Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications

This research aims to produce thermostable alkaline protease from Bacillus subtilis isolated from La Hot Spring, Terengganu, Malaysia. The study was also conducted to determine the optimum conditions for protease production and stability by considering several parameters including pH, temperature and salt concentration. All seven bacteria were screened on skim milk agar overnight at 37 °C. Three strains with the highest proteolytic activity were identified in protease specific medium. The thermostable alkaline protease had an optimum temperature of 60 °C which achieved 85.73, 82.90 and 83.05 U/mL of protease activity for the three strains respectively. Furthermore, the strains exhibited significant activity of more than 90% from their original activity. Meanwhile, the optimum pH for protease production was pH 9 with the protease activity of 76.76, 79.71 and 88.39 U/mL for TB4, TB6 and TB9 strains, respectively. Proteases were found stable at pH 9 where the loss did not exceed 30% of its original activity. Collectively, all of the data emphasised that proteases from B. subtilis were alkaline thermostable proteases in accordance with a recent report. The finding highlights the viability of the proteases for biotechnological and industrial applications.

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Metabolic engineering and synthetic biology employing Lactococcus lactis and Bacillus subtilis cell factories

Current Opinion in Biotechnology ◽

10.1016/j.copbio.2019.01.007 ◽

2019 ◽

Vol 59 ◽

pp. 1-7 ◽

Cited By ~ 8

Author(s):

Amanda Y van Tilburg ◽

Haojie Cao ◽

Sjoerd B van der Meulen ◽

Ana Solopova ◽

Oscar P Kuipers

Keyword(s):

Bacillus Subtilis ◽

Metabolic Engineering ◽

Synthetic Biology ◽

Lactococcus Lactis ◽

Subtilis Cell ◽

Cell Factories

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Applications of Bacillus subtilis Spores in Biotechnology and Advanced Materials

Applied and Environmental Microbiology ◽

10.1128/aem.01096-20 ◽

2020 ◽

Vol 86 (17) ◽

Author(s):

Xiaopei Zhang ◽

Amal Al-Dossary ◽

Myer Hussain ◽

Peter Setlow ◽

Jiahe Li

Keyword(s):

Bacillus Subtilis ◽

Large Scale ◽

Genetic Manipulation ◽

Industrial Applications ◽

Advanced Materials ◽

Content Type ◽

Interdisciplinary Approaches ◽

Food Shortages ◽

Material Sciences ◽

Basic Studies

ABSTRACT The bacterium Bacillus subtilis has long been an important subject for basic studies. However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large‐scale fermentation, superior capacity for protein secretion, and generally recognized as safe (GRAS) status. In addition, as the metabolically dormant form of B. subtilis, its spores have attracted great interest due to their extreme resistance to many environmental stresses, which makes spores a novel platform for a variety of applications. In this review, we summarize both conventional and emerging applications of B. subtilis spores, with a focus on how their unique characteristics have led to innovative applications in many areas of technology, including generation of stable and recyclable enzymes, synthetic biology, drug delivery, and material sciences. Ultimately, this review hopes to inspire the scientific community to leverage interdisciplinary approaches using spores to address global concerns about food shortages, environmental protection, and health care.

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Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

Applied and Environmental Microbiology ◽

10.1128/aem.71.4.1899-1908.2005 ◽

2005 ◽

Vol 71 (4) ◽

pp. 1899-1908 ◽

Cited By ~ 15

Author(s):

Helga Westers ◽

Peter G. Braun ◽

Lidia Westers ◽

Haike Antelmann ◽

Michael Hecker ◽

...

Keyword(s):

Bacillus Subtilis ◽

Protease Inhibitor ◽

Bactericidal Activity ◽

Industrial Applications ◽

High Potential ◽

Proteolytic Degradation ◽

Wild Type ◽

Factor Production ◽

Protease Deficient ◽

High Level

ABSTRACT Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.

