Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars

doi:10.1016/S1872-2067(25)64675-4

Abstract

Abstract:

Multienzyme cascades enable the sequential synthesis of complex chemicals by combining multiple catalytic processes in one pot, offering considerable time and cost savings compared to a series of separate batch reactions. However, challenges related to coordination and regulatory interplay among multiple enzymes reduce the catalytic efficiency of such cascades. Herein, we genetically programmed a scaffold framework that selectively and orthogonally recruits enzymes as designed. The system was then used to generate multienzyme complexes of D-allulose 3-epimerase (DAE), ribitol dehydrogenase (RDH), and formate dehydrogenase (FDH) for rare sugar production. This scaffolded multienzymatic assembly achieves a 10.4-fold enhancement in the catalytic performance compared to its unassembled counterparts, obtaining allitol yield of more than 95%. Molecular dynamics simulations revealed that shorter distances between neighboring enzymes in scaffold-mounted complexes facilitated the transfer of reaction intermediates. A dual-module catalytic system incorporating (1) scaffold-bound complexes of DAE, RDH, and FDH and (2) scaffold-bound complexes of alcohol dehydrogenase and NADH oxidase expressed intracellularly in E. coli was used to synthesize D-allulose from D-fructose. This system synthesized 90.6% D-allulose from 300 g L⁻¹ D-fructose, with a space-time yield of 13.6 g L⁻¹ h⁻¹. Our work demonstrates the programmability and versatility of scaffold-based strategies for the advancement of multienzyme cascades.

Key words: Multienzymatic cascade reaction, Protein scaffold, Multienzymatic complexes, Nanoreactors, Molecular dynamics simulation

Xin Gao, Guangyao Tang, Jiajun Yan, Senbiao Fang, Kangming Tian, Fuping Lu, Hui-Min Qin. Programmed protein scaffold for multienzyme assembly empowering the biosynthesis of rare sugars[J]. Chinese Journal of Catalysis, 2025, 72: 95-105.

×
share this article

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.cjcatal.com/EN/10.1016/S1872-2067(25)64675-4

https://www.cjcatal.com/EN/Y2025/V72/I5/95

Figures/Tables 6

Fig. 1. Scaffolded strategy to assemble a tri-enzymatic cascade system. (a) Schematic representation of the three enzymes involved in the production of allitol. (b) Design and construction of multienzymatic assemblies on the protein scaffold. MALDI-TOF MS analysis (c) and dynamic light scattering analysis (d) of the purified SpyTag-DAE, SnoopTag-RDH, PDZlig-FDH, protein scaffold, and the assemblies. The molecular weights of the SnoopTag-RDH, SpyTag-DAE, PDZlig-FDH, protein scaffolds, and the assemblies were approximately 28.46, 32.90, 41.40, 55.70, and 159.34 kDa, respectively.

Fig. 2. Catalytic performance of the tri-enzymatic cascade reaction. (a) Allitol yield at the molar ratios were 1:1:2 and 1:1:3 of DAE, RDH, and FDH for the assembled system and the unassembled free system. (b) RMSD values of the assembled and unassembled models in 800-ns MD simulations. Changes in the spatial positions of three catalytic units both in the unassembled free systems (c) and assembled systems (d) from 800-ns MD simulations. Changes in distance triggered by the movement of spatial phases in both assembled and unassembled free systems were analyzed. The transfer of intermediates in this biosynthetic pathway was analyzed in both an unassembled free system (e) and assembled system (f) using six-phase-node steered MD simulations.

Fig. 3. Design and characterization of immobilized tri-enzyme assemblies. (a) Schematic of the preparation of co-immobilized DAE/RDH/FDH-scaffold on nanocrystals (DRF-S@nanocrystals) and investigation of the bioconversion process. Physical and chemical characterization of DRF-S@nanocrystals using FT-IR spectroscopy (b), and XRD analysis (c). (d) Confocal fluorescence microscopy images of DRF-S@nanocrystals. To identify DAE, RDH, and FDH, they were labeled with rhodamine B isothiocyanate (RhBITC), fluorescein isothiocyanate (FITC), and coumarin, respectively.

Fig. 4. Catalytic performance of the multienzyme system. (a) Specific activity and allitol yield of the immobilized forms (DRF-S@nanocrystals) and scaffolded forms (DRF-S) with D-fructose concentrations of 10-300 g/L. (b) Relative activities of DRF-S@nanocrystals and DRF-S were assessed after incubation at various temperatures and pH values. c) Relative activity and allitol yield of DRF-S@nanocrystals during 10 consecutive reaction cycles. d) Storage stability of DRF-S and DRF-S@nanocrystals.

