Bacillus megaterium infection presenting as pulmonary alveolar proteinosis, a case report.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a special clinical presentation mostly associated with autoimmune disorders. Here we report a rare case of PAP secondary to infection of Bacillus megaterium. CASE PRESENTATION: A 58-year-old woman presented with intermittent cough and dyspnea for half a year. Chest CT scan showed "crazy paving" pattern. B. megaterium was identified by percutaneous CT-guided needle biopsy. She continuously received antimicrobial treatment since the diagnosis and follow-up examination suggested great improvement. CONCLUSIONS: To our knowledge, this is the first case of B. megaterium infection presented with PAP pattern in healthy individuals. Attention should be paid on the secondary causes including rare pathogen infection when patients presented with PAP syndrome.

Protein Gas Vesicles of Bacillus megaterium as Enhancers of Ultrasound-Induced Transcriptional Regulation.

Gas vesicles (GVs) are large cylindrical gas-filled protein assemblies found in diverse aquatic bacteria that enable their adaptation of buoyancy. GVs have already been used as ultrasound contrasting agents. Here, we investigate GVs derived from Bacillus megaterium, aiming to minimize the number of accessory Gvps within the GV gene cluster and demonstrate the use of GVs as enhancers of acoustic radiation force administered by ultrasound. Three (GvpR, GvpT, and GvpU) out of 11 genes in the cluster were found to be dispensable for functional GV formation, and their omission resulted in narrower GVs. Two essential proteins GvpJ and GvpN were absent from recently determined GV structures, but GvpJ was nevertheless found to be tightly bound to the cylindrical part of GVs in this study. Additionally, the N-terminus of GvpN was observed to play an important role in the formation of mature GVs. The binding of engineered GvpC fromAnabaena flos-aquae to HEK293 cells via integrins enhanced the acoustic force delivered by ultrasound and resulted in an increased Ca2+ influx into cells. Coupling with a synthetic Ca2+-dependent signaling pathway GVs efficiently enhanced cell stimulation by ultrasound, which expands the potentials of noninvasive sonogenetics cell stimulation.

[Effects of Different Microbial Fertilizers on Soil Quality and Maize Yield in Coastal Saline Soil].

Microbial fertilizers have the characteristics of high efficiency and environmental protection in improving saline soils, and the application of functional microbial fertilizers is of great significance for the green abatement of saline barriers and the improvement of soil quality in coastal areas. The experiment was based on moderately saline soil in the coastal area of Hebei Province, with corn as the indicator crop, on the basis of conventional chemical fertilizer application. Different microbial fertilizer treatments, namely, T1 （conventional chemical fertilizer 750 kg·hm-2 + compound microbial agent 75 kg·hm-2）, T2 （conventional chemical fertilizer 750 kg·hm-2 + Bacillus megaterium 300 kg·hm-2）, T3 （conventional chemical fertilizer 750 kg·hm-2 + B. mucilaginosus 300 kg·hm-2）, T4 （conventional chemical fertilizer 750 kg·hm-2 + organic silicon fertilizer 600 kg·hm-2）, T5 （conventional chemical fertilizer 750 kg·hm-2 + bio-organic fertilizer 600 kg·hm-2）, T6 （conventional fertilizer 750 kg·hm-2 + active microalgae 15 kg·hm-2）, and CK （only fertilizer 750 kg·hm-2）, were used for these seven treatments, to study the effects of different microbial fertilizers on soil nutrients, salinity, bacterial community, and corn yield and economic efficiency during two critical periods （V12 stage and maturity stage） of corn. The results showed that compared with that in CK, T1 significantly increased soil total nitrogen （TN） and available phosphorus （AP） contents during the whole growth period. Over the whole reproductive period, soil organic matter （OM） at maturity increased by 10.35% over the V12 stage compared to that in CK, but there was no significant difference between treatments. Compared with that in CK, T5 and T6 significantly reduced soil total salinity and Ca2+ content during the whole growth period by an average of 14.51%-18.48% and 24.25%-25.51%. T1 significantly increased the bacterial diversity index over the whole growth period by 45.16% compared to that in CK. The dominant soil phyla were Actinobacteria, Proteobacteria, Acidobacteria, and Chloroflexi, and the dominant genera were Bacillus and Geminicoccaceae. The most abundant functions of the bacterial community in the study area were chemoheterotrophy and aerobic chemoheterotrophy, with average relative abundances of 28.89% and 27.11%, and T3 and T6 significantly improved soil N cycling function. The results of redundancy analysis （RDA） indicated that Na+, SO42-, pH, and EC were important factors driving the structure of the bacterial community, and correlation heatmaps showed that Na+, SO42-, pH, and EC were significantly and positively correlated mainly with the phylum Planctomycetota, whereas soil OM and TN were significantly and positively correlated with Cyanobacteria. Compared with that in CK, T6 increased the relative abundance of Cyanobacteria and optimized the bacterial community structure during the whole growth period. Using recommended dosages of bacterial fertilizers T1 and T6 increased maize yield by 7.31%-24.83% and economic efficiency by 9.05%-23.23%, respectively. The preliminary results of soil chemical properties and yield correlation analysis revealed that EC, AP, HCO3-, and Mg2+ were the obstacle factors limiting soil productivity in coastal areas. In conclusion, the use of the compound bacterial agent （T1） and active microalgae （T6） at the recommended dosage can significantly enhance soil nutrients, reduce salinity, and improve the structural diversity of soil bacterial communities, which not only ensures the increase in maize yield and efficiency but also realizes the efficient use of microbial fertilizers and the improvement of soil quality.

