Host engineering for improved valerolactam production in Pseudomonas putida

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 produ...

Full description

Saved in:
Bibliographic Details
Published inbioRxiv
Main Authors Thompson, Mitchell, Valencia, Luis E, Blake-Hedges, Jacquelyn, Cruz-Morales, Pablo, Velasquez, Alexandria, Pearson, Allison, Sermeno, Lauren, Sharpless, William, Benites, Veronica, Chen, Yan, Baidoo, Edward, Petzold, Christopher J, Deutschbauer, Adam, Keasling, Jay D
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 06.06.2019
Subjects
Aromatic compounds
Biodegradation
Caprolactam
Carbon sources
Gene deletion
Hydrolase
Lignin
Lysine
Metabolic engineering
Metabolism
Proteomics
Pseudomonas putida
Online AccessGet full text

Cover

More Information
Summary: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. 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 yield 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.
DOI:10.1101/660795