Abstract
BackgroundOxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes.ResultsA gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli, further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter.ConclusionsA SARP activator, OtcR, was identified in oxy cluster of S. rimosus; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0231-7) contains supplementary material, which is available to authorized users.
Highlights
Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus
Discovery of Streptomyces antibiotic regulatory protein (SARP) regulator OtcR The SARP regulator Ctc11 from the ctc gene cluster of S. aureofaciens was found to activate the expression of oxy cluster in heterologous host S. coelicolor CH999 [13], which strongly suggests that the expression of oxy cluster in S. rimosus is positively regulated by a native SARP regulator
The amino acid sequence of OtcR is closely related to Ctc11. They both have an N terminal DNA-binding domain and a C terminal bacterial transcriptional activation domain (BTAD), but lack the other extra domains usually found in members of the SARP regulators, such as an ATPase domain or tetratricopeptide repeat (TPR) domain [20,21]
Summary
Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. OTC is an aromatic polyketide antibiotic commercially produced by Streptomyces rimosus. The OTC biosynthetic gene cluster is located in a 34 kb segment of S. rimosus genome flanked by two putative resistance genes (otrA and otrB) [2]. The complete sequence of the OTC biosynthetic gene cluster was not released to the public, so in 2006, Tang’s group systematically re-sequenced the. For the regulation of OTC biosynthesis, there is a paucity of curated information, only one annotated regulatory gene otrR was reported which is divergently transcribed from the resistance gene otrB [11]. It could be involved in the regulation of otrB, which encodes a MFS family efflux pump probably involved in the export of OTC [11]
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