Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.

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%.

The transformation of fluorobenzene (FB) by whole cell expressing toluene-4-monooxygenase (T4MO) resulted in the formation of various hydroxylated products. The predominant product was either 4-fluorophenol (4FP) or 4-fluorocatechol (4Fcat) depending on the ratio of biocatalyst to substrate concentration. The transformation of 1 mM FB by whole cells (1.5 mg CDW/ml) gave a 52% yield of 4Fcat as a single product. The yield of 4Fcat was improved 1.6-fold (80%) by adding 10 mM ascorbic acid to the biotransformations. A combination of two biocatalysts (whole cells expressing T4MO and cell free mushroom tyrosinase) also resulted in the transformation of FB (5 mM) to higher concentrations of 4Fcat (1.8 mM) compared to a whole cell biotransformation alone. However, mixed products were formed and the yield of 4Fcat from FB was lower using the two-step (tandem) method (27%) compared to the use of whole cells of P. mendocina KR1 alone (80%).

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