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Title: Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440

Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Furthermore, heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. In order to improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, -35 sequences, and -10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-foldmore » range of protein expression levels. One additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.« less
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  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
Publication Date:
Grant/Contract Number:
Published Article
Journal Name:
Metabolic Engineering Communications
Additional Journal Information:
Journal Volume: 5; Journal Issue: C; Journal ID: ISSN 2214-0301
Research Org:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC); USDOE Office of Energy Efficiency and Renewable Energy (EERE), Bioenergy Technologies Office (EE-3B)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; Pseudomonas putida; Genetic engineering; Promoter library; Site-specific recombinase; Gene expression
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1394234