title: Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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 analysis, a single mutation (C→T) at position 4690 was found, and we confirmed that this single mutation was responsible for the increased plasmid copy number. The effectiveness of the isolated high-copy-number plasmid for the overproduction of recombinant proteins was successfully demonstrated with two protein models Glutathione-S-transferase (GST) and α-amylase.
  
  
  CONCLUSIONS
  The high-copy number plasmid was successfully isolated by FACS-based high-throughput screening of a plasmid library in L. citreum. The isolated plasmid could be a useful genetic tool for high-level gene expression in Leuconostoc, and for extending the applications of this useful bacteria to various areas in the dairy and pharmaceutical industries.
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus PROGRAM, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. yeonjeong@kaist.ac.kr.
authors.forename: Yeon Jeong
authors.initials: YJ
authors.lastname: Son
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus PROGRAM, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. ajryu@kaist.ac.kr.
authors.forename: Ae Jin
authors.initials: AJ
authors.lastname: Ryu
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju, 28644, Republic of Korea. liling1113@hanmail.net.
authors.forename: Ling
authors.initials: L
authors.lastname: Li
authors.affiliation: Division of Animal, Horticultural and Food Sciences, Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju, 28644, Republic of Korea. namsoo@cbnu.ac.kr.
authors.forename: Nam Soo
authors.initials: NS
authors.lastname: Han
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus PROGRAM, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. kjjeong@kaist.ac.kr.|Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. kjjeong@kaist.ac.kr.
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007934:Leuconostoc / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins