自诱导策略在麦角硫因生物合成中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0575

摘要/Abstract

摘要：

【目的】旨在挖掘细菌途径ERG合成潜力，为之后对细菌途径关键酶改造，提高ERG产量提供实验依据。【方法】在大肠杆菌中引入耻垢分枝杆菌ERG合成途径，创建阳性对照工程菌RE。通过摇瓶发酵比较常规诱导法与自诱导法ERG产量；随后对底盘细胞RE的His和Cys途径进行改造，增强其前体氨基酸内源合成能力，得到菌株RE-CH；使用RE-CH菌株在10 L罐上建立自诱导发酵工艺进行发酵放大研究，并对补料策略进行优化；最后，改变调控策略，增加菌体密度，在30 L罐上进行发酵，以期增加ERG产量。【结果】相比常规诱导法，自诱导法ERG产量提高2.8倍；发酵验证表明新工程菌（RE-CH）ERG合成能力得到增强；10 L罐优化补料策略后，ERG产量达到1.1 g/L；30 L罐调整调控策略后，发酵95.5 h，ERG的产量达到4.3 g/L。【结论】利用细菌ERG合成途径发酵ERG产量与真菌途径产量相当，且使用优化后的发酵工艺相比已报道的真菌途径发酵工艺发酵周期缩短约33%。

关键词: 麦角硫因, 大肠杆菌, 耻垢分枝杆菌, 发酵工程, 代谢工程, 自诱导, 细菌合成途径

Abstract:

【Objective】The aim of this study is to explore the ERG synthesis potential of the bacterial pathway and to provide an experimental basis for the subsequent modification of the key enzymes of the bacterial pathway to improve ERG production.【Method】Introduction of the ERG synthesis pathway from Mycobacterium smegmatis to Escherichia coli Rosetta2（DE3）, a positive control strain RE was created. This was followed by a comparison of the ERG production yield between the conventional induction method and the self-induction method. Subsequently, the His and Cys pathways in the RE strain were modified to enhance the endogenous synthesis of precursor Enhanced its precursor amino acids, and the strain RE-CH was obtained. The amino acid in-situ synthesis capacity was obtained for the strain RE-CH. The RE-CH strain was employed in a 10 L bioreactor to establish self-induction fermentation process, which was then scaled up for fermentation optimization. Finally, the regulatory strategy was altered to enhance cell density, and fermentation was conducted in a 30 L bioreactor to boost the production of ERG.【Result】The results demonstrated that the yield of ERG produced by the self-induction fermentation process was 2.8 times higher than that produced by the conventional induction method. The fermentation verification indicated that the newly developed strain（RE-CH）demonstrated the enhanced ERG synthesis capabilities. The synthetic ability of the strain（RE-CH）was enhanced. Following optimization of the feeding strategy in a 10 L reactor, the yield of ERG reached 1.1 g/L. In a 30 L reactor, the control strategy was adjusted, resulting in a fermentation period of 95.5 h and an ERG yield of 4.3 g/L.【Conclusion】It can be concluded that the fermentation of ERG via the bacterial pathway is comparable to that via the fungal pathway, and that the optimized fermentation process is approximately 33% shorter than the previously reported fungal pathway fermentation process.

Key words: ergothioneine, Escherichia coli, Mycobacterium smegmatis, fermentation engineering, metabolic engineering, self-induction, bacterial synthesis pathway

饶峻, 赵晨, 李端华, 廖豪, 黄加雨, 王辂. 自诱导策略在麦角硫因生物合成中的应用[J]. 生物技术通报, 2025, 41(1): 333-346.

RAO Jun, ZHAO Chen, LI Duan-hua, LIAO Hao, HUANG Jia-yu, WANG Lu. Application of Auto-induction Strategy in Ergothioneine Biosynthesis[J]. Biotechnology Bulletin, 2025, 41(1): 333-346.

