Metabolomics methods for the synthetic biology of secondary metabolism.

Many microbial secondary metabolites are of high biotechnological value for medicine, agriculture, and the food industry. Bacterial genome mining has revealed numerous novel secondary metabolite biosynthetic gene clusters, which encode the potential to synthesize a large diversity of compounds that have never been observed before. The stimulation or "awakening" of this cryptic microbial secondary metabolism has naturally attracted the attention of synthetic microbiologists, who exploit recent advances in DNA sequencing and synthesis to achieve unprecedented control over metabolic pathways. One of the indispensable tools in the synthetic biology toolbox is metabolomics, the global quantification of small biomolecules. This review illustrates the pivotal role of metabolomics for the synthetic microbiology of secondary metabolism, including its crucial role in novel compound discovery in microbes, the examination of side products of engineered metabolic pathways, as well as the identification of major bottlenecks for the overproduction of compounds of interest, especially in combination with metabolic modeling. We conclude by highlighting remaining challenges and recent technological advances that will drive metabolomics towards fulfilling its potential as a cornerstone technology of synthetic microbiology.

Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor.

BACKGROUND: The transition from exponential to stationary phase in Streptomyces coelicolor is accompanied by a major metabolic switch and results in a strong activation of secondary metabolism. Here we have explored the underlying reorganization of the metabolome by combining computational predictions based on constraint-based modeling and detailed transcriptomics time course observations. RESULTS: We reconstructed the stoichiometric matrix of S. coelicolor, including the major antibiotic biosynthesis pathways, and performed flux balance analysis to predict flux changes that occur when the cell switches from biomass to antibiotic production. We defined the model input based on observed fermenter culture data and used a dynamically varying objective function to represent the metabolic switch. The predicted fluxes of many genes show highly significant correlation to the time series of the corresponding gene expression data. Individual mispredictions identify novel links between antibiotic production and primary metabolism. CONCLUSION: Our results show the usefulness of constraint-based modeling for providing a detailed interpretation of time course gene expression data.

The dynamic architecture of the metabolic switch in Streptomyces coelicolor.

BACKGROUND: During the lifetime of a fermenter culture, the soil bacterium S. coelicolor undergoes a major metabolic switch from exponential growth to antibiotic production. We have studied gene expression patterns during this switch, using a specifically designed Affymetrix genechip and a high-resolution time-series of fermenter-grown samples. RESULTS: Surprisingly, we find that the metabolic switch actually consists of multiple finely orchestrated switching events. Strongly coherent clusters of genes show drastic changes in gene expression already many hours before the classically defined transition phase where the switch from primary to secondary metabolism was expected. The main switch in gene expression takes only 2 hours, and changes in antibiotic biosynthesis genes are delayed relative to the metabolic rearrangements. Furthermore, global variation in morphogenesis genes indicates an involvement of cell differentiation pathways in the decision phase leading up to the commitment to antibiotic biosynthesis. CONCLUSIONS: Our study provides the first detailed insights into the complex sequence of early regulatory events during and preceding the major metabolic switch in S. coelicolor, which will form the starting point for future attempts at engineering antibiotic production in a biotechnological setting.