Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem
Abstract
:1. Introduction
2. Results and Discussion
2.1. Selection of Mutational Residues
2.2. Single-Point and Combinatorial Mutation of FLS
2.3. Acetaldehyde Tolerance Evaluation of FLS and Its Variants
2.4. Kinetic Parameters
2.5. In Silico Structure Analysis of Wild FLS and Its Variant I28V/L482E
2.6. Fed-Batch Synthesis of 2,3-Butanediol from Ethanol by a Modified Four-Enzyme Synthetic Biosystem
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Bacterial Growth, Recombinant Protein Expression and Purification
3.3. Mutant Library Construction for FLS
3.4. Preliminary Screening of Mutant Library
3.5. Substrate Tolerance Analysis of Beneficial Variants
3.6. Enzyme Activity and Kinetic Parameter Assays
3.7. Fed-Batch Synthesis of 2,3-BD from Ethanol and GC Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Enzyme | Km (mM) a | kcat (s−1) a | kcat/Km(s−1 M−1) | Ref. |
---|---|---|---|---|
FLS | 58.46 ± 2.32 | 0.45 ± 0.04 | 7.69 | This study |
FLS:I28V | 81.95 ± 3.61 | 3.33 ± 0.21 | 40.59 | This study |
FLS:L482E | 50.95 ± 3.22 | 0.49 ± 0.06 | 9.66 | This study |
FLS:F484A | 53.28 ± 2.52 | 1.74 ± 0.12 | 32.56 | This study |
FLS:I28V/L482E | 375.55 ± 6.84 | 8.76 ± 0.13 | 23.34 | This study |
FLS:L482S | 47.45 ± 1.20 | 0.63 ± 0.01 | 13.30 | [27] |
Enzyme | Method | Substrate | 2,3-BD (g/L) | Ref. |
---|---|---|---|---|
The lysate from E. coli expressing ALS/ALDC/BDH | Cell-free | Glucose | 82.00 | [10] |
BDH, NOX and Vitreoscilla hemoglobin | Whole-cell | Acetoin | 38.41 | [11] |
E. coli expressing BDH | Whole-cell | Diacetyl | 26.80 | [13] |
Bacillus subtilis expressing acetoin reductase and NOX | Whole-cell | Acetoin | 74.50 | [14] |
The lysates from E. coli expressing ALS/ALDC/BDH and cyanobacteria | Cell-free | Starch | 13.52 | [42] |
EtDH:D46G, FLS:I28V/L482E, NOX and BDH:S199A | Cell-free | Ethanol | 124.83 | This study |
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Zhang, J.; Lin, H.; Zheng, C.; Yang, B.; Liang, M.; Lin, Y.; Zhang, L. Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem. Molecules 2024, 29, 3934. https://doi.org/10.3390/molecules29163934
Zhang J, Lin H, Zheng C, Yang B, Liang M, Lin Y, Zhang L. Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem. Molecules. 2024; 29(16):3934. https://doi.org/10.3390/molecules29163934
Chicago/Turabian StyleZhang, Jiming, Hui Lin, Chaosong Zheng, Bin Yang, Miao Liang, Yi Lin, and Liaoyuan Zhang. 2024. "Efficient 2,3-Butanediol Production from Ethanol by a Modified Four-Enzyme Synthetic Biosystem" Molecules 29, no. 16: 3934. https://doi.org/10.3390/molecules29163934