Efficient fermentative production of l-theanine by Corynebacterium glutamicum
- 79 Downloads
l-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. l-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in l-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of l-theanine in Corynebacterium glutamicum, an industrially safe host. For l-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of l-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an l-glutamate overproducing strain, to produce l-theanine upon ethylamine addition to the hosts. Second, the l-glutamate exporter in host cells was disrupted, which markedly increased the l-theanine titer in GDK-9 cells and almost eliminated the accumulation of l-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated l-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived l-serine/l-alanine decarboxylases; however, these enzymes showed no l-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L l-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of l-theanine achieved using ethylamine supplementation.
Keywordsl-Theanine l-Glutamate Ethylamine ATP Fermentation γ-Glutamylmethylamide synthetase Corynebacterium glutamicum
This work was financially supported by the National Key Research and Development Program of China (2018YFA0900304), National Natural Science Foundation of China (31700037, 31500026), and Postdoctoral Research Foundation of China (2016M601269).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Conway J (2019) Value of the global tea market from 2017 to 2024 (in billion U.S. dollars). https://www.statista.com/statistics/326384/global-tea-beverage-market-size. Accessed 9 July 2019
- Li YJ, Sun LC, Feng J, Wu RF, Xu QY, Zhang CL, Chen N, Xie XX (2016) Efficient production of α-ketoglutarate in the gdh deleted Corynebacterium glutamicum by novel double-phase pH and biotin control strategy. Bioprocess Biosyst Eng 39(6):967–976. https://doi.org/10.1007/s00449-016-1576-y CrossRefPubMedGoogle Scholar
- Liu ZW, Wu ZJ, Li H, Wang YX, Zhuang J (2017) L-theanine content and related gene expression: novel insights into theanine biosynthesis and hydrolysis among different tea plant (Camellia sinensis L.) tissues and cultivars. Front Plant Sci 8:498. https://doi.org/10.3389/fpls.2017.00498
- Tachiki T, Yamada T, Mizuno K, Ueda M, Shiode J, Fukami H (1998) γ-Glutamyl transfer reactions by glutaminase from Pseudomonas nitroreducens IFO12694 and their application for the syntheses of theanine and γ-glutamylmethylamide. Biosci Biotechnol Biochem 62:1279–1283. https://doi.org/10.1271/bbb.62.1279 CrossRefPubMedGoogle Scholar
- Tochikura T, Tachiki T (1983) Microbial production of theanine-related compounds. JP Patent 58040094Google Scholar
- Wang Q, Min C, Zhu FF, Xin YQ, Zhang SQ, Luo L, Yin ZM (2011) Production of bioactive γ-glutamyl transpeptidase in Escherichia coli using SUMO fusion partner and application of the recombinant enzyme to L-theanine synthesis. Curr Microbiol 62:1535–1541. https://doi.org/10.1007/s00284-011-9891-7 CrossRefPubMedGoogle Scholar
- Wang Y, Cao GQ, Xu DY, Fan LW, Wu XY, Ni XM, Zhao SX, Zheng P, Sun JB, Ma Y (2018) A novel L-glutamate exporter of Corynebacterium glutamicum. Appl Environ Microb 84(6). https://doi.org/10.1128/AEM.02691-17
- Yamamoto S, Uchimura K, Wakayama M, Tachiki T (2004) Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30: an enzyme usable for production of theanine by coupling with the alcoholic fermentation system of baker’s yeast. Biosci Biotechnol Biochem 68(9):1888–1897. https://doi.org/10.1271/bbb.68.1888 CrossRefPubMedGoogle Scholar
- Yamamoto S, Wakayama M, Tachiki T (2008) Cloning and expression of Methylovorus mays No. 9 gene encoding γ-glutamylmethylamide synthetase: an enzyme usable in theanine formation by coupling with the alcoholic fermentation system of baker’s yeast. Biosci Biotechnol Biochem 72(1):101–109. https://doi.org/10.1271/bbb.70462 CrossRefPubMedGoogle Scholar
- Zhang CL, Li YJ, Ma J, Liu Y, He JL, Li YZ, Zhu FZ, Meng J, Zhan JJ, Li ZX, Zhao L, Ma Q, Fan XG, Xu QY, Xie XX, Chen N (2018) High production of 4-hydroxyisoleucine in Corynebacterium glutamicum by multistep metabolic engineering. Metab Eng 49:287–298. https://doi.org/10.1016/j.ymben.2018.09.008 CrossRefPubMedGoogle Scholar