The DNA fragments containing an entire otc gene clusters (Additional file 1: Figure S1) were cloned directly from the genomic DNA of the HP0508 strain using a single-step cloning approach based on TAR cloning in yeast [19]. The HP0508 strain was cultivated in TSB liquid medium. Genomic DNA was isolated following standard procedures [40], and was digested using SpeI as an overnight reaction at 37 °C. The digested DNA fragments were precipitated, washed with 70 % ethanol, and dissolved in 100 µL 20 % Tris-EDTA buffer. The primers OTC hook-up_F, OTC hook-up_R, OTC hook-down_F and OTC hook-down_R were use to amplify two homologous regions bordering the otc gene cluster by PCR (Phusion High-Fidelity DNA Polymerase; Thermo Scientific), both of which were ~ 1 kb in size. The two homologs were then digested by BamHI and KpnI and simultaneously cloned into the pYAC-ΦC31-Ts vector. Prior to TAR cloning, the pYAC-ΦC31-Ts vector with the two homologous ‘hooks’, was linearized with SpeI (Additional file 1: Figure S1). Preparation of the yeast spheroplast was following a previous protocol [41], and TAR cloning of the otc gene cluster from genomic DNA was performed following the protocol [19, 42].

The presence of an entire otc cluster cloned into the pYAC-ΦC31-Ts-OTC in the yeast transformants was confirmed initially by colony PCR screening using four strategic sequence-tagged site primer pairs specific for amplification of otc gene cluster sequences (STS-OT, STS-OS, STS-OH, STS-OP; Additional file1: Figure S1, Additional file 1: Table S1), which were located in the middle and extremities of the otc gene cluster. The transformants that showed positive PCR amplicons with all four sequence-tagged-site primer pairs were further analyzed by restriction analysis after re-transformation of the plasmid from the yeast transformants into E. coli DH10β. Plasmid DNA that presumably contained the entire otc gene cluster was isolated, and restriction analysis was completed from each individual E. coli transformant. Plasmid DNA was isolated from several independent E. coli transformants to confirm their genetic stabilities in both yeast and E. coli. All of the analyzed plasmid clones showed the same restriction enzyme pattern with SacI restriction enzyme, corresponding to the expected restriction enzyme pattern of the cloned genomic fragment containing the entire otc gene cluster. Nonmethylated DNA of the pYAC-ΦC31-Ts-OTC plasmid was isolated from E. coli ET12567 and used for further transformation into different S. rimosus strains.

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