Sensor-Enabled Alleviation of Product Inhibition in Chorismate Pyruvate-Lyase

• DOI: 10.1021/acssynbio.8b00465
• Publication Date: 2019-03-12T21:57:10Z
• Authors: Ramesh K. Jha; Niju Narayanan; Naresh Pandey; Jeremy M. Bingen; Theresa L. Kern; Christopher W. Jo...; Charlie E. M. Str...; Gregg T. Beckham; Scott P. Hennelly; Taraka Dale
• Keywords: Pseudomonas putida; Product inhibition; Metabolic Engineering; High-Throughput Screening; Bioproduction

Product inhibition is a frequent bottleneck in industrial enzymes, and testing mutations to alleviate product via traditional methods remains challenging as many variants need be tested against multiple substrate concentrations. Further, screening are conducted vitro, resulting enzyme may perform differently vivo the context of whole-cell metabolism regulation. In this study, we address these two problems by establishing high-throughput method an industrially relevant enzyme, chorismate pyruvate-lyase (UbiC). First, engineered highly specific, genetically encoded biosensor for 4-hydroxybenzoate (4HB) host, Pseudomonas putida KT2440. We subsequently applied detect activity heterologously expressed UbiC that converts into 4HB pyruvate. By using benzoate surrogate inhibits without activating biosensor, were able efficiently create screen diversified library with reduced inhibition. Introduction improved variant experimental production strain precursor cis,cis-muconic acid (muconate), enabled >2-fold yield improvement glucose muconate conversion when new was from plasmid 60% increase same genomically integrated strain. Overall, work demonstrates coupling catalysis biosensing, can identified, result increased titers downstream molecule interest.