Bacillus Subtilis Expression System Research Articles

A small antimicrobial peptide derived from a Burkholderia bacterium exhibits a broad-spectrum and high inhibiting activities against crop diseases.

Crop diseases cause significant quality and yield losses to global crop products each year and are heavily controlled by chemicals along with very limited antibiotics composed of small molecules. However, these methods often result in environmental pollution and pest resistance, necessitating the development of new bio-controlling products to mitigate these hazards. To identify effective antimicrobial peptides (AMPs) considered as potential sources of future antibiotics, AMPs were screened from five bacterial strains showing antagonism against a representative phytopathogenic fungus (Rhizoctonia Solani) through the Bacillus subtilis expression system, which has been developed for identifying bacterial AMPs by displaying autolysis morphologies. A total of 5000 colonies were screened, and five displaying autolysis morphologies showed antagonism against R. solani. A novel AMP with the strongest antagonism efficiency was determined and tentatively named HR2-7, which is composed of 24 amino acids with an alpha-helical structure. HR2-7 has strong and broad-spectrum antimicrobial activity, tested against 10 g-positive and -negative bacteria and four phytopathogenic fungi by contact culture in plates with minimal lethal concentrations of 4.0 μM. When applied as purified peptide or in fermented B. subtilis culture solution, HR2-7 showed strong controlling efficiency on plants against diverse fungal and bacterial pathogens. Based on current understanding, HR2-7 is recognized as the first AMP derived from an agricultural antagonistic bacterium. It exhibits wide-ranging and notable antimicrobial efficacy, offering a supplementary approach for managing plant diseases, in addition to conventional chemical pesticides and antibiotics.

The use of Bacillus subtilis as a cost-effective expression system for production of Cholera Toxin B fused factor VIII epitope regions applicable for inducing oral immune tolerance.

Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.

Isolation and Optimization of a Broad-Spectrum Synthetic Antimicrobial Peptide, Ap920-WI, from Arthrobacter sp. H5 for the Biological Control of Plant Diseases.

Antimicrobial peptides (AMPs) are naturally occurring molecules found in various organisms that can help to defend against invading microorganisms and reduce the likelihood of drug resistance development. This study focused on the isolation of new AMPs from the genome library of a Gram-positive bacterium called Arthrobacter sp. H5. To achieve this, we used the Bacillus subtilis expression system and employed bioinformatics techniques to optimize and modify the peptides, resulting in the development of a new synthetic antimicrobial peptide (SAMP). Ap920 is expected to be a new antimicrobial peptide with a high positive charge (+12.5). Through optimization, a new synthetic antimicrobial peptide, Ap920-WI, containing only 15 amino acids, was created. Thereafter, the antimicrobial and antifungal activities of Ap920-WI were determined using minimum inhibitory concentration (MIC) and the concentration for 50% of maximal effect (EC50). The Ap920-WI peptide was observed to target the outer membrane of fungal hyphae, leading to inhibition of growth in Rhizoctonia Solani, Sclerotinia sclerotiorum, and Botrytis cinerea. In plants, Ap920-WI showed significant antifungal activity and inhibited the infestation of S. sclerotiorum on rape leaves. Importantly, Ap920-WI was found to be safe for mammalian cells since it did not show any hemolytic activity against sheep red blood cells. Overall, the study found that the new synthetic antimicrobial peptide Ap920-WI exhibits broad-spectrum activity against microorganisms and may offer a new solution for controlling plant diseases, as well as hold potential for drug development.

A novel antimicrobial peptide screened by a Bacillus subtilis expression system, derived from Larimichthys crocea Ferritin H, exerting bactericidal and parasiticidal activities.

Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective antimicrobial activities and multiple function mechanisms against pathogenic microorganisms. In this study, a novel AMP containing 51 amino acids, named Lc1687, was screened from the large yellow croaker (Larimichthys crocea) via a B. subtilis system. Bioinformatics and circular dichroism (CD) analyses showed that Lc1687 is a novel anionic amphiphilic α-helical peptide, which was derived from the C-terminal of a Ferritin heavy subunit. The recombinant Lc1687 (named rLc1687) purified from Escherichia coli exhibited strong activities against Gram-positive (Gram+) bacterium Staphylococcus aureus, Gram-negative (Gram-) bacteria Vibrio vulnificus, V. parahaemolyticus, and Scuticociliatida. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) revealed the possible function mechanisms of this peptide, which is to target and disrupt the bacterial cell membranes, including pore-forming, loss of fimbriae, and cytoplasm overflow, whereas gel retardation assay revealed that peptide Lc1687 cannot bind bacterial DNA. The peptide stability analysis showed that rLc1687 acts as a stable antimicrobial agent against Gram+ and Gram- bacteria at temperatures ranging from 25 to 100°C, pH 3-12, and UV radiation time ranging from 15 to 60 min. A hemolytic activity assay confirmed that this peptide may serve as a potential source for clinical medicine development. Taken together, Lc1687 is a novel AMP as it is a firstly confirmed Ferritin fragment with antimicrobial activity. It is also a promising agent for the development of peptide-based antibacterial and anti-parasitic therapy.

Isolation of Potato Endophytes and Screening of Chaetomium globosum Antimicrobial Genes.

Antimicrobial peptides (AMPs) have natural antibacterial activities that pathogens find difficult to overcome. As a result of this occurrence, AMPs can act as an important substitute against the microbial resistance. In this study, we used plate confrontation tests to screen out 20 potential endophytes from potato tubers. Among them, endophyte F5 was found to significantly inhibit the growth of five different pathogenic fungi. Following that, phylogenetic analysis revealed that the internal transcribed spacer (ITS) sequences were 99% identical to Chaetomium globosum corresponding sequences. Thereafter, the Bacillus subtilis expression system was used to create a C. globosum cDNA library in order to isolate the resistance genes. Using this approach, the resistance gene screening technology in the indicator bacteria built-in library was used to identify two antimicrobial peptides, CgR2150 and CgR3101, with broad-spectrum antibacterial activities. Furthermore, the results showed that CgR2150 and CgR3101 have excellent UV, thermal, and enzyme stabilities. Also, these two peptides can significantly inhibit the growth of various bacteria (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Clavibacter michiganensis, and Clavibacter fangii) and fungi (Fusarium graminearum, Rhizoctonia solani, and Botrytis cinerea). Scanning electron microscopy (SEM) observations revealed that CgR2150 and CgR3101 peptides act against bacteria by disrupting bacterial cell membranes. Moreover, hemolytic activity assay showed that neither of the two peptides exhibited significant hemolytic activity. To conclude, the antimicrobial peptides CgR2150 and CgR3101 are promising in the development of a new antibacterial agent and for application in plant production.

Antimicrobial Peptides from Plants: A cDNA-Library Based Isolation, Purification, Characterization Approach and Elucidating Their Modes of Action.

Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plants’ immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimicrobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or homologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.

Isolation of Antimicrobial Genes from Oryza rufipogon Griff by Using a Bacillus subtilis Expression System with Potential Antimicrobial Activities.

Antimicrobial genes are distributed in all forms of life and provide a primary defensive shield due to their unique broad-spectrum resistance activities. To better isolate these genes, we used the Bacillus subtilis expression system as the host cells to build Oryza rufipogon Griff cDNA libraries and screen potential candidate genes from the library at higher flux using built-in indicator bacteria. We observed that the antimicrobial peptides OrR214 and OrR935 have strong antimicrobial activity against a variety of Gram-positive and Gram-negative bacteria, as well as several fungal pathogens. Owing to their high thermal and enzymatic stabilities, these two peptides can also be used as field biocontrol agents. Furthermore, we also found that the peptide OrR214 (MIC 7.7–10.7 μM) can strongly inhibit bacterial growth compared to polymyxin B (MIC 5–25 μM) and OrR935 (MIC 33–44 μM). The cell flow analysis, reactive oxygen burst, and electron microscopy (scanning and transmission electron microscopy) observations showed that the cell membranes were targeted by peptides OrR214 and OrR935, which revealed the mode of action of bacteriostasis. Moreover, the hemolytic activity, toxicity, and salt sensitivity experiments demonstrated that these two peptides might have the potential to be used for clinical applications. Overall, OrR214 and OrR935 antimicrobial peptides have a high-throughput bacteriostatic activity that acts as a new form of antimicrobial agent and can be used as a raw material in the field of drug development.

Maltose Induced Expression of Cecropin AD by SUMO Technology in Bacillus subtilis WB800N.

