family of thermostable fungal cellulases created by structure-guided recombination

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 14; pp. 5610 - 5615
• Main Authors: Heinzelman, Pete, Snow, Christopher D, Wu, Indira, Nguyen, Catherine, Villalobos, Alan, Govindarajan, Sridhar, Minshull, Jeremy, Arnold, Frances H
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 07.04.2009; National Acad Sciences
• Subjects: Acetates; Adenylate cyclase; Amino acid sequence; Amino acids; Biochemistry; Biological Sciences; Biomass; Catalysis; cellobiohydrolase; Cellulase; cellulases; Cellulases - genetics; Cellulose; cellulose 1,4-beta-cellobiosidase; Chimeras; enzymatic hydrolysis; enzyme activity; Enzyme Stability; Enzymes; Fungal Proteins - genetics; Gels; gene expression; Genes; genetic engineering; genetic recombination; genetically engineered microorganisms; Genetics; half life; Half lives; heat inactivation; heat stability; Hot Temperature; Humicola; Humicola insolens; Hydrolysis; inactivation; Inventories; Libraries; Mathematical models; pH effects; phosphoric acid; Protein Engineering - methods; Recombinant Fusion Proteins; Recombination; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sodium; Sugars; Temperature; Temperature effects; Thermal stability; thermophilic fungi; Yeast
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0901417106

SCHEMA structure-guided recombination of 3 fungal class II cellobiohydrolases (CBH II cellulases) has yielded a collection of highly thermostable CBH II chimeras. Twenty-three of 48 genes sampled from the 6,561 possible chimeric sequences were secreted by the Saccharomyces cerevisiae heterologous host in catalytically active form. Five of these chimeras have half-lives of thermal inactivation at 63 °C that are greater than the most stable parent, CBH II enzyme from the thermophilic fungus Humicola insolens, which suggests that this chimera collection contains hundreds of highly stable cellulases. Twenty-five new sequences were designed based on mathematical modeling of the thermostabilities for the first set of chimeras. Ten of these sequences were expressed in active form; all 10 retained more activity than H. insolens CBH II after incubation at 63 °C. The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 °C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes. Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.