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Background
The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production poten...
Citations
... Bacillus subtilis 168 (BS168) is a preferred microbial strain for the production of MK-7, owing to several distinct advantages. First, B. subtilis has been granted Generally Recognized as Safe (GRAS) status by the U.S. Food and Drug Administration (FDA), attesting to its safety for applications in food, feed, and pharmaceuticals [5,6]. Second, it serves as a promising bacterial chassis for secondary metabolite biosynthesis [7,8]. ...
Background
Menaquinone-7 (MK-7) is a valuable vitamin K2 produced by Bacillus subtilis. Although many strategies have been adopted to increase the yield of MK-7 in B. subtilis, the effectiveness of these common approaches is not high because long metabolic synthesis pathways and numerous bypass pathways competing for precursors with MK-7 synthesis. Regarding the modification of bypass pathways, studies of common static metabolic engineering method such as knocking out genes involved in side pathway have been reported previously. Since byproductsphenylalanine(Phe), tyrosine (Tyr), tryptophan (Trp), folic acid, dihydroxybenzoate, hydroxybutanone in the MK-7 synthesis pathway are indispensable for cell growth, the complete knockout of the bypass pathway restricts cell growth, resulting in limited increase in MK-7 synthesis. Dynamic regulation via quorum sensing (QS) provides a cost-effective strategy to harmonize cell growth and product synthesis, eliminating the need for pricey inducers. SinR, a transcriptional repressor, is crucial in suppressing biofilm formation, a process closely intertwined with MK-7 biosynthesis. Given this link, we targeted SinR to construct a dynamic regulatory system, aiming to modulate MK-7 production by leveraging SinR’s regulatory influence.
Results
A modular PhrC-RapC-SinR QS system is developed to dynamic regulate side pathway of MK-7. In this study, first, we analyzed the SinR-based gene expression regulation system in B. subtilis 168 (BS168). We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrC-RapC-SinR QS system to dynamically control the synthesis of Phe, Tyr, Trp, folic acid, dihydroxybenzoate, hydroxybutanone in MK-7 synthesis in BS168. Cell growth and efficient synthesis of the MK-7 production can be dynamically balanced by this QS system. Using this system to balance cell growth and product fermentation, the MK-7 yield was ultimately increased by 6.27-fold, from 13.95 mg/L to 87.52 mg/L.
Conclusion
In summary, the PhrC-RapC-SinR QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which has potential applicability to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.
... Its emergence as a model organism in biotechnology is supported by numerous processes that have received GRAS rating by the FDA and QPS assessment by EFSA (Leuschner et al., 2010;de Boer Sietske and Diderichsen, 1991). This includes its application in food production, such as historically in soy bean fermentation for thousands of years (Zweers et al., 2008). Because of their natural ability to secrete large amounts of homologous proteins like amylases and proteases into the medium and its good and prototrophic growth on cheap carbon sources, bacilli are generally used for the production of hydrolases (Gu et al., 2018). ...
Proteins face an obstacle race on their way to successful folding. Chaperones facilitate the proper folding of proteins by ensuring they remain on the correct path toward their final tertiary structure. In bacilli, the PrsA chaperone is essential for the correct folding and stabilization of proteins within the cell wall. Overexpression of the PrsA chaperone has been shown to improve the successful folding and secretion of many biotechnologically relevant secreted enzymes. This resulted in a double benefit: firstly, it promotes the efficient release of properly folded enzymes from the cell wall, and second, it reduces the folding stress for the cell, thereby enhancing the overall fitness of the production organism. This paper presents a workflow in which different wild-type PrsA molecules in Bacillus subtilis are co-expressed with different amylases having different signal peptides and promoters. To achieve this, six genome-reduced strains and nine PrsA proteins were systematically selected based on their cultivation performance and the production of two reference amylases. Following strain selection and deletion of major extracellular proteases, several hundred individual strains were created and screened using a stepwise and modular automation approach combined with amplicon sequencing. In addition to providing the key learnings from the workflow, it was revealed that no single PrsA molecule consistently improved amylase production, but genetic constructs combining different elements showed up to a 10-fold variation in yield. Among the screened constructs, the signal peptides YdjM and YvcE demonstrated the best performance.
