利用代谢工程在酿酒酵母中高效合成2-萘乙醇

doi:10.13560/j.cnki.biotech.bull.1985.2024-0225

摘要/Abstract

摘要：

【目的】 为实现生物合成重要化合物2-萘乙醇，探索利于2-萘乙醇合成的基因组合。【方法】 向酿酒酵母BY4741发酵液中外源添加2-萘丙氨酸，运用高效液相色谱法进行产物检测；绘制BY4741的生长曲线和产量曲线，添加不同浓度2-萘丙氨酸到BY4741发酵液中并检测BY4741生长和生产情况；对BY4741进行代谢工程改造，构建过表达Ehrlich途径6个基因的质粒并转化进BY4741中；发酵6株改造菌株，检测产物产量。【结果】 发酵液中检测到了推测产物2-萘乙醇，代谢工程改造菌株相较于野生型BY4741菌株产量均有所提升，其中过表达ARO9和ARO10基因使得2-萘乙醇的产量在外源添加1 mmol/L 2-萘丙氨酸的情况下达到0.258 mmol/L，转化率达到野生型的2.3倍。【结论】 在酿酒酵母中实现了2-萘乙醇的生物合成，探索出ARO9和ARO10是利于2-萘乙醇生物合成的基因组合，为工业生产2-萘乙醇提供了一种新的生物合成方法。

关键词: 2-萘乙醇, 代谢工程, 生物合成, 酿酒酵母, Ehrlich途径

Abstract:

【Objective】 To elucidate the biosynthesis of 2-naphthaleneethanol and explore the gene combination beneficial to the synthesis of 2-naphthaleneethanol. 【Method】 2-Naphthylalanine was added to the fermentation broth of Saccharomyces cerevisiae BY4741 and the product was detected by HPLC. The growth curve and yield curve of BY4741 were explored. After adding different concentrations of 2-naphthylalanine into the fermentation broth of BY4741, the growth and production level were detected. The plasmids overexpressing six genes of Ehrlich pathway were constructed and transformed into BY4741. Six modified strains were fermented and the product yield was detected. 【Result】 2-Naphthaleneethanol was detected in the fermentation broth as expected. Compared with the wild type BY4741, the yields of the metabolically engineered strains were all improved. Overexpressing ARO9 and ARO10 made the yield of 2-naphthaleneethanol reached 0.258 mmol/L when exogenous 2-naphthylalanine was added, and the transformation rate reached 2.3 times that of the wild type. 【Conclusion】 The biosynthesis of 2-naphthaleneethanol was achieved in S. cerevisiae. ARO9 and ARO10 were found to be the optimal gene combinations beneficial to the biosynthesis of 2-naphthaleneethanol. This study provides a new biosynthesis method for industrial production of 2-naphthaleneethanol.

Key words: 2-naphthaleneethanol, metabolism, biosynthesis, Saccharomyces cerevisiae, Ehrlich pathway

何玙冰, 付振浩, 李仁瀚, 刘秀霞, 刘春立, 杨艳坤, 李业, 白仲虎. 利用代谢工程在酿酒酵母中高效合成2-萘乙醇[J]. 生物技术通报, 2024, 40(7): 99-107.

HE Yu-bing, FU Zhen-hao, LI Ren-han, LIU Xiu-xia, LIU Chun-li, YANG Yan-kun, LI Ye, BAI Zhong-hu. Efficient Biosynthesis of 2-Naphthaleneethanol in Metabolically Engineered Saccharomyces cerevisiae[J]. Biotechnology Bulletin, 2024, 40(7): 99-107.

图/表 8

参考文献 33

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名称Name	序列Sequence（5'-3'）
ARO8_F	GCATCGTCTCATCGGTCTCATATGACTTTACCTGAATCAAAAGACTTTTCTT
ARO8_R	ATGCCGTCTCAGGTCTCAGGATCCTTTGGAAATACCAAATTCTTCGTATAAAGT
ARO9_F	GCATCGTCTCATCGGTCTCATATGACTGCTGGTTCTGCC
ARO9_R	ATGCCGTCTCAGGTCTCAGGATCCACTTTTATAGTTGTCAAAAAATTCTTTTATGCC
ARO10_F	GCATCGTCTCATCGGTCTCATATGGCACCTGTTACAATTGAAAAGT
ARO10_R	ATGCCGTCTCAGGTCTCAGGATCCTTTTTTATTTCTTTTAAGTGCCGCTGC
PDC1_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATTACTTTGGGTAAATATTTGT
PDC1_R	ATGCCGTCTCAGGTCTCAGGATCCTTGCTTAGCGTTGGTAGCAG
PDC5_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATAACCTTAGGTAAATATTTATTTGAA
PDC5_R	ATGCCGTCTCAGGTCTCAGGATCCTTGTTTAGCGTTAGTAGCGGC
PDC6_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATTACTCTTGGAAAATACTT
PDC6_R	ATGCCGTCTCAGGTCTCAGGATCCTTGTTTGGCATTTGTAGCGG
HO5'_F	CACATCATTTTCGTGGATCC
HO5'_R	ACAGCGATGGAACTTACGGC
HO3'_F	TATCGTGTTGCATCTGCGGC
HO3'_R	CTTTGGACTTAAAATGGCGT

