Community Research and Development Information Service - CORDIS

Final Report Summary - 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 relevant compounds currently used in our society as biofuels, fragrances, drugs, pigments or nutraceuticals. Despite their unquestionable value for humanity, natural sources for most of them are limited and chemical biosynthesis is often expensive and environmentally damaging. However, in the last decades our ability to use biological sources for technological applications (biotechnology) has reached a maturity that offers an alternative to still keep using these compounds without compromising natural sources or having detrimental effects on our environment. Biotechnological production of isoprenoids represents a more sustainable approach to meet the actual and increasing demand.

Despite their astonishing diversity both at the structural and functional level, all isoprenoids are derived from the same two small universal building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In nature, two different pathways are able to produce IPP and DMAPP. The mevalonate (MVA) pathway is present mainly in Eukaryotes (including humans) and Achaea, whereas the 2-methyl 3-erythritol-4-phosphate (MEP) pathway is found in most bacteria (including many important pathogens) and the chloroplasts of plant cells. However, cells from living organisms have evolved for millions of years with the main objective of specie perpetuation building up biological constraints to these two pathways (and many others) to ensure the required supply of building blocks to optimize survival but avoiding unnecessary accumulation representing a waste of precious resources. Living organisms are programed to produce what they require, not what humans require from them. A paradigmatic example is a compound called taxol, which shows anticarcinogenic activity and is currently used to fight a variety of human cancers. Taxol is an isoprenoid-based compound naturally produced by the slow-growing tree Taxus brevifolia (or Pacific yew). However, the amount that the tree produces is so little that between 2 and 4 200-year-old trees are required to extract a single dose for human treatments.

A hundred years ago humans could only take what nature was providing. However, since we understood and developed our ability to reshape living organisms by modifying its genetic information new possibilities has emerged. We now are able to readapt dedicated organisms (basically but not only fast-growing microorganisms) to make them produce high amounts of compounds of interest for humans like taxol. In the last couple of decades lots of efforts had been focussed on readapting the MVA and MEP pathways to boost the production of the small building blocks and hence the bio-production of relevant isoprenoids in dedicated microorganisms. Mathematical calculations predict a 10% higher maximum yield of the MEP pathway, making it a more desirable target for biotechnology applications. However, the MEP pathway has been revealed to contain a lot of evolutionary constraints that control the amount of small building blocks that are naturally produced in living cells.

The primary objective of Biohelp was to identify and disentangle all these constraints of the MEP pathway that limits its productivity in regular living cells. We used the most advanced laboratory technologies in combination with computational calculations to study the characteristics of every single step of this pathway. By combining these two sources of information we were able to understand the many pathway constraints and consequently remove or overcome them to unlock the ability to produce the building blocks and hence the isoprenoids. We applied the new information obtained from the project studies to generate dedicated microorganisms producing high amounts of B-carotene (the orange pigment colouring carrots) and lycopene (the red pigment colouring tomato fruits) but this could be applied to many other isoprenoids like taxol. Today we are one step closer to be able to keep providing treatment with taxol to more people and in a more affordable way without depleting our natural resources.

Biohelp took advantage of all the previous and the new information obtained during the project about all the constraints that apply to the MEP pathway to understand the limitations naturally occurring on each step not in isolation but in the context of the cell as a whole entity where virtually everything is interconnected to build up those complex and incredible machines of life. Biohelp translated all these laboratory knowledge into computational information to build up a virtual MEP pathway on a computer than can now be analysed and used to predict the response of this pathway to external changes. This represents a powerful tool that will allow hypothesis validation without experimental work in a near future.
However, sometimes increasing the amount of the desired isoprenoid does not depend only on the ability to increase the building blocks for its production and other factors limit their accumulation. This is the case of B-carotene and lycopene. Their accumulation is toxic for the dedicated microorganism when produced to a high concentration. To alleviate this problem Biohelp has assembled parts from different organisms that, when working in an orchestrated manner, add a new ability to the microorganism. The upgraded bacteria can export the produced compound outside the cell preventing toxic effects but also facilitating its posterior purification.

The secondary objective of BioHelp is focused on drug discovery. The indiscriminate and excessive use of antibiotics has led to a rapid increase of resistant pathogens and the general decline in their efficacy. 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 increasing number of multi-resistant pathogens highlights the need to revitalize drug discovery against new and unrelated drug targets. The MEP pathway is one of the most promising targets for the development of new antibiotics. The presence and essential role of the MEP pathway in most bacteria combined with its absence in humans (producing isoprenoids using the MVA pathway) allows the identification of new antibiotics against pathogens with no undesired effects.

A protein called DXR commonly catalyses one of the reactions required to complete the MEP pathway. We recently identified a new alternative enzyme of the MEP pathway present in some microorganisms including pathogens such as Brucella and Bartonella. We called it DXR-II. DXR-II catalyses the same reaction as DXR but they are different enough between them to make feasible the identification and development of new enzyme-specific antibiotics that should have no side effects in humans (because do not affect the MVA pathway) but also protect the beneficial microorganisms present in the human gut (which use DXR instead of DXR-II).

BioHelp has been focused on the identification of new drugs that selectively inhibit DXR-II. We used computational analysis to virtually identify potential structures that could then be produced as drugs and we also developed a method for a rapid screening to identify candidates that act on DXR-II but not DXR from the vast amount of chemical compounds available from nature and chemical synthesis. This new method has been optimized to screen extensive chemical libraries against both enzymes. We identified small molecules having the desired effect that will be further developed into actual 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.

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