Metabolic engineering of S. cerevisiae for the production of flavonoids
Doctoral thesis, 2023
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.
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.
microbial cell factories
high-value products
sustainability
cinnamic acid
plant natural products
Genetic engineering
yeast
KC-salen, Kemigården 4, Chalmers
Opponent: Professor Paola Branduardi, Università degli Studi di Milano-Bicocca, Italy
Author
Marta Tous Mohedano
Chalmers, Life Sciences, Systems and Synthetic Biology
Optimization of Pinocembrin Biosynthesis in Saccharomyces cerevisiae
ACS Synthetic Biology,;Vol. 12(2023)p. 144-152
Journal article
Tous-Mohedano, M., Konzock, O., Yu, R., and Chen, Y. Improving the aromatic acid tolerance of S. cerevisiae by transcriptomics analysis and transcription factor engineering
Fine-tuning of p-coumaric acid synthesis to increase (2S)-naringenin production in yeast
Metabolic Engineering,;Vol. 79(2023)p. 192-202
Journal article
Optimizing yeast for high-level production of kaempferol and quercetin
Microbial Cell Factories,;Vol. 22(2023)
Journal article
Flavonoids are natural products commonly found in plants. There are more than 9000 known flavonoids compounds. Many of these present a wide range of appealing characteristics like anticancer, antioxidant, or cardioprotective properties. Flavonoids are therefore interesting as drug candidates to treat common diseases such as cancer, cardiovascular and neurodegenerative diseases. Currently, flavonoids are mainly extracted directly from plants. However, the amount that plants produce naturally is very small. Furthermore, harsh purification methods have to be used and this is not environmentally friendly.
Microorganisms, such as the yeast S. cerevisiae, present an alternative opportunity to produce flavonoids. S. cerevisiae, known as baker’s yeast, has been traditionally used to produce beer, wine, and bread. In the last decades and with the development of genetic engineering, yeast has also been used as a microbial cell factory to produce commodity chemicals, fuel substitutes, pharmaceutical drugs, and natural products.
The research included in this thesis optimizes the production of two types of flavonoids: pinocembrin, naringenin in S. cerevisiae. By genetic engineering, I introduced efficient enzymes that catalyse the production of the flavonoids, increased the cellular supply of precursor molecules, and eliminated or regulated biological pathways in yeast that competed with the flavonoid production. I also addressed challenges that limit flavonoid production. For example, in the pinocembrin synthesis pathway, S. cerevisiae is exposed to toxic flavonoid precursor (cinnamic acid). To solve this problem, we looked into the genetic response of S. cerevisiae when it grows in the presence of the toxic compound cinnamic acid and overexpress or delete genes that mitigated the toxicity. In the naringenin pathway, we found that yeast secretes the important precursor p-coumaric acid and therefore cannot utilize it effectively for naringenin production. Therefore, we developed a method to control the pathway and increased the availability of the precursor. Finally, we scaled up flavonoid production from shake flask to 1-L bioreactors in several flavonoid production strains, taking a first step from lab bench towards the construction of microbial factories.
Altogether this thesis demonstrates the potential of S. cerevisiae as an alternative to conventional extraction-based processes to produce flavonoids and presents several strategies to optimize their production in yeast.
Microorganisms, such as the yeast S. cerevisiae, present an alternative opportunity to produce flavonoids. S. cerevisiae, known as baker’s yeast, has been traditionally used to produce beer, wine, and bread. In the last decades and with the development of genetic engineering, yeast has also been used as a microbial cell factory to produce commodity chemicals, fuel substitutes, pharmaceutical drugs, and natural products.
The research included in this thesis optimizes the production of two types of flavonoids: pinocembrin, naringenin in S. cerevisiae. By genetic engineering, I introduced efficient enzymes that catalyse the production of the flavonoids, increased the cellular supply of precursor molecules, and eliminated or regulated biological pathways in yeast that competed with the flavonoid production. I also addressed challenges that limit flavonoid production. For example, in the pinocembrin synthesis pathway, S. cerevisiae is exposed to toxic flavonoid precursor (cinnamic acid). To solve this problem, we looked into the genetic response of S. cerevisiae when it grows in the presence of the toxic compound cinnamic acid and overexpress or delete genes that mitigated the toxicity. In the naringenin pathway, we found that yeast secretes the important precursor p-coumaric acid and therefore cannot utilize it effectively for naringenin production. Therefore, we developed a method to control the pathway and increased the availability of the precursor. Finally, we scaled up flavonoid production from shake flask to 1-L bioreactors in several flavonoid production strains, taking a first step from lab bench towards the construction of microbial factories.
Altogether this thesis demonstrates the potential of S. cerevisiae as an alternative to conventional extraction-based processes to produce flavonoids and presents several strategies to optimize their production in yeast.
Infrastructure
Chalmers Infrastructure for Mass spectrometry
Subject Categories
Food Science
Biological Sciences
Bioinformatics and Systems Biology
Roots
Basic sciences
Areas of Advance
Health Engineering
ISBN
978-91-7905-883-8
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5349
Publisher
Chalmers
KC-salen, Kemigården 4, Chalmers
Opponent: Professor Paola Branduardi, Università degli Studi di Milano-Bicocca, Italy