Combining Metabolic Engineering and Synthetic Biology Approaches for the Production of Abscisic Acid in Yeast
Doctoral thesis, 2023
Nature presents us with a myriad of complex and diverse molecules. Many of these molecules prove to be useful to humans and find applications as pharmaceuticals, biofuels, agrochemicals, cosmetic ingredients or food additives. One highly promising natural product with a broad range of potential applications is the terpenoid abscisic acid (ABA). ABA fulfils a pivotal role in higher plants by regulating various developmental processes as well as abiotic stress responses. However, ABA is also produced in many other organisms, including humans. It appears to be a ubiquitous and evolutionary conserved signalling molecule throughout nature.
Genetically engineered microorganisms, referred to as microbial cell factories, can be a sustainable source of natural products. In this thesis, a cell factory for the heterologous production of ABA was established and optimized employing the yeast Saccharomyces cerevisiae.
Cell factory development is an inherently time-consuming process. As an enabling technology for subsequent work on the ABA cell factory, we expanded the modular cloning toolkit for yeast and made it more applicable for common genetic engineering tasks (Paper I). The ABA biosynthetic pathway of Botrytis cinerea was used to construct an ABA-producing S. cerevisiae strain (Paper II). The activity of two B. cinerea proteins, BcABA1 and BcABA2, was found to limit ABA titers. Two optimization approaches were devised for the following studies. Firstly, various rational engineering targets were explored, of which the native yeast gene PAH1 was identified as the most promising candidate (Paper III). Knockdown of PAH1 benefited ABA production without affecting growth. Secondly, platform strains for screening BcABA1 and BcABA2 enzyme libraries were developed, which utilize an ABA biosensor and enable a high throughput screening approach (Paper IV).
In this work, we combined metabolic engineering and synthetic biology approaches for the heterologous production of ABA, and furthermore provided tools and insights that will be useful beyond the scope of this project.
Genetically engineered microorganisms, referred to as microbial cell factories, can be a sustainable source of natural products. In this thesis, a cell factory for the heterologous production of ABA was established and optimized employing the yeast Saccharomyces cerevisiae.
Cell factory development is an inherently time-consuming process. As an enabling technology for subsequent work on the ABA cell factory, we expanded the modular cloning toolkit for yeast and made it more applicable for common genetic engineering tasks (Paper I). The ABA biosynthetic pathway of Botrytis cinerea was used to construct an ABA-producing S. cerevisiae strain (Paper II). The activity of two B. cinerea proteins, BcABA1 and BcABA2, was found to limit ABA titers. Two optimization approaches were devised for the following studies. Firstly, various rational engineering targets were explored, of which the native yeast gene PAH1 was identified as the most promising candidate (Paper III). Knockdown of PAH1 benefited ABA production without affecting growth. Secondly, platform strains for screening BcABA1 and BcABA2 enzyme libraries were developed, which utilize an ABA biosensor and enable a high throughput screening approach (Paper IV).
In this work, we combined metabolic engineering and synthetic biology approaches for the heterologous production of ABA, and furthermore provided tools and insights that will be useful beyond the scope of this project.
metabolic engineering
Botrytis cinerea
cell factory
abscisic acid
synthetic biology
biosensor
sesquiterpenoid
standardization
Saccharomyces cerevisiae
Author
Maximilian Otto
Chalmers, Life Sciences, Systems and Synthetic Biology
Abscisic acid (ABA) is a chemical that is found in surprisingly many organisms. It appears to function as a universal signalling molecule. ABA has numerous potential applications in agriculture and medicine. In plants, it can regulate processes like fruit ripening and make crop plants more resilient to droughts. In humans, ABA appears to reduce inflammation and regulate blood glucose levels, characteristics that could benefit patients with chronic inflammatory diseases or type-2-diabetes. However, ABA is produced only in miniscule amounts in nature and another source of the molecule is required for it to be used on a broad scale.
Progress in the field of genetic engineering allows us to modify microorganisms, such as baker’s yeast, to produce valuable molecules that they would not produce naturally. In this thesis, we developed new genetic engineering tools and used them to construct ABA-producing yeast strains. However, the productivity of the strains needs to be improved for them to become a sustainable source of ABA in the future. We explored different strategies to enhance the performance of the strains and found that interfering with their native lipid metabolism benefits ABA production. We furthermore developed strains that can sense and report ABA concentrations in vivo using a biosensor. The biosensor strains will be useful tools to discover improved enzyme variants, which can increase the cell factory’s productivity further.
This thesis lays the groundwork for ABA production in yeast and, in addition, provides valuable tools and insights that will be useful for other genetic engineering projects.
Progress in the field of genetic engineering allows us to modify microorganisms, such as baker’s yeast, to produce valuable molecules that they would not produce naturally. In this thesis, we developed new genetic engineering tools and used them to construct ABA-producing yeast strains. However, the productivity of the strains needs to be improved for them to become a sustainable source of ABA in the future. We explored different strategies to enhance the performance of the strains and found that interfering with their native lipid metabolism benefits ABA production. We furthermore developed strains that can sense and report ABA concentrations in vivo using a biosensor. The biosensor strains will be useful tools to discover improved enzyme variants, which can increase the cell factory’s productivity further.
This thesis lays the groundwork for ABA production in yeast and, in addition, provides valuable tools and insights that will be useful for other genetic engineering projects.
Subject Categories
Industrial Biotechnology
Biological Sciences
ISBN
978-91-7905-767-1
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5233
Publisher
Chalmers