Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening of Enzymes for HB Production from 1,2-BD
2.2. Analysis of Substrate Stereospecificity
2.3. Enzymatic Properties of (2R, 3R)-BDH and (2S, 3S)-BDH for 1,2-BD Mixture
2.4. Construction of Whole Cell Biocatalysts
2.5. HB Production from 1,2-BD Mixture by Whole-Cell Catalysis
2.6. Effects of VHB Expression on the Production of HB from 1,2-BD Mixture
3. Materials and Methods
3.1. Strains, Plasmids, Primers, and Chemicals
3.2. Recombinant Enzyme Expression and Purification
3.3. Enzyme Assays
3.4. Analysis of Substrate Stereospecificity
3.5. Construction of Whole Cell Biocatalysts
3.6. HB Production from 1,2-BD Mixture by Whole-Cell Catalysis
3.7. Effect of VHB Co-Expression on HB Production from 1,2-BD Mixture
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Enzyme | Specific Activity (U/mg) |
---|---|
(2R, 3R)-BDH | 5.02 ± 0.31 |
(2S, 3S)-BDH | 0.57 ± 0.07 |
GDH | 2.28 ± 0.13 |
meso-BDH | 0.41 ± 0.05 |
CaADH | 0.23 ± 0.01 |
CaADH:S199A | 0.90 ± 0.13 |
CbADH | 1.43 ± 0.24 |
Strains | (S)-1,2-BD (mM) | (R)-1,2-BD (mM) | Substrate Stereospecifity | HB (mM) |
---|---|---|---|---|
1,2-BD mixture standard | 45.00 ± 1.17 | 55.00 ± 0.64 | ND | 0 |
E. coli (pET28a) as control | 44.37 ± 2.34 | 54.13 ± 3.41 | ND | 1.32 ± 0.71 |
E. coli (pET-gdh) | 45.71 ± 0.97 | 31.25 ± 1.35 | (R)-1,2-BD | 23.75 ± 0.72 |
E. coli (pET-ssbdh) | 4.46 ± 1.44 | 51.37 ± 1.74 | (S)-1,2-BD | 40.54 ± 0.61 |
E. coli (pET-mbdh) | 23.64 ±0.79 | 55.42 ± 2.11 | (S)-1,2-BD | 21.36 ± 0.92 |
E. coli (pET-rrbdh) | 43.41 ± 1.31 | 2.61 ± 0.93 | (R)-1,2-BD | 51.09 ± 0.57 |
E. coli (pET-Cbadh) | 32.34 ± 0.91 | 56.72 ± 0.85 | (S)-1,2-BD | 12.66 ± 0.64 |
E. coli (pET-Caadh) | 36.23 ± 0.94 | 54.01 ± 0.69 | (S)-1,2-BD | 8.77 ± 0.78 |
E. coli (pET-Caadh:S199A) | 34.42 ± 1.44 | 52.34 ± 1.34 | (S)-1,2-BD | 10.58 ± 0.83 |
E. coli Strains | HB (mM) |
---|---|
E. coli (pET28a) | 2.08 ± 0.64 |
E. coli (pET-ssbdh) | 76.78 ± 5.27 |
E. coli (pET-ssbdh-nox) | 124.59 ± 7.53 |
E. coli (pET-rrbdh) | 158.91 ± 7.41 |
E. coli (pET-rrbdh-nox) | 202.98 ± 5.67 |
Strains or Plasmids | Genotype or Properties | Source |
---|---|---|
Strains | - | - |
Serratia sp. T241 | Wild type | Laboratory stock |
E. coli DH5α | F−,φ80d/lacZΔM15, Δ(lacZYA-argF)U169, deoR, recA1, endA1, hsdR17(rk-mk+), phoA, supE44, λ−, thi-1, gyrA96, relA1 | Tiangen Biotech |
E. coli BL21(DE3) | F-, ompT, hsdSB(rB-mB-), gal(λ c I 857, ind1, Sam7, nin5, lacUV-T7 gene1), dcm(DE3) | Tiangen Biotech |
Plasmids | - | - |
pET28a | Kmr; expression vector | Laboratory stock |
pET-nox | Kmr; nox in pET28a | Laboratory stock |
pBR322-vgb | AMPr; pBR322 vector containing the vgb gene | Laboratory stock |
pET-ssbdh | Kmr; ssbdh in pET28a | This study |
pET-mbdh | Kmr; mbdh in pET28a | This study |
pET-rrbdh | Kmr; rrbdh in pET28a | This study |
pET-gdh | Kmr; gdh in pET28a | This study |
pET-Cbadh | Kmr; Cbadh in pET28a | This study |
pET-Caadh | Kmr; Caadh in pET28a | This study |
pET-Caadh:S199A | Kmr; Caadh:S199A in pET28a | This study |
pET-rrbdh-nox | Kmr; rrbdh and nox in pET28a | This study |
pET-rrbdh-nox-vgb | Kmr; rrbdh, nox, and vgb in pET28a | This study |
pET-ssbdh-nox | Kmr; ssbdh and nox in pET28a | This study |
pET-ssbdh-nox-vgb | Kmr; ssbdh, nox, and vgb in pET28a | This study |
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Lin, H.; Xu, J.; Sun, W.; Hu, W.; Gao, H.; Hu, K.; Qiu, J.; Huang, B.; Zhang, L. Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli. Catalysts 2021, 11, 1184. https://doi.org/10.3390/catal11101184
Lin H, Xu J, Sun W, Hu W, Gao H, Hu K, Qiu J, Huang B, Zhang L. Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli. Catalysts. 2021; 11(10):1184. https://doi.org/10.3390/catal11101184
Chicago/Turabian StyleLin, Hui, Jiayin Xu, Wenlian Sun, Wujia Hu, Huifang Gao, Kaihui Hu, Junzhi Qiu, Binbin Huang, and Liaoyuan Zhang. 2021. "Efficient 1-Hydroxy-2-Butanone Production from 1,2-Butanediol by Whole Cells of Engineered E. coli" Catalysts 11, no. 10: 1184. https://doi.org/10.3390/catal11101184