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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
  • 59 Downloads

Abstract

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.

Keywords

Cysteine In vitro metabolic engineering Thermophilic enzyme Kinetic model 

Notes

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.

Funding

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)

References

  1. Atomi H (2005) Recent progress towards the application of hyperthermophiles and their enzymes. Curr Opin Chem Biol 9:166–173CrossRefGoogle Scholar
  2. Awano N, Wada M, Mori H, Nakamori S, Takagi H (2005) Identification and functional analysis of Escherichia coli cysteine desulfhydrases. Appl Environ Microbiol 71:4149–4152CrossRefGoogle Scholar
  3. Dassler T, Maier T, Winterhalter C, Bock A (2000) Identification of a major facilitator protein from Escherichia coli involved in efflux of metabolites of the cysteine pathway. Mol Microbiol 36:1101–1112CrossRefGoogle Scholar
  4. Denk D, Bock A (1987) L-Cysteine biosynthesis in Escherichia coli: nucleotide sequence and expression of the serine acetyltransferase (cysE) gene from the wild-type and a cysteine-excreting mutant. J Gen Microbiol 133:515–525Google Scholar
  5. Ding YR, Ronimus RS, Morgan HW (2001) Thermotoga maritima phosphofructokinases: expression and characterization of two unique enzymes. J Bacteriol 183:791–794CrossRefGoogle Scholar
  6. Franke I, Resch A, Dassler T, Maier T, Bock A (2003) YfiK from Escherichia coli promotes export of O-acetylserine and cysteine. J Bacteriol 185:1161–1166CrossRefGoogle Scholar
  7. Harris CL (1981) Cystine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme. J Bacteriol 145:1031–1035Google Scholar
  8. Hold C, Billerbeck S, Panke S (2016) Forward design of a complex enzyme cascade reaction. Nat Commun 7:12971CrossRefGoogle Scholar
  9. Honda K, Hara N, Cheng M, Nakamura A, Mandai K, Okano K, Ohtake H (2016) In vitro metabolic engineering for the salvage synthesis of NAD. Metab Eng 35:114–120CrossRefGoogle Scholar
  10. Honda K, Kimura K, Ninh PH, Taniguchi H, Okano K, Ohtake H (2017) In vitro bioconversion of chitin to pyruvate with thermophilic enzymes. J Biosci Bioeng 124:296–301CrossRefGoogle Scholar
  11. Hunt S (1985) Degradation of amino acids accompanying in vitro protein hydrolysis. In: Barrett GC (ed) Chemistry and biochemistry of the amino acids. Springer, pp 376–398.  https://doi.org/10.1007/978-94-009-4832-7_12
  12. Jagura-Burdzy G, Kredich NM (1983) Cloning and physical mapping of the cysB region of Salmonella typhimurium. J Bacteriol 155:578–585Google Scholar
  13. Kredich NM (1992) The molecular basis for positive regulation of cys promoters in Salmonella typhimurium and Escherichia coli. Mol Microbiol 6:2747–2753CrossRefGoogle Scholar
  14. Kredich NM, Tomkins GM (1966) The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem 241:4955–4965Google Scholar
  15. Liang ZX, Lee T, Resing KA, Ahn NG, Klinman JP (2004) Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase. Proc Natl Acad Sci U S A 101:9556–9561CrossRefGoogle Scholar
  16. Liao H, Myung S, Zhang YH (2012) One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP. Appl Microbiol Biotechnol 93:1109–1117CrossRefGoogle Scholar
  17. Liu H, Fang G, Wu H, Li Z, Ye Q (2018) L-Cysteine production in Escherichia coli based on rational metabolic engineering and modular strategy. Biotechnol J 13:1700695CrossRefGoogle Scholar
  18. Marsh JJ, Lebherz HG (1992) Fructose-bisphosphate aldolases: an evolutionary history. Trends Biochem Sci 17:110–113CrossRefGoogle Scholar
  19. Mino K, Ishikawa K (2003) A novel o-phospho-L-serine sulfhydrylation reaction catalyzed by o-acetylserine sulfhydrylase from Aeropyrum pernix K1. FEBS Lett 551:133–138CrossRefGoogle Scholar
  20. Ninh PH, Honda K, Sakai T, Okano K, Ohtake H (2015) Assembly and multiple gene expression of thermophilic enzymes in Escherichia coli for in vitro metabolic engineering. Biotechnol Bioeng 112:189–196CrossRefGoogle Scholar
