Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum

Microbial Cell Factories
Yeon Jeong SonKi Jun Jeong

Abstract

Leuconostoc is a hetero-fermentative lactic acid bacteria, and its importance is widely recognized in the dairy industry. However, due to limited genetic tools including plasmids for Leuconostoc, there has not been much extensive research on the genetics and engineering of Leuconostoc yet. Thus, there is a big demand for high-copy-number plasmids for useful gene manipulation and overproduction of recombinant proteins in Leuconostoc. Using an existing low-copy plasmid, the copy number of plasmid was increased by random mutagenesis followed by FACS-based high-throughput screening. First, a random library of plasmids was constructed by randomizing the region responsible for replication in Leuconostoc citreum; additionally, a superfolder green fluorescent protein (sfGFP) was used as a reporter protein. With a high-speed FACS sorter, highly fluorescent cells were enriched, and after two rounds of sorting, single clone exhibiting the highest level of sfGFP was isolated. The copy number of the isolated plasmid (pCB4270) was determined by quantitative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy number (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysi...Continue Reading

References

Jun 29, 1999·Current Opinion in Microbiology·W M de Vos
Feb 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·P S DaughertyG Georgiou
Feb 7, 2003·Applied and Environmental Microbiology·Ki Jun Jeong, Sang Yup Lee
Aug 7, 2003·Applied and Environmental Microbiology·Wilbert SybesmaJeroen Hugenholtz
Dec 21, 2004·Journal of Biotechnology·Hans Peter Sørensen, Kim Kusk Mortensen
May 21, 2005·Plasmid·Junghyun ParkJeongho Kim
Oct 13, 2006·Proceedings of the National Academy of Sciences of the United States of America·K MakarovaD Mills
Sep 26, 2009·Chembiochem : a European Journal of Chemical Biology·Guangyu Yang, Stephen G Withers
Sep 29, 2011·Journal of Microbiology and Biotechnology·Gan-Erdene OtgonbayarNam Soo Han
Oct 26, 2011·Microbial Cell Factories·Willem M de Vos
Dec 14, 2012·Microbial Cell Factories·Elena García-Fruitós
May 2, 2013·Biotechnology and Bioengineering·Sung Sun YimKi Jun Jeong
Jun 25, 2015·Microbiology Spectrum·Joshua Lilly, Manel Camps
Aug 19, 2015·Journal of Microbiology and Biotechnology·Seung Kee ChoNam Soo Han

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Citations

Nov 16, 2018·Journal of the Science of Food and Agriculture·Fatma Chaari, Semia Ellouz Chaabouni
Mar 11, 2017·BioMed Research International·Subash C B GopinathSuresh V Chinni
Jan 23, 2021·Archives of Microbiology·Muhammad Adeel FarooqShumaila Mumtaz
Feb 20, 2021·Journal of Biological Engineering·Phuong N L VoDokyun Na

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Methods Mentioned

BETA
PCR
FACS
electrophoresis
Assay
protein folding
X-ray

Software Mentioned

ImageJ
LightCycler

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