The P _alkBFGHJKL Promoter Is under Carbon Catabolite Repression Control in Pseudomonas oleovorans but Not in Escherichia coli alk ⁺ Recombinants

Abstract

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.

Carbon catabolite repression inPseudomonas: optimizing metabolic versatility and interactions with the environment

“…However, not all organic acids can induce a CCR effect and, even more intriguingly, some of them have a clear effect in some cases, but not in others. For example, citrate allows a good expression of the P. putida GPo1 OCT plasmid alkane degradation pathway (Yuste et al, 1998;Staijen et al, 1999), but has a repressive effect on the P. putida H phenol degradation genes (Müller et al, 1996) and on the P. putida CA-3 pathway for styrene assimilation (O'Leary et al, 2001). This may be due to specific differences in the bacterial strains or in the genes analysed or the experimental conditions used.…”

Positively regulated bacterial expression systems

“…comm.). The AlkS/PalkB expression system is not subject to catabolite repression in E. coli (Staijen et al, 1999). AlkS/PalkB has been used for tightly controlled and high-level expression of recombinant xylene oxidase for biotransformation of styrene into (S)-styrene oxide (Panke et al, 1999), and it has also been shown to function well for induced production of recombinant proteins under HCDC of E. coli (Makart et al, 2007).…”

Carbon metabolism and product inhibition determine the epoxidation efficiency of solvent-tolerantPseudomonas sp. strain VLB120ΔC

“…Biocatalysts based on bacterial wild-type strains, which are able to assimilate hydrocarbons as sole source of carbon and energy, are often tolerant to toxic effects of such growth substrates and their oxidized metabolites (Ramos et al, 2002) and often show high oxidation activities ). Yet, diverse enzymes and regulatory systems involved in the assimilation of hydrocarbons (Jimenez et al, 2002;Nelson et al, 2002) may cause degradation of the products of interest, byproduct formation, and catabolite repression (Santos et al, 2000;Staijen et al, 1999). As a result, metabolic pathway engineering and biochemical process engineering are often crucial for the development of a viable process based on hydrocarbon degraders.…”