Cecropin AD (CAD) is a hybrid peptide composed of 37 amino acids with the characters of strong antibacterial, antitumor properties and no hemolytic activity, which was regarded as a promising antibiotic candidate. Thus, a safe method to produce Cecropin AD is necessary to be found. In the study, Bacillus subtilis WB800N was employed as host strain. The CAD coding sequence fused with the signal peptide of SPsacB, the 6 × His gene and the gene of small ubiquitin-like modifier were cloned into the maltose-inducible vector pGJ148. Under the induction by 6% maltose, the recombinant fusion protein was successfully expressed and detected in culture substrate. An optimized amount (26.4mg/L) of the recombinant CAD was purified of culture supernatant. After purification and digestion, the recombinant CAD was harvested about 4.5mg/L with a purity of 93%. Recombinant CAD exhibited similar antimicrobial activity with synthetic CAD. This shows that the production of CAD in maltose-induced Bacillus subtilis expression system is a relatively safe method, which is vital for the application of CAD in animal husbandry production.

Production and Physicochemical Properties of Food-Grade High-Molecular-Weight Lactobacillus Inulin.

Inulin has been widely applied in food, pharmaceuticals, and many other fields because of its versatile physicochemical properties and physiological functions. Previous research showed that inulosucrase from microorganisms could produce higher-molecular-weight inulin than vegetal inulin. Herein, a food-grade recombinant Bacillus subtilis expression system was constructed to produce inulosucrase from Lactobacillus gasseri DSM 20604 without antibiotic resistance genes. The produced inulosucrase was used to biosynthesize inulin with an average molecular weight of 5.8 × 106 Da. The physicochemical properties of the produced Lactobacillus inulin were evaluated including microstructure, thermal characteristics, crystallinity, rheological behaviors, and storage stability. By comparing with vegetal inulin and other polymers, the biosynthesized microbial inulin showed some superior properties, such as better gel-forming capability and storage stability in aqueous solution than vegetal inulin. These results opened up possibilities for further investigations aimed at developing microbial inulin in the food industry.

Cell Membrane-Interrupting Antimicrobial Peptides from Isatis indigotica Fortune Isolated by a Bacillus subtilis Expression System.

The situation of drug resistance has become more complicated due to the scarcity of plant resistance genes, and overcoming this challenge is imperative. Isatis indigotica has been used for the treatment of wounds, viral infections, and inflammation for centuries. Antimicrobial peptides (AMPs) are found in all classes of life ranging from prokaryotes to eukaryotes. To identify AMPs, I. indigotica was explored using a novel, sensitive, and high-throughput Bacillus subtilis screening system. We found that IiR515 and IiR915 exhibited significant antimicrobial activities against a variety of bacterial (Xanthomonas oryzae, Ralstonia solanacearum, Clavibacter michiganensis, and C. fangii) and fungal (Phytophthora capsici and Botrytis cinerea) pathogens. Scanning electron microscope and cytometric analysis revealed the possible mechanism of these peptides, which was to target and disrupt the bacterial cell membrane. This model was also supported by membrane fluidity and electrical potential analyses. Hemolytic activity assays revealed that these peptides may act as a potential source for clinical medicine development. In conclusion, the plant-derived novel AMPs IiR515 and IiR915 are effective biocontrol agents and can be used as raw materials in the drug discovery field.

Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

Characterization and high-efficiency secreted expression in Bacillus subtilis of a thermo-alkaline \u03b2-mannanase from an alkaliphilic Bacillus clausii strain S10

