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Citrate Supplementation Modulates Medium Viscosity and Poly-γ-Glutamic Acid Synthesis by Engineered B. subtilis 168
Frederik Völker1, Kyra Hoffmann2, Birthe Halmschlag1
1Institute of Applied Microbiology-iAMB Aachen Biology and Biotechnology-ABBt RWTH Aachen University Aachen Germany.
Engineering in Life Sciences
|March 6, 2025
Summary
Citrate significantly enhances poly-γ-glutamic acid (γ-PGA) production in Bacillus subtilis by influencing glutamate metabolism and reducing medium viscosity. Even with limited uptake due to carbon catabolite repression, citrate impacts growth and biopolymer yield.
Area of Science:
- Biotechnology and microbial fermentation
- Biopolymer synthesis and characterization
- Metabolic engineering and regulation
Background:
- Poly-γ-glutamic acid (γ-PGA) is an industrially valuable biopolymer produced by Bacillus species.
- Co-feeding carbon and nitrogen sources, particularly glycerol and citrate, is crucial for γ-PGA production.
- Citrate's role in fueling biomass and tricarboxylic acid (TCA) cycle precursors is recognized, but its direct impact on γ-PGA synthesis and medium properties requires further elucidation.
Purpose of the Study:
- To investigate the influence of sole citrate presence on Bacillus subtilis growth, γ-PGA production, and culture viscosity.
- To elucidate the mechanism by which citrate affects γ-PGA synthesis and medium rheology.
- To identify regulatory pathways influenced by citrate availability during biopolymer production.
Main Methods:
- Cultivation of Bacillus subtilis 168 with varying citrate concentrations in the production medium.
- Measurement of γ-PGA titer, biomass, and culture broth viscosity.
- Proteome analysis to identify changes in protein abundance under different citrate availabilities.
Main Results:
- High citrate concentration led to a 6-fold increase in γ-PGA titer compared to low citrate levels, despite limited citrate import due to carbon catabolite repression (CCR).
- Citrate influenced the fate of imported glutamate rather than serving as a direct precursor for γ-PGA synthesis.
- Citrate depletion caused a significant spike in culture broth viscosity, while its presence reduced viscosity by interacting with divalent cations.
Conclusions:
- Citrate supplementation is a key strategy for optimizing γ-PGA production yields and controlling medium viscosity in Bacillus subtilis.
- Citrate impacts intracellular glutamate metabolism and potentially triggers unexplored regulatory mechanisms affecting glutamate utilization.
- Understanding citrate's metabolic and regulatory effects provides avenues for enhancing industrial γ-PGA production efficiency and consistency.
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