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Title: 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:
 [1];  [2];  [3];  [4];  [5];  [5];  [5];  [5];  [5];  [5];  [5];  [5];  [6];  [7]
  1. 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
  2. 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
  3. Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division and Dept. of Chemistry
  4. 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
  5. Joint BioEnergy Inst., Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems & Engineering Division
  6. 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
  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/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
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:
Journal Article: Published Article
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Name: Metabolic Engineering Communications; Journal ID: ISSN 2214-0301
Publisher:
Elsevier
Country of Publication:
United States
Language:
English

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. United States: 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. United States. 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., and Keasling, Jay D. Sat . "Omics-driven identification and elimination of valerolactam catabolism in Pseudomonas putida KT2440 for increased product titer". United States. doi: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},
issn = {2214-0301},
number = ,
volume = ,
place = {United States},
year = {2019},
month = {8}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.mec.2019.e00098

Figures / Tables:

Figure 1 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 » growth rates (1/hr) were 0.29 on glucose, 0.29 on 5AVA, and 0.21 on valerolactam. (C) RB-TnSeq analysis of genome fitness assays of P. putida libraries grown on either 5AVA or valerolactam as a sole carbon source. Red oval shows the predicted fitness result of a valerolactam hydrolase. (D) Results of shotgun proteomics of proteins found in the supernatant of P. putida grown on either 10mM glucose or 10mM valerolactam as a sole carbon source. Venn diagram shows the number of proteins with an exponentially modified protein abundance index (emPAI) relative abundance above 0.1 shared or unique to each carbon source (E) Table shows the most abundant proteins specific to grown on valerolactam. OplA (Q88H50_PSEPK) and OplB (Q88H51_PSEPK) are in bold.« less

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Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.