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

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.

Activation of microbial secondary metabolic pathways: Avenues and challenges

“…The titres of SMs produced in E. coli have been generally low. Other promising heterologous hosts for production of prokaryotic SMs include Myxococcus xanthus , Pseudomonas putida [ 170 ] and Bacillus subtilis [ 171 ].…”

Accelerated genome engineering of Pseudomonas putida by I‐SceI―mediated recombination and CRISPR‐Cas9 counterselection

“…One possible solution could be to implement conditional origins of replication in the current plasmid system, e.g. temperature‐sensitive derivatives of oriV(RK2) (Valla et al ., ) that has been shown to function in P. putida (Choi et al ., ). This will be of particularly value for the curing of plasmid DNA from cells that are severely impaired in growth and are thus less prone to lose plasmids during proliferation.…”

“…Such recombinase‐based genome‐editing approaches have recently been complemented by the design of a RecET system for P . putida (Choi et al ., ; Choi and Lee, ), and Aparicio et al . () further honed the CRISPR‐Cas9 counterselection strategy, combining it with Ssr‐based recombination for genome engineering.…”

Protocols for yTREX/Tn5‐based gene cluster expression in Pseudomonas putida

“…It should be noted that highly sophisticated tools are available to achieve an accurate, marker‐less and efficient chromosomal integration of biosynthetic gene clusters in the P. putida chromosome, for example via phage recombinases (Choi et al ., ). At the same time, a broad variety of calibrated constitutive and inducible promoters has been established (Zobel et al ., ; Calero et al ., ; Elmore et al ., ; Nikel and de Lorenzo, ).…”