RESUMEN
Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From â¼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa , Pruebas de Enzimas , Genoma Fúngico , Mutación , Ingeniería de Proteínas , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Celulosa 1,4-beta-Celobiosidasa/química , Celulosa 1,4-beta-Celobiosidasa/clasificación , Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Genoma Fúngico/genética , Ingeniería de Proteínas/métodos , Especificidad por Sustrato , Talaromyces/enzimología , Talaromyces/genética , Trichoderma/enzimología , Trichoderma/genética , Trichoderma/metabolismo , BiocatálisisRESUMEN
Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle.