Perspectives for the Genetic Engineering of Hydrocarbon Oxidizing Bacteria
Hydrocarbons have many different structures, but these microbial substrates have two general characteristics which influence the nature of oxidation pathways. (1) Hydrocarbons are usually hydrophobic molecules, and oxidizing activities are consequently located in cellular membranes. (2) Hydrocarbons are composed of several basic structural units (such as aromatic rings and aliphatic chains). In order to break down these composite substrates, oxidation pathways are organized into segments that are conveniently labeled “throats” and “stomachs”. Throats contain the initial activities which convert a specific hydrocarbon substrate into one of a few common intermediates, such as fatty acids or catechols. Stomachs contain the subsequent activities which transform the intermediates into the organic acid substrates of central metabolic pathways. Typical stomachs include β-oxidation and catechol ring fission pathways. Detailed genetic analysis has been carried out for a few hydrocarbon oxidation pathways, including the plasmidborne alk and xyl systems for oxidation of n-alkanes and of toluene or xylenes. In both cases, there is duplication of plasmic-and chromosome-encoded activities at certain steps of the oxidation pathway. Induction specificities in both systems are often more important than enzyme specificities in determining growth phenotypes on different substrates. Oxidation of halogenated aromatic substrates, such as chlorobenzoic acid or the pesticide 24D, has been the subject of less detailed genetic study, but haloaromatic pathways illustrate the same basic principles as those for non-halogenated substrates: (i) the need to match throats and stomachs, and (ii) the importance of proper pathway regulation to ensure substrate catabolism. The use of broad host-range cloning vectors means that recombinant DNA methods are available for use with hydrocarbon-oxidizing gram-negative bacteria. However, detailed consideration of the possible rate-limiting steps in hydrocarbon oxidation indicates that cloning and overproduction of particular enzymes may often not be successful in strain improvement for specific biodegradations or bioconversions. A particular problem can be the toxicity of membrane hydrocarbon-oxidizing proteins when they are produced at very high levels.