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 chromosomeReport as inadecuate




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 - Download this document for free, or read online. Document in PDF available to download.

Microbial Cell Factories

, 10:64

First Online: 05 August 2011Received: 13 May 2011Accepted: 05 August 2011

Abstract

BackgroundPlasmid-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.

ResultsA 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 mpr 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 mpr cassette was carried on a multi-copy plasmid.

ConclusionA 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.

AbbreviationsAntRantibiotic resistance marker

AprEalkaline serine protease subtilisin

bpbase pairs

CCAcarbon catabolite activation

CCCcarbon catabolite control

CCRcarbon catabolite repression

Cmchloramphenicol

CmCm resistance

crecatabolite responsive element

CSLcorn steep liquor

Emerythromycin

EmEm resistance

GSPglutamyl-specific protease, the mpr gene protein product

marker-less straina bacterial strain that does not carry AntR in its genome

NprEneutral protease

PCRpolymerase chain reaction

Prppromoter of the B. amyloliquefaciens A-50 rplU-rpmA genes

Tertranscription terminator of the B. amyloliquefaciens pheA gene

mpr cassetteexpression cassette where the structural portion of the B. amyloliquefaciens A-50 mpr gene is sandwiched between Prp and Ter

SDS-PAGEsodium dodecyl sulphate polyacrilamide gel electrophoresis

-denotes a plasmid-carrying strain.

Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-10-64 contains supplementary material, which is available to authorized users.

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Author: Yurgis AV Yomantas - Elena G Abalakina - Lyubov I Golubeva - Lyubov Y Gorbacheva - Sergey V Mashko

Source: https://link.springer.com/







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