Identification and enzyme studies of rare amino acid biosynthesis from Streptomyces cattleya

Streptomyces--Metabolism;Microbiological synthesis;Toxins--Synthesis;Amino acids--Synthesis;Isomerases

Chan, K. K. Jason ; O'Hagan, David ; O'Hagan, David

University:University of St Andrews

Department:Chemistry (School of)

关键词: Streptomyces--Metabolism;    Microbiological synthesis;    Toxins--Synthesis;    Amino acids--Synthesis;    Isomerases;   

Others  :  https://research-repository.st-andrews.ac.uk/bitstream/handle/10023/4478/JasonChanPhDThesis.pdf?sequence=3&isAllowed=y

来源: DR-NTU

【 摘 要 】

This thesis is focussed on the biosynthesis of three toxins: fluoroacetate, 4-fluoro-L-theronine and β-ethynyl-L-serine which are biosynthesised by the soil bacteria Streptomyces cattleya. The two fluorinated metabolites originate from a common biosynthetic pathway and the thesis describes studies carried out on an aldose-ketose isomerase enzyme of the pathway. The biosynthetic origin of β-ethynyl-L-serine is not known. A total synthesis of this acetylenic amino acid is descibed along with the development of a new analytical method for identifying the metabolite and for future isotope-labelling based biosynthetic studies.Chapter 1 presents the background of this research. It is focussed on the biosynthesis of fluoroacetate and 4-fluoro-L-threonine by S. cattleya and it also introduces alkyne-containing natural products and their biosynthesis.Chapter 2 describes the work carried out on crystallisation of the aldose-ketose isomerase of the fluorometabolite pathway in S. cattleya. Crystals of the isomerase were obtained and they were diffracted by X-ray, however a structure could not be solved.Chapter 3 contains site-directed mutagenesis studies of the isomerase from S. cattleya.Chapter 4 describes an enantioselective total synthesis of β-ethynyl-L-serine. A robust analytical technique based on derivatisation using 'Click' chemistry and LC-MS was developed for the detection of this amino acid directly from the fermentation broth.Chapter 5 details the experimental procedures for compounds synthesised in this thesis and the biological procedures for gene cloning and protein purification.