Manipulating The Expression Of BulZ And Its Target Gene Product To Increase Tacrolimus Production

So far,regulatory factors and mechanism of tacrolimus biosynthesis have been poorly investigated in S.tsukubaensis.In most Streptomyces species,antibiotic production and/or morphological differentiation can be affected by the signal molecule GBL.In this study,the regulatory mechanism of the SARP family regulator BulZ in tacrolimus biosynthesis and morphological differentiation of S.tsukubaensis was explored.A knockout of bulZ resulted in a 47.5% decrease of tacrolimus production and a delay of spore differentiation.We identified that BulZ,a SARP family regulator,acted as a positive regulator for spore differentiation and tacrolimus production.Using quantitative real-time PCR(qRT-PCR)analysis and electrophoretic mobility shift assays(EMSAs),it was found that BulZ directly activated the transcription of itself and bulS2,a putative GBL synthetase.Moreover,bulS2 was shown to play a positive role in tacrolimus biosynthesis.At the same time,BulZ was able to indirectly regulate the expression of the cluster-linked activator genes tcs7 and fkbN,as well as the GBL receptor gene bulR1.Co-overexpression of bulZ and its target gene bulS2 improved tacrolimus production by 36% compared to the control strain,reaching 324 mg/L.Moreover,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 sporation.Our results provide evidence for a role of BulZ as a pleiotropic activator for tacrolimus biosynthesis and spore differentiation.The insights obtained in this study will help further provide new avenues for further improvement of tacrolimus production.