Backgroundβ-Mannanase catalyzes the cleavage of β-1,4-linked internal linkages of mannan backbone randomly to produce new chain ends. Alkaline and thermostable β-mannanases provide obvious advantages for many applications in biobleaching of pulp and paper, detergent industry, oil grilling operation and enzymatic production of mannooligosaccharides. However, only a few of them are commercially exploited as wild or recombinant enzymes, and none heterologous and secretory expression of alkaline β-mannanase in Bacillus subtilis expression system was reported. Alkaliphilic Bacillus clausii S10 showed high β-mannanase activity at alkaline condition. In this study, this β-mannanase was cloned, purified and characterized. The high-level secretory expression in B. subtilis was also studied.ResultsA thermo-alkaline β-mannanase (BcManA) gene encoding a 317-amino acid protein from alkaliphilic Bacillus clausii strain was cloned and expressed in Escherichia coli. The purified mature BcManA exhibited maximum activity at pH 9.5 and 75 °C with good stability at pH 7.0–11.5 and below 80 °C. BcManA demonstrated high cleavage capability on polysaccharides containing β-1,4-mannosidic linkages, such as konjac glucomannan, locust bean gum, guar gum and sesbania gum. The highest specific activity of 2366.2 U mg−1 was observed on konjac glucomannan with the Km and kcat value of 0.62 g l−1 and 1238.9 s−1, respectively. The hydrolysis products were mainly oligosaccharides with a higher degree of polymerization than biose. BcManA also cleaved manno-oligosaccharides with polymerization degree more than 3 without transglycosylation. Furthermore, six signal peptides and two strong promoters were used for efficiently secreted expression optimization in B. subtilis WB600 and the highest extracellular activity of 2374 U ml−1 with secretory rate of 98.5% was obtained using SPlipA and P43 after 72 h cultivation in 2 × SR medium. By medium optimization using cheap nitrogen and carbon source of peanut meal and glucose, the extracellular activity reached 6041 U ml−1 after 72 h cultivation with 6% inoculum size by shake flask fermentation.ConclusionsThe thermo-alkaline β-mannanase BcManA showed good thermal and pH stability and high catalytic efficiency towards konjac glucomannan and locust bean gum, which distinguished from other reported β-mannanases and was a promising thermo-alkaline β-mannanase for potential industrial application. The extracellular BcManA yield of 6041 U ml−1, which was to date the highest reported yield by flask shake, was obtained in B. subtilis with constitutive expression vector. This is the first report for secretory expression of alkaline β-mannanase in B. subtilis protein expression system, which would significantly cut down the production cost of this enzyme. Also this research would be helpful for secretory expression of other β-mannanases in B. subtilis.

A novel expression vector for the secretion of abaecin in Bacillus subtilis.

This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 μM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 μM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.

Stepwise optimization of a low-temperature Bacillus subtilis expression system for "difficult to express" proteins.

In order to improve the overproduction of "difficult to express" proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called "downstream box" sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5'-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins.

Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis.

The intein expression system has been widely applied in Escherichia coli to express various proteins and peptides. However, the removal of endotoxin from the recombinant proteins expressed in E. coli is very difficult and therefore complicates the purification process. In this study, we constructed an intein-based expression vector for an antimicrobial peptide (cathelicidin from Bungarus fasciatus) and expressed the intein fusion peptide in a Bacillus subtilis expression system. The fusion peptide was secreted into the culture medium, identified by Western blot and purified by affinity chromatography and intein self-cleavage in just one step. Approximately, 0.5mg peptide was obtained from 1 litre of culture medium. The purified peptide showed antimicrobial activity. Our results indicate that the intein expression system may be a safe and efficient method to produce soluble peptides and proteins in B. subtilis.

Expressing antimicrobial peptide cathelicidin-BF in Bacillus subtilis using SUMO technology

Small ubiquitin-related modifier (SUMO) technology has been widely used in Escherichia coli expression systems to produce antimicrobial peptides. However, E. coli is a pathogenic bacterium that produces endotoxins and can secrete proteins into the periplasm, forming inclusion bodies. In our work, cathelicidin-BF (CBF), an antimicrobial peptide purified from Bungarus fasciatus venom, was produced in a Bacillus subtilis expression system using SUMO technology. The chimeric genes his-SUMO-CBF and his-SUMO protease 1 were ligated into vector pHT43 and expressed in B. subtilis WB800N. Approximately 22 mg of recombinant fusion protein SUMO-CBF and 1 mg of SUMO protease 1 were purified per liter of culture supernatant. Purified SUMO protease 1 was highly active and cleaved his-SUMO-CBF with an enzyme-to-substrate ratio of 1:40. Following cleavage, recombinant CBF was further purified by affinity and cation exchange chromatography. Peptide yields of ~3 mg/l endotoxin-free CBF were achieved, and the peptide demonstrated antimicrobial activity. This is the first report of the production of an endotoxin-free antimicrobial peptide, CBF, by recombinant DNA technology, as well as the first time purified SUMO protease 1 with high activity has been produced from B. subtilis. This work has expanded the application of SUMO fusion technology and may represent a safe and efficient way to generate peptides and proteins in B. subtilis.

