Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (11): 360-372.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0858
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WANG Wen-tao(), FENG Qi, LIU Chen-guang(), BAI Feng-wu, ZHAO Xin-qing
Received:
2023-09-04
Online:
2023-11-26
Published:
2023-12-20
Contact:
LIU Chen-guang
E-mail:tenebrae@sjtu.edu.cn;cg.liu@sjtu.edu.cn
WANG Wen-tao, FENG Qi, LIU Chen-guang, BAI Feng-wu, ZHAO Xin-qing. Redox-sensitive Genetic Parts Improve the Tolerance of Yeast to Lignocellulosic Hydrolysate Inhibitors[J]. Biotechnology Bulletin, 2023, 39(11): 360-372.
名称 Name | 描述 Description | 来源 Source |
---|---|---|
E. coli DH5α | 质粒构建 | 实验室保存 |
S. cerevisiaeS288C | 宿主细胞 | 购买于ATCC |
SC-PGK1 | S288C, HO-yEGFP | 实验室保存 |
SC-TRR1 | S288C, HO-TRR1 | 本工作构建 |
SC-TRX2 | S288C, HO-TRX2 | 本工作构建 |
SC-MET16 | S288C, HO-MET16 | 本工作构建 |
SC-SOD | S288C, HO-SOD | 本工作构建 |
SC-CTT | S288C, HO-CTT | 本工作构建 |
SC-IDP | S288C, HO-IDP | 本工作构建 |
SC-GLR | S288C, HO-GLR | 本工作构建 |
SC-ADH | S288C, HO-ADH | 本工作构建 |
SC-GRE | S288C, HO-GRE | 本工作构建 |
SC-AC | S288C, HO-AC | 本工作构建 |
Table 1 Strains used
名称 Name | 描述 Description | 来源 Source |
---|---|---|
E. coli DH5α | 质粒构建 | 实验室保存 |
S. cerevisiaeS288C | 宿主细胞 | 购买于ATCC |
SC-PGK1 | S288C, HO-yEGFP | 实验室保存 |
SC-TRR1 | S288C, HO-TRR1 | 本工作构建 |
SC-TRX2 | S288C, HO-TRX2 | 本工作构建 |
SC-MET16 | S288C, HO-MET16 | 本工作构建 |
SC-SOD | S288C, HO-SOD | 本工作构建 |
SC-CTT | S288C, HO-CTT | 本工作构建 |
SC-IDP | S288C, HO-IDP | 本工作构建 |
SC-GLR | S288C, HO-GLR | 本工作构建 |
SC-ADH | S288C, HO-ADH | 本工作构建 |
SC-GRE | S288C, HO-GRE | 本工作构建 |
SC-AC | S288C, HO-AC | 本工作构建 |
名称 Name | 描述 Description | 来源 Source |
---|---|---|
GP-SOD | PTRR1-SOD1-TADH1 | 本工作构建 |
GP-CTT | PTRR1-CTT1-TADH1 | 本工作构建 |
GP-IDP | PTRR1-IDP1-TADH1 | 本工作构建 |
GP-GLR | PTRR1-GLR1-TADH1 | 本工作构建 |
GP-ADH | PMET16-ADH6-TADH1 | 本工作构建 |
GP-GRE | PMET16-GRE2-TADH1 | 本工作构建 |
GP-AC | PTRR1-SOD1-TADH1- PMET16-ADH6-TADH1 | 本工作构建 |
Table 2 Genetic parts used
名称 Name | 描述 Description | 来源 Source |
---|---|---|
GP-SOD | PTRR1-SOD1-TADH1 | 本工作构建 |
GP-CTT | PTRR1-CTT1-TADH1 | 本工作构建 |
GP-IDP | PTRR1-IDP1-TADH1 | 本工作构建 |
GP-GLR | PTRR1-GLR1-TADH1 | 本工作构建 |
GP-ADH | PMET16-ADH6-TADH1 | 本工作构建 |
GP-GRE | PMET16-GRE2-TADH1 | 本工作构建 |
GP-AC | PTRR1-SOD1-TADH1- PMET16-ADH6-TADH1 | 本工作构建 |
名称 Name | 成分 Ingredient | 培养微生物 Cultured microorganism | 备注 Remark |
---|---|---|---|
LB | 5 g/L酵母粉、10 g/L胰蛋白胨和 10 g/L NaCl | 大肠杆菌 E. coli | 固体培养基添加20 g/L琼脂粉 |
YPD生长 | 10 g/L酵母粉、20 g/L蛋白胨和 20 g/L葡萄糖 | 酿酒酵母 S. cerevisiae | |
YPD发酵 | 3 g/L酵母粉、4 g/L蛋白胨和 100 g/L葡萄糖 | 酿酒酵母 S. cerevisiae |
Table 3 Names and composition of the media used
名称 Name | 成分 Ingredient | 培养微生物 Cultured microorganism | 备注 Remark |