Efficient Immobilization of heavy metals using newly synthesized magnetic nanoparticles and some bacteria in a multi-metal contaminated soil.

Simultaneous application of modified Fe3O4 with biological treatments in remediating multi-metal polluted soils, has rarely been investigated. Thus, a pioneering approach towards sustainable environmental remediation strategies is crucial. In this study, we aimed to improve the efficiency of Fe3O4 as adsorbents for heavy metals (HMs) by applying protective coatings. We synthesized core-shell magnetite nanoparticles coated with modified nanocellulose, nanohydrochar, and nanobiochar, and investigated their effectiveness in conjunction with bacteria (Pseudomonas putida and Bacillus megaterium) for remediating a multi-metal contamination soil. The results showed that the coatings significantly enhanced the immobilization of heavy metals in the soil, even at low doses (0.5%). The coating of nanocellulose had the highest efficiency in stabilizing metals due to the greater variety of surface functional groups and higher specific surface area (63.86 m2 g-1) than the other two coatings. Interestingly, uncoated Fe3O4 had lower performance (113.6 m2 g-1) due to their susceptibility to deformation and oxidation. The use of bacteria as a biological treatment led to an increase in the stabilization of metals in soil. In fact, Pseudomonas putida and Bacillus megaterium increased immobilization of HMs in soil successfully because of extracellular polymeric substances and intensive negative charges. Analysis of metal concentrations in plants revealed that Ni and Zn accumulated in the roots, while Pb and Cd were transferred from the roots to the shoots. Treatment Fe3O4 coated with modified nanocellulose at rates of 0.5 and 1% along with Pseudomonas putida showed the highest effect in stabilizing metals. Application of coated Fe3O4 for in-situ immobilization of HMs in contamination soils is recommendable due to their high metal stabilization efficiency and suitability to apply in large quantities.

Tuning the peroxidase activity of artificial P450 peroxygenase by engineering redox-sensitive residues.

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium. The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

UV protection and insecticidal activity of microencapsulated Vip3Ag4 protein in Bacillus megaterium.

In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.

Sequence-Guided Redesign of an Omega-Transaminase from Bacillus megaterium for the Asymmetric Synthesis of Chiral Amines.

ω-Transaminases (ω-TAs) are attractive biocatalysts asymmetrically catalyzing ketones to chiral amines. However, poor non-native catalytic activity and substrate promiscuity severely hamper its wide application in industrial production. Protein engineering efforts have generally focused on reshaping the substrate-binding pockets of ω-TAs. However, hotspots around the substrate tunnel as well as distant sites outside the pockets may also affect its activity. In this study, the ω-TA from Bacillus megaterium (BmeTA) was selected for engineering. The tunnel mutation Y164F synergy with distant mutation A245T which was acquired through a multiple sequence alignment showed improved soluble expression, a 3.7-fold higher specific activity and a 19.9-fold longer half-life at 45 °C. Molecule Dynamics simulation explains the mechanism of improved catalytic activity, enhanced thermostability and improved soluble expression of BmeTAY164F/A245T(2 M). Finally, the resting cells of 2 M were used for biocatalytic processes. 450 mM of S-methoxyisopropylamine (S-MOIPA) was obtained with an ee value of 97.3 % and a conversion rate of 90 %, laying the foundation for its industrial production. Mutant 2 M was also found to be more advantageous in catalyzing the transamination of various ketones. These results demonstrated that sites that are far away from the active center also play an important role in the redesign of ω-TAs.

