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

Leis B, Angelov A, Mientus M, Li H, Pham VT, Lauinger B, Bongen P, Pietruszka J, Gonçalves LG, Santos H, Liebl W - Front Microbiol (2015)

Bottom Line: We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Technische Universität München Freising, Germany.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: In order to classify the gene products identified in our metagenomic screenings, we performed multiple sequence alignments with known esterases and lipases from families I to VIII (Arpigny and Jaeger, 1999). Recently, the number of known lipolytic enzyme families has been extended by two new families, termed LipS and LipT (Chow et al., 2012). From the positive fosmids identified in E. coli (AZ2-4-B6 and M12-4-D9), both encoding α/β-hydrolase ORFs were identified as esterase class V and LipS family protein, respectively. The lipolytic enzymes discovered by using T. thermophilus for functional library screening, EstA2 and EstB1, could not be assigned to any known esterase/lipase family (Figure 4).


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

Leis B, Angelov A, Mientus M, Li H, Pham VT, Lauinger B, Bongen P, Pietruszka J, Gonçalves LG, Santos H, Liebl W - Front Microbiol (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4389547&req=5

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.
Mentions: In order to classify the gene products identified in our metagenomic screenings, we performed multiple sequence alignments with known esterases and lipases from families I to VIII (Arpigny and Jaeger, 1999). Recently, the number of known lipolytic enzyme families has been extended by two new families, termed LipS and LipT (Chow et al., 2012). From the positive fosmids identified in E. coli (AZ2-4-B6 and M12-4-D9), both encoding α/β-hydrolase ORFs were identified as esterase class V and LipS family protein, respectively. The lipolytic enzymes discovered by using T. thermophilus for functional library screening, EstA2 and EstB1, could not be assigned to any known esterase/lipase family (Figure 4).

Bottom Line: We scored a greater number of active esterase clones in the thermophilic bacterium than in the mesophilic 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Technische Universität München Freising, Germany.

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.

No MeSH data available.


Related in: MedlinePlus