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The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine

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Abstract

Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.

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Abbreviations

LDH:

Leucine dehydrogenase

FDH:

Formate dehydrogenase

TMA:

Trimethylpyruvic acid

Coh:

Cohesin module

Doc:

Dockerin module

CBM:

Carbohydrate-binding module

References

  1. Li, J., Pan, J., Zhang, J., & Xu, J. H. (2014). Stereoselective synthesis of L-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. Journal of Molecular Catalysis B: Enzymatic, 105, 11–17.

    Article  CAS  Google Scholar 

  2. Jin, J. Z., Chang, D. L., & Zhang, J. (2011). Discovery and application of new bacterial strains for asymmetric synthesis of L-tert-butyl leucine in high enantioselectivity. Applied Biochemistry and Biotechnology, 164, 376–385.

    Article  CAS  Google Scholar 

  3. Bommarius, A. S., Schwarm, M., & Drauz, K. (1998). Biocatalysis to amino acid-based chiral pharmaceuticals—examples and perspectives. Journal of Molecular Catalysis B: Enzymatic, 5, 1–11.

    Article  CAS  Google Scholar 

  4. Liu, W., Ma, H., Luo, J., Shen, W., Xu, X., Li, S., et al. (2014). Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli. Biochemical Engineering Journal, 91, 204–209.

    Article  CAS  Google Scholar 

  5. Liu, W., Luo, J., Zhuang, X., Shen, W., Zhang, Y., Li, S., et al. (2014). Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium. Biochemical Engineering Journal, 83, 116–120.

    Article  CAS  Google Scholar 

  6. Chen, R., Chen, Q., Kim, H., Siu, K. H., Sun, Q., Tsai, S. L., et al. (2014). Biomolecular scaffolds for enhanced signaling and catalytic efficiency. Current Opinion in Biotechnology, 28, 59–68.

    Article  CAS  Google Scholar 

  7. Idan, O., & Hess, H. (2013). Engineering enzymatic cascades on nanoscale scaffolds. Current Opinion in Biotechnology, 24, 606–611.

    Article  CAS  Google Scholar 

  8. You, C., & Zhang, Y. H. P. (2014). Annexation of a high-activity enzyme in a synthetic three-enzyme complex greatly decreases the degree of substrate channeling. ACS Synthetic Biology, 3, 380–386.

    Article  CAS  Google Scholar 

  9. Demishtein, A., Karpol, A., Barak, Y., Lamed, R., & Bayer, E. A. (2010). Characterization of a dockerin-based affinity tag: application for purification of a broad variety of target proteins. Journal of Molecular Recognition, 23, 525–535.

    Article  CAS  Google Scholar 

  10. Liu, F., Banta, S., & Chen, W. (2013). Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production. Chemical Communications, 49, 3766–3768.

    Article  CAS  Google Scholar 

  11. Fontes, C. M., & Gilbert, H. J. (2010). Cellulosomes: highly efficient nanomachines designed to designed to deconstruct plant cell wall complex carbohydrates. Annual Review of Biochemistry, 79, 655–681.

    Article  CAS  Google Scholar 

  12. Hyeon, J. E., Jeon, S. D., & Han, S. O. (2013). Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications. Biotechnology Advances, 31, 936–944.

    Article  CAS  Google Scholar 

  13. You, C., Myung, S., & Zhang, Y. H. P. (2012). Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. Angewandte Chemie, International Edition, 51, 8787–8790.

    Article  CAS  Google Scholar 

  14. You, C., Chen, H., Myung, S., Sathitsuksanoh, N., Ma, H., Zhang, X. Z., et al. (2013). Enzymatic transformation of nonfood biomass to starch. Proceedings of the National Academy of Sciences, 110, 7182–7187.

    Article  CAS  Google Scholar 

  15. Zhang, Y. H. P., Cui, J. B., Lynd, L. R., & Kuang, L. R. (2006). A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules, 7, 644–648.

    Article  CAS  Google Scholar 

  16. Gao, S. H., You, C., Renneckar, S., Bao, J., & Zhang, Y. H. P. (2014). New insights into enzymatic hydrolysis of heterogeneous cellulose by using carbohydrate-binding module 3 containing GFP and carbohydrate- binding module 17 containing CFP. Biotechnology for Biofuels, 7.

  17. Carvalho, A. L., Dias, F. M. V., Prates, J. A. M., Nagy, T., Gilbert, H. J., Davies, G. J., et al. (2003). Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex. Proceedings of the National Academy of Sciences of the United States of America, 100, 13809–13814.

    Article  CAS  Google Scholar 

  18. Chiang, C. J., Lin, L. J., Wang, Z. W., Lee, T. T., & Chao, Y. P. (2014). Design of a noncovalently linked bifunctional enzyme for whole-cell biotransformation. Process Biochemistry, 49, 1122–1128.

