Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer
Abstract
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.
- Authors:
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE)
- OSTI Identifier:
- 1564441
- Alternate Identifier(s):
- OSTI ID: 1570232
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Published Article
- Journal Name:
- Metabolic Engineering Communications
- Additional Journal Information:
- Journal Name: Metabolic Engineering Communications Journal Volume: 9 Journal Issue: C; Journal ID: ISSN 2214-0301
- Publisher:
- Elsevier
- Country of Publication:
- Netherlands
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
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., and Keasling, Jay D.. Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer. Netherlands: N. p., 2019.
Web. doi:10.1016/j.mec.2019.e00098.
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.. Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer. Netherlands. https://doi.org/10.1016/j.mec.2019.e00098
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., and Keasling, Jay D.. Sun .
"Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer". Netherlands. https://doi.org/10.1016/j.mec.2019.e00098.
@article{osti_1564441,
title = {Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer},
author = {Thompson, Mitchell G. and Valencia, Luis E. and Blake-Hedges, Jacquelyn M. and Cruz-Morales, Pablo and Velasquez, Alexandria E. and Pearson, Allison N. and Sermeno, Lauren N. and Sharpless, William A. and Benites, Veronica T. and Chen, Yan and Baidoo, Edward E. K. and Petzold, Christopher J. and Deutschbauer, Adam M. and Keasling, Jay D.},
abstractNote = {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.},
doi = {10.1016/j.mec.2019.e00098},
journal = {Metabolic Engineering Communications},
number = C,
volume = 9,
place = {Netherlands},
year = {2019},
month = {12}
}
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https://doi.org/10.1016/j.mec.2019.e00098
https://doi.org/10.1016/j.mec.2019.e00098
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Figure 1: Identification of the P. putida valerolactam hydrolase: (A) Route of valerolactam catabolism through the L-lysine catabolic route of P. putida (B) Growth of P. putida in minimal medium supplemented with either 10mM glucose, 5AVA, or valerolactam. Shaded area represents the 95% confidence interval (cI), n = 3. Maximalmore »
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Catabolism of biogenic amines in Pseudomonas species
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