Scaling Up the Production of Recombinant Antimicrobial Plantaricin E from a Heterologous Host, Escherichia coli

Abstract

Enhanced production of heterologously expressed plantaricin (plnE) from Escherichia coli BL21 (DE3) was achieved from a small- to large-scale batch culture. Starting from a 15-ml shake-flask culture grown in Luria-Bertani (LB) broth, the protein expression could be scaled up using 50 ml, 100 ml, 1 l, and 2 l batch culture. Using similar condition, plantaricin E (PlnE) was successfully expressed in a 30-l stirred fermenter. The protein was expressed as TRX-(His)6-fusion protein and separated by Ni(2+) affinity chromatography. Growth in two complex media, LB and Terrific broth (TB), was optimized and compared for the production of PlnE, which was higher in LB in comparison with that of TB. In the fermenter, 140 and 180 mg of PlnE could be produced from 12 l of culture volume at 30 and 25 °C, respectively. The yield of heterologously purified PlnE was found to be 1.2-1.5%, which was much higher in comparison with the plantaricins produced from the native strain of Lactobacillus plantarum (0.3-0.7%). Overproduction of PlnE with the help of heterologous expression can overcome the constraint of the low yield from producer strain and provides an easy and low-cost strategy for large-scale production.

Heterologous Expression of Biopreservative Bacteriocins With a View to Low Cost Production

“…Therefore, there are no general rules. Pal and Srivastava ( 2015b ) found higher Plantaricin E yield in LB than in TB, while Kuthning et al ( 2015 ) found the best-producing conditions for Bliα and Bliβ lichenicidin peptides was medium M compared to 2 × YT, SB, TB, and LB-Kelly.…”

“…In addition, the use of fusion protein partners can increase the expression level, enhance protein solubility and assist in correct folding and disulfide bond formation (Ingham and Moore, 2007 ). Common fusion partners used for bacteriocin production include the cellulose binding domain (CBD cenA ) (Klocke et al, 2005 ), maltose-binding protein (MBP) (Quadri et al, 1997 ; Kim et al, 2006 ), thiorredoxin (Trx) (Gibbs et al, 2004 ; Richard et al, 2004 ; Beaulieu et al, 2007 ; Yildirim et al, 2007 ; Jasniewski et al, 2008 ; Caetano et al, 2011a ; Liu et al, 2011 ; Pal and Srivastava, 2014 , 2015a , b ; Jiang et al, 2016 ; Mustopa et al, 2016 ; Meng et al, 2017 ; Tang et al, 2018 ) and the small ubiquitin-related modifier SUMO (Wang et al, 2013 ).…”

“…The cyanogen bromide (CNBr) chemical cleavage strategy has been used to release the mature PlnE and PlnF (Fimland et al, 2008 ) and PlnJ and PlnK (Rogne et al, 2009 ), as has also been described for production of carnobacteriocin B2 and BM1 (Jasniewski et al, 2008 ) and piscicolin 126 (Gibbs et al, 2004 ). However, the most common approach to release recombinant bacteriocins is to include a sequence, between the signal peptide and the bacteriocin, recognized by Factor Xa (Quadri et al, 1997 ; Kawai et al, 2003 ; Klocke et al, 2005 ; Moon et al, 2006 ; Ingham and Moore, 2007 ; Rogne et al, 2008 ; Shi et al, 2011 ), trypsin (Shi et al, 2012 ; Himes et al, 2016 ), thrombin (Klocke et al, 2005 ), enterokinases (Beaulieu et al, 2007 ; Jasniewski et al, 2008 ; Liu et al, 2011 ; Pal and Srivastava, 2014 , 2015a , b ; Jiang et al, 2016 ; Meng et al, 2016 ; Tang et al, 2018 ), and SUMO proteases (Wang et al, 2013 ). Alternatively, the use of intein fusions has been described for the cloning and expression of self-cleaving fusion forms of unmodified bacteriocins under appropriate buffer conditions (Ingham et al, 2005 ).…”

Recent achievements and perspectives for large-scale recombinant production of antimicrobial peptides

“…In the past five years, there are several published attempts to produce AMPs using fermenters at scales and productivity larger than that of using shake flasks aforementioned. E. coli has been utilized to produce AMPs including plantaricin E (Pal and Srivastava 2015) and Pa-MAP 2 (Sousa et al 2016), as well as P. pastoris for MP1102 (Zhang et al 2015b), NZ17074 (Wang et al 2014), and apidaecin (Cao et al 2018;Chen et al 2017), and B. subtilis for gageomacrolactins (Tareq et al 2013). Unlike growing cells in shake-flasks, critical parameters are controlled in fermenters including temperature, pH, and dissolved oxygen (DO).…”

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

“…for bulk production of chemicals, metabolites and enzymes [ 23 , 28 ] as well as for in situ delivery of vaccines [ 8 , 11 , 12 , 32 , 50 ]. Anti-microbial features, such as plantaricin production, are also of growing importance [ 33 ].…”