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A high-throughput screening strategy for accurate quantification of menaquinone based on fluorescence-activated cell
Yan Liu1,2, Zheng-Lian Xue3, Shao-Peng Chen2
1College of Biochemical Engineering, Anhui Polytechnic University, 241000, Wuhu, China.
Journal of Industrial Microbiology & Biotechnology
|March 23, 2016
Summary
Researchers developed a novel fluorescence-activated cell sorting (FACS) method to efficiently screen bacterial strains for high menaquinone (vitamin K2) production. This technique uses rhodamine 123 staining to quantify vitamin K2 content, improving high-throughput screening.
Area of Science:
- Microbiology
- Biotechnology
- Analytical Chemistry
Background:
- Efficient screening of bacterial strains for high yield of menaquinone (vitamin K2, MK) is crucial for industrial applications.
- Existing methods may lack the speed and accuracy required for high-throughput evaluation of mutant libraries.
- Developing quantitative methods to assess MK accumulation in bacteria is essential for strain improvement.
Purpose of the Study:
- To develop a novel, quantitative method using fluorescence-activated cell sorting (FACS) for enhanced screening efficiency and accuracy of high-yield MK bacterial strains.
- To optimize a staining protocol that correlates fluorescence signals with MK content.
- To establish a high-throughput evaluation method for identifying bacterial mutants with increased MK-accumulating properties.
Main Methods:
- Optimization of a staining technique using rhodamine 123 (Rh123), a dye reflecting membrane potential, to link MK content with fluorescence signals.
- Adjustment of staining and incubation conditions, including the use of 10% sucrose and MgCl2 pre-treatment, to improve Rh123 uptake and cell viability.
- Application of FACS for rapid assaying of large cell populations (50,000 cells in <1 hour) and correlation of fluorescence intensity with MK content measured by HPLC.
Main Results:
- An optimized staining protocol was established, maximizing fluorescence signal differences between spontaneous and MK-accumulating cells.
- The optimized conditions, including 10% sucrose and MgCl2 pre-treatment, enhanced Rh123 uptake and cell viability.
- The FACS method demonstrated a linear response between mean fluorescence and HPLC-measured MK content, validating its quantitative accuracy.
Conclusions:
- A novel and effective staining protocol using FACS was developed for the high-throughput evaluation of bacterial strains, specifically Flavobacterium sp. mutants, for increased MK accumulation.
- This method significantly enhances screening efficiency and accuracy compared to traditional approaches.
- The developed protocol serves as a valuable reference for screening other industrial microbial strains based on their accumulation properties.
