Escherichia coli strain for efficiently synthesizing ergothioneine and application of escherichia coli strain in fermentation production of ergothioneine
By optimizing the ergothioneine biosynthetic enzyme, a highly efficient Escherichia coli production strain was constructed, solving the problem of low ergothioneine production efficiency, achieving efficient fermentation production and cost reduction, and increasing the yield to 20.7 g/L.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-12
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies for ergothioneine production have low efficiency and high costs, making it difficult to meet the needs of widespread application.
By screening and directed evolution to optimize the enzymes involved in the three-step biosynthesis of ergothioneine, a highly efficient Escherichia coli production strain was constructed. This included optimizing S-adenosylmethionine synthase, glutathione reductase, and ergothioneine synthase, thereby increasing the SAM concentration and the efficiency of reducing oxidized glutathione to reduced glutathione, thus achieving efficient fermentation production of ergothioneine.
It significantly increased the fermentation yield of ergothioneine from 8.6 g/L to 20.7 g/L, and reduced production costs.
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of synthetic biology and bioengineering, and particularly relates to construction of an Escherichia coli strain for high-efficiency synthesis of ergothioneine. BACKGROUND
[0002] Ergothioneine is the most broad-spectrum natural active oxygen scavenger, and has wide prospects in the fields of cosmetics, health products, food, etc. Fungal strains (such as Auricularia edible fungi) as natural strains for synthesis of ergothioneine have been replaced by model strains (including Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, etc.) transformed with ergothioneine synthetases (such as EGT1 and EGT2) due to the problem of too low ergothioneine yield. Through optimization, the fermentative production of ergothioneine has exceeded 10 g / L (CN120249341A). However, further improvement of the production strain of ergothioneine to improve the production efficiency of ergothioneine and reduce the cost to benefit the general public is still the future development direction of the fermentative production of ergothioneine.
[0003] The biosynthesis of ergothioneine includes three steps of histidine methylation, formation of sulfoxide intermediate, and reduction cleavage of the sulfoxide intermediate to synthesize ergothioneine. The histidine methylation is the rate-limiting step, and thus the optimization of histidine methylation is the core to improve the fermentative yield of ergothioneine. SUMMARY
[0004] The present application first screens and optimizes the enzymes for the three-step biosynthesis of ergothioneine through directed evolution, and obtains ergothioneine synthetase 1 cpEGT1-M capable of efficiently performing histidine methylation and formation of sulfoxide intermediate, and ergothioneine synthetase 2 clEGT2-M capable of efficiently catalyzing cleavage of the sulfoxide intermediate, constructs the Escherichia coli production strain E1 of ergothioneine, and obtains 8.6 g / L of ergothioneine through fermentation. It is found that the concentration of S-adenosylmethionine (SAM) of the strain E1 is much lower than that of other model strains (such as Saccharomyces cerevisiae), and thus the present application screens and optimizes the S-adenosylmethionine synthetase through directed evolution, and obtains S-adenosylmethionine synthetase ctMetK-M, and constructs the Escherichia coli production strain E2 of ergothioneine. The strain E2 greatly improves the concentration of SAM, and improves the fermentative yield of ergothioneine to 15.3 g / L. Then, the present application further screens and optimizes the glutathione reductase through directed evolution, and obtains glutathione reductase pfGR-M, and constructs the Escherichia coli production strain E3 of ergothioneine. PfGR-M improves the efficiency of reduction of oxidized glutathione to reduced glutathione, accelerates the efficiency of further reduction of the cleavage product of the sulfoxide intermediate to ergothioneine, and further improves the fermentative yield of ergothioneine to 20.7 g / L.
[0005] In order to achieve the purpose of the present application, the technical solution adopted is: An S-adenosylmethionine synthetase ctMetK-M, the amino acid sequence of which is shown as SEQ ID NO: 1.
[0006] In a preferred embodiment of the present application, the nucleotide sequence of the coding gene (ctMetK-M) of the S-adenosylmethionine synthetase ctMetK-M is shown as SEQ ID NO: 2.
[0007] A glutathione reductase pfGR-M, the amino acid sequence of which is shown as SEQ ID NO: 3.
[0008] In a preferred embodiment of the present application, the nucleotide sequence of the coding gene (pfGR-M) of the glutathione reductase pfGR-M is shown as SEQ ID NO: 4.
[0009] An ergothioneine synthetase 1 cpEGT1-M, the amino acid sequence of which is shown as SEQ ID NO: 5.
[0010] In a preferred embodiment of the present application, the nucleotide sequence of the coding gene (cpEGT1-M) of the ergothioneine synthetase 1 cpEGT1-M is shown as SEQ ID NO: 6.
[0011] An ergothioneine synthetase 2 clEGT2-M, the amino acid sequence of which is shown as SEQ ID NO: 7.
[0012] In a preferred embodiment of the present application, the nucleotide sequence of the coding gene (clEGT2-M) of the ergothioneine synthetase 2 clEGT2-M is shown as SEQ ID NO: 8.
[0013] An expression vector for synthesizing the S-adenosylmethionine synthetase MetK-M, the expression vector being used for expressing the S-adenosylmethionine synthetase MetK-M.
[0014] An expression vector for synthesizing the glutathione reductase pfGR-M, the expression vector being used for expressing the glutathione reductase pfGR-M.
[0015] An expression vector for synthesizing the ergothioneine synthetase 1 cpEGT1-M, the expression vector being used for expressing the ergothioneine synthetase 1 cpEGT1-M.
[0016] An expression vector for synthesizing ergothioneine synthase 2clEGT2-M, the expression vector being used to express ergothioneine synthase 2clEGT2-M.