... As a model organism of Bacillus subtilis, the expression system has been widely used in the production of industrial enzymes, the synthesis of metabolites, and basic research [1][2][3]. Compared with other expression systems, it has the advantages of a fast growth rate, strong protein secretion ability, clear genetic background, and foodgrade safe microorganisms, which are ideal host bacteria for the expression of foreign genes [1][2][3]. ...
... As a model organism of Bacillus subtilis, the expression system has been widely used in the production of industrial enzymes, the synthesis of metabolites, and basic research [1][2][3]. Compared with other expression systems, it has the advantages of a fast growth rate, strong protein secretion ability, clear genetic background, and foodgrade safe microorganisms, which are ideal host bacteria for the expression of foreign genes [1][2][3]. ...
Background
As an important prokaryotic model organism, Bacillus subtilis has been widely used in the industrial production of a variety of target products. The efficient secretion of target products has always been the main purpose of industrial microbial technology. The modification of gene regulatory networks is an important technical means to construct a factory of microbial cells that efficiently secretes target products. However, the regulatory network of the efficient expression of foreign genes in B. subtilis has not been studied at the translation level.
Results
In this study, Ribo-seq and RNA-seq technology were used to study the changes in differentially expressed genes during the efficient secretion of the protease PB92 by B. subtilis WB600, and the results revealed the gene regulatory network related to efficient secretion of foreign protein. The results revealed that the correlation between the differentially expressed genes of B. subtilis at the transcription and translation levels was only 0.5354. Forty-one common (transcription and translation) and 436 unique (translation) key differential gene sets that may be related to the efficient secretion of foreign proteins were revealed. KEGG enrichment analysis of these key gene sets revealed that they were involved mainly in the cell motility and central metabolic regulatory network of B. subtilis.
Conclusion
Our study provides important guidance for the construction of cell factories and metabolic networks for the efficient secretion of target products by B. subtilis.
... B. subtilis is a gram-positive nonpathogenic and generally regarded as safe (GRAS) bacterium [1], and is widely used as cell factory for the industrial-scale production of enzymes for the detergents, food, beverages, paper and pharmaceutical industries [2][3][4][5]. To date, approximately 60% (in weight) of the commercially available enzymes are produced by Bacillus species [2]. ...
Background
Bacillus subtilis is widely used for industrial enzyme production due to its capacity to efficiently secrete proteins. However, secretion efficiency of enzymes varies widely, and optimizing secretion is crucial to make production commercially viable. Previously, we have shown that overexpression of the xylanase XynA lowers expression of Clp protein chaperones, and that inactivation of CtsR, which regulates and represses clp transcription, increases the production of XynA. In the current study, we examined whether the same is the case for overexpression of the α-amylase AmyM from Geobacillus stearothermophilus by B. subtilis, and why XynA shows a different timing of secretion compared to AmyM.
Results
Transcriptome analyses revealed that B. subtilis cells overexpressing AmyM exhibited a distinct profile compared to XynA overexpressing cells, however there were also similarities and in both cases expression of CtsR controlled genes was downregulated. In contrast to XynA, inactivation of CtsR did not improve AmyM production. Upregulation of other protein chaperones, including GroEL/ES and DnaJ/K, by inactivating their transcriptional repressor HrcA, had almost no effect on XynA yields and in fact considerably lowered that of AmyM. Despite using the same promoter, the production of XynA peaks well before AmyM reaches its optimal secretion rate. Transcriptome and ribosome profiling indicated that this is neither related to transcription nor to translation regulation. We show that the reduced secretion in the stationary phase is partially due to the activity of secreted proteases, but also due to the activity of the intracellular protease LonA. The absence of this protein resulted in a 140% and 20% increased production for XynA and AmyM, respectively.
Conclusion
The combination of transcriptome and ribosome profiling offered important information to determine at which cellular level production bottlenecks occurred. This helped us to identify LonA protease as an important factor influencing enzyme production yields in B. subtilis.
... Bacillus subtilis is recognized for its ability to secrete various enzymes for industrial and laboratory applications, growing well on inexpensive substrates due to its adaptive metabolism and physiological features [42]. The single-cell membrane of B. subtilis has been reported to facilitate protein secretion, downstream processing, and cost reduction compared with other bacteria [43]. Ultimately, B. subtilis is generally recognized as safe (GRAS) among Bacillus and other microbes. ...
