Identification Of SACE₇040 And SACE₀012, Members Of TetR Family Related To The Morphological Differentiation Of Saccharopolyspora Erythraea

Saccharopolyspora erythraea, a mycelium-forming actinomycete, is used for the industrial-scale production of erythromycin A. Currently, it has been suggested that engineering of regulatory networks that control the biosynthesis of bioactive secondary metabolites is an important strategy to enhance the production of various industrial compounds. However, the regulatory mechanism of erythromycin biosynthetic is poorly understood and only one global regulator BldD which is essential for both erythromycin biosynthesis and morphological differentiation, has been reported to date. The TetR family is a common class of transcriptional regulators that is widely distributed among bacteria. It has attracted widespread attention because the characterized members of the family regulate a wide range of cellular activities. Genome analysis reveals that Sac. erythraea contains 101 TetR-like transcriptional regulators, but few of them have been characterized in detail. The SACE₀012 disruption mutant could form aerial mycelium earlier than its original strain A226, demonstrating that SACE₀012 was a regulator related to the morphological differentiation of Sac. erythraea. In addition, SACE₇040 was deleted in the bldD-disrupted mutant, and interestingly the defect in aerial development exhibited by the bldD mutant could be overcome by SACE₇040 disruption, implying the crosstalking of SACE₇040 and BldD for morphogenesis in Sac. erythraea.SACE₀012, SACE₀820, SACE₁255 and SACE₁632 gene mutants were constructed via a linearized fragment homologous recombination strategy. Transformants were selected by growing on R3M agar medium flooded with thiostrepton (30μg/mL). When grown on R3M agar medium, the SACE₀012 mutant differed from A226 in the rate of aerial mycelium formation. Compared with the original strain,ΔSACE₀012 mutant formed aerial mycelium at least 12 h earlier. However, erythromycin production was not apparently changed in the mutant.In addition, we inactivate SACE₇040 gene inΔbldD mutant. The linearized fragment homologous recombination strategy was used to replace SACE₇040 with the thiostrepton resistance gene in AbldD mutant. When AbldD/ASACE₇040 was grown on R3M medium, a white layer of fuzz that was suggestive of aerial hyphae appeared on the colony surface, showing that the double disruption mutant colonies restored aerial mycelium production. This finding provides evidence for the existence of a biological relevance of SACE₇040 and bldD.To confirm whether the observed phenotype was due to the SACE₇040 deletion in bldD mutant, pZMW-7040 containing a single copy of SACE₇040 was introduced into theΔbldD/ΔSACE₇040 mutant. The resulting strain,ΔbldD/ΔSACE₇040 pZMW-7040 failed to form aerial mycelium. This bald phenotype was also characteristic of the bldD mutant, suggesting that complementation of the double deletion mutant with SACE₇040 restored the bldD mutant phenotype. However, complementation with the same plasmid lacking SACE₇040 maintained the previous phenotype.The finding here provides a starting point for understanding the regulation of morphological differentiation in Sac. erythraea which is a poorly understood process. Furthermore, the availability of Sac. erythraea genome information makes it possible to identify gene function in this organism, and thus there is good reason to believe that many more important new regulators of erythromycin biosynthesis and morphological differentiation await discovery.