Immobilization of Pb(II) by Bacillus megaterium-based microbial-induced phosphate precipitation (MIPP) considering bacterial phosphorolysis ability and Ca-mediated alleviation of lead toxicity.

Inappropriate handling of lead (Pb)-containing wastewater that is produced as a result of smelting activities threatens the surrounding environment and human health. The microbial-induced phosphate precipitation (MIPP) technology was applied to immobilize Pb2+ in an aqueous solution considering bacterial phosphorolysis ability and Ca-mediated alleviation of lead toxicity. Pb immobilization was accompanied by sample characterization in order to explore the inherent mechanism that affected the immobilization efficiency. Results showed that Ca2+ use elevated the immobilization efficiency through the prevention of bacterial physisorption and chemisorption, an enhancement to the phosphatase activity and the degree of SGP hydrolysis, and the provision of nucleation sites for Pb2+ to attach. The formation of the Pb-GP complex helped the bacteria to maintain its activity at the commencement of catalyzing SGP hydrolysis. The nucleated minerals that were precipitated in a columnar shape through a directional stacking manner under MIPP featured higher chemical stability compared to non-nucleated minerals. As a result, there were three pathways, namely, bacterial physisorption, bacterial chemisorption, and substrate chelation, applied for Pb immobilization. The immobilization efficiency of 99.6% is achieved by precipitating bioprecipitates including Pb5(PO4)3Cl, Pb10(PO4)6Cl2, and Ca2Pb3(PO4)3Cl. The findings accentuate the potential of applying the MIPP technology to Pb-containing wastewater remediation.

Efficacy of DAP coated with bacterial strains and their metabolites for soil phosphorus availability and maize growth.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P2O5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Synbiotic Bacillus megaterium DSM 32963 and n-3 PUFA Salt Composition Elevates Pro-Resolving Lipid Mediator Levels in Healthy Subjects: A Randomized Controlled Study.

Beneficial health effects of omega-3 polyunsaturated fatty acids (n-3 PUFA) are partly attributed to specialized pro-resolving mediators (SPMs), which promote inflammation resolution. Strategies to improve n-3 PUFA conversion to SPMs may, therefore, be useful to treat or prevent chronic inflammatory disorders. Here, we explored a synbiotic strategy to increase circulating SPM precursor levels. Healthy participants (n = 72) received either SynΩ3 (250 mg eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) lysine salts; two billion CFU Bacillus megaterium; n = 23), placebo (n = 24), or fish oil (300 mg EPA plus DHA; N = 25) capsules daily for 28 days in a randomized, double-blind placebo-controlled parallel 3-group design. Biomarkers were assessed at baseline and after 2 and 28 days of intervention. The primary analysis involved the comparison between SynΩ3 and placebo. In addition, SynΩ3 was compared to fish oil. The synbiotic SynΩ3 comprising Bacillus megaterium DSM 32963 and n-3 PUFA salts significantly increased circulating SPM precursor levels, including 18-hydroxy-eicosapentaenoic acid (18-HEPE) plus 5-HEPE, which was not achieved to this extent by fish oil with a similar n-3 PUFA content. Omega-3 indices were increased slightly by both SynΩ3 and fish oil. These findings suggest reconsidering conventional n-3 PUFA supplementation and testing the effectiveness of SynΩ3 particularly in conditions related to inflammation.

The role and mechanism of Bacillus megaterium strain A14 in inhibiting the cadmium uptake by peanut plants in acidic red soil.

