초록
<P>The lactic acid bacteria (LAB) <I>Leuconostoc citreum</I> 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 <I>L</I>. <I>citreum</I>, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1<SUP>st</SUP> cistron) followed by target genes (2<SUP>nd</SUP> cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in <I>L</I>. <I>citreum</I> was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2<SUP>nd</SUP> cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P<SUB>710V4</SUB>) were successfully isolated. The usefulness of the engineered BCD with P<SUB>710V4</SUB> 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.</P>