2.3. AP-3 Resistance Evaluation

Resistance evaluation on solid medium was performed with petri dishes (d = 3.5 cm). Before resistance evaluation, glycerol stocks of WXR-24 (high-yield AP-3 producer of A. pretiosum) and WXR-30 (ftsZ-overexpressing mutant of A. pretiosum) were diluted with 20% glycerol to have around 100 colony forming units (CFU) per 1 μL. For resistance evaluation, each Petri dish was filled with 4 mL YMG solid medium containing different concentrations of AP-3. Then 20 μL diluted stock of WXR-24 or WXR-30 were spread on the dishes. The dishes were incubated at 30 °C for 72 h and taken out for pictures and colony counting.

To evaluate strain resistance in liquid medium, WXR-24 and WXR-30 were initially cultivated on YMG plates for two days, followed by consecutive cultivation in S1 and S2 media. Then 150 μL of S2 was transferred into a well of 24-deep-well plate, in which 1.5 mL fermentation medium containing different concentrations of AP-3 was added. For growth comparison of WXR-24 and WXR-30, the S2 cultures of the two strains were diluted to obtain similar CFUs before inoculation. Strain resistance was evaluated by growth determination from day 1 to day 5 using a microplate reader.

For AP-3 supplementation, AP-3 was dissolved in DMSO, and the solution was added to the solid or liquid media before inoculation. Media supplied with same amount of DMSO was used as a control. The final concentration of DMSO was 1.5% (v/v).