Applying optogenetics in Synechococcus sp. PCC 7002

Forbes, Liam (2025) Applying optogenetics in Synechococcus sp. PCC 7002. PhD thesis, University of Glasgow.

Abstract

Cyanobacteria can be utilised in biotechnology as environmentally sustainable cell machines to convert CO2 into a diverse range of biochemicals. However, a lack of molecular tools available for precise and dynamic control of gene expression hinders metabolic engineering and often contributes to low product titres. Optogenetic tools enable light-regulated control of gene expression with high tunability and reversibility, though their application in cyanobacteria species with high industrial potential is limited. In this study, two different optogenetic systems were characterised in Synechococcus sp. PCC 7002, applications for metabolic engineering were tested, and a transcriptomics study was performed to pinpoint novel optogenetic tools. Firstly, the green/red lightresponsive CcaS/CcaR optogenetic system originating from Synechocystis sp. PCC 6803 was expressed in Synechococcus sp. PCC 7002 and performance was monitored by measuring its regulation of Green Fluorescent Protein (GFP). Characterisation of activation and deactivation was performed under a range of different light wavelengths and intensities using both fluorescence spectrometry and qRT-PCR. The performance of the CcaS/CcaR system was further improved by making targeted genetic modifications to the pCpcG2 output promoter. Secondly, the blue light-regulated YF1/FixJ optogenetic system of noncyanobacterial origin was expressed in Synechococcus sp. PCC 7002. The system demonstrated poor performance with minimal increase in system output in response to blue light or darkness. To evaluate the use of optogenetics for metabolic engineering, the optimised CcaS/CcaR system was tested for lightcontrolled heterologous production of mannitol and L-alanine. While the former was successful, the latter failed despite careful troubleshooting. Finally, to identify new potential optogenetic tools from Synechococcus sp. PCC 7002, RNA sequencing was used to identify genes differentially expressed in blue and orange light. A variety of genes were identified, including transcriptional regulators and kinases which could have roles in wavelength-specific signalling pathways. Overall, this work lays a critical foundation for the use of optogenetics to increase control over metabolic pathways and improve the production of commercially valuable compounds in an industrially promising cyanobacteria species.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH345 Biochemistry; Q Science > QH Natural history > QH426 Genetics
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
• Supervisor's Name: Amtmann, Professor Anna and Christie, Professor John
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-84891
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 12 Feb 2025 16:05
• Last Modified: 12 Feb 2025 16:06
• Thesis DOI: 10.5525/gla.thesis.84891
• URI: https://theses.gla.ac.uk/id/eprint/84891