The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Dieter Jahn - One of the best experts on this subject based on the ideXlab platform.
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getting the big beast to work systems biotechnology of Bacillus Megaterium for novel high value proteins
Journal of Biotechnology, 2013Co-Authors: Claudia Korneli, Rebekka Biedendieck, Dieter Jahn, Florian David, Christoph WittmannAbstract:The high industrial relevance of the soil bacterium Bacillus Megaterium as host for recombinant proteins is driving systems-wide analyses of its metabolic and regulatory networks. The present review highlights novel systems biology tools available to unravel the various cellular components on the level of metabolic and regulatory networks. These provide a rational platform for systems metabolic engineering of B. Megaterium. In line, a number of interesting studies have particularly focused on studying recombinant B. Megaterium in its industrial bioprocess environment thus integrating systems metabolic engineering with systems biotechnology and providing the full picture toward optimal processes.
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erratum to transcriptome profiling of degu expression reveals unexpected regulatory patterns in Bacillus Megaterium and discloses new targets for optimizing expression
Applied Microbiology and Biotechnology, 2011Co-Authors: Claudia Borgmeier, Kristina Hoffmann, Rebekka Biedendieck, Dieter Jahn, Friedhelm MeinhardtAbstract:The first whole transcriptome assessment of a Bacillus Megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. Megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.
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polysaccharide synthesis of the levansucrase sacb from Bacillus Megaterium is controlled by distinct surface motifs
Journal of Biological Chemistry, 2011Co-Authors: Christian P Strube, Dieter Jahn, Arne Homann, Martin Gamer, Juergen Seibel, Dirk W HeinzAbstract:Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus Megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (kcat) and substrate affinity (Km) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.
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systems biology of recombinant protein production using Bacillus Megaterium
Methods in Enzymology, 2011Co-Authors: Rebekka Biedendieck, Claudia Borgmeier, Friedhelm Meinhardt, Simon Stammen, Christoph Wittmann, Boyke Bunk, Christian Scherling, Dieter JahnAbstract:The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus Megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. Megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. Megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. Megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. Megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data.
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high yield intra and extracellular protein production using Bacillus Megaterium
Applied and Environmental Microbiology, 2010Co-Authors: Simon Stammen, Rebekka Biedendieck, Martin Gamer, Claudia Korneli, Ezequiel Francolara, Britta Katrin Muller, Dieter JahnAbstract:The Bacillus Megaterium protein production system based on the inducible promoter of the xyl operon (P(xylA)) was systematically optimized. Multiple changes in basic promoter elements, such as the -10 and -35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g(CDW)) or 124 mg liter(-1). In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter(-1), corresponding to 36.8 mg protein per g(CDW). Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. Megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter(-1) (5.3 to 6.6 mg liter(-1)), was observed for strains where the Tfh export was facilitated by a codon-optimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter(-1), 7.7 mg liter(-1), was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.
Rebekka Biedendieck - One of the best experts on this subject based on the ideXlab platform.
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prototyping of Bacillus Megaterium genetic elements through automated cell free characterization and bayesian modelling
bioRxiv, 2016Co-Authors: Simon J Moore, Rebekka Biedendieck, J Macdonald, Sarah Wienecke, Nicolas Kylilis, Karen M Polizzi, Paul S FreemontAbstract:Automation and factorial experimental design together with cell-free in vitro transcription-translation systems offers a new route to the precise characterization of regulatory components. This now presents a new opportunity to illuminate the genetic circuitry from arcane microbial chassis, which are difficult to assess in vivo. One such host, Bacillus Megaterium, is a giant microbe with industrial potential as a producer of recombinant proteins at gram per litre scale. Herein, we establish a B. Megaterium cell-free platform and characterize a refactored xylose-repressor circuit using acoustic liquid handling robotics to simultaneously monitor 324 reactions in vitro. To accurately describe the system, we have applied a Bayesian statistical approach to infer model parameters by simultaneously using information from multiple experimental conditions. These developments now open up a new approach for the rapid and accurate characterization of genetic circuitry using cell-free reactions from unusual microbial cell chasses for bespoke applications.
