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

Abstract

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 (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 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.

Enhanced production of recombinant proteins in Corynebacterium glutamicum by construction a bicistronic gene expression system

“…The strongest three bicistronic promoters shown in our study remained higher potency than the control when expressing different proteins, so we inferred that the forecistron sequence could be developed as an independent expression enhancer. Furthermore more parameters, such as the length [36] and the amino acid composition [37] of the first cistron need to be investigated to evaluate its improvement ability in combination with different promoters, UTR, SD sequences [19,38], target genes, and different hosts-the designing principles established in E.coli may not be fully applicable to C. glutamicum, as the 24 promoters in our study showed a completely different intensity trend between the two hosts.…”

“…Solutions by optimizing the TIR/UTR sequence for each protein target is very tedious and timeconsuming [18][19][20]. To build a vector with good compatibility of expression of various proteins, a bicistronic design (BCD) expression strategy can be considered.…”

“…(2) translation efficiency is facilitated by the coupled translation designing of linking area of the two coding sequences (CDS): ribosome herein initiates the translation process immediately after it dropped off from the stop codon of the first cistron-usually a sequence-economic and easily translated polypeptide [22]. Although the mechanism and influencing factors of the bicistronic expression system have not been fully elucidated so far, there are still some successful expression cases using bicistronic expression patterns in E. coli [21,23,24] and lactic acid bacteria [19]. Our previous work [25,26] also demonstrated that the expression level and translation efficiency of the endogenous bicistronic promoters could be stronger than the respective monocistronic promoters for gene expression in C. glutamicum.…”

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

“…; Jang et al . ). Using the host and its vector series, we further developed a dsrC ‐ mutant strain producing no dextran in sucrose medium (Jin et al .…”

“…The minimized shuttle vector showed a broad host spectrum covering various LAB species such as L. mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lb. reuteri, Streptococcus thermophilus, Weissella confusa and Oenococcus oeni, and thus it has been used to express heterologous genes in several studies Cho et al 2015;Jang et al 2018). Using the host and its vector series, we further developed a dsrCmutant strain producing no dextran in sucrose medium (Jin et al 2014), a foodgrade expression system by employing a bile salt hydrogenase gene (bsh) (Cho et al 2015) and a high-copy number plasmid (pCB4270) system screened using fluorescenceactivated cell sorting (FACS) technique (Son et al 2016).…”