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The Role of Synthetic Biology in the Design of Microbial Cell Factories for Biofuel Production

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/601834 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 20

Author(s):

Verónica Leticia Colin ◽

Analía Rodríguez ◽

Héctor Antonio Cristóbal

Keyword(s):

Synthetic Biology ◽

Industrial Applications ◽

Microbial Cell ◽

Biodiesel Production ◽

Biofuel Production ◽

Attractive Alternative ◽

Efficient Production ◽

Microbial Cell Factories ◽

Cell Factories

Insecurity in the supply of fossil fuels, volatile fuel prices, and major concerns regarding climate change have sparked renewed interest in the production of fuels from renewable resources. Because of this, the use of biodiesel has grown dramatically during the last few years and is expected to increase even further in the future. Biodiesel production through the use of microbial systems has marked a turning point in the field of biofuels since it is emerging as an attractive alternative to conventional technology. Recent progress in synthetic biology has accelerated the ability to analyze, construct, and/or redesign microbial metabolic pathways with unprecedented precision, in order to permit biofuel production that is amenable to industrial applications. The review presented here focuses specifically on the role of synthetic biology in the design of microbial cell factories for efficient production of biodiesel.

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Cloning and Expression of theγ-Polyglutamic Acid Synthetase GenepgsBCA inBacillus subtilisWB600

BioMed Research International ◽

10.1155/2016/3073949 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

Biaosheng Lin ◽

Zhijuan Li ◽

Huixia Zhang ◽

Jiangwen Wu ◽

Maochun Luo

Keyword(s):

Bacillus Subtilis ◽

Fermentation Broth ◽

Recombinant Expression ◽

Product Yield ◽

Industrial Applications ◽

Cloning And Expression ◽

Polyglutamic Acid ◽

Recombinant Bacteria ◽

Expression Product ◽

Clone And Express

To clone and express theγ-polyglutamic acid (γ-PGA) synthetase genepgsBCA inBacillus subtilis, a pWB980 plasmid was used to construct and transfect the recombinant expression vector pWB980-pgsBCA intoBacillus subtilisWB600.PgsBCAwas expressed under the action of a P43 promoter in the pWB980 plasmid. Our results showed that the recombinant bacteria had the capacity to synthesizeγ-PGA. The expression product was secreted extracellularly into the fermentation broth, with a product yield of 1.74 g/L or higher.γ-PGA samples from the fermentation broth were purified and characterized. Hydrolysates ofγ-PGA presented in single form, constituting simple glutamic acid only, which matched the characteristics of the infrared spectra of theγ-PGA standard, and presented as multimolecular aggregates with a molecular weight within the range of 500–600 kDa. Expressing theγ-PGA synthetase genepgsBCAinB. subtilissystem has potential industrial applications.

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Simultaneous Multiplex Genome Engineering via Accelerated Natural Transformation in Bacillus subtilis

Frontiers in Microbiology ◽

10.3389/fmicb.2021.714449 ◽

2021 ◽

Vol 12 ◽

Author(s):

Aihua Deng ◽

Zhaopeng Sun ◽

Tiantian Wang ◽

Di Cui ◽

Lai Li ◽

...

Keyword(s):

Bacillus Subtilis ◽

Synthetic Biology ◽

High Throughput Screening ◽

Large Scale ◽

Genome Engineering ◽

Natural Transformation ◽

Efficient Design ◽

Accelerated Evolution ◽

One Step ◽

Tyrosine Biosynthesis

Multiplex engineering at the scale of whole genomes has become increasingly important for synthetic biology and biotechnology applications. Although several methods have been reported for engineering microbe genomes, their use is limited by their complex procedures using multi-cycle transformations. Natural transformation, involving in species evolution by horizontal gene transfer in many organisms, indicates its potential as a genetic tool. Here, we aimed to develop simultaneous multiplex genome engineering (SMGE) for the simple, rapid, and efficient design of bacterial genomes via one-step of natural transformation in Bacillus subtilis. The transformed DNA, competency factors, and recombinases were adapted to improved co-editing frequencies above 27-fold. Single to octuplet variants with genetic diversity were simultaneously generated using all-in-one vectors harboring multi-gene cassettes. To demonstrate its potential application, the tyrosine biosynthesis pathway was further optimized for producing commercially important resveratrol by high-throughput screening of variant pool in B. subtilis. SMGE represents an accelerated evolution platform that generates diverse multiplex mutations for large-scale genetic engineering and synthetic biology in B. subtilis.

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Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.800265 ◽

2021 ◽

Vol 9 ◽

Author(s):

Waseem Ayoub Malik ◽

Saleem Javed

Keyword(s):

Bacillus Subtilis ◽

Biochemical Characterization ◽

Biomass Conversion ◽

Industrial Applications ◽

Partial Purification ◽

Subtilis Strain ◽

Complex Nature ◽

Structural Modifications ◽

Optimum Activity

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

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