- Title
- Type III polyketide biosynthesis in cyanobacteria
- Creator
- Larsen, Joachim Steen
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Type III polyketide synthases (PKSs) are small homodimeric enzymes responsible for the biosynthesis of a wide range of ecologically and industrially relevant compounds. They carry out the decarboxylative Clasien condensation of extender substrate molecules to a covalently bound starter substrate in an iterative way. Type III PKSs have mainly been studied in plants and heterotrophic bacteria. This thesis aimed to investigate the distribution and diversity of type III PKS biosynthesis pathways and products in cyanobacteria, photosynthetic bacteria renowned for their production of bioactive compounds. Phylogenetic analysis of type III PKS biosynthesis gene clusters from published cyanobacterial genomes showed that they have evolved into three distinct classes – one class similar to (7.7)paracyclophane-like biosynthesis gene clusters, one class similar to hierridin biosynthesis gene clusters and a novel class associated with cytochrome b5 genes. In silico structural analysis of representative protein models and previously elucidated crystal structurers showed distinct differences between bacterial, plant, fungal and cyanobacterial type III PKSs. Two novel type III PKS biosynthesis gene clusters were prioritized for further analysis; the 3.6 kb, four ORF rms gene cluster from Raphidiopsis mediterranea Skuja FSS1-150/1, which uniquely encodes a prenyltransferase, and the 26 kb 12 ORF mks cluster from Microcystis aeruginosa PCC 7806, a hybrid type I/III PKS gene cluster, inversely regulated to microcystin (hepatotoxin) biosynthesis. The type III PKS encoded within the rms cluster (RmsB) was heterologously expressed and purified to homogeneity for in vitro biochemical studies. The results showed that RmsB utilises long acyl-CoAs as substrates. Interestingly, mutagenesis of the active site residues did not abolish activity. Instead, the substrate specificity changed from myristoyl-CoA to decanoyl-CoA. This suggests that RmsB uses a different reaction mechanism to plant and bacterial type III PKSs. We subsequently cloned the entire rms cluster and heterologously expressed it in E. coli to investigate the metabolites being produced. LC-MS analysis showed that the engineered E. coli strain produces four novel compounds, including a prenylated herridin. The type III PKS encoded within the mks cluster (MksG), was heterologously expressed and purified to homogeneity for in vitro biochemical studies. The results showed that MksG can utilize a range of acyl-CoAs to produce diverse metabolites. Heterologous expression of the entire mks cluster in E. coli resulted in the production of multiple novel compounds, when analysed via HPLC, however, only two compounds could be identified by subsequent LC-MS analysis. One being a methyl-alkylresorcinol and the other being a proposed dimerisation of this compound. The process of 2 dimerisation is still unknown as the mks cluster does not contain the canonical genes for dimerisation. Overall, the results from this thesis have expanded knowledge of this understudied group of enzymes, including the distribution, diversity, structure, reaction mechanisms, substrate specificity, and reaction products of cyanobacterial type III PKSs from two distinct phylogenetic lineages. This knowledge can be used to drive the research within this field and to identify novel compounds of ecological and biotechnological relevance.
- Subject
- cyanobacteria; polyketide synthases; heterologous expression; type III polyketide synthases (PKSs)
- Identifier
- http://hdl.handle.net/1959.13/1479657
- Identifier
- uon:50347
- Rights
- Copyright 2023 Joachim Steen Larsen
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT01 | Thesis | 8 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 233 KB | Adobe Acrobat PDF | View Details Download |