engineering aimed at improving its thermostability (Heinzelman et al, 2009;Ito et al, 2013), and we have also recently applied random mutagenesis to this enzyme (Tachioka et al, 2016). Here, we report the structure of the CD of PcCel6A in its apo form and in complex with the ligand cellobiose.…”

“…3b), than the serine residues that are found in other homologous fungal structures which originally have a serine instead of cysteine (2.6-2.7 Å in Chaetomium thermophilum Cel6A and CcCel6C), which is consistent with their discussion. Ito and coworkers also improved the thermostability of PcCel6A by 16 cumulative mutations (Ito et al, 2013). The largest stabilization of 1.2 C was achieved by a mutation of Met257 to isoleucine, and the authors deduced from modelling PcCel6A that an exchange to more hydrophobic residues stabilizes the research communications Acta Cryst.…”

Robust enzyme design: Bioinformatic tools for improved protein stability

“…The best variant showed 9.1°C increase in the mid-point of thermal denaturation and contained eight consensus mutations. In a similar study, five thermophilic proteins were aligned to predict 41 consensus mutations in the catalytic domain of cellobiohydrolase II from Phanerochaete chrysosporium [37]. After the first round of screening, 13 advantageous single and double mutations were identified which were randomly recombined and screened to identify a variant with significantly increased thermostability.…”

Bacterial genomes: what they teach us about cellulose degradation

“…Large-scale domain mixing experiments will be conducted to determine best combinations of CBM, linker and GH family members [80][81][82][83][84]. These experiments will not only provide important information on the relationship between structure and activity, but also provide new, high activity cellulases to reduce the cost of producing biofuels.…”