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Applied Microbiology and Biotechnology Aug 2021Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications... (Review)
Review
Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. KEY POINTS: • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.
Topics: Bacillus megaterium; Beauty; Biotechnology; Recombinant Proteins; Vitamin B 12
PubMed: 34263356
DOI: 10.1007/s00253-021-11424-6 -
Microbial Cell Factories Jan 2024Silk proteins have emerged as versatile biomaterials with unique chemical and physical properties, making them appealing for various applications. Among them, spider...
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.
Topics: Silk; Bacillus megaterium; Escherichia coli; Recombinant Proteins; Bioreactors
PubMed: 38279170
DOI: 10.1186/s12934-024-02304-5 -
Brazilian Journal of Microbiology :... Mar 2021As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain...
As a key precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG) was mainly produced from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain (Bacillus spp.) with a low conversion rate for decades. The aim of this study was to enhance the 2-KLG production by co-culturing K. vulgare and Bacillus megaterium using three-stage temperature control (TSTC) strategy. By investigating the temperature effect on the 2-KLG fermentation, the optimum temperatures for the growths of K. vulgare and B. megaterium were 32 °C and 29 °C, respectively, while the optimum temperature for 2-KLG production was 35 °C. We developed a TSTC process: the temperature was kept at 32 °C during the first 16 h of fermentation, then decreased to 29 °C for the following 14 h, and maintained at 35 °C to the end of fermentation. By using this new process, the productivity and yield of 2-KLG from L-sorbose were obtained at 2.19 ± 0.19 g/L/h and 92.91 ± 1.02 g/L in 20-L fermentors for 5 batches, respectively, which were 22.35% and 6.02% higher than that of the control treatment (the single temperature of 29 °C). The increased cell density of K. vulgare during the exponential phase and the enhanced SDH activity (increased by 25.18% at 36 h, 17.14% at 44 h) in the production stage might be the reasons for enhanced 2-KLG conversion rate and yield. Our results demonstrated the feasibility of the TSTC strategy for 2-KLG production.
Topics: Bacillus megaterium; Bacteriological Techniques; Bioreactors; Culture Media; Fermentation; Rhodobacteraceae; Sorbose; Sugar Acids; Temperature
PubMed: 33145708
DOI: 10.1007/s42770-020-00396-w -
Applied Microbiology and Biotechnology Oct 2007Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of... (Review)
Review
Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a "toolbox" of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.
Topics: Bacillus megaterium; Genetic Engineering; Industrial Microbiology; Plasmids; Recombinant Proteins; Soil Microbiology
PubMed: 17657486
DOI: 10.1007/s00253-007-1089-3 -
New Biotechnology Mar 2021Due to high manufacturing costs, industrial production and application of bio-based polyhydroxyalkanoates (PHA) as bioplastics remain below the expected potential....
Effects of nutrient and oxygen limitation, salinity and type of salt on the accumulation of poly(3-hydroxybutyrate) in Bacillus megaterium uyuni S29 with sucrose as a carbon source.
Due to high manufacturing costs, industrial production and application of bio-based polyhydroxyalkanoates (PHA) as bioplastics remain below the expected potential. Improving yields and productivities during biotechnological production will contribute to eliminating existing shortcomings and should therefore be a priority in process development with new strains and substrates. The present study investigates key parameters such as different nutrient and oxygen limitation strategies and the salinity and type of salt to determine their impact on growth and poly(3-hydroxybutyrate) (P(3HB)) formation behaviour of Bacillus megaterium. The oxygen-limiting conditions applied resulted in a longer process duration and were found to be least effective with regard to P(3HB) content in the biomass. A higher P(3HB) content of 0.42 g g was achieved when nitrogen was limited compared to 0.34 g g under phosphate-limiting conditions; however, sucrose utilization was better when phosphate was limited. Replacing NaCl by KCl and evaluating different concentrations ranging from 0.08 to 1.7 mol L in the process medium showed that B. megaterium has a higher tolerance to KCl as the biomass and P(3HB) formation was increased to 0.48 g g compared to 0.36 g g. The combination of applying KCl instead of NaCl together with phosphorous limitation significantly increased P(3HB) productivity to 0.25 g L h compared to 0.09 g L h. It can be concluded that the effective utilization of sucrose as a carbon source requires a combination of high nitrogen and low phosphorous concentration and a salt content of 0.6 g L KCl for P(3HB) production with B. megaterium uyuni S29.
Topics: Bacillus megaterium; Culture Media; Hydroxybutyrates; Nutrients; Oxygen; Polyesters; Salinity; Salts; Sucrose
PubMed: 33278638
DOI: 10.1016/j.nbt.2020.11.012 -
Microbiology (Reading, England) May 1994It is evident that B. megaterium is an intriguing organism because of its biochemical versatility, its wide distribution ecologically, its ability to undergo... (Review)
Review
It is evident that B. megaterium is an intriguing organism because of its biochemical versatility, its wide distribution ecologically, its ability to undergo sporulation, and its usefulness as an industrial production strain and expression host. With the progress in genetics and the availability of molecular tools such as new transposons, vectors and efficient transformation, an understanding of some of the organization and regulation of many genes is increasing rapidly. Such recent discoveries as the ability of oxetanocin to combat some medically significant, recalcitrant viruses further demonstrates that there is much to be learned and much to benefit from continued study of B. megaterium.
