Application of Auto-induction Strategy in Ergothioneine Biosynthesis

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

Abstract

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

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.

Figures/Tables 15

References 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