---|---|---|---|
LB | 5 g/L酵母粉、10 g/L胰蛋白胨和 10 g/L NaCl | 大肠杆菌 E. coli | 固体培养基添加20 g/L琼脂粉 |
YPD生长 | 10 g/L酵母粉、20 g/L蛋白胨和 20 g/L葡萄糖 | 酿酒酵母 S. cerevisiae | |
YPD发酵 | 3 g/L酵母粉、4 g/L蛋白胨和 100 g/L葡萄糖 | 酿酒酵母 S. cerevisiae |
启动子 Promoter | 长度 Length/bp | GC含量 GC content/% | Yap1结合位点 Yap1 binding site |
---|---|---|---|
PTRR1 | 545 | 39 | 2 |
PTRX2 | 275 | 38 | 3 |
PMET16 | 418 | 34 | 3 |
Table 4 Sequence analysis of the promoters
启动子 Promoter | 长度 Length/bp | GC含量 GC content/% | Yap1结合位点 Yap1 binding site |
---|---|---|---|
PTRR1 | 545 | 39 | 2 |
PTRX2 | 275 | 38 | 3 |
PMET16 | 418 | 34 | 3 |
启动子 Promoter | EL | 抑制物:H2O2Inhibitor: H2O2 | 抑制物:糠醛 Inhibitor: Furfural | 抑制物:5-HMF Inhibitor: 5-HMF | |||
---|---|---|---|---|---|---|---|
最大RS值 Max RS value | 对应浓度 Corresponding concentration | 最大RS值 Max RS value | 对应浓度 Corresponding concentration | 最大RS值 Max RS value | 对应浓度 Corresponding concentration | ||
PTRR1 | 15.77 | 2.84 | 1 mmol/L | 1.64 | 10 mmol/L | 1.21 | 30 mmol/L |
PTRX2 | 16.76 | 2.80 | 1 mmol/L | 2.40 | 10 mmol/L | 1.11 | 30 mmol/L |
PMET16 | 1.26 | 1.27 | 1 mmol/L | 4.19 | 20 mmol/L | 1.54 | 30 mmol/L |
Table 5 Response performances of the promoters
启动子 Promoter | EL | 抑制物:H2O2Inhibitor: H2O2 | 抑制物:糠醛 Inhibitor: Furfural | 抑制物:5-HMF Inhibitor: 5-HMF | |||
---|---|---|---|---|---|---|---|
最大RS值 Max RS value | 对应浓度 Corresponding concentration | 最大RS值 Max RS value | 对应浓度 Corresponding concentration | 最大RS值 Max RS value | 对应浓度 Corresponding concentration | ||
PTRR1 | 15.77 | 2.84 | 1 mmol/L | 1.64 | 10 mmol/L | 1.21 | 30 mmol/L |
PTRX2 | 16.76 | 2.80 | 1 mmol/L | 2.40 | 10 mmol/L | 1.11 | 30 mmol/L |
PMET16 | 1.26 | 1.27 | 1 mmol/L | 4.19 | 20 mmol/L | 1.54 | 30 mmol/L |
菌株 Strain | 基因元件转录水平变化倍数 Fold-change in transcription level of gene element | 代谢速率变化(相比野生型) Metabolic rate change(Compared to wild type) | |||
---|---|---|---|---|---|
与无胁迫比较 Compared with no stress | 胁迫下相比野生型 Compared to wild type under stress | 葡萄糖消耗速率 Glucose consumption rate/% | 乙醇生产速率 Ethanol production rate/% | 比生长速率 Specific growth rate/% | |
SC-ADH | 3.84 | 1.12 | 27.23 | 39.99 | 20.46 |
SC-GRE | 12.28 | 2.10 | 5.85 | 26.04 | -0.16 |
Table 9 Metabolic rate changes in recombinant strains under 5-HMF stress
菌株 Strain | 基因元件转录水平变化倍数 Fold-change in transcription level of gene element | 代谢速率变化(相比野生型) Metabolic rate change(Compared to wild type) | |||
---|---|---|---|---|---|
与无胁迫比较 Compared with no stress | 胁迫下相比野生型 Compared to wild type under stress | 葡萄糖消耗速率 Glucose consumption rate/% | 乙醇生产速率 Ethanol production rate/% | 比生长速率 Specific growth rate/% | |
SC-ADH | 3.84 | 1.12 | 27.23 | 39.99 | 20.46 |
SC-GRE | 12.28 | 2.10 | 5.85 | 26.04 | -0.16 |
