Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700

Abstract

The efficient production of antimicrobial peptides (AMPs) for clinical applications has attracted the attention of the scientific community. To develop a novel microbial cell factory for the efficient biosynthesis of a cecropin A-melittin mutant (CAM-W), a recombinant Bacillus subtilis WB700 expression system was genetically modified with a novel vector, including a fusion gene encoding CAM-W, the autoprotease EDDIE and the signal peptide SacB under the control of the maltose-inducible promoter Pglv. A total of 159 mg of CAM-W was obtained from 1 L of fermentation supernatant. The purified CAM-W showed a consistent size with the expected molecular weight of 3.2 kDa. Our findings suggest that this novel expression system can be used as a powerful tool for the efficient production of CAM-W.

Encapsulin carrier proteins for enhanced expression of antimicrobial peptides

“…However, recombinant AMP production in bacterial hosts such as Escherichia coli is hindered by the bactericidal nature of these peptides, which can lead to host toxicity and/or proteolysis (Li, ; Parachin, Mulder, Viana, Dias, & Franco, ). Several strategies have been developed to overcome these limitations, including using alternative hosts (Cao et al, ; Ji et al, ), employing secretion (S. J. Lee, Park, Han, Kim, & Reeves, ), and expressing AMPs as tandem multimers (Tian, Dong, Yang, Teng, & Wang, ) or within inclusion bodies (J. H. Lee et al, ). The most common strategy is to fuse AMPs to carrier proteins (e.g., small ubiquitin‐related modifier [SUMO], thioredoxin A [Trx], glutathione S‐transferase [GST]) that mask peptide toxicity and reduce proteolytic susceptibility (Li, ).…”

High copy number and highly stable Escherichia coli–Bacillus subtilis shuttle plasmids based on pWB980

“…At present, the majority of available B. subtilis expression plasmids are derived from pC194 [10][11][12], pLS1 [13,14] and pUB110 [3,15]. In order to improve transformation efficiency of plasmids, a lot of E. coli-B.…”

Cecropin A-melittin mutant is a potential therapeutic agent in treating bacterial gastroenteritis

“…For the assays, the HT-29 cells were first starved for 24 h in serum-free medium and then were seeded in a 24-well plate (Nunc, Germany) at 1 × 10 5 cells/well. At subconfluency, the medium was replaced, and the cells were incubated with serial CAM-W dilutions (CAM-W sample was obtained by using the recombinant Bacillus subtilis strain stored in our laboratory with reported method [11]) of 300, 150, 75, 40, 20, 10, 5, 2.5, 1.2, 0.6, and 0.3 mg/L in a volume of 100 μl for 24 h [13]. Cell viability was assessed in exposed cultures by using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium thiazolyl blue assay (MTT, Roche Diagnostics, Germany).…”

“…Like other biosynthesized AMPs, such as sublancin [8,9] and cecropin AD [10], CAM-W was just recently recombinantly produced in a Bacillus subtilis host [11]. The present study investigated the potential of recombinant CAM-W in treating bacterial gastroenteritis, including the in vitro cytotoxicity towards a human colorectal adenocarcinoma cell line HT-29, acute oral toxicity, the bioavailability, and the in vivo antibacterial activity using a Shiga toxin-producing E. coli (STEC) infection mice model.…”