名称Name	序列Sequence（5'-3'）
ARO8_F	GCATCGTCTCATCGGTCTCATATGACTTTACCTGAATCAAAAGACTTTTCTT
ARO8_R	ATGCCGTCTCAGGTCTCAGGATCCTTTGGAAATACCAAATTCTTCGTATAAAGT
ARO9_F	GCATCGTCTCATCGGTCTCATATGACTGCTGGTTCTGCC
ARO9_R	ATGCCGTCTCAGGTCTCAGGATCCACTTTTATAGTTGTCAAAAAATTCTTTTATGCC
ARO10_F	GCATCGTCTCATCGGTCTCATATGGCACCTGTTACAATTGAAAAGT
ARO10_R	ATGCCGTCTCAGGTCTCAGGATCCTTTTTTATTTCTTTTAAGTGCCGCTGC
PDC1_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATTACTTTGGGTAAATATTTGT
PDC1_R	ATGCCGTCTCAGGTCTCAGGATCCTTGCTTAGCGTTGGTAGCAG
PDC5_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATAACCTTAGGTAAATATTTATTTGAA
PDC5_R	ATGCCGTCTCAGGTCTCAGGATCCTTGTTTAGCGTTAGTAGCGGC
PDC6_F	GCATCGTCTCATCGGTCTCATATGTCTGAAATTACTCTTGGAAAATACTT
PDC6_R	ATGCCGTCTCAGGTCTCAGGATCCTTGTTTGGCATTTGTAGCGG
HO5'_F	CACATCATTTTCGTGGATCC
HO5'_R	ACAGCGATGGAACTTACGGC
HO3'_F	TATCGTGTTGCATCTGCGGC
HO3'_R	CTTTGGACTTAAAATGGCGT

名称 Name	携带基因/特征 Characteristics of carried gene	大肠杆菌抗性标记 E. coli marker	部件类型/使用的部件 Type/type used	来源 Origin
pYTK001	Part plasmid entry vector	CamR	entry vector	Lee等^[16]
pYTK002	ConLS	CamR	1	Lee等^[16]
pYTK003	ConL1	CamR	1	Lee等^[16]
pYTK004	ConL2	CamR	1	Lee等^[16]
pYTK009	pTDH3	CamR	2	Lee等^[16]
pYTK011	pPGK1	CamR	2	Lee等^[16]
pYTK048	Spacer	CamR	234	Lee等^[16]
pYTK051	tENO1	CamR	4	Lee等^[16]
pYTK052	tSSA1	CamR	4	Lee等^[16]
pYTK067	ConR1	CamR	5	Lee等^[16]
pYTK068	ConR2	CamR	5	Lee等^[16]
pYTK072	ConRE	CamR	5	Lee等^[16]
pYTK095	AmpR-ColE1	AmpR	678	Lee等^[16]
pBL602	Integration（HO-loc）HIS3 GFP Dropout	KanR	15678	实验室保藏
pBL617	ARO8	CamR	3	实验室保藏
pBL618	ARO9	CamR	3	实验室保藏
pBL621	ARO10	CamR	3	实验室保藏
pBL623	PDC1	CamR	3	实验室保藏
pBL624	PDC5	CamR	3	实验室保藏
pBL625	PDC6	CamR	3	实验室保藏
pBL883	ConL1-Spacer-ConRE	AmpR	pYTK003, pYTK048, pYTK072, pYTK095	本研究构建
pBL884	ConL2-Spacer-ConRE	AmpR	pYTK004, pYTK048, pYTK072, pYTK096	本研究构建
pBL885	ConLS-pPGK1-ARO8-tENO1-ConR1	AmpR	pYTK002, pYTK011, pYTK051, pYTK067, pYTK095, pBL617	本研究构建
pBL886	ConLS-pPGK1-ARO9-tENO1-ConR1	AmpR	pYTK002, pYTK011, pYTK051, pYTK067, pYTK095, pBL618	本研究构建
pBL889	ConL1-pTDH3-ARO10-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL621	本研究构建
pBL890	ConL1-pTDH3-PDC1-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL623	本研究构建
pBL891	ConL1-pTDH3-PDC5-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL624	本研究构建
pBL892	ConL1-pTDH3-PDC6-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL625	本研究构建
pBL909	Integration（HO-loc）HIS3 pPGK1-ARO8-tENO1	KanR	pBL602, pBL883, pBL885	本研究构建
pBL910	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1	KanR	pBL602, pBL883, pBL886	本研究构建
pBL961	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-ARO10-tSSA1	KanR	pBL602, pBL884, pBL886, pBL889	本研究构建
pBL962	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC1-tSSA1	KanR	pBL602, pBL884, pBL886, pBL890	本研究构建
pBL963	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC5-tSSA1	KanR	pBL602, pBL884, pBL886, pBL891	本研究构建
pBL964	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC6-tSSA1	KanR	pBL602, pBL884, pBL886, pBL892	本研究构建

