Characterization Of TtmRⅠ, TtmRⅡ TtmRⅢ And TtmRⅣ Gene In The Tetramycin Biosynthetic Gene Cluster From Streptomyces Ahygroscopicus

Tetramycin, a 26-member tetraene microlide, and nystatin, a 38-member tetraene microlide were produced by Streptomyces ahygroscopicus H42 as secondary metabolistes. Four putative regulatory genes ttmRⅠ, ttmRⅡ, ttmRⅢ and ttmRⅣ located in the tetramycin biosynthetic gene cluster were found in Streptomyces ahygroscopicus, which were difference from the analogue pimaricin regulatory genes pimM and pimR, but were similar to those genes in nystatin biosynthesis pathway, nysRⅠ, nysRⅡ, nysRⅢ and nysRIV. This work was focused on the regulatory mechanism during tetramycin biosynthesis by use of gene disruption, complementation, expression, RT-PCR, and EMSA.Database analysis of putative regulatory gene products showed that TtmRI, TtmRⅡ, and TtmRIII belonged to the LAL transcriptional regulators family, while TtmRIV belonged to the PAS-LuxR transcriptional regulators family. Four gene disruption strains, AttmRI, AttmRⅡ, AttmRIII, and AttmRIV, were got by in frame deletion of the four putative regulatory genes. The production of tetramycin was reduced in AttmRI, AttmRⅡ, AttmRⅢ strains, while it was disappeared in AttmRIV strain. The complementation of them restored their production in different degree. As a couple of competitors in using precursors, when the biosynthetic pathway of tetramycin was blocked, the precursors were redirected to the pathway of nystatin. So the production of nystatin Al was significantly enhanced. Antibiotic biosynthesis genes were also generally controlled as operons by the regulators in other Streptomyces strains. Five operons, ttmK-C, ttmG-F-SO, ttmS2-S3-S4, ttmRI-RII-RIII, and ttmA-B-P, were found in the tetramycin gene cluster by the RT-PCR analysis, while for other genes, each gene to be a transcribe unit. The low-level transcription level was found for most genes in the gene cluster in the four mutants. Thus, these four proteins are a group of positive regulators in the transcription of the tetramycin gene cluster in S. ahygroscopicus. In addition, the AttmRⅡ showed reduction of the grey pigment. So the TtmRII had pleiotropic effects on secondary metabolism and development, and the others were the path-way specific regulators. It was the first LAL regulator encoded by regulatory gene in biosynthesis gene cluster. Results of electrophoretic mobility shift assays showed that TtmRI, TtmRII, TtmRIII, and TtmRⅣ regulate the transcription of the target genes by binding to the putative promoters of them in the tetramycin pathway. These phenomenons indicated that these regulators regulated these genes directly. The production of tetramycin was decreased following the increasing copy of ttmRLAL (ttmRⅠ, ttmRⅡ, and ttmRⅢ), with ermEp* promoter, indicating that there was a limiting amount of ttmRLAL for the production of tetramycin. Increasing a copy of ttmRIV, with ermEp* promoter in the strain H42 boosted tetramycin A production by 3.3 fold. Binding the results of RT-PCR and EMSA showed the four regulatory genes regulate each other and auto-regulate. So the four regulators formed a complex regulatory net to control the tetramycin biosynthetic pathway strictly. Analysis of the tetramycin biosynthesis regulation enriched the knowledge of the regulation of the antibiotic biosynthesis.