22Noda Institute for Scientific Research GRANT
2014 Research Grant
Outline of Research Result

Ergothioneine production by methylotrophic bacteria
Akio Tani
Institute of Plant Science and Resources, Okayama University

Research aims
Methylobacterium species are methylotrophic bacteria
belonging to Alphaproteobacteria, and have been studied indetail as a model for bacterial methylotrophy. Recent research studies have revealed that plants emit huge amountsof methanol and that these methylotrophic bacteria are oneof the major bacterial species inhabiting the plant surface.1)
Furthermore, inoculation of the methylotrophic bacteriamay promote plant growth.2) During our research on themechanism of plant growth promotion, we found throughmetabolome analysis that one of the species accumulated
ergothioneine (EGT; Fig. 1). In this study, we aimed to investigate the physiological role of EGT accumulation in thespecies and the feasibility of fermentative production ofEGT.
Methods
Methylobacterium aquaticum strain 22A was used as amodel in this study.3) The strain was isolated from the bryophyte Racomitrium japonicum, the complete genome ofwhich has been sequenced.4) A plasmid, pK18mobSacB,
was used for generating a gene deletion mutant of thisstrain. HPLC was used for EGT quantification. Aminoacids were analyzed with an amino acid analyzer.

accumulated in the cell compared with other amino acids(Fig. 2). We also found that most Methylobacterium species
could synthesize EGT, with strain 22A being one of thebest strains among them. The cultivation condition was optimized by changing the concentration of the nitrogensources, iron, lanthanoid, and amino acids. As a result, theproductivity reached up to 1 mg/100 mL culture medium(1.2 mg/g wet weight cells, 6.3 mg/g dry weight cells)
using 2% methanol as a carbon source in one month of cultivation. The productivity overwhelmed that of Pleurotus
osteatus (oyster mushroom).5)
EGT biosynthesis genes were first identified as a genecluster (egtABCDE) in Mycobacterium species.6) We foundthe homologous genes to be scattered among the genome ofstrain 22A, except for egtB and egtD that were clustered. A
deletion mutant of egtBD was generated, which completelyabolished the productivity of EGT. The mutant (∆egtBD)
showed more severe susceptibility to heat shock and UV irradiation and higher resistance to hydrogen peroxide than
the wild type. We also found that EGT could be degraded
by UV irradiation, and the intracellular EGT content decreased upon UV irradiation of the mutant cells. In addition, the mutant was more susceptible to sunlight irradiation. However, the mutant could grow on methanol, suggesting that EGT is not involved in methylotrophy.
Conclusion

Results
In the metabolome analysis of strain 22A cells grown on
methanol, EGT was found as a most prominent peak bycapillary electrophoresis-mass spectrometry. A quantification method based on HPLC analysis was established.
Methanol was found to be the best substrate for EGT production among other carbon sources, such as ethanol, succinate, and glucose. EGT was the most abundant amino acid

Fig. 1.

Ergothioneine

Taken together, we showed that it is possible to produce
EGT from Methylobacterium species utilizing methanol asa carbon source. High-cell density cultivation using theFig. 2.

Intracellular amino acids in strain 22A.

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genus is possible, with a yield of 100 g dry weight cells/
liter.7) We are still optimizing the production by examining
the high-cell density cultivation, screening microorganisms
with higher productivity, optimizing the cultivation conditions, and promoting the gene expression. In this study, we
found that EGT accumulates in high amounts when thestrain is grown on methanol, but EGT is not involved inmethylotrophy. Since the mutant showed higher susceptibility to sunlight, EGT would appear to be an importantamino acid for the genus to inhabit the plant surface.
References
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