Engineering of a chromogenic enzyme screening system based on an auxiliary indole‐3‐carboxylic acid monooxygenase

• Published in: MicrobiologyOpen (Weinheim) Vol. 8; no. 8; pp. e00795 - n/a
• Main Authors: Časaitė, Vida, Sadauskas, Mikas, Vaitekūnas, Justas, Gasparavičiūtė, Renata, Meškienė, Rita, Skikaitė, Izabelė, Sakalauskas, Mantas, Jakubovska, Jevgenija, Tauraitė, Daiva, Meškys, Rolandas
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.08.2019; John Wiley and Sons Inc; Wiley
• Subjects: Aldehyde dehydrogenase; Aldehyde Dehydrogenase - genetics; Aldehyde Dehydrogenase - isolation & purification; Aldehydes; Amides; amidohydrolase; Amidohydrolases - genetics; Amidohydrolases - isolation & purification; Bacteria; Biocatalysts; Biotechnology; Carboxylic acids; Chromogenic Compounds - metabolism; Cloning; Cloning, Molecular; Dehydrogenase; Dehydrogenases; Deoxyribonucleic acid; DNA; E coli; Enzymes; Escherichia coli - genetics; functional screening; Gene Expression; Genes; Genetic engineering; Genetic Testing - methods; Genetics, Microbial - methods; Indigo; Indigo Carmine - metabolism; Indoles; Indoles - metabolism; indole‐3‐carboxylic acid monooxygenase; metagenomics; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - isolation & purification; Monooxygenase; Original; Oxidation; Oxidation-Reduction; Plasmids; Rhodococcus; Rhodococcus - genetics; Screening; Substrate specificity; Substrates; Vectors (Biology); Xanthine dehydrogenase; Lithuania; United States > US; Switzerland; amidohydrolase; indole-3-carboxylic acid monooxygenase; aldehyde dehydrogenase; metagenomics; functional screening
• ISSN: 2045-8827; 2045-8827
• DOI: 10.1002/mbo3.795

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. We present a high‐throughput approach for screening of aldehyde dehydrogenases or other enzymes, based on a pro‐chromogenic substrate, which is transformed into an insoluble indigo dye. Indole‐3‐carboxylic acid monooxygenase as auxiliary enzyme and indole‐3‐carboxaldehyde as a primary substrate were used for screening. The clones expressing aldehyde dehydrogenases produce indole‐3‐carboxylic acid, which is converted to indigo and is monitored as blue colonies on agar plates.