title: 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.
authors.affiliation: Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.forename: Weixia
authors.initials: W
authors.lastname: Gao
authors.forename: Jun
authors.initials: J
authors.lastname: Feng
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.forename: Yulian
authors.initials: Y
authors.lastname: He
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Cao
authors.forename: Qiang
authors.initials: Q
authors.lastname: Li
authors.forename: Yang
authors.initials: Y
authors.lastname: Sun
authors.forename: Chao
authors.initials: C
authors.lastname: Yang
authors.forename: Cunjiang
authors.initials: C
authors.lastname: Song
authors.forename: Shufang
authors.initials: S
authors.lastname: Wang
mesh_terms: D001407:Bacillus / Q000235:genetics*; D017353:Gene Deletion; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D011099:Polyglutamic Acid / Q000378:metabolism*; D012641:Selection, Genetic
chemical_list: D011099:Polyglutamic Acid