Additional file 1 of Multiple copies of the oxytetracycline gene cluster in selected Streptomyces rimosus strains can provide significantly increased titers
Creators: name: Špela Pikl
name: Rincón, Andrés Felipe Carrillo
name: Slemc, Lucija
name: Dušan Goranovič
name: Avbelj, Martina
name: Krešimir Gjuračić
name: Sucipto, Hilda
name: Stare, Katja
name: Špela Baebler
name: Šala, Martin
name: Meijin Guo
name: Luzhetskyy, Andriy
name: Petković, Hrvoje
name: Magdevska, Vasilka
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Genetics
subject: Molecular Biology
subject: Evolutionary Biology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: FOS: Earth and related environmental sciences
subject: Ecology
subject: 69999 Biological Sciences not elsewhere classified
subject: Computational Biology

description: Additional file 1: Table S1: Primers used in this study, and their respective sequences. RT-qPCR TaqMan MGB probes (P) were labeled with FAM at 5’, and nonfluorescent quencher (NFQ) at 3’. Table S2: Relative gene expression data for a reference (16S rRNA) and 5 target genes. Relative copy numbers (obtained by quantification using standard curve), relative copy numbers, normalized to the reference gene and relative normalized copy numbers, scaled to the lowest expression value for each individual gene (presented in Figure 5), are shown. Relative copy numbers are not comparable between genes. Table S3: Strains and plasmids used in this study. Figure S1. Schematic presentation of the plasmid containing the entire otc gene cluster, obtained via a single-step transformation-associated recombination cloning approach. The homologies used to clone the entire gene cluster are located externally at both ends of the otc gene cluster, and are labeled in blue. The S. cerevisiae/ E. coli/ Streptomyces shuttle vector is approximately 7400 bp long, each homology for homologous recombination is approximately 1 kb long, and otc cluster is approximately 28 kb long. The black arrows indicate primers used to screen for the correct plasmid. The red arrows above left and right homology, Hook-Up and Hook-Do, respectively, indicate primers used to create Southern blotting probes, which were used for otc cluster identification in Figure 4. Figure S2: SacI restriction analysis of individual plasmid constructs that were assumed to contain the entire otc gene cluster. The restriction pattern of pYAC-ΦC31-Ts-OTC (38408 bp) digested with SacI, demonstrated seven bands of 11638 bpand 10515 bp (these two appear as one band on the agarose gel), 5029 bp, 4373 bp, 4298 bp, 1885 bp, and 670 bp clones. (a-e) Transformants obtained from genomic DNA of the individual yeast transformants previously selected by colony PCR. (a1-a4) Individual plasmid rescue clones from E. coli. Figure S3:Morphological properties of the wild-type S. rimosus ATCC 10970 strain, the M4018 medium OTC-producing strain, and the HP0508 high OTC-producing strain, and of the same strains with an entire otc cluster deleted. (A, B) Upper of the MS plates, showing S. rimosus ATCC 10970, M4018, and HP0508 (A), and showing the S. rimosus ΔOTC mutants (B). (C, D) Bottom of MS plates, showing of S. rimosus ATCC 10970, M4018 and HP0508 (C), and showing the S. rimosus ΔOTC mutants (D). All three of the strains show slightly darker brown mycelia, as observed at the bottom of the plate. The colour has been correlated with OTC production; however, the pigment is of an unknown source, and its intensity does not directly correspond to the OTC titer, as the M4018 strain, which is a medium OTC-producer, shows the most intense dark color. The dark pigmentation disappears following otc gene cluster deletion. All three of the strains produce white spores on top of the mycelia; however, sporulation is reduced with higher OTC production, and HP0508 shows the lowest number of spores, indicating non-homogenous growth of the spores (Figure S3A, B). Figure S4: HPLC analysis of the OTC produced by different S. rimosus strains and their respective OTC deletion mutants. (A) ATCC 10970. (B) ATCC 10970ΔOTC. (C) M4018. (D) 15883S. (E) HP0508. (F) HP0508ΔOTC. The arrows indicate OTC with a retention time of approximately 18.2 min. OTC was quantified using a standard and calculated from the area under curve. Figure S5: OTC titer following complementation of the S. rimosus ATCC10970 ΔOTC, 15883S, and HP0508 ΔOTC deletion mutants with an entire otc gene cluster. Blue bars, relative OTC titers of the parent S. rimosus strains: ATCC 10970, M4018, HP0508. Orange bars, relative OTC titers of the complemented ΔOTC mutants. Figure S6: OTC titers from the independent transformants carrying the pYAC-ΦC31-Ts-h control plasmid (i.e., without the otc cluster) in S. rimosus ATCC 10970, M4018, and HP0508. Blue bars, relative OTC titers of the parent S. rimosus strains: ATCC 10970, M4018, HP0508. Orange bars, relative OTC titers of the independent pYAC-ΦC31-Ts-h transformants. The significance of the variability was tested with two-tailed Mann-Whitney tests. No significant variability was detected for the ATCC 10970 strain transformed with the control plasmid (P &gt; 0.05), while significant variability was detected for the M4018 and HP0508 transformants with the control (P