The genetic stability of M4018::otc transformant O51 obtained that showed significant increases in OTC titer and containing multiple copies of the chromosomally integrated otc gene cluster was tested with two independent procedures: OTC titer measurements and PFGE analysis.

The genetic stability of M4018::otc was tested by monitoring the OTC titers in the M4018 strain, the M4018::otc O51 transformant, and seven colonies that were derived from colony O51 and M4018::YAC-h transformants. Seven independent and morphologically stable O51-derived colonies were obtained by streaking colony O51 on MS medium to single colonies. Seven single colonies were afterwards patched on MS media to obtain more biomass. A plug of spores from patches on MS plates were inoculated in 5 mL TSB medium without and with thiostrepton, and were cultivated at 28 °C with 220 rpm shaking. After 24 h, 10 % of the culture was used to inoculate 5 mL GOTC production medium, which was then cultivated at 28 °C with 220 rpm shaking and at 60 % humidity, for 5 days. After 5 days the OTC was extracted from the production broth and analyzed as described in Sect. "High performance liquid chromatography".

Finally here, the genetic stability of the selected M4018::otc strains was tested by analysis of the genome via PFGE. Seven colonies derived from the O51 transformant, as described above, were inoculated with a plug from sporulating colonies on MS medium in 5 mL TSB medium, and cultivated at 28 °C with 220 rpm shaking. After 24 h cultivation, the transformants were analyzed on PFGE after restriction with XhoI or AseI rare cutter enzymes, as described in Sect. "Pulse-field gel electrophoresis". The M4018::otc O51 transformant was taken as the control.

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