A detailed discussion of further scale-up of macrocyclization of tetratryptomycin A (3) with CttpC is described below (Figure ). The macrocyclization process was monitored by LC-MS while tetratryptomy-cin A (3) was catalyzed by CttpC. The findings revealed that upon adding 1 mg of tetratryptomycin A (3) to 50 ml of S. coelicolor M1146 culture expressing CttpC in a Meyer flask, cyctryptomycin A (1) was the major product obtained, while cyctryptomycin B (2) was the minor product after a 24-hour incubation. Further experiments were conducted on a 10 mg scale, revealing that after 14 days, the starting material 3 was almost depleted, and cyctryptomycin A (1) was the major product obtained, with cyctryptomycin B (2) being the minor product. To further increase the scale, macrocyclization was performed using 30 mg of tetratryptomycin A (3), and after 14 days, the starting material 3 was observed, and although the reaction was not completed, the desired compounds cyctryptomycin A (1) and cyctryptomycin B (2) were obtained in the similar ratio on 10 mg scale. Conversely, on 50 mg scale, the concentration of 3 may have been too high, and after 14 days, a significant amount of starting material 3 remained, resulting in a poor conversion rate of the reaction. Based on these results, it is evident that the most efficient method was to prepare a 50 ml S. coelicolor M1146 culture in a Meyer flask and add 30 mg of tetratrypto-mycin A (3) for macrocyclization, using CttpC as a catalyst. It is worth mentioning that macrocyclization can be performed on a 3.54 g (= 30 mg × 118 batches) scale of tetratryptomycin A (3) in one cycle by simultaneously stirring 118 Meyer flasks in an incubator during the scale-up. The temperature in the incubator was also investigated, and it became clear that it is crucial to maintain the temperature between 28 °C and 30 °C. Conversely, if the temperature exceeds 35 °C, the growth of S. coelicolor M1146 expressing CttpC was inhibited.