Charactrisation of amino acid biosynthetic pathways in Synechocystis sp. PCC 6803

Steward, Harry (2023) Charactrisation of amino acid biosynthetic pathways in Synechocystis sp. PCC 6803. Masters thesis, University of East Anglia.

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Abstract

Cyanobacteria are one of the most important phyla of organisms on the planet, accounting for 20-30% of global carbon fixation[1]. Cyanobacteria have a wide array of potential applications in biotechnology. Key to unlocking that potential is gaining a deeper understanding of fundamental biological processes in cyanobacteria, most notably in model species such as Synechocystis sp. PCC 6803 (Synechocystis). Much of our current understanding of this model organism’s central metabolism is based solely on identifying genes with homologs characterised in model heterotrophs. This includes amino acid biosynthesis, which includes several uncharacterised pathways that could be unique to cyanobacteria. A comprehensive understanding of amino acid biosynthesis will enhance their industrial utilisation. To characterise genes encoding enzymes involved in amino acid biosynthesis, this study aims to generate a novel method for generating auxotrophic mutants in Synechocystis. Multiple approaches were trialled. Initially mutants were generated using increasing concentrations of kanamycin, eliminating slr0036 as an essential gene. I then attempted to segregate genes by supplementing media with single amino acids. This approach proved unsuccessful, likely due to the absence of importers or insufficient transport of amino acids into the cytosol. I then attempted to leverage the OppA oligopeptide importer, to import short chain oligopeptides into the cell where they could then be catabolised into their constituent amino acids. This method for compensating for the loss of function of the gene, allowed for successful generation of the slr1312 and slr2035 auxotrophic mutants. I tried to allow for import of single amino acids by introducing high affinity Escherichia coli (E. coli) K12 amino acid importers into Synechocystis, however due to time constraints I was unable to finish this. This study provides an invaluable technique for characterisation of genes encoding proteins involved in amino acid biosynthesis and identification of novel enzymes involved in novel cyanobacterial pathways.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	11 Jun 2024 11:01
Last Modified:	11 Jun 2024 11:01
URI:	https://ueaeprints.uea.ac.uk/id/eprint/95568
DOI:

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