Fig. 1 Growths of the recombinant strains under stress-free and oxidative conditions A: Growth under no stress. B: Glucose consumption under no stress. C: Ethanol production under no stress. D: Growth under oxidative condition. E: Glucose consumption under oxidative condition. F: Ethanol production under oxidative condition
菌株 Strain | 基因元件转录水平变化倍数 Fold-change in transcription level of gene element | 代谢速率变化(相比野生型) Metabolic rate change(Compared to wild type) | |||||
---|---|---|---|---|---|---|---|
与无胁迫比较 Compared with no stress | 胁迫下相比野生型 Compared to wild type under stress | 葡萄糖消耗速率 Glucose consumption rate | 乙醇生产速率 Ethanol production rate | 比生长速率 Specific growth rate | |||
SC-CTT | 2.42 | 130 | 30.23% | 32.79% | 32.51% | ||
SC-SOD | 4.34 | 5.33 | 6.47% | 4.37% | 3.79% | ||
SC-IDP | 1.34 | 1.13 | 13.68% | 12.02% | 15.12% | ||
SC-GLR | 1.57 | 6.56 | 11.67% | 16.34% | 15.03% |
Table 6 Transcriptional level changes of genetic parts and metabolic rate changes in recombinant strains
菌株 Strain | 基因元件转录水平变化倍数 Fold-change in transcription level of gene element | 代谢速率变化(相比野生型) Metabolic rate change(Compared to wild type) | |||||
---|---|---|---|---|---|---|---|
与无胁迫比较 Compared with no stress | 胁迫下相比野生型 Compared to wild type under stress | 葡萄糖消耗速率 Glucose consumption rate | 乙醇生产速率 Ethanol production rate | 比生长速率 Specific growth rate | |||
SC-CTT | 2.42 | 130 | 30.23% | 32.79% | 32.51% | ||
SC-SOD | 4.34 | 5.33 | 6.47% | 4.37% | 3.79% | ||
SC-IDP | 1.34 | 1.13 | 13.68% | 12.02% | 15.12% | ||
SC-GLR | 1.57 | 6.56 | 11.67% | 16.34% | 15.03% |
菌株 Strain | ROS荧光强度 ROS fluorescence intensity |
---|---|
WT | 2017±373 |
SC-CTT | 1221±48 |
SC-SOD | 1401±185 |
SC-IDP | 1428±66 |
SC-GLR | 1465±104 |
Table 7 ROS levels in strains
菌株 Strain | ROS荧光强度 ROS fluorescence intensity |
---|---|
WT | 2017±373 |
SC-CTT | 1221±48 |
SC-SOD | 1401±185 |
SC-IDP | 1428±66 |
SC-GLR | 1465±104 |
Fig. 2 Growths of the recombinant strains under stress-free, furfural stress and 5-HMF stress conditions A: Growth under no stress. B: Glucose consumption under no stress. C: Ethanol production under no stress. D: Growth curve under 3 g/L furfural stress. E: Growth curve under 4 g/L furfural stress. F: Shock experiment with 4 g/L furfural. G: Growth under 5-HMF stress. H: Glucose consumption under 5-HMF stress. I: Ethanol production under 5-HMF stress
菌株 Strain | 糠醛胁迫下ROS荧光强度 ROS fluorescence intensity under furfural stress | 5-HMF胁迫下ROS荧光强度 ROS fluorescence intensity under 5-HMF stress |
---|---|---|
WT | 1118±219 | 1281±395 |
SC-ADH | 1200±182 | 913±23 |
SC-GRE | 1618±149 | 1135±203 |
Table 8 ROS levels under furfural stress and 5-HMF stress
菌株 Strain | 糠醛胁迫下ROS荧光强度 ROS fluorescence intensity under furfural stress | 5-HMF胁迫下ROS荧光强度 ROS fluorescence intensity under 5-HMF stress |
