Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

• Published in: Applied microbiology and biotechnology Vol. 89; no. 6; pp. 1867 - 1875
• Main Authors: Coulombel, Lydie, Nolan, Louise C., Nikodinovic, Jasmina, Doyle, Evelyn M., O’Connor, Kevin E.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01-03-2011; Springer; Springer Nature B.V
• Subjects: Applied Genetics and Molecular Biotechnology; Biocatalysts; Bioconversions. Hemisynthesis; Biological and medical sciences; Biomedical and Life Sciences; Bioreactors; Biosynthesis; Biotechnology; Biotransformation; Catechols - metabolism; E coli; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - metabolism; Fermentation; Fundamental and applied biological sciences. Psychology; Gene amplification; Gene expression; Glucose; Life Sciences; Methods. Procedures. Technologies; Microbial Genetics and Genomics; Microbiology; Microorganisms; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; NMR; Nuclear magnetic resonance; Phenols; Phenols - metabolism; Phenylacetates - metabolism; Plasmids; Solvent extraction; Studies; Temperature; Time Factors; Trace elements; 4-Halogenated catechols; 4-Hydroxyphenylacetate 3-hydroxylase; Biotransformation; Scale-up process; Enzyme; Escherichia coli; Hydroxylase; Pyrocatechol; Dimensioning; Phenols; Bacteria; Oxidoreductases; Operating process; Enterobacteriaceae
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-2969-5

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%.