In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation....In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.展开更多
Microbial transglutaminase(TGase)is a protein that is secreted in a mature form and finds wide applications in meat products,tissue scaffold crosslinking,and textile engineering.Streptomyces mobaraensis is the only li...Microbial transglutaminase(TGase)is a protein that is secreted in a mature form and finds wide applications in meat products,tissue scaffold crosslinking,and textile engineering.Streptomyces mobaraensis is the only licensed producer of TGase.However,increasing the production of TGase using metabolic engineering and heterologous expression approaches has encountered challenges in meeting industrial demands.Therefore,it is necessary to identify the regulatory networks involved in TGase biosynthesis to establish a stable and highly efficient TGase cell factory.In this study,we employed a DNA-affinity capture assay and mass spectrometry analysis to discover several transcription factors.Among the candidates,eight were selected and found to impact TGase biosyn-thesis.Notably,SMDS_4150,an AdpA-family regulator,exhibited a significant influence and was hence named AdpASm.Through electrophoretic mobility shift assays,we determined that AdpASm regulates TGase biosynthesis by directly repressing the transcription of tg and indirectly inhibiting the transcription of SMDS_3961.The latter gene encodes a LytR-family positive regulator of TGase biosynthesis.Additionally,AdpASm exhibited negative regulation of its own transcription.To further enhance TGase production,we combined the overexpression of SMDS_3961 with the repression of SMDS_4150,resulting in a remarkable improvement in TGase titer from 28.67 to 52.0 U/mL,representing an 81.37%increase.This study establishes AdpA as a versatile regulator involved in coordinating enzyme biosynthesis in Streptomyces species.Furthermore,we elucidated a cascaded regulatory network governing TGase production.展开更多
基金Supported by the National Basic Research Program of China (Grant No. 2003CB114205)
文摘In Streptomyces griseus, AdpA, the key transcriptional activator in the A-factor regulatory cascade, switches on the transcription of multiple genes required for secondary metabolism and morphological differentiation. Streptomyces avermitilis also contains an ortholog of adpA, which is named adpA-a. To clarify the in vivo function of adpA-a, an adpA-a-disrupted strain was constructed by double crossover recombination. No difference in avermectin production was found between the adpA-a-disruptant and the wild-type strain. However, this disruptant neither formed spores nor produced melanin and its phenotype was restored to the original wild-type by a single copy of the adpA-a gene integrated into the chromosome. This report shows that adpA-a is involved in regulation of morphological differentiation and melanin production in S. avermitilis.
基金supported by the National Key R&D Program of China(2021YFC2100600,2019YFA0905400)the Na-tional Natural Science Foundation of China(31830104)Science and Technology Commission of Shanghai Municipality(19430750600,19JC1413000,17JC1403600).
文摘Microbial transglutaminase(TGase)is a protein that is secreted in a mature form and finds wide applications in meat products,tissue scaffold crosslinking,and textile engineering.Streptomyces mobaraensis is the only licensed producer of TGase.However,increasing the production of TGase using metabolic engineering and heterologous expression approaches has encountered challenges in meeting industrial demands.Therefore,it is necessary to identify the regulatory networks involved in TGase biosynthesis to establish a stable and highly efficient TGase cell factory.In this study,we employed a DNA-affinity capture assay and mass spectrometry analysis to discover several transcription factors.Among the candidates,eight were selected and found to impact TGase biosyn-thesis.Notably,SMDS_4150,an AdpA-family regulator,exhibited a significant influence and was hence named AdpASm.Through electrophoretic mobility shift assays,we determined that AdpASm regulates TGase biosynthesis by directly repressing the transcription of tg and indirectly inhibiting the transcription of SMDS_3961.The latter gene encodes a LytR-family positive regulator of TGase biosynthesis.Additionally,AdpASm exhibited negative regulation of its own transcription.To further enhance TGase production,we combined the overexpression of SMDS_3961 with the repression of SMDS_4150,resulting in a remarkable improvement in TGase titer from 28.67 to 52.0 U/mL,representing an 81.37%increase.This study establishes AdpA as a versatile regulator involved in coordinating enzyme biosynthesis in Streptomyces species.Furthermore,we elucidated a cascaded regulatory network governing TGase production.
