耐高温酿酒酵母的构建与高温耐受机制解析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0417

摘要/Abstract

摘要：

旨在构建优良的高温耐受酿酒酵母菌株，并探究其高温耐受机制。通过CRISPR/Cas9技术在絮凝性工业酿酒酵母KF-7中敲除ASP3（编码 L-天冬酰胺酶II）并进一步高表达CRZ1（编码具有锌指结构的转录因子Crz1p），通过比较转录组解析重组菌株的高温耐受机制。结果显示，在44℃高温条件下，ASP3敲除菌株KAS11利用98.36 g/L葡萄糖产生43.68 g/L乙醇。在KAS11基础上高表达CRZ1后，菌株KASCR7发酵105.37 g/L葡萄糖产48.02 g/L乙醇。与KF-7相比，两个重组菌株的乙醇产量分别提升了4.77%和15.18%。比较转录组分析结果表明，在高温胁迫下，重组菌株的核糖体生物合成及翻译相关基因受到抑制，而热休克蛋白基因以及NAD⁺、NADH、嘌呤、甘油、脯氨酸等合成相关基因受到诱导，这些响应可能共同导致重组菌株的高温耐受性提升。研究结果可为构建高温耐受酿酒酵母菌株提供优良菌株资源和理论基础。

关键词: 酿酒酵母, 燃料乙醇, 胁迫耐受, 高温发酵, 比较转录组

Abstract:

This work aims to construct Saccharomyces cerevisiae strains with excellent high-temperature tolerance and investigate the high-temperature tolerance mechanisms of the strains. CRISPR/Cas9 technology was used to knock out ASP3 in flocculating industrial S. cerevisiae strain KF-7 and further highly expressed CRZ1（Encoding transcription factor Crz1p with zinc finger structure）. And the high-temperature tolerance mechanism of the recombinant strains was revealed through comparative transcriptomic analysis. The results showed that the ASP3-knockout strain KAS11 utilized 98.36 g/L glucose and produced 43.68 g/L ethanol at 44℃. After CRZ1 high expression based on KAS11, strain KASCR7 produced 48.02 g/L ethanol from 105.37 g/L glucose. Compared with KF-7, the ethanol production of the two recombinant strains increased by 4.77% and 15.18%, respectively. Comparative transcriptomic analysis revealed that genes involved in ribosome biogenesis and translation significantly repressed in the recombinant strains under high-temperature stress. In contrast, heat shock protein genes as well as genes involved in biosynthesis of NAD⁺, NADH, purine, glycerol, and proline were significantly induced. These responses may collectively lead to the enhanced high-temperature tolerance of the recombinant strains. The results may provide excellent strain resources and theoretical basis for the construction of excellent high-temperature tolerant S. cerevisiae strains.

Key words: Saccharomyces cerevisiae, fuel ethanol, stress tolerance, high-temperature fermentation, comparative transcriptome

孙言秋, 谢采芸, 汤岳琴. 耐高温酿酒酵母的构建与高温耐受机制解析[J]. 生物技术通报, 2023, 39(11): 226-237.

SUN Yan-qiu, XIE Cai-yun, TANG Yue-qin. Construction and Mechanism Analysis of High-temperature Resistant Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2023, 39(11): 226-237.

图/表 15

参考文献 34

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质粒Plasmid	描述Description	来源Source
Cas9-NAT	Ampr; Cas9; NAT1	[11]
pMEL13-CRZ1	Ampr; 2 μm orign, KanMX, gRNA-CRZ1	[9]
pMEL13-ASP3	Ampr; 2 μm orign, KanMX, gRNA-ASP3	[9]

质粒Plasmid	描述Description	来源Source
Cas9-NAT	Ampr; Cas9; NAT1	[11]
pMEL13-CRZ1	Ampr; 2 μm orign, KanMX, gRNA-CRZ1	[9]
pMEL13-ASP3	Ampr; 2 μm orign, KanMX, gRNA-ASP3	[9]

