Establishment of an efficient transformation protocol and its application in 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 7.1×10(4) 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.
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China.
Yang
Y
Liu
Hua
H
Zheng
GuiHua
G
Zhan
Wen
W
Qin
Li
L
Tian
WenLi
W
Li
D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000737:chemistry; D001407:Bacillus / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics; D018274:Electroporation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism; D060847:Metabolic Engineering; D010957:Plasmids / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics; D014169:Transformation, Bacterial; D014871:Water Microbiology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D018942:Macrolides; C453562:macrolactin A; C082662:phosphopantetheinyl transferase; D049452:Green Fluorescent Proteins; D017855:Transferases (Other Substituted Phosphate Groups)