Manipulating the expression of SARP family regulator BulZ and its target gene product to increase tacrolimus production.

Tacrolimus (FK506), an effective immunosuppressant, is widely used in the treatment of autoimmune diseases. In this study, we identified that BulZ, a Streptomyces antibiotic regulatory protein (SARP) family regulator, acted as a positive regulator for spore differentiation and tacrolimus production. A knockout of bulZ resulted in a 47.5% decrease of tacrolimus production and a delay of spore differentiation. Using quantitative real-time PCR (qRT-PCR) analysis and electrophoretic mobility shift assays (EMSAs), it was found that BulZ directly activated the transcriptions of bulZ and bulS2, a putative γ-butyrolactone (GBL) synthetase, and bulS2 was shown to play a positive role in tacrolimus biosynthesis. Meanwhile, BulZ was able to indirectly regulate the transcriptions of the cluster-linked activator genes tcs7 and fkbN, as well as the GBL receptor gene bulR1. STSU_RS22595, which encoded a WhiB family transcriptional regulator, was found to be a previously unknown potential target gene of BulZ based on a whole-genome search of the conserved sequence (5'-TSVAVVVNVNBTSRAGNN-3') of the SARP-binding motifs. Although overexpression of STSU_RS22595 did not result in an obvious enhancement of tacrolimus yield, STSU_RS22595 might have important effects on the spore differentiation process. Finally, co-overexpression of bulZ and its target gene bulS2 improved tacrolimus production by 36% compared to the control strain, reaching 324 mg/L. The insights obtained in this study will help further elucidate the regulatory mechanism of tacrolimus biosynthesis and provide new avenues for further improvement of tacrolimus production.