Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

doi:10.1007/s11427-014-4632-3

	Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain; Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain
	Liu Yang 1; Zheng Hua 1; Zhan GuiHua 1; Qin Wen 1; Tian Li 2,3; Li WenLi 1
刊名	SCIENCE CHINA-LIFE SCIENCES ; SCIENCE CHINA-LIFE SCIENCES
	2014-06 ; 2014-06
卷号	57 期号:6 页码:627-635
关键词	marine-derived marine-derived Bacillus marinus Bacillus marinus electroporation electroporation macrolactin macrolactin green fluorescent protein green fluorescent protein phosphopantetheinyl transferase phosphopantetheinyl transferase
ISSN号	1674-7305 ; 1674-7305
DOI	10.1007/s11427-014-4632-3 ; 10.1007/s11427-014-4632-3
英文摘要	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 10(3)-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-1 MgCl2 further improved the efficiency by additional 2-fold, with a maximum value of 7.1x10(4) cfu/mu g 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.; 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 10(3)-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-1 MgCl2 further improved the efficiency by additional 2-fold, with a maximum value of 7.1x10(4) cfu/mu g 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.
资助项目	National Key Technologies Research and Development Program[2011BAE06B04] ; National Key Technologies Research and Development Program[2011BAE06B04]
WOS研究方向	Life Sciences & Biomedicine - Other Topics ; Life Sciences & Biomedicine - Other Topics
语种	英语 ; 英语
出版者	SCIENCE PRESS ; SCIENCE PRESS
WOS记录号	WOS:000337087900011 ; WOS:000337087900011
内容类型	期刊论文
源URL	[http://ir.fio.com.cn/handle/2SI8HI0U/3827]
专题	自然资源部第一海洋研究所
作者单位	1.Ocean Univ China, Sch Med & Pharm, Key Lab Marine Drugs, Minist Educ China, Qingdao 266003, Peoples R China; 2.State Ocean Adm, Inst Oceanog 1, Qingdao 266061, Peoples R China; 3.Qingdao Univ Sci & Technol, Qingdao 266042, Peoples R China
推荐引用方式 GB/T 7714	Liu Yang,Zheng Hua,Zhan GuiHua,et al. Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain, Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain[J]. SCIENCE CHINA-LIFE SCIENCES, SCIENCE CHINA-LIFE SCIENCES,2014, 2014,57, 57(6):627-635, 627-635.
APA	Liu Yang,Zheng Hua,Zhan GuiHua,Qin Wen,Tian Li,&Li WenLi.(2014).Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain.SCIENCE CHINA-LIFE SCIENCES,57(6),627-635.
MLA	Liu Yang,et al."Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain".SCIENCE CHINA-LIFE SCIENCES 57.6(2014):627-635.