New Frontiers in Screening for Microbial Biocatalysts Edited by K. Kieslich, C.P. van der Beek, J.A.M. de Bont and W.J.J, van den Tweel 211 © 1998 Elsevier Science B.V. All rights reserved. Alkane hydroxylase systems in Pseudomonas aeruginosa strains able to grow on n-octane Jan B. van Beilen, Liesbeth Veenhoff and Bernard Witholt Institute of Biotechnology, ETH-H6nggerberg, CH-8093 Zurich, Switzerland 1. SUMMARY Pseudomonas strains able to grow on n-octane were selected from different strain collections, and compared with P. oleovorans GPol with respect to the genetics of alkane metabolism in these strains. We found that the enzymes involved in alkane oxidation in four P. aeruginosa strains are virtually identical to the alkane hydroxylase system of P. oleovorans GPol. PCR-cloning and sequencing showed that the differences are limited to a few nucleotide substitutions outside the open- reading frames. 2. INTRODUCTION Many bacteria are able grow on medium chain-length alkanes. Of these strains, only P. oleovorans TF4-1L (GPol) has been studied in detail with respect to both the genetics and enzymology of alkane metabolism (Fig. 1) (1), also because this strain has proven to be a versatile biocatalyst (2, and references therein). Figure 1. Organization of the P. oleovoransfl/fc-genes,and func- tion of the encoded proteins. The alkane hydroxylase system con- sists of AlkB (alkane hydrox- ylase), AlkG (rubredoxin) and AlkT (rubredoxin reductase). AlkH and AlkJ are aldehyde and alcohol dehydrogenases, respec- tively, AlkK is an acyl-CoA syn- thetase, AlkL is an outer-mem- brane protein of unknown func- tion, AlkF is a non-functional rubredoxin and AlkS regulates expression of the alkBFGHJKL operon. XJ outer membrane AlkL periplasmic space alkB FG H