Production of ergothioneine by Methylobacterium species

Abstract

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur-containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize EGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 μg/100 ml culture (1200 μg/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

Keywords: Methylobacterium species; antioxidant; ergothioneine; glutathione; methanol; methylotroph; reactive oxygen species.

Figure 1

Amino acid content in strain 22A cells grown on methanol (0.5%) for 7 days. EGT was quantified by an HPLC and other amino acids were quantified using an amino acid analyzer. Glutamine and asparagine could not be analyzed by the analyzer. The data are presented as the mean ± standard deviation (SD) (n = 3).

Figure 2

EGT content in selected Methylobacterium type strains. The strains were grown on 3% methanol in MM, and subjected to intracellular EGT quantification after 7 days of cultivation. The experiment was done in triplicate. The data are presented as the mean ± SD (n = 3). Bar, EGT content (μg/g wet weight cell), and circles, cell yield (mg wet weight).

Figure 3

EGT productivity of Methylobacterium sp. strain 22A. The strain was grown on 0.5% (circles), 1% (diamonds), 2% (squares), and 3% (triangles) methanol in a 100 ml MM for 38 days. The experiment was done in triplicate. The data are presented as the mean ± SD (n = 3).

Figure 4

EGT productivity of Methylobacterium sp. strain 22A grown on various carbon sources and media. The experiment was done in triplicate using 5 ml media. The data are presented as the mean ± SD (n = 3). Bar, EGT production (μg/5 ml culture), and circles, cell yield (mg wet weight).

Figure 5

(A) HPLC chromatograms of (a) standard EGT (100 μM), (b) extract of strain 22A wild-type cells, and (c) extract of Δegt cells. The arrow indicates the EGT peak detected at 6.2 min. The HPLC analysis condition is described in the text. (B) EGT productivity of Δegt, ΔegtComp, and WTegtDup cells. Two independent transformants of WTegtDup isolates were tested. The cells were grown on 0.5% methanol in MM medium for 1 week and subjected to EGT quantification. Bar, EGT production (μg/5 ml culture), and circles, cell yield (mg wet weight). The data are presented as the mean ± SD (n = 5). p-values were generated by analysis of variance (ANOVA) using the Dunnett's multiple comparison test with the control (the wild type) for EGT production.

Figure 6

Phenotypic characterization of Δegt mutant. (A) Heat shock assay at 46°C (closed symbols, wild type, and open symbols, Δegt); (B) UV (253 nm) resistance assay; (C) sunlight-resistance assay; (D–F), disk diffusion assay using hydrogen peroxide, methylglyoxal, and diamide, respectively. All experiments except (C) that was done in quintuplicate, were done in quadruplicate. The data are presented as the mean ± SD (n = 4). Experimental conditions are written in the text.

Figure 7

Growth of strain 22A-rif and Δegt inoculated onto A. thaliana seeds. After 27 days of inoculation, the shoots were sampled and subjected to CFU determination using selective media. The data are presented as mean ± SD (n = 5).