APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli Anett Kirschner & Josef Altenbuchner & Uwe T. Bornscheuer Received: 23 January 2007 / Revised: 18 February 2007 / Accepted: 19 February 2007 / Published online: 9 March 2007 # Springer-Verlag 2007 Abstract The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expres- sion levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C n ) to the corresponding primary alcohols (C n-2 ) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons. Keywords Baeyer–Villiger monooxygenase . Esterase . Alcohol dehydrogenase . Aliphatic alcohols . Pseudomonas fluorescens . Degradation pathway Introduction Aliphatic hydrocarbons are assimilated by a wide variety of microorganisms including genera of bacteria, yeast, fungi and even algae (Britton 1984; Leahy and Colwell 1990). In nature, two major pathways for the degradation of acyclic hydrocarbons evolved (Fig. 1). The terminal oxidation (pathway 1) of these compounds to carboxylic acids is more frequently found, whilst significantly less examples for the sub-terminal oxidation were reported so far (Ashraf et al. 1994; Forney and Markovetz 1970; Whyte et al. 1998). The sub-terminal oxidation (pathway 2) involves a monooxygenase-mediated hydroxylation of the alkane to a secondary alcohol that is then oxidised by an alcohol dehydrogenase to the corresponding ketone. A Baeyer– Villiger monooxygenase (BVMO) can convert this ketone into an ester that can be hydrolysed by an esterase, resulting in a carboxylic acid and a primary alcohol. The latter is after- wards oxidised to a carboxylic acid (following pathway 1), which can be further degraded by β-oxidation. Alkane-degrading microorganisms are of great interest with respect to the environment, as they are able to naturally remove the aftereffects of oil spills and other hydrocarbon pollutions not only in marine environment and fresh water but also in contaminated soil (Atlas 1981; Leahy and Colwell 1990; Margesin and Schinner 1997, 2001). Furthermore, monooxygenases capable of hydroxylating chemically inert hydrocarbons are also valuable biocatalysts for organic synthesis (Urlacher and Schmid 2006). Previously, we reported on the identification, cloning and characterization of a lactone-specific esterase (EstF1) from Pseudomonas fluorescens DSM 50106 (Khalameyzer et al. 1999). Further sequencing of the EstF1-encoding fragment from the genomic library (GenBank accession number AF090329) identified additional open reading frames encoding for a putative alcohol dehydrogenase (adhF1), a putative BVMO (bmoF1) and a putative alkane hydroxylase (ahyF1; Fig. 2). The alcohol dehydrogenase (AdhF1) and the BVMO (BmoF1) could be functionally Appl Microbiol Biotechnol (2007) 75:1095–1101 DOI 10.1007/s00253-007-0902-3 A. Kirschner : U. T. Bornscheuer (*) Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany e-mail: uwe.bornscheuer@uni-greifswald.de J. Altenbuchner Institute of Industrial Genetics, Stuttgart University, Allmandring 31, 70569 Stuttgart, Germany