In this study, the affinity potentials of biosynthesized and functionalized iron oxide nanoparticles (IONPs), gold nanoparticles (AuNPs), and iron-gold nanocomposites (Fe3O4-AuNCs) harnessed to target enteric pathogens were evaluated. Colloidal IONPs and AuNPs biosynthesized by Bacillus subtilis were monitored and characterized with UV–Vis spectroscopy, Fourier transform infrared (FTIR), scanning electron microscope (SEM), energy dispersive X-ray (EDX), and X-ray diffractometer (XRD). The synthesized nanoparticles were functionalized independently and as nanocomposites with the ligand (ethylenediaminetetraacetic acid (EDTA)) and polymers (polyethyleneimine (PEI) and polyethylene glycol (PEG)). Affinity capture of the derived functionalized nanoparticles and nanocomposites was evaluated on enteric bacteria and monitored at 540–600 nm wavelengths. Colloidal suspensions of the biosynthesized IONPs and AuNPs showed UV–Vis absorption peaks at 261 and 548 nm and mean distribution sizes of 72 and 83 nm, respectively. The energy dispersive X-ray (EDX) analysis showed 48.59% zerovalent iron oxide and 51.26% zerovalent gold. X-ray diffractometer (XRD) reported Bragg’s peaks corresponding with the crystal lattice of iron oxide and gold nanoparticles. Capped Fe3O4-AuNCs functionalized with PEI had a significantly higher ratio of zerovalent gold (75.56%) compared to capped Fe3O4-AuNCs functionalized with PEG (61.40%); however, an increased zerovalent iron oxide (20.00%) than PEI-functionalized Fe3O4-AuNCs (7.40%). Capped Fe3O4-AuNCs showed significant binding affinities with Bacillus cereus, Salmonella typhi, Staphylococcus aureus, and Klebsiella aerogenes. The results show that ligands and polymers could enhance Fe3O4-AuNCs’ ability to interact and bind to enteric bacterial pathogen’s negatively charged cell wall (teichoic acid) and outer membrane (lipopolysaccharides). Biosynthesized and functionalized IONPs, AuNPs, and Fe3O4-AuNCs provide cost-effective and feasible nano-based detection of enteric bacteria.
... As reported by U.S. Food and Drug Administration center, B. subtilis is a food-grade strain and has no safety concerns. In addition, B. subtilis owns high ability to secret recombinant proteins and it is easy to design several genetic manipulations because of its de nite genetic background [21][22][23]. Choosing B. subtilis as the expression host for BcMFAse extracellular production is bene cial to basic research and industrial application. ...
In order to meet the desire of maltopentaose (G5) in industrial application, we developed a glycerol-inducible expression system in Bacillus subtilis to overexpress maltooligosaccharide-forming α-amylase from Bacillus cereus ATCC 14579 (BcMFAse). Verifying the glycerol-inducible promoter, optimizing fermentation conditions, comparing homologous promoter and constructing double translation initiation sites were studied. Results shown that the optimal induced time for glycerol-inducible promoter is at 8 h, the optimal induced concentration of glycerol is 1% and the optimized fermentation medium was consisted of 2% tryptone, 0.6% yeast exact, 1% NaCl and 0.6% casein hydrolysate with highest BcMFAse activity (~1549.9 U/mL) promoted by P GlpD in 500 mL triangular flask. Comparing to the homologous promoter, P GlpDL from Bacillus paralicheniformis A4-3 exhibited stronger ability to promoted the expression of BcMFAse and the maximum BcMFAse activity was ~2364.6 U/mL. The BcMFAse activity achieved ~3137.5 U/mL by constructing double translation initiation sites (TISs) at 5´-untranslated region(5´-UTR) of promoter P GlpDL . This study provided a high-efficiency way for overexpressing the BcMFAse in B. subtilis , which would economically producing G5 on industry.
... El género Bacillus ha recibido la designación de caballo de batalla industrial por estar entre los microorganismos más utilizados para la producción a gran escala de proteínas recombinantes, aminoácidos y productos químicos finos (Westers H, 2003;Straight PD, et al., 2006). No será inapropiado llamarlos "fábrica de células" por el gran potencial que poseen (Zweers JC, 2008). ...
