High-Throughput Screens and Selections for Microbially Produced Diacids

Production of renewably sourced industrial chemicals using microbial production processes requires extensive strain engineering to improve strain titers (g/L), productivities (g/L/hr), and yields (g-product/g-feedstock). Microbial strains are typically optimized by screening for incremental improvements in production using high-throughput screening or selection assays; however, this remains exceedingly difficult for the vast majority of chemicals currently being targeted for microbial production. Design and implementation of cost-effective, high-throughput screening and selection methods would greatly accelerate R & amp;D efforts and improve prospects for successful commercialization of microbially produced industrial chemicals. Lygos is addressing this problem through construction of a biosensor for detection of dicarboxylic acids (C4-C7 chain-lengths). Dicarboxylic acids are a multi-billion dollar class of compounds being extensively investigated as targets for microbial production; furthermore, the technology could be readily extrapolated toward a wide range of industrially important chemicals. In Phase I of this SBIR grant application, biosensor(s) for the target diacids are constructed and characterized. The biosensors will be constructed using diacid-responsive transcription factors; they will be subsequently characterized as spectrophotometric assays measuring diacid titers from production strains in 96-well plate format. In Phase II, large libraries ( & gt10,000 strains) of diacid-producing microbes will be constructed and assayed for improved productivities using the biosensor screen. Specific product targets in Phase II implementation include succinic, adipic, or pimelic acids. Success of this technology at Phase I and Phase II grant levels will accelerate commercialization of microbially produced diacids and ensure cost-competitiveness with petrochemical routes.