# A High-Throughput Screening Strategy for Bacillus subtilis Producing Menaquinone-7 Based on Fluorescence-Activated Cell Sorting

## TL;DR

This paper introduces a fast method to find high-producing Bacillus subtilis strains for MK-7 using fluorescence and sorting techniques.

## Contribution

A novel HTS strategy using Rhodamine 123 and fluorescence-activated cell sorting to screen for MK-7 production in Bacillus subtilis.

## Key findings

- A linear correlation (R2 = 0.9646) was found between MK-7 content and fluorescence intensity.
- The mutant AR03-27 showed an 85.65% increase in MK-7 yield compared to the original strain.
- Mutants with higher MK-7 production had mutations in membrane transport-related genes.

## Abstract

Menaquinone-7 (MK-7) is recognized for its important biological activity, and Bacillus subtilis 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 (R2 = 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.