supplier. Let's talk about it today. Ergothioneine (EGT) is a natural antioxidant that cannot be synthesized by the human body itself. It has unique cellular physiological protective functions such as scavenging free radicals, detoxification, maintenance of DNA biosynthesis, normal cell growth, cellular immunity, anti-radiation, whitening and anti-aging. (EGT) it has a broad application prospect in food, cosmetics, functional food and biomedical industries. But at the same time, as a natural antioxidant, it is safe and non-toxic, which also makes it a hot spot in the current industry development.
include natural biological extraction, chemical synthesis and biosynthesis [3-4]. The chemical synthesis method is difficult to prepare and expensive, and the safety can not be guaranteed. Natural bioextraction method has low yield and high cost. It limits the application of ergothioneine. Its biosynthesis method has the advantages of low cost and easy availability of raw materials.
A variety of natural mycorrhizal fungi have been found to be capable of synthesizing ergothioneine. These include enoki mushrooms, shiitake mushrooms, shiitake mushrooms, reishi mushrooms, porcini mushrooms, Agaricus bisporus mushrooms, amygdalus mushrooms, and golden-topped sidearm mushrooms.
When the carbon source was 25 g/L fructose and 1 g/L aspartic acid, the yield reached 1.89 mg/g DW. When 2 mmol/L methionine was added to the medium, the ergothioneine production in the mycelium was 1.8 times higher after 15 d of fermentation. To study the process of ergothioneine production by fermentation of almond mushroom mycelium in 10 L tank scale, 4 mmol/L histidine, 1 mmol/L cysteine and 1 mmol/L methionine were added to the culture medium.
in the mycelium on the 18th-20th day of fermentation was about 64.2 mg/L. The optimal carbon and nitrogen sources for ergothioneine fermentation were glucose and tryptone, which were further optimized by the medium optimization. The ergothioneine was prepared by deep fermentation using golden-topped siderophore mycelium. At the same time, 8 mmol/L cysteine, 4 mmol/L histidine and 0.5 mmol/L methionine were added, and the content of ergothioneine in mycelium reached 97.69 mg/L at 16 d of fermentation.
On the basis of the effects of amino acids, precursors and exogenous nutritional factors on the synthesis and accumulation of ergothioneine, a flat mushroom strain with high ergothioneine production was screened. And a deep fermentation control strategy for high-intensity accumulation of ergothioneine was established. In addition, since most of the ergothioneine synthesized by microorganisms accumulates intracellularly, it can only be detected and utilized by moving it to extracellularly. In order to meet the requirements for production safety and product safety in several fields, a total aqueous phase leaching process for intracellular ergothioneine has also been established [5].
Based on the theoretical guidance of ergothioneine biosynthetic pathway and key enzyme analysis, the heterologous expression of ergothioneine biosynthetic enzymes was realized in some model microorganisms. This constructed a series of ergothioneine-engineered bacteria, and the fermentation was optimized to increase the yield of by 120-fold.
The gene for the key enzyme was cloned into E. coli and the engineered bacteria were applied to a batch replenishment culture in a fermenter. By optimizing the fermentation medium, the yield of ergothioneine reached 657 mg/L after 216 h of fermentation, indicating that the key enzyme and its related regulation could affect the efficient synthesis of ergothioneine.
In addition to the regulation of key enzymes, the modification and optimization of the synthesis pathway of precursor aminosine is also an effective way to improve the synthesis efficiency of ergothioneine. Two different sources of ergothioneine synthesis pathways were heterologously expressed in Escherichia coli, which could improve ergothioneine production. By this modification strategy, the accumulation of ergothioneine reached 710.53 mg/L for 108 h using a 3 L tank for replenishment batch fermentation.
Alternatively, a high cysteine-producing strain was obtained by activating the cysteine synthesis and secretion system in E. coli. The synthesis key enzyme gene was cloned into this strain and the gene affecting methionine synthesis was knocked out. Using the modified strain to ferment in a 3 L fermenter with supplements such as histidine, methionine, and Na2S2O3, the yield of reached 1.3 g/L for 216 h. This is the highest fermentation level of ergothioneine preparation by engineered fungal fermentation publicly reported.
The synthesis of fungal ergothioneine involves two key enzymes. This could greatly simplify the metabolic regulation of the synthetic pathway. In the future, it may become the main focus of heterologous synthesis of ergothioneine. The ergothioneine synthesis gene of Pulsatilla vulgaris was heterologously expressed in Aspergillus oryzae. Ergothioneine was prepared by solid-state fermentation using this engineered bacterium, and the yield of after fermentation optimization was 231 mg/kg. 20-fold increase in the level of ergothioneine synthesis was achieved over the previous fermentation.
The highest fermentation level of ergothioneine produced by natural mycobacteria was over 500 mg/L. The fermentation period was usually 10-20 d. The fermentation period of the engineered bacterium could be controlled to 3-9 d. The highest fermentation level was 1.3 g/L. However, the product needs to be purified and used.
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