图/表 15

参考文献 47

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ERG合成途径来源 ERG synthesis pathway source	关键酶^* Key enzymes^*	工程菌 Engineering bacteria	发酵工艺 Fermentation process	诱导方式 Induction method	发酵周期Fermentation period/h	菌体密度 Bacterial density	ERG产量ERG production/（g·L^-1）	ERG产率 ERG productivity/（g·L^-1·h^-1）	参考文献 Reference
Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	73	-	0.6	8.2	[25]
Bacteria	W	Corynebacterium glutamicum	Fed-batch baffled flasks	-	120	OD₆₀₀>80	0.1	0.8	[26]
Bacteria	W	E. coli	Baffled flasks	Manual addition of inducers	192	OD₆₀₀=14.0	0.7	3.4	[24]
Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	216	OD₆₀₀≈55	1.3	6.1	[23]
Fungi	W	Yarrowia lipolytica	Fed-batch jar fermentation	-	168	OD₆₀₀>150	7.3	43.5	[27]
Fungi	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	143	OD₆₀₀=130	4.3	30.3	[3]
Fungi	W	Yarrowia lipolytica	Fed-batch jar fermentation	-	220	60.6 g/L CDW	1.6	7.4	[28]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	96	OD₆₀₀>75	2.6	27.4	[29]
Fungi+Bacteria	M	E. coli	Fed-batch jar fermentation	Manual addition of inducers	94	OD₆₀₀≈100	5.4	57.4	[30]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	108	OD₆₀₀=45.7	0.7	6.6	[31]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	108	OD₆₀₀=65	2	18.6	[32]

ERG合成途径来源 ERG synthesis pathway source	关键酶^* Key enzymes^*	工程菌 Engineering bacteria	发酵工艺 Fermentation process	诱导方式 Induction method	发酵周期Fermentation period/h	菌体密度 Bacterial density	ERG产量ERG production/（g·L^-1）	ERG产率 ERG productivity/（g·L^-1·h^-1）	参考文献 Reference
Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	73	-	0.6	8.2	[25]
Bacteria	W	Corynebacterium glutamicum	Fed-batch baffled flasks	-	120	OD₆₀₀>80	0.1	0.8	[26]
Bacteria	W	E. coli	Baffled flasks	Manual addition of inducers	192	OD₆₀₀=14.0	0.7	3.4	[24]
Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	216	OD₆₀₀≈55	1.3	6.1	[23]
Fungi	W	Yarrowia lipolytica	Fed-batch jar fermentation	-	168	OD₆₀₀>150	7.3	43.5	[27]
Fungi	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	143	OD₆₀₀=130	4.3	30.3	[3]
Fungi	W	Yarrowia lipolytica	Fed-batch jar fermentation	-	220	60.6 g/L CDW	1.6	7.4	[28]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	96	OD₆₀₀>75	2.6	27.4	[29]
Fungi+Bacteria	M	E. coli	Fed-batch jar fermentation	Manual addition of inducers	94	OD₆₀₀≈100	5.4	57.4	[30]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	108	OD₆₀₀=45.7	0.7	6.6	[31]
Fungi+Bacteria	W	E. coli	Fed-batch jar fermentation	Manual addition of inducers	108	OD₆₀₀=65	2	18.6	[32]

