Ergothioneine biosynthesis: The present state and future prospect

Abstract

Ergothioneine (ERG), a rare natural thio-histidine derivative with potent antioxidant properties and diverse biological functions, is widely utilized in food processing, cosmetics, pharmaceuticals, and nutritional supplements. Current bioproduction methods for ERG primarily depend on fermenting edible mushrooms. However, with the advancement in synthetic biology, an increasing number of genetically engineered microbial hosts have been developed for ERG production, including Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Given the involvement of multiple precursor substances in ERG synthesis, it is crucial to employ diverse strategies to regulate the metabolic flux of ERG synthesis. This review comprehensively evaluates the physiological effects and safety considerations associated with ERG, along with the recent advancements in catalytic metabolic pathway for ERG production using synthetic biology tools. Finally, the review discusses the challenges in achieving efficient ERG production and the strategies to address these challenges using synthetic biology tools. This review provides a literature analysis and strategies guidance for the further application of novel synthetic biology tools and strategies to improve ERG yield.

Keywords: Chassis microbes; Ergothioneine; Fermentation; Metabolic engineering; Recombinant microorganism; Synthetic biology.

Fig. 1

Application prospect of ergothioneine in different industries.

Fig. 2

Two tautomeric forms of ERG.

Fig. 3

Antioxidant properties of ERG.

Fig. 4

Two primary modalities of in vitro biocatalysis and transformation. In a pure enzyme catalytic system, it is necessary to add specific substrates (such as l-cysteine, l-histidine, and l-methionine), cofactors (such as ATP and pyridoxal-5′-phosphate (PLP)), and precise proportions of enzyme compositions. In a whole-cell catalytic system, only the addition of a specific substrate and the expression of the enzyme-catalyzed system within whole cells are required.

Fig. 5

Exploration, modification, screening, and analysis of key enzymes in the synthetic pathway. Using various databases to screen and align key enzymes in the ERG biosynthetic pathway; Utilizing computer simulations to predict possible beneficial mutation sites, and employing kits to introduce random mutations; Relying on HPLC detection methods or HTS to identify beneficial mutants; Performing functional analysis of molecular dynamics and kinetic parameters on the identified beneficial mutants.

Fig. 6

There are three main ERG biosynthetic pathways. (A) Anaerobic biosynthetic pathway, it occurs mainly in Chlorobium limicola, catalyzed by EanA and EanB. (B) Eukaryotic biosynthesis pathway, it occurs mainly in Neurospora crassa, catalyzed by Egt1 and Egt2. (C) Prokaryotic biosynthesis pathway, it occurs mainly in Mycobacterium smegmatis, catalyzed by five enzymatic steps, involving EgtA, EgtB, EgtC, EgtD, and EgtE.

Fig. 7

Applications of intracellular biosensors. (A) Transcription factor-based biosensors: Transcription factors can recognize and bind to specific molecules. When combined, they activate or inhibit the expression of downstream genes. Through changes in reporter gene expression, fluorescent signals are detected and output, allowing for the measurement of the target metabolite concentration. (B) Ribosome switch-based biosensors: Ribosomal switches typically consist of specific RNA domains that bind to target molecules. Upon binding, the RNA structure changes, affecting the translation efficiency of the ribosome on the mRNA. Detection of target molecules is achieved through changes in the yield of downstream products. (C) Protein-based biosensors: Sensor proteins specifically recognize and bind to target molecules. Upon binding, the conformation or function of the protein changes. This change can be converted into a detectable signal through various methods (e.g., fluorescence change, enzyme activity change), enabling the detection of the target molecule concentration.

Fig. 8

Strategies for Ergothioneine Synthesis. Modification of key enzymes in the ERG synthesis pathway. Enhance the supply of precursors for ERG synthesis (blue and red genes) and block competing metabolic pathways (green genes) to balance cell growth and promote ERG accumulation using CRISPR-derived genome editing technology. Construct a biosensor to regulate the synthesis of cofactors and coordinate cell growth required for ERG synthesis. Strengthen the precursor intake and ERG secretion pathways to promote ERG accumulation and efflux.