Protocols for RecET-based markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

Abstract

Pseudomonas putida has emerged as a promising host for the production of chemicals and materials thanks to its metabolic versatility and cellular robustness. In particular, P. putida KT2440 has been officially classified as a generally recognized as safe (GRAS) strain, which makes it suitable for the production of compounds that humans directly consume, including secondary metabolites of high importance. Although various tools and strategies have been developed to facilitate metabolic engineering of P. putida, modification of large genes/clusters essential for heterologous expression of natural products with large biosynthetic gene clusters (BGCs) has not been straightforward. Recently, we reported a RecET-based markerless recombineering system for engineering P. putida and demonstrated deletion of multiple regions as large as 101.7 kb throughout the chromosome by single rounds of recombineering. In addition, development of a donor plasmid system allowed successful markerless integration of heterologous BGCs to P. putida chromosome using the recombineering system with examples of - but not limited to - integrating multiple heterologous BGCs as large as 7.4 kb to the chromosome of P. putida KT2440. In response to the increasing interest in our markerless recombineering system, here we provide detailed protocols for markerless gene knockout and integration for the genome engineering of P. putida and related species of high industrial importance.

Figure 1

Design of homology arms (HAs) and construction of donor DNAs. A. Scheme of designing 100‐bp HAs and primers for the construction of linear donor dsDNAs. B. Scheme of designing ~1‐kb HAs and primers for the construction of donor plasmids for gene knockout and adaptor plasmids for cloning genes to be integrated. Dashed lines in (A) and (B) indicate sequence homologies and directionality among different constructs. Names of primers mentioned in the protocols (Table S3) are presented next to the primers with some fixed sequences, if available. bla, ampicillin resistance gene; tetA(C), tetracycline resistance gene; HA_L & HA_R, HAs placed left and right to the tetA(C) gene; MCS, multiple cloning site. pUC, pUC origin of replication; p15A, p15A origin of replication. The primer sequences are shown again in Table S3 for better visibility.

Figure 2

Scheme of markerless recombineering of P. putida using the RecET and Cre vectors. This figure shows more detailed procedures of what we previously described (Choi et al., 2018). A. Overall procedure of markerless recombineering of P. putida described in subsection ‘Recombineering of P. putida for gene knockout and integration’ of protocols. Bold numbers indicate corresponding steps of the protocol. Refer to the steps in the protocol for detailed description. For steps x to xv, ampicillin may be added to the plates to maintain the RecET vector for subsequent rounds of recombineering. Dashed lines indicate removal of corresponding element from recombinant strains during incubation. Ap, ampicillin; Km, kanamycin; Tc, tetracycline. B. Vector map of the RecET vector pJB658‐recET. bla, ampicillin resistance gene; RK2, RK2 origin of replication; trfA, gene coding replicase of RK2 origin. C. Vector map of the Cre vector pRK2Cre. aph(3ʹ)‐Ia, kanamycin resistance gene; trfAts, temperature‐sensitive version of the trfA gene.

Figure 3

Examples of markerless recombineering in P. putida. A. Chromosomal regions of P. putida KT2440 examined for markerless deletion using the RecET recombineering system. Red, blue, and purple marks indicate regions knocked out using linear donor dsDNAs, donor plasmid, and both of them respectively. This figure includes the results of the previously reported (Choi et al., 2018) and updated genome engineering. B. Recombinant P. putida strains ΔpvdD::EGFP, ΔpvdD::Flaviolin, and ΔpvdD::Violacein harbouring heterologous biosynthetic genes/clusters of EGFP, flaviolin, and violacein on the chromosome (Table S2) show green, brown, and purple colours of each product respectively. These results have already been reported in our previous paper (Choi et al., 2018), but we took a new picture of engineered strains for better understanding of the protocols in this paper.

Figure 4

Effect of HA length on recombineering efficiency. The number of tetracycline (Tc)‐resistant colonies appeared after recombineering using 1 μg of donor plasmids pTetSac‐ΔpvdD100::Adaptor, pTetSac‐ΔpvdD200::Adaptor, pTetSac‐ΔpvdD400::Adaptor, pTetSac‐ΔpvdD600::Adaptor, pTetSac‐ΔpvdD800::Adaptor, and pTetSac‐ΔpvdD::Adaptor harbouring pairs of 0.1‐, 0.2‐, 0.4‐, 0.6‐, 0.8‐, and 1.0‐kb HAs respectively (Table S1). Successful knockout of the pvdD gene of all eight colonies randomly selected from each knockout experiment – or all colonies for experiments with less than eight colonies – was verified. The values and error bars represent means and standard deviations of colony counts from triplicate experiments, respectively, while all the actual three data points are also shown.