CN-116396878-B - Lithospermum lipolyticum strain for synthesizing ergothioneine and application thereof

CN116396878BCN 116396878 BCN116396878 BCN 116396878BCN-116396878-B

Abstract

The invention discloses a yeast strain for synthesizing ergothioneine and application thereof, belonging to the technical fields of genetic engineering and biological engineering. The present invention achieves heterologous synthesis of ergothioneine by expressing Trichoderma parareesei-derived TrEGT and TrEGT2 in yarrowia lipolytica. By screening the different promoter drives TrEGT and TrEGT, the best combination of ergothioneine synthesis genes was obtained. The gene combination is further integrated in multiple copies, and the synthesis enhancement of precursor histidine improves the ability of understanding the synthesis of ergothioneine by the yarrowia lipolytica, so that the fermentation yield of the shake flask reaches 1200+/-12.3 mg/L and the fermentation yield on a 5L fermentation tank reaches 8.6g/L. The invention integrates the heterologous metabolic pathway into the yarrowia lipolytica to realize the high-efficiency production of the ergothioneine, thereby being beneficial to realizing the low-cost large-scale production of the ergothioneine and the product thereof.

Inventors

ZHOU JINGWEN
LIU MENGSU
CHEN JIAN
ZENG WEIZHU
HU LINFENG

Assignees

江南大学

Dates

Publication Date: 20260505
Application Date: 20230418

Claims (5)

1. A yeast engineering strain for synthesizing ergothioneine is characterized by taking a yeast Po1f for synthesizing ergothioneine as an original strain, expressing ergothioneine synthetic protein 1 TrEGT1 from Trichoderma parareesei and expressing cyclohexyl cysteine sulfoxide lyase TrEGT2 from Trichoderma parareesei, wherein the amino acid sequence of the ergothioneine synthetic protein 1 is shown as SEQ ID NO.1, the amino acid sequence of the cyclohexyl cysteine sulfoxide lyase is shown as SEQ ID NO.2, trEGT and TrEGT2 are integrated on a genome in multiple copies, and the promoters for regulating TrEGT and TrEGT comprise a strong promoter TEFin or a strong promoter Hp4d, the nucleotide sequence of the strong promoter TEFin is shown as SEQ ID NO.5, the nucleotide sequence of the strong promoter Hp4d is shown as SEQ ID NO.6, the yeast engineering strain for synthesizing ergothioneine is also integrated with a histidine synthase Gene YlHis, and the nucleotide sequence of the Gene YlHis is shown as GeneID 2905745.
2. A method for producing ergothioneine, characterized in that the engineering strain of yarrowia lipolytica according to claim 1 is cultured in a fermentation medium at 28-30 ℃ for a period of time.
3. The method of claim 2, wherein the fermentation process is further fed.
4. The method according to claim 3, wherein the feed is glucose fed so that the glucose concentration in the fermentation system is 0.5 to 1 g/L.
5. Use of the yeast engineering strain of yarrowia lipolytica of claim 1 or the method of any of claims 2-4 in the production of ergothioneine-containing products.

Description

Lithospermum lipolyticum strain for synthesizing ergothioneine and application thereof Technical Field The invention relates to a yeast strain of yarrowia lipolytica for synthesizing ergothioneine and application thereof, belonging to the technical fields of genetic engineering and biological engineering. Background Ergothioneine (ERG) is a thiol-containing histidine derivative that protects cells from oxidative damage caused by excess Reactive Oxygen Species (ROS). ERG is predominantly present in thioketone tautomers at physiological pH conditions, with a relatively high reduction potential (-60 mV), making it more stable and resistant to autoxidation than other thiol-containing antioxidants (e.g., glutathione). ERG is considered to have beneficial effects on human health, such as anti-inflammatory, anti-aging, antidepressant properties and the ability to prevent ultraviolet injury, as a powerful antioxidant and cytoprotective agent. Recently, ERG has been evaluated as an additive material for foods, cosmetics, etc., which will increase the market demand of ERG and the search for a production process of ERG. ERG is commonly found in higher organisms such as plants and mammals, but it is biosynthesized only by bacteria and fungi such as cyanobacteria, actinomycetes, basidiomycete mushrooms, and the like. For humans, the main dietary source of ERG is mushrooms. Studies have shown that eating mushrooms can reduce the risk of cognitive functional diseases and cancers. ERG is a compound that is presumed to cause this effect due to its antioxidant properties. In the transgenic caenorhabditis elegans model of alzheimer's disease with human beta-amyloid accumulation, ERG has been shown to improve health and longevity. Thus ERG is a prime candidate for new dietary supplements for health aging. At present, only chemically synthesized ERG is commercially available. However, toxic byproducts of the vitamin or nutraceutical chemical synthesis process can cause consumer safety problems and the stringent purification process exacerbates manufacturing costs. In contrast, the microbial fermentation method for synthesizing ergothioneine has the advantage of more green environmental protection. Ergothioneine is biosynthesized from the precursors histidine, cysteine and S-adenosylmethionine. In the fungal pathway, histidine is methylated three times by Egt1 to form histidine trimethyl inner salt, then Egt1 enzyme links cysteine to form cyclohexyl cysteine sulfoxide, then cyclohexyl cysteine sulfoxide lyase Egt2 dissociates ammonium pyruvate from the intermediate, and finally sulfur is reduced to produce ERG. The step of synthesis of ergothioneine in bacteria is relatively complex, the disadvantage of the bacterial route is that five enzymes are used instead of two, and ATP is used to form other pathway intermediates including glutamate, which must be cleaved off in subsequent steps. In addition, escherichia coli as a pathogenic microorganism cannot be used for production in the related fields such as foods and medicines. De novo synthesis of ergothioneine has been achieved by expressing the ergothioneine synthesis pathway in microbial hosts, but yields are low and difficult for industrial scale production. The yarrowia lipolytica is taken as a typical unconventional oleaginous yeast, is an internationally recognized safe and reliable microorganism production host, has complete genetic operation tools, also has a powerful reduction system, and is an ideal ergothioneine production host. Disclosure of Invention The invention provides a yeast engineering strain for synthesizing ergothioneine, which expresses ergothioneine synthesized protein 1TrEGT1 from Trichoderma parareesei in a starting strain and expresses cyclohexyl cysteine sulfoxide lyase TrEGT2 from Trichoderma parareesei. In one embodiment, the amino acid sequence of the ergothioneine synthetic protein 1 is shown as SEQ ID NO.1, and the amino acid sequence of the cyclohexylcysteine sulfoxide lyase is shown as SEQ ID NO. 2. In one embodiment, the starting strain is Saccharomyces lipolytica Po1f, disclosed in the paper "Combining 26srDNA and the Cre-loxP System for Iterative Gene Integration and Efficient Marker Curation in Yarrowia lipolytica". In one embodiment, the yarrowia lipolytica is a starting strain of yarrowia lipolytica Po1f, yarrowia lipolytica E129, yarrowia lipolytica E150, yarrowia lipolytica Po1d, yarrowia lipolytica Po1g or yarrowia lipolytica Po1 h. In one embodiment, the TrEGT and TrEGT2 are expressed episomally, or are expressed integrally on the genome, by plasmids. In one embodiment, the TrEGT and TrEGT genes are expressed using pYlxp as an expression vector. In one embodiment, the TrEGT and TrEGT2 are integrated on the genome. In one embodiment, the TrEGT and TrEGT2 multiple copies are integrated on the genome, with a copy number of 3 or greater. In one embodiment, the TrEGT and TrEGT2 are integrated at the 26S rDNA site of the genome. In one embodi