Biotechnol Lett (2007) 29:1045–1050 DOI 10.1007/s10529-007-9365-y
ORIGINAL RESEARCH PAPER
Use of Pseudomonas mendocina, or recombinant Escherichia coli cells expressing toluene-4monooxygenase, and a cell-free tyrosinase for the synthesis of 4-fluorocatechol from fluorobenzene Louise C. Nolan Æ Kevin E. O’Connor
Received: 4 December 2006 / Revised: 1 March 2007 / Accepted: 2 March 2007 / Published online: 11 April 2007 Springer Science+Business Media B.V. 2007
Abstract 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%).
L. C. Nolan K. E. O’Connor (&) School of Biomolecular and Biomedical Science, Centre for Synthesis and Chemical Biology, Conway Institute for Biomolecular and Biomedical Research, National University of Ireland, University College Dublin, Ardmore house, Belfield, Dublin 4, Republic of Ireland e-mail:
[email protected]
Keywords Fluorobenzene 4-Fluorocatechol Pseudomonas mendocina KR1 Toluene-4monooxygenase Tyrosinase
Introduction Substituted catechols (diphenols) are important aromatic compounds and are widely used as synthons in industrial processes such as the manufacture of plastics, polymers, drugs and dyes (Allouche et al. 2004; Held et al. 1998; Pialis and Saville 1998; Tao et al. 2004a, b). Site-specific hydroxylation of the aromatic ring for the production of these compounds can be a challenging process using conventional organic synthesis (Allouche et al. 2004; Robinson et al. 1992; Tao et al. 2004a, b). These reactions often employ expensive starting materials and exhibit poor regiospecificity resulting in low product yield (Held et al. 1998; Robinson et al. 1992; Tao et al. 2004a). This limitation has lead to increased interest in the microbial synthesis of these compounds (Allouche et al. 2004; Fishman et al. 2004; Tao et al. 2004a, b). Pseudomonas mendocina KR1 expresses a toluene-4-monooxygenase (T4MO) that hydroxylates toluene to its 4-substituted phenolic derivative p-cresol (Whited and Gibson 1991a, b; Yen and Blatt 1989). In addition, strain KR1 transforms a number of phenyl compounds, such as
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benzene and nitrobenzene, to their phenolic derivatives (Fishman et al. 2004; Tao et al. 2004b; Yen and Blatt 1989). Furthermore, T4MO wild type and engineered strains hydroxylate phenol and nitrophenol to very low levels of catechol and 4-nitrocatechol, respectively, (2% of total products by wild type enzyme, 8% of total products by engineered enzyme) (Tao et al. 2004b; Fishman et al. 2004). Tyrosinase (EC 1.14.18.1) is a cofactor-independent copper enzyme that catalyses the regiospecific hydroxylation of monophenols to diphenols (monophenolase activity) followed by the subsequent oxidation of diphenols (catechols) to highly reactive and unstable o-quinones (diphenolase activity) (Fig. 1) (Battaini et al. 2002; Claus and Decker 2006; Espı´n et al. 2001). However, in the presence of reducing agents such as ascorbic acid o-quinones are reduced back to the catechol with the coincident formation of inactive dehydroascorbic acid (Fig. 1) (Battaini et al. 2002; Espı´n et al. 2001). Tyrosinase has been reported to produce important catechols such as 3,4-dihydroxyphenylalanine (L-DOPA) and hydroxytyrosol from tyrosine and tyrosol respectively under reducing conditions (Espı´n et al. 2001; Pialis and Saville 1998). The purpose of this study is to investigate: (A) the ability of whole cells expressing T4MO activity, and (B) a combination of cells expressing T4MO activity and cell free tyrosinase preparation to transform the cheap arene, fluorobenzene (FB), to a potentially valuable 4-substituted catechol as the major or sole product (4-fluorocatechol (4Fcat)) (Fig. 1) (Brooks et al. 2004; Espı´n et al. 2001; Lynch et al. 1997). Since cells expressing T4MO convert benzene and nitrobenzene to their equivalent catechols (Fishman et al.
F
F T4MO
F
F
Ascorbic acid
T4MO
OH OH
Fluorobenzene
4-Fluorophenol
OH
4-Fluorocatechol
O O
4-Fluoroquinone
Fig. 1 Biotransformation reaction catalysed by toluene-4monooxygenase and tyrosinase. The conversion of o-quinone back to catechol is catalysed by ascorbic acid with the subsequent formation of dehydroascorbic acid
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2004; Tao et al. 2004a) but with a low efficiency (
