title: Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning.
abstract: Metagenome cloning has become a powerful tool to exploit the biocatalytic potential of microbial communities for the discovery of novel biocatalysts. In a novel variant of direct expression cloning, metagenomic DNA was isolated from compost by a modified direct lysis method, purified by size exclusion chromatography and cloned into an expression vector allowing bidirectional transcription. Transformation of Escherichia coli DH5alpha resulted in a metagenomic expression library with an average insert size of 3.2 kb. To estimate the functional diversity of the constructed library, it was screened by different approaches based on functional heterologous expression. A large number of active clones were identified, including lipolytic enzymes, amylases, phosphatases and dioxygenases. Molecular analysis of one important class of industrial biocatalysts, the lipolytic enzymes, confirmed the novelty and dissimilarity of all recovered genes, which exhibited only limited similarity to known enzymes. Equally, the novelty of another three genes encoding phosphatase or dioxygenase activity, respectively, was shown. These results demonstrate the suitability of this direct cloning approach, which comprised a dual-orientation expression vector and a simple one-step DNA purification method, for the efficient discovery of numerous active novel clones. By this means it provides an efficient way for the rapid generation of large libraries of hitherto unknown enzyme candidates which could be screened for different specific target reactions.
authors.affiliation: Fraunhofer Institute for Interfacial Engineering and Biotechnology, Institute for Interfacial Engineering, University of Stuttgart, Nobelstrasse 12, D-70569 Stuttgart, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Lämmle
authors.forename: Hubert
authors.initials: H
authors.lastname: Zipper
authors.forename: Michael
authors.initials: M
authors.lastname: Breuer
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
authors.forename: Christiane
authors.initials: C
authors.lastname: Buta
authors.forename: Herwig
authors.initials: H
authors.lastname: Brunner
authors.forename: Steffen
authors.initials: S
authors.lastname: Rupp
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D004798:Enzymes / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005786:Gene Expression Regulation; D016680:Genome, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012988:Soil Microbiology
chemical_list: D004798:Enzymes