Regulatory Mechanisms Of Validamycin Biosynthesis In Streptomyces Hygroscopicus Var.Jinggangensis

Validamycin A(VAL-A) is a C7 N aminocyclitol antibiotic produced by Streptomyces hygroscopicus var. jinggangensis, which has been widely used as antifungal agent against rice sheath blight disease in Asia. Additionally, VAL-A and its transformed product valienamine are considered to be pharmaceutically important precursors for the synthesis of voglibose, an effective drug for treating insulin-independent diabetes. In 2005, the whole gene cluster responsible for validamycin biosynthesis was cloned, and eight essential genes were identified in three operons. Subsequently, the mechanism of VAL-A biosynthesis has been investigated in S. hygroscopicus 5008(5008). In 2012, the complete genome sequences of 5008 and industrial strain TL01 were clarified by our laboratory. VAL-A biosynthesis was proven to be affected by γ-butyrolactone and temperature. However,the understanding on the regulatory principles of VAL-A biosynthesis remains very limited.We initially observed that the sequences of VAL-A biosynthetic gene cluster in5008 and TL01 are almost identical. However, TL01 has a 137-bp deletion in the valK-val A intergenic(hereafter valK-valA-int) promoter region. Deletion of the137-bp segment in 5008 resulted in a dramatic increase of VAL-A yield by 2-fold,and the transcription of valK and valA were enhanced. Moreover, In situ insertion of the 137-bp segment into TL01 led to a significant reduction of VAL-A production by80 % and the expression of val K and valA by 80 %. This work demonstrated that the137-bp segment acts as a cis-element negatively involved in the VAL-A biosynthesis by repressing the transcription of VAL-A biosynthetic genes.Subsequently, we showed that GlnR, a global regulator usually involved innitrogen metabolism, is specifically associated with valK-val A-int promoter region by DNA-affinity chromatography and MS-based protein identification. EMSA and DNase I footprinting assays revealed two GlnR binding sites in this promoter region.Targeted disruption of glnR in 5008 led to a significant increase in the transcription of VAL-A structural genes, albeit the VAL-A production was reduced by 80 % and the sporulation of the mutant was impaired. Compared with the wild-type 5008,site-directed mutagenesis of GlnR Binding Site I improved VAL-A production by1.5-fold, whereas the mutation of GlnR Binding Site II resulted in a 50 % reduction of VAL-A yield. Moreover, tandem mutation of Site I in the Site II mutant led to a 66 %increase of VAL-A production. Meanwhile, the mutation of GlnR Binding Site II in TL01 also led to a significant reduction of VAL-A production by 20 %. The result suggested that GlnR not only serves as a negative regulator by binding Site I, but also as a positive regulator by binding Site II for VAL-A biosynthesis. Furthermore,overexpression of glnR in the Site I mutant JG45 improved VAL-A production for41 % compared with the control strain containing the vector at 48 h. Meanwhile,overexpression of glnR in TL01 also led to an increase of VAL-A production by 40 %at 48 h. Therefore, the obtained data illustrated a novel regulatory feature of the global regulator GlnR.Previous studies revealed that the biosynthesis of VAL-A was thermo-regulated in 5008. Based on the transcriptome of 5008 cultured at 30°C and 37°C in industrial medium, we proved that a SARP-family regulator(SHJG0322) is a positive regulator involved in the thermo-regulation of VAL-A biosynthesis. Then we compared the transcriptomes of strain 5008 and JG27(△SHJG0322 mutant) cultured at 37°C in industrial medium using DNA microarray. There are 51 markedly down-regulated DEGs, and 21 significantly up-regulated DEGs, which are mostly involved in carbon metabolism, nitrogen metabolism, and transcriptional regulation. The obtained data suggested that SHJG0322 plays an important role in the regulation of primary metabolism and certain signal transduction pathways. Among those differentially expressed transcriptional regulators in JG27, the transcription of a global regulatorencoding gene afsS was down-regulated by 80 %, whose deletion in 5008 reduced VAL-A production by 70 %. These results indicated that afsS is positively involved in the thermo-regulation of VAL-A biosynthesis.Moreover, among 22 markedly differentially expressed regulators at 37°C in5008, it was found that a two-component regulator gene SHJG6961 was up-regulated by 8.9-fold at 37°C. Subsequent gene inactivation study demonstrated that deletion of SHJG6961 in TL01 reduced VAL-A production by 31.4 % at 37°C. These results implied that two-component regulatory system SHJG6960/SHJG6961 might act as a temperature sensor in the thermo-regulation of VAL-A biosynthesis. Furthermore, two heat-shock protein genes(SHJG4359 and SHJG8393), an ECF σ factor gene(SHJG4152), and a stress response σ factor gene(SHJG2902) were up-regulated at37°C, whose inactivation all resulted in reduced VAL-A production at 30°C and 37°C.These results implied that the thermo-regulation of VAL-A biosynthesis might be closely connected with the heat-shock regulatory system of actinobacteria.