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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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-02-01
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| Series: | Microorganisms |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-2607/13/3/536 |
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| Summary: | 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. |
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| ISSN: | 2076-2607 |