Actinomycetes are a rich source of secondary metabolites and the origin of half the antibiotics in current use. Sequencing of a number of actinomycete genomes has revealed that most strains have the potential of producing numerous bioactive metabolites. It is estimated that less than 10% of their potential has been explored to date. Accessing this rich source of bioactives has proven difficult due to the complex regulation of secondary metabolism in actinomycetes. During the developmental cycle, a transient stage of growth arrest, known as the ’metabolic switch’, marks the transition from primary to secondary metabolism. Given that the metabolic switch is required for production, and presumably dictates the subset of metabolites produced, understanding the molecular mechanism underpinning this cellular event is of major importance to biotechnology.
Saccharopolyspora erythraea (S. erythraea) is a soil-dwelling filamentous actinomycete used for the industrial production of erythromycin, one of the most popular antibiotics in human medicine. Despite extensive industrial exploitation of S. erythraea, its metabolism remains largely unexplored, with more than 20 potentially bioactive secondary metabolites of unknown chemistry and function. In this thesis, a systems biology platform was developed and used to delineate the control mechanisms around the metabolic switch in S. erythraea.
A set of systems biology tools was developed to explore the developmental cycle of S. erythraea in bioreactors at transcriptional, translational and post-translational level. First, a genome-scale metabolic network was constructed and used to increase erythromycin production by 50% using a precursor supply strategy (chapter II). Reconstruction of the metabolic network highlighted a large number of singletons and the presence of ‘uncommon’ pathways. These results suggested that a re-annotation of the genome was required. Thus, we proceeded to sequence mRNA across the developmental cycle in bioreactors. RNA-seq data was combined with LC-MS/MS proteomics and in silico simulations to report the first functional genome annotation of an actinomycete. Through this process we confirmed the presence of 164 hypothetical proteins and annotated 59 new proteins that mapped to previously un-annotated regions (chapter III). Furthermore, deep characterisation of the transcriptome across the developmental cycle enabled the study of transcriptional dynamics of genomic macro domains, and to identify a targeted mRNA degradation phenomenon during the metabolic switch of S. erythraea. Moreover, using quantitative phosphoproteomics, we described potential regulatory events on enzymes from central metabolism which are involved in secondary metabolic functions. More importantly, we identified for the first time dynamic phosphorylation of proteins involved in the metabolic switch. Finally, we used the collected biological data sets to propose and discuss a cellular mechanism of the metabolic switch of S. erythraea.
The work presented in this thesis represents the first attempt to systematically characterize and understand the mechanisms underpinning the control of the metabolic switch in S. erythraea. This work is a relevant contribution to the current knowledge of the biology of S. erythraea, and provides a solid foundation for the design of strategies to improve the production of erythromycin and to discover new bioactive products.