Genetic tools for reliable gene expression and recombineering in Pseudomonas putida

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.

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida

“…The robust catabolic pathways of P. putida , while useful for producing valuable molecules from diverse carbon sources, can also serve as an obstacle to achieving high product titers as it can often metabolize the desired products [26]. Given recent advances in gene editing techniques [27–31] and our ability to rapidly assay for gene function with transposon site sequencing [17,18,32], engineering non-model hosts like P. putida for industrial applications has become less challenging.…”

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

“…For comparison, the ColE1/pRO1600 origins of replication used in the previous experiments (13 copies per cell in P. aeruginosa) (Farinha and Kropinski, 1990) were replaced by the broad-host range replication origin RSF1010. Respective copy numbers are high and mostly host-independent (Meyer, 2009), with 130 ± 40 copies reported for P. putida KT2440 (Cook et al, 2018). The RSF1010 origin of replication was tested in combination with all three RBSs, measuring growth rate, Cyp content, and whole-cell activity (Supplementary Table S2).…”

Restoration of wild-type motility to flagellin-knockoutEscherichia coli

“…In the second stage, the p15a replication origin was replaced by RK2 in each of the three medium copy plasmid variants. RK2 was amplified from pBb(RK2)1k-GFPuv [12], which was a gift from Brian Pfleger (University of Wisconsin-Madison), using Q5 DNA polymerase and primers RK2ori_Nhe_F and RK2ori_Hin_R ( Table 2 ). The product was digested with NheI and HindIII, gel purified and ligated into the gel purified plasmid backbones, which had been digested with the same enzymes.…”