KMID : 0545120200300050785
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Journal of Microbiology and Biotechnology 2020 Volume.30 No. 5 p.785 ~ p.792
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Production of L-Theanine Using Escherichia coli Whole-Cell Overexpressing ¥ã-Glutamylmethylamide Synthetase with Baker¡¯s Yeast
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Yang Soo-Yeon
Han Yeong-Hoon Park Ye-Lim Park Jun-Young No So-Young Jeong Da-Ham Park Sae-Rom Park Hyung-Yeon Kim Woo-Seong Seo Seung-Oh Yang Yung-Hun
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Abstract
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L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from Methylovorus mays No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::gmas was selected for strains. The optimal temperature, pH, and metal ion concentration were 50oC, 7, and 5 mM MnCl2, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Baker¡¯s yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.
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KEYWORD
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5L-theanine, whole-cell biocatalyst, baker&rsquo, s yeast, ATP regeneration
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