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Engineering Bacillus subtilis J46 for efficient utilization of galactose through adaptive laboratory evolution.

Jae Woong Choi1, Nho-Eul Song1, Sang-Pil Hong1

  • 1Research Group of Traditional Food, Korea Food Research Institute, 245, Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, 55365, Republic of Korea.

AMB Express
|January 28, 2024
PubMed
Summary

Adaptive laboratory evolution significantly enhanced galactose utilization in Bacillus subtilis, increasing growth rates and bio-product yields. Key genetic mutations in araR and glcR were identified as drivers of this improvement.

Keywords:
Adaptive laboratory evolutionBacillus subtilisGalactoseLeloir pathwayReverse engineering

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Area of Science:

  • Microbial Engineering
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Bacillus subtilis possesses natural pathways for galactose metabolism.
  • Enhancing microbial galactose utilization is crucial for producing valuable bio-products.
  • Adaptive Laboratory Evolution (ALE) offers a strategy to improve microbial metabolic capabilities.

Purpose of the Study:

  • To enhance galactose utilization in Bacillus subtilis using ALE.
  • To identify the genetic and metabolic alterations responsible for improved galactose metabolism.
  • To assess the impact of enhanced metabolism on bio-product formation.

Main Methods:

  • Bacillus subtilis strains underwent approximately 5000 generations of ALE in a galactose-rich environment.
  • Whole-genome sequencing was performed on evolved strains to identify genetic mutations.
  • Reverse engineering was used to confirm the advantageous mutations in specific genes (araR and glcR).
  • Specific growth rates and bio-product (protease, β-galactosidase) yields were measured.

Main Results:

  • ALE resulted in a significant increase in specific growth rate on galactose (0.319 h⁻¹ compared to 0.03 h⁻¹ in wild-type).
  • Whole-genome sequencing identified 63 single nucleotide polymorphisms (SNPs) in the evolved strain BSGA14.
  • Two specific mutations in the araR and glcR genes were identified as crucial for enhanced growth.
  • The engineered strain BSGALE4, with these two mutations, showed comparable growth rates to the evolved strain.
  • Evolved strains demonstrated increased productivity of protease and β-galactosidase.

Conclusions:

  • ALE is an effective method for enhancing galactose metabolism in Bacillus subtilis.
  • Specific mutations in araR and glcR play a key role in improving galactose utilization.
  • This study provides insights into microbial engineering for industrial applications involving enhanced galactose conversion.