Identification of Thermoregulatory Factors Involved in Validamycin Biosynthesis and Reconstitution of a Thermoregulated Expression System

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.