Production of L-Theanine Using Escherichia coli Whole-Cell Overexpressing γ-Glutamylmethylamide Synthetase with Baker’s Yeast

Soo Yeon Yang, Yeong Hoon Han, Ye Lim Park, Jun Young Park, So Young No, Daham Jeong, Saerom Park, Hyung Yeon Park, Wooseong Kim, Seung Oh Seo, Yung Hun Yang

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)785-792
Number of pages8
JournalJournal of Microbiology and Biotechnology
Volume30
Issue number5
DOIs
StatePublished - 28 May 2020

Bibliographical note

Funding Information:
This work was supported by Research Program to solve social issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234) and National Research Foundation of Korea (NRF) (NRF-2017R1D1A1B03033594, NRF-2019M3E6A1103979 and NRF-2019R1F1A1058805). In addition, this work was also supported by the polar academic program (PAP, PE18900). This paper was also supported by Konkuk University Researcher Fund in 2019. Consulting service from the Microbial Carbohydrate Resource Bank (MCRB, Korea) was kindly appreciated.

Publisher Copyright:
Copyright© 2020 by The Korean Society for Microbiology and Biotechnology

Keywords

  • ATP regeneration
  • Baker’s yeast
  • L-theanine
  • Whole-cell biocatalyst

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