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getting the big beast to work systems biotechnology of Bacillus Megaterium for novel high value proteins
Journal of Biotechnology, 2013Co-Authors: Claudia Korneli, Rebekka Biedendieck, Dieter Jahn, Florian David, Christoph WittmannAbstract:The high industrial relevance of the soil bacterium Bacillus Megaterium as host for recombinant proteins is driving systems-wide analyses of its metabolic and regulatory networks. The present review highlights novel systems biology tools available to unravel the various cellular components on the level of metabolic and regulatory networks. These provide a rational platform for systems metabolic engineering of B. Megaterium. In line, a number of interesting studies have particularly focused on studying recombinant B. Megaterium in its industrial bioprocess environment thus integrating systems metabolic engineering with systems biotechnology and providing the full picture toward optimal processes.
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erratum to transcriptome profiling of degu expression reveals unexpected regulatory patterns in Bacillus Megaterium and discloses new targets for optimizing expression
Applied Microbiology and Biotechnology, 2011Co-Authors: Claudia Borgmeier, Kristina Hoffmann, Rebekka Biedendieck, Dieter Jahn, Friedhelm MeinhardtAbstract:The first whole transcriptome assessment of a Bacillus Megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. Megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.
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systems biology of recombinant protein production using Bacillus Megaterium
Methods in Enzymology, 2011Co-Authors: Rebekka Biedendieck, Claudia Borgmeier, Friedhelm Meinhardt, Simon Stammen, Christoph Wittmann, Boyke Bunk, Christian Scherling, Dieter JahnAbstract:The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus Megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. Megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. Megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. Megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. Megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data.
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high yield intra and extracellular protein production using Bacillus Megaterium
Applied and Environmental Microbiology, 2010Co-Authors: Simon Stammen, Rebekka Biedendieck, Martin Gamer, Claudia Korneli, Ezequiel Francolara, Britta Katrin Muller, Dieter JahnAbstract:The Bacillus Megaterium protein production system based on the inducible promoter of the xyl operon (P(xylA)) was systematically optimized. Multiple changes in basic promoter elements, such as the -10 and -35 region and the ribosome-binding site, resulted in an 18-fold increase of protein production compared to the production of the previously established system. The production in shaking-flask culture of green fluorescent protein (Gfp) as a model product led to 82.5 mg per g cell dry weight (g(CDW)) or 124 mg liter(-1). In fed-batch cultivation, the volumetric protein yield was increased 10-fold to 1.25 g liter(-1), corresponding to 36.8 mg protein per g(CDW). Furthermore, novel signal peptides for Sec-dependent protein secretion were predicted in silico using the B. Megaterium genome. Subsequently, leader peptides of Vpr, NprM, YngK, YocH, and a computationally designed artificial peptide were analyzed experimentally for their potential to facilitate the secretion of the heterologous model protein Thermobifida fusca hydrolase (Tfh). The best extracellular protein production, 5,000 to 6,200 U liter(-1) (5.3 to 6.6 mg liter(-1)), was observed for strains where the Tfh export was facilitated by a codon-optimized leader peptide of YngK and by the signal peptide of YocH. Further increases in extracellular protein production were achieved when leader peptides were used in combination with the optimized expression system. In this case, the greatest extracellular enzyme amount of 7,200 U liter(-1), 7.7 mg liter(-1), was achieved by YocH leader peptide-mediated protein export. Nevertheless, the observed principal limitations in protein export might be related to components of the Sec-dependent protein transport system.
Friedhelm Meinhardt - One of the best experts on this subject based on the ideXlab platform.
