Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase.
Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1-2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5-20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.
School of Biomolecular and Biomedical Sciences, University College Dublin, Belfield, Dublin, Ireland.
Lydie
L
Coulombel
Louise C
LC
Nolan
Jasmina
J
Nikodinovic
Evelyn M
EM
Doyle
Kevin E
KE
O'Connor
D019149:Bioreactors; D001711:Biotransformation; D002396:Catechols / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D010648:Phenylacetates / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D002396:Catechols; D010636:Phenols; D010648:Phenylacetates; C026246:4-hydroxyphenylacetate; C072305:4-fluorocatechol; C106880:4-fluorophenol; D006899:Mixed Function Oxygenases