caa a22 4500 605500460 CHVBK 20210128100556.0 cr unu---uuuuu 210128e20150601xx s 000 0 eng 10.1007/s00253-015-6485-5 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6485-5 A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35 [Elektronische Daten] [Zhaopeng Sun, Aihua Deng, Ting Hu, Jie Wu, Qinyun Sun, Hua Bai, Guoqiang Zhang, Tingyi Wen] Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine. However, it is difficult to perform genetic manipulations using the endogenous recombination machinery. In many bacteria, phage recombineering systems have been employed to improve recombineering frequencies. To date, an efficient phage recombineering system for B. subtilis has not been reported. Here, we, for the first time, identified that GP35 from the native phage SPP1 exhibited a high recombination activity in B. subtilis. On this basis, we developed a high-efficiency GP35-meditated recombineering system. Taking single-stranded DNA (ssDNA) as a recombineering substrate, ten recombinases from diverse sources were investigated in B. subtilis W168. GP35 showed the highest recombineering frequency (1.71 ± 0.15 × 10−1). Besides targeting the purine nucleoside phosphorylase gene (deoD), we also demonstrated the utility of GP35 and Beta from Escherichia coli lambda phage by deleting the alpha-amylase gene (amyE) and uracil phosphoribosyltransferase gene (upp). In all three genetic loci, GP35 exhibited a higher frequency than Beta. Moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with B. subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed. The results suggested that closer kinship to B. subtilis resulted in higher frequency in B. subtilis. In conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology. Springer-Verlag Berlin Heidelberg, 2015 Bacillus subtilis nationallicence Recombinase nationallicence Recombineering frequency nationallicence PCR-based ssDNA nationallicence Sun Zhaopeng CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Deng Aihua CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Hu Ting CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Wu Jie CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Sun Qinyun CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Bai Hua CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Zhang Guoqiang CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Wen Tingyi CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5151-5162 0175-7598 99:12<5151 2015 99 253 https://doi.org/10.1007/s00253-015-6485-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6485-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sun Zhaopeng CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Deng Aihua CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Hu Ting CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Wu Jie CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Sun Qinyun CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Bai Hua CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Zhang Guoqiang CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 700 1- Wen Tingyi CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, 100101, Beijing, China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/12(2015-06-01), 5151-5162 0175-7598 99:12<5151 2015 99 253