Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway

Abstract

Background: Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy.

Results: Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions.

Conclusions: Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.

Keywords: Biosynthesis; Ergothioneine; Escherichia coli; Heterologous expression; Neurospora crassa; Trichoderma reesei.

Fig. 1

HPLC analysis of ERG content of T. reesei conidia and mycelia. “ERG” indicates the ERG standards (50 mg/L)

Fig. 2

The biosynthetic pathway of ergothioneine (ERG) and the domain prediction of Tregt1 and Tregt2. A Conserved domain predictions of Tregt1 (NCBI Reference Sequence: XP_006968620) and Tregt2 (NCBI Reference Sequence: XP_006968735) from T. reesei. B The biosynthetic pathway of ERG in Neurospora crassa (solid arrow) and Mycobacterium smegmatis (dashed arrow). The ERG biosynthesis in N. crassa requires only two main enzymes (Egt1 and Egt2) while in M. smegmatis, a gene cluster egtABCDE is responsible for ERG biosynthesis. Egt1 is a bifunctional enzyme that catalyzes the first two steps: the addition of three methyl groups in l-histidine to form hercynine, and the formation of C–S bond between l-cysteine (l-Cys) and hercynine to form hercynylcysteine sulfoxide. M. smegmatis sulfoxide synthase utilizes γ-glutamylcysteine (γGC) but not cysteine as sulfur donor. l-Met, l-methionine; SAM, S-adenosylmethionine; l-Cys, l-cysteine; l-Glu, l-glutamic acid

Fig. 3

Plasmid profiles, protein expression and ERG biosynthesis. A Schematic drawing of plasmids expressing tregt1 and/or tregt2 used for transforming E. coil BW25113. B The production of ERG by 48-h whole cell catalysis using the recombinant strains BW-tregt1, BW-tregt2 and BW-tregt1-tregt2. Data in the figure are mean values (n = 3 biological replicates). C Detection of Tregt1 and/or Tregt2 expression in recombinant E. coli by SDS-PAGE. M. Protein marker; 1. BW-pBAD (control); 2. BW-tregt1; 3. BW-tregt2; 4. BW-tregt1-tregt2

Fig. 4

ERG production of the recombinant strains in a 2-L jar fermenter. The production of ERG using the recombinant strains BW-tregt1-tregt2 A and BW-ncegt1-ncegt2 B during high-cell-density fermentation. Data in the figure are mean values (n = 3 biological replicates)