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  • 1
    Online Resource
    Online Resource
    Springer Science and Business Media LLC ; 2020
    In:  Microbial Cell Factories Vol. 19, No. 1 ( 2020-12)
    In: Microbial Cell Factories, Springer Science and Business Media LLC, Vol. 19, No. 1 ( 2020-12)
    Abstract: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. Results The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10 T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (P pps ) against the strong constitutive promoter P veg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (P pps ::P veg ) did not increase the plipastatin yield. Afterwards, deletion of surfactin ( srfAA-AD ) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. Conclusions This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.
    Type of Medium: Online Resource
    ISSN: 1475-2859
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2020
    detail.hit.zdb_id: 2091377-1
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  • 2
    Online Resource
    Online Resource
    Frontiers Media SA ; 2020
    In:  Frontiers in Bioengineering and Biotechnology Vol. 8 ( 2020-11-26)
    In: Frontiers in Bioengineering and Biotechnology, Frontiers Media SA, Vol. 8 ( 2020-11-26)
    Abstract: The anaerobic growth of B. subtilis to synthesize surfactin poses an alternative strategy to conventional aerobic cultivations. In general, the strong foam formation observed during aerobic processes represents a major obstacle. Anaerobic processes have, amongst others, the distinct advantage that the total bioreactor volume can be exploited as foaming does not occur. Recent studies also reported on promising product per biomass yields. However, anaerobic growth in comparison to aerobic processes has several disadvantages. For example, the overall titers are comparably low and cultivations are time-consuming due to low growth rates. B. subtilis JABs24, a derivate of strain 168 with the ability to synthesize surfactin, was used as model strain in this study. Ammonium and nitrite were hypothesized to negatively influence anaerobic growth. Ammonium with initial concentrations up to 0.2 mol/L was shown to have no significant impact on growth, but increasing concentrations resulted in decreased surfactin titers and reduced nitrate reductase expression. Anaerobic cultivations spiked with increasing nitrite concentrations resulted in prolonged lag-phases. Indeed, growth rates were in a similar range after the lag-phase indicating that nitrite has a neglectable effect on the observed decreasing growth rates. In bioreactor cultivations, the specific growth rate decreased with increasing glucose concentrations during the time course of both batch and fed-batch processes to less than 0.05 1/h. In addition, surfactin titers, overall Y P/X and Y P/S were 53%, ∼42%, and ∼57% lower than in serum flask with 0.190 g/L, 0.344 g/g and 0.015 g/g. The Y X/S , on the contrary, was 30% lower in the serum flask with 0.044 g/g. The productivities q were similar with ∼0.005 g/(g⋅h). However, acetate strongly accumulated during cultivation and was posed as further metabolite that might negatively influence anaerobic growth. Acetate added to anaerobic cultivations in a range from 0 g/L up to 10 g/L resulted in a reduced maximum and overall growth rate μ by 44% and 30%, respectively. To conclude, acetate was identified as a promising target for future process enhancement and strain engineering. Though, the current study demonstrates that the anaerobic cultivation to synthesize surfactin represents a reasonable perspective and feasible alternative to conventional processes.
    Type of Medium: Online Resource
    ISSN: 2296-4185
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2020
    detail.hit.zdb_id: 2719493-0
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  • 3
    In: AMB Express, Springer Science and Business Media LLC, Vol. 11, No. 1 ( 2021-12)
    Abstract: A novel approach targeting self-inducible surfactin synthesis under oxygen-limited conditions is presented. Because both the nitrate (NarGHI) and nitrite (NasDE) reductase are highly expressed during anaerobic growth of B. subtilis , the native promoter P srfA of the surfactin operon in strain B. subtilis JABs24 was replaced by promoters P narG and P nasD to induce surfactin synthesis anaerobically. Shake flask cultivations with varying oxygen availabilities indicated no significant differences in native P srfA expression. As hypothesized, activity of P narG and P nasD increased with lower oxygen levels and surfactin was not produced by P srfA ::P narG as well as P srfA ::P nasD mutant strains under conditions with highest oxygen availability. P narG showed expressions similar to P srfA at lowest oxygen availability, while maximum value of P nasD was more than 5.5-fold higher. Although the promoter exchange P srfA ::P narG resulted in a decreased surfactin titer at lowest oxygen availability, the strain carrying P srfA ::P nasD reached a 1.4-fold increased surfactin concentration with 696 mg/L and revealed an exceptional high overall Y P/X of 1.007 g/g. This value also surpassed the Y P/X of the reference strain JABs24 at highest and moderate oxygen availability. Bioreactor cultivations illustrated that significant cell lysis occurred when the process of “anaerobization” was performed too fast. However, processes with a constantly low agitation and aeration rate showed promising potential for process improvement, especially by employing the strain carrying P srfA ::P nasD promoter exchange. Additionally, replacement of other native promoters by nitrite reductase promoter P nasD represents a promising tool for anaerobic-inducible bioprocesses in Bacillus .
