Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli

Abstract

Ergothioneine (ERG), a unique thiol compound, is suggested to function as an antioxidant and cytoprotectant. Despite several recent attempts to produce ERG using various organisms, its yield was still very low and the costs remained high. Since the level of ERG produced depends strictly on the availability of three distinct precursor amino acids (l-cysteine (Cys), l-histidine, and l-methionine (Met)), metabolic engineering for enhancement of the flux toward ERG biosynthesis is required. Herein, we took advantage of a high-Cys production system using Escherichia coli cells, in which Cys biosynthesis and excretion were activated, and applied it to the fermentative production of ERG from glucose. The Cys overproduction in E. coli cells carrying the egtBCDE genes from Mycobacterium smegmatis was effective for ERG production. Furthermore, coexpression of the egtA gene, which encodes γ-glutamylcysteine synthetase that synthesizes the γ-glutamylcysteine used as a sulfur source of ERG biosynthesis, enhanced ERG production even though E. coli intrinsically has γ-glutamylcysteine synthetase. Additionally, disruption of the metJ gene that encodes the transcriptional repressor involved in Met metabolism was effective in further increasing the production of ERG. Finally, we succeeded in the high-level production of 1.31 g/L ERG in a fed-batch culture process using a jar fermenter.

Alterations of the gut ecological and functional microenvironment in different stages of multiple sclerosis

“…In the laboratory, the fecal samples were frozen and stored at −80 °C until use. Sulfur metabolomic analysis was performed by Sulfur Index service in Japan with liquid chromatography coupled to a tandem mass spectrometry (LC-MS/MS) system as described previously ( 54 , 55 ). Briefly, the sulfur-containing compounds in the samples were extracted by adding methanol and converted to fluorescent derivatives with monobromobimane.…”

The biology of ergothioneine, an antioxidant nutraceutical

“…In a follow-up study, the authors expressed egtA from M. smegmatis and it had a positive effect on ERG production. Furthermore, they enhanced cysteine and S-adenosine methionine biosynthesis and obtained 1•3 g/l or ERG in a fed-batch fermentation (116) , achieving currently the highest titre reported for heterologous ERG production. Recently, we reported the engineering of baker's yeast Saccharomyces cerevisiae for the production of ERG (117) .…”

Engineering the yeastSaccharomyces cerevisiaefor the production of L-(+)-ergothioneine

“…Recently, another group managed to produce 1.3 g/L ergothioneine using E. coli as a production organism (Tanaka et al, 2019). They used a cysteine hyperproducing strain, which was also engineered for increased methionine production.…”

“…Production of ergothioneine in microbial cell factories would provide a sustainable low-cost alternative to its current manufacturing processes. So far, fermentative ERG production has been reported in bacteria and filamentous fungi, including Methylobacterium aquaticum strain 22A (Alamgir et al, 2015), Aureobasidium pullulans (Fujitani et al, 2018), Rhodotorula mucilaginosa (Fujitani et al, 2018) , cyanobacteria (Pfeiffer et al, 2011), Aspergillus oryzae (Takusagawa et al, 2019) and Escherichia coli (Osawa et al, 2018; Tanaka et al, 2019). To the best of our knowledge, there are no reports on ergothioneine production in baker’s yeast, which is the preferred host for the production of nutraceuticals (Huang et al, 2008; Li and Borodina, 2014; Yuan and Alper, 2019).…”