Increasing demands for alternative fuels such as bioethanol has driven the development of more efficient processes. In particular, the use of microbial consortia is a promising approach to dramatically reduce costs and to improve fuel production directly from renewable biomass. However, regulation and coordination of different populations within the consortia remain challenging since currently available intracellular sensing circuits are incapable of responding to the two key substrates, cellulose and hemicellulose, that are present exclusively in the extracellular medium.  To meet these challenges, we developed a new synthetic extracellular sensing circuit to control yeast behaviors by reconstitution of extracellular protein sensors. These synthetic fusion proteins are designed to trigger split intein-mediated reconstitution of functional α-factor peptide analogues in response to nutrient conditions. The reconstituted α-factors bind onto Ste2p, a native yeast G protein-coupled receptor, and activate the yeast mating response and transcriptional induction. By integrating this native mating signaling pathway with heterologous gene expression cassettes, we can control timing and level of induction to direct phenotypic behaviors by applying our split intein fusion proteins in yeast cultures. This synthetic control strategy will enable regulation of yeast functions in a wide range of applications, including consolidated bioprocessing.

Synthetic extracellular sensing circuit by intein-mediated reconstitution of yeast mating factor

251st National Meeting (2016)

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