Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we...Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987,which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases.The transformation efficiency was greatly enhanced 103-fold by using unmethylated plasmid to bypass modification-restriction barrier,and using glycine betaine to protect cells from electrical damages during electroporation.Addition of HEPES and 2 mmol L?1MgCl2 further improved the efficiency by additional 2-fold,with a maximum value of 7.1×104 cfu/μg pHT3101.To demonstrate the feasibility and efficiency of the protocol,a green fluorescent protein reporter system was constructed;furthermore,phosphopantetheinyl transferase gene sfp,which is essential to the biosynthesis of polyketides and nonribosomal peptides,was overexpressed in B-9987,leading to increased production of macrolactin A by about 1.6-fold.In addition,this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6,indicating it could be a reference for other undomesticated Bacillus strains.To our knowledge,this is the first report regarding the transformation of marine-derived Bacillus strain.展开更多
Natural product discovery is pivot for drug development,however,this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways.We recently developed a highly efficient ...Natural product discovery is pivot for drug development,however,this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways.We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins.By applying this approach in the Streptomyces alboniger NRRL B-1832,we herein identified three cryptic nucleosides products,including one known puromycin A and two new derivatives(puromycin B and C).The biosynthesis of these products doesn't require the involvement of carrier protein,indicating the phosphopantetheinyl transferase(PPtase)indeed plays a fundamental regulatory role in metabolites biosynthesis.These results demonstrate that the PPtasebased approach have a much broader effective scope than the previously assumed carrier proteininvolving pathways,which will benefit future natural products discovery and biosynthetic studies.展开更多
基金supported by grants from the National Natural Science Foundation of China (31070072,31171201)the Program for New Century Excellent Talents in University (NCET-0900717)partially supported by the National Key Technologies Research and Development Program (2011BAE06B04)
文摘Marine-derived Bacillus strains have been proved to be a very promising source for natural product leads.However,transformation of environmental strains is much more difficult than that of domesticated strains.Here,we report the development of an efficient and robust electroporation-based transformation system for marine-derived Bacillus marinus B-9987,which is a macrolactin antibiotics producer and a very promising biological control agent against fungal plant diseases.The transformation efficiency was greatly enhanced 103-fold by using unmethylated plasmid to bypass modification-restriction barrier,and using glycine betaine to protect cells from electrical damages during electroporation.Addition of HEPES and 2 mmol L?1MgCl2 further improved the efficiency by additional 2-fold,with a maximum value of 7.1×104 cfu/μg pHT3101.To demonstrate the feasibility and efficiency of the protocol,a green fluorescent protein reporter system was constructed;furthermore,phosphopantetheinyl transferase gene sfp,which is essential to the biosynthesis of polyketides and nonribosomal peptides,was overexpressed in B-9987,leading to increased production of macrolactin A by about 1.6-fold.In addition,this protocol is also applicable to marine-derived Bacillus licheniforms EI-34-6,indicating it could be a reference for other undomesticated Bacillus strains.To our knowledge,this is the first report regarding the transformation of marine-derived Bacillus strain.
基金This work was financially supported by NSFC(Nos.31322002,31500049 and 31270119,81760633)the State Key Laboratory of Microbial Metabolism(MMLKF17-08).
文摘Natural product discovery is pivot for drug development,however,this endeavor is often challenged by the wide inactivation or silence of natural products biosynthetic pathways.We recently developed a highly efficient approach to activate cryptic/silenced biosynthetic pathways through augmentation of the phosphopantetheinylation of carrier proteins.By applying this approach in the Streptomyces alboniger NRRL B-1832,we herein identified three cryptic nucleosides products,including one known puromycin A and two new derivatives(puromycin B and C).The biosynthesis of these products doesn't require the involvement of carrier protein,indicating the phosphopantetheinyl transferase(PPtase)indeed plays a fundamental regulatory role in metabolites biosynthesis.These results demonstrate that the PPtasebased approach have a much broader effective scope than the previously assumed carrier proteininvolving pathways,which will benefit future natural products discovery and biosynthetic studies.
