High-Yield Intra- and Extracellular Protein Production Using
            <i>Bacillus megaterium</i>

Stammen, Simon; Müller, Boje; Korneli, Claudia; Biedendieck, Rebekka; Gamer, Martin; Franco‐Lara, Ezequiel; Jahn, Dieter

doi:10.1128/aem.00431-10

Cited by 82 publications

(76 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Only a few proteases have experimentally been found to be secreted, including metalloprotease InhA, extracellular protease Vpr, aminopeptidase YwaD, and neutral protease NprM (67). The gene for the major extracellular protease NprM was deleted from production strain MS941 (69), which allowed up to 1.2 g/liter functional proteins to be recovered from the medium (4,20,25,54,66). These data clearly demonstrate the advantage of B. megaterium strains for recombinant protein production.…”

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

confidence: 82%

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

et al. 2011

Self Cite

View full text Add to dashboard Cite

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.

show abstract

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

confidence: 82%

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

et al. 2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…The intracellular B. megaterium protein production system, which is inducible by xylose, gave 1.25 g eGFP liter Ϫ1 , corresponding to a product yield of 3.7% (3.68 g eGFP per 100 g CDW) in fed-batch fermentations. In addition, Stammen et al (37) used antibiotics until the end of the fermentation process, with the result that no plasmids were lost during the fermentation process.…”

Section: Discussionmentioning

confidence: 99%

Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation

Wenzel

Muller

Siemann‐Herzberg

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

A novel technically compliant expression system was developed for heterologous protein production in Bacillus subtilis with the aim of increasing product yields at the same time as decreasing production costs. Standard systems involve the positively regulated manP promoter of the mannose operon, which led to relatively high product yields of 5.3% (5.3 g enhanced green fluorescent protein [eGFP] per 100 g cell dry weight [CDW]) but required large quantities of mannose to induce the reactions, thus rendering the system's technical application rather expensive. To improve this situation, mutant B. subtilis strains were used: the ⌬manA (mannose metabolism) strain TQ281 and the ⌬manP (mannose uptake) strain TQ356. The total amount of inducer could be reduced with TQ281, which, however, displayed sensitivity to mannose. An inducer-independent self-induction system was developed with TQ356 to further improve the cost efficiency and product yield of the system, in which glucose prevents induction by carbon catabolite repression. To create optimal self-induction conditions, a glucose-limited process strategy, namely, a fed-batch process, was utilized as follows. The initiation of self-induction at the beginning of the glucose-restricted transition phase between the batch and fed-batch phase of fermentation and its maintenance throughout the glucose-limiting fed-batch phase led to a nearly 3-fold increase of product yield, to 14.6%. The novel B. subtilis self-induction system thus makes a considerable contribution to improving product yield and reducing the costs associated with its technical application.The Gram-positive soil bacterium Bacillus subtilis is an attractive host for the production of various industrial proteins, such as amylases, proteases, and lipases. It is classified as a generally recognized as safe (GRAS) organism due to its lack of pathogenicity and absence of endotoxins. Further advantages of B. subtilis are its direct secretion of proteins into the medium via the Sec and Tat protein secretion machineries (40), capacity for genetic manipulation, easy handling, short processing times, and application in large-scale industrial production of proteins, for example, laundry enzymes and riboflavin (34). Homologous proteins can be produced in the order of several grams per liter, but only a few milligrams per liter of proteins can be produced from heterologous sources (34,35). When it comes to the biotechnological production of heterologous proteins, care must be taken to enable the reliable and inexpensive production of high yields of desired proteins. Priority was given to the development of a novel, technically compliant bacterial gene expression system that would be both cheap and highly reliable. Regulated promoters used in expression vectors can be induced by sugars, isopropyl-␤-D-thiogalactopyranoside (IPTG), temperature shifts, acid shock, or ethanol (27,28,29,35). If such systems are to be technically exploited, ways need to be found to circumvent the high price of inducers required. Autoinducible s...

show abstract

“…(3). Moreover, 99 tRNA-coding genes and 409 genes related to heterologous protein secretion systems were identified (8,15).…”

mentioning

confidence: 99%

Complete Genome Sequence of the Industrial Strain Bacillus megaterium WSH-002

Zhang

et al. 2011

J Bacteriol

View full text Add to dashboard Cite

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.Bacillus megaterium, a potential industrial strain, has exhibited many advantages in the industrial production of recombinant proteins (1, 8) and vitamins (2). In this study, B. megaterium WSH-002, the companion strain to improve Ketogulonicigenium vulgare growth and 2-keto-L-gulonic acid synthesis in the vitamin C industry (12, 16), was sequenced.The B. megaterium WSH-002 genome was sequenced by a combined strategy of the Sanger shotgun approach and 454 single-end sequencing technology. A genomic library containing a 5-kb insert was constructed, and 9,604 single-end reads were generated using the Sanger shotgun method, giving 15.0-fold coverage of the genome. Using the 454 Newbler (454 Life Sciences, Branford, CT), about 97% of the 258,353 single-end reads were assembled into two large scaffolds, including 62 contigs. The relationship of the contigs was determined by multiplex PCR, and the gaps between the contigs were closed by PCR amplification, primer walking, or shotgun sequencing with an ABI 3730 sequencer. The complete genome of WSH-002 achieves an error rate of less than 1 in the range of a 10-kb sequence through sequence assembly and quality assessment by the Phred/Phrap/Consed software package (6). Protein-coding genes were predicted by combining the results obtained with Glimmer 3.02 (4) and ZCURVE (7), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (10

show abstract

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

Cited by 82 publications

References 35 publications

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

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

Self-Inducible Bacillus subtilis Expression System for Reliable and Inexpensive Protein Production by High-Cell-Density Fermentation

Complete Genome Sequence of the Industrial Strain Bacillus megaterium WSH-002

Contact Info

Product

Resources

About