Induction of Bacillus subtilis expression system using environmental stresses and glucose starvation

The application of safe and cheap inducers is important in the field of fermentation technology, which persuades employing new expression systems. In this study, a Bacillus subtilis expression system was induced by applying starvation and environmental stresses to produce xylanase. The expression plasmid harbors SigB-dependent ohrB promoter. The target gene was expressed by inoculating the recombinant strain into glucose-limited synthetic medium resulting in a sharp increase of xylanase activity at the end of logarithmic growth phase. The recombinant strain was able to express the xylanase enzyme 14-fold higher than that of the control one. The induction was also performed by exposing the recombinant strain to NaCl and ethanol stresses, and heat shock; the strain growing in LB showed 5-, 15- and 6-fold increases in xylanase activity, respectively. The best induction using environmental stresses was achieved by applying the salt stress in the synthetic medium. The maximum expression for NaCl and ethanol stresses occurred after 40 min of induction. All observed inductions were related to activation of SigB protein causing expression of the SigB-dependent xylanase gene. This SigB-dependent expression system can be considered as a biotechnology tool and an alternative to eliminate the cost of conventional inducers.

Extracellular production of cycloisomaltooligosaccharide glucanotransferase and cyclodextran by a protease-deficient Bacillus subtilis host–vector system

A cycloisomaltooligosaccharide (CI; cyclodextran) production system was developed using a Bacillus subtilis expression system for the cycloisomaltooligosaccharide glucanotransferase (CITase) gene. The CITase gene of Bacillus circulans T-3040, along with the α-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, was cloned into the Bacillus expression vector pUB110 and subsequently expressed in B. subtilis strain 168 and its alkaline (aprE) and neutral (nprE) protease-deficient strains. The recombinant CITase produced by the protease-deficient strains reached 1U/mL in the culture supernatant within 48h of cultivation, which was approximately 7.5 times more than that produced by the industrial CITase-producing strain B. circulans G22-10 derived from B. circulans T-3040. When aprE- and nprE-deficient B. subtilis 168 harboring the CITase gene was cultured with 10% dextran 40 for 48h, 17% of the dextran in the culture was converted to CIs (CI-7 to CI-12), which was approximately three times more than that converted by B. circulans G22-10 under the same dextran concentration. The B. subtilis host-vector system enabled us to produce CIs by direct fermentation of dextran along with high CITase production, which was not possible in B. circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase.

Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation

A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ΔmanA (mannose metabolism) strain TQ281 and the ΔmanP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.

Development of a Bacillus subtilis expression system using the improved Pglv promoter

BackgroundB. subtilis is an important organism in the biotechnological application. The efficient expression system is desirable in production of recombinant gene products in B. subtilis. Recently, we developed a new inducible expression system in B. subtilis, which directed by B. subtilis maltose utilization operon promoter Pglv. The system demonstrated high-level expression for target proteins in B. subtilis when induced by maltose. However, the system was markedly repressed by glucose. This limited the application of the system as a high-expression tool in biotechnology field. The aim of this study was to further improve the Pglv promoter system and enhance its expression strength.ResultsHere, site-directed mutagenesis was facilitated to enhance the expression strength of Pglv. The transcription level from four mutants was increased. Production of β-Gal from the mutants reached the maximum 1.8 times as high as that of wildtype promoter. When induced by 5% maltose, the production of β-Gal from two mutants reached 14.3 U/ml and 13.8 U/ml, 63.5% and 57.5% higher than wildtype promoter (8.8 U/ml) respectively. Thus, site-directed mutagenesis alleviated the repression of glucose and improved the expression activity. To further improve the promoter system, the B. subtilis expression host was reconstructed, in which B. subtilis well-characterized constitutive promoter P43 replaced the promoter of the glv operon in B. subtilis chromosome through a double crossover event. The β-galactosidase production from the improved system (21.1 U/mL) increased compared to that from origin system. Meanwhile, the repression caused by glucose was further alleviated.ConclusionsIn this study, we obtained a mutated promoter Pglv-M1 through site-directed mutagenesis, which demonstrated high expression strength and alleviated the repression caused by glucose. Moreover, we alleviated the repression and enhanced the expression activity of the Pglv-M1 promoter system via reconstruction of the B. subtilis host. Thus, we provided a valuable expression system in B. subtilis.