In vitro production of cysteine from glucose
- 55 Downloads
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.
KeywordsCysteine In vitro metabolic engineering Thermophilic enzyme Kinetic model
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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
- Harris CL (1981) Cystine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme. J Bacteriol 145:1031–1035Google Scholar
- 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
- Jagura-Burdzy G, Kredich NM (1983) Cloning and physical mapping of the cysB region of Salmonella typhimurium. J Bacteriol 155:578–585Google Scholar
- Kredich NM, Tomkins GM (1966) The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. J Biol Chem 241:4955–4965Google Scholar
- Punekar NS (2018) Enzymes: catalysis, kinetics and mechanisms. Springer. https://doi.org/10.1007/978-981-13-0785-0
- 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
- 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
- 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
- Zhang YHP (2010) Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations: Challenges and opportunities. Biotechnol Bioeng 105:663–677Google Scholar