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erratum to transcriptome profiling of degu expression reveals unexpected regulatory patterns in Bacillus Megaterium and discloses new targets for optimizing expression
Applied Microbiology and Biotechnology, 2011Co-Authors: Claudia Borgmeier, Kristina Hoffmann, Rebekka Biedendieck, Dieter Jahn, Friedhelm MeinhardtAbstract:The first whole transcriptome assessment of a Bacillus Megaterium strain provides unanticipated insights into the degSU regulon considered to be of central importance for exo-enzyme production. Regulatory patterns as well as the transcription of degSU itself deviate from the model organism Bacillus subtilis; the number of DegU-regulated secretory enzymes is rather small. Targets for productivity optimization, besides degSU itself, arise from the unexpected DegU-dependent induction of the transition-state regulator AbrB during exponential growth. Induction of secretion-assisting factors, such as the translocase subunit SecY or the signal peptidase SipM, promote hypersecretion. B. Megaterium DegSU transcriptional control is advantageous for production purposes, since the degU32 constitutively active mutant conferred hypersecretion of a heterologous Bacillus amyloliquefaciens amylase without the detrimental rise, as for B. subtilis and Bacillus licheniformis, in extracellular proteolytic activities.
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systems biology of recombinant protein production using Bacillus Megaterium
Methods in Enzymology, 2011Co-Authors: Rebekka Biedendieck, Claudia Borgmeier, Friedhelm Meinhardt, Simon Stammen, Christoph Wittmann, Boyke Bunk, Christian Scherling, Dieter JahnAbstract:The Gram-negative bacterium Escherichia coli is the most widely used production host for recombinant proteins in both academia and industry. The Gram-positive bacterium Bacillus Megaterium represents an increasingly used alternative for high yield intra- and extracellular protein synthesis. During the past two decades, multiple tools including gene expression plasmids and production strains have been developed. Introduction of free replicating and integrative plasmids into B. Megaterium is possible via protoplasts transformation or transconjugation. Using His(6)- and StrepII affinity tags, the intra- or extracellular produced proteins can easily be purified in one-step procedures. Different gene expression systems based on the xylose controlled promoter P(xylA) and various phage RNA polymerase (T7, SP6, K1E) driven systems enable B. Megaterium to produce up to 1.25g of recombinant protein per liter. Biomass concentrations of up to 80g/l can be achieved by high cell density cultivations in bioreactors. Gene knockouts and gene replacements in B. Megaterium are possible via an optimized gene disruption system. For a safe application in industry, sporulation and protease-deficient as well as UV-sensitive mutants are available. With the help of the recently published B. Megaterium genome sequence, it is possible to characterize bottle necks in the protein production process via systems biology approaches based on transcriptome, proteome, metabolome, and fluxome data. The bioinformatical platform (Megabac, http://www.megabac.tu-bs.de) integrates obtained theoretical and experimental data.
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Bacillus Megaterium from simple soil bacterium to industrial protein production host
Applied Microbiology and Biotechnology, 2007Co-Authors: Patricia S Vary, Friedhelm Meinhardt, Rebekka Biedendieck, Tobias Fuerch, Manfred Rohde, Wolfdieter Deckwer, Dieter JahnAbstract: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.
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identification and functional characterization of a type i signal peptidase gene of Bacillus Megaterium dsm319
Applied Microbiology and Biotechnology, 2004Co-Authors: Hannes Nahrstedt, K D Wittchen, M A Rachman, Friedhelm MeinhardtAbstract:The sipM gene of Bacillus Megaterium encoding a type I signal peptidase (SPase) was isolated and structurally characterized. RNA analysis revealed a transcript size in accordance with a bicistronic operon comprising sipM and an adjacent open reading frame. Inactivation of sipM by targeted gene disruption could not be achieved, indicating its essential role for cell viability since there might be no other type I SPase of major importance present in B. Megaterium. Plasmid-assisted amplification of the gene resulted in an increase in activity of the heterologous glucanase used as an extracellular reporter, suggesting a potential bottleneck for protein secretion within this species.
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cloning expression and carbon catabolite repression of the bamm gene encoding β amylase of Bacillus Megaterium dsm319
Applied Microbiology and Biotechnology, 2001Co-Authors: Jinsook Lee, K D Wittchen, C Stahl, Jan Strey, Friedhelm MeinhardtAbstract:The bamM gene from Bacillus Megaterium DSM319 encoding an extracellular β-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 °C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE – retaining unchanged the amino acid sequence – provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.
