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Identification of novel biomass-degrading enzymes from genomic dark matter: Poplulating genomic sequence space with functional annotation

Lookup NU author(s): Professor Hans-Peter Klenk


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Although recent nucleotide sequencing technologies have significantly enhanced our understanding of microbial genomes, the function of ∼35% of genes identified in a genome currently remains unknown. To improve the understanding of microbial genomes and consequently of microbial processes it will be crucial to assign a function to this “genomic dark matter.” Due to the urgent need for additional carbohydrate-active enzymes for improved production of transportation fuels from lignocellulosic biomass, we screened the genomes of more than 5,500 microorganisms for hypothetical proteins that are located in the proximity of already known cellulases. We identified, synthesized and expressed a total of 17 putative cellulase genes with insufficient sequence similarity to currently known cellulases to be identified as such using traditional sequence annotation techniques that rely on significant sequence similarity. The recombinant proteins of the newly identified putative cellulases were subjected to enzymatic activity assays to verify their hydrolytic activity towards cellulose and lignocellulosic biomass. Eleven (65%) of the tested enzymes had significant activity towards at least one of the substrates. This high success rate highlights that a gene context-based approach can be used to assign function to genes that are otherwise categorized as “genomic dark matter” and to identify biomass-degrading enzymes that have little sequence similarity to already known cellulases. The ability to assign function to genes that have no related sequence representatives with functional annotation will be important to enhance our understanding of microbial processes and to identify microbial proteins for a wide range of applications. Biotechnol. Bioeng. 2014;111: 1550–1565.

Publication metadata

Author(s): Piao H, Froula J, Du C, Kim TW, Hawley ER, Bauer S, Wang Z, Ivanova N, Clark DS, Klenk HP, Hess M

Publication type: Article

Publication status: Published

Journal: Biotechnology and Bioengineering

Year: 2014

Volume: 111

Issue: 8

Pages: 1550-1565

Print publication date: 01/08/2014

Online publication date: 28/05/2014

Acceptance date: 24/03/2014

ISSN (print): 0006-3592

ISSN (electronic): 1097-0290

Publisher: John Wiley & Sons, Inc.


DOI: 10.1002/bit.25250


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