The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally coregulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction.

Cellulases belong to the large family of glycosyl hydrolases (GHs) and are produced by a variety of bacteria and fungi. These extracellular enzymes act as endoglucanases (EGs), cellobiohydrolases or beta-glucosidases. In this paper, we describe molecular screening for EGs from the GH family 12. Using three homologous sequence boxes deduced from five previously known members of the family, we analysed 22 cellulase-producing fungal strains obtained from a diverse area of the fungal kingdom. Polymerase chain reactions using degenerate primers designed to the homologous protein boxes were used to identify the family 12 homologues. Several fungi showed the presence of multiple versions of the gene, while amino acid sequence analysis showed diversity in 15 novel members of the family, ranging from 26% to 96% similarity. Our sequence analysis shows that the phylogenetic tree of family 12 EGs can be divided into four subfamilies: 12-1 (fungal group I), 12-2 (fungal group II), 12-3 ( Streptomyces group in which Rhodothermus marinus fits) and 12-4 ( Thermophiles group). Erwinia carotovora may form a new subgroup.

Secreted mixtures of cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion. Here, we enhanced the thermal stability of Cel7A through directed evolution. Sites with increased thermal stability properties were combined, and a Cel7A variant (FCA398) was obtained, which exhibited a 10.4 °C increase in and a 44-fold greater half-life compared with the wild-type enzyme. This Cel7A variant contains 18 mutated sites and is active under application conditions up to at least 75 °C. The X-ray crystal structure of the catalytic domain was determined at 2.1 Å resolution and showed that the effects of the mutations are local and do not introduce major backbone conformational changes. Molecular dynamics simulations revealed that the catalytic domain of wild-type Cel7A and the FCA398 variant exhibit similar behavior at 300 K, whereas at elevated temperature (475 and 525 K), the FCA398 variant fluctuates less and maintains more native contacts over time. Combining the structural and dynamic investigations, rationales were developed for the stabilizing effect at many of the mutated sites.

Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5P ends of 21 888 cDNA clones were sequenced from which a unique set of over 5000 were also 3P sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.

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