Characterisation of Amino Acid Biosynthetic Pathways in Synechocystis sp. PCC 6803 via Analysis of Auxotrophic Mutants

Jones, Joshua (2023) Characterisation of Amino Acid Biosynthetic Pathways in Synechocystis sp. PCC 6803 via Analysis of Auxotrophic Mutants. Masters thesis, University of East Anglia.

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Abstract

Cyanobacteria are an important phylum which play a key role in ecological processes such as nitrogen and carbon fixation, in addition to being a potential renewable platform for production of high value chemical precursors. Many stains of cyanobacteria also produce notable natural products such as herbicides, anti-microbial, and anti-fungal compounds. To utilise cyanobacteria more effectively, greater understanding of central metabolism would aid engineering strategies for redirection of metabolic flux to secondary metabolites. Even in well studied cyanobacteria such as Synechocystis sp. PCC 6803 (Synechocystis), many genes involved in the biosynthesis of amino acids, precursors for a range of chemicals and natural products, remain uncharacterised. In this study I attempted to characterise genes encoding for amino acid biosynthetic enzymes via generation of auxotrophic mutants, a process whereby a target gene is deleted while mutants are cultivated on media containing the metabolite synthesised by the deleted target pathway. An initial approach at generating auxotrophic mutants on BG11 media supplemented exclusively with a single amino acid yielded only partially segregated mutants. This suggests that native Synechocystis amino acid transporters were either absent, or inefficient at importing amino acids to compensate for loss of gene function. A second approach successfully obtained auxotrophic mutants in multiple amino acid biosynthesis pathways by using BG11 media supplemented with an oligopeptide mix. This is the first study in which an auxotrophic mutant was generated in Synechocystis using these methods. This thesis establishes and discusses methods of generating auxotrophic mutants to provide insight for further characterisation of proteins involved in amino acid biosynthesis.

Item Type:	Thesis (Masters)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	15 Oct 2024 07:58
Last Modified:	15 Oct 2024 07:58
URI:	https://ueaeprints.uea.ac.uk/id/eprint/97024
DOI:

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