Systems Biology of Recombinant Protein Production Using Bacillus megaterium

Biedendieck, Rebekka; Borgmeier, Claudia; Bunk, Boyke; Stammen, Simon; Scherling, Christian; Meinhardt, Friedhelm; Wittmann, Christoph; Jahn, Dieter

doi:10.1016/b978-0-12-385118-5.00010-4

Cited by 60 publications

(55 citation statements)

References 58 publications

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“…The protoplast transformation of B. megaterium was performed as outlined previously [82]. The protoplast transformation of B. licheniformis was performed according to Waschkau et al [47].…”

Section: Methodsmentioning

confidence: 99%

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

2017

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BackgroundDifferent strains of the genus Bacillus are versatile candidates for the industrial production and secretion of heterologous proteins. They can be cultivated quite easily, show high growth rates and are usually non-pathogenic and free of endo- and exotoxins. They have the ability to secrete proteins with high efficiency into the growth medium, which allows cost-effective downstream purification processing. Some of the most interesting and challenging heterologous proteins are recombinant antibodies and antibody fragments. They are important and suitable tools in medical research for analytics, diagnostics and therapy. The smallest conventional antibody fragment with high-affinity binding to an antigen is the single-chain fragment variable (scFv). Here, different strains of the genus Bacillus were investigated using diverse cultivation systems for their suitability to produce and secret a recombinant scFv.ResultsExtracellular production of lysozyme-specific scFv D1.3 was realized by constructing a plasmid with a xylose-inducible promoter optimized for Bacillus megaterium and the D1.3scFv gene fused to the coding sequence of the LipA signal peptide from B. megaterium. Functional scFv was successfully secreted with B. megaterium MS941, Bacillus licheniformis MW3 and the three Bacillus subtilis strains 168, DB431 and WB800N differing in the number of produced proteases. Starting with shake flasks (150 mL), the bioprocess was scaled down to microtiter plates (1250 µL) as well as scaled up to laboratory-scale bioreactors (2 L). The highest extracellular concentration of D1.3 scFv (130 mg L−1) and highest space–time-yield (8 mg L−1 h−1) were accomplished with B. subtilis WB800N, a strain deficient in eight proteases. These results were reproduced by the production and secretion of a recombinant penicillin G acylase (Pac).ConclusionsThe genus Bacillus provides high potential microbial host systems for the secretion of challenging heterologous proteins like antibody fragments and large proteins at high titers. In this study, the highest extracellular concentration and space–time-yield of a recombinant antibody fragment for a Gram-positive bacterium so far was achieved. The successful interspecies use of the here-designed plasmid originally optimized for B. megaterium was demonstrated by two examples, an antibody fragment and a penicillin G acylase in up to five different Bacillus strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0625-9) contains supplementary material, which is available to authorized users.

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“…The protoplast transformation of B. megaterium was performed as outlined previously [82]. The protoplast transformation of B. licheniformis was performed according to Waschkau et al [47].…”

Section: Methodsmentioning

confidence: 99%

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

2017

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“…To confirm the successful cloning of Cm R gene cassette into pWH1520, the clones were double digested with KpnI and SpeI (Fast digest, 37°C, 4 h). Further, the vector-construct was transformed into B. megaterium protoplasts following the method reported by Biedendieck et al (2011). After transformation, the protoplasts were plated on Tet-LB-agar plate and incubated at 37°C to check for positive clones.…”

Section: Cloning Of CM R Gene Cassette Into Vector Pwh1520mentioning

confidence: 99%

Studies on the production of shikimic acid using the aroK knockout strain of Bacillus megaterium

Ghosh¹,

Mohan

Banerjee³

2016

World J Microbiol Biotechnol

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Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu(®)) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.

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“…1: Bacillus megaterium-Vektoren. Ausgangsshuttlevektor (A) und Variationen des Vektors bezüglich Promotor (Xylose-, Saccharoseinduzierbar; Phagen-Promotoren T7, K1E, SP6) (B) [5,8,10], Affinitätstag (C) [7,10], Signalsequenz (SP) (D) [5,10], Systeme zur Sekretion und Reinigung (E) [3,5] …”

Section: Bacillus-expressionssystemeunclassified

“…By using these systems, recombinant proteins can be produced and secreted in the grams per litre scale. 10.1007/s12268-014-0501-7 © Springer-Verlag 2014 ó Prokaryotische Mikroorganismen werden für die Produktion rekombinanter Proteine im großen Maßstab genutzt. Vorteile liegen in kurzen Generationszeiten, hohen Biomassekonzentrationen, Wachstum auf günstigen Substraten und einer einfachen genetischen Handhabung.…”

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Bacillus megaterium — ein Produktionssystem für rekombinante Proteine

et al. 2014

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During the past years plasmid systems for recombinant protein production using Bacillus megaterium were developed. These systems are based on multicopy plasmids with functional elements for cloning in Escherichia coli and induced gene expression in B. megaterium. Different promoters, signals for protein secretion and affinity tags for purification arranged in a high variety of combinations are available. By using these systems, recombinant proteins can be produced and secreted in the grams per litre scale.

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Systems Biology of Recombinant Protein Production Using Bacillus megaterium

Cited by 60 publications

References 58 publications

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

Recombinant production of the antibody fragment D1.3 scFv with different Bacillus strains

Studies on the production of shikimic acid using the aroK knockout strain of Bacillus megaterium

Bacillus megaterium — ein Produktionssystem für rekombinante Proteine

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