Abstract
L-tert-Leucine is an unnatural amino acid that is a key intermediate for the synthesis of several important drugs. The L-tert-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase and formate dehydrogenase. In this study, recombinant strains of Escherichia coli expressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH), respectively, and the strain co-expressing the two enzymes were constructed. The activity for the two enzymes of the cell extraction from different recombinant strains was determined. L-tert-Leucine was successfully synthesized by the recombinant strains, and the yield in different conditions was compared. The production of L-tert-Leucine was the highest when cell extraction of strains containing pLeuDH and pFDH, 1 mL cells extract could produce 4.5 mg L-tert-Leucine, while 1 mL whole cells could only produce 1.05 mg L-tert-Leucine. The yield of L-tert-Leucine was 3.375 mg/mL cell extraction of the strain containing pLeuDHFDH when NAD was added, while the yield fell to 1.635 mg/mL when the whole cell was used.
J. Bai and Y. Song—Co-first author.
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References
Bea HS, Park HJ, Lee SH, Yun H (2011) Kinetic resolution of aromatic β-amino acids by-transaminase. Chem Commun 47:5894–5896
Allenmark S, Lamm B (2001) A useful route to (R)—and (S)-tert-leucine. Chirality 13:43–47
Bommarius AS, Schwarm M, Drauz K (1998) Biocatalysisto amino acid-based chiral pharmaceuticals—examples and perspectives. J Mol Catal B Enzym 5:1–11
Wandrey C, Bossow B (1986) Continuous cofactor regeneration-utilisation of polymer bound NAD (H) for the production of optically active acids. Biotechnol Bioind 3:813
Ohshima T, Soda K (1989) Thermostable amino acid dehydrogenases: applications and gene cloning. Trends Biotechnul 7:210–214
Bommarius AS, Schwarm M, Sfingl K, Kottenhahn M, Huthmacher K, Drauz K (1995) Synthesis and use of enantiomerically pure tert-leucine. Tetrahedron Asymmetry 6:2851–2888
Manuel RK, Luis ES, Ericka PF, Anne G (2011) Strategy for the extraction of yeast DNA from artisan agave must for quantitative PCR analysis. Biosci Bioeng 112:518–521
Ohshima T, Nishida N, Bakthavatsalam S, Kataoka K, Takada H, Yoshimura T, Esaki N, Soda K (1994) The purification, characterization, cloning and sequencing of the gene for a halo stable and thermo stable leucine dehydrogenase from Thermo actionmyces intermedius. Eur J Biochem 222:305–312
Galkin A, Kulakova L, Tishkov V, Esaki N, Soda K (1995) Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacteriumvaccae N10. Appl Microbiol Biotechnol 44:479–483
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Kuroda S, Tanizawa K, Sakamoto Y, Tanaka H, Soda K (1990) Alanine dehydrogenase from two Bacillus species with distinct thermo stabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD (P)-dependent dehydrogenases. Biochemistry 29:1009–1015
Ohshima T, Nishida N, Bakthavatsalam S, Kataoka K, Takada H, Yoshimura T, Esaki N, Soda K (1994) The purification, characterization, cloning and sequencing of the gene for a halo stable and thermo stable leucine dehydrogenase from Thermo actinomyces intermedius. Eur J Biochem 222:305–312
Takada H, Yoshimura T, Ohshima T, Esaki N, Soda K (1991) Thermostable phenylalanine dehydrogenase of Thermo actinomyces intermedius: cloning, expression, and sequencing of its gene. J Biochem 109:371–376
Tishkov VI, Galkin AG, Gladyshev VN, Karzanov VV, Egorov AM (1992) Analysis of gene structure, optimization of expression in E. coli and properties of recombinant formate dehydrogenase of bacterium Pseudomonassp. 101. Biotechnology (Russia) 5:52–59
Mädje K, Schmölzer K, Nidetzky B, Kratzer R (2012) Host cell and expression engineering for development of an E. coli ketoreductase catalyst: enhancement of formate dehydrogenase activity for regeneration of NADH. Microb Cell Fact 11:1–7
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Bai, J., Song, Y., Luo, X., Yang, H., Du, W., Zhang, T. (2015). Construction of L-tert-Leucine Producing Strain by Expressing Heterologous Leucine Dehydrogenase and Formate Dehydrogenase in Escherichia coli . In: Zhang, TC., Nakajima, M. (eds) Advances in Applied Biotechnology. Lecture Notes in Electrical Engineering, vol 333. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-46318-5_3
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DOI: https://doi.org/10.1007/978-3-662-46318-5_3
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