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Efficient fermentative production of l-theanine by Corynebacterium glutamicum

  • Hongkun Ma
  • Xiaoguang Fan
  • Ningyun Cai
  • Dezhi Zhang
  • Guihong Zhao
  • Ting Wang
  • Rui Su
  • Meng Yuan
  • Qian Ma
  • Chenglin Zhang
  • Qingyang Xu
  • Xixian Xie
  • Ning Chen
  • Yanjun LiEmail author
Biotechnological products and process engineering
  • 79 Downloads

Abstract

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.

Keywords

l-Theanine l-Glutamate Ethylamine ATP Fermentation γ-Glutamylmethylamide synthetase Corynebacterium glutamicum 

Notes

Funding information

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.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_10255_MOESM1_ESM.pdf (1.5 mb)
ESM1 (PDF 1495 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Hongkun Ma
    • 1
  • Xiaoguang Fan
    • 1
    • 2
    • 3
  • Ningyun Cai
    • 1
  • Dezhi Zhang
    • 1
  • Guihong Zhao
    • 1
  • Ting Wang
    • 1
  • Rui Su
    • 1
  • Meng Yuan
    • 1
  • Qian Ma
    • 1
    • 2
    • 3
  • Chenglin Zhang
    • 1
    • 2
    • 3
  • Qingyang Xu
    • 1
    • 2
    • 3
  • Xixian Xie
    • 1
    • 2
    • 3
  • Ning Chen
    • 1
    • 2
    • 3
  • Yanjun Li
    • 1
    • 2
    • 3
    Email author
  1. 1.College of BiotechnologyTianjin University of Science and TechnologyTianjinChina
  2. 2.Key Laboratory of Industrial Fermentation Microbiology, Ministry of EducationTianjin University of Science and TechnologyTianjinChina
  3. 3.National and Local United Engineering Lab of Metabolic Control Fermentation TechnologyTianjin University of Science and TechnologyTianjinChina

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