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.

Figure 1

Structure of the pHE52mpr plasmid carrying the mpr^B.amy cassette. P_rp - promoter of the rplU-rpmA genes from B. amyloliquefaciens A-50; Ter - Rho-independent transcription terminator of the pheA gene. The BglII-site-ended (boldface) followed by 5'-portion of B. amyloliquefaciens A-50 mpr gene is shown. In this part, SD-sequence and ATG-codon are marked as boldface and capital letters, respectively. Two putative cre elements, which are homologous to the known [45-47] consensus sequence, are also indicated.

Figure 2

Scheme of the targeted mpr^B.amy-cassette integration into the JE852 chromosome (aprE851 is shown as an example of the target gene).

Figure 3

Cells plated on skim-milk test plates: The dependence of extracellular protease activity on the copy number of the integrated mpr^B.amy cassettes. 1.-clone of B. subtilis JE852; 2. and 3.-1 and 3 copies of mpr^B.amy-cassette integrated in the chromosome of JE852; 4.-clone of the plasmid-carrier strain B. subtilis JE852/pHE52mpr.

Figure 4

Southern hybridization of the chromosomal DNAs and SDS-PAGE of extracellular proteins secreted by the strains with different copy-number (N) of the integrated mprB.amy-cassettes.A – Hybridization with DNAs isolated from cells carrying (indicated as 1.; 2.; 3; etc. (N = 1: lane 1 (1.); N = 2: lane 2 (1.+2.); N = 4: lane 3 (1.+2.+3.+4.); and N = 7: lane 4 (1.+2.+3.+4.+5.+6.+7.) and lane 6 (1.+2.+3.+4.+5.+6.+8.). B SDS-PAGE of extracellular proteins secreted by the strains with: N = 0 (lane 2), N = 1 (lane 3), N = 2 (lane 4), N = 3 (lane 5), N = 9 (lane 8) and N = 10 (lane 9) mpr^B.amy cassettes in the chromosome or carrying the multi-copy-number recombinant plasmid pHE52mpr (lanes 6 and 10). Lanes 1 and 7-reference proteins with molecular mass given in kDa. The mature 220 amino acid B. amiloliquefaciens GSP has a molecular mass of about 23.7 kDa. The indicated extracellular GSP presented slightly lower electrophoretic mobility corresponding to 27-28 kDa; this was previously documented for the mature GSP from B. subtilis [17].

Figure 5

The general scheme of the mpr^B.amy-Cm^R-cassette integration into random points of the B. subtilis chromosome.