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

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Shengyue Ji ◽  
Weili Li ◽  
Abdul Rasheed Baloch ◽  
Meng Wang ◽  
Hengxin Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Fusion Gene ◽  
Expression System ◽  
Inducible Promoter ◽  
Clinical Applications ◽  
Cell Factory ◽  
Efficient Production ◽  
Microbial Cell Factory ◽  
Gene Encoding ◽  
Cecropin A

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 P glv . 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.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis

2020 ◽  
Vol 66 (1) ◽  
pp. 39-45
Author(s):  
Marzieh Rezaei ◽  
Mohammad Rabbani Khorasgani ◽  
Sayyed Hamid Zarkesh Esfahani ◽  
Rahman Emamzadeh ◽  
Hamid Abtahi
Keyword(s):  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Fusion Gene ◽  
Brucella Melitensis ◽  
Expression System ◽  
Interleukin 2 ◽  
Cell Factory ◽  
Promising Alternative ◽  
Alternative Expression ◽  
Restriction Sites

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Stable episomal expression system under control of a stress inducible promoter enhances the immunogenicity of Bacillus subtilis as a vector for antigen delivery

Vaccine ◽  
2006 ◽  
Vol 24 (15) ◽  
pp. 2935-2943 ◽  
Author(s):  
Juliano D. Paccez ◽  
Wilson B. Luiz ◽  
Maria E. Sbrogio-Almeida ◽  
Rita C.C. Ferreira ◽  
Wolfgang Schumann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Expression System ◽  
Inducible Promoter ◽  
Antigen Delivery

scholarly journals Efficient Production of Polymyxin in the Surrogate Host Bacillus subtilis by Introducing a ForeignectBGene and Disrupting theabrBGene

2012 ◽  
Vol 78 (12) ◽  
pp. 4194-4199 ◽  
Author(s):  
Soo-Young Park ◽  
Soo-Keun Choi ◽  
Jihoon Kim ◽  
Tae-Kwang Oh ◽  
Seung-Hwan Park
Keyword(s):  
Bacillus Subtilis ◽  
Paenibacillus Polymyxa ◽  
Biosynthetic Gene Cluster ◽  
Upstream Region ◽  
Efficient Production ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Gene Encoding ◽  
Content Type ◽  
Surrogate Host

ABSTRACTIn our previous study,Bacillus subtilisstrain BSK3S, containing a polymyxin biosynthetic gene cluster fromPaenibacillus polymyxa, could produce polymyxin only in the presence of exogenously addedl-2,4-diaminobutyric acid (Dab). The dependence of polymyxin production on exogenous Dab was removed by introducing anectBgene encoding the diaminobutyrate synthase ofP. polymyxainto BSK3S (resulting in strain BSK4). We found, by observing the complete inhibition of polymyxin synthesis when thespo0Agene was knocked out (strain BSK4-0A), that Spo0A is indispensable for the production of polymyxin. Interestingly, theabrB-spo0Adouble-knockout mutant, BSK4-0A-rB, and the singleabrBmutant, BSK4-rB, showed 1.7- and 2.3-fold increases, respectively, in polymyxin production over that of BSK4. These results coincided with the transcription levels ofpmxAin the strains observed by quantitative real-time PCR (qRT-PCR). The AbrB protein was shown to bind directly to the upstream region ofpmxA, indicating that AbrB directly inhibits the transcription of polymyxin biosynthetic genes. The BSK4-rB strain, producing high levels of polymyxin, will be useful for the development and production of novel polymyxin derivatives.

scholarly journals Cloning and expression of LTB in Escherichia coli and Bacillus subtilis

2013 ◽  
Vol 16 (1) ◽  
pp. 13-22 ◽  
Author(s):  
Trang Thi Phuong Phan ◽  
Anh Le Tuan Nguyen ◽  
Hoang Duc Nguyen
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Fusion Protein ◽  
Expression System ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
E Coli ◽  
B Subunit ◽  
The Common

LTB is the B subunit of heat labile toxins (LT) in Escherichia coli ETEC. This subunit is non-toxic but has a high immune response. Therefore, LTB is considered a suitable antigen for partial vaccine against the diarrhea caused by E. coli ETEC. The most important component of partial vaccine is antigen protein. Nowadays, with the advancement of recombinant protein technology, these antigens are mainly produced by the common bacterial expression system as E. coli. However, the recombinant proteins produced by E. coli are often miscellaneous with enterotoxins, which should be removed from pharmaceutical products. Thus, the production of antigen proteins in other expression system without endotoxins like Bacillus subtilis is in attention. We conducted the experiments of cloning and expressing LTB using a novel pHT plasmid that allow the protein to be expressed in both of E. coli and B. subtilis. We were successful to generate plasmid pHT326 and express the gene encoding for the fusion protein of LTB and LysSN-6xHis-TEV in B. subtilis and E. coli. The binding of fusion protein on the columns that have affinity with His-tag was confirmed. This result is about to be applied for the development of partial vaccine aganst the diarrhea as well as the development of some diagnostic kits for ETEC in food or medical waste and kits to detect antibodies against LTB in animals.

scholarly journals Metabolic engineering Escherichia coli for efficient production of icariside D2

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Xue Liu ◽  
Lingling Li ◽  
Jincong Liu ◽  
Jianjun Qiao ◽  
Guang-Rong Zhao
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Carbon Sources ◽  
Gene Expression Pattern ◽  
Fine Tuning ◽  
Cell Factory ◽  
Efficient Production ◽  
Uridine Diphosphate ◽  
Microbial Cell Factory ◽  
E Coli

