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CN-121991915-A - Mutant for synthesizing ergothioneine and application thereof

CN121991915ACN 121991915 ACN121991915 ACN 121991915ACN-121991915-A

Abstract

The invention discloses a mutant for synthesizing ergothioneine and application thereof, and relates to the technical fields of genetic engineering and synthetic biology, wherein the mutant comprises mutant ergothioneine synthetase and mutant cysteine sulfoxide lyase, the amino acid sequence of the mutant ergothioneine synthetase is selected from any one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, and the amino acid sequence of the mutant ergothioneine synthetase is selected from any one of SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11. The invention obtains the mutant ergothioneine synthase and the mutant cysteine sulfoxide lyase with high activity, and uses the two mutants obtained by screening for producing ergothioneine, thereby having the advantages of high acid production, short fermentation time and the like, greatly saving material cost and being beneficial to industrial production.

Inventors

  • WU SHENGLIANG
  • LIANG YANMING
  • WU ZIJIAN
  • LIU ZIHAO
  • Zan Fengjun

Assignees

  • 绵阳晟氏健康科技有限公司

Dates

Publication Date
20260508
Application Date
20260123

Claims (6)

  1. 1. A mutant for synthesizing ergothioneine is characterized by comprising mutant ergothioneine synthetase and mutant cysteine sulfoxide lyase, wherein the amino acid sequence of the mutant ergothioneine synthetase is selected from any one of SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, and the amino acid sequence of the mutant ergothioneine synthetase is selected from any one of SEQ ID No. 9, SEQ ID No. 10 and SEQ ID No. 11.
  2. 2.A gene encoding the mutant ergothioneine synthase according to claim 1.
  3. 3. A dual expression vector comprising a gene encoding the mutant ergothioneine synthase of claim 1 and a gene encoding the mutant cysteine sulfoxide lyase.
  4. 4. An engineering bacterium is characterized by being obtained by taking E.coli W3110 as an initial strain, knocking out tnaA and yciW genes, adding a sequence shown in SEQ ID No. 12 to enhance expression of metK genes to obtain a chassis strain, and transferring the double expression vector of claim 3 into the chassis strain to obtain the engineering bacterium.
  5. 5. Use of a mutant according to claim 1 for the synthesis of ergothioneine.
  6. 6. A method of synthesizing ergothioneine, comprising the steps of: Inoculating the seed solution containing the engineering bacteria of claim 4 into a fermentation tank, and performing initial culture under the conditions that the pH is regulated to 6.8-7.0, the temperature is 37 ℃, the rotating speed is 100 rpm/min, the ventilation amount is 8m <3 >/h, the dissolved oxygen is controlled to be 20% -40%, after the dissolved oxygen rebounds, feeding the first material, the feeding acceleration is 30 ml/(L.h), when the OD 600 is 25-30 ℃, adding the inducer at one time, slowly reducing the temperature to 30 ℃, feeding the second material, feeding the first material, 40 ml/(L.h), culturing for 80h, and then placing the material into the tank; The formula of the feed is 800g/L glucose, 20g/L magnesium sulfate heptahydrate and 3g/L defoamer; The second formula of the feed comprises 20g/L of L-histidine, 40g/L of L-methionine, 15.5g/L of L-cysteine hydrochloride and 0.3g/L of pyridoxal phosphate; The inducer comprises 25g/L IPTG and 300 g/L-arabinose.

Description

Mutant for synthesizing ergothioneine and application thereof Technical Field The invention relates to the technical fields of genetic engineering and synthetic biology, in particular to a mutant for synthesizing ergothioneine and application thereof. Background Ergothioneine (Ergothioneine, ERG) is a natural amino acid with a very unique thiazoline ring structure, and is known as "antioxidant king", "longevity vitamins" and "cell guard". In recent years, it has received unprecedented attention in the fields of skin care products, health care products and medical research. The excellent efficacy of ergothioneine stems from its unique mechanism of action. The most core capability of the antioxidant is strong antioxidant capability, can efficiently neutralize various reactive oxygen Radicals (ROS) and reactive nitrogen Radicals (RNS), and can regenerate other antioxidants (such as vitamin C, vitamin E, glutathione and the like) to form a strong antioxidant network. Secondly, it can be specifically enriched in mitochondria, directly protect mitochondrial DNA and membrane structures from oxidative damage, ensure stable cell energy supply, thereby delaying cell aging, and inhibit activation of key inflammatory signal pathways such as nuclear factor kB (NF-kB), thereby reducing the production of pro-inflammatory cytokines (such as TNF-alpha, IL-6, IL-1 beta) and radically helping to alleviate chronic inflammation. Ergothioneine cannot be synthesized in human body by itself, and is mainly supplemented by some diets including mushrooms, meats, grains and the like, but natural-source ergothioneine is very limited and rare, and cannot meet the increasingly-expanded market demands. The main method for synthesizing ergothioneine at present is through chemical synthesis and biosynthesis, more chemical synthesis is in laboratory research and development stage, and can not be used for industrial production, the biosynthesis mainly depends on microbial fermentation method, and a method for producing ergothioneine and enzyme preparation simultaneously by using Trichoderma reesei is mainly used for fermentation extraction, for example, CN120699781A discloses a Cordyceps militaris strain with high yield of ergothioneine and application thereof, the highest yield of mutant Cordyceps militaris strain reaches 3.833mg/g after precursor substances are added, the fermentation time is longer and reaches 7 days, the yield is general, CN120555527A discloses a method for producing ergothioneine and enzyme preparation simultaneously by using Trichoderma reesei, and the highest shaking bottle yield reaches 250mg/L, and the yield is very low. Therefore, the strain after natural screening and mutation has low yield and cannot meet the industrial production standard. Therefore, related workers try to construct ergothioneine engineering bacteria by using model strains, such as CN112251392A discloses a genetic engineering strain for producing ergothioneine and application thereof, an ergothioneine operator egtBCDE and sulfoxide synthase mutant are integrated in an escherichia coli host MG1655, continuous fermentation is carried out in a 5L fermentation tank for 52h, the yield is up to 2.9g/L, CN117844722A discloses a genetic engineering strain and application thereof, genes such as EgtB, egtD, egtE and the like are constructed and expressed in vibrio natrii, the culture is carried out in a 6L fermentation tank for 60h, the yield is up to 4.55g/L, CN120099041A discloses a lipolysis yarrowia strain for synthesizing ergothioneine and application thereof, the lipolysis yarrowia strain is used as an original strain for transformation, semi-rational transformation is carried out on key enzyme TrEGT1, and continuous fermentation is carried out in a 5L fermentation tank for 168h, so as to achieve the maximum yield of 10.3g/L. In general, fermentation is performed by modifying chassis cells through prokaryotic or eukaryotic strains, so that on one hand, the fermentation yield is not high and cannot be expanded to industrial production, and on the other hand, the fermentation time is too long although the yield can break through 10g/L, so that a large amount of material resources and labor cost are consumed, and the industrial production is not facilitated. Disclosure of Invention In order to solve the defects in the prior art, the invention provides a mutant for synthesizing ergothioneine and application thereof, which improves the yield and shortens the fermentation time. In order to achieve the object of the invention, the following scheme is adopted: first, the following description is provided: The ergothioneine synthetase is derived from non-alfalfa variant fusarium (Verticillium nonalfalfae), the sequence shown in SEQ ID No. 1 is the original gene sequence of the ergothioneine synthetase, and the sequence shown in SEQ ID No. 2 is the original amino acid sequence of the ergothioneine synthetase. The cysteine sulfoxide lyase is derived from Altern