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Genetic tools for reliable gene expression and recombineering in Pseudomonas putida

  • Metabolic Engineering and Synthetic Biology - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Pseudomonas putida is a promising bacterial host for producing natural products, such as polyketides and nonribosomal peptides. In these types of projects, researchers need a genetic toolbox consisting of plasmids, characterized promoters, and techniques for rapidly editing the genome. Past reports described constitutive promoter libraries, a suite of broad host range plasmids that replicate in P. putida, and genome-editing methods. To augment those tools, we have characterized a set of inducible promoters and discovered that IPTG-inducible promoter systems have poor dynamic range due to overexpression of the LacI repressor. By replacing the promoter driving lacI expression with weaker promoters, we increased the fold induction of an IPTG-inducible promoter in P. putida KT2440 to 80-fold. Upon discovering that gene expression from a plasmid was unpredictable when using a high-copy mutant of the BBR1 origin, we determined the copy numbers of several broad host range origins and found that plasmid copy numbers are significantly higher in P. putida KT2440 than in the synthetic biology workhorse, Escherichia coli. Lastly, we developed a λRed/Cas9 recombineering method in P. putida KT2440 using the genetic tools that we characterized. This method enabled the creation of scarless mutations without the need for performing classic two-step integration and marker removal protocols that depend on selection and counterselection genes. With the method, we generated four scarless deletions, three of which we were unable to create using a previously established genome-editing technique.

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Acknowledgements

This study was supported by research grants from the National Science Foundation (CBET-114678, MCB-1716594) and US-AID (PEER 3-195). T.B.C. and D.K.C. are recipients of NIH Biotechnology Training Program Fellowships (NIGMS 5 T32 GM08349). J.M.R. was supported by an NSF Graduate Research Fellowship (DGE-1256259). S.A.L. is the recipient of a fellowship from the Promega Corporation through the Dane County Youth Apprenticeship Program in Biotechnology. The authors would like to thank Dr. Yalun Arafin and Dr. Fransiskus Ivan for their assistance with the project.

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Authors and Affiliations

  1. Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, USA

    Taylor B. Cook, Jacqueline M. Rand, Wasti Nurani, Dylan K. Courtney, Sophia A. Liu & Brian F. Pfleger

  2. Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, USA

    Brian F. Pfleger

  3. Waunakee High School, Waunakee, USA

    Sophia A. Liu

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  1. Taylor B. Cook
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Correspondence to Brian F. Pfleger.

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Cook, T.B., Rand, J.M., Nurani, W. et al. Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. J Ind Microbiol Biotechnol 45, 517–527 (2018). https://doi.org/10.1007/s10295-017-2001-5

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