The ability to utilise biosynthetic enzymes and biosynthetic pathways to produce natural products or chiral building blocks has inherent advantages over traditional synthetic chemistry methodology. Due to the high degree of enantioselectivity biosynthetic assembly lines produce their target compounds, in addition to the selectivity in which enzymes catalyse reactions make them an appealing route to numerous pharmaceutically relevant compounds or building blocks. This thesis focuses on elucidating biosynthesis pathways and expanding substrate scope of biosynthetic enzymes and pathways to create natural product analogues or building blocks via biocatalytic or biosynthetic methodologies. A key factor in designing peptide ligands as pharmaceutically relevant compounds is the conformational rigidity of the peptide. By decreasing conformation freedom, the available topographies available is limited and thus, the entropic cost of binding to a molecular target in vivo is reduced. Work within this thesis has focused on broadening the substrate scope of tryptophan synthase from Salmonella typhimurium, StTrpS, past substitutions on the indole ring, utilising L-threonine as the β-hydroxy amino acid source resulting in the biocatalytic synthesis of (2S, 3S) β-methyl tryptophan as a single enantiomer. In addition, a range of other indole derivatives can be incorporated using this methodology, such as L amino acid oxidases, or halogenase enzymes gives access to a range of β-methyl tryptophan derivatives. By expanding the substrate scope of biosynthetic enzymes, such as Cfl ligase from S. scabies it is possible to gain insight into their tolerance and therefor potential to be installed into a pathway to create novel natural product analogues in vivo. It was found that in addition to its native substrates, a range of structurally similar CFA mimics, including the plant signalling hormone jasmonic acid can successfully be ligated to a range of non-polar amino acids. Future work on obtaining an enzymatic crystal structure with a synthetic AMP mimic will allow site directed mutagenesis to be conducted in the future to further expand substrate scope or increase activity. Due to the complexity of bioactive natural products, an attractive route to the synthesis of analogues would be to use the native biosynthetic assembly line. By expressing only the core NRPS genes within a heterologous host, Streptomyces coelicolor, it was possibly to feed in exocellular tryptophan analogues to the TxtAB containing heterologous host for the biosynthetic production of thaxtomin analogues. Whilst adenylation domains are highly specific for their cognate amino acid, it was possible to create 4 thaxtomin D analogues using this host. In addition, by combining StTrpS and TxtAB within the heterologous host, the feeding of 4-substituted indoles allowed access to thaxtomin analogues. This route negates the need for a symmetric synthesis and allows the one access to thaxtomin analogues from indole in a single fermentation step. Elucidating biosynthetic pathways allows insight into how nature can biosynthesise complex diverse natural products. Despite malonomycin first being isolated in the 1970âs, very little is known about its biosynthesis. Prior work within the research group focusing on bioinformatics led to the discovery of the gene cluster responsible for malonomycin biosynthesis. Contained within the gene cluster is a putative vitamin K dependent carboxylase. Through gene knock out experiments and compound isolation and characterisation, this putative carboxylase was found to be responsible for the carboxylation and work is continuing on in vitro characterisation of this novel bacterial enzyme.
Date of Award | 1 Aug 2018 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Jason Micklefield (Supervisor) & Lu Shin Wong (Supervisor) |
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Biosynthesis of Natural Products: Investigation of Biosynthesis pathways, expanding the Substrate Scope and in vitro Characterisation of Biosynthetic Enzymes
Francis, D. (Author). 1 Aug 2018
Student thesis: Phd