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

• Published in: Scientific reports Vol. 8; no. 1; pp. 8852 - 11
• Main Authors: Jang, Seung Hoon, Cha, Ji Won, Han, Nam Soo, Jeong, Ki Jun
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 11.06.2018; Nature Publishing Group
• Subjects: 631/553/2698; 631/61/185; Bacteria; Fermented food; Flow cytometry; Food industry; Glutathione transferase; Green fluorescent protein; Growth hormone; Humanities and Social Sciences; Lactic acid; Lactic acid bacteria; multidisciplinary; Protein sorting signals; Proteins; Science; Science (multidisciplinary); α-Amylase
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-018-27091-z

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.