Metabolite biosensors for cell factory development
Doktorsavhandling, 2021
Through synergy with natural sciences and engineering disciplines, biotechnology has become a broad, interdisciplinary, scientific field with many applications. One such application is the sustainable production of industrially relevant products using living systems such as microorganisms. Transforming microorganisms to cell factories is, however, a labour-intensive and cost-ineffective process, requiring many years of extensive research. Several fields together known as systems metabolic engineering, including synthetic biology, have greatly facilitated the process of customizing microorganisms to benefit human interests. Among several emerging tools are metabolite biosensors, which can be employed in high-throughput screening endeavours for identifying productive cells and in dynamic pathway regulation for optimizing metabolic systems. Developing and engineering metabolite biosensors to fit a certain application is, however, challenging.
This thesis focuses on different aspects of utilizing and engineering metabolite-responsive transcription factor-based biosensors for facilitating the development of Saccharomyces cerevisiae as a cell factory. To that end, we improved the dynamic range of a malonyl-CoA-responsive biosensor by i) evaluating different binding site locations of the bacterial transcription factor FapR within different yeast promoters and by ii) using a chimeric transcription factor based on a native repressor system from S. cerevisiae. Furthermore, we suggest the possibility of using the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 system to facilitate biosensor development by guiding binding site positioning. We also employed an acyl-CoA-responsive biosensor based on the bacterial transcription factor FadR to screen for genes boosting the fatty acyl-CoA levels, which are precursors for industrially relevant compounds such as fatty alcohols. The possibility of developing fatty acid-responsive biosensors based on other transcription factors, including the endogenous transcription factor Mga2, has also been addressed. Finally, we looked into the potential of developing an alkane-responsive biosensor based on a system from Yarrowia lipolytica. Overall, this thesis provides answers, discussions and potential future directions on using and engineering metabolite biosensors for cell factory development.
This thesis focuses on different aspects of utilizing and engineering metabolite-responsive transcription factor-based biosensors for facilitating the development of Saccharomyces cerevisiae as a cell factory. To that end, we improved the dynamic range of a malonyl-CoA-responsive biosensor by i) evaluating different binding site locations of the bacterial transcription factor FapR within different yeast promoters and by ii) using a chimeric transcription factor based on a native repressor system from S. cerevisiae. Furthermore, we suggest the possibility of using the CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 system to facilitate biosensor development by guiding binding site positioning. We also employed an acyl-CoA-responsive biosensor based on the bacterial transcription factor FadR to screen for genes boosting the fatty acyl-CoA levels, which are precursors for industrially relevant compounds such as fatty alcohols. The possibility of developing fatty acid-responsive biosensors based on other transcription factors, including the endogenous transcription factor Mga2, has also been addressed. Finally, we looked into the potential of developing an alkane-responsive biosensor based on a system from Yarrowia lipolytica. Overall, this thesis provides answers, discussions and potential future directions on using and engineering metabolite biosensors for cell factory development.
malonyl-CoA
metabolite-responsive transcription factor-based biosensors
Saccharomyces cerevisiae
alkanes
fatty acyl-CoA
synthetic biology
Författare
Yasaman Dabirian
Chalmers, Biologi och bioteknik, Systembiologi
Sustainability has become a household word in many parts of the world. Sustainable development is necessary in order to ensure that we meet our needs of the present without preventing future generations to meet their own needs. To achieve this, three pillars are often carefully considered, including environmental, economic and social needs. There are several research areas with major focus on sustainable development, one being biotechnology. With industrial biotechnology, many of our daily products and necessities can be produced using microorganisms, such as the well-known baker’s yeast Saccharomyces cerevisiae used in making bread and brewing beer. In contrast to petroleum-driven production of various compounds and chemicals, microbial production offers an environmentally friendly and sustainable alternative to produce chemicals, bioplastics, pharmaceuticals and biofuels using renewable resources. Microorganisms used for bio-based production are often referred to as cell factories. Cell factories can contribute to a sustainable development by i) reducing our dependency on petroleum, lowering the emission of greenhouse gases (environmental), ii) moving towards a circular economy (economic), where waste products are recycled and reused as opposed to being disposed in landfills and iii) producing pharmaceuticals that are either too complex to be produced using merely chemical synthesis or too expensive to be afforded by people in developing countries (social).
The challenge, however, lies in creating robust cell factories that allow for cost-competitive production. Despite great advances in biological engineering, it still takes immense amount of research, effort and resources to develop a cell factory that is suitable for industrial production. This is mainly due to the complexity of living systems and their highly dynamic metabolism, making biological engineering unpredictive and trial-and-error part of the everyday process. Therefore, instead of targeting specific parts of the genome, researchers are moving more towards approaches where many different combinations and genetic variants are randomly created simultaneously. However, to be able to analyze all different variants, it is necessary to have tools that allows for efficient analysis. Genetically encoded biosensors, also known as metabolite biosensors, are promising tools for efficiently analysing a large number of different cells. Ideally, metabolite biosensors would transform any sought after cellular changes to clear output signals, such as a fluorescent signal or improved growth fitness, enabling fast identification of promising cell factories using less time and resources. In reality, however, developing and optimizing a biosensor for a specific purpose is challenging.
The main work in this thesis has been focused on developing metabolite-responsive transcription factor-based biosensors, specifically for fatty molecules such as fatty acids and alkanes as these are industrially relevant compounds. Overall, this thesis highlights both the challenges and potentials of biosensors.
The challenge, however, lies in creating robust cell factories that allow for cost-competitive production. Despite great advances in biological engineering, it still takes immense amount of research, effort and resources to develop a cell factory that is suitable for industrial production. This is mainly due to the complexity of living systems and their highly dynamic metabolism, making biological engineering unpredictive and trial-and-error part of the everyday process. Therefore, instead of targeting specific parts of the genome, researchers are moving more towards approaches where many different combinations and genetic variants are randomly created simultaneously. However, to be able to analyze all different variants, it is necessary to have tools that allows for efficient analysis. Genetically encoded biosensors, also known as metabolite biosensors, are promising tools for efficiently analysing a large number of different cells. Ideally, metabolite biosensors would transform any sought after cellular changes to clear output signals, such as a fluorescent signal or improved growth fitness, enabling fast identification of promising cell factories using less time and resources. In reality, however, developing and optimizing a biosensor for a specific purpose is challenging.
The main work in this thesis has been focused on developing metabolite-responsive transcription factor-based biosensors, specifically for fatty molecules such as fatty acids and alkanes as these are industrially relevant compounds. Overall, this thesis highlights both the challenges and potentials of biosensors.
Ämneskategorier
Industriell bioteknik
ISBN
978-91-7905-436-6
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4903
Utgivare
Chalmers