生物技术通报 ›› 2023, Vol. 39 ›› Issue (11): 360-372.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0858
收稿日期:
2023-09-04
出版日期:
2023-11-26
发布日期:
2023-12-20
通讯作者:
刘晨光,男,博士,副教授,研究方向:微生物代谢工程与生物质炼制;E-mail: cg.liu@sjtu.edu.cn作者简介:
王文韬,男,研究方向:微生物电化学和电发酵;E-mail: tenebrae@sjtu.edu.cn
基金资助:
WANG Wen-tao(), FENG Qi, LIU Chen-guang(), BAI Feng-wu, ZHAO Xin-qing
Received:
2023-09-04
Published:
2023-11-26
Online:
2023-12-20
摘要:
纤维素乙醇作为一种清洁可再生的绿色能源,具有良好的应用前景。然而酿酒酵母利用木质纤维素原料生产乙醇的发酵过程易受多种抑制物胁迫的影响,因此提高其胁迫耐受性具有重要意义。本研究在细胞内设计了一种氧化还原敏感型基因元件,通过生物传感器Yap1感应胞内氧化还原状态,以调控抗胁迫基因智能表达。首先,分析了Yap1调控的天然内源启动子PTRR1、PTRX2和PMET16对木质纤维素水解液中典型抑制物的响应强度。其次,根据不同胁迫种类组合相应启动子与抗胁迫的效益基因,构建氧化还原敏感型基因元件提高了酿酒酵母的胁迫耐受性。最后,将表现较好的基因元件GP-CTT和GP-ADH串联整合到一起构建了双基因元件系统,在5-HMF和H2O2双重胁迫下细胞的死亡率与野生型相比下降了69.6%。相较于单基因元件GP-CTT,双基因元件整合菌株的比生长速率、葡萄糖消耗速率和乙醇生产速率分别提高了64.2%、60.1%和58.9%,重组菌株过氧化氢酶的酶活力提高了40.2%。本研究通过理性设计氧化还原敏感型基因元件的遗传回路,强化胞内关键抗氧化酶和醛降解途径,系统地提高了酿酒酵母的胁迫耐受性,为动态地提高酵母鲁棒性提供了新的见解。
王文韬, 冯颀, 刘晨光, 白凤武, 赵心清. 氧化还原敏感型基因元件增强酵母木质纤维素水解液抑制物胁迫耐受性[J]. 生物技术通报, 2023, 39(11): 360-372.
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 |
表1 使用的菌株
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 |
表2 使用的基因元件
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 |
表3 使用的培养基及组成成分
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 |
表4 启动子序列分析
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 |
表5 启动子的响应表现
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 |
表9 5-HMF胁迫下重组菌株的代谢速率变化
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 |
图1 无胁迫与氧化胁迫下重组菌株的生长情况 A:无胁迫下的生长曲线;B:无胁迫下的葡萄糖消耗曲线;C:无胁迫下的乙醇生产曲线;D:氧化胁迫下的生长曲线;E:氧化胁迫下的葡萄糖消耗曲线;F:氧化胁迫下的乙醇生产曲线
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% |
表6 基因元件转录水平变化以及重组菌株的代谢速率变化
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 |
表7 菌株的ROS水平
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 |
图2 无胁迫、糠醛胁迫以及5-HMF胁迫条件下重组菌株的生长情况 A:无胁迫下生长曲线;B:无胁迫下葡萄糖消耗曲线;C:无胁迫下乙醇生产曲线;D:3 g/L 糠醛胁迫的生长曲线;E:4 g/L 糠醛胁迫的生长曲线;F:4 g/L 糠醛的冲击实验;G:5-HMF胁迫下生长曲线;H:5-HMF胁迫下葡萄糖消耗曲线;I:5-HMF胁迫下乙醇生产曲线
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 |
表8 糠醛与5-HMF胁迫下的ROS水平
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 |
表10 双基因元件应对复杂胁迫的性能提升(与野生型比较)
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% |
表11 双基因元件应对复杂胁迫的性能提升(延滞期添加抑制物,与SC-CTT比较)
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% |
图4 美蓝染色后的光学显微形态 A:野生型;B:SC-AC(照片在400 ×光学显微镜下拍摄)
Fig. 4 Optical microscopic images of cells stained with methylene blue A: Wild-type. B: SC-AC(The photos were taken under 400 × optical microscope)
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 |
表12 延滞期胁迫组重组菌株的生理状态表征
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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