Prospecting for novel biocatalysts in a soil metagenome

Abstract

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.

FIG.1.

(A) Agarolytic activities of the microbial community studied on 1.5% agar plates. The plates were incubated at room temperature for 4 to 5 days. (B) Dendrogram of the 16S rRNA genes identified in the agarolytic bacterial community, showing the relationship to the closest known relatives. Evolutionary distances and the phylogenetic relationships were calculated from the aligned sequences and were corrected for multiple base changes at single positions by the method of Jukes and Cantor (18). The phylogenetic tree was calculated by using the software package Phylip. Only high-quality DNA sequences from the 16S rRNA clones were included in the calculations, and the hypervariable regions in the 16S rRNA molecule were excluded. Scale bar = 10 nucleotide substitutions per 100 nucleotides. (C) Immunological detection of the agarase AguB: Western blot of Ni-NTA-purified AguB obtained by using a six-His-specific polyclonal antibody. Lane AguB, protein extract of E. coli AguB after Ni-NTA purification; lane control, E. coli BL21(DE3) with empty pET19B after Ni-NTA purification; lane M, marker (prestained SeeBlue Plus2; Invitrogen, San Diego, Calif.). Approximately 3.0 μg of total protein per lane was loaded onto the corresponding SDS-polyacrylamide gel.

FIG. 2.

Physical maps of the central parts of five of the different cosmids isolated in this work. The arrows indicate the locations and the directions of transcription of the ORFs on the different cosmids (pVIIC10, pXIVA3, pIIIE5, pXB11, pXE1, and pIIIF2). ORFs encoding potential agarases are indicated by red arrows. Identical agarase genes are indicated by the same letters (a to f) in parentheses; the agarases designated e and d differ in their N-terminal domains but are identical at their C termini. Possible genes with potential value in biotechnology are shown in color. Possible functions of the indicated ORFs are shown in Table 2, together with the GenBank accession numbers. A conserved agarolytic gene cluster is enclosed in a box. ORFs uvs072, uvs093, and uvs085 in this conserved cluster code for the same 785-amino-acid protein and are indicated by gray arrows; contig ends are indicated by two slashes, and the vertical arrows indicate the positions of the two 31-bp inverted repeats. Additional data, including the results of similarity searches and the lengths of the predicted ORFs, are available on the web at http://www.gwdg.de/∼biofilm/genemining.htm.