Improving flavonoid production in Saccharomyces cerevisiae using synthetic biology tools

Abstract: Flavonoids are plant secondary metabolites and represent one of the largest classes of natural products. Due to their health-beneficial properties, they have found potential applications in foods, beverages, cosmetics, and pharmaceuticals. Currently, their production is based on extraction from plant material. However, the low abundance of flavonoids in nature hinders efficient extraction and purification, thus inhibiting their market expansion. Chemical synthesis, although possible, relies on the use of harmful chemicals, harsh operating conditions, and high energy consumption. Metabolic engineering of microorganisms to develop so-called “microbial cell factories” has gained increasing attention as a more efficient and sustainable way to produce a variety of chemicals – including flavonoids. Saccharomyces cerevisiae (baker’s yeast) is one of the most well-studied and widely applied eukaryotic organisms for this endeavor. The fact that yeast shares cellular similarities to plants makes it a suitable host for the heterologous expression of flavonoid biosynthetic pathways. In this thesis, I present our efforts to improve the production of flavonoids in S. cerevisiae through the development and application of several synthetic biology tools. First, transcription factor-based biosensors for the isoflavonoid genistein and the flavonoid precursor p -coumaroyl-CoA were established. The latter sensor was used to devise a dynamic regulation strategy for the production of naringenin, a central flavanone and precursor for many flavanone derivatives. Cell growth was improved and naringenin titers were increased significantly. Next, a malonate assimilation pathway was implemented in yeast to enhance the supply of malonyl-CoA, an important precursor for all flavonoid compounds. By expressing a heterologous malonate transporter and malonyl-CoA synthetase, I constructed strains able to grow on externally supplied malonate. The malonate transporter was further evolved through targeted in vivo mutagenesis and beneficial mutations were identified through growth-based enrichment under selective conditions. Lastly, the production of the dihydrochalcone phloretin was explored. Its biosynthesis was accompanied by substantial byproduct formation and product degradation in the yeast cultivation medium. Different strategies, including enzyme scaffolding and antioxidant supplementation, were investigated to improve yeast-based production. Taken together, I addressed some significant challenges within microbial flavonoid production and showcased how synthetic biology tools may overcome these obstacles.