The AlkB Monooxygenase of Pseudomonas oleovorans

Abstract

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 F! oleovorans and the E. coli alk' recombinants HBlOl (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 HBlOl (pGEc47) produces similar amounts of AlkB as I? 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 F! oleovorans, E. coli HBlOl (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 pg . mg protein-' . h-', respectively. The half-lives of AlkB were estimated to be 80, 3 and 15 for l? oleovorans, E. coli HBlOl (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.

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

“…putida SH41. Pseudomonas putida GPo1 (ATCC 29347) is known to contain one alkB gene copy on the pOCT plasmid, which is present in 1–2 copies per cell (Staijen et al. 1997).…”

Xylene Monooxygenase Catalyzes the Multistep Oxygenation of Toluene and Pseudocumene to Corresponding Alcohols, Aldehydes, and Acids in Escherichia coli JM101

“…However, activity has been limited, and this is probably due to insufficient expression of the xyl genes in E. coli. The alk regulatory system of P. oleovorans GPo1 is not subject to catabolite repression in E. coli, and expression of the alk genes in glucose grown E. coli W3110 via the alk regulatory system permits accumulation of the membrane located alkane hydroxylase AlkB up to 10 -15% of total cell protein (29,30). The alk regulatory system has therefore been used to increase the volumetric activities of E. coli recombinants producing (S)-styrene, based on a 5-fold increase of the expression of xylMA via the alk regulatory system, resulting in styrene oxidation activities of up to 91 units/g of CDW (21).…”

“…To follow product formation over time cell aliquots were incubated with the same substrate for different time periods (5,10,20,30,40, and 80 min). The reactions were stopped as described for the whole cell activity assays and analyzed.…”

Monooxygenases as biocatalysts: Classification, mechanistic aspects and biotechnological applications

“…Proposed catalytic cycle of pterin-dependent monooxygenases (Fitzpatrick, 2003). Staijen et al, 1997;Staijen et al, 2000;Bühler et al, 2000). The ability of these biocatalysts to hydroxylate (non-)activated carbon atoms for biocatalytic applications has been widely studied (van Beilen et al, 1994;Wubbolts et al, 1994;Bühler et al, 2002;Bertrand et al, 2005).…”