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Applied and Environmental Microbiology, October 2003, p. 6235-6242, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6235-6242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Prospecting for Novel Biocatalysts in a Soil Metagenome

S. Voget,1 C. Leggewie,2 A. Uesbeck,1 C. Raasch,3 K.-E. Jaeger,2 and W. R. Streit1*

Institut für Mikrobiologie und Genetik, Universität Göttingen,1 Laboratorium für Genomanalyse der Universität Göttingen, D-37077 Göttingen,3 Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, D-52425 Jülich, Germany2

Received 20 March 2003/ Accepted 18 July 2003

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an {alpha}-amylase (amyA), a 1,4-{alpha}-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

* Corresponding author. Mailing address: Institut für Mikrobiologie und Genetik, Universität Göttingen, Grisebachstr. 8, 37077 Göttingen, Germany. Phone: (49) 551-393775. Fax: (49) 551-393793. E-mail: wstreit{at}gwdg.de.

Applied and Environmental Microbiology, October 2003, p. 6235-6242, Vol. 69, No. 10
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.10.6235-6242.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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