Development of a markerless gene deletion system for Bacillus subtilis based on the mannose phosphoenolpyruvate-dependent phosphotransferase system

Abstract

To optimize Bacillus subtilis as a production strain for proteins and low molecular substances by genome engineering, we developed a markerless gene deletion system. We took advantage of a general property of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), in particular the mannose PTS. Mannose is phosphorylated during uptake by its specific transporter (ManP) to mannose 6-phosphate, which is further converted to fructose 6-phosphate by the mannose-6-phosphate isomerase (ManA). When ManA is missing, accumulation of the phosphorylated mannose inhibits cell growth. This system was constructed by deletion of manP and manA in B. subtilis D6, a 168 derivative strain with six large deletions of prophages and antibiotic biosynthesis genes. The manP gene was inserted into an Escherichia coli plasmid together with a spectinomycin resistance gene for selection in B. subtilis. To delete a specific region, its up-and downstream flanking sites (each of approximately 700 bp) were inserted into the vector. After transformation, integration of the plasmid into the chromosome of B. subtilis by single cross-over was selected by spectinomycin. In the second step, excision of the plasmid was selected by growth on mannose. Finally, excision and concomitant deletion of the target region were verified by colony PCR. In this way, all nine prophages, seven antibiotic biosynthesis gene clusters and two sigma factors for sporulation were deleted and the B. subtilis genome was reduced from 4215 to 3640 kb. Despite these extensive deletions, growth rate and cell morphology remained similar to the B. subtilis 168 parental strain. INTRODUCTIONBacillus subtilis is an important producer of industrial enzymes, such as amylases and proteases, with favourable features including high protein secretion capability, GRAS (generally recognized as safe) status, natural competence, a long tradition in fermentation and an extensive existing knowledge base of its genetics, physiology and biochemistry. Up-to-date information is available on SubtiWiki (Michna et al., 2014). Strain optimization to remove adverse properties such a sporulation, lysogenic prophages, production of antibiotics and extracellular proteases (Krishnappa et al., 2013;Stein, 2005;Westers et al., 2003Westers et al., , 2004 or to introduce new metabolic routes, requires powerful tools for genetic engineering.An easy method for chromosomal deletions is to construct deletion cassettes comprising an antibiotic resistance marker flanked by sequences that normally flank the region to be deleted. The successful integration of the cassette via homologous recombination can then be selected by the antibiotic resistance. For multiple modifications of the chromosome, recognition sites for sitespecific recombinases, such as Xer/dif, Flp/FRT or Cre/ loxP (Bloor & Cranenburgh, 2006;Hoang et al., 1998;Marx & Lidstrom, 2002), allow the precise excision of the marker gene, and the cassette can be used again. The risk of unintended large chromosomal deletions or inversions can be avoide...

Large-scale reduction of the Bacillus subtilis genome: consequences for the transcriptional network, resource allocation, and metabolism

“…E. coli NM538 (Frischauf et al 1983) was used for plasmid multimerization to enhance B. subtilis transformation efficiency. The genome reduced B. subtilis IIG-Bs20 (Wenzel and Altenbuchner 2015) derived from Δ6 (Westers et al 2003) formed the starting point of all further chromosomal deletions. The genotypes of the deletion strains B. subtilis PG10, are shown in Supplemental Table S1.…”

“…Deletion formation in B. subtilis Δ6 Deletions in B. subtilis Δ6 were done in a markerless way using a selection/counter-selection strategy as described previously (Wenzel and Altenbuchner 2015). Briefly, two fragments of ∼700 bp are amplified from the upstream and downstream regions of the target sequence, fused to each other, and inserted into the nonreplicative suicide vectors pJOE6743.1 (Wenzel and Altenbuchner 2015) or pJOE8739.1.…”

“…In order to construct a genome-reduced B. subtilis strain in which all remaining genes are essential for the survival of the cell and the genome integrity as well as to identify a minimal gene set required for life, we used a fast and reliable marker-free deletion system for a stepwise reduction of the B. subtilis Δ6 genome (Westers et al 2003;Wenzel and Altenbuchner 2015). In order to avoid problems with loss of growth or genetic competence, we decided at an advanced stage of the project to continue with two independent strain lineages (Fig.…”

“…After integration of the vector into the chromosome via homologous recombination, counter-selection was performed by growing the cells first in LB liquid medium supplemented with 0.5% mannose and then on LB agar plates with mannose. Colonies with the desired deletions were identified by PCR (Wenzel and Altenbuchner 2015).…”

“…Briefly, two fragments of ∼700 bp are amplified from the upstream and downstream regions of the target sequence, fused to each other, and inserted into the nonreplicative suicide vectors pJOE6743.1 (Wenzel and Altenbuchner 2015) or pJOE8739.1. The latter vector contains the ccdB gene under the E. coli rhamnose promoter.…”