Construction and Mechanism Analysis of High-temperature Resistant Saccharomyces cerevisiae

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

Abstract

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

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.

Figures/Tables 15

References 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