Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine

Microbial Cell Factories - 2021
Liping Liao1, Yonghui Zhang1, Yali Wang1, Yousi Fu1, Aihui Zhang1, Ruodian Qiu1, Shuhao Yang1, Baishan Fang1
1Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, People's Republic of China

Tóm tắt

Abstract Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.

Từ khóa


Tài liệu tham khảo

Luo W, Zhu J, Zhao Y, Zhang H, Yang X, Liu Y, Rao Z, Yu X. Cloning and expression of a novel leucine dehydrogenase: characterization and l-tert-Leucine production. Front Bioeng Biotech. 2020;8:186.

Jiang W, Fang B. Synthesizing Chiral Drug Intermediates by Biocatalysis. Appl Biochem Biotech. 2020;192:146–79.

Jin JZ, Chang DL, Zhang J. Discovery and Application of New Bacterial Strains for Asymmetric Synthesis of l-Tert-Butyl Leucine in High Enantioselectivity. Appl Biochem Biotech. 2011;164:376–85.

Liu W, Ma H, Luo J, Shen W, Xu X, Li S, Hu Y, Huang H. Efficient synthesis of l-tert-leucine through reductive amination using leucine dehydrogenase and formate dehydrogenase coexpressed in recombinant E. coli. Biochem Eng J. 2014;91:204–9.

Kragl U, Vasic-Racki D, Wandrey C. Continuous production of l-tert-leucine in series of two enzyme membrane reactors: modelling and computer simulation. Bioprocess Eng. 1996;14:291–7.

Hong E, Cha M, Yun H, Kim B. Asymmetric synthesis of l-tert-leucine and l-3-hydroxyadamantylglycine using branched chain aminotransferase. J Mol Catal B-Enzym. 2010;66:228–33.

Stelkes-Ritter U, Beckers G, Bommarius A, Drauz K, Günther K, Kottenhahn M, Schwarm M, Kula MR. Kinetics of peptide amidase and its application for the resolution of racemates. Biocatal Biotransfor. 1997;15:205–19.

Agosta E, Caligiuri A, D’Arrigo P, Servi S, Tessaro D, Canevotti R. Enzymatic approach to both enantiomers of N-Boc hydrophobic amino acids. Tetrahedron-Asymmetr. 2006;17:1995–9.

Liu W, Luo J, Zhuang X, Shen W, Zhang Y, Li S, Hu Y, Hu Y. Efficient preparation of enantiopure l-tert-leucine through immobilized penicillin G acylase catalyzed kinetic resolution in aqueous medium. Biochem Eng J. 2014;83:116–20.

Turner NJ, Winterman JR, McCague R, Parratt J, Taylor S. Synthesis of homochiral l-(S)-tert-leucine via a lipase catalysed dynamic resolution process. Tetrahedron Lett. 1995;36:1113–6.

Bommarius AS, Schwarm M, Drauz K. Biocatalysis to amino acid-based chiral pharmaceuticals—examples and perspectives. J Mol Catal B-Enzym. 1998;5:1–11.

Menzel A, Werner H, Altenbuchner J, Gröger H. From enzymes to “designer bugs” in reductive amination: a new process for the synthesis of l-tert-leucine using a whole cell-catalyst. Eng Life Sci. 2004;4:573–6.

Li J, Pan J, Zhang J, Xu JH. Stereoselective synthesis of l-tert-leucine by a newly cloned leucine dehydrogenase from Exiguobacterium sibiricum. J Mol Catal B-Enzym. 2014;105:11–7.

Liese A, Seelbach K, Wandrey C. Industrial Biotransformations. 2nd ed. Weinheim: Wiley-VCH; 2006.

Dunn MF, Niks D, Ngo H, Barends TR, Schlichting I. Tryptophan synthase: the workings of a channeling nanomachine. Trends Biochem Sci. 2008;33:254–64.

Hyeon JE, Jeon SD, Han SO. Cellulosome-based, Clostridium-derived multi-functional enzyme complexes for advanced biotechnology tool development: advances and applications. Biotechnol Adv. 2013;31:936–44.

