Metabolic engineering of S. cerevisiae for the production of flavonoids

Sammanfattning: The interest in the production of natural products such as flavonoids has been increasing during the last decade. Flavonoids have several attractive bioactivities including antitumoral, antioxidant or antimicrobial properties. To produce these high-value products, we usually recur to chemical synthesis or plant extraction. However, these two options are costly and not environmentally friendly, and microbial production is therefore preferred. S. cerevisiae is a thoroughly characterized model organism with a wide range of available tools for engineering, making it an ideal organism for this challenge.   The aim of this thesis was to apply different strategies to engineer S.  cerevisiae to establish and optimize the production of the flavonoids pinocembrin and naringenin, and their derivatives. Different approaches were used: different heterologous genes were screened, their copy number was increased to achieve the highest production, the competing pathways were eliminated, and the precursors availability was increased. Furthermore, the bottlenecks of the pathways were identified. For pinocembrin production, I established that the accumulation of the toxic intermediate cinnamic acid limits production. Therefore, the transcriptional changes that S. cerevisiae undergoes under aromatic acid stress were investigated. My findings indicate that by employing transcription factor engineering it is possible to develop strains that are tolerant to aromatic compounds that can be utilized for the production of valuable natural products. When analysing the naringenin biosynthetic pathway it was found that the distribution of the pathway intermediates in the cell is a major issue. The spatiotemporal distribution of p-coumaric acid (a key pathway intermediate) and naringenin was assessed and it was determined that p-coumaric acid accumulates extracellularly and cannot be fully utilized. Therefore, a dual dynamic control system that combines a malonyl-CoA biosensor regulator and an RNAi strategy was established, to autonomously control the synthesis of p-coumaric acid and downregulate the fatty acid pathways that compete directly for the precursor malonyl-CoA. Finally, the production of naringenin and pinocembrin derivatives was established including kaempferol, quercetin and baicalein which present valuable bioactivities.   Overall, this thesis employs diverse strategies for constructing and optimizing yeast factories for flavonoid production.