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

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

doi:10.1016/j.mec.2019.e00098

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

Metab Eng Commun. 2019 Aug 10:9:e00098. doi: 10.1016/j.mec.2019.e00098. eCollection 2019 Dec.

Authors

Mitchell G Thompson^{1

2

3}, Luis E Valencia^{1

2

4}, Jacquelyn M Blake-Hedges^{1

2

5}, Pablo Cruz-Morales^{1

2

6}, Alexandria E Velasquez^{1

2}, Allison N Pearson^{1

2}, Lauren N Sermeno^{1

2}, William A Sharpless^{1

2}, Veronica T Benites^{1

2}, Yan Chen^{1

2}, Edward E K Baidoo^{1

2}, Christopher J Petzold^{1

2}, Adam M Deutschbauer^{3

7}, Jay D Keasling^{1

2

4

8

9

10}

Affiliations

¹ Joint BioEnergy Institute, 5885 Hollis Street, Emeryville, CA, 94608, USA.
² Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
³ Department of Plant and Microbial Biology, University of California, Berkeley, CA, 94720, USA.
⁴ Joint Program in Bioengineering, University of California, Berkeley/San Francisco, CA, 94720, USA.
⁵ Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
⁶ Centro de Biotecnologia FEMSA, Instituto Tecnologico y de Estudios Superiores de Monterrey, Mexico.
⁷ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁸ Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA.
⁹ The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.
¹⁰ Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China.

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 (Zhang et al., 2017). 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 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 engineering efforts.