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.