    Type of Medium: Online Resource
    ISSN: 2191-0855
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2021
    detail.hit.zdb_id: 2621432-5
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  • 4
    In: AMB Express, Springer Science and Business Media LLC, Vol. 13, No. 1 ( 2023-05-27)
    Abstract: The complex regulatory network in Bacillus , known as quorum sensing, offers many opportunities to modify bacterial gene expression and hence to control bioprocesses. One target regulated by this mechanism is the activity of the P srfA promoter, which is engaged in the formation of lipopeptide surfactin. It was hypothesised that deletion of rapC , rapF and rapH , encoding for prominent Rap-phosphatases known to affect P srfA activity, would enhance surfactin production. Therefore, these genes were deleted in a sfp + derivative of B. subtilis 168 with subsequent evaluation of quantitative data. Up to the maximum product formation of the reference strain B. subtilis KM1016 after 16 h of cultivation, the titers of the rap deletion mutants did not exceed the reference. However, an increase in both product yield per biomass Y P/X and specific surfactin productivity q surfactin was observed, without any considerable effect on the ComX activity. By extending the cultivation time, a 2.7-fold increase in surfactin titer was observed after 24 h for strain CT10 (Δ rapC ) and a 2.5-fold increase for CT11 (Δ rapF ) compared to the reference strain KM1016. In addition, Y P/X was again increased for strains CT10 and CT11, with values of 1.33 g/g and 1.13 g/g, respectively. Interestingly, the effect on surfactin titer in strain CT12 (Δ rapH ) was not as distinct, although it achieved the highest promoter activity (P srfA - lacZ ). The data presented support the possibility of involving the quorum sensing system of Bacillus in bioprocess control as shown here on the example of lipopeptide production.
    Type of Medium: Online Resource
    ISSN: 2191-0855
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2621432-5
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  • 5
    In: Microbiology Resource Announcements, American Society for Microbiology, Vol. 8, No. 38 ( 2019-09-19)
    Abstract: Bacillus velezensis UTB96 was isolated from soil based on its antifungal activity. Whole-genome sequencing of strain UTB96 provided further information about its secondary metabolite gene clusters. Compared to the well-known strain FZB42, UTB96 lacks an IS 3 element and a type I restriction endonuclease.
    Type of Medium: Online Resource
    ISSN: 2576-098X
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 2968655-6
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  • 6
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 201, No. 9 ( 2019-05)
    Abstract: Bacillus subtilis phosphorylates sugars during or after their transport into the cell. Perturbation in the conversion of intracellular phosphosugars to the central carbon metabolites and accumulation of phosphosugars can impose stress on the cells. In this study, we investigated the effect of phosphosugar stress on B. subtilis . Preliminary experiments indicated that the nonmetabolizable analogs of glucose were unable to impose stress on B. subtilis . In contrast, deletion of manA encoding mannose 6-phosphate isomerase (responsible for conversion of mannose 6-phosphate to fructose 6-phosphate) resulted in growth arrest and bulged cell shape in the medium containing mannose. Besides, an operon encoding a repressor (GlcR) and a haloic acid dehalogenase (HAD)-like phosphatase (PhoC; previously YwpJ) were upregulated. Integration of the P glcR -lacZ cassette into different mutational backgrounds indicated that P glcR is induced when (i) a manA -deficient strain is cultured with mannose or (ii) when glcR is deleted. GlcR repressed the transcription of glcR-phoC by binding to the σ A -type core elements of P glcR . An electrophoretic mobility shift assay showed no interaction between mannose 6-phosphate (or other phosphosugars) and the GlcR-P glcR DNA complex. PhoC was an acid phosphatase mainly able to dephosphorylate glycerol 3-phosphate and ribose 5-phosphate. Mannose 6-phosphate was only weakly dephosphorylated by PhoC. Since deletion of glcR and phoC alone or in combination had no effect on the cells during phosphosugar stress, it is assumed that the derepression of glcR-phoC is a side effect of phosphosugar stress in B. subtilis . IMPORTANCE Bacillus subtilis has different stress response systems to cope with external and internal stressors. Here, we investigated how B. subtilis deals with the high intracellular concentration of phosphosugars as an internal stressor. The results indicated the derepression of an operon consisting of a repressor (GlcR) and a phosphatase (PhoC). Further analysis revealed that this operon is not a phosphosugar stress response system. The substrate specificity of PhoC may indicate a connection between the glcR-phoC operon and pathways in which glycerol 3-phosphate and ribose 5-phosphate are utilized, such as membrane biosynthesis and teichoic acid elongation.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 7
    In: MicrobiologyOpen, Wiley, Vol. 10, No. 5 ( 2021-10)
