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

Thompson, Mitchell G.; Valencia, Luis E.; Blake-Hedges, Jacquelyn M.; Cruz-Morales, Pablo; Velasquez, Alexandria E.; Pearson, Allison N.; Sermeno, Lauren N.; Sharpless, William A.; Benites, Veronica T.; Chen, Yan; Baidoo, Edward E. K.; Petzold, Christopher J.; Deutschbauer, Adam M.; Keasling, Jay D.

doi:10.1016/j.mec.2019.e00098

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

Journal Article · 10 August 2019 · Metabolic Engineering Communications

DOI: https://doi.org/10.1016/j.mec.2019.e00098 · OSTI ID:1564441

Thompson, Mitchell G. ^[1]; Valencia, Luis E. ^[2]; Blake-Hedges, Jacquelyn M. ^[3]; Cruz-Morales, Pablo ^[4]; Velasquez, Alexandria E. ^[5]; Pearson, Allison N. ^[5]; Sermeno, Lauren N. ^[5]; Sharpless, William A. ^[5]; Benites, Veronica T. ^[5]; Chen, Yan ^[5]; Baidoo, Edward E. K. ^[5]; Petzold, Christopher J. ^[5]; Deutschbauer, Adam M. ^[6]; Keasling, Jay D. ^[7]

Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology
Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Univ. of California, Berkeley/San Francisco, CA (United States). Joint Program in Bioengineering
Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division and Dept. of Chemistry
Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Inst. Tecnologica y de Estudios Superiores de Monterrey, Monterrey (Mexico). Centro de Biotecnologia FEMSA
Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division
Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division
Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division; Univ. of California, Berkeley/San Francisco, CA (United States). Joint Program in Bioengineering; Univ. of California, Berkeley, CA (United States). Dept. of Chemical and Biomolecular Engineering; Technical Univ. of Denmark (Denmark), The Novo Nordisk Foundation Center for Biosustainability; Shenzhen Inst. for Advanced Technologies, Shenzhen (China). Inst. for Synthetic Biology, Center for Synthetic Biochemistry

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 titer of valerolactam from undetectable after 48h 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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1564441

Alternate ID(s):: OSTI ID: 1570232

Journal Information:: Metabolic Engineering Communications, Vol. 9, Issue C; ISSN 2214-0301

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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