The Analysis of Genetically Engineered Polyketide Metabolites

Kearney, Dominic (2000). The Analysis of Genetically Engineered Polyketide Metabolites. MPhil thesis The Open University.



Experience has shown that genetically engineered, truncated polyketide synthases sometimes gave very low production levels (as low as l-2mg per litre broth culture). To understand how the protein producing them works, it is important to be able to characterise the metabolites. To do this effectively these small quantities of product must be isolated from the broth so that NMR spectra may be obtained.
Within this thesis is described details of the initial analysis by GC-MS, the separation and subsequent isolation by preparative HPLC-MS and the interpretation of the NMR spectra. Work is also described relating to the pre-HPLC cleanup of biological extracts including flash silica chromatography and size exclusion chromatography. One of the compounds characterised has proven to have different stereochemistry to that expected based on previous knowledge. The structure, including stereochemistry of this new compound was obtained by a combination of a process of elimination (spiking studies) and by NMR spectra.
A comparative study of the effectiveness of HPLC-MS and GC-MS for the accurate quantification of triketide lactones has shown that GC-MS is the more preferable method having lower detection limits for this particular group of compounds.
The synthesis of a Ʌ-lactone is also described (this was used as a standard in both quantification and spiking studies).

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