Metabolic engineering of an adipic acid sythesis pathway

Metabolic engineering - defined as the knowledge based redirection of metabolic fluxes - still deserves significant fundamental research before broad industrial applications in biotechnology are feasible. Especially the design of new pathways, via new intermediate metabolites, poses unusual stress situations on the physiology of engineered strains, e.g. by toxic intermediates and/or end products. This project aims at understanding and eventually engineering of cell physiological reactions to the production of adipic acid in microorganisms, a substance not naturally synthesized in these organisms. A metabolic pathway will be engineered comprising the hexanoic acid synthase from Aspergillus parasiticus and the mono-oxygenase from Pseudomonas putida. This new pathway is particularly challenging as it includes hexanoic acid as toxic metabolite. The project refers therefore to important questions about robustness of the metabolic network of the host cells, which will be analyzed in a comparative way in two bacterial and two yeast species. Quantitative physiological analyses should reveal how robust the metabolic network of the cells is, reacting to the engineered pathway. Apart from the production of hexanoic and adipic acid, the intracellular pH and reactive oxygen species (ROS) shall be determined. The intracellular pH will show how robust the regulation of this parameter is, when an increased amount of acids is produced intracellulary. ROS are on one hand markers for stress with a lot of implications, such as apoptosis in the case of the eukaryotic yeasts. On the other hand they might be formed (and cause problems) by the heterologous mono-oxygenase system. Introduction of appropriate scavengers might solve such a problem in the future if it was to be identified. Final evaluation of the improved strains will allow to choose the right organism to set out for the development of a biotechnological production process of adipic acid in the future. Adipic acid is used for the manufacture of important polymers as nylon-66. So far most of it is produced from benzene via cyclohexane/cyclohexanol that is oxidised by nitric acid. This is disadvantageous in that the process depends on petroleum derived carbon and it is an enormous source of environmental pollution. A biotechnological approach for adipic acid production, starting from a renewable carbon source, completely avoiding the use of environmentally problematic substances is therefore highly desirable.

Microbial conversion of renewable resources into chemicals and fuels is one of the key technologies for the future. This project led the basis for the microbial production of the Nylon precursor adipic acid from glucose. To this end we suggest an artificial metabolic pathway combining activities from two different bacteria: In a first step an intermediate is formed from glucose. This intermediate is then in a second step converted into adipic acid, which is an industrially important product, because it is the basis for Nylon production. (About 8.9 % of all manmade fibers are made of Nylon.) Peculiarly, the identified microorganism for the first step is Megasphaera elsdenii, a bacterium which has been isolated from the rumen of cow and sheep. While this organism has been known for some time and research regarding its beneficial health effects for ruminants has been ongoing, the biotechnological potential of this bacterium has not been elucidated so far. Within this project we obtained the genome sequence of M. elsdenii and were able to deduce the biosynthetic pathway to the adipic acid intermediate. The second step is catalyzed by Pseudomonas putida, a bacterium which is known for its capability to degrade petrol derived environmental pollutants. The feasibility of the proposed metabolic pathway has been successfully shown, setting the basis for further developments.