Elucidation of gene regulatory mechanisms in ageing bacterial colonies and tool development for cell factory optimization

Ida Lauritsen

Research output: Book/ReportPh.D. thesis

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Bacteria are indispensible model organisms for studying fundamental biology and their metabolic pathways are being engineered for the purpose of creating high-performance microbial cell factories and moving towards a more sustainable society. However, due to unpredictability, lack of robustness, and complexity of biological systems, more insight into cellular behavior and evolvability in extreme physiological conditions is required in addition to the development of efficient and flexible molecular engineering tools.

This PhD thesis aims at addressing two sides of biology for improving microbial cell factory performance: applied engineering and fundamental biological knowledge. The possibilities to regulate processes that convert DNA to protein and bacterial genome editing technologies are explained as well as regulatory mechanisms of sugar utilization and the role of the wellstudied global transcription factor, the cyclic AMP receptor protein (CRP), in the model organism Escherichia coli (E. coli). Cellular conditions in stationary phase and transcriptional responses upon starvation are described due to a recent experimental evolution study that revealed CRP to be a mutational hotspot in dormant and carbon-starved bacterial cells.

The work presented in this thesis covers the development of two CRISPR-Cas9-based synthetic biology tools for microbial cell factory optimization that i) can modulate two steps of the information flow from DNA to functional proteins and ii) selectively remove unwanted plasmids used in molecular biology and engineering. The CRiPi technology enabled interference with uninvestigated essential genes from E. coli and the one-step curing platform pFREE demonstrated efficient curing in both E. coli and the soil-bacterium Pseudomonas putida. In a manuscript of this thesis, CRP activity was inhibited by the pyrimidine nucleosides, cytidine and uridine, that accumulate during carbon starvation. The surprising new connection between CRP and the pyrimidine metabolism suggests an alternative way to reduce global CRP-cAMP-dependent transcription for balancing nitrogen and carbon metabolism in response to carbon deprivation and ageing. We envision that the work presented in this thesis can provide the engineering tools and basis for further investigations of regulatory mechanisms in changing environmental conditions that hopefully can be explored for designing improved and more stable microbial cell factories in the future.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages245
Publication statusPublished - 2020


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