Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

@en

For millennia, humans have used microbes to produce industrial products of social and economical value through fermentation processes. In recent years, the application of engineering principles to microbiology have dramatically expanded our ability to modify and optimize microbes for the production of a wide variety of commercial products from renewable feedstocks: food and commodity chemicals, to biofuels and fine chemicals such as pharmaceuticals, fragrances, cosmetics or dyes. The use of microbial bioprocesses for the production of natural products represents an attractive and sustainable alternative to current industrial production methods, which mainly rely on chemical synthesis and/ or extraction from native producers. Advanced biomanufacturing technologies would not only provide sustainable economic benefits (by reducing the monetary cost of production of useful chemicals), but also offer social and environmental benefits. Synthetic biology has allowed engineering the production of many industrial compounds within microbes that do not naturally produce them – this is called “heterologous microbial biosynthesis”. In addition to replacing current manufacturing processes, heterologous microbial biosynthesis likely offers the only viable platform to produce certain natural products at industrial scales. Indeed, many relevant compounds cannot be viably manufactured through chemical synthesis, and/or are produced at undetectable/insufficient levels in native organisms. However, many heterologous bioprocesses remain in their infancy to fully enable an economically viable delivery of relevant natural products to the market. In order to build and sustain the promise of a bioeconomy for the 21st century, metabolic engineering is under pressure to continue to provide largescale, sustainable and cost-competitive bioprocesses that meet global needs. In this thesis, we focus on the development of microbial strains to accelerate the microbial production of 2 different families of high-value compounds of prominent biotechnological relevance within the established microbial chassis Escherichia coli: antibiotics and isoprenoids. The fight against antimicrobial resistance is considered one of the greatest public health challenges of the 21st century. Recent technologies have uncovered new antibiotics that, if harnessed, might help alleviate this crisis. However, most of these new antibiotic compounds are far too complex for economical chemical synthesis, and are naturally produced by unculturable and/or genetically intractable microbes. Developing new heterologous microbial platforms for antibiotic production may be an efficient solution for harnessing the clinical potential of these molecules and their commercialization. Isoprenoids represent one of the largest families of natural compounds (over 50,000 molecules) with an incredible number of practical uses, and of great commercial value: from high-value compounds such as many pharmaceuticals, fragrances and flavors, to commodity chemicals such as solvents, rubber or advanced biofuels. We focus in particular on relevant obstacles associated with the development of proof-of-principle strains for the laboratory-scale production of these high-value chemicals. @en

Carbapenems, a family of β-lactam antibiotics, are among the most powerful bactericidal compounds in clinical use. However, as rational engineering of native carbapenem-producing microbes is not currently possible, the present carbapenem supply relies upon total chemical synthesis of artificial carbapenem derivatives. To enable access to the full diversity of natural carbapenems, we have engineered production of a simple carbapenem antibiotic within Escherichia coli. By increasing concentrations of precursor metabolites and identifying a reducing cofactor of a bottleneck enzyme, we improved productivity by 60-fold over the minimal pathway and surpassed reported titers obtained from carbapenem-producing Streptomyces species. We stabilized E. coli metabolism against antibacterial effects of the carbapenem product by artificially inhibiting membrane synthesis, which further increased antibiotic productivity. As all known naturally occurring carbapenems are derived from a common intermediate, our engineered strain provides a platform for biosynthesis of tailored carbapenem derivatives in a genetically tractable and fast-growing species.

@en