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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

(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).
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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).

Mentions: After screening of the generated libraries in E. coli using a plate assay at 60°C, only two halo-forming colonies were identified on tributyrin agar plates, one from the Azores sample (termed AZ2-4-B6) and one from the compost sample (M12-4-D9). In order to perform the screening in T. thermophilus, the BL03 strain was transformed with 83 fosmid pools, each prepared from 96 fosmid clones. The transformant pools obtained in this way were examined for their ability to grow on minimal medium plates supplemented with tributyrin. A total of 13 pools showed substantial growth on these plates (namely fosmid pools AZ2-4; AZ3-14; AZ3-25; AZ3-30; AZ3-32; AZ3-33; AZ3-37; AZ3-38; AZ3-47; AZ4-2; M12-3; M12-4; and M12-6). From these candidate pools, single fosmid transformations were performed in 96 well plate format. Again, growth on minimal medium was monitored, and positive clones were selected. After re-streaking them two more times to confirm the stability of their phenotype, six fosmid-containing T. thermophilus clones could be isolated from five different fosmid pools. They stably grew on minimal medium agar supplemented with tributyrin, and hydrolysis halos on tributyrin substrate plates were monitored in comparison with the BL03 strain carrying an empty fosmid (Figure 1A). In addition, the esterase activity of crude cell extracts obtained from the respective strains was measured with various pNP-substrates (Figure 1B). Almost all fosmid clones showed preferences for medium to short acyl chain fatty acid pNP-esters. Crude extract from the strain AZ3-14-D2 exhibited the highest activities of all crude extracts tested. This extract was most active over a broad range of pNP-substrates tested and its long-chain palmitate fatty acid hydrolysis was approximately three-fold higher than in the BL03 empty fosmid control. The two fosmids that conferred tributyrase activity in E. coli, AZ2-4-B6 and M12-4-D9, were also found to confer activity in T. thermophilus when tested in pNP-assays. Fosmid M12-4-D9 could not be identified by heterologous growth complementation screenings in T. thermophilus BL03, but showed higher activity on pNP-C8 compared to E. coli. In general, the pNP-substrate preference of the fosmid-encoded esterases for short-chain acyl esters was in agreement with the observed capabilities of halo formation on tributyrin agar plates.


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)

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

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

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).
Mentions: After screening of the generated libraries in E. coli using a plate assay at 60°C, only two halo-forming colonies were identified on tributyrin agar plates, one from the Azores sample (termed AZ2-4-B6) and one from the compost sample (M12-4-D9). In order to perform the screening in T. thermophilus, the BL03 strain was transformed with 83 fosmid pools, each prepared from 96 fosmid clones. The transformant pools obtained in this way were examined for their ability to grow on minimal medium plates supplemented with tributyrin. A total of 13 pools showed substantial growth on these plates (namely fosmid pools AZ2-4; AZ3-14; AZ3-25; AZ3-30; AZ3-32; AZ3-33; AZ3-37; AZ3-38; AZ3-47; AZ4-2; M12-3; M12-4; and M12-6). From these candidate pools, single fosmid transformations were performed in 96 well plate format. Again, growth on minimal medium was monitored, and positive clones were selected. After re-streaking them two more times to confirm the stability of their phenotype, six fosmid-containing T. thermophilus clones could be isolated from five different fosmid pools. They stably grew on minimal medium agar supplemented with tributyrin, and hydrolysis halos on tributyrin substrate plates were monitored in comparison with the BL03 strain carrying an empty fosmid (Figure 1A). In addition, the esterase activity of crude cell extracts obtained from the respective strains was measured with various pNP-substrates (Figure 1B). Almost all fosmid clones showed preferences for medium to short acyl chain fatty acid pNP-esters. Crude extract from the strain AZ3-14-D2 exhibited the highest activities of all crude extracts tested. This extract was most active over a broad range of pNP-substrates tested and its long-chain palmitate fatty acid hydrolysis was approximately three-fold higher than in the BL03 empty fosmid control. The two fosmids that conferred tributyrase activity in E. coli, AZ2-4-B6 and M12-4-D9, were also found to confer activity in T. thermophilus when tested in pNP-assays. Fosmid M12-4-D9 could not be identified by heterologous growth complementation screenings in T. thermophilus BL03, but showed higher activity on pNP-C8 compared to E. coli. In general, the pNP-substrate preference of the fosmid-encoded esterases for short-chain acyl esters was in agreement with the observed capabilities of halo formation on tributyrin agar plates.

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