Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Abstract

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

Keywords: Thermus thermophiles; comparative screening; functional metagenomics; novel metagenomic esterases; novel screening host.

Figure 1

(A) Growth complementation screening results of T. thermophilus BL03 transformed with metagenomic fosmid DNA (upper scan picture) and corresponding tributyrase halo formation capabilities on substrate plates (lower scan picture). (B) Tributyrase activity of 8 single fosmids in E. coli EPI300 (upper part of the table) and T. thermophilus BL03 (below). In this representation, the increase of halo formation (black bars, halo area was calculated as described in the Materials and Methods section) on 1% (v/v) tributyrin substrate plates after 3 days of incubation at 60°C is shown (from triplicate measurements, n = 3; error bars indicate the standard deviation). In T. thermophilus, heterologous growth complementation results after 3 days of growth at 60°C on minimal medium are depicted. Comparative pNP-activity data from crude extracts (specific activity mU × mg⁻¹) is shown for each single fosmid clone as average values from duplicate measurements (± standard deviation).

Figure 2

SDS-PAGE analysis of the purification steps of his-tagged EstA2 (A) and EstB1 (B). Lanes M, protein ladder; Lanes 1, empty vector control; lanes 2, crude lysate; lanes 3, flow-through; Lanes 4–5, eluate fractions of target proteins. Predicted sizes of the proteins with C-terminal his-tag fusions are 18.7 kDa for EstA2 and 64.6 kDa for EstB1, respectively.

Figure 3

Characterization of the purified esterase-active proteins EstA2 and EstB1. (A) Substrate specificity of various acyl chain length pNP-esters. (B) Temperature and (C) pH optimum. The assays were performed in 50 mM phosphate buffer (pH 6–7) or 50 mM Tris-HCl buffer (pH 7.2–8.8) under optimal activity parameters (80°C for EstA2 and 75°C for EstB1, respectively) against 1.25 mM pNP-butyrate. 0.065–1.3 μg of purified enzyme was used for the assays. Data represents average values and standard deviations (error bars).

Figure 4

Topologic view of a similarity tree of all lipolytic esterase and lipase protein families I to VIII (according to Arpigny and Jaeger, 1999), including the recently defined families LipS and LipT as well as classified, but still unknown protein families (Chow et al., 2012) and the new esterases from our functional metagenomic screenings (AZ2-4-B6, M12-4-D9, EstA2, EstB1). The data was generated from multiple sequence alignments of selected representative protein sequences (according to Chow et al., 2012) using the ClustalW algorithm (Boc et al., 2012). Tree reconstruction and visualization was performed with MEGA Version 5.2 (Tamura et al., 2011). The metagenomic proteins identified in this study are marked with black arrows. The length of the tree branches does not represent the phylogenetic distance between the protein sequences.