Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

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Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable industrial strains producing heterologous bioproducts, requires development of a more efficient method. Current methods rely on time-consuming homologous recombination techniques and transposon-mediated random insertions. Here we report a RecET recombineering system for markerless integration of heterologous genes into the P. putida chromosome. The efficiency and capacity of the recombineering system were first demonstrated by knocking out various genetic loci on the P. putida chromosome with knockout lengths widely spanning 0.6-101.7 kb. The RecET recombineering system developed here allowed successful integration of biosynthetic gene clusters for four proof-of-concept bioproducts, including protein, polyketide, isoprenoid, and amino acid derivative, into the target genetic locus of P. putida chromosome. The markerless recombineering system was completed by combining Cre/lox system and developing efficient plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest.
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Issue Date
2018-05
Language
English
Article Type
Article
Citation

METABOLIC ENGINEERING, v.47, pp.463 - 474

ISSN
1096-7176
DOI
10.1016/j.ymben.2018.05.003
URI
http://hdl.handle.net/10203/242628
Appears in Collection
CBE-Journal Papers(저널논문)
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