Additional file 1 of Systems biology of industrial oxytetracycline production in Streptomyces rimosus: the secrets of a mutagenized hyperproducer
Creators: name: Beganovic, Selma
name: Rückert-Reed, Christian
name: Sucipto, Hilda
name: Shu, Wei
name: Gläser, Lars
name: Patschkowski, Thomas
name: Struck, Ben
name: Kalinowski, Jörn
name: Luzhetskyy, Andriy
name: Wittmann, Christoph
Publisher: name: figshare
Year: 2024
Subjects: subject: Microbiology
subject: FOS: Biological sciences
subject: Genetics

description: Additional file 1: Fig. S1. Impact of the nutrient environment on oxytetracycline production in S. rimosus. Production by strains ATCC 10970 and HP126 in complex starch-based medium (A) and production by strain ATCC 10970 in minimal medium with different sugars as carbon sources (10 g L−1) (B). The data represent the final oxytetracycline titer at the end of cultivation after 120 h. n = 3. The complex starch-based medium did not appear to be optimal for the planned systems biology analyses. It could not be ruled out that the two strains use the contained proportions of soybean meal and corn steep liquor differently, which would have significantly complicated the interpretation of the multiomics data [4]. Therefore, a minimal, fully defined medium was desired for the subsequent investigations, which could reveal the phenotypic differences between the strains [23]. A mineral medium containing a limiting amount of phosphate to trigger the formation of oxytetracycline [63] was tested for this purpose. The basic salt mixture was amended with the following different sugars as the sole carbon and energy source to identify the most suitable setup: five hexoses (galactose, glucose, fructose, fucose, rhamnose), two pentoses (arabinose, xylose), a sugar alcohol (mannitol), and a disaccharide (maltose). Test tubes filled with 1 mL of medium were used to screen for the substrate that led to the best production in the wild type (B). Notably, the strain produced measurable amounts of oxytetracycline on all tested carbon sources, revealing a broad substrate spectrum. The highest titer (17 mg L−1) was observed on mannitol. It was almost twice as high as that achieved on the hexose sugars. In comparison, oxytetracycline formation on the two pentoses and maltose was even smaller (between 1 and 2 mg L−1), indicating that the carbon source substantially impacted production efficiency. Based on the outcome, mannitol appeared most suitable. A test tube culture with strain HP126 on mannitol-based medium yielded 200 mg L−1 oxytetracycline, almost tenfold more than that observed for the wild type. Obviously, the minimal medium with sugar alcohol reflected the different production performance of both strains very well, as desired for the planned systems biology comparison. This medium was therefore selected for further studies. Generally, all test tube and shake flask cultures revealed excellent reproducibility (Fig. 2). Fig. S2. Heatmap of the sample-to-sample distances. The read count data were fed to DESeq2 [77] to calculate normalized read counts. After regularized log transformation was performed with blind dispersion estimation, the sample-to-sample distances were calculated and used for hierarchical clustering, which was then visualized using pheatmap [86]. Samples were taken from cultures of S. rimosus ATCC 10970 (R7), its mutagenized oxytetracycline-overproducing derivative HP126 (HP) and the chassis strain HP126 ∆v3 (DV3) after 24 h. n = 3. Fig. S3. Statistical evaluation of gene expression profiles using PCA. For the calculation of normalized read counts, the raw read count data were processed by DESeq2 [87], including regularized log transformation (with blind dispersion estimation enabled). Subsequently, PCA was performed and visualized using ggplot2 [88]. Samples were taken from cultures of S. rimosus ATCC 10970 (R7), its mutagenized oxytetracycline-overproducing derivative HP126 (HP) and the chassis strain HP126 ∆v3 (DV3) after 24 h. n = 3. Table S1. Copy number of the genes related to major catabolic and anabolic processes in S. rimosus ATCC10970, S. rimosus HP126 and S. rimosus HP126 Δv3. Table S2. BlastP identification of morphology regulators and their expression levels in S. rimosus HP126 and S. rimosus HP126 Δv3.