    Article  CAS  Google Scholar 

  19. Voronov-Goldman, M., Levy-Assaraf, M., Yaniv, O., Wisserman, G., Jindou, S., Borovok, I., et al. (2014). Structural characterization of a novel autonomous cohesin from Ruminococcus flavefaciens. Acta Crystallographica Section F-Structural Biology Communications, 70, 450–456.

    Article  CAS  Google Scholar 

  20. Adams, J. J., Webb, B. A., Spencer, H. L., & Smith, S. P. (2005). Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: calcium-induced effects on conformation and target recognition. Biochemistry, 44, 2173–2182.

    Article  CAS  Google Scholar 

  21. Lineweaver, H. (1985). Citation classic—the determination of enzyme dissociation-constants. Current Contents/Life Sciences, 19–19.

  22. Li, H. M., Zhu, D. M., Hyatt, B. A., Malik, F. M., Biehl, E. R., & Hua, L. (2009). Cloning, protein sequence clarification, and substrate specificity of a leucine dehydrogenase from Bacillus sphaericus ATCC4525. Applied Biochemistry and Biotechnology, 158, 343–351.

    Article  CAS  Google Scholar 

  23. You, C., & Zhang, Y. H. P. (2013). Self-assembly of synthetic metabolons through synthetic protein scaffolds: one-step purification, co-immobilization, and substrate channeling. ACS Synthetic Biology, 2, 102–110.

    Article  CAS  Google Scholar 

  24. You, C., Zhang, X. Z., & Zhang, Y. H. P. (2012). Mini-scaffoldin enhanced mini-cellulosome hydrolysis performance on low-accessibility cellulose (Avicel) more than on high-accessibility amorphous cellulose. Biochemical Engineering Journal, 63, 57–65.

    Article  CAS  Google Scholar 

  25. Schirwitz, K., Schmidt, A., & Lamzin, V. S. (2007). High-resolution structures of formate dehydrogenase from Candida boidinii. Protein Science, 16, 1146–1156.

    Article  CAS  Google Scholar 

  26. Katoh, R., Ngata, S., Ozawa, A., Ohshima, T., Kamekura, M., & Misono, H. (2003). Purification and characterization of leucine dehydrogenase from an alkaliphilic halophile, Natronobacterium magadii MS-3. Journal of Molecular Catalysis B: Enzymatic, 23, 231–238.

    Article  CAS  Google Scholar 

  27. Gao, X., Yang, S., Zhao, C., Ren, Y., & Wei, D. (2014). Artificial multienzyme supramolecular device: highly ordered self-assembly of oligomeric enzymes in vitro and in vivo. Angewandte Chemie, International Edition, 53, 14027–14030.

    Article  CAS  Google Scholar 

  28. Srere, P. A. (1987). Complexes of sequential metabolic enzymes. Annual Review of Biochemistry, 56, 89–124.

    Article  CAS  Google Scholar 

  29. Moller, B. L. (2010). Dynamic metabolons. Science, 330, 1328–1329.

    Article  Google Scholar 

  30. Fierobe, H. P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belaich, A., et al. (2002). Degradation of cellulose substrates by cellulosome chimeras substrate targeting versus proximity of enzyme components. Journal of Biological Chemistry, 277, 49621–49630.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors are grateful to Professor Y.-H. Percival Zhang in Virginia Tech and Jibin Sun in Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences for their technical assistance and gifting plasmids. This work was supported by the National Natural Science Foundation of China (No. 41176111, No. 41306124), the State Key Program of National Natural Science Foundation of China (No. 21336009), and the Fundamental Research Funds for the Central Universities (No. 2013121029).

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Authors and Affiliations

  1. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, People’s Republic of China

    Jixue Lu, Yonghui Zhang, Dongfang Sun, Wei Jiang, Shizhen Wang & Baishan Fang

  2. The Key Lab for Synthetic Biotechnology of Xiamen City, Xiamen University, Xiamen, Fujian, 361005, People’s Republic of China

    Shizhen Wang & Baishan Fang

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  1. Jixue Lu
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  2. Yonghui Zhang
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  3. Dongfang Sun
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  4. Wei Jiang
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Correspondence to Shizhen Wang or Baishan Fang.

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Jixue Lu and Yonghui Zhang contributed equally to this work and should be considered co-first authors.

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Lu, J., Zhang, Y., Sun, D. et al. The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine. Appl Biochem Biotechnol 180, 1180–1195 (2016). https://doi.org/10.1007/s12010-016-2160-2

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  • DOI: https://doi.org/10.1007/s12010-016-2160-2

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