Las bacterias son microorganismos procariotas que han estado presentes desde el inicio de la vida en la Tierra; y el género Bacillus, descubierto desde los inicios de la Microbiología, se considera un género fascinante por su amplia distribución en el planeta, fácil cultivo a nivel laboratorio, así como por su relevancia para la biosíntesis de metabolitos de importancia en la vida actual, tales como la producción de bioplásticos, péptidos antimicrobianos, enzimas, bioinsecticidas, etc. Por lo tanto, el objetivo del presente escrito es describir la relevancia del género Bacillus, en diversos ámbitos científicos y tecnológicos.
... At present, DNA recombination technology is continuously improving, and B. subtilis has been investigated as a potential vaccine expression vector in the treatment and prevention of various diseases caused by bacteria, viruses, and parasites, as it effectively induces immune responses in the body. Compared with the commonly used Escherichia coli expression vectors, B. subtilis can survive in extreme environments, produce a large amount of protein, and secrete it into the culture medium [34]; moreover, as a probiotic, it can improve animal intestinal health. ...
The H9N2 subtype of the avian influenza virus (AIV) is one of the main subtypes of low pathogenic AIV, and it seriously affects the poultry breeding industry. Currently, vaccination is still one of China’s main strategies for controlling H9N2 avian influenza. In this study, we selected MW548848.1 on the current popular main branch h9.4.2.5 as the reference strain, and we optimized the amino acid sequence of HA1 to make it suitable for expression in Bacillus subtilis. The B. subtilis expression vector showed good safety and stress resistance; therefore, this study constructed a recombinant B. subtilis expressing H9N2 HA1 protein and evaluated its immunogenicity in mice. The following results were obtained: the sIgA level of HA1 protein in small intestine fluid and the IgG level of PHT43-HA1/B. subtilis in serum were significantly improved (P < 0.01); PHT43-HA1/B. subtilis can cause a special immune response in mice; and cytokine detection interferon-gamma (IFN-γ) (P < 0.05) and Interleukin 2 (IL-2) (P < 0.01) expressions significantly increased. Additionally, the study found that PHT43-HA1/B. subtilis can alleviate the attack of H9N2 AIV in the spleen, lungs, and small intestine of mice. This study was the first to use an oral recombinant B. subtilis-HA1 vaccine candidate, and it provides theoretical data and technical reference for the creation of a new live vector vaccine against H9N2 AIV.
... Owing to its superior enzyme secretion capacity and safe use, the bacterium Bacillus subtilis and its close relatives are extensively employed for the industrialscale production of enzymes for the detergent, food, paper and pharmaceuticals industries [1][2][3][4]. Many genetic strategies have been applied to optimize B. subtilis strains for the production of heterologous proteins, including the use of strong promoters, optimizing ribosomal binding sequences, and inactivating its main extracellular proteases [5][6][7][8][9]. ...
Background
The bacterium Bacillus subtilis is extensively used for the commercial production of enzymes due to its efficient protein secretion capacity. However, the efficiency of secretion varies greatly between enzymes, and despite many years of research, optimization of enzyme production is still largely a matter of trial-and-error. Genome-wide transcriptome analysis seems a useful tool to identify relevant secretion bottlenecks, yet to this day, only a limited number of transcriptome studies have been published that focus on enzyme secretion in B. subtilis. Here, we examined the effect of high-level expression of the commercially important enzyme endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq.
Results
Using the novel gene-set analysis tool GINtool, we found a reduced activity of the CtsR regulon when XynA was overproduced. This regulon comprises several protein chaperone genes, including clpC, clpE and clpX, and is controlled by transcriptional repression. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. When we abolished this negative feedback, by inactivating the repressor CtsR, the XynA production increased by 25%.
Conclusions
Overproduction of enzymes can reduce the pool of Clp protein chaperones in B. subtilis, presumably due to negative feedback regulation. Breaking this feedback can improve enzyme production yields. Considering the conserved nature of Clp chaperones and their regulation, this method might benefit high-yield enzyme production in other organisms.
... The Gram-positive bacterium, Bs, is an effective expression system for foreign proteins due to the structural differences in the outer cell membrane compared to the Gram-negative bacterium, E. coli (Su et al. 2020;Zweers et al. 2008). The gene lin encoding the Linocin 18 protein (bacteriocin) from the bacterium Br. linens M18 has been successfully expressed in E. coli (Valdes-Stauber and Scherer 1996). ...
Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N.
Key Points
• Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified
• It defined the autocidal activity of Linocin M18 (encapsulating) protein
• Identified the possible killing mechanism of the encapsulins
