Fig 1.
Plasmid maps of pCNA, pSNK, and pBDC.
The helper plasmids pCNA and pSNK, which could be eliminated by incubation at 42°C, were obtained upon insertion of I-CreI and I-SceI, respectively, into pKOBEG. The bla cassette was obtained from pET3b (Novagen), and the kan cassette was obtained from pET28a (Novagen). The p15A ori and cat cassettes of the landing pad plasmid pBDC were obtained from pACYCDuet-1 (commercial plasmid purchased from EMD Biosciences). IC and IS represent the I-CreI endonuclease recognition site and I-SceI endonuclease recognition site, respectively.
Fig 2.
The landing pad plasmid and donor plasmid used for genomic replacement.
(a) The landing pad regions of plasmids pBDC-Yi and pBDC-Yri. (b) Linearized vector map of donor plasmid pBRIS-GC. RCS indicates the reverse complementary sequence.
Fig 3.
Strategy for markerless deletion and genomic replacement.
(a) Progress of markerless deletion. In the first step, the pCNA and pBDC-Xd plasmids were transformed into E. coli DH1. With the addition of l-arabinose, pCNA expressed I-CreI endonuclease and λ-Red recombinases. The plasmid pBDC-Xd was digested by I-CreI endonuclease to release the landing pad fragment, which was the substrate for recombination. The landing pad fragment integrated into the E. coli chromosome. In the second step, the helper plasmid pSNK providing I-SceI endonuclease function was transformed into the host. Finally, the chromosome was cleaved by I-SceI endonuclease at the integration site. Recombination with the two homology regions, 50 bp of the right side of the left homology arm and left side of the right homology arm, led to chromosome repair with DSB-mediated recombination and resulted in a clean deletion. (b) Events of identifying markerless deletion and genomic replacement. In the protocol for markerless deletion, samples were verified by PCR with the primers X-0/X-1 (X = 1, 2, 5, 7, 8, 19, 55, or 63). Positive colonies were about 2300 bp in the first step and about 1000 bp in the second step. In the protocol for genomic replacement, samples were verified by PCR with the primers Y-0/Y-1 (Y = 8, 23, 57, or 64). Positive colonies were about 2300 bp in the first step and about 4500 bp in the second step. (c) Genomic replacement protocol. pBDC-Yi was employed as a landing pad plasmid in the first step. In the second step, the host cells were transformed with pSNK and pBRIS-GC (donor plasmid). pSNK expressed I-SceI endonuclease and λ-Red recombinases when l-arabinose was added. The donor plasmid and chromosome were cleaved at I-SceI endonuclease recognition sites. The integration of the donor fragment was accomplished by the expression of the λ-Red system.
Fig 4.
Chromosomal modification positions.
(a) Eight nonessential regions were chosen for markerless deletion. (b) GC flux was integrated into four positions, symmetrically distributed throughout the genome. The arrows denote the direction of gene expression.
Table 1.
Efficiency of pCNA/pSNK mediated deletions.
Table 2.
Strains used for lycopene production.