名称 Name	携带基因/特征 Characteristics of carried gene	大肠杆菌抗性标记 E. coli marker	部件类型/使用的部件 Type/type used	来源 Origin
pYTK001	Part plasmid entry vector	CamR	entry vector	Lee等^[16]
pYTK002	ConLS	CamR	1	Lee等^[16]
pYTK003	ConL1	CamR	1	Lee等^[16]
pYTK004	ConL2	CamR	1	Lee等^[16]
pYTK009	pTDH3	CamR	2	Lee等^[16]
pYTK011	pPGK1	CamR	2	Lee等^[16]
pYTK048	Spacer	CamR	234	Lee等^[16]
pYTK051	tENO1	CamR	4	Lee等^[16]
pYTK052	tSSA1	CamR	4	Lee等^[16]
pYTK067	ConR1	CamR	5	Lee等^[16]
pYTK068	ConR2	CamR	5	Lee等^[16]
pYTK072	ConRE	CamR	5	Lee等^[16]
pYTK095	AmpR-ColE1	AmpR	678	Lee等^[16]
pBL602	Integration（HO-loc）HIS3 GFP Dropout	KanR	15678	实验室保藏
pBL617	ARO8	CamR	3	实验室保藏
pBL618	ARO9	CamR	3	实验室保藏
pBL621	ARO10	CamR	3	实验室保藏
pBL623	PDC1	CamR	3	实验室保藏
pBL624	PDC5	CamR	3	实验室保藏
pBL625	PDC6	CamR	3	实验室保藏
pBL883	ConL1-Spacer-ConRE	AmpR	pYTK003, pYTK048, pYTK072, pYTK095	本研究构建
pBL884	ConL2-Spacer-ConRE	AmpR	pYTK004, pYTK048, pYTK072, pYTK096	本研究构建
pBL885	ConLS-pPGK1-ARO8-tENO1-ConR1	AmpR	pYTK002, pYTK011, pYTK051, pYTK067, pYTK095, pBL617	本研究构建
pBL886	ConLS-pPGK1-ARO9-tENO1-ConR1	AmpR	pYTK002, pYTK011, pYTK051, pYTK067, pYTK095, pBL618	本研究构建
pBL889	ConL1-pTDH3-ARO10-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL621	本研究构建
pBL890	ConL1-pTDH3-PDC1-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL623	本研究构建
pBL891	ConL1-pTDH3-PDC5-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL624	本研究构建
pBL892	ConL1-pTDH3-PDC6-tSSA1-ConR2	AmpR	pYTK003, pYTK009, pYTK052, pYTK068, pYTK095, pBL625	本研究构建
pBL909	Integration（HO-loc）HIS3 pPGK1-ARO8-tENO1	KanR	pBL602, pBL883, pBL885	本研究构建
pBL910	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1	KanR	pBL602, pBL883, pBL886	本研究构建
pBL961	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-ARO10-tSSA1	KanR	pBL602, pBL884, pBL886, pBL889	本研究构建
pBL962	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC1-tSSA1	KanR	pBL602, pBL884, pBL886, pBL890	本研究构建
pBL963	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC5-tSSA1	KanR	pBL602, pBL884, pBL886, pBL891	本研究构建
pBL964	Integration（HO-loc）HIS3 pPGK1-ARO9-tENO1 pTDH3-PDC6-tSSA1	KanR	pBL602, pBL884, pBL886, pBL892	本研究构建

名称Name	基因型Genotype	来源Origin
BY4741	MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0	Brachmann等^[18]
yBL_909	[BY4741]Holoc::pBL_909	本研究构建
yBL_910	[BY4741]Holoc::pBL_910	本研究构建
yBL_961	[BY4741]Holoc::pBL_961	本研究构建
yBL_962	[BY4741]Holoc::pBL_962	本研究构建
yBL_963	[BY4741]Holoc::pBL_963	本研究构建
yBL_964	[BY4741]Holoc::pBL_964	本研究构建