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Title: Understanding and improving the fluorescent reporter iLOV for applications in biotechnology
Author: Kjeldsen, Annemette
ISNI:       0000 0005 0286 8716
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2021
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In recent years, engineering of Light, Oxygen and Voltage (LOV) domains have resulted in the development of flavin based fluorescent proteins (FbFP). The improved FbFP iLOV is a flavin mononucleotide (FMN) dependent fluorescent protein with great promise as a smaller and potentially more stable oxygen-independent alternative to the well-stablished Green Fluorescent Protein (GFP). Particularly, within industrial biotechnology the properties of the FbFPs can be advantageous for in vivo bioprocess optimisation. In this project, iLOV and two closely related FbFPs, LOVC426A and phiLOV2.1, have been studied. These demonstrate different fluorescent properties, and it has been hypothesised that this relates to differences in protein dynamics. This is best studied by nuclear magnetic resonance (NMR). Preparation of iLOV for this purpose revealed unreported self-association tendencies of the protein, resulting in broadening of the resonances and low-quality spectra. The zwitterionic detergent CHAPS was found to disrupt the self-association, resulting in high quality spectra and allowing for near complete backbone resonance assignment of the three AtLOV2 derived FbFPs. The dimerisation propensity of the three FbFPs was further explored by gel filtration analysis, revealing that iLOV appear to populate three states: a monomeric form, a stable dimer, and an intermediate, less stable presumed dimeric species. Using X-filtered NMR, the interaction between iLOV and CHAPS was probed to identify a series of hydrophobic residues across the main β-sheet. Point mutations at these sites yielded several novel iLOV variants with reduced self-association without loss of fluorescence. The most promising candidate, iLOV5xβ, was further characterised, and compared with iLOV, phiLOV2.1 and LOVC426A. The utility of iLOV as a transcriptional reporter in E. coli was investigated using constitutive and inducible promoters. Expression of iLOV appeared to slow E. coli growth primarily in the lag phase of growth, with this effect being less severe when the novel variant iLOV5xβ was expressed. To develop an iLOV-based in vivo biosensor for the anaerobic production of succinate in E. coli, the native response system DcuS/R was utilised to design three types of biosensors. Finally, iLOV was used as a fluorescent reporter for production of the antimicrobial peptide (AMP) epidermicin NI01. This enabled screening of transformants for jackpot clones by both direct screening and FACS. The iLOV-tag both improved expression of the target and eased the development of purification protocols. A P. pastoris strain with yields suitable for commercial production of the AMP was identified by these methods.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: QH345 Biochemistry