[0017] A recombinant strain containing all or part of the expression vector as described above, or having integrated polynucleotide sequences encoding the S-adenosylmethionine synthase ctMetK-M, glutathione reductase pfGR-M, ergothioneine synthase 1 cpEGT1-M, and ergothioneine synthase 2 clEGT2-M into its genome.
[0018] In a preferred embodiment of the present invention, the recombinant strain contains the polynucleotide sequences of S-adenosylmethionine synthase ctMetK-M, glutathione reductase pfGR-M, ergothioneine synthase 1 cpEGT1-M, and / or ergothioneine synthase 2clEGT2-M.
[0019] In a preferred embodiment of the present invention, the recombinant strain is prepared by the following method: In the first step, strain E1 was prepared by transforming E. coli BL21(DE3) into expression vectors of ergothioneine synthase 1 cpEGT1-M and ergothioneine synthase 2clEGT2-M.
[0020] The second step involves converting strain E1 into expression vectors for S-adenosylmethionine synthase ctMetK-M and glutathione reductase pfGR-M to prepare strain E3.
[0021] In a preferred embodiment of the present invention, recombinant strain E3 is fermented to prepare ergothioneine according to the following steps: The first step involved fermenting the recombinant strain in a fermentation medium for 4 hours at a temperature of 37 °C and a dissolved oxygen level of 20%.
[0022] The second step involves continuing fermentation for 4 hours with the addition of feed culture medium, at a fermentation temperature of 30 °C and a dissolved oxygen level of 20%.
[0023] The third step involved adding IPTG and continuing fermentation for 62 hours with the addition of feed medium and amino acid supplementation. The fermentation temperature was 30 °C and the dissolved oxygen level was 20%.
[0024] The amino acid replenishment solution for the fermentation was ultimately supplemented with histidine 11 g / L, cysteine 9 g / L, and methionine 20 g / L.
[0025] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.
Claims
1. An S-adenosylmethionine synthetase ctMetK-M, characterized in that, The amino acid sequence of the S-adenosylmethionine synthetase ctMetK-M is shown as SEQ ID NO:
1.
2. The S-adenosylmethionine synthetase MetK-M according to claim 1, characterized in that, The gene encoding the S-adenosylmethionine synthase ctMetK-M ( ctMetK-M The nucleotide sequence is shown in SEQ ID NO:
2.
3. A glutathione reductase pfGR-M, characterized in that, The amino acid sequence of the glutathione reductase pfGR-M is shown as SEQ ID NO:
3.
4. The glutathione reductase pfGR-M according to claim 3, wherein, The nucleotide sequence of the encoding gene (pfGR-M) of the glutathione reductase pfGR-M is shown as SEQ ID NO:
4.
5. A ergothioneine synthase 1 cpEGT1-M, characterized in that, The amino acid sequence of the ergothioneine synthetase 1 cpEGT1-M is shown as SEQ ID NO:
5.
6. Ergothioneine synthase 1 cpEGT1-M according to claim 5, characterized in that, The nucleotide sequence of the encoding gene (cpEGT1-M) of the ergothioneine synthetase 1 cpEGT1-M is shown as SEQ ID NO:
6.
7. An ergothioneine synthase 2 clEGT2-M, characterized in that, The amino acid sequence of the ergothioneine synthetase 2 clEGT2-M is shown as SEQ ID NO:
7.
8. The ergothioneine synthase 2 clEGT2-M of claim 7, wherein, The nucleotide sequence of the encoding gene (clEGT2-M) of the ergothioneine synthetase 2 clEGT2-M is shown as SEQ ID NO:
8.
9. The expression vector of the S-adenosylmethionine synthetase MetK-M of claim 1, which is used for expressing the S-adenosylmethionine synthetase MetK-M.
10. The expression vector of the glutathione reductase pfGR-M of claim 3, which is used for expressing the glutathione reductase pfGR-M.
11. The expression vector of the ergothioneine synthetase 1 cpEGT1-M of claim 5, which is used for expressing the ergothioneine synthetase 1 cpEGT1-M.
12. The expression vector of the ergothioneine synthetase 2 clEGT2-M of claim 7, which is used for expressing the ergothioneine synthetase 2 clEGT2-M.
13. A recombinant strain, which contains the expression vector of claims 9-12, or has integrated into its genome a polynucleotide sequence encoding the S-adenosylmethionine synthetase ctMetK-M of claim 1, the glutathione reductase pfGR-M of claim 3, the ergothioneine synthetase 1 cpEGT1-M of claim 5, or the ergothioneine synthetase 2 clEGT2-M of claim 7.
14. The recombinant bacterial strain of claim 13, wherein The recombinant strain is Escherichia coli; preferably, BL21(DE3).
15. A method of biosynthetic production of ergothioneine, characterized in that, The recombinant strain of claim 13 is used for fermentation to prepare ergothioneine according to the following steps: 1) The recombinant strain is fermented in a fermentation medium for 4 h at a temperature of 37 °C and with a dissolved oxygen of 20%. 16.2) The fermentation is continued for 4 h at a temperature of 30 °C and with a dissolved oxygen of 20% with dropwise addition of a feed medium. 17.3) After addition of IPTG, the fermentation is continued for 62 h at a temperature of 30 °C and with a dissolved oxygen of 20% with dropwise addition of a feed medium and an amino acid supplement.
18. The amino acid supplement of the fermentation is finally supplemented with histidine 11 g / L, cysteine 9 g / L, and methionine 20 g / L.
Citation Information
Patent Citations
Microorganism for efficiently producing ergothioneine as well as preparation method and application thereof
CN120249341A