Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase
Abstract Here, we present a proof‐of‐principle for a new high‐throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme‐coding genes were selected, including membe...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-08-01
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| Series: | MicrobiologyOpen |
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| Online Access: | https://doi.org/10.1002/mbo3.795 |
| _version_ | 1818548756703870976 |
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| author | Vida Časaitė Mikas Sadauskas Justas Vaitekūnas Renata Gasparavičiūtė Rita Meškienė Izabelė Skikaitė Mantas Sakalauskas Jevgenija Jakubovska Daiva Tauraitė Rolandas Meškys |
| author_facet | Vida Časaitė Mikas Sadauskas Justas Vaitekūnas Renata Gasparavičiūtė Rita Meškienė Izabelė Skikaitė Mantas Sakalauskas Jevgenija Jakubovska Daiva Tauraitė Rolandas Meškys |
| author_sort | Vida Časaitė |
| collection | DOAJ |
| description | Abstract Here, we present a proof‐of‐principle for a new high‐throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme‐coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro‐chromogenic substrate, which is transformed by the target enzyme to indole‐3‐carboxylic acid. The later compound is converted to indoxyl by a newly identified indole‐3‐carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme‐producing colonies turn blue. Two types of pro‐chromogenic substrates have been tested. Indole‐3‐carboxaldehydes and the amides of indole‐3‐carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine‐tuning of the pro‐chromogenic substrate allows screening enzymes with the desired substrate specificity. |
| first_indexed | 2024-12-12T08:24:26Z |
| format | Article |
| id | doaj.art-ec7f509282114674ba255b5cbbde38f8 |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-12T08:24:26Z |
| publishDate | 2019-08-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-ec7f509282114674ba255b5cbbde38f82022-12-22T00:31:18ZengWileyMicrobiologyOpen2045-88272019-08-0188n/an/a10.1002/mbo3.795Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenaseVida Časaitė0Mikas Sadauskas1Justas Vaitekūnas2Renata Gasparavičiūtė3Rita Meškienė4Izabelė Skikaitė5Mantas Sakalauskas6Jevgenija Jakubovska7Daiva Tauraitė8Rolandas Meškys9Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaDepartment of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center Vilnius University Vilnius LithuaniaAbstract Here, we present a proof‐of‐principle for a new high‐throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme‐coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro‐chromogenic substrate, which is transformed by the target enzyme to indole‐3‐carboxylic acid. The later compound is converted to indoxyl by a newly identified indole‐3‐carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme‐producing colonies turn blue. Two types of pro‐chromogenic substrates have been tested. Indole‐3‐carboxaldehydes and the amides of indole‐3‐carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine‐tuning of the pro‐chromogenic substrate allows screening enzymes with the desired substrate specificity.https://doi.org/10.1002/mbo3.795aldehyde dehydrogenaseamidohydrolasefunctional screeningindole‐3‐carboxylic acid monooxygenasemetagenomics |
| spellingShingle | Vida Časaitė Mikas Sadauskas Justas Vaitekūnas Renata Gasparavičiūtė Rita Meškienė Izabelė Skikaitė Mantas Sakalauskas Jevgenija Jakubovska Daiva Tauraitė Rolandas Meškys Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase MicrobiologyOpen aldehyde dehydrogenase amidohydrolase functional screening indole‐3‐carboxylic acid monooxygenase metagenomics |
| title | Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase |
| title_full | Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase |
| title_fullStr | Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase |
| title_full_unstemmed | Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase |
| title_short | Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase |
| title_sort | engineering of a chromogenic enzyme screening system based on an auxiliary indole 3 carboxylic acid monooxygenase |
| topic | aldehyde dehydrogenase amidohydrolase functional screening indole‐3‐carboxylic acid monooxygenase metagenomics |
| url | https://doi.org/10.1002/mbo3.795 |
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