High-throughput Screening High-yield Bacitracin Strain from Bacillus licheniformis DW2

doi:10.13560/j.cnki.biotech.bull.1985.2020-0080

Abstract

Abstract: Bacitracin,as secondary metabolite with complex biotransformation pathway,is a widely used broad-spectrum peptide antibiotic. An industrial strain Bacillus licheniformis DW2 was used for this study,and strains of high-yield bacitracin were obtained using the Atmospheric and Room Temperature Plasma（ARTP）mutagenesis and an established high-throughput screening strategy based on flow cytometry（FCM）. Finally,the 5 best performing strains with regard to bacitracin yield were determined by screening,and they were 8#6D,7#5E,6#7E,5#8D,and 2#5F. The yield increased by 20.9%,20.9%,20.8%,19.7%,and 22.2%,respectively,compared with that of starting DW2 strain. The highest titer of the bacitracin reached 912 U/mL. With the help of high-flow sorting by flow cytometry,high-throughput screening characterized of simple and safe can quickly increase bacitracin production.

Key words: bacitracin, ARTP, biuret coloration, flow cytometry, multi-well microtiter plates.

MI Liang-bo, ZENG Wei-zhu, HUANG Ke-xue, WANG De-ming, DU Guo-cheng, ZHOU Jing-wen, CHEN Jian. High-throughput Screening High-yield Bacitracin Strain from Bacillus licheniformis DW2[J]. Biotechnology Bulletin, 2020, 36(7): 90-96.

References

[1] Potts AR, Psurek T, Jones C, et al.Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifier[J]. J Pharm Biomed Anal, 2012, 70:619-623.
[2] Pavli V, Kmetec V.Pathways of chemical degradation of polypeptide antibiotic bacitracin[J]. Biol Pharm Bull, 2006, 29:2160-2167.
[3] Ming LJ, Epperson JD.Metal binding and structure-activity relationship of the metalloantibiotic peptide bacitracin[J]. J Inorg Biochem, 2002, 91:46-58.
[4] Qin W, Hui Z, Xia W, et al.Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2[J]. Appl Biochem Biotechnol, 2017, 183:1146-1157.
[5] Xu C, Lv F, Bie X, et al.Optimization of bacitracin a high-yield industrial fermentation medium[J]. Science & Technology of Food Industry, 2016, 37:197.
[6] Wang Q, Zheng H, Wan X, et al.Optimization of inexpensive agricultural by-products as raw materials for bacitracin production in Bacillus licheniformis DW2[J]. Appl Biochem Biotechnol, 2017, 183:1146-1157.
[7] Deng K, Ji ZX, Chen SW.Effect of dissolved oxygen on bacitracin fermentation by Bacillus licheniformis DW2[J]. Chinese Journal of Antibiotics, 2009, 34:664-668.
[8] Pranita AG, Ashok VG, Sneha L.Optimization of bacitracin production from Bacillus licheniformis NCIM 2536[J]. Int J Curr Microbiol Appl Sci, 2014, 3(9):819-829.
[9] Jia R, Zhang C, Lu F, et al.Breeding and comparative analysis of high-yield bacitracin-producing Bacillus licheniformis by genome shuffling[J]. Food Science, 2016, 37:135-140.
[10] Rey MW, Ramaiya P, Nelson BA, et al.Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species[J]. Genome Biol, 2004, 5:12.
[11] Zhu S, Cai DB, Liu ZW, et al.Enhancement of bacitracin production by NADPH generation via overexpressing glucose-6-phosphate dehydrogenase Zwf in Bacillus licheniformis[J]. Appl Biochem Biotechnol, 2019, 187:1502-1514.
[12] 李阳, 吴非, 蔡冬波, 等. 地衣芽胞杆菌DW2中敲除氨基酸转运蛋白基因yhdG提高杆菌肽产量[J]. 生物工程学报, 2018, 34(6):916-927.
[13] Zhu J, Cai DB, Xu HX, et al.Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis[J]. Systems & Synthetic Biology, 2018, 3:236-243.
[14] Ikeda H, Nonomiya T, Usami M, et al.Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis[J]. P Natl Acad Sci USA, 1999, 96:9509-9514.
[15] Liu L, Tao J, Bi L.Quantitative analysis of buckwheat protein with biuret method[J]. Food Science, 2004, 25:258-261.
[16] Shi L, Chen XS, Tang WY, et al.Combination of FACS and homologous recombination for the generation of stable and high-expression engineered cell lines[J]. PLoS One, 2014, 9:11.
[17] Wang D, Wang Q, Qiu YM, et al.Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2[J]. Res Microbiol, 2017, 168:515-523.
[18] Shu CC, Wang D, Guo J, et al.Analyzing AbrB-knockout effects through genome and transcriptome sequencing of Bacillus licheniformis DW2[J]. Front Microbiol, 2018, 9:11.
[19] Liu WS, Jiang RR.Combinatorial and high-throughput screening approaches for strain engineering[J]. Appl Microbiol Biotechnol, 2015, 99:2093-2104.