Periodic Reporting for period 1 - METABOLON (Molecular understanding of metabolic complexes: Towards engineering the next generation of synthetic biosystems) Période du rapport: 2018-09-01 au 2020-08-31 Résumé du contexte et des objectifs généraux du projet Natural products have found applications as pharmaceuticals, flavors and fragrances. Yet, their application is limited due to low availability and inefficient chemical synthesis. Production of these compounds in engineered microbes can provide a sustainable solution. However, current methods only go as far as the reconstruction of natural biosynthetic pathways in heterologous systems, lacking the effectiveness of natural hosts. Thus, many important compounds (e.g. taxol) escape industrial production, as their biosynthesis involves many steps and intricate regulation. Pathways imported into microbial hosts frequently encounter suboptimal catalytic activities, accumulation of labile or toxic intermediates and undesired by-products. Therefore, future production platforms are envisioned to mimic the host environment to achieve optimal enzyme function and pathway regulation. This will enable formation of multi-molecular complexes (metabolons) responsible for efficient metabolic channeling and coordinated processing of substrates and intermediates. Research on the regulation of biosynthetic pathways is still in its infancy. In terpenoid biosynthesis this concept is even less advanced, as no clear evidence of metabolon formation is reported to date. Understanding how natural products are produced has important implications for the pharmaceutical and food industry that will help to cope with global emergencies and to fulfill a fundamental need of mankind for new drugs and valuable compounds. This project addressed imperative issues in contemporary biotechnology such as the production of highly complex structures required for potent bioactivity and the urgent need to replace harmful synthetic chemistry methods with cheap and green technologies. Its outcomes aimed to provide insides for engineering advanced cell factories that will enable low-cost, scalable and less time-consuming processes. As the bioeconomy era unfolds, yeast emerge as robust cell factories for production of a broad range of products from medicines to small molecules, enzymes and vaccines, components of human breast milk, heme for meat substitutes, bioplastics, biomaterials and even biofuels. The research described here also help address basic scientific questions on the molecular regulation and biochemical understanding of the role of membrane composition on functionality of membrane-associated proteins of plant origin that may be involved in formation of yet questionable metabolic complexes or metabolons.The biosynthetic pathway leading to the potent antioxidant carnosic acid involves several successive oxidation events catalyzed by two cytochrome P450 enzymes (CYPs). This pathway is highly suited as a read out of the effect of membrane composition on membrane-bound complex performance in yeast. Capitalizing on the existing expertise in reconstruction of carnosic acid in yeast, I applied an interdisciplinary approach that involve metabolic engineering, lipid biochemistry and biophysics to study the role of membrane lipid composition on the CYPs activity and the assembly of the metabolic complex involved in this pathway. The overall goal of this project was to decipher the molecular limitations of reconstructing this complex in yeast cells and to by-pass current restrictions to achieve improved production yields for further applications. Thus, I aimed to mimic the phospholipid composition of plant ER membrane in yeast and evaluate the activity of the two plant CYPs expressed in this host and their interactions with a suitable reductase (POR). Moreover, I aimed to identify the composition, protein stoichiometry and dynamics of the putative complex and the possible interdependence of the presence of CYP/POR partners on metabolon formation, function and regulation. The last objective was to develop an improved yeast production platform for carnosic acid and related compounds by using the knowledge developed in this project. Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent I examined the role of membrane phospholipid composition on the performance of carnosic acid pathway when reconstructed in yeast. The downstream enzymes involved in the last steps of carnosic acid biosynthesis are cytochrome P450s (CYPs) anchored in the ER-membrane and require activity of an appropriate CYP reductase for electron transfer. Comparing the lipid composition between ER membrane of plants and yeast, I identified significant differences in several lipid species that could play a major role in the function of membrane bound enzymes, such as CYPs and CYP reductases. Thus, I overexpressed genes encoding rationally selected enzymes to modulate lipid biosynthesis in yeast membranes. Using these strains, I engineered CYP-driven oxidation of carnosic acid precursors and measured the influence on the introduced lipid modifications on carnosic acid pathway productivity. Several modifications were identified to have a beneficial effect on the synthesis of carnosic acid and its oxygenated precursors. The best producing strain enabled 3,5-fold increased yield versus the control strain with native membrane.To understand the molecular aspects of these findings we prepared yeast microsomes expressing labeled POR and CYPs with the fluorescent proteins EGFP and mCherry, respectively. The obtained fractions were used to perform Fluorescence resonance energy transfer (FRET) microscopy of single microsome that confirmed changes in the interactions of POR-EGFP and CYP-mCherry in strains with engineered membrane versus wild type yeast. The engineered strains were further exploited to produce other related molecule, some of which new-to-nature compounds. Among these, I identified several compounds with anti-diabetic activity. The results of this project are currently used for preparation of two manuscripts reporting on engineering the membrane lipid composition for improved activity of cytochrome P450s and on expansion of diterpene chemical space towards discovery of efficient bioactivities. Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent) Carnosic acid (CA) is a phenolic diterpene with antioxidant properties used in food products susceptible to degradation and rancidity, such as processed meat and poultry, salad dressings, seasonings, snacks, nuts, soup bases, edible fats and oils, natural foods, and pet foods. The major source for commercial extraction of CA is rosemary. Increasing demand for nutraceuticals is contributing to the growing market of rosemary extract across the globe, which is expected to reach USD 270 million by the end of 2025. Thus, there is an urgent need for sustainable and cost-effective ways to produce CA. I performed fermentor scale cultivation and achieved the highest reported to date yield of CA and related compounds. In addition, I used the lipid modified strains to produce related compounds in high amounts, which enabled structural NMR characterization and bioactivity analysis. Thus, I identified seven new-to-nature molecules of which two exhibited improved inhibitory potency against PTP1B. I foresee the commencement of biological synthesis in yeast as the method of choice for stable, functional supply chains of chemicals. Thus, I anticipate a strong societal impact of this project, as lowering the production cost of high-value compounds will translate into better availability of goods, better treatments, etc. More broadly, this project will propel the science developed by the proposed research towards higher Technology Readiness Levels to maximize benefits for EU and worldwide as a whole.