  21. Owusu RK, Cowan DA (1989) Correlation between microbial protein thermostability and resistance to denaturation in aqueous:organic solvent two-phase systems. Enzym Microb Technol 11:568–574CrossRefGoogle Scholar
  22. Pennacchio A, Pucci B, Secundo F, La Cara F, Rossi M, Raia CA (2008) Purification and characterization of a novel recombinant highly enantioselective short-chain NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus. Appl Environ Microbiol 74:3949–3958CrossRefGoogle Scholar
  23. Petroll K, Kopp D, Care A, Bergquist PL, Sunna A (2019) Tools and strategies for constructing cell-free enzyme pathways. Biotechnol Adv 37:91–108CrossRefGoogle Scholar
  24. Punekar NS (2018) Enzymes: catalysis, kinetics and mechanisms. Springer.  https://doi.org/10.1007/978-981-13-0785-0
  25. Restiawaty E, Iwasa Y, Maya S, Honda K, Omasa T, Hirota R, Kuroda A, Ohtake H (2011) Feasibility of thermophilic adenosine triphosphate-regeneration system using Thermus thermophilus polyphosphate kinase. Process Biochem 46:1747–1752CrossRefGoogle Scholar
  26. Rollin JA, del Campo JM, Myung S, Sun F, You C, Bakovic A, Castro R, Chandrayan SK, Wu CH, Adams MWW, Senger RS, Zhang YHP (2015) High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling. Proc Natl Acad Sci 112:4964–4969CrossRefGoogle Scholar
  27. Sørensen MA, Pedersen S (1991) Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli. J Bacteriol 173:5244–5246CrossRefGoogle Scholar
  28. Sperl JM, Sieber V (2018) Multienzyme cascade reactions –status and recent advances. ACS Catal 8:2385–2396CrossRefGoogle Scholar
  29. Sugimoto E, Pizer LI (1968) The mechanism of end product inhibition of serine biosynthesis. I. Purification and kinetics of phosphoglycerate dehydrogenase. J Biol Chem 243:2081–2089Google Scholar
  30. Takagi H, Ohtsu I (2016) L-Cysteine metabolism and fermentation in microorganisms. In: Yokota A, Ikeda M (eds) Advances in biochemical engineering/biotechnology, vol 159. Springer Nature, pp 129–151.  https://doi.org/10.1007/10_2016_29
  31. Takumi K, Nonaka G (2016) Bacterial cysteine-inducible cysteine resistance system. J Bacteriol 198:1384–1392CrossRefGoogle Scholar
  32. Takumi K, Ziyatdinov MK, Samsonov V, Nonaka G (2017) Fermentative production of cysteine by Pantoea ananatis. Appl Environ Microbiol 83:e02502–e02516CrossRefGoogle Scholar
  33. Wada M, Awano N, Haisa K, Takagi H, Nakamori S (2002) Purification, characterization and identification of cysteine desulfhydrase of Corynebacterium glutamicum, and its relationship to cysteine production. FEMS Microbiol Lett 217:103–107CrossRefGoogle Scholar
  34. Wilding KM, Schinn SM, Long EA, Bundy BC (2018) The emerging impact of cell-free chemical biosynthesis. Curr Opin Biotechnol 53:115–121CrossRefGoogle Scholar
  35. Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S, Nishino K, Yamaguchi A, Takagi H (2006) Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli. Appl Environ Microbiol 72:4735–4742CrossRefGoogle Scholar
  36. Ye X, Honda K, Sakai T, Okano K, Omasa T, Hirota R, Kuroda A, Ohtake H (2012) Synthetic metabolic engineering-a novel technology for designing a chimeric metabolic pathway. Microb Cell Factories 11:120CrossRefGoogle Scholar
  37. Yokoyama S, Hirota H, Kigawa T, Yabuki T, Shirouzu M, Terada T, Ito Y, Matsuo Y, Kuroda Y, Nishimura Y, Kyogoku Y, Miki K, Masui R, Kuramitsu S (2000) Structural genomics projects in Japan. Nat Struct Biol 7:943–945CrossRefGoogle Scholar
  38. You C, Shi T, Li Y, Han P, Zhou A, Zhang YP (2017) An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch. Biotechnol Bioeng 114:1855–1864CrossRefGoogle Scholar
  39. Zhang YHP (2010) Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities. Biotechnol Bioeng 105:663–677Google Scholar
  40. Zhu F, Zhong X, Hu M, Lu L, Deng Z, Liu T (2014) In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli. Biotechnol Bioeng 111:1396–1405CrossRefGoogle Scholar

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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