title: A Sense of Balance: Experimental Investigation and Modeling of a Malonyl-CoA Sensor in Escherichia coli.
abstract: Production of value-added chemicals in microorganisms is regarded as a viable alternative to chemical synthesis. In the past decade, several engineered pathways producing such chemicals, including plant secondary metabolites in microorganisms have been reported; upscaling their production yields, however, was often challenging. Here, we analyze a modular device designed for sensing malonyl-CoA, a common precursor for both fatty acid and flavonoid biosynthesis. The sensor can be used either for high-throughput pathway screening in synthetic biology applications or for introducing a feedback circuit to regulate production of the desired chemical. Here, we used the sensor to compare the performance of several predicted malonyl-CoA-producing pathways, and validated the utility of malonyl-CoA reductase and malonate-CoA transferase for malonyl-CoA biosynthesis. We generated a second-order dynamic linear model describing the relation of the fluorescence generated by the sensor to the biomass of the host cell representing a filter/amplifier with a gain that correlates with the level of induction. We found the time constants describing filter dynamics to be independent of the level of induction but distinctively clustered for each of the production pathways, indicating the robustness of the sensor. Moreover, by monitoring the effect of the copy-number of the production plasmid on the dose-response curve of the sensor, we managed to coarse-tune the level of pathway expression to maximize malonyl-CoA synthesis. In addition, we provide an example of the sensor's use in analyzing the effect of inducer or substrate concentrations on production levels. The rational development of models describing sensors, supplemented with the power of high-throughput optimization provide a promising potential for engineering feedback loops regulating enzyme levels to maximize productivity yields of synthetic metabolic pathways.
authors.affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences , Szeged , Hungary.
authors.forename: Tamás
authors.initials: T
authors.lastname: Fehér
authors.affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; Paris Diderot University , Paris , France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Libis
authors.affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; Research Programme on Biomedical Informatics (GRIB), Department of Experimental and Health Sciences, Hospital del Mar Medical Research Institute (IMIM), Universitat Pompeu Fabra , Barcelona , Spain ; SYNBIOCHEM Center, Manchester Institute of Biotechnology, School of Chemistry, University of Manchester , Manchester , UK.
authors.forename: Pablo
authors.initials: P
authors.lastname: Carbonell
authors.affiliation: Institute of Systems and Synthetic Biology, University of Evry Val d'Essonne , Evry , France ; SYNBIOCHEM Center, Manchester Institute of Biotechnology, School of Chemistry, University of Manchester , Manchester , UK.
authors.forename: Jean-Loup
authors.initials: JL
authors.lastname: Faulon
keywords: dynamic pathway regulation; fluorescent reporter circuit; high-throughput screening; malonyl-CoA; sensor–actuator circuit; synthetic regulatory circuit