Engineering of expression vector for efficient protein production in Leuconostoc citreum

Abstract

Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that produces lactic acid as a final end product in fermentation. Traditionally, LAB have long been used in the production of fermented dairy, meat and vegetable products as well as in wine and sourdough production. In addition, some species of LAB can produce health-related molecules such as antimicrobial peptides or bacteriocins that are used as biopreservative agents in foods. Recently, LAB have attracted attentions as a promising microbial cell factory and as mucosal delivery vehicles. Leuconostoc belongs to LAB is a non-sporulating, low G+C content, and a hetero-fermentative bacteria. Leuconostoc also plays important role in fermented food industry and has a broad spectrum of product such as lactic acid, alcohol, and aromatic compound. However, despite its importance in food and biotechnology industries, there has been little effort to develop genetic tools for engineering of the bacteria. In this study, I tried to engineer the expression vector system for enhancement of gene expression in L. citreum. For this purpose, I introduced bicistronic design (BCD) expression system into L. citreum. After the expression of target gene was observed in this expression system, Shine-Dalgarno (SD) sequence in the plasmid was engineered. SD2 library was constructed using super-folder green fluorescent protein (sfGFP) as a reporter, and highly fluorescent clones were isolated by FACS screening. The improvement of gene expression by the isolated strong SD2 was demonstrated with three recombinant proteins. Next, synthetic promoter library derived $P_{710}$ promoter was constructed in the engineered plasmid with strong SD2. The synthetic promoter library that induces sfGFP was screened by FACS. As a result, I could successfully isolate strong promoter and verify the strong promoter with two recombinant proteins. The expression system engineered with the promoter and SD2 showed about 1.6 times higher $\alpha$-amylase productivity than the previous expression system. Therefore, the expression vector system developed in this study will be useful for engineering of L. citreum in the future.