Metabolic Engineering of Bacillus subtilis for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains
2.2. Media and Reagents
2.3. Strain Construction
2.4. Fermentation
2.5. Quantification of Cell Density and γ-PGA
2.6. Genome-Scale Metabolic Modeling
2.7. Statistical Analysis and Visualization
3. Results
3.1. Evaluation of γ-PGA Production from Glycerol Using Strains with Engineered Accumulation of Polymer Precursors in the swrA+ degU32Hy Background
3.1.1. Selection of Glycerol Concentration in Fermentation Media
3.1.2. Fermentation of Crude Glycerol
3.1.3. Fermentations in Glycerol-Based Media with Different Glutamate Concentrations
3.2. Engineering the γ-PGA Biosynthetic Operon Regulation Using a Synthetic Promoter
3.2.1. γ-PGA Production in Glucose-Based Media
3.2.2. γ-PGA Production in Glycerol-Based Media
3.3. Metabolic Engineering of the Inducible γ-PGA Production Strain to Remove Competing Pathways for Precursor Accumulation and Enhance Glutamate Metabolism
3.3.1. In Silico Identification of Overexpression Targets
3.3.2. Introducing a sucCD Knockout and/or racE Overexpression in the Physpank-pgs Strain Background
3.3.3. Scale-Up in Bioreactor Experiments
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strain | Relevant Genotype 1 | Reference |
---|---|---|
PB5383 | swrA+ degU32Hy (Smr) | [42] |
PB5691 | swrA+ degU32Hy (Smr) ΔodhAB (Catr) | [32] |
PB5716 | swrA+ degU32Hy (Smr) ΔsucCD (Tetr) | [32] |
PB5741 | swrA+ ywsC::RBSspoVG:sfGFP_Physpank::ywsC; (Emr) | [47] |
PB5763 | swrA+ ywsC::RBSspoVG:sfGFP_Physpank::ywsC; (Emr) racE::Pspac::racE (Catr) | This study |
PB5765 | swrA+ ywsC::RBSspoVG:sfGFP_Physpank::ywsC; (Emr) ΔsucCD (Tetr) | This study |
PB5764 | swrA+ ywsC::RBSspoVG:sfGFP_Physpank::ywsC; (Emr) racE::Pspac::racE (Catr) ΔsucCD (Tetr) | This study |
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Pasotti, L.; Massaiu, I.; Magni, P.; Calvio, C. Metabolic Engineering of Bacillus subtilis for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock. Fermentation 2024, 10, 319. https://doi.org/10.3390/fermentation10060319
Pasotti L, Massaiu I, Magni P, Calvio C. Metabolic Engineering of Bacillus subtilis for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock. Fermentation. 2024; 10(6):319. https://doi.org/10.3390/fermentation10060319
Chicago/Turabian StylePasotti, Lorenzo, Ilaria Massaiu, Paolo Magni, and Cinzia Calvio. 2024. "Metabolic Engineering of Bacillus subtilis for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock" Fermentation 10, no. 6: 319. https://doi.org/10.3390/fermentation10060319
APA StylePasotti, L., Massaiu, I., Magni, P., & Calvio, C. (2024). Metabolic Engineering of Bacillus subtilis for the Production of Poly-γ-Glutamic Acid from Glycerol Feedstock. Fermentation, 10(6), 319. https://doi.org/10.3390/fermentation10060319