PT - JOURNAL ARTICLE AU - Matthew R. Incha AU - Mitchell G. Thompson AU - Jacquelyn M. Blake-Hedges AU - Allison N. Pearson AU - Matthias Schmidt AU - Adam M. Deutschbauer AU - Jay D. Keasling TI - Leveraging host metabolism for bisdemethoxycurcumin production in <em>Pseudomonas putida</em> AID - 10.1101/753889 DP - 2019 Jan 01 TA - bioRxiv PG - 753889 4099 - http://biorxiv.org/content/early/2019/08/31/753889.short 4100 - http://biorxiv.org/content/early/2019/08/31/753889.full AB - Pseudomonas putida is a saprophytic bacterium with robust carbon metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase lyase (Ech), resulted in ‘drop-in’ production of the type III polyketide bisdemethoxycurcumin.