Genome engineering using a synthetic gene circuit in Bacillus subtilis

Abstract

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications.

Figure 1.

A synthetic gene circuit containing two repressible promoters and two repressor genes for a counter-selectable marker system during genome engineering of B. subtilis.

Figure 2.

Strategy for constructing the B. subtilis 168 Trp⁺ strain using a synthetic gene circuit. The integration vector contains the B. subtilis trpC gene harboring three missing bases (ATT), the P_xyl-lacI cassette and the neomycin-resistant gene (neo). N and C represent N- and C-terminal parts of the B. subtilis trpC gene. The helper plasmid contains a xylR repressor gene, replication origin (rep) for Bacillus and the P_spac–cat fusion cassette. The integration vector was inserted into the B. subtilis 168 chromosome by single crossover integration. In vivo recombination between the N or C fragments under the presence of xylose and chloramphenicol (Cm) resulted in the deletion of the P_xyl-lacI cassette and neo. The helper plasmid was removed to construct the B. subtilis Trp⁺ strain.

Figure 3.

(A) Insertion of nucleotides into the trpC gene of B. subtilis 168 to construct the B. subtilis 168 Trp⁺ strain. The numbers indicate nucleic acid sequence positions relative to the first nucleotide of the start codon. (B) Growth of B. subtilis 168 Trp⁺ harboring the helper plasmid. Growth inhibition of the Trp⁺ strain on TSA containing neomycin indicated that the P_xyl-lacI cassette and neo were deleted during in vivo recombination. Growth on TSA containing chloramphenicol revealed the presence of the helper plasmid. The Trp⁺ strain can be grown on a defined medium without a tryptophan supplement. (C) Growth of the Trp⁻ (black circles) and Trp⁺ (black squares) strains in a defined medium with or without a tryptophan supplement.

Figure 4.

Deletion of nucleotides in the aprE gene (A) and point mutation in the nprE gene (B) of Bacillus subtilis 168 Trp⁺ strain to construct strains lacking AprE (AprE⁻) and NprE (NprE⁻). The numbers indicate nucleic acid sequence positions relative to the first nucleotide of the start codon. The point mutation created a stop codon in the NprE gene. (C) The protease assay of B. subtilis Trp⁺, AprE⁻ and NprE⁻ strains and a comparison of them with the WB800N strain in which eight extracellular proteases were deleted.

Figure 5.

(A) Gene structures of wild-type (BS168) and deletion mutant of amyE gene (BS168 ΔamyE). Arrows above the genes indicate primer binding sites. (B) Amylase assay of BS168 (1) and BS ΔamyE (2) on an LB agar plate containing 1% starch. (C) PCR analyses of BS168 (1) and BS ΔamyE (2) with the indicated primer sets. Expected sizes of PCR products were shown in the table.

Figure 6.

(A) Gene structures of wild-type (BS168) and deletion mutant of pps operon (BS168 Δpps). Arrows above the genes indicate primer binding sites. (B) PCR analyses of BS168 (1) and BS Δpps (2) with the indicated primer sets. Expected sizes of PCR products were shown in the table.