Abstract Background Icariside D2 is a plant-derived natural glycoside with pharmacological activities of inhibiting angiotensin-converting enzyme and killing leukemia cancer cells. Production of icariside D2 by plant extraction and chemical synthesis is inefficient and environmentally unfriendly. Microbial cell factory offers an attractive route for economical production of icariside D2 from renewable and sustainable bioresources. Results We metabolically constructed the biosynthetic pathway of icariside D2 in engineered Escherichia coli. We screened the uridine diphosphate glycosyltransferases (UGTs) and obtained an active RrUGT3 that regio-specifically glycosylated tyrosol at phenolic position to exclusively synthesize icariside D2. We put heterologous genes in E. coli cell for the de novo biosynthesis of icariside D2. By fine-tuning promoter and copy number as well as balancing gene expression pattern to decrease metabolic burden, the BMD10 monoculture was constructed. Parallelly, for balancing pathway strength, we established the BMT23–BMD12 coculture by distributing the icariside D2 biosynthetic genes to two E. coli strains BMT23 and BMD12, responsible for biosynthesis of tyrosol from preferential xylose and icariside D2 from glucose, respectively. Under the optimal conditions in fed-batch shake-flask fermentation, the BMD10 monoculture produced 3.80 g/L of icariside D2 using glucose as sole carbon source, and the BMT23–BMD12 coculture produced 2.92 g/L of icariside D2 using glucose–xylose mixture. Conclusions We for the first time reported the engineered E. coli for the de novo efficient production of icariside D2 with gram titer. It would be potent and sustainable approach for microbial production of icariside D2 from renewable carbon sources. E. coli–E. coli coculture approach is not limited to glycoside production, but could also be applied to other bioproducts.

Recent Advances in Recombinant Protein Production by Bacillus subtilis

2020 ◽  
Vol 11 (1) ◽  
pp. 295-318 ◽  
Author(s):  
Kang Zhang ◽  
Lingqia Su ◽  
Jing Wu
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Genetic Manipulation ◽  
Microbial Cell ◽  
Secretory Proteins ◽  
Cell Factory ◽  
Microbial Cell Factory ◽  
Recent Advances

Bacillus subtilis has become a widely used microbial cell factory for the production of recombinant proteins, especially those associated with foods and food processing. Recent advances in genetic manipulation and proteomic analysis have been used to greatly improve protein production in B. subtilis. This review begins with a discussion of genome-editing technologies and application of the CRISPR–Cas9 system to B. subtilis. A summary of the characteristics of crucial legacy strains is followed by suggestions regarding the choice of origin strain for genetic manipulation. Finally, the review analyzes the genes and operons of B. subtilis that are important for the production of secretory proteins and provides suggestions and examples of how they can be altered to improve protein production. This review is intended to promote the engineering of this valuable microbial cell factory for better recombinant protein production.

scholarly journals Generation of recombinant Bacillus subtilis expressing Porcine Epidemic Diarrhea Virus (PEDV) S1 protein in vegetative cell

2018 ◽  
Vol 7 (2.10) ◽  
pp. 50
Author(s):  
Paradon Jorasa ◽  
Phenjun Mekvichitsaeng ◽  
Yaowaluck Maprang Roshorm
Keyword(s):  
Bacillus Subtilis ◽  
Porcine Epidemic Diarrhea Virus ◽  
Vegetative Cell ◽  
Fusion Gene ◽  
Pcr Amplification ◽  
High Yield ◽  
Efficient Production ◽  
Fusion Genes ◽  
Diarrhea Virus ◽  
Porcine Epidemic Diarrhea

Porcine epidemic diarrhea virus (PEDV) is a mucosal (gut surface) pathogen that causes severe diarrhea in piglets; thus, a vaccine capable of inducing gut-mucosal immune response is crucial for controlling PEDV infection. Bacillus subtilis has been considered a choice for vaccine delivery to the gut mucosa. In this study, we aimed to generate recombinant B. subtilis that can produce PEDV S1 protein in vegetative cell. Two promoters, PrrnO and PgsiB-PsecA, were selected for an early and high yield expression of PEDV S1 gene in B. subtilis vegetative cell and germinating spore. Promoters, PrrnO and PgsiB-PsecA, were linked to the 5’ end of the fusion gene pgsA-PEDVS1 and the fusion genes were then inserted into plasmid pDG1662. Recombinant B. subtilis strains were generated by integrating the fusion genes into B. subtilis 168 chromosome via double crossover homologous recombination. PCR amplification and amylase activity assay confirmed integration of the fusion genes into B. subtilis chromosome at amyE locus. Expression of the pgsA-PEDVS1 in B. subtilis vegetative cells germinating from spores was then studied at 2, 4, 8 and 16 hours of culture. Tested by western blot analysis, although only cleaved products of PgsA-PEDVS1 protein were observed, expression levels of pgsA-PEDVS1 under the control of both promoters were comparable at all time points. Importantly, PgsA-PEDVS1 protein could be detected as early as 2 hours after spore inoculation in LB medium. This study suggests that both PgsiB-PsecA and PrrnO promoters can be used for efficient production of PEDV S1 in germinating spore and vegetative cell and may be applicable for expression of other heterologous genes in B. subtilis vegetative cell.  

scholarly journals A Derepression System Based on the Bacillus subtilis Sporulation Pathway Offers Dynamic Control of Heterologous Gene Expression

2007 ◽  
Vol 73 (7) ◽  
pp. 2390-2393 ◽  
Author(s):  
Reindert Nijland ◽  
Jan-Willem Veening ◽  
Oscar P. Kuipers
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Dynamic Control ◽  
Inducible Promoter ◽  
Heterologous Gene ◽  
Gene Regulatory

ABSTRACT By rewiring the sporulation gene-regulatory network of Bacillus subtilis, we generated a novel expression system relying on derepression. The gene of interest is placed under the control of the abrB promoter, which is active only when Spo0A is absent, and Spo0A is controlled via an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter.

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