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 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.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.
Highlights
Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants
Primers for the amplification of mpr by polymerase chain reaction (PCR), mpr-F/R, were designed based on the available B. amyloliquefaciens ZB42 genome sequence (GenBank/EMBL accession number NC_009725) [38]
We showed that glutamic acid-specific protease (GSP) production was significantly increased during fermentation of the B. subtilis JE852/pHE52mpr strain on TYS6C media, in which starch was the main carbon source
Summary
Plasmid-less, engineered Bacillus strains have several advantages over plasmid-carrier variants Their stability and potential ecological safety make them of use in industrial applications. Gram-positive bacteria are widely used for biotechnology applications, including vaccine delivery [1,2,3] and in situ production of anti-infective protectants [4] and microbicides [5] These microorganisms serve as largescale producers of nucleotides, vitamins, ribose, poly-gglutamic acids [6], absorbents [7], and insecticides [8]. B. subtilis GSP, encoded by the mpr gene, is synthesized as an inactive pre-pro-peptide This precursor is subsequently processed by the Sip and Bpr proteases, and mature extracellular GSP have a length of 220 amino acids [17]. Though they were initially a subject of basic science investigation [18,19,20], some GSPs (from B. licheniformis in particular [21]) are being utilized in commercial applications such as food production [22,23]
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