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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0858

摘要/Abstract

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P_TRR1、P_TRX2和P_MET16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。

关键词: 氧化还原电位, 基因元件, 酿酒酵母, 胁迫耐受性, 木质纤维素, 燃料乙醇

Abstract:

Cellulosic ethanol, as a clean and renewable green energy, has promising application prospects. However, the fermentation process of Saccharomyces cerevisiae using lignocellulose to produce ethanol is susceptible to various stresses，thus improving the stress tolerance of fermenting microorganisms has become an essential topic in the field of biorefinery. In this work, we attempted to engineer cells to use the output of the internal redox-sensitive biosensor Yap1 to drive intracellular metabolic reactions（including ROS metabolism and aldehyde degradation）, which will improve the tolerance of S. cerevisiae to reactive oxygen species and aldehyde inhibitors. Firstly, we analyzed the sequences of several native endogenous promoters（P_TRR1, P_TRX2, and P_MET16）in S. cerevisiae that can be regulated by the redox-sensitive biosensor Yapl and their response strengths to typical inhibitors in lignocellulosic hydrolysates. Secondly, we designed redox-sensitive genetic parts in order to improve the stress resistance of S. cerevisiae by combining the promoters with corresponding resistance genes. Finally, we tried to integrate the better performing GP-CTT and GP-ADH in tandem to construct a dual-genetic parts system, which was named GP-AC. The integration greatly improved the ability of S. cerevisiae to cope with multiple complex stresses, and the cell death rate decreased by 69.6% under the coexistence of aldehyde and oxidation stress. Compared with GP-CTT, the specific growth rate, glucose consumption rate and ethanol production rate of GP-AC increased by 64.2%, 60.1%, and 58.9%, respectively. At the same time, the catalase activity of the recombinant strain increased by 40.2% compared with the single genetic part. In conclusion, this study systematically improved the stress tolerance of S. cerevisiae by rationally designing the genetic circuit of redox-sensitive genetic parts and strengthening the intracellular vital antioxidant enzymes and aldehyde degradation pathways. It provides new insights for rationally designing and constructing feedback genetic circuits to dynamically improve yeast robustness.

Key words: redox-potential, genetic parts, Saccharomyces cerevisiae, stress tolerance, lignocellulose, fuel ethanol

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.

图/表 17

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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
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	P_TRR1-SOD1-T_ADH1	本工作构建
GP-CTT	P_TRR1-CTT1-T_ADH1	本工作构建
GP-IDP	P_TRR1-IDP1-T_ADH1	本工作构建
GP-GLR	P_TRR1-GLR1-T_ADH1	本工作构建
GP-ADH	P_MET16-ADH6-T_ADH1	本工作构建
GP-GRE	P_MET16-GRE2-T_ADH1	本工作构建
GP-AC	P_TRR1-SOD1-T_ADH1- P_MET16-ADH6-T_ADH1	本工作构建

名称 Name	描述 Description	来源 Source
GP-SOD	P_TRR1-SOD1-T_ADH1	本工作构建
GP-CTT	P_TRR1-CTT1-T_ADH1	本工作构建
GP-IDP	P_TRR1-IDP1-T_ADH1	本工作构建
GP-GLR	P_TRR1-GLR1-T_ADH1	本工作构建
GP-ADH	P_MET16-ADH6-T_ADH1	本工作构建
GP-GRE	P_MET16-GRE2-T_ADH1	本工作构建
GP-AC	P_TRR1-SOD1-T_ADH1- P_MET16-ADH6-T_ADH1	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