类型 Type	名称 Name	描述 Description	来源 Source
Plasmid	pET-28a	Expression vector, Kan^r	Lab stock
	pET28a-egtABCDE	pET-28a containing egtABCDE	This work
	pET28a-egtA	pET-28a containing egtA	This work
	pET28a-egtB	pET-28a containing egtB	This work
	pET28a-egtC	pET-28a containing egtC	This work
	pET28a-egtD	pET-28a containing egtD	This work
	pET28a-egtE	pET-28a containing egtE	This work
	pACYC184	Expression vector, Cm^r, Tc^r	General Biol
	pACYC184-cysE-serA	pACYC184 containing cysE^{T167A, G203S, T234S, P252L, M256Q} and serA^T410stop	This work
	pACYC184-hisG	pACYC184 containing hisG^{S143F, ∆209-281}	This work
	pACYC184-hisG-cysE-serA	pACYC184 containing cysE^{T167A, G203S, T234S, P252L, M256Q}, serA^T410stopand hisG^{S143F, ∆209-281}	This work
Strain	E. coli Rosetta2（DE3）	Expression host	Lab stock
	RE	E. coli Rosetta2（DE3）harboring pET28a-egtABCDE	This work
	REA	E. coli Rosetta2（DE3）harboring pET28a-egtA	This work
	REB	E. coli Rosetta2（DE3）harboring pET28a-egtB	This work
	REC	E. coli Rosetta2（DE3）harboring pET28a-egtC	This work
	RED	E. coli Rosetta2（DE3）harboring pET28a-egtD	This work
	REE	E. coli Rosetta2（DE3）harboring pET28a-egtE	This work
	RE-C	RE harboring pACYC184-cysE-serA	This work
	RE-H	RE harboring pACYC184-hisG	This work
	RE-CH	RE harboring pACYC184-hisG-cysE-serA	This work

类型 Type	名称 Name	描述 Description	来源 Source
Plasmid	pET-28a	Expression vector, Kan^r	Lab stock
	pET28a-egtABCDE	pET-28a containing egtABCDE	This work
	pET28a-egtA	pET-28a containing egtA	This work
	pET28a-egtB	pET-28a containing egtB	This work
	pET28a-egtC	pET-28a containing egtC	This work
	pET28a-egtD	pET-28a containing egtD	This work
	pET28a-egtE	pET-28a containing egtE	This work
	pACYC184	Expression vector, Cm^r, Tc^r	General Biol
	pACYC184-cysE-serA	pACYC184 containing cysE^{T167A, G203S, T234S, P252L, M256Q} and serA^T410stop	This work
	pACYC184-hisG	pACYC184 containing hisG^{S143F, ∆209-281}	This work
	pACYC184-hisG-cysE-serA	pACYC184 containing cysE^{T167A, G203S, T234S, P252L, M256Q}, serA^T410stopand hisG^{S143F, ∆209-281}	This work
Strain	E. coli Rosetta2（DE3）	Expression host	Lab stock
	RE	E. coli Rosetta2（DE3）harboring pET28a-egtABCDE	This work
	REA	E. coli Rosetta2（DE3）harboring pET28a-egtA	This work
	REB	E. coli Rosetta2（DE3）harboring pET28a-egtB	This work
	REC	E. coli Rosetta2（DE3）harboring pET28a-egtC	This work
	RED	E. coli Rosetta2（DE3）harboring pET28a-egtD	This work
	REE	E. coli Rosetta2（DE3）harboring pET28a-egtE	This work
	RE-C	RE harboring pACYC184-cysE-serA	This work
	RE-H	RE harboring pACYC184-hisG	This work
	RE-CH	RE harboring pACYC184-hisG-cysE-serA	This work

引物名称 Primer name	引物序列 Primer sequence（5'-3'）
EgtA-F	ATATACCATGGCCCTGCCGG
EgtA-R	CCCAAGCTTTCCTTCTTACAGTTCACCTTTTGCC
EgtB-F	CATGCCATGGAACTGATTGCACGTGAAACCCTGG
EgtB-R	CCCAAGCTTCTCCTTCTTACACATCCCATGCC
EgtC-F	CATGCCATGGAATGTCGCCATGTTGCCTGGCT
EgtC-R	CCCAAGCTTCTTCTTACAGCGGGGTAACAAC
EgtD-F	CATGCCATGGAAACCCTGAGCCTGGCCAATTATC
EgtD-R	CCCAAGCTTCCTTCTTAACGAACTGCCAGGC
EgtE-F	CATGCCATGGAAATGCTGGCACAGCAGTGGCGTGA
EgtE-R	CCCAAGCTTAGCTTTTACGGTGCTTCACGC
HisG-F	ATAAAATATTTCTAGTTTTTTTCATATGCCTGACGGAGTTCACAC
HisG-R	TGCACTGAAATCTAGTTAATTCTGTGCATGCAGAATACCCT