Community Research and Development Information Service - CORDIS

Periodic Report Summary 1 - BIOHELP (Revealing the hidden secrets of the MEP pathway to engineer new bio-resources for humanity)

Isoprenoids are a vast family of natural compounds produced in all free-living forms of life. They play essential roles in fundamental processes like photosynthesis, membrane integrity, and antioxidant protection; but many of them are also industrially relevant compounds commercially used as biofuels, fragrances, drugs, pigments or nutraceuticals. However, natural sources for most of them are limited and chemical biosynthesis is often expensive and environmentally damaging. Biotechnological production of isoprenoids is a more sustainable approach to meet the actual and increasing demand.
Despite their astonishing variety both at the structural and functional level, all isoprenoids are derived from the C5 universal building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In nature, two chemically unrelated pathways are responsible for the synthesis of IPP and DMAPP. The mevalonate (MVA) pathway is present mainly in Eukaryotes and Achaea, whereas the 2-methyl 3-erythritol-4-phosphate (MEP) pathway is found in most bacteria (including many important pathogens). In plants both pathways coexist, but in different subcellular compartments: the MVA pathway is located in the cytosol and the MEP is located in the plastids.
In the last couple of decades metabolic engineering efforts had been focussed on both the MVA and MEP pathways to improve the bio-production of relevant isoprenoids. To date, the most successful approaches have been achieved by improving the MVA pathway, whereas engineering the MEP pathway has only provided partial success. However, in silico calculations predict a 10% higher maximum yield of the MEP pathway, making it a more attractive target. The MEP pathway has been revealed to be tightly regulated and current efforts to boost the pathway flux to optimize the production of industrially-relevant compounds are still far from the predicted maximum yields.
The primary objective of Biohelp is to identify and disentangle the multi-layer regulatory factors that currently limit the use of this pathway by using a new approach combining wet lab and in silico modelling. The project employs an adaptive, systems level strategy to determine the in vivo kinetic parameters in a MEP pathway model to understand the pathway regulation and consequently establish the conditions to maximize the flux. Those conditions will be applied for the production of carotenoids as representatives of industrially-relevant compounds but will also be of application for many other isoprenoids.
We are developing a new methodology to incorporate kinetic, regulatory and metabolic controls to understand the relative contribution of each step and the overall metabolic flux through the pathway in the context of the cell as a whole entity. All this in vivo data is currently used to generate a model using a novel Bayesian approach whereby a model is first constructed using prior in vitro data to constrain the solution space and then refined by incorporating all our new in vivo data producing a more natural evolution of the model.
The secondary objective of BioHelp is focused on drug discovery. The indiscriminate and excessive use of current antibiotics has led to a broad scale community resistance and the general decline in their efficacy. The increasing number of superbugs and the current usage of reservoir antibiotics highlight the need to revitalize drug discovery against new and unrelated drug targets.
In its 2014 report the WHO declared that antibiotic resistance is no longer a prediction, but is happening right now across the world. Without urgent action, the world is heading towards a post-antibiotic era, in which common infections, which have been treatable, can once again kill.
The differential distribution of the MVA (present in humans) and MEP (present in bacteria) pathways makes the MEP pathway one of the most promising targets for the development of new drugs against pathogens with no undesired effects on humans. However, to date, only one compound, Fosmidomycin (a specific inhibitor of DXR, the first committed step of the MEP pathway), has reached the clinical phase.
We recently identified a new enzyme, DXR-II, showing no homology to DXR but able to catalyze exactly the same biochemical reaction. DXR-II has been identified in the genome of a large number of microorganisms, including pathogens such as Brucella and Bartonella. The active sites of DXR and DXR-II do not resemble each other, making feasible the identification and development of new enzyme-specific antibiotics that should have no side effects in humans and also protect the beneficial flora present in the human gut. Through BioHelp, we aim to identify new drugs that selectively inhibit DXR-II and begin in vitro validation of these drugs. This will lead to the development of a new generation of antibiotics for treatment of human pathogens that cause highly prevalent diseases in the third world without side effects on beneficial bacteria.
We are using computational protein modelling to identify potential ligand-binding pockets and library screening to identify candidates that dock to DXR-II but not DXR. A new ligand-protein interaction method based on high-resolution native protein mass spectrometry has been optimized to screen extensive chemical libraries against both enzymes and establish the foundations for basic structures of small molecules to be further developed into actual drugs.

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