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

Microbial Cell Factories

Research

Ae Jin Ryu¹  Yeon Jeong Son¹  Ki Jun Jeong²  Ling Li³  Nam Soo Han³ 

[1] Department of Chemical and Biomolecular Engineering, BK21 Plus PROGRAM, KAIST, 291 Daehak-ro, Yuseong-gu, 34141, Daejeon, Republic of Korea;Department of Chemical and Biomolecular Engineering, BK21 Plus PROGRAM, KAIST, 291 Daehak-ro, Yuseong-gu, 34141, Daejeon, Republic of Korea;Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, 34141, Daejeon, Republic of Korea;Division of Animal, Horticultural and Food Sciences, Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, 28644, Cheongju, Republic of Korea;

关键词: Lactic acid bacteria;    Leuconostoc citreum;    High copy plasmid;    Plasmid engineering;    FACS;   

DOI  :  10.1186/s12934-015-0400-8

 received in 2015-11-08, accepted in 2015-12-16,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundLeuconostoc 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.ResultsUsing 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.ConclusionsThe 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.

【 授权许可】

CC BY   
© Son et al. 2016