A markerless gene replacement method for B. amyloliquefaciens LL3 and its use in genome reduction and improvement of poly-γ-glutamic acid production

Abstract

We herein adapted a markerless gene replacement method by combining a temperature-sensitive plasmid pKSV7 with a counterselectable marker, the upp gene encoding uracil phosphoribosyltransferase (UPRTase), for the poly-γ-glutamic acid (γ-PGA)-producing strain Bacillus amyloliquefaciens LL3. Deletion of the upp gene conferred LL3 5-fluorouracil (5-FU) resistance. Sensitivity to 5-FU was restored when LL3 Δupp was transformed with pKSV7-based deletion plasmid which carries a functional allele of the upp gene of Bacillus subtilis 168. These observations allowed us to adapt a two-step plasmid integration and excision strategy to perform markerless deletion of genes of interest. Deletion plasmid harboring a mutant allele of the target gene was first integrated in the genome by culturing cells under nonpermissive conditions for pKSV7 replication. Single-crossover recombinants were then grown without antibiotics to aid the second recombinational event. 5-FU was used to select for double-crossover recombinants with plasmid evicted from the chromosome. The resulting recombinants either harbored the wild-type or mutated allele of the target gene and could be identified by PCR and DNA sequencing. Using this method, we successively removed the amyA gene and a 47-kb fragment of the bae cluster from the genome of LL3, with higher efficiency compared with previous reports. We also investigated the effects of a transcriptional regulator, RocR, on γ-PGA production and cell growth. Specific γ-PGA production of the rocR mutant was increased by 1.9-fold, which represents a new way to improve γ-PGA production.

Deletion of genes involved in glutamate metabolism to improve poly-gamma-glutamic acid production in B. amyloliquefaciens LL3

“…We previously reported that the deletion of RocR, a transcriptional regulator in glutamate metabolism, may have contributed to the increase of γ-PGA production in Bacillus amyloliquefaciens LL3 [24]. Wu et al [23] also reported that by directing more carbon flux distribution toward glutamate synthesis by the presence of additives, γ-PGA production was increased.…”

“…The precipitate was centrifuged at 4,000×g (4 °C) and then dialyzed against distilled water and lyophilized to obtain γ-PGA. [24].…”

“…Glutamic acid-independent poly-γ-glutamic acid (γ-PGA) producing strain [9] LL3 ΔU LL3 carrying an in-frame deletion in the upp gene [24] LL3 ΔUR LL3 ΔU carrying an in-frame deletion in the rocR gene [24] LL3 …”

“…A upp-based markerless gene replacement method was used to perform in-frame deletion of the target genes [24].…”

“…B. amyloliquefaciens LL3 and its derivatives were transformed using the high-osmolarity electroporation method as described previously [24].…”