TY - JOUR T1 - Host engineering for improved valerolactam production in <em>Pseudomonas putida</em> JF - bioRxiv DO - 10.1101/660795 SP - 660795 AU - Mitchell G. Thompson AU - Luis E. Valencia AU - Jacquelyn M. Blake-Hedges AU - Pablo Cruz-Morales AU - Alexandria E. Velasquez AU - Allison N. Pearson AU - Lauren N. Sermeno AU - William A. Sharpless AU - Veronica T. Benites AU - Yan Chen AU - Edward E. K. Baidoo AU - Christopher J. Petzold AU - Adam M. Deutschbauer AU - Jay D. Keasling Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/06/06/660795.abstract N2 - Pseudomonas putida is a promising bacterial chassis for metabolic engineering given its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorous metabolism can also be a hindrance when it can naturally metabolize products produced from engineered pathways. Herein we show that P. putida is able to use valerolactam as a sole carbon source, as well as degrade caprolactam. Lactams represent important nylon precursors, and are produced in quantities exceeding one million tons per year[1]. To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing (RB-TnSeq) and shotgun proteomics to identify the oplBA locus as the likely responsible amide hydrolase that initiates valerolactam catabolism. Deletion of the oplBA genes prevented P. putida from growing on valerolactam, prevented the degradation of valerolactam in rich media, and dramatically reduced caprolactam degradation under the same conditions. Deletion of oplBA, as well as pathways that compete for precursors L-lysine or 5-aminovalerate, increased the titer of valerolactam from undetectable after 48 hours of production to ~90 mg/L. This work may serve as a template to rapidly eliminate undesirable metabolism in non-model hosts in future metabolic engineering efforts. ER -