Marco Malten - One of the best experts on this subject based on the ideXlab platform.
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metabolic engineering of cobalamin vitamin b12 production in Bacillus Megaterium
Microbial Biotechnology, 2010Co-Authors: Rebekka Biedendieck, Evelyne Deery, Marco Malten, Heiko Barg, Boyke Bunk, Janhenning Martens, Helen K Leech, Martin J Warren, Dieter JahnAbstract:Cobalamin (vitamin B(12)) production in Bacillus Megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B(12) biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG(A)cbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B(12) riboswitch upstream of the cbiXJCDETLFGAcysG(A)cbiYbtuR operon and the recombinant production of three different vitamin B(12) binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B(12)-dependent feedback inhibition. All these strategies increased cobalamin production in B. Megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B(12) concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.
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plasmid system for the intracellular production and purification of affinity tagged proteins in Bacillus Megaterium
Biotechnology and Bioengineering, 2007Co-Authors: Rebekka Biedendieck, Marco Malten, Wolfdieter Deckwer, Yang Yang, Dieter JahnAbstract:A multiple vector system for the intracellular high-level production of affinity tagged recombinant proteins in Bacillus Megaterium was developed. The N- and C-terminal fusion of a protein of interest to a Strep II and a His 6 -tag is possible. Corresponding genes are expressed under the control of a xylose-inducible promoter in a xylose isomerase deficient host strain. The exemplatory protein production of green fluorescent protein (GFP) showed differences in produced and recovered protein amounts in dependence of the employed affinity tag and its N- or C-terminal location. Up to 9 mg GFP per liter shake flask culture were purified using one-step affinity chromatography. Integration of a protease cleavage site into the recombinant fusion protein allowed tag removal via tobacco etch virus (TEV) protease or Factor Xa treatment and a second affinity chromatographic step. Up to 274 mg/L culture were produced at 52 g CDW/L using a glucose limited fedbatch cultivation. GFP production and viability of the production host were followed by flow cytometry.
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a Bacillus Megaterium plasmid system for the production export and one step purification of affinity tagged heterologous levansucrase from growth medium
Applied and Environmental Microbiology, 2006Co-Authors: Marco Malten, Rebekka Biedendieck, Martin Gamer, Annchristin Drews, Simon Stammen, Klaus Buchholz, Lubbert Dijkhuizen, Dieter JahnAbstract:A multiple vector system for the production and export of recombinant affinity-tagged proteins in Bacillus Megaterium was developed. Up to 1 mg/liter of a His6-tagged or Strep-tagged LactoBacillus reuteri levansucrase was directed into the growth medium, using the B. Megaterium esterase LipA signal peptide, and recovered by one-step affinity chromatography.
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production and secretion of recombinant leuconostoc mesenteroides dextransucrase dsrs in Bacillus Megaterium
Biotechnology and Bioengineering, 2005Co-Authors: Marco Malten, Wolfdieter Deckwer, Rajan Hollmann, Dieter JahnAbstract:Leuconostoc mesenteroides dextransucrase DsrS was recombinantly produced in Bacillus Megaterium and exported into the growth medium. For this purpose a plasmid-based xylose-inducible gene expression system was optimized via introduction of a multiple cloning site and an encoded optimal B. Megaterium ribosome binding site. A cre mediating glucose-dependent catabolite repression was removed. Recombinant DsrS was found in the cytoplasm and exported via its native leader sequence into the growth medium. Elimination of the extracellular protease NprM increased extracellular DsrS concentrations by a factor of 4 and stabilized the recombinant protein for up to 12 h. Cultivation in a semi-defined medium resulted in a further doubling of extracellular DsrS concentration up to an activity of 65 Units/L. To develop an industrial process a high cell density cultivation of B. Megaterium was established yielding cell dry weights of up to 80 g/L. After induction of dsrS expression high specific (362 Units/g) and volumetric (28,600 Units/L) activities of dextran free DsrS were measured. However, using high cell density cultivation, most DsrS was found cell-associated indicating current limitations of the production process. A protease accessibility assay identified the major limitation of DsrS production at the level of protein folding. Intracellular misfolding of DsrS hampered DsrS export via the SEC pathway at high cell densities. The subsequent use of a semi-defined mineral medium and the induction of DsrS production at lower cell densities increased protein export efficiency remarkably, but also led to extracellular DsrS aggregation. Further optimization strategies for the production of recombinant DsrS in B. Megaterium are discussed.
