Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium

Biedendieck, Rebekka; Beine, Rafael; Gamer, Martin; Jordan, Eva; Buchholz, Klaus; Seibel, Jürgen; Dijkhuizen, Lubbert; Malten, Marco; Jahn, Dieter

doi:10.1007/s00253-006-0756-0

Cited by 33 publications

(18 citation statements)

References 22 publications

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“…In order to maximize the capacity of B. megaterium for the secretion of exoenzymes in high amounts into the environment, a series of vectors employing the signal peptides (SP) of the B. megaterium extracellular esterase LipA (Malten et al 2006) and penicillin G acylase (PGA) (Biedendieck et al 2007a) was developed for the recombinant production of extracellular proteins. 5…”

Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

“…Levansucrase from Lactobacillus reuteri strain 121 (van Hijum et al 2001;van Hijum et al 2004) was secreted best using the leader peptide of B. megaterium LipA (Biedendieck et al 2007a). More (Fig.…”

Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

“…The resulting B. megaterium strain MS941 (Tab. 3) proved to be useful for efficient secretion of heterologous proteins like a dextransucrase (DsrS) from Leuconostoc mesenteroides, a L. reuteri levansucrase (Lev), the B. megaterium penicillin G amidase (PGA), and a T. fusca 5 hydrolase (TFH) (Biedendieck et al 2007a;Malten et al 2006;Malten et al 2005a;Yang et al 2006;Yang et al 2007). Concomitant expression of the gene encoding the major signal-peptidase SipM of B. megaterium strain DSM319 additionally enhanced recombinant protein secretion (Biedendieck et al 2007a;Malten et al 2005b;Nahrstedt et al 2004).…”

Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

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Bacillus megaterium—from simple soil bacterium to industrial protein production host

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Biedendieck

Fuerch

et al. 2007

Appl Microbiol Biotechnol

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Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

Section: Plasmids For the Production And Purification Of Extracellulamentioning

confidence: 99%

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Bacillus megaterium—from simple soil bacterium to industrial protein production host

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Biedendieck

Fuerch

et al. 2007

Appl Microbiol Biotechnol

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“…Biotechnological use of B. megaterium for protein production. One major advantage of B. megaterium for industrial protein production is its ability to secrete proteins directly into the growth medium (2,4,37,45). B. megaterium employs Sec and Tat systems for this purpose; the relevant genes were all detected in the genomes.…”

Section: Vol 193 2011 Genome Sequences Of B Megaterium Qm B1551 Anmentioning

confidence: 99%

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

et al. 2011

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Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B 12 , penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B 12 through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second ␤-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.

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“…DNA sequences encoding C-or N-terminal affinity tags were added (5,16). Moreover, various plasmids for Secdependent secretion of recombinant, heterologous proteins were constructed (3,17). In general, exoproteins which are secreted via the Sec pathway are synthesized as protein precursors with N-terminal leader sequences.…”

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High-Yield Intra- and Extracellular Protein Production Using Bacillus megaterium

Stammen

Müller

Korneli

et al. 2010

Appl Environ Microbiol

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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 codonoptimized 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.

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Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium

Cited by 33 publications

References 22 publications

Bacillus megaterium—from simple soil bacterium to industrial protein production host

Bacillus megaterium—from simple soil bacterium to industrial protein production host

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

High-Yield Intra- and Extracellular Protein Production Using Bacillus megaterium

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