Hirakawa H, Haga T, Nagamune T. Artificial protein complexes for biocatalysis. Top Catal. 2012;55:1124–37.

Gao X, Yang S, Zhao C, Ren Y, Wei D. Artificial Multienzyme supramolecular device: highly ordered self-assembly of oligomeric enzymes in vitro and in vivo. Angew Chem Int Edit. 2014;53:14027–30.

Zhang Y, Wang Y, Wang S, Fang B. Engineering bifunctional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration. Eng Life Sci. 2017;17:989–96.

Zhang YHP. Substrate channeling and enzyme complexes for biotechnological applications. Biotechnol Adv. 2011;29:715–25.

Zhuang X, Zhang A, Qiu S, Tang C, Zhao S, Li H, Zhang Y, Wang Y, Wang B, Fang B, Hong W. Coenzyme coupling boosts charge transport through single bioactive enzyme junctions. iScience. 2020;23:101001.

Yang X, Mu X, Nie Y, Xu Y. High efficient co-expression of leucine dehydrogenase and glucose dehydrogenase in Escherichia coli. Acta Microbiologica Sinica. 2016;56:1709–18.

Liechti G, Singh R, Rossi PL, Gray MD, Adams NE, Maurell AT. Chlamydia trachomatis dapF encodes a bifunctional enzyme capable of both D-glutamate racemase and diaminopimelate epimerase Activities. Mbio. 2018;9:e00204–18.

Böttcher T. From molecules to life: quantifying the complexity of chemical and biological systems in the Universe. J Mol Evol. 2018;86:1–10.

Wichmann R, Vasic-Racki D. Cofactor regeneration at the lab scale. Adv biochem Eng Biot. 2005;92:225–60.

Conrado RJ, Varner JD, DeLisa MP. Engineering the spatial organization of metabolic enzymes: mimicking nature’s synergy. Curr Opin Biotech. 2008;19:492–9.

Lu P, Feng MG. Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl Microbiol Biot. 2008;79:579–87.

Huang Z, Ye F, Zhang C, Chen S, Chen Y, Wu J, Togo M, Xing X. Rational design of a tripartite fusion protein of heparinase I enables one-step affinity purification and real-time activity detection. J Biotechnol. 2013;163:30–7.

Wang S, Cui GZ, Song XF, Feng Y, Cui Q. Efficiency and stability enhancement of Cis-epoxysuccinic acid hydrolase by fusion with a carbohydrate binding module and immobilization onto cellulose. Appl Biochem Biotech. 2012;168:708–17.

Bülow L. Preparation of artificial bifunctional enzymes by gene fusion. Biochem Soc Symp. 1990;57:123–33.

Hölsch K, Weuster-Botz D. Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins. Biotechnol Appl Bioc. 2010;56:131–40.

Fu J, Yang YR, Johnson-Buck A, Liu M, Liu Y, Walter NG, Woodbury NW, Yan H. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat Nanotechnol. 2014;9:531–6.

Aevarsson A, Seger K, Turley S, Sokatch JR, Hol WG. Crystal structure of 2-oxoisovalerate and dehydrogenase and the architecture of 2-oxo acid dehydrogenase multienzyme complexes. Nat Struct Biol. 1999;6:785–92.

Grogan G. Synthesis of chiral amines using redox biocatalysis. Curr Opin Chem Biol. 2018;43:15–22.

Kurbanoglu EB, Zilbeyaz K, Taskin M, Kurbanoglu NI. Total production of (R)-3,5-bistrifluoromethylphenyl ethanol by asymmetric reduction of 3,5-bis(trifluoromethyl)-acetophenone in the submerged culture of Penicillium expansum isolate. Tetrahedron-Asymmetr. 2009;20:2759–63.

Lu J, Zhang Y, Sun D, Jiang W, Wang S, Fang B. The development of leucine dehydrogenase and formate dehydrogenase bifunctional enzyme cascade improves the Biosynthsis of l-tert-Leucine. Appl Biochem Biotech. 2016;180:1180–95.

Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22:195–201.

Thông tin
Thông tin xuất bản

Microbial Cell Factories

Thông tin tác giả