According to the computational chemistry of the most stable conformation, the two tryptophan sites of tetratryptomycin A (3) are located a long distance from each other, making macrocyclization a challenging task. In fact, as mentioned above, more than 100 screenings employing chemical and bio-oxidation (e.g., chicken and pig liver) conditions did not result in macrocyclization, as the tryptophan moiety was oxidized to oxindole as a byproduct and halogenated. On the other hand, the CttpC enzyme catalyzes the macrocyclization of tetratryptomycin A (3) to afford cyctryptomycin A and B (1-2). 1 Therefore, we investigated the conditions under which the specific gene for this essential enzyme, CttpC, could be introduced into S. coelicolor M1146 and cultivated in large quantities. After investigating various culture conditions, it was found that the most effective method was to place approximately 6 L of S. coelicolor M1146 culture medium containing the specific gene for CttpC and 3.54 g of tetratryptomycin A (3) in 118 Meyer flasks of 50 ml each and react at 28-30 °C (see the Supporting Information). This largescale culture successfully grew 154 g of S. coelicolor M1146 expressing CttpC in one cycle (see the photo in Scheme 1B). Ensuring adequate oxygen was especially important for the scalable preparation of S. coelicolor M1146. Specifically, it was crucial to avoid the utilization of Meyer flasks (3 L or 5 L) that were excessively voluminous. In fact, after scrutinizing the conditions for mass culture, we endeavored to cultivate S. coelicolor M1146 in sizable Meyer flasks (3 L or 5 L), but the number of S. coelicolor M1146 colonies observed was insufficient and the cultivation of the organisms was unsuccessful. Furthermore, the inclusion of tetratryptomycin A (3) did not result in a reaction or any progress in macrocyclization. Since stirring efficiency is important for the culture of S. coelicolor M1146, it is likely that large Meyer flasks such as 3 L or 5 L were not sufficient to ensure oxygenation (see the Supporting Information). After careful examination of various culture conditions, it was interesting to note that many colonies of S. coelicolor M1146 were observed in relatively small Meyer flasks, such as 125 ml and 50 ml, and that the culture progressed well (Scheme 1B). Notably, this method proceeded well on a 3.5 g scale and finally led to the synthesis of cyctetryptomycin A and B (1-2) in 8 steps.