RRC ID 56349
Author Choi KR, Cho JS, Cho IJ, Park D, Lee SY.
Title Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida.
Journal Metab Eng
Abstract 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.
Volume 47
Pages 463-474
Published 2018-5
DOI 10.1016/j.ymben.2018.05.003
PII S1096-7176(18)30166-6
PMID 29751103
MeSH DNA Transposable Elements Gene Expression* Genetic Engineering / methods* Homologous Recombination Microorganisms, Genetically-Modified* / genetics Microorganisms, Genetically-Modified* / metabolism Multigene Family* Pseudomonas putida* / genetics Pseudomonas putida* / metabolism Recombinant Proteins / biosynthesis Recombinant Proteins / genetics
IF 7.808
Times Cited 14
Resource
Prokaryotes E. coli Cloning Vector pJB658