Jian Chen - One of the best experts on this subject based on the ideXlab platform.
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sporulation and spore stability of Bacillus Megaterium enhance ketogulonigenium vulgare propagation and 2 keto l gulonic acid biosynthesis
Bioresource Technology, 2012Co-Authors: Yibo Zhu, Jie Liu, Jingwen Zhou, Jian ChenAbstract:Abstract Bacillus spp. is widely used as the companion bacterium in the two-step biosynthesis of 2-keto- l -gulonic acid (2-KLG), which is the direct precursor in the production of vitamin C by Ketogulonigenium vulgare. To understand the effects of sporulation and spore stability on 2-KLG production, the spo0A and spoVFA deletion mutants of Bacillus Megaterium were constructed. The sorbose conversion rates of spo0A and spoVFA mutant co-culture systems were 33% and 70% lower, respectively, than that of the wild-type co-culture system. In addition, K. vulgare cell numbers in the two mutant systems declined by 15% and 49%, respectively, compared to the value in the wild-type system. Correlation analysis indicated that the 2-KLG concentration is positively related to sorbose dehydrogenase activity and the K. vulgare cell number. This study demonstrated that sporulation and spore stability of the wild-type companion play key roles in the enhancement of K. vulgare propagation and 2-KLG biosynthesis.
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complete genome sequence of the industrial strain Bacillus Megaterium wsh 002
Journal of Bacteriology, 2011Co-Authors: Liming Liu, Jing Zhang, Wei Zou, Zhemin Zhou, Jie Liu, Lei Wang, Jian ChenAbstract:ABSTRACT Bacillus Megaterium, an industrial strain, has been widely used in protein production and the vitamin C industry. Here we reported a finished, annotated, and compared 4.14-Mbp high-quality genome sequence of B. Megaterium WSH-002, which is the companion strain for Ketogulonicigenium vulgare in the vitamin C industry and is stocked in our laboratory.
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development of chemically defined media supporting high cell density growth of ketogulonicigenium vulgare and Bacillus Megaterium
Bioresource Technology, 2011Co-Authors: Jing Zhang, Liming Liu, Jie Liu, Jingwen Zhou, Kejie Chen, Jian ChenAbstract:Abstract The immediate precursor of L-ascorbic acid, or vitamin C, is 2-keto- l -gulonic acid (2-KLG). This is commonly produced commercially by Ketogulonicigenium vulgare and Bacillus Megaterium , using corn steep liquor powder (CSLP) as an organic nitrogen source. In this study, the effects of the individual CSLP components (amino acids, vitamins, and metal elements) on 2-KLG production were evaluated, with the aim of developing a complete, chemically defined medium for 2-KLG production. Forty components of CSLP were analyzed, and key components were correlated to biomass, 2-KLG productivity, and consumption rate of L-sorbose. Glycine had the greatest effect, followed by serine, biotin, proline, nicotinic acid, and threonine. The combination of 0.28 g L −1 serine, 0.36 g L −1 glycine, 0.18 g L −1 threonine, 0.28 g L −1 proline, 0.19 g L −1 nicotinic acid, and 0.62 mg L −1 biotin in a chemically defined medium produced the highest maximum biomass concentration (4.2 × 10 9 cfu mL −1 ), 2-KLG concentration (58 g L −1 ), and yield (0.76 g g −1 ) after culturing for 28 h.