---|---|---|
WT | 1118±219 | 1281±395 |
SC-ADH | 1200±182 | 913±23 |
SC-GRE | 1618±149 | 1135±203 |
菌株 Strain | 添加抑制物时期 Inhibitor addition period | 葡萄糖消耗速率 Glucose consumption rate/% | 乙醇生产速率 Ethanol production rate/% | 比生长速率 Specific growth rate/% |
---|---|---|---|---|
SC-ADH | 无胁迫 | 1.4 | 4.6 | 3.3 |
对数期 | 4.63 | 1.16 | 4.62 | |
稳定期 | 3.08 | 5.90 | 0.25 | |
SC-CTT | 无胁迫 | 3.4 | -0.1 | 1.1 |
对数期 | 17.4 | 5.68 | 17.8 | |
稳定期 | 1.07 | 3.10 | 1.68 | |
SC-AC | 无胁迫 | 6.7 | -0.8 | 0.7 |
对数期 | 31.0 | 41.7 | 24.3 | |
稳定期 | 3.07 | 4.86 | 1.26 |
Table 10 Performance improvements of dual genetic parts in response to complex stress(Compared to the wild-type)
菌株 Strain | 添加抑制物时期 Inhibitor addition period | 葡萄糖消耗速率 Glucose consumption rate/% | 乙醇生产速率 Ethanol production rate/% | 比生长速率 Specific growth rate/% |
---|---|---|---|---|
SC-ADH | 无胁迫 | 1.4 | 4.6 | 3.3 |
对数期 | 4.63 | 1.16 | 4.62 | |
稳定期 | 3.08 | 5.90 | 0.25 | |
SC-CTT | 无胁迫 | 3.4 | -0.1 | 1.1 |
对数期 | 17.4 | 5.68 | 17.8 | |
稳定期 | 1.07 | 3.10 | 1.68 | |
SC-AC | 无胁迫 | 6.7 | -0.8 | 0.7 |
对数期 | 31.0 | 41.7 | 24.3 | |
稳定期 | 3.07 | 4.86 | 1.26 |
菌株 Strain | 总生物量 Total biomass | 葡萄糖消耗速率 Glucose consumption rate | 乙醇生产速率 Ethanol production rate | 比生长速率 Specific growth rate |
---|---|---|---|---|
SC-AC | 11.5% | 60.1% | 58.9% | 64.2% |
Table 11 Performance improvements of dual genetic parts in response to complex stress(Adding inhibitors during the lag phase, compared to SC-CTT strain)
菌株 Strain | 总生物量 Total biomass | 葡萄糖消耗速率 Glucose consumption rate | 乙醇生产速率 Ethanol production rate | 比生长速率 Specific growth rate |
---|---|---|---|---|
SC-AC | 11.5% | 60.1% | 58.9% | 64.2% |
菌株 Strain | ROS level | NAD+/ NADH | NADP+/ NADPH | ATP content/ (nmol·mg-1Protein) | Total GSH content/(mmol·mg-1Protein) | SOD enzyme activity/(U·mg-1Protein) | CAT enzyme activity/(U·mg-1Protein) |
---|---|---|---|---|---|---|---|
SC-CTT | 324±5 | 0.32±0.04 | 0.26±0.02 | 11.54±0.33 | 16.98±3.29 | 295±15 | 26.10±1.22 |
SC-AC | 312±2 | 0.30±0.14 | 0.28±0.01 | 8.34±0.52 | 23.37±4.45 | 242±98 | 36.60±0.09 |
Table 12 Characterization of the physiological state of the recombinant strains in the lag phase stress group
菌株 Strain | ROS level | NAD+/ NADH | NADP+/ NADPH | ATP content/ (nmol·mg-1Protein) | Total GSH content/(mmol·mg-1Protein) | SOD enzyme activity/(U·mg-1Protein) | CAT enzyme activity/(U·mg-1Protein) |
---|---|---|---|---|---|---|---|
SC-CTT | 324±5 | 0.32±0.04 | 0.26±0.02 | 11.54±0.33 | 16.98±3.29 | 295±15 | 26.10±1.22 |
SC-AC | 312±2 | 0.30±0.14 | 0.28±0.01 | 8.34±0.52 | 23.37±4.45 | 242±98 | 36.60±0.09 |
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