Recombinant Chlorobenzene Dioxygenase from Pseudomonas sp. P51: A Biocatalyst for Regioselective Oxidation of Aromatic Nitriles Selcuk Yildirim, a Telma T. Franko, b Roland Wohlgemuth, c Hans-Peter E. Kohler, d Bernard Witholt, a Andreas Schmid e, * a Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, CH-8093 Zurich, Switzerland b School of Chemical Engineering, UNICAMP, P.O. Box 6066, 13081-970, Campinas, Brazil c Fluka Chemie AG, Biochemistry Department, Industriestr. 25, 4700 Buchs, Switzerland d EAWAG, Swiss Federal Institute of Environmental Science and Technology, 8600 Dübendorf, Switzerland e Department of Biochemical & Chemical Engineering, University of Dortmund, Emil-Figge-Strasse 66, 44227 Dortmund, Germany Fax: ( þ 49)-231-755-7382, e-mail: andreas.schmid@bci.uni-dortmund.de Received: February 1, 2005; Accepted: March 29, 2005 Abstract: An efficient biocatalyst was developed for the cis-dihydroxylation of aromatic nitriles. The chlor- obenzene dioxygenase (CDO) genes of Pseudomonas sp. strain P51 were cloned under the strict control of the Palk promoter of Pseudomonas putida GPo1. Es- cherichia coli JM101 cells carrying the resulting plas- mid pTEZ30 were used for the biotransformation of benzonitrile in a 2-L stirred tank bioreactor. Use of a stable expression system resulted in an average spe- cific activity and an average volumetric productivity of 1.47 U/g cdw and 120 mg of product/h/L, respec- tively. The values represent a three-fold increase com- pared to the results of the similar biotransformations with E. coli JM101 (pTCB144) where the genes of CDO were expressed under the control of lac promot- er. The productivity of the cis-dihydroxylation process was limited by product toxicity. Removal of the prod- ucts at toxic concentrations by means of an external charcoal column resulted in an additional increase in product concentration by 43%. E. coli JM101 (pTEZ30) was further used for the regio- and stereo- specific dihydroxylations of various monosubstituted benzonitriles, benzyl cyanide, and cinnamonitrile. Bio- transformations resulted in products with 42.9 – 97.1% enantiomeric excess. Initial enzymatic activities and isolated yields were obtained in the range of 1.7 – 4.7 U/g cdw and of 3 –62%, respectively. Keywords: aromatic nitriles; asymmetric synthesis; bi- ocatalysis; biotransformations; dihydroxylation; in situ product removal Introduction The use of enzymes as catalyst in organic synthesis pro- vides new routes to synthesize previously undescribed compounds. [1–3] Asymmetric dihydroxylation of aro- matic compounds by bacterial dioxygenases [4,5] (Fig- ure 1) is such a reaction, where the product cis-dihydro- diols are valuable chiral synthons used in organic syn- theses. [6–8] Next to catalyst selectivity, process efficiency depends on turnover frequency, total turnover number and volumetric productivity. [9] The rates of cis-dihydrox- ylation of non-natural (non-physiological) substrates are generally relatively low, compared to the rates ach- ieved with natural substrates such as toluene for TDO, naphthalene for NDO and chlorobenzene for CDO. [10,11] Recombinant biocatalysts circumvent such limitations of (mutated) wild-type cells. Volumetric pro- ductivities can be increased and total reaction times can Figure 1. Structural organization of chlorobenzene dioxyge- nase and designation of the protons and carbons of cis-dihy- drodiols formed by chlorobenzene dioxygenase. Gene prod- ucts; tcbAa: large subunit of terminal oxygenase, tcbAb: small subunit of terminal oxygenase, tcbAc: ferredoxin, tcbAd: re- ductase. FULL PAPERS 1060  2005 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/adsc.200505075 Adv. Synth. Catal. 2005, 347, 1060 – 1072