Study On GDM Biosynthetic Regulatory Genes

Streptomyces hygrscopicus 17997 was isolated as a geldanamycin producer from a Chinese soil by the Institute of Medicinal Biotechnology, CAMS. Geldanamycin biosynthetic gene cluster has been cloned from S. hygroscopicus 17997, in which two regulatory genes, gdmRⅠand gdmRⅡ, were found, and their deduced products were identified homologous with LuxR transcriptional regulatory proteins. Disruption of gdmRⅠand gdmRⅡgenes, respectively, resulted in completely loss of the GDM production, which suggested that they all played the positive regulatory role in GDM biosynthesis.The growth pattern studies showed that the exponential phase of S. hygroscopicus 17997 was from 24 to 60h, and the lag phase was from 60 to 120h. The typical trophophase (12-48h) and idiophase (48-96h) could be distinguished in S. hygroscopicus 17997 fermentation course.The results were in agreement with the GDM biosynthesis cycle. Semi-quantitative RT-PCR was carried out to investigate the transcription activities of the GDM biosynthetic genes in S. hygroscopicus 17997 and the two regulatory gene disruptants. The results suggested the transcription of gdmRⅠand gdmRⅡappeared at 72h, reached peak at 96h, and after 96h decreased gradually, which were in agreement with the time of initiation of biosynthesis GDM in S. hygroscopicus 17997. And both of gdmRⅠand gdmRⅡgenes positively controlled the transcription of the genes involved in polyketide formation of GDM biosynthesis, but not the gdmN (carbamyltransferase) gene, a post-PKS modification gene of GDM biosynthesis.The gdmRⅠand gdmRⅡpromoter, P_gdmRⅠ-P_gdmRⅡ regions were deduced according to bio-informatics analysis. The different length fragments of both deduced promoter regions (P_{gdmRⅠ^--1-V}:739bp, 649bp, 451bp, 278bp, 152bp; P_{gdmRⅡ^-Ⅰ-V}: 331bp, 293bp, 233bp, 186bp, 120bp) were generated by PCR and constructed in the streptomyces promoter-probe plasmid pIJ486, separately, taking ermE promoter as positive control and pIJ486 itself as negative control. The resultant plasmids were introduced into the S. lividans TK24 and the kanamycin (Km) resistance level of the transformants was considered to roughly estimate the transcription level of these promoter regions. The results showed that Km resistance level of P_{gdmRⅠ-Ⅰ-V} was 250μg/ml, 300μg/ml, 200μg/ml, 50μg/ml, 0μg/ml, respectively. It is suggested that P_gdmRⅠ might be located at -649 and -278 or -152 of the upstream of the gdmRⅠORF. The Krn resistance level of P_{gdmRⅡ-Ⅰ-V} was apparently350μg/ml, 250μg/ml, 200μg/ml, 500μg/ml, 0μg/ml, respectively. In the same experimental condition the ermE promoter activity was reached to 500μg/ml. Rather high level of Km resistance in the region between -331 and -120 and highest Km resistance in the region between -186 and -120 suggested that possible existence of divergent transcription promoters localized upstream of the gdmRⅡORF. In -331 to -120 region two promoters with same transcription direction were present. And in -186 region might be a promoter with convergent transcription existed upstream of the gdmRⅡORF. Intensive studies will be required to clarify the phenomenon.In this studies the relationships of the regulatory genes gdmRⅠ,gdmRⅡand GDM biosynthesis at the transcriptional level were discussed, and the two regulatory gene promoter regions were roughly localized.. It is of great importance both for a fundamental understanding of the regulatory mechanisms of GDM biosynthesis and for the further rational improvement of GDM producers.