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Enzyme and Microbial Technology Apr 2023A novel glycoside hydrolase family 26 β-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products...
A novel glycoside hydrolase family 26 β-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35-328) and PpMan26A-Δ205 (aa 206-656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The K and k values for konjac gum were 2.13 mg/mL and 416.66 s, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3-8. CBM35pp exerted strong binding activity toward mannans but without β-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 β-mannanases and expands the potential industrial application of PpMan26A.
Topics: beta-Mannosidase; Paenibacillus polymyxa; Oligosaccharides; Mannans; Substrate Specificity; Hydrolysis
PubMed: 36680817
DOI: 10.1016/j.enzmictec.2023.110197 -
Plant Physiology and Biochemistry : PPB Nov 2020In recent decades, drought has become a global problem for food security and agricultural production. A variety of strategies have been developed to enhance drought...
Plant growth-promoting rhizobacterium, Paenibacillus polymyxa CR1, upregulates dehydration-responsive genes, RD29A and RD29B, during priming drought tolerance in arabidopsis.
In recent decades, drought has become a global problem for food security and agricultural production. A variety of strategies have been developed to enhance drought tolerance, but largely unsuccessful since most drought-responsive genes (DRGs) stimulate a stomata closure and in turn suppress plant growth and yield. To access if and/or how plants could enhance drought tolerance without trading off growth and development, we screened and isolated a plant growth-promoting rhizobacterium, Paenibacillus polymyxa CR1, capable of 1) priming drought tolerance and concurrently 2) increasing root growth in plants, e.g., Arabidopsis and soybean. In parallel, we uncovered that P. polymyxa CR1 3) induces the expression of two DRGs, Response to Desiccation (RD)29A and RD29B, 4) of which pattern upregulations are controlled by a diurnal rhythm. Besides, RD29A and RD29B act as 5) 'memory' genes; their transcript levels are increased to a greater extent when plants encountered P. polymyxa CR1 for the second time compared to an initial exposure. In line with these findings, T-DNA insertion mutant Arabidopsis of RD29A or RD29B displayed enhanced susceptibility to drought, without any change in stomata behaviors or growth rates, than wild-type plants. Hence, we conclude that RD29A or RD29B are unique, efficacious generic materials that can potentially aid in upgrading the plants own survival capacity against drought without reducing yield potential.
Topics: Arabidopsis; Arabidopsis Proteins; Cold Shock Proteins and Peptides; Dehydration; Droughts; Gene Expression Regulation, Plant; Paenibacillus polymyxa; Plants, Genetically Modified; Stress, Physiological
PubMed: 32947123
DOI: 10.1016/j.plaphy.2020.08.049 -
Journal of Biotechnology Apr 2019Paenibacillus polymyxa is a rhizobacterium that has attracted substantial attention due to its ability to produce functional metabolites and promote plant growth....
Paenibacillus polymyxa is a rhizobacterium that has attracted substantial attention due to its ability to produce functional metabolites and promote plant growth. Metabolic and genetic improvements in this species will benefit research and other applications of the bacterium. However, a suitable gene expression system has not been established in this species. In this study, a promoter trap system based on a green fluorescent protein and a chloramphenicol-resistance gene was developed to isolate native promoters of P. polymyxa SC2-M1 to regulate gene expression. Through high-throughput screening, the novel promoter P was identified, sequenced, and subsequently characterized. Promoter P is a strong, continuous expression system containing the typical -10 and -35 motifs regions. Its effective sequence was evaluated and then cascaded to improve the promotion efficiency. To further verify the existence of P, a heterogenous xylose isomerase was expressed by P in P. polymyxa SC2-M1. In the resulting strain, the amount of xylose consumed was increased by 2.5 g/L during the 78 h fermentation period. Meanwhile, the production levels of lactate and acetate increased. It was confirmed that promoter P could effectively mediate gene expression in P. polymyxa SC2-M1 and will further benefit the quantitative monitoring of gene expression in P. polymyxa.
Topics: Cloning, Molecular; Gene Expression; Metabolic Engineering; Paenibacillus polymyxa; Plasmids; Promoter Regions, Genetic; Recombinant Proteins
PubMed: 30831123
DOI: 10.1016/j.jbiotec.2019.02.002 -
Frontiers in Bioengineering and... 2022Production of some antimicrobial peptides by bacterial producers is a resource-intensive process, thus, using inexpensive growth media and simplifying antimicrobial...
