Dataset Information

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome.

ABSTRACT: BACKGROUND: Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid. RESULTS: A plasmid-less B. subtilis JE852-based strain secreting glutamyl-specific protease (GSP-the protein product of the mpr gene from B. amyloliquefaciens) was constructed that exhibits decreased levels of other extracellular proteases. Ten copies of an mprB.amy cassette in which the GSP gene was placed between the promoter of the B. amyloliquefaciens rplU-rpmA genes and the Rho-independent transcription terminator were ectopically inserted into designated (3 copies) and random (7 copies) points in the recipient chromosome. The resulting strain produced approximately 0.5 g/L of secreted GSP after bacterial cultivation in flasks with starch-containing media, and its performance was comparable to an analogous strain in which the mprB.amy cassette was carried on a multi-copy plasmid. CONCLUSION: A novel strategy for ectopically integrating a cassette into multiple random locations in the B. subtilis chromosome was developed. This new method is based on the construction of DNA fragments in which the desired gene, marked by antibiotic resistance, is sandwiched between "front" and "back" portions of random chromosomal DNA restriction fragments. These fragments were subsequently inserted into the targeted sites of the chromosome using double-cross recombination. The construction of a marker-free strain was achieved by gene conversion between the integrated marked gene and a marker-less variant carried by plasmid DNA, which was later removed from the cells.

SUBMITTER: Yomantas YA

PROVIDER: S-EPMC3166918 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Publications

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome.

Yomantas Yurgis Av YA Abalakina Elena G EG Golubeva Lyubov I LI Gorbacheva Lyubov Y LY Mashko Sergey V SV

Microbial cell factories 20110805

<h4>Background</h4>Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants. Specifically, their stability and potential ecological safety make them of use in industrial applications. As a rule, however, it is necessary to incorporate many copies of a key gene into a chromosome to achieve strain performance that is comparable to that of cells carrying multiple copies of a recombinant plasmid.<h4>Results</h4>A plasmid-less B. subtilis JE852-based strain secr ...[more]

PMID: 21819557

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Project description:UnlabelledBacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial model Bacillus subtilis as a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.ImportanceThe identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells.

Dataset Information

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome.

Publications

Overproduction of Bacillus amyloliquefaciens extracellular glutamyl-endopeptidase as a result of ectopic multi-copy insertion of an efficiently-expressed mpr gene into the Bacillus subtilis chromosome.

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