Enhanced production of ergothioneine in Aspergillus oryzae

Abstract

Ergothioneine (EGT) is a rare amino acid with potent antioxidant and anti-inflammatory properties, with a wide range of applications in food, cosmetics, and medicine. In the present study, Aspergillus oryzae, a common edible fungus, was engineered as an optimal host for EGT production. Moreover, two endogenous genes involved in EGT biosynthesis were characterized. The homolog AoEgt1 was shown to be localized in the vacuoles, whereas the homolog AoEgt2 was found in the peroxisomes. Overexpression of EGT biosynthetic genes from different organisms enhanced EGT production, yielding 15.17 mg EGT/g of dry weight. Using glucose as the carbon source and supplementing methionine (Met) as a precursor further increased EGT production to 20.03 mg EGT/g of dry weight, constituting an eight-fold increase compared to the wild-type strain. This study discusses the successful construction of a high-yielding A. oryzae strain for EGT biosynthesis, providing a novel strategy for efficient EGT synthesis. KEY POINTS: • Two newly described homologs, AoEgt1 and AoEgt2, were identified in A. oryzae. • AoEgt1 and AoEgt2 were found to contribute to EGT biosynthesis. • EGT production was significantly increased by overexpression of Egt1 and Egt2. • Glucose and Met supplementation in the medium increased EGT production.

Keywords: Agrobacterium transformation; Aspergillus oryzae; Neurospora crassa; Ergothioneine; Filamentous fungus.

Fig. 1

Two different biosynthetic pathways of ergothioneine (EGT). The EGT biosynthesis in N. crassa requires only two main enzymes (Egt1 and Egt2) (Bello et al. ; Hu et al. 2014); however, in Mycobacterium smegmatis, a gene cluster EgtABCDE is responsible for the biosynthesis of EGT (Seebeck 2010). Met, methionine; SAM, S-adenosylmethionine; Glu, glutamic acid

Fig. 2

Phylogenetic tree analysis of Egt1 (a) and Egt2 (b). The two A. oryzae enzymes are marked by a black dot. The numbers on the right of the figures represent the results of sequence comparison results in % identity between A. oryzae and other fungi

Fig. 3

Subcellular localization of AoEgt1 and AoEgt2 in A. oryzae. a Fluorescence observation of transformants under 40-fold microscope. From top to bottom: pEX2B-Mcherry/AoΔhisBΔpyrG, AoEgt1/AoΔhisBΔpyrG, and AoEgt2/AoΔhisBΔpyrG. b From top to bottom: The mycelium of AoΔhisBΔpyrG transformed with pEX1H-GFP, AoVam3-GFP, respectively; transformants of AoVam3-GFP co-transformed with AoEgt1-Mcherry, GFP-PTS1/AoΔhisBΔpyrG, and GFP-PTS1 co-transformed with AoEgt2-Mcherry. Left to right: differential interference contrast (DIC); GFP fluorescence image; Mcherry fluorescence image; DIC, GFP, and Mcherry merged image

Fig. 4

EGT detection in overexpressed strains. a The EGT contents of the wild-type and overexpressed strains. b HPLC analysis of EGT contents of A. oryzae strains. WT, the wild-type strain; DW, dry weight. Statistical analyses were performed using one-way ANOVA; the asterisk “**” indicates significant difference compared with the control group (P < 0.01)

Fig. 5

The optimal carbon sources for EGT biosynthesis. a Comparison of different carbon sources for EGT biosynthesis. b HPLC analysis of EGT contents of A. oryzae with different carbon sources. DW, dry weight. Statistical analyses were performed using one-way ANOVA; the asterisk “**” indicates significant difference compared with the control group (P < 0.01)

Fig. 6

Effects of amino acid supplementation on EGT contents and fungal colony. a Effects of His, Cys, and Met on EGT contents. b Effects of different Met concentrations on EGT contents. c Effects of different Met concentrations on fungal colony. His, histidine; Cys, cysteine; Met, methionine; WT, the wild-type strain; DW, dry weight. Statistical analyses were performed using one-way ANOVA; the asterisk “**” indicates significant difference compared with the control group (P < 0.01)

Fig. 7

EGT contents in DPY medium supplemented with 0.4% Met (treatment) and based on DPY medium (CK). WT, the wild-type strain; DW, dry weight. Statistical analyses were performed using one-way ANOVA; the asterisk “***” indicates significant difference compared with the control group (P < 0.001)