AIMS: This study explores the potential of cadmium (Cd)-resistant bacteria, specifically Bacillus megaterium A14, to decrease Cd accumulation in peanuts, a crop susceptible to metal uptake from contaminated soils, by understanding the bacterium's impact on plant Cd absorption mechanisms. METHODS AND RESULTS: Through pot experiments, we observed that A14 inoculation significantly increased peanut biomass under Cd stress conditions, primarily by immobilizing the metal and reducing its bioavailability. The bacterium effectively changed the distribution of Cd, with a notable 46.53% reduction in the exchangeable fraction, which in turn limited the expression of genes related to Cd transport in peanuts. Additionally, A14 enhanced the plant's antioxidant response, improving its tolerance to stress. Microbial analysis through 16S sequencing demonstrated that A14 inoculation altered the peanut rhizosphere, particularly by increasing populations of Firmicutes and Proteobacteria, which play crucial roles in soil remediation from heavy metals. CONCLUSION: The A14 strain effectively counters Cd toxicity in peanuts, promoting growth through soil Cd sequestration, root barrier biofilm formation, antioxidant system enhancement, suppression of Cd transport genes, and facilitation of Cd-remediating microorganisms.

Enhancing the catalytic efficiency of M32 carboxypeptidase by semi-rational design and its applications in food taste improvement.

BACKGROUND: Carboxypeptidase is an exopeptidase that hydrolyzes amino acids at the C-terminal end of the peptide chain and has a wide range of applications in food. However, in industrial applications, the relatively low catalytic efficiency of carboxypeptidases is one of the main limiting factors for industrialization. RESULTS: The study has enhanced the catalytic efficiency of Bacillus megaterium M32 carboxypeptidase (BmeCPM32) through semi-rational design. Firstly, the specific activity of the optimal mutant, BmeCPM32-M2, obtained through single-site mutagenesis and combinatorial mutagenesis, was 2.2-fold higher than that of the wild type (187.9 versus 417.8 U mg-1), and the catalytic efficiency was 2.9-fold higher (110.14 versus 325.75 s-1 mmol-1). Secondly, compared to the wild type, BmeCPM32-M2 exhibited a 1.8-fold increase in half-life at 60 °C, with no significant changes in its enzymatic properties (optimal pH, optimal temperature). Finally, BmeCPM32-M2 significantly increased the umami intensity of soy protein isolate hydrolysate by 55% and reduced bitterness by 83%, indicating its potential in developing tasty protein components. CONCLUSION: Our research has revealed that the strategy based on protein sequence evolution and computational residue mutation energy led to an improved catalytic efficiency of BmeCPM32. Molecular dynamics simulations have revealed that a smaller substrate binding pocket and increased enzyme-substrate affinity are the reasons for the enhanced catalytic efficiency. Furthermore the number of hydrogen bonds and solvent and surface area may contribute to the improvement of thermostability. Finally, the de-bittering effect of BmeCPM32-M2 in soy protein isolate hydrolysate suggests its potential in developing palatable protein components. © 2024 Society of Chemical Industry.

PHB Production by Bacillus megaterium LSRB 0103 Using Cornstarch and Urea.

Polyhydroxybutyrates (PHBs) are biopolymers that are good green alternative for synthetic carbon-based polymers, and are also one of the most researched members of the Polyhydroxyalkanoates (PHA) family. In this study, a gram-positive bacterial strain Bacillus megaterium LSRB 0103 was isolated from Pallikaranai Marshland, Chennai, India. Primary screening using Sudan Black dye revealed the presence of intracellular PHB granules. Minimal Davis Media (MDM) which was used or PHB production gave a yield of 0.7107 g/L. Subsequently, using response surface methodology (RSM), a central composite design (CCD) model was designed for media optimization having cornstarch, urea, and pH as independent variables. The findings of the CCD model were fitted into a second-order polynomial equation. The RSM model predicted the maximum PHB yield of 0.93 g/L, at these independent variable levels, cornstarch, 5 g/L; urea, 2.1 g/L; and pH 7.0; while the experimental PHB yield was 0.94 g/L, with a percentage error of 1.1%. This study is the first-time report of production of PHB by Bacillus megaterium using cornstarch and urea as substrate.

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity-Based Probes.

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY-family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol-aziridine activity-based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol-aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5-probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin-probe enables SQase capture and identification by proteomics. The Cy5-probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT-qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

Bacillus megaterium: Evaluation of Chemical Nature of Metabolites and Their Antioxidant and Agronomics Properties.

