Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer

Abstract Pseudomonas putidais a promising bacterial chassis for metabolic engineeringgiven its ability to metabolize a wide array of carbon sources, especially aromatic compounds derived from lignin. However, this omnivorousmetabolism 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 (Zhang et al., 2017). To better understand this metabolism we use a combination of Random Barcode Transposon Sequencing(RB-TnSeq) and shotgun proteomicsto 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 degradationunder 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 h 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 engineeringefforts.