Topics: Bacillus megaterium; Classification; Industrial Microbiology; Spores, Bacterial
PubMed: 8025666
DOI: 10.1099/13500872-140-5-1001 -
Molecules (Basel, Switzerland) Jul 2023Wild-type cytochrome P450 CYP102A1 from is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high... (Review)
Review
Wild-type cytochrome P450 CYP102A1 from is a highly efficient monooxygenase for the oxidation of long-chain fatty acids. The unique features of CYP102A1, such as high catalytic activity, expression yield, regio- and stereoselectivity, and self-sufficiency in electron transfer as a fusion protein, afford the requirements for an ideal biocatalyst. In the past three decades, remarkable progress has been made in engineering CYP102A1 for applications in drug discovery, biosynthesis, and biotechnology. The repertoire of engineered CYP102A1 variants has grown tremendously, whereas the substrate repertoire is avalanched to encompass alkanes, alkenes, aromatics, organic solvents, pharmaceuticals, drugs, and many more. In this article, we highlight the major advances in the past five years in our understanding of the structure and function of CYP102A1 and the methodologies used to engineer CYP102A1 for novel applications. The objective is to provide a succinct review of the latest developments with reference to the body of CYP102A1-related literature.
Topics: NADPH-Ferrihemoprotein Reductase; Cytochrome P-450 Enzyme System; Oxidation-Reduction; Electron Transport; Bacterial Proteins; Bacillus megaterium
PubMed: 37513226
DOI: 10.3390/molecules28145353 -
International Microbiology : the... Mar 2013To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human...
To further our understanding of the virulence potential of Bacillus megaterium strains, cell association and invasion assays were conducted in vitro by infecting human enterocytes (Caco-2 cells) with 53 strains of this bacterium isolated from honey. Two series of experiments were performed: (i) necrosis and cell detachment assays with the supernatants of bacterial culture filtrates from 16-h cultures and (ii) adhesion/invasion assays in which cultured enterocytes incubated with bacteria from 3-h cultures were resuspended in Dulbecco's modified Eagle's medium and chloramphenicol. The detachment of Caco-2 cells was evaluated by staining the cells with crystal violet. Necrosis was assessed by fluorescence microscopy of cells labeled with propidium iodide. Association (adhesion plus invasion) was determined by plate counts and invasion in an aminoglycoside protection assay. The results showed that spent culture supernatants detached and necrotized Caco-2 cells in a strain-dependent manner. Seven out of 53 B. megaterium filtered culture supernatants caused complete cell detachment. Suspensions of these same bacterial strains adhered and invaded enterocytes in 2-h infection experiments. To our knowledge, this is the first report on the interaction between B. megaterium and intestinal epithelial Caco-2 cells.
Topics: Bacillus megaterium; Bacterial Adhesion; Caco-2 Cells; Enterocytes; Honey; Host-Pathogen Interactions; Humans; Necrosis; Species Specificity; Virulence
PubMed: 24151779
DOI: 10.2436/20.1501.01.177 -
Proceedings of the National Academy of... May 2018Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression...
Native cell-free transcription-translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, , is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription-translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription-translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
Topics: Bacillus megaterium; Cell-Free System; Models, Biological; Protein Biosynthesis; Transcription, Genetic
PubMed: 29666238
DOI: 10.1073/pnas.1715806115 -
Nature Communications Aug 2017Bacteria have developed various mechanisms by which they sense, interact, and kill other bacteria, in an attempt to outcompete one another and survive. Here we show that...
Bacteria have developed various mechanisms by which they sense, interact, and kill other bacteria, in an attempt to outcompete one another and survive. Here we show that Bacillus subtilis can kill and prey on Bacillus megaterium. We find that Bacillus subtilis rapidly inhibits Bacillus megaterium growth by delivering the tRNase toxin WapA. Furthermore, utilizing the methionine analogue L-azidohomoalanine as a nutrient reporter, we provide evidence of nutrient extraction from Bacillus megaterium by Bacillus subtilis. Toxin delivery and nutrient extraction occur in a contact-dependent manner, and both activities are abolished in the absence of the phosphodiestrase YmdB, shown previously to mediate intercellular nanotube formation. Furthermore, we detect the localization of WapA molecules to nanotubes. Thus, we propose that Bacillus subtilis utilizes the same nanotube apparatus in a bidirectional manner, delivering toxin and acquiring beneficial cargo, thereby maximally exploiting potential niche resources.Bacteria can exchange nutrients and macromolecules through tubular membranous structures called nanotubes. Here, the authors show that Bacillus subtilis can kill and prey on Bacillus megaterium by delivering a toxin and extracting nutrients in a nanotube-dependent manner.
Topics: Antibiosis; Antigens, Bacterial; Bacillus megaterium; Bacillus subtilis; Bacterial Proteins; Bacterial Toxins; Nanotubes; Phosphoric Diester Hydrolases
PubMed: 28827522
DOI: 10.1038/s41467-017-00344-7