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Updated: Aug 1, 2025
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Single-cell Microfluidic Analysis of Bacillus subtilis
Published on: January 26, 2018
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Characterization of a Riboflavin-Producing Mutant of Bacillus subtilis Isolated by Droplet-Based Microfluidics
Fan Xu1,2,3, Chuan Liu2,3,4, Miaomiao Xia2
1School of Chemical Engineering, Hebei University of Technology, Tianjin 300131, China.
Microorganisms
|April 28, 2023
Summary
Droplet-based microfluidics efficiently screens for improved riboflavin production in Bacillus subtilis. This method identified a superior producer, U3, with significantly enhanced riboflavin output and identified key genetic mutations for overproduction.
Area of Science:
- Biotechnology
- Microbiology
- Synthetic Biology
Background:
- Bacillus subtilis is a key industrial strain for riboflavin production.
- High-throughput screening methods are crucial for strain improvement but are underdeveloped for riboflavin production in B. subtilis.
- Droplet-based microfluidics enables single-cell encapsulation and fluorescence-based screening.
Purpose of the Study:
- To establish an efficient, high-throughput screening method for improving riboflavin production in B. subtilis using droplet-based microfluidics.
- To identify novel B. subtilis strains with enhanced riboflavin production capabilities.
- To elucidate the genetic basis of riboflavin overproduction.
Main Methods:
- Application of droplet-based microfluidics for high-throughput screening of a B. subtilis random mutation library.
- Flask and fed-batch fermentation to evaluate riboflavin production and biomass of selected strains.
- Whole genome sequencing and comparative analysis to identify mutations in high-producing strains.
- Genetic manipulation to confirm the role of identified mutations in riboflavin overproduction.
Main Results:
- A superior riboflavin producer, strain U3, was selected using droplet-based microfluidics screening.
- Strain U3 exhibited higher riboflavin production (24.3 g/L, an 18% increase) and yield (8.7 g/100g glucose, a 19% increase) compared to the parent strain S1 in fed-batch fermentation.
- Whole genome sequencing identified mutations in sinR and icd genes in strain U3, which were confirmed to enhance riboflavin production when introduced into the parent strain.
Conclusions:
- Droplet-based microfluidics is a powerful tool for the efficient screening and improvement of industrial B. subtilis strains for enhanced riboflavin production.
- The identified sinR and icd mutations are key genetic determinants for riboflavin overproduction in B. subtilis.
- This study provides valuable protocols and insights for developing next-generation riboflavin-producing microbial factories.
