Biosynthesis of new bioactive molecules by synthetic biology – Mutabiosyntome

Bioactive molecules are essential for human activities such as animal and human health and agriculture. These molecules are either isolated from natural sources (natural products) or synthesised in laboratories (synthetic molecules). In human pharmacopeia, natural products are particularly represented in the anticancer and antimicrobial drug categories, whereas synthetic molecules have been so far the major source of analgesic, anti-allergic or anti-inflammatory molecules. Recent studies have shown that the chemical spaces populated by these two types of molecules differ significantly. Thus, to obtain new bioactive molecules it would be interesting to fill the gap between the chemical space of natural products and of synthetic chemicals.
Our project aims at increasing the chemical diversity of natural products using a synthetic biology approach, i.e. using micro-organisms to produce of “unnatural” natural products. Natural product synthetic biology is based on two complementary approaches, combinatorial biosynthesis and mutasynthesis. Combinatorial biosynthesis uses original combinations of biosynthetic genes from various natural product clusters to generate natural product analogues. Mutasynthesis is based on the feeding of microbial strains blocked in the biosynthesis of an intermediate with chemically synthesized intermediate analogues, resulting in “unnatural natural products”.
Although combinatorial biosynthesis and mutasynthesis approaches have been explored during the last decades, they have encountered various degrees of success. This is probably due in part to our limited understanding of the key determinants influencing the outcomes of such approaches. With our project we aim to identify these key factors using two families of molecules, the pyrrolamides and the aminoglycosides, as model systems. The structural architecture of all nonribosomal peptide synthetases (NRPSs) involved in pyrrolamide assembly isolated so far (standalone domains and modules) and the diversity of precursors incorporated make of pyrrolamides appealing candidates for a combinatorial biosynthetic studies. Aminoglycosides are an important class of broad-spectrum antibiotics with some biosynthetic enzymes exhibiting a certain degree of substrate flexibility, a good starting point for mutasynthesis. In addition, in spite of the many aminoglycoside biosynthetic gene clusters reported, very few attempts to generate new aminoglycosides through combinatorial biosynthesis or mutasynthesis have been made so far.
The project will first involve the construction of biological tools (isolation and characterisation of new pyrrolamide biosynthetic gene clusters, pyrrolamide/aminoside precursor biosynthetic gene cassettes, aminoglycoside biosynthetic gene deletion mutants and engineered pyrrolamide NRPS genes) and chemical tools (synthesis of pyrrolamide and aminoside mutasynthons). These tools will next be used to explore factors that can be expected to impact the feasibility of combinatorial biosynthesis and mutasynthesis approaches and to obtain new pyrrolamide and aminoglycoside derivatives that will be tested for their biological activities. Altogether, this project is expected to contribute to the development of new tools and methodologies for combinatorial biosynthesis and mutasynthesis. It will also result in new derivatives of pyrrolamides and aminoglycosides. Finally, it will improve our knowledge of pyrrolamide and aminoglycoside biosyntheses.