A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting
Menaquinone-7 (MK-7) is recognized for its important biological activity, and <i>Bacillus subtilis</i> is the preferred strain for its fermentative production. However, the limited phenotypic diversity among high-yielding strains complicates the development of rapid screening methods. To...
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2025-02-01
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| author | Lina Yang Can Tang Yan Cui Jianhua Zhang |
| author_facet | Lina Yang Can Tang Yan Cui Jianhua Zhang |
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| description | Menaquinone-7 (MK-7) is recognized for its important biological activity, and <i>Bacillus subtilis</i> is the preferred strain for its fermentative production. However, the limited phenotypic diversity among high-yielding strains complicates the development of rapid screening methods. To address this, we utilized the effect of MK-7 on transmembrane potential to develop a high-throughput screening (HTS) strategy for efficiently identifying strains with improved MK-7 production. Among various membrane potential fluorescent dyes tested, Rhodamine 123 was selected for quantifying intracellular MK-7 levels due to its effective staining and minimal impact on cell growth. By optimizing pretreatment protocols and staining conditions, we established an HTS protocol that combines fluorescence-activated cell sorting with HPLC to identify strains with increased MK-7 production. A linear correlation was observed between mean MK-7 content and average fluorescence intensity (R<sup>2</sup> = 0.9646). This approach was applied to mutant libraries generated through atmospheric room temperature plasma mutagenesis. After three cycles of mutagenesis and screening, the mutant AR03-27 was identified, showing an 85.65% increase in MK-7 yield compared to the original SJTU2 strain. Resequencing analysis revealed that the top three mutants contained mutations in genes related to membrane transport, suggesting their potential role in enhancing MK-7 yield. |
| format | Article |
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| language | English |
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| spelling | doaj-art-e572232c87424b28a21c5e24eb7cdee22025-08-20T01:48:53ZengMDPI AGMicroorganisms2076-26072025-02-0113353610.3390/microorganisms13030536A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell SortingLina Yang0Can Tang1Yan Cui2Jianhua Zhang3School of Agriculture and Biology, Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, ChinaSchool of Agriculture and Biology, Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, ChinaSchool of Agriculture and Biology, Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, ChinaSchool of Agriculture and Biology, Bor S. Luh Food Safety Research Center, Shanghai Jiao Tong University, Shanghai 200240, ChinaMenaquinone-7 (MK-7) is recognized for its important biological activity, and <i>Bacillus subtilis</i> is the preferred strain for its fermentative production. However, the limited phenotypic diversity among high-yielding strains complicates the development of rapid screening methods. To address this, we utilized the effect of MK-7 on transmembrane potential to develop a high-throughput screening (HTS) strategy for efficiently identifying strains with improved MK-7 production. Among various membrane potential fluorescent dyes tested, Rhodamine 123 was selected for quantifying intracellular MK-7 levels due to its effective staining and minimal impact on cell growth. By optimizing pretreatment protocols and staining conditions, we established an HTS protocol that combines fluorescence-activated cell sorting with HPLC to identify strains with increased MK-7 production. A linear correlation was observed between mean MK-7 content and average fluorescence intensity (R<sup>2</sup> = 0.9646). This approach was applied to mutant libraries generated through atmospheric room temperature plasma mutagenesis. After three cycles of mutagenesis and screening, the mutant AR03-27 was identified, showing an 85.65% increase in MK-7 yield compared to the original SJTU2 strain. Resequencing analysis revealed that the top three mutants contained mutations in genes related to membrane transport, suggesting their potential role in enhancing MK-7 yield.https://www.mdpi.com/2076-2607/13/3/536vitamin K<sub>2</sub>ARTP mutant librarieshigh yield mutant selectionfluorescence intensityresequencing |
| spellingShingle | Lina Yang Can Tang Yan Cui Jianhua Zhang A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting Microorganisms vitamin K<sub>2</sub> ARTP mutant libraries high yield mutant selection fluorescence intensity resequencing |
| title | A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting |
| title_full | A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting |
| title_fullStr | A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting |
| title_full_unstemmed | A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting |
| title_short | A High-Throughput Screening Strategy for <i>Bacillus subtilis</i> Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting |
| title_sort | high throughput screening strategy for i bacillus subtilis i producing menaquinone 7 based on fluorescence activated cell sorting |
| topic | vitamin K<sub>2</sub> ARTP mutant libraries high yield mutant selection fluorescence intensity resequencing |
| url | https://www.mdpi.com/2076-2607/13/3/536 |
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