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 (1(st) cistron) followed by target genes (2(nd) 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 2(nd) 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 α-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.