An interesting example of the application of recombinant whole-cell biocatalysis is the conversion of 2-hydroxybiphenyl (2-phenylphenol) to 2,3-dihydroxybiphenyl
2-Hydroxybiphenyl 3-monooxygenase (E.c. 188.8.131.52)
Figure 15.7 Production of 3-substituted catechols using a designer biocatalyst.
or 3-phenylcatechol (Figure 15.7). Catechols are important building blocks for the chemical and pharmaceutical industries. However, their chemical synthesis is cumbersome. Especially the synthesis of 3-substituted catechols requires numerous agents with low environmental compatibility (organometallic reagents, HBr) and energy intensive reaction conditions (low temperature). At the same time, catechol and some of its derivatives are central metabolites in the microbial catabolism of aromatic compounds.36 Besides naturally occurring aromatics such as tyrosine and phenylalanine, nonbiological aromatic solvents and numerous polycyclic aromatic hydrocarbons are also readily degraded or modified by bacteria, with the genus Pseudomonas playing an important role in such turnover of aromatics in nature.
Pseudomonas azelaica HBP1 is a prominent example of interesting aromatic compound degradation.37 The strain readily degrades 2-phenylphenol—a man-made compound that has been widely used as a food-protecting agent and as a germicide. The initial step of 2-phenylphenol transformation by the strain is formation of 3-phenylcatechol by a 2-hydroxybiphenyl 3-monooxygenase (Figure 15.7).38 This enzyme has a broad substrate range and oxidizes numerous other 2-substituted phenols to corresponding 3-substituted catechols in a highly regioselective cofactor-dependent reaction. However, the strain cannot be used for catechol synthesis, because reaction products are instantly broken down. Hence, the 2-hydroxybiphenyl 3-monooxygenase gene was cloned and expressed in E. coli JM101.39 The resulting biocatalyst E. coli JM101 [pHBP461] efficiently overproduces the monooxygenase but does not degrade the products formed, which makes this strain a promising candidate for synthesis of 3-substituted catechols.
A major challenge that had to be met arose from the extreme bactericidal properties of phenols and catechols. Most microorganisms are poisoned at phenol or catechol concentrations in the 0.1-1 g/L range. The biocatalyst E. coli JM101 [pHBP461] is no exception, and was inactivated by 200 mg/L of both 2-phenylphenol and 3-phenylcatechol.40 Furthermore, 3-substituted catechols are of limited stability in aerated aqueous solutions and form multimeric humic-acid-like structures as unwanted side products. 3-Phenylcatechol, for instance, has a half-life time of only 14 h at pH 7.2.
Last but not least, catechols are highly water-soluble (the water solubility of catechol is approximately 1 g per 2.3 mL of water), which makes it difficult to directly extract them in situ from reaction media with organic, water immiscible solvents. Nevertheless, extraction of catechols from aqueous systems with hydro-phobic polymers such as the polystyrene-based resin Amberlite XAD-4 is
straightforward. We have therefore employed XAD-4 to combine biocatalytic synthesis with simultaneous product extraction. The system (Figure 15.8) comprises a continuously stirred tank reactor, a starting material feed pump, a product recovery loop with a (semi-) fluidized bed of XAD-4, and a pump to circulate the entire reaction mixture through the loop.40 Preliminary studies indicated that XAD-4 had no detrimental effects on E. coli JM101 (pHBP461), hence, separation of biomass and reaction liquid prior to catechol extraction was not required. The biocatalytic reaction was carried out at very low concentrations of the toxic substrate and product. This was achieved by feeding the substrate at a rate lower than the potential bioconversion rate in the reactor.
This assured that all substrate fed to the bioreactor was instantly converted to product: no substrate accumulated in the bioreactor. To prevent accumulation of product in the reactor, the reactor contents were circulated through the external fluidized-bed module, which contained Amberlite XAD-4 resin. All product adsorbed to the resin, while cells and medium components passed through the bed and back into the bioreactor, ready to convert more of the substrate newly fed into the bioreactor.
Figure 15.9 shows that this approach worked quite nicely: the substrate was added to a total concentration of 2.5 g/L, but neither substrate nor product accumulated in the bioreactor medium.30 Without a product recovery loop the product concentration (3-phenylcatechol) did not exceed 0.4 g/L, because of biocatalyst deactivation (results not shown). With the loop, 2-phenylphenol and 3-phenylcatechol concentrations remained below 0.1 g/L. Therefore, cell viability and biocatalytic activity were maintained, as indicated by the constantly low dissolved oxygen tension in the aerated reactor. As a result product yields (based on the 3-phenylcatechol eluted from the product sink) increased by one order of magnitude.40
The system was used for the preparative scale synthesis of numerous catechols. Table 15.2 shows that a half dozen other phenols could be converted to the corresponding catechols. In each case, 1 to 2 g of substrate was converted with yields
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