Streptomyces coelicolor is the model organism for the genus Streptomyces, which produces many bioactive secondary metabolites with clinical applications. Based on work done in Escherichia coli, the glyoxylate shunt was thought to be the main anapleurotic pathway in S. coelicolor during growth on fatty acids and therefore an important pathway in providing precursors for secondary metabolism. The S. coelicolor genome contains genes for a second anapleurotic pathway, the ethylmalonyl-CoA pathway. The relative importance of both to anapleurosis in streptomycete metabolism was unclear. The function of the glyoxylate shunt was investigated in this thesis using sequence analysis, genetic manipulation, transcriptomics and mathematical modelling. Analysis of orthologues of aceA, ccr and genes encoding tricarboxylic acid (TCA) cycle genes revealed that all are subject to a similar level of purifying selection pressure. The operons of the glyoxylate shunt and the ethylmalonyl-CoA pathway share a 15 bp palindromic motif in their upstream sequences, which was also found upstream of other genes. This suggests an overlap in regulation and thus an overlap in function. The sequence analysis is contradicted by results of experiments with an aceA⁻ aceB1⁻ mutant, which did not display a phenotype during growth on Tween 40, a model carbon source for fatty acids. Results obtained by total RNA sequencing indicate that the ethylmalonyl-CoA pathway is the main anapleurotic pathway during growth of S. coelicolor on fatty acids whereas expression of the glyoxylate shunt is minimal. This apparent contradiction is resolved by hypothesising that the ethylmalonyl-CoA pathway is the main anapleurotic pathway, but that the glyoxylate shunt provides a backup when acyl-CoA thioesters are withdrawn from the ethylmalonyl-CoA pathway for secondary metabolite biosynthesis.Enzymes of the isocitrate branchpoint were isolated following heterologous expression and analysed. The resulting kinetic parameters, as well as their specific activities measured during growth on Tween 40 and additional data from literature, were used to set up a mathematical model of the TCA cycle and the glyoxylate shunt. Simulations of this model predicted that, as growth proceeds from early to mid and late exponential phase, the relative concentrations of TCA cycle intermediates changed from promoting gluconeogenesis to accomodating secondary metabolism. Further model refinement is needed using data on the flux through the ethylmalonyl- CoA pathway as these were unavailable at the time of writing.
|Date of Award||1 Jan 2014|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Paul Herron (Supervisor) & Paul Hoskisson (Supervisor)|