Development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum

The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P-710V4) were successfully isolated. The usefulness of the engineered BCD with P-710V4 and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and alpha-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.