Bacillus is a class of spore-producing Gram-positive bacteria that produce a variety of antimicrobial substances with different structures and functions. The application of the antimicrobial substances produced by Bacillus can effectively inhibit the activity of harmful bacteria and fungi and promote the sustainable development of green agriculture. The antimicrobial substances produced by Bacillus mainly include proteins, lipopeptides, polyketones, and polypeptides. This paper reviews the synthesis gene clusters, synthesis pathways, structures, and mechanisms of various antimicrobial substances produced by Bacillus and discusses the challenges in the industrial application of these antimicrobial substances. Furthermore, this paper clarifies the future research and development focuses and prospects the application prospects, and provides comprehensive theoretical support for the in-depth research and wide application of the antimicrobial substances produced by Bacillus.
Bacillus/genetics* ; Anti-Infective Agents/metabolism* ; Bacterial Proteins/genetics* ; Antimicrobial Peptides/biosynthesis* ; Lipopeptides/biosynthesis*

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.
Antimicrobial Cationic Peptides ; biosynthesis ; Antineoplastic Agents ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

Bactericidal/permeability-increasing protein (BPI) can bind to and specifically neutralize lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria. In order to evaluate potent LPS-neutralizing activity of bovine BPI, the full-length coding sequence (1 449bp) or 714 bp N-terminal coding sequence (BPI714) of bovine BPI was transfected into mHEK293 cells and the expression of LPS-induced inflammatory cytokines was studied. First, we constructed the lentiviral expression vectors and generated mHEK293 cells stably expressing recombinant bovine BPI or BPI714. Then, we detected the expression of IL-8, IL-1β, TNF-α, NF-κB-1 and NF-κB-2 genes by real-time PCR at 0, 1, 3, 6, 12, 24, 36 and 48 h post of LPS induction in cells with or without recombinant bovine BPI or BPI714 ectopic expression, respectively. In response to LPS, the robust abundance of inflammatory cytokines including IL-8, IL-1β, TNF-α and NF-κB-2 was observed in wild type mHEK293 cells at eachtime point. On the contrary, mRNA abundance of IL-8, TNF-α and NF-κB-2 in transfected mHEK293 cells showed no significant changes at each indicated time point. Our results demonstrated that recombinant bovine full length BPI or BPI714 down-regulated the expression of inflammatory cytokines and revealed that either of bovine BPI or BPI714 was able to inhibit the immune respond stimulated by LPS. This study provides evidence for further investigating the mechanisms and application of BPI/LPS-neutralizing activity and also documents a reliable approach for analysis of the efficacy of antibacterial proteins.
Animals ; Antimicrobial Cationic Peptides ; chemistry ; Blood Proteins ; chemistry ; Cattle ; Cytokines ; biosynthesis ; HEK293 Cells ; Humans ; Interleukins ; biosynthesis ; Lipopolysaccharides ; chemistry ; NF-kappa B ; biosynthesis ; Transfection ; Tumor Necrosis Factor-alpha ; biosynthesis

Due to their potential application as novel antibiotics, antimicrobial peptides are attracting much attention. Large quantities of highly purified peptides are crucial to basic and clinical studies. Natural resources of antimicrobial peptides are limited and hard to purify, chemical synthesis is of high-cost and unstable, so recombinant expression of antimicrobial peptides is a cost-effective way. Escherichia coli has been the most widely used as host to express antimicrobial peptides with fusion protein, which can not only avoid the lethal effect towards the host, but also protect the peptide from degradation by proteases. Combined with our research, the present article reviews the progress of fusion vector, cleavage methods and optimization options for antimicrobial peptides production with fusion protein in Escherichia coli.
Antimicrobial Cationic Peptides ; biosynthesis ; Escherichia coli ; metabolism ; Genetic Vectors ; Recombinant Fusion Proteins ; biosynthesis

Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; Humans ; Microbial Sensitivity Tests ; Muramidase ; biosynthesis ; genetics ; Peptides, Cyclic ; biosynthesis ; genetics ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside (IPTG). We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 degrees C for 4 h, there was the highest expression level of fusion protein HIS-TmAMP1m in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m had better stability and remained higher antibacterial activities when incubated at 100 degrees C for 10 h, repeated freeze thawing at -20 degrees C, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMP1m has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.
Animals ; Anti-Infective Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Tenebrio ; chemistry

In the current study, we synthesized a 16-residue-long peptide VR with the aim of inspecting the feasibility to design beta-hairpin-like antimicrobial peptide. The peptide was designed by alternating arrangement of arginine and valine and linking two stranded antiparallel beta-sheet with a short loop segment (DPG) and a disulfide bridge. Antimicrobial and hemolytic activities were investigated. Melittin was chosen as a control peptide. We also tested bactericidal kinetics and salt sensitivity. Results show that VR had similar antibacterial activity compared with melittin. However, VR displayed much less hemolytic activity than melittin. These results suggest that VR had higher cell selectivity than melittin. The antibacterial activity of VR was not inhibited in the presence of 25 and 50 mmol/L NaCl. VR still possessed antibacterial activity in the presence of 100 mmol/L NaCl. Collectively, the de novo peptide VR displayed high antimicrobial activity, low hemolytic activity, and salt resistant, indicating that VR was a promising candidate for novel antimicrobial applications.
Anti-Infective Agents ; chemistry ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; chemistry ; pharmacology ; Arginine ; chemistry ; Bacteria ; drug effects ; Drug Design ; Escherichia coli ; drug effects ; Hemolysis ; drug effects ; Microbial Sensitivity Tests ; Staphylococcus aureus ; drug effects ; Valine ; chemistry

Antimicrobial peptides (AMPs) are of great significance in the field of food, feed and medicine due to their wide spectrum of antimicrobial activity and new mechanism of action different from conventional antibiotics. AMPs production from natural sources is usually limited, and chemical synthesis is not economically practical, especially for the production of long peptides. Therefore, heterologous expression of AMPs has been widely studied as an alternative, and fusion expression plays an important role in increasing production. The present review mainly focuses on the types and bioactivities of AMPs. In addition, the recent strategies to the most commonly used carrier proteins for fusion expression of AMPs and prospects for future research were also discussed.
Anti-Infective Agents ; metabolism ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; biosynthesis ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Thioredoxins ; biosynthesis ; genetics

OBJECTIVETo clone tenecin gene, an antibacterial peptide gene, from Tenebrio molitor for its prokaryotic expression and explore the molecular mechanism for regulating the expression of antibacterial peptide in Tenebrio molitor larvae.

METHODSThe antibacterial peptide was induced from the larvae of Tenebrio molitor by intraperitoneal injection of Escherichia coli DH-5α (1×10(8)/ml). RT-PCR was performed 72 h after the injection to clone Tenecin gene followed by sequencing and bioinformatic analysis. The recombinant expression vector pET-28a(+)-Tenecin was constructed and transformed into E. coli BL21(DE3) cells and the expression of tenecin protein was observed after IPTG induction.

RESULTSTenecin expression was detected in transformed E.coli using SDS-PAGE after 1 mmol/L IPTG induction. Tenecin gene, which was about 255 bp in length, encoded Tenecin protein with a relative molecular mass of 9 kD. Incubation of E.coli with 80, 60, 40, and 20 µg/ml tenecin for 18 h resulted in a diameter of the inhibition zone of 25.1∓0.03, 20.7∓0.06, 17.2∓0.11 and 9.3∓0.04 mm, respectively.

CONCLUSIONSTenecin protein possesses strong antibacterial activity against E. coli DH-5α, which warrants further study of this protein for its potential as an antibacterial agent in clinical application.

Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Cloning, Molecular ; Escherichia coli ; drug effects ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insect Proteins ; biosynthesis ; genetics ; Larva ; chemistry ; Microbial Sensitivity Tests ; Molecular Sequence Data ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Tenebrio ; chemistry

Antimicrobial Cationic Peptides ; biosynthesis ; Hepacivirus ; pathogenicity ; Hepcidins ; Iron ; metabolism ; Liver ; chemistry ; metabolism ; pathology