Artificial scaffolding proteins for engineering natural product biosynthetic pathways – SCAFFOLD-ART

Natural products (NPs) are small bioactive molecules produced by living organisms, often microorganisms. They are at the origin of many drugs. The diversity of NPs has not been exhaustively explored nor exploited. In many fields (anti-infectives, anti-tumor, ...) new bioactive molecules are actively sought, notably among NPs. Synthetic biology, which aims at rationally modifying living organisms to give them new functions, can contribute to this search for bioactive molecules. In the area of NPs, synthetic biology can contribute to the discovery of novel NPs by expressing NP biosynthetic gene clusters which are silent in laboratory conditions, to the improvement of NP biosynthetic yields, or to the biosynthesis of new NPs by the reprogramming of biosynthetic assembly lines. However, synthetic biology of NPs is still in its infancy and novel and powerful tools are needed to facilitate its development.
The biosynthesis of NPs involves an often complex succession of enzyme-catalyzed steps. Each step can be performed independently of others. However the enzymes of a metabolic pathway can be organized in space in order to improve the overall pathway’s efficiency. Synthetic biology approaches aim at bringing together and spatially organizing in the cell the enzymes involved in the same pathway. This can be done by fusing catalytic domains with binding domains so that different enzymes bind to the same molecule (DNA, RNA, protein). These scaffolding approaches require the development of powerful, modular and easy-to-use tools. We propose to use a new family of artificial proteins, alphaReps to develop new scaffolding tools. AlphaReps are folded, extremely stable, and not prone to aggregation. They can therefore be easily fused to other protein partners. AlphaReps have a curved solenoid shape whose concave region forms a hypervariable surface of interaction. It is possible to select alphaRep capable of specifically binding any target protein, natural or artificial, arbitrarily chosen. It is thus possible to generate, in a fast and efficient manner, a set of pairs of orthogonal proteins, each one interacting specifically with a single partner. We propose to develop novel systems to spatially organize biosynthetic enzymes fused with alphaRep. This will position these enzymes close to each other, increasing the efficiency of natural biosynthetic pathways . Moreover, this will allow the design of new pathways, and the production of new compounds, by the co-localization of enzymes originating from different pathways (combinatorial biosynthesis). These tools will be tested with peptidic NP biosynthetic pathways involving cyclodipeptide synthases or non-ribosomal peptide synthetases. We will study the impact of scaffolding enzyme providing precursors together with the enzymes assembling these precursors, when there is a strong competition between different pathways for these precursors. The effect of scaffolding on the efficiency of a complex pathway will also be tested in a heterologous host bacterium as well as in the original host. Finally, the impact of scaffolding in combinatorial biosynthetic approaches will be evaluated. In this project, new tools for synthetic biology will be designed, constructed and tested in different microorganisms, taking as models the biosynthetic pathways of NPs, some of which have antibiotic activities. In addition to these tools, this project will provide information on the elements important for the optimization of a biosynthetic pathway.