Abstract
Maltol derivatives are utilized in a variety of fields due to their metal-chelating abilities, and modification of the 2-methyl side chain is known to effectively expand their functional diversity. In the present study, microbial enzymes were screened for hydroxylating activity towards the 2-methyl group in a maltol derivative, 3-benzyloxy-2-methyl-4-pyrone (BMAL). Novosphingobium sp. SB32149 was found to have the ability to convert BMAL into 3-benzyloxy-2-hydroxymethyl-4-pyrone (BMAL-OH). The enzymes responsible, a cytochrome P450 monooxygenase (P450nov), a ferredoxin (FDXnov), and a ferredoxin reductase (FDRnov), were identified in the SB32149 strain. In the reaction with recombinant Escherichia coli expressing P450nov, FDXnov, and FDRnov, BMAL-OH was successfully produced from BMAL. Moreover, using the directed evolution approach, four amino acid substitutions, L188P/F218L/L237M in P450nov and A10T in FDXnov, were found to enhance BMAL-OH production. Consequently, up to 5.2 g/L BMAL-OH was obtained from 8.0 g/L BMAL by bioconversion using a 250-mL jar fermenter, indicating that this strain may be useful for synthesis of maltol derivatives which could have potential applications in various fields.
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Acknowledgements
We would like to thank MicroBiopharm Japan Co., Ltd. for providing an E. coli library expressing 230 types of bacterial P450s, the P450-expressing vectors pCbCa and pAbAc, and E. coli BL21(DE3) lacking the gene encoding the multidrug efflux pump tolC.
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Kozono, I., Mihara, K., Minagawa, K. et al. Engineering of the cytochrome P450 monooxygenase system for benzyl maltol hydroxylation. Appl Microbiol Biotechnol 101, 6651–6658 (2017). https://doi.org/10.1007/s00253-017-8414-2
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DOI: https://doi.org/10.1007/s00253-017-8414-2