title: A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell sorting.
abstract: To enhance the screening efficiency and accuracy of a high-yield menaquinone (vitamin K2, MK) bacterial strain, a novel, quantitative method by fluorescence-activated cell sorting (FACS) was developed. The staining technique was optimized to maximize the differences in fluorescence signals between spontaneous and MK-accumulating cells. The fluorescence carrier rhodamine 123 (Rh123), with its ability to reflect membrane potential, proved to be an appropriate fluorescent dye to connect the MK content with fluorescence signal quantitatively. To promote adequate access of the fluorescent molecule to the target and maintain higher cell survival rates, staining and incubation conditions were optimized. The results showed that 10 % sucrose facilitated uptake of Rh123, while maintaining a certain level of cell viability. The pre-treatment of cells with MgCl2 before staining with Rh123 also improved cell viability. Using FACS, 50 thousands cells can easily be assayed in less than 1 h. The optimized staining protocol yielded a linear response for the mean fluorescence against high performance liquid chromatography-measured MK content. We have developed a novel and useful staining protocol in the high-throughput evaluation of Flavobacterium sp. mutant libraries, using FACS to identify mutants with increased MK-accumulating properties. This study also provides reference for the screening of other industrial microbial strains.
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China.|Key Lab of Ion Beam Bioengineering, Chinese Academy of Science, 230031, Hefei, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China. xuezl@ahpu.edu.cn.
authors.forename: Zheng-Lian
authors.initials: ZL
authors.lastname: Xue
authors.affiliation: Key Lab of Ion Beam Bioengineering, Chinese Academy of Science, 230031, Hefei, China.
authors.forename: Shao-Peng
authors.initials: SP
authors.lastname: Chen
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Wang
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China.
authors.forename: Wei-Liang
authors.initials: WL
authors.lastname: Gong
authors.affiliation: Key Lab of Ion Beam Bioengineering, Chinese Academy of Science, 230031, Hefei, China. zhengzhiming2012@gmail.com.
authors.forename: Zhi-Ming
authors.initials: ZM
authors.lastname: Zheng
mesh_terms: D003470:Culture Media; D005417:Flavobacterium / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D057166:High-Throughput Screening Assays; D007218:Industrial Microbiology; D050296:Microbial Viability; D015203:Reproducibility of Results; D013194:Staining and Labeling; D024482:Vitamin K 2 / Q000032:analysis*
chemical_list: D003470:Culture Media; D005456:Fluorescent Dyes; D024482:Vitamin K 2
keywords: Fluorescence-activated cell sorting; High-throughput; Menaquinone; Quantification