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

A multistep conversion system composed of phenol hydroxylase (PHIND) 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 (PHIND) 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 center dot L-1 cell dry weight) and pH 8.0, respectively. For the first step conversion, the formation rate of 3-methylcatechol (0.29 mu mol center dot L-1 center dot min(-1)center dot mg(-1) cell dry weight) from o-cresol was similarly with that of methylcatechols (0.28 mu mol center dot L-1 center dot min(-1)center dot mg(-1) cell dry weight) from m-cresol by strain PHIND. For the second step conversion, strain BphC(LA-4) showed higher formation rate (0.83 mu mol center dot L-1 center dot min(-1)center dot 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 mu mol center dot L-1 center dot min(-1)center dot mg(-1)cell 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.