Multistep conversion of cresols by phenol hydroxylase and 2,3-dihydroxy-biphenyl 1,2-dioxygenase

Abstract

A multistep conversion system composed of phenol hydroxylase (PH_IND) and 2,3-dihydroxy-biphenyl 1,2-dioxygenase (BphC_LA-4) was used to synthesize methylcatechols and semialdehydes from o- and m-cresol for the first time. Docking studies displayed by PyMOL predicted that cresols and methylcatechols could be theoretically transformed by this multistep conversion system. High performance liquid chromatography mass spectrometry (HPLC-MS) analysis also indicated that the products formed from multistep conversion were the corresponding 3-methylcatechol, 4-methylcatechol, 2-hydroxy-3-methyl-6-oxohexa-2,4-dienoic acid (2-hydroxy-3-methyl-ODA) and 2-hydroxy-5-methyl-6-oxohexa-2,4-dienoic acid (2-hydroxy-5-methyl-ODA). The optimal cell concentrations of the recombinant E. coli strain BL21 (DE3) expressing phenol hydroxylase (PH_IND) and 2,3-dihydroxy-biphenyl 1,2-dioxygenase (BphC_LA-4) and pH for the multistep conversion of o- and m-cresol were 4.0 (g·L^-1 cell dry weight) and pH 8.0, respectively. For the first step conversion, the formation rate of 3-methylcatechol (0.29 μmol·L^-1·min^-1·mg^-1 cell dry weight) from o-cresol was similarly with that of methylcatechols (0.28 μmol·L^-1·min^-1·mg^-1 cell dry weight) from m-cresol by strain PH_IND. For the second step conversion, strain BphC_LA-4 showed higher formation rate (0.83 μmol·L^-1·min^-1·mg^-1 cell dry weight) for 2-hydroxy-3-methyl-ODA and 2-hydroxy-5-methyl-ODA from m-cresol, which was 1.1-fold higher than that for 2-hydroxy-3-methyl-ODA (0.77 μmol·L^-1·min^-1·mg^-1cell dry weight) from o-cresol. The present study suggested the potential application of the multistep conversion system for the production of chemical synthons and high-value products.

Keywords

multistep conversion / cresols / phenol hydroxylase / 2 / 3-dihydroxybiphenyl 1 / 2-dioxygenase / methylcatechols

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Acknowledgements

The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China (Grant Nos. 51078054, 51108120, and 51178139) and the National Creative Research Group from the National Natural Science Foundation of China (No. 51121062).

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