Function-Based Metagenomic Library Screening and Heterologous Expression Strategy for Genes Encoding Phosphatase Activity

Abstract

The release of phosphate from inorganic and organic phosphorus compounds can be mediated enzymatically. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation and diagnostic analysis. Metagenomic approaches provide access to novel phosphatase-encoding genes. Here, we describe a function-based screening approach for rapid identification of genes conferring phosphatase activity from small-insert and large-insert metagenomic libraries derived from various environments. This approach bears the potential for discovery of entirely novel phosphatase families or subfamilies and members of known enzyme classes hydrolyzing phosphomonoester bonds such as phytases. In addition, we provide a strategy for efficient heterologous phosphatase gene expression.

Functional Metagenomics Reveals an Overlooked Diversity and Novel Features of Soil-Derived Bacterial Phosphatases and Phytases

“…For function-based screening of metagenomic libraries, we used our recently described method (18). Small‐insert libraries were constructed using the plasmid pCR-XL-TOPO as vector (Invitrogen GmbH, Karlsruhe, Germany) and Escherichia coli DH5α [F – φ80 lacZ Δ M15 Δ( lacZYA - argF ) U169 recA1 endA1 hsdR17 (r K – m K + ) phoA supE44 λ – thi - 1 gyrA96 relA1 ] as screening host.…”

“…The genes pho07 and pho18 carried by plasmids pLP07 and pLP18, respectively, were selected for heterologous expression with the pET-20b (+) (V5-epitope/His tag) vector (Merck KGaA, Darmstadt, Germany) as recommended by Villamizar et al for metagenome-derived phosphatases (18). Crude extracts containing the target proteins were derived from the expression strain E.…”

“…Crude extracts containing the target proteins were derived from the expression strain E. coli BL21 and filtered as described by Villamizar et al (18). For purification of the proteins, the filtered crude extracts were then transferred to nickel columns (Protino2000 Ni-Ted columns; Macherey and Nagel, Düren, Germany).…”

“…In this study, we use a function-based screening approach (18) to identify environmental phosphatases/phytases. By using soil metagenomes as a source, we were able to recover novel genes encoding phosphatases with phytase activity.…”

Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-β-Lactamase Superfamily Domain, Associated with Phytase Activity

“…In order to induce phytase activity, phytate (2.5 g/liter) was used as the phosphorus source and 25 μg/ml of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as the indicator. Clones with phosphatase/phytase activity turned from white to dark blue within 48 h (48, 49).…”