Utilizing genetically encoded biosensors for monitoring microbial stress at different scales

High-throughput screening (HTS) combined with metabolite responsive biosensors has significantly speeded up the identification of high producer strains. At large-scales, organisms are exposed to various industrial stresses, which can lead to a significant loss of productivity due to cellular stress. Therefore, deciphering molecular mechanisms required to withstand this environmental stress is important for increasing fermentation-based productivity. Despite the importance of understanding industrial stress, synthetic biology and cell factory design rarely address this issue up front. To address this need, genetically encoded biosensors for monitoring microbial stress were combined with state-of-the-art HTS approaches. Such novel methods expand the scope of HTS approaches by allowing important internal parameters like pH or the redox environment to be easily monitored in a hands-off, continuous, and high-throughput manner. For these exact reasons, such biosensors are indispensable tools that facilitate physiological studies from plate-based to shaker-flask all the way through bioreactor cultivations, thus linking environmental stresses to the cellular stress response.

Chapter 1 introduces the background behind the multidisciplinary research topics essential for the development of microbial bioprocesses. Common types of cellular stress, arising during the transition from the initial screens to large-scale bioprocesses are discussed, and appropriate genetically encoded biosensors to adequately assess vital internal parameters are introduced. This will provide the context for the aim of the present PhD project.

Chapter 2 describes the application of a pH-sensitive fluorescent protein (mCherryEA) as genetically encoded biosensor that allows for real-time analysis of the internal pH in Escherichia coli colonies on agar plates to facilitate robot-assisted phenotypic screenings. The applicability of the novel screening approach was demonstrated by a proof-of-concept screen of a transposon (Tn) derived mutant library of E. coli. Identification of the Tn-insertion sites in mutants with altered internal pH levels revealed that the transposon was inserted into genes known to be associated with pH-stress adaptation. Insertion into rssB (encoding for the adaptor protein RssB which mediates the proteolytic degradation of the general stress response regulator RpoS), resulted in an accumulation of RpoS which in turn modulated the pH-homeostatic capacity of E. coli.

Chapter 3 describes the development of a novel technique to facilitate the analysis of biosensor signals from arrayed colonies. Optical analysis can be challenging as imaging devices need to be equipped with appropriate filter modules. To overcome this, a microplate reader-based method for the biosensor analysis in arrayed colonies was developed. In contrast to current imaging technologies, the monochromatic technology allows for high flexibility with respect to different fluorescence wavelengths which can be detected and thus the application of a broad range of different genetically encoded biosensors.

Chapter 4 reveals the importance of the non-enzymatic antioxidant mycothiol (MSH) in Corynebacterium glutamicum in maintaining a reducing redox environment. Aerobic bioreactor cultivations revealed a high susceptibility of a mutant strain lacking MSH towards oxidative stress (C. glutamicum ΔmshC). To further investigate this, the genetically encoded biosensor Mrx1-roGFP2 was applied as an analytical tool to measure intracellular redox states. During aerobic bioreactor cultivations, an oxidative redox shift of the biosensor protein Mrx1- roGFP2 in C. glutamicum was observed, indicating the protective function of MSH to resist against oxidative stress.

Chapter 5 investigated the applicability of impedance flow cytometry (IFC) as an alternative tool to assess the cell viability by comparing it with the conventional plating method. This facilitated the assessment of cell viability from non- growing but glutamate producing C. glutamicum cultures during micro-fermentations.

Chapter 6 describes the development of a combinatorial biosensor system that monitors oxidative stress and DNA-damage at the same time in one cell. Oxidative stress is known to induce DNA damage and genetically encoded biosensors have been developed to target both types of stress separately using the redox biosensor protein Mrx-roGFP2 and the transcriptional biosensor P_{recA_}e2-crimson. In this chapter, the compatibility of a combination of both sensor types is demonstrated both in vitro and in vivo in C. glutamicum. The applicability of this novel sensor system was tested during shaker-flask cultivations upon applying artificial oxidants, which resulted in an immediate oxidative redox shift measured via the biosensor protein Mrx1-roGFP2, followed by an induction of the DNA-damage response. This was seen to be even more pronounced for the MSH-deficient mutant strain C. glutamicum ΔmshC, known for its susceptibility towards oxidative stress.

Chapter 7 critically reviews different modelling concepts applied in industrial biotechnology. It focuses on coupling different metabolic models to computational fluid dynamics (CFD) as a modelling concept to link environmental stress with cellular physiology. This chapter discusses how genetically encoded biosensors can be used with different scale-down approaches towards the development of digital models suitable for both cell factory design and process optimization at industrial scales in the future.

Chapter 8 is dedicated to an overall discussion of the presented PhD thesis with concluding aspects, which puts the work in a larger context.