Production of some antimicrobial peptides by bacterial producers is a resource-intensive process, thus, using inexpensive growth media and simplifying antimicrobial extraction and down-stream processing are highly desirable. Acid whey, a dairy industry waste, is explored as a medium for production of broad-spectrum antimicrobials from selected bacteriocinogenic bacteria. Neutralized and yeast extract-supplemented acid whey was suitable for production of antimicrobials by four tested strains, but OSY-EC was the most prolific antimicrobial producer. Concentrating synthesized antimicrobials during culture incubation using beads of polymeric adsorbent resin, followed by solvent extraction and freeze-drying, resulted in antimicrobials-rich powder (AMRP). Under these conditions, OSY-EC produced paenibacillin, polymyxin E, and fusaricidin, which are active against Gram-positive and Gram-negative bacteria and fungi, respectively. When media containing 2x and 4x minimum inhibitory concentrations of AMRP were inoculated with and , microbial populations decreased by ≥4-log CFU ml in tryptic soy broth and ≥3.5-log CFU ml in milk. The antimicrobial mechanism of action of AMRP solutions was attributed to the disruption of cytoplasmic membrane of indicator strains, and . These findings exemplify promising strategies for valorization of acid whey microbial bioreactions to yield potent antimicrobials.
PubMed: 35646844
DOI: 10.3389/fbioe.2022.869778 -
Trends in Biotechnology Apr 2022Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic... (Review)
Review
Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic engineering ensures efficient and fast tailoring of microbial metabolism for a wide range of bioconversions. However, improving intrinsic properties such as tolerance, handling, growth, and substrate consumption rates is still challenging. At the same time, synthetic biology tools are becoming easier applicable and transferable to nonmodel organisms. These trends have resulted in the exploitation of new and unconventional microbial systems with sophisticated properties, which render them promising hosts for the bio-based industry. Here, we highlight the metabolic and cellular capabilities of representative prokaryotic newcomers and discuss the potential and drawbacks of these hosts for industrial application.
Topics: Biotechnology; Genetic Engineering; Metabolic Engineering; Synthetic Biology
PubMed: 34482995
DOI: 10.1016/j.tibtech.2021.08.003 -
RNA Biology Nov 2021Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able...
Nine distinct classes of self-cleaving ribozymes are known to date, of which the pistol ribozyme class was discovered only 5 years ago. Self-cleaving ribozymes are able to cleave their own phosphodiester backbone at a specific site with rates much higher than those of spontaneous RNA degradation. Our study focuses on a bioinformatically predicted pistol ribozyme from the bacterium . We provide a biochemical characterization of this ribozyme, which includes an investigation of the effect of various metal ions on ribozyme cleavage and a kinetic analysis of ribozyme activity under increasing Mg concentrations and pH. Based on the obtained results, we discuss a possible catalytic role of divalent metal ions. Moreover, we investigated the ligation activity of the pistol ribozyme - an aspect that has not been previously analysed for this ribozyme class. We determined that the pistol ribozyme is almost fully cleaved at equilibrium with the ligation rate constant being nearly 30-fold lower than the cleavage rate constant. In summary, we have characterized an additional representative of this recently discovered ribozyme class isolated from . We expect that our biochemical characterization of a pistol representative in a cultivatable, genetically tractable organism will support our future investigation of the biological roles of this ribozyme class in bacteria.
Topics: Biocatalysis; Catalytic Domain; Computational Biology; Kinetics; Models, Molecular; Nucleic Acid Conformation; Paenibacillus polymyxa; RNA, Catalytic
PubMed: 33622172
DOI: 10.1080/15476286.2021.1874706 -
Microbiology Resource Announcements Mar 2020DSM 292 was originally isolated from soil in 1947 due to its ability to produce antibiotics. The low proteolytic properties of strain DSM 292 warrant its examination as...
DSM 292 was originally isolated from soil in 1947 due to its ability to produce antibiotics. The low proteolytic properties of strain DSM 292 warrant its examination as a host for heterologous protein production. Here, we report the draft genome sequence of DSM 292 as established by Illumina MiSeq paired-end sequencing.
PubMed: 32165383
DOI: 10.1128/MRA.00071-20 -
Molecules (Basel, Switzerland) Nov 2023Glycosidases are essential for the industrial production of functional oligosaccharides and many biotech applications. A novel β-galactosidase/α-L-arabinopyranosidase...
