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KMID : 0613820180280060656
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Journal of Life Science
2018 Volume.28 No. 6 p.656 ~ p.662
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Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin
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Jung Hye-Sook
Jung Hae-Bin
Kim Hee-Sook
Kim Chang-Gyeom
Lee Jin-Ho
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Abstract
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Flavin-containing monooxygenases from Corynebacterium (cFMOs) were mutagenized based on homology modeling to develop variants with an enhanced indigoid production capability. The four mutants, F170Y, A210G, A210S, and T326S, which fused to a maltose-binding protein (MBP), were constructed, and their biochemical properties were characterized. Of these, purified MBP-T326S required a higher concentration of exogenous FAD (100 mM) than the wild-type MBP-cFMO for optimal activity and showed a 3.8-fold increase in the kcat/Km value at 100 μM FAD compared to that of MBP-cFMO at 2 μM FAD. The indole oxygenase activities of MBP-T326S decreased to 63-77% compared to that of the MBP-cFMO In addition, MBP-T326S displayed a very low level of futile NADPH oxidase activities (21-24%) in the absence of a substrate. Mutant proteins except for T326S displayed similar Km and increased kcat/Km values compared to the wild-type. MBP-F170Y and -A210S mutants showed elevated indole oxygenase activity higher than 3.1- and 2.9-fold, respectively, in comparison with MBP-cFMO. When indigoid production was carried out in LB broth with 2.5 g/l of tryptophan, Escherichia coli expressing cFMO produced 684 mg/l of indigo and 104 mg/l of indirubin, while cells harboring T326S produced 1,040 mg/l of indigo and 112 mg/l of indirubin. The results indicate that the production of indigo was 13% higher when compared to a previous report in which an E. coli expressing FMO from Methylophaga produced 920 mg/l of indigo. The protein engineering of cFMO based on homology modeling provided a more rational strategy for developing indigoid-producing strains.
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KEYWORD
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Corynebacterium glutamicum, flavin-containing monooxygenase, indigoid, protein engineering, T326S mutant
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