Identification of Thermoregulatory Factors Involved in Validamycin Biosynthesis and Reconstitution of a...

Thermoregulatory systems are rarely utilized in the synthetic biology of Streptomyces. In Streptomyces hygroscopicus TL01, the previous finding of enhanced validamycin biosynthesis at 37°C indicated the involvement of a thermoregulatory system. In this study, the P_valA (P_KA) promoter was thermoregulated in Streptomyces albus J1074, and its regulation was determined by a GC-rich DNA clamp. P_KA-binding regulatory proteins were identified through DNA affinity chromatography. Overexpression of SHJG_5852 and SHJG_8253 in S. albus resulted in the P_KA strength ratios of 2.61 and 4.34 at 37°C and 30°C, respectively. In contrast, the overexpression of SHJG_6392 led to an inhibited transcription of the P_KA at 30°C. A thermoregulatory system was reconstituted in S. albus J1074 with a shorter but more efficient promoter derived from the P_KA, SHJG_5852, and SHJG_6392. With this system, the induced indigoidine titer at 37°C was 3.56 times that at 30°C. In summary, we identified thermoregulatory factors involved in validamycin biosynthesis and reconstituted a system for the thermoregulated production of actinobacterial natural products.