Bacillus megaterium is particularly known for its abundance in soils and its plant growth promotion. To characterize the metabolites excreted by this specie, we performed successive liquid/liquid extractions from bacteria culture medium with different polarity solvents (cyclohexane, dichloromethane, ethyl acetate and butanol) to separate the metabolites in different polarity groups. The extracts were characterized regarding their total phenolic content, the amount of reducing sugar, the concentration of primary amines and proteins, their chromatographic profile by HPLC-DAD-ELSD and their chemical identification by GC-MS. Among the 75 compounds which are produced by the bacteria, 19 identifications were for the first time found as metabolites of B. megaterium and 23 were described for the first time as metabolites in Bacillus genus. The different extracts containing B. megaterium metabolites showed interesting agronomic activity, with a global inhibition of seed germination rates of soya, sunflower, corn and ray grass, but not of corn, compared to culture medium alone. Our results suggest that B. megaterium can produce various metabolites, like butanediol, cyclic dipeptides, fatty acids, and hydrocarbons, with diverse effects and sometimes with opposite effects in order to modulate its response to plant growth and adapt to various environmental effects. These findings provide new insight into bioactive properties of this species for therapeutic uses on plants.

High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Production and secretion of recombinant spider silk in Bacillus megaterium.

BACKGROUND: Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider silk, known for its exceptional mechanical strength, has attracted considerable attention. Recombinant production of spider silk represents the most promising route towards its scaled production; however, challenges persist within the upstream optimization of host organisms, including toxicity and low yields. The high cost of downstream cell lysis and protein purification is an additional barrier preventing the widespread production and use of spider silk proteins. Gram-positive bacteria represent an attractive, but underexplored, microbial chassis that may enable a reduction in the cost and difficulty of recombinant silk production through attributes that include, superior secretory capabilities, frequent GRAS status, and previously established use in industry. RESULTS: In this study, we explore the potential of gram-positive hosts by engineering the first production and secretion of recombinant spider silk in the Bacillus genus. Using an industrially relevant B. megaterium host, it was found that the Sec secretion pathway enables secretory production of silk, however, the choice of signal sequence plays a vital role in successful secretion. Attempts at increasing secreted titers revealed that multiple translation initiation sites in tandem do not significantly impact silk production levels, contrary to previous findings for other gram-positive hosts and recombinant proteins. Notwithstanding, targeted amino acid supplementation in minimal media was found to increase production by 135% relative to both rich media and unaltered minimal media, yielding secretory titers of approximately 100 mg/L in flask cultures. CONCLUSION: It is hypothesized that the supplementation strategy addressed metabolic bottlenecks, specifically depletion of ATP and NADPH within the central metabolism, that were previously observed for an E. coli host producing the same recombinant silk construct. Furthermore, this study supports the hypothesis that secretion mitigates the toxicity of the produced silk protein on the host organism and enhances host performance in glucose-based minimal media. While promising, future research is warranted to understand metabolic changes more precisely in the Bacillus host system in response to silk production, optimize signal sequences and promoter strengths, investigate the mechanisms behind the effect of tandem translation initiation sites, and evaluate the performance of this system within a bioreactor.

PHB production by Bacillus megaterium strain MNSH1-9K-1 using low-cost media.

Plastics are widely used for diverse applications due to their versatility. However, their negative impact on ecosystems is undeniable due to their long-term degradation. Thus, there is a rising need for developing eco-friendlier alternatives to substitute fossil-based plastics, like biopolymers. PHA are synthesized intracellularly by microorganisms under stressful conditions of growth and have similar characteristics to conventional polymers, like their melting point, transition temperatures, crystallinity, and flexibility. Although it is feasible to use biopolymers for diverse industrial applications, their elevated production cost due to the supplies needed for microbiological procedures and the low productivity yields obtained have been the main limiting factors for their commercial success. The present study assessed the ability of Bacillus megaterium strain MNSH1-9K-1 to produce biopolymers using low-cost media from different kinds of fruit-peel residues. The results show that MNSH1-9K-1 can produce up to 58 g/L of PHB when grown in a medium prepared from orange-peel residues. The data obtained provide information to enhance the scalability of these kinds of biotechnological processes.