Development of high copy plasmid for enhanced protein production in Leuconostoc citreum

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 muta-genesis followed by FACS-based high-throughput screening. First, a random library of plasmids was con-structed by randomizing the region responsible for replication in Leuconostoc citreum

additionally, a su-perfolder 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 quan-titative PCR (qPCR). It was found that the isolated plasmid has approximately a 30-fold higher copy num-ber (approx. 70 copies per cell) than that of the original plasmid. From the sequence analysis, a single mu-tation (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 Glutathi-one-S-transferase (GST) and α -amylase. In couclusion, 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.