Improving flavonoid production in Saccharomyces cerevisiae using synthetic biology tools
Doctoral thesis, 2023
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.
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.
metabolic engineering
biosensors
in vivo directed evolution
enzyme scaffolding
yeast
KC-salen, Kemigården 4, Chalmers
Opponent: Prof. Mattheos Koffas, Rensselaer Polytechnic Institute, Troy, NY, USA
Author
Dany Liu
Chalmers, Life Sciences, Systems and Synthetic Biology
A p-Coumaroyl-CoA Biosensor for Dynamic Regulation of Naringenin Biosynthesis in Saccharomyces cerevisiae
ACS Synthetic Biology,;Vol. In Press(2022)
Journal article
A highly selective cell-based fluorescent biosensor for genistein detection
Engineering Microbiology,;Vol. 3(2023)
Journal article
Skrekas, C., Liu, D., Sun, C., Brack, Y., Bornscheuer, U. T., Siewers, V., David, F. In vivo evolution of malonate transport in Saccharomyces cerevisiae.
Liu, D., Siewers, V. Addressing challenges in yeast-based phloretin production.
Flavonoids are plant compounds with interesting health-beneficial properties. They can be used as additives in foods and beverages, as dietary supplements, as cosmetic ingredients, and have potential applications as pharmaceuticals. Current flavonoid production is mainly based on extraction from plant material. As these molecules do not occur in high concentrations in nature, extraction and purification processes are often inefficient and expensive.
A newer approach to obtain flavonoids, as well as many other chemicals, is to use genetically engineered microorganisms. By introducing plant genes into the microbe’s genome, it is possible to change its metabolism toward the production of the desired flavonoid compound. Unlike plants, microbes such as yeasts and bacteria grow fast and are easy to cultivate in bioreactors. Therefore, this concept, termed metabolic engineering, can increase production rates and yields, as well as product concentrations, compared to plant-based extraction. While many studies have proven that microbes can be engineered to successfully synthesize flavonoids, strains and bioprocesses must be further optimized to ensure economically viable production.
In this thesis, I present different approaches to improve yeast-based flavonoid production using synthetic biology tools. First, so-called biosensors were developed. They can be used to detect intracellular concentrations of intermediate compounds or final products and thereby aid in the finding of high-producer strains, or even to dynamically regulate the synthesis pathway. Second, a directed evolution method was explored to obtain mutant strains for efficient uptake of an important flavonoid precursor molecule. Finally, the assembly of specific enzymes using a scaffolding approach was investigated to further enhance production performance. Collectively, these results demonstrate the immense potential of microbial flavonoid production as a promising alternative to conventional extraction-based processes.
A newer approach to obtain flavonoids, as well as many other chemicals, is to use genetically engineered microorganisms. By introducing plant genes into the microbe’s genome, it is possible to change its metabolism toward the production of the desired flavonoid compound. Unlike plants, microbes such as yeasts and bacteria grow fast and are easy to cultivate in bioreactors. Therefore, this concept, termed metabolic engineering, can increase production rates and yields, as well as product concentrations, compared to plant-based extraction. While many studies have proven that microbes can be engineered to successfully synthesize flavonoids, strains and bioprocesses must be further optimized to ensure economically viable production.
In this thesis, I present different approaches to improve yeast-based flavonoid production using synthetic biology tools. First, so-called biosensors were developed. They can be used to detect intracellular concentrations of intermediate compounds or final products and thereby aid in the finding of high-producer strains, or even to dynamically regulate the synthesis pathway. Second, a directed evolution method was explored to obtain mutant strains for efficient uptake of an important flavonoid precursor molecule. Finally, the assembly of specific enzymes using a scaffolding approach was investigated to further enhance production performance. Collectively, these results demonstrate the immense potential of microbial flavonoid production as a promising alternative to conventional extraction-based processes.
Synthetic microbial consortia-based platform for flavonoids production using synthetic biology (Synbio4Flav)
European Commission (EC) (EC/H2020/814650), 2019-01-01 -- 2023-02-28.
Driving Forces
Sustainable development
Subject Categories
Biochemistry and Molecular Biology
Areas of Advance
Life Science Engineering (2010-2018)
ISBN
978-91-7905-880-7
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5346
Publisher
Chalmers
KC-salen, Kemigården 4, Chalmers
Opponent: Prof. Mattheos Koffas, Rensselaer Polytechnic Institute, Troy, NY, USA