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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

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.
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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.

Mentions: From all candidate esterase-encoding ORFs, we selected EstA2 and EstB1 for expression, purification and characterization using T. thermophilus as an expression host. Expression of these ORFs in pMK18 (de Grado et al., 1999) led to significant pNP-butyrate-cleaving activity (measured at 60°C). Further investigation revealed that short-chain acyl esters were the preferred substrates, such as pNP-butyrate, that was hydrolyzed most optimally around 80–85°C and pH 8.0. EstA2 (encoded on fosmid AZ3-33-C12) resulted in a significant activity increase in the extracellular fraction, resulting in a 1.54-fold induction of activity, while EstB1 showed a 1.53-fold increased activity level compared to 338.4 ± 4.8 mU/mg protein in BL03 with the empty pMK18 vector. However, no recombinant protein band could be detected by SDS-PAGE analysis. We then attempted to produce C-terminally tagged versions of both proteins in E. coli by using the pET21a(+) expression vector. For both proteins, purification using heat treatment followed by IMAC gravity flow columns resulted in functional protein preparations (Figure 2).


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)

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

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

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.
Mentions: From all candidate esterase-encoding ORFs, we selected EstA2 and EstB1 for expression, purification and characterization using T. thermophilus as an expression host. Expression of these ORFs in pMK18 (de Grado et al., 1999) led to significant pNP-butyrate-cleaving activity (measured at 60°C). Further investigation revealed that short-chain acyl esters were the preferred substrates, such as pNP-butyrate, that was hydrolyzed most optimally around 80–85°C and pH 8.0. EstA2 (encoded on fosmid AZ3-33-C12) resulted in a significant activity increase in the extracellular fraction, resulting in a 1.54-fold induction of activity, while EstB1 showed a 1.53-fold increased activity level compared to 338.4 ± 4.8 mU/mg protein in BL03 with the empty pMK18 vector. However, no recombinant protein band could be detected by SDS-PAGE analysis. We then attempted to produce C-terminally tagged versions of both proteins in E. coli by using the pET21a(+) expression vector. For both proteins, purification using heat treatment followed by IMAC gravity flow columns resulted in functional protein preparations (Figure 2).

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