Insights Into The Regulatory Role Of TetR Family In Secondary Metabolism Of Streptomyces Sp. LZ35

Streptomyces sp. LZ35, a geldanamycin-producing marine strain, was isolated from the soil collected at intertidal belt in Xiamen. Previously, our research group has already sequenced the whole genome of this strain. Bioinformatic analysis indicated that the genome of LZ35contained45secondary metabolite gene clusters, while only ten types of compounds had been isolated from this strain. It can be inferred that secondary metabolism in LZ35is stringently regulated by the precise and sophisticated regulatory network, as a result of which the expression of some secondary metabolite gene clusters is in quite low level or even silenced. TetR family is the largest regulatory family in actinomycetes, which accounts for nearly2%of total proteins encoded in LZ35, indicating its significance in regulatory of secondary metabolism. Understanding regulatory mechanisms of TetR family in LZ35may be of great value in exploiting new compounds in this strain.Bioinformatic analysis showed that LZ35₇692was a TetR-type regulator, which was specifically similar to ArpA, the receptor for quorum sensing molecule A-factor in S. griseus. Meanwhile, LZ35₇693, upstream of LZ35₇692, resembled the A-factor synthase AfsA. In this thesis, in-depth study was carried out to elucidate the function of LZ35₇692on secondary metabolism regulation in LZ35. Firstly, LZ35₇692was overexpressed and purified in vitro, yielding His-tagged recombinant LZ35₇692with high purity. Although an effort was made to crystalize LZ35₇692, only some needle crystals were obtained in the end, which was insufficient to collect fine X-ray diffraction data. Subsequently, electrophoretic mobility shift assay was used to demonstrate that LZ35₇692could bind to the intergenic region between LZ35₇692and LZ35₇693and the binding could be eliminated by fermentation broth of LZ35wild type. Meanwhile, a PCR-based system was employed to obtain LZ35₇692disruption mutant. The analysis of secondary metabolites by HPLC showed that, in contrast to wild type strain, different chromatogram peaks were detected in LZ35A7692mutant, suggesting that LZ35₇692may repress biosynthesis of compounds corresponding to different HPLC peaks.Besides, LZ35₇693and encoding gene of the other A-factor synthase LZ35₈405were disrupted respectively. The growth of LZ35△7693mutant was inhibited compared with wild type strain in YEME media. However, no obvious difference was found the two mutants and wild type strain via HPLC analysis.Finally, through HPLC analysis of gene disruption mutants of eight TetR family proteins located in secondary metabolite gene clusters, LZ35Aheng△3050and LZ35△heng△6514mutants were found to exhibit manifest differences in HPLC peaks in comparison with LZ35Aheng strain, revealing their negative roles in secondary metabolism in LZ35.