We tested the synthesis and in vivo function of the inducible alkane hydroxylase of Pseudomonas oleovorans GPo1 in several Escherichia coli recombinants. The enzyme components (AlkB, AlkG and AlkT) were synthesized at various rates in different E. coli hosts, which after induction produced between twofold and tenfold more of the Alk components than did P. oleovorans. The enzyme components were less stable in recombinant E. coli hosts than in P. oleovorans. In addition, the specific activity of the alkane mono-oxygenase component AlkB was five or six times lower in E. coli than in P. oleovorans. Evidently, optimal functioning of the hydroxylase system requires factors or a molecular environment that are available in Pseudomonas but not in E. coli. These factors are likely to include correct interactions of AlkB with the membrane and incorporation of iron into the AlkG and AlkB apoproteins.Keywords: alkane hydroxylase; expression; Pseudomonas oleovorans; rubredoxin reductase; rubredoxin.Alkane hydroxylase is an inducible enzyme system that carries out the first oxidation step in the utilization of alkanes by the oil-consuming Pseudomonas oleovorans GPo1 (TF4-1L; ATCC 29347) [1], and as such plays an important role in the environment. In addition, the alkane hydroxylase system is able to carry out a wide range of stereoselective and regioselective oxidation reactions, giving it considerable commercial potential as a biocatalyst [2]. The alkane hydroxylase system ( Fig. 1) consists of a mono-oxygenase, a rubredoxin, and a rubredoxin reductase [3,4]. The enzyme system and other proteins involved in alkane degradation are encoded by the alkBFGHJKL and alkST genes [5,6] located on the OCT plasmid [7]. The mono-oxygenase component, encoded by alkB, is an integral cytoplasmic membrane protein [8±10], and is the best studied example of a large class of structurally related non-heme iron proteins [11]. Rubredoxin, encoded by alkG [12,13], and rubredoxin reductase, encoded by alkT [14,15], are cytoplasmic proteins. The three Alk proteins are present in P. oleovorans in markedly non-stoichiometric amounts: the in vivo molar proportions of AlkB, AlkG and AlkT have been estimated to be close to 50 : 10 : 1 [16]. The alk genes were previously cloned in pLAFR1. We have introduced the resulting plasmid pGEc47 into Escherichia coli hosts, and after induction such recombinants are able to oxidize alkanes [17]. However, despite considerable overexpression of alkane mono-oxygenase in some E. coli hosts [18], the most active recombinants showed in vivo alkane-oxidation rates no higher than that of the native host strain [19]. To understand why this is so, we followed the synthesis and degradation of the three individual enzyme components and related this to the specific activity of each of these components in vivo, in both E. coli recombinants and the native host.In this paper we show that each of the three Alk proteins was generally synthesized in larger amounts in the E. coli alk 1 recombinants than in P. oleovorans, in relative amo...
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.
The alk genes are located on the OCT plasmid ofPseudomonas oleovorans and encode an inducible pathway for the utilization of n-alkanes as carbon and energy sources. We have investigated the influence of alternative carbon sources on the induction of this pathway in P. oleovorans andEscherichia coli alk + recombinants. In doing so, we confirmed earlier reports that induction of alkane hydroxylase activity in pseudomonads is subject to carbon catabolite repression. Specifically, synthesis of the monooxygenase component AlkB is repressed at the transcriptional level. The alk genes have been cloned into plasmid pGEc47, which has a copy number of about 5 to 10 per cell in both E. coli and pseudomonads.Pseudomonas putida GPo12 is a P. oleovoransderivative cured of the OCT plasmid. Upon introduction of pGEc47 in this strain, carbon catabolite repression of alkane hydroxylase activity was reduced significantly. In cultures of recombinant E. coli HB101 and W3110 carrying pGEc47, induction of AlkB and transcription of the alkB gene were no longer subject to carbon catabolite repression. This suggests that carbon catabolite repression of alkane degradation is regulated differently inPseudomonas and in E. coli strains. These results also indicate that P alkBFGHJKL , the P alk promoter, might be useful in attaining high expression levels of heterologous genes in E. coligrown on inexpensive carbon sources which normally trigger carbon catabolite repression of native expression systems in this host.
The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47). AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain.
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