    Abstract: Bacillus subtilis is described as a promising production strain for lipopeptides. In the case of B . subtilis strains JABs24 and DSM10 T , surfactin and plipastatin are produced. Lipopeptide formation is controlled, among others, by the DegU response regulator. The activating phospho‐transfer by the DegS sensor kinase is stimulated by the pleiotropic regulator DegQ, resulting in enhanced DegU activation. In B .  subtilis 168, a point mutation in the degQ promoter region leads to a reduction in gene expression. Corresponding reporter strains showed a 14‐fold reduced expression. This effect on degQ expression and the associated impact on lipopeptide formation was examined for B . subtilis JABs24, a lipopeptide‐producing derivative of strain 168, and B . subtilis wild‐type strain DSM10 T , which has a native degQ expression. Based on the stimulatory effects of the DegU regulator on secretory protease formation, the impact of degQ expression on extracellular protease activity was additionally investigated. To follow the impact of degQ , a deletion mutant was constructed for DSM10 T , while a natively expressed degQ version was integrated into strain JABs24. This allowed strain‐specific quantification of the stimulatory effect of degQ expression on plipastatin and the negative effect on surfactin production in strains JABs24 and DSM10 T . While an unaffected degQ expression reduced surfactin production in JABs24 by about 25%, a sixfold increase in plipastatin was observed. In contrast, degQ deletion in DSM10 T increased surfactin titer by threefold but decreased plipastatin production by fivefold. In addition, although significant differences in extracellular protease activity were detected, no decrease in plipastatin and surfactin produced during cultivation was observed.
    Type of Medium: Online Resource
    ISSN: 2045-8827 , 2045-8827
    URL: Issue
    Language: English
    Publisher: Wiley
    Publication Date: 2021
    detail.hit.zdb_id: 2661368-2
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  • 8
    Online Resource
    Online Resource
    American Society for Microbiology ; 2016
    In:  Journal of Bacteriology Vol. 198, No. 20 ( 2016-10-15), p. 2887-2896
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 198, No. 20 ( 2016-10-15), p. 2887-2896
    Abstract: Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes ( ganA and ganB ), two transporter component genes ( ganQ and ganP ), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB ). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the −35 box of the σ A -type promoter of P gan , which is located upstream of ganS . IMPORTANCE Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis . Nevertheless, the B. subtilis utilization system of galactan, which is found as the side chain of the rhamnogalacturonan type I complex in pectin, has remained partially studied. Here, we investigated the galactan utilization system consisting of the ganSPQAB operon and its regulator ganR . This study improves our knowledge of the carbohydrate degradation systems of B. subtilis , especially the pectin degradation systems. Moreover, the galactan-degrading enzymes may be exploited for the production of galacto-oligosaccharides, which are used as prebiotic substances in the food industry.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 9
    In: AMB Express, Springer Science and Business Media LLC, Vol. 11, No. 1 ( 2021-12)
    Abstract: Wild-type cultivations are of invaluable relevance for industrial biotechnology when it comes to the agricultural or food sector. Here, genetic engineering is hardly applicable due to legal barriers and consumer’s demand for GMO-free products. An important pillar for wild-type cultivations displays the genus Bacillus . One of the challenges for Bacillus cultivations is the global ComX-dependent quorum sensing system. Here, molecular process control can serve as a tool to optimize the production process without genetic engineering. To realize this approach, quantitative knowledge of the mechanism is essential, which, however, is often available only to a limited extent. The presented work provides a case study based on the production of cyclic lipopeptide surfactin, whose expression is in dependence of ComX, using natural producer B. subtilis DSM 10  T . First, a surfactin reference process with 40 g/L of glucose was performed as batch fermentation in a pilot scale bioreactor system to gain novel insights into kinetic behavior of ComX in relation to surfactin production. Interestingly, the specific surfactin productivity did not increase linearly with ComX activity. The data were then used to derive a mathematic model for the time course of ComX in dependence of existing biomass, biomass growth as well as a putative ComX-specific protease. The newly adapted model was validated and transferred to other batch fermentations, employing 20 and 60 g/L glucose. The applied approach can serve as a model system for molecular process control strategies, which can thus be extended to other quorum sensing dependent wild-type cultivations.
    Type of Medium: Online Resource
    ISSN: 2191-0855
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2021
    detail.hit.zdb_id: 2621432-5
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  • 10
    Online Resource
    Online Resource
    Armenian Green Publishing Co. ; 2015
    In:  Iranian Journal of Biotechnology Vol. 13, No. 4 ( 2015-12-28), p. 17-24
    In: Iranian Journal of Biotechnology, Armenian Green Publishing Co., Vol. 13, No. 4 ( 2015-12-28), p. 17-24
    Type of Medium: Online Resource
    ISSN: 1728-3043 , 2322-2921
    Language: English
    Publisher: Armenian Green Publishing Co.
    Publication Date: 2015
    detail.hit.zdb_id: 2223669-7
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