引物用途 Usage of primer	引物名称Name	引物序列 Primer sequence（5'-3'）
扩增带CRZ1同源臂的修复片段	CRZ1 RF F	GGGCTGAAAAGTACATCCGCGCATTTAACAATTGCTAAGCCACACACCATAGCTTCAAAATG
扩增带CRZ1同源臂的修复片段	CRZ1 RF R	TAGTCATGTAGGAAGCCATATTTCCGTTGCTGAATGACATTTTGTAATTAAAACT
高表达CRZ1验证引物	CRZ1 V_P F	GCTTTGACTGCACTTTAGCTTAG
高表达CRZ1验证引物	CRZ1 V_P R	ATTATTACGTCTGTAAGCGC
敲除ASP3修复片段	ASP3 RF F	AGAGCAAATGTTGGCTCGCTATTCTTTTGTAAGCAATCTGGTACTCACCAACCTCCAACTAGCCTGATCAGTGACTTTTCATCACACTGTGTTTTTATATAGTTCTTAGTAGTAAATATA
敲除ASP3修复片段	ASP3 RF R	TATATTTACTACTAAGAACTATATAAAAACACAGTGTGATGAAAAGTCACTGATCAGGCTAGTTGGAGGTTGGTGAGTACCAGATTGCTTACAAAAGAATAGCGAGCCAACATTTGCTCT
敲除ASP3验证引物	ASP3 V_P F	TATCAGACCCTTCAGCACGT
敲除ASP3验证引物	ASP3 V_P R	TGACACTGCTCAAGGGATAA

引物用途 Usage of primer	引物名称Name	引物序列 Primer sequence（5'-3'）
扩增带CRZ1同源臂的修复片段	CRZ1 RF F	GGGCTGAAAAGTACATCCGCGCATTTAACAATTGCTAAGCCACACACCATAGCTTCAAAATG
扩增带CRZ1同源臂的修复片段	CRZ1 RF R	TAGTCATGTAGGAAGCCATATTTCCGTTGCTGAATGACATTTTGTAATTAAAACT
高表达CRZ1验证引物	CRZ1 V_P F	GCTTTGACTGCACTTTAGCTTAG
高表达CRZ1验证引物	CRZ1 V_P R	ATTATTACGTCTGTAAGCGC
敲除ASP3修复片段	ASP3 RF F	AGAGCAAATGTTGGCTCGCTATTCTTTTGTAAGCAATCTGGTACTCACCAACCTCCAACTAGCCTGATCAGTGACTTTTCATCACACTGTGTTTTTATATAGTTCTTAGTAGTAAATATA
敲除ASP3修复片段	ASP3 RF R	TATATTTACTACTAAGAACTATATAAAAACACAGTGTGATGAAAAGTCACTGATCAGGCTAGTTGGAGGTTGGTGAGTACCAGATTGCTTACAAAAGAATAGCGAGCCAACATTTGCTCT
敲除ASP3验证引物	ASP3 V_P F	TATCAGACCCTTCAGCACGT
敲除ASP3验证引物	ASP3 V_P R	TGACACTGCTCAAGGGATAA

培养基Culture medium	组分Components	pH
LB+Kana	5 g/L酵母浸出粉，10 g/L蛋白胨，10 g/L NaCl，0.1 g/L Kana	7.0
LB+NAT	5 g/L酵母浸出粉，10 g/L蛋白胨，10 g/L NaCl，0.05 g/L NAT	7.0
2% YPD	10 g/L酵母浸出粉，20 g/L蛋白胨，20 g/L葡萄糖	自然
2% YPD+NAT	10 g/L酵母浸出粉，20 g/L蛋白胨，20 g/L葡萄糖，0.05 g/L NAT	自然
2% YPD+G418	10 g/L酵母浸出粉，20 g/L 蛋白胨，20 g/L葡萄糖，0.1 g/L G418	自然
2% YPD+NAT+G418	10 g/L酵母浸出粉，20 g/L蛋白胨，20 g/L葡萄糖，0.05 g/L NAT，0.1 g/L G418	自然
5% YPD	10 g/L酵母浸出粉，20 g/L蛋白胨，50 g/L葡萄糖	自然
15% YPD	10 g/L酵母浸出粉，20 g/L蛋白胨，150 g/L葡萄糖	自然