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:

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.

Publication Date:

Sun Dec 01 00:00:00 EST 2019

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