The AlkB Monooxygenase of Pseudomonas oleovorans

We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk+ recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli. The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amounts of AlkB as P. oleovorans (1.5-2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB. The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic-labeling and immunological techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 microg x mg protein(-1) x h(-1), respectively. The half-lives of AlkB were estimated to be 80, 3 and 15 for P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47), respectively. Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates. The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.