Glycosidases are essential for the industrial production of functional oligosaccharides and many biotech applications. A novel β-galactosidase/α-L-arabinopyranosidase (PpBGal42A) of the glycoside hydrolase family 42 (GH42) from KF-1 was identified and functionally characterized. Using NPG as a substrate, the recombinant PpBGal42A (77.16 kD) was shown to have an optimal temperature and pH of 30 °C and 6.0. Using NPαArap as a substrate, the optimal temperature and pH were 40 °C and 7.0. PpBGal42A has good temperature and pH stability. Furthermore, Na, K, Li, and Ca (5 mmol/L) enhanced the enzymatic activity, whereas Mn, Cu, Zn, and Hg significantly reduced the enzymatic activity. PpBGal42A hydrolyzed NP-β-D-galactoside and NP-α-L-arabinopyranoside. PpBGal42A liberated galactose from β-1,3/4/6-galactobiose and galactan. PpBGal42A hydrolyzed arabinopyranose at C20 of ginsenoside Rb2, but could not cleave arabinofuranose at C20 of ginsenoside Rc. Meanwhile, the molecular docking results revealed that PpBGal42A efficiently recognized and catalyzed lactose. PpBGal42A hydrolyzes lactose to galactose and glucose. PpBGal42A exhibits significant degradative activity towards citrus pectin when combined with pectinase. Our findings suggest that PpBGal42A is a novel bifunctional enzyme that is active as a β-galactosidase and α-L-arabinopyranosidase. This study expands on the diversity of bifunctional enzymes and provides a potentially effective tool for the food industry.
Topics: Paenibacillus polymyxa; Lactose; Molecular Docking Simulation; Galactose; Glycoside Hydrolases; Cloning, Molecular; beta-Galactosidase; Hydrogen-Ion Concentration; Substrate Specificity; Paenibacillus
PubMed: 38005185
DOI: 10.3390/molecules28227464 -
Biochemical and Biophysical Research... Oct 2018Accompanied by benefits from horizontally transferred genes, bacteria have to face the risk of the invasion of dangerous genes. Bacteria often use the...
Accompanied by benefits from horizontally transferred genes, bacteria have to face the risk of the invasion of dangerous genes. Bacteria often use the restriction-modification (R-M) system, which is consisted of methyl transferase (MEase) and restrictase (REase), to protect self-DNA and defend against foreign DNA. Paenibacillus polymyxa, widely used as growth promoting rhizobacteria in agriculture, can also produce compounds of medical and industrial interests. It is unclear whether R-M systems exist in P. polymyxa. In this study, we used a shuttle plasmid with epigenetic modification from different bacteria to explore R-M systems in P. polymyxa. We found that DNA which is methylated by DNA adenine methyltransferase (Dam) in E. coli was strongly restricted, indicating the presence of a Dam-methylation-dependent R-M system in P. polymyxa. Whereas, DNA from a damE. coli strain was also moderately restricted, indicating the presence of a Dam-methylation-independent R-M system. Degradation of plasmid DNA with Dam methylation by cell-free protein extract of P. polymyxa provides additional evidence for the presence of Dam-methylation-dependent R-M system. Taken together, our work showed that there are two different types of R-M system in P. polymyxa, providing a foundation for the study of innate immunity in P. polymyxa and for the development of genetic engineering tools in P. polymyxa.
Topics: Bacillus subtilis; Cell-Free System; DNA; DNA Methylation; DNA Restriction-Modification Enzymes; Epigenesis, Genetic; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Genetic Engineering; Paenibacillus polymyxa; Plasmids; Site-Specific DNA-Methyltransferase (Adenine-Specific)
PubMed: 30224061
DOI: 10.1016/j.bbrc.2018.09.016 -
Applied and Environmental Microbiology Apr 2020The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by increases medium viscosity, which in turn presents considerable technical and...
The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in , was successfully disrupted. The levansucrase null mutant produced 2.5 ± 0.1 and 1.2 ± 0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7 ± 2.5 and 3.1 ± 0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1 ± 0.8 and 6.5 ± 0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0 ± 0.1 and 4.6 ± 0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges. Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic species is perhaps one of the most vigorously pursued, tools for engineering species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of were developed. Overall, although our study shows that levan is not the only EPS produced by , it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.
Topics: Bacterial Proteins; Butylene Glycols; Fermentation; Gene Silencing; Genes, Bacterial; Hexosyltransferases; Paenibacillus polymyxa; Polysaccharides, Bacterial
PubMed: 32144108
DOI: 10.1128/AEM.00196-20