Metagenomics: an inexhaustible access to nature's diversity.

The chemical industry has an enormous need for innovation. To save resources, energy and time, currently more and more established chemical processes are being switched to biotechnological routes. This requires white biotechnology to discover and develop novel enzymes, biocatalysts and applications.

Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis.

A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-aminopenicillanic acid (6-APA) and activated D-phenylglycine derivatives. We directed changes in amino acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: alpha R160, alpha F161 and beta F24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method.

Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases.

To obtain new amidases of biocatalytic relevance, we used microorganisms indigenous to different types of soil and sediment as a source of DNA for the construction of environmental gene banks, following two different strategies. In one case, DNA was isolated from soil without preceding cultivation to preserve a high degree of (phylo)genetic diversity. Alternatively, DNA samples were obtained from enrichment cultures, which is thought to reduce the number of clones required to find a target enzyme.

Quantifying the accessibility of the metagenome by random expression cloning techniques.

The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion.

Efficient recovery of environmental DNA for expression cloning by indirect extraction methods.

Using direct and cell extraction-based (indirect) isolation methods, DNA was obtained from environmental samples with largely differing characteristics (loam soil, sand soil, sediment, activated sludge, and compost) and evaluated with respect to the comprised bacterial diversity and its suitability for expression cloning in Escherichia coli. Indirect DNA extraction methods yielded 10 to 100-fold lower amounts of DNA than direct procedures, but the bacterial diversity of DNA recovered by indirect means was distinctly higher as shown by denaturing gradient gel electrophoresis. Furthermore, much lower amounts of eukaryotic DNA were co-extracted if cell extraction-based methods were used (<8% of eukaryotic DNA by indirect methods versus 61-93% by direct lysis protocols).