SBIR-STTR Award

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.

Dicarboxylic acids are important industrial chemicals currently produced from petroleum using ecologically damaging manufacturing methods; furthermore, dicarboxylic acid prices are rising with increasing petroleum feedstock prices. Lygos is developing microbial hosts for the cost-competitive production of dicarboxylic acids from renewable feedstocks, including cellulosic hydrolysate. The combined Phase I/II approach is the development and use of a high-throughput dicarboxylic acid screen to assay for improved dicarboxylic acid production from engineered microbes. In Phase I, we constructed and characterized three dicarboxylic acid screens applicable for short- to medium-chain microbially produced diacids (up to C7). The screens are based on microbial sensing of exogenous dicarboxylic acid and an associated increase in a colorimetric reporter output. The dicarboxylic acid screen will now be implemented as a high-throughput assay for improved dicarboxylic acid production in engineered microbes. Combinatorial libraries of engineered proteins and metabolic pathways will be tested for improvements in strain yield, titer, and productivity. Dicarboxylic acid production in the evolved strain will be also demonstrated using a cellulosic hydrolysate sugar source. Commercial Applications and Other

Benefits:
Dicarboxylic acids are high-value chemicals used in the manufacturing of commodity and specialty goods. Lygos evolved microbial production platform will enable the cost- competitive production of diacids from renewable feedstocks, providing both an economic and environmental benefit. A key aspect of the grant effort is the production of diacids using cellulose-derived sugars.