In vitro production of cysteine from glucose

  • Yohei Hanatani
  • Makoto Imura
  • Hironori Taniguchi
  • Kenji Okano
  • Yoshihiro Toya
  • Ryo Iwakiri
  • Kohsuke HondaEmail author
Biotechnological products and process engineering


Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.


Cysteine In vitro metabolic engineering Thermophilic enzyme Kinetic model 


Author contributions

MI, RI, and KH conceived and designed the experiments. YH, MI, HT, and KO carried out the experiments. YH, and YT performed kinetic modeling and optimization analysis. YH, MI, and KH analyzed data. YH, and KH wrote the manuscript.


This work was partly supported by the Japan Science and Technology Agency (JST), A-STEP Stage II program, and the Japan Society for the Promotion of Science (JSPS) KAKENHI grant (17K07720).

Compliance with ethical standards

Conflict of interest

MI, RI, and KH are inventors of pending patent applications related to this work.

Ethical statement

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

Supplementary material

253_2019_10061_MOESM1_ESM.pdf (741 kb)
ESM 1 (PDF 740 kb)


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Copyright information

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

Authors and Affiliations

  1. 1.Department of Biotechnology, Graduate School of EngineeringOsaka UniversitySuitaJapan
  2. 2.Bio Science Research Center, Mitsubishi Corporation Life Sciences Ltd.SaikiJapan
  3. 3.Department of Bioinformatic Engineering, Graduate School of Information Science and TechnologyOsaka UniversitySuitaJapan

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