The pYAC-ΦC31-Ts-OTC plasmid (see Sect. "Cloning of the entire otc gene cluster from S. rimosus HP0508 by the single-step transformation associated recombination approach".) that contained the entire otc gene cluster was transformed into the ATCC 10970, M4018 or HP0508 strains via conjugation [39], as described in Sect. "Strains and plasmids". Exconjugants were selected, cultivated, and analyzed as described in Sects. "Media and culture conditions" and "High performance liquid chromatography". Sixteen morphologically stable M4018 transformants were selected for two re-test fermentations, to evaluate the consistency of the OTC production. In the first re-test fermentation method as described in Sect. "Media and culture conditions" was used. In the second re-test fermentation a plug from a sporulating colony on MS medium was inoculated into 5 mL GOTC-V medium and cultivated at 28 °C with 220 rpm shaking, for 24 h. After 24 h, 25 mL production medium (in a 250 mL glass flask) was inoculated with 10 % inoculum from seed cultures and cultivated at 28 °C with 220 rpm shaking and 60 % humidity for 5 days. The broth was then acidified and the OTC extracted as described in Sect. "High performance liquid chromatography". This test was repeated twice. Out of 16 M4018::otc transformants, six were selected for further analysis of genomic stability of the integrated plasmid and identification of the otc copies inserted into the S. rimosus genome.

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