Fig. 5. Biosynthesis of D-allulose using an organized bi-enzyme system. (a) Schematic diagram of the parallel reactions that generate D-allulose. (b) Cartoon images illustrating how ADH and NOX are clustered differently in the two strains. (c) TEM images of the Strains 1 and 2. Scale bar: 100 nm. (d) Scaffold-mediated protein clustering in E. coli cells in vivo was investigated using confocal laser scanning microscopy (CLSM). Representative images of mCherry- labeled SpyTag-NOX and GFP-labeled PDZlig-ADH, as well as a schematic diagram of the design and construction of Strains 1-1 and 2-1.

Fig. 6. (a) Schematic illustration of the dual-modular catalytic strategy utilized in this work for the production of D-allulose from D-fructose. (b) D-allulose production in Strains 1 and 2 in the time period from 30 to 240 min. (c) Specific activity profiles of Strains 1 and 2 in the allitol concentration range of 3 to 50 g L-1. (d) Production of D-allulose from D-fructose using the dual-modular catalytic strategy. Error bars indicate the standard deviations (n = 3).

References

[1]	W. Kang, T. Ma, M. Liu, J. Qu, Z. Liu, H. Zhang, B. Shi, S. Fu, J. Ma, L. T. F. Lai, S. He, J. Qu, S. Wing-Ngor Au, B. Ho Kang, W. C. Yu Lau, Z. Deng, J. Xia, T. Liu, Nat. Commun., 2019, 10, 4248.
[2]	Z. Zhang, L. Fang, F. Wang, Y. Deng, Z. Jiang, A. Li, Angew. Chem. Int. Ed., 2023, 62, e202215935.
[3]	S. M. Jo, F. R. Wurm, K. Landfester, Angew. Chem. Int. Ed., 2021, 60, 7728-7734.
[4]	A. I. Benítez-Mateos, D. Roura Padrosa, F. Paradisi, Nat. Chem., 2022, 14, 489-499.
[5]	J. Vanderstraeten, Y. Briers, Biotechnol. Adv., 2020, 44, 107627.
[6]	N. C. Dubey, B. P. Tripathi, ACS Appl. Bio Mater., 2021, 4, 1077-1114.
[7]	Y. Xu, X. Wang, C. Zhang, X. Zhou, X. Xu, L. Han, X. Lv, Y. Liu, S. Liu, J. Li, G. Du, J. Chen, R. Ledesma-Amaro, L. Liu, Nat. Commun., 2022, 13, 3040.
[8]	P. Zhou, H. Liu, X. Meng, H. Zuo, M. Qi, L. Guo, C. Gao, W. Song, J. Wu, X. Chen, W. Chen, L. Liu, Angew. Chem. Int. Ed., 2023, 62, e202215778.
[9]	W. Kang, X. Ma, H. Zhang, J. Ma, C. Liu, J. Li, H. Guo, D. Wang, R. Wang, B. Li, C. Xue, J. Am. Chem. Soc., 2024, 146, 6686-6696.
[10]	A. Ledesma-Fernandez, S. Velasco-Lozano, J. Santiago-Arcos, F. López-Gallego, A. L. Cortajarena, Nat. Commun., 2023, 14, 2587.
[11]	Y. Liu, S. Gonen, T. Gonen, T. O. Yeates, Proc. Natl. Acad. Sci. U. S. A., 2018, 115, 3362-3367.
[12]	C. J. Delebecque, A. B. Lindner, P. A. Silver, F. A. Aldaye, Science, 2011, 333, 470-474.
[13]	G. B. Chu, W. Y. Li, X. X. Han, H. H. Sun, Y. Han, G. Y. Zhi, D. H. Zhang, Small, 2023, 19, 2301413.
[14]	Z. Liu, S. Cao, M. Liu, W. Kang, J. Xia, ACS Nano, 2019, 13, 11343-11352.
[15]	C. Y. Tan, H. Hirakawa, R. Suzuki, T. Haga, F. Iwata, T. Nagamune, Angew. Chem. Int. Ed., 2016, 55, 15002-15006.
[16]	J. Qu, S. Cao, Q. Wei, H. Zhang, R. Wang, W. Kang, T. Ma, L. Zhang, T. Liu, S. Wing-Ngor Au, F. Sun, J. Xia, ACS Nano, 2019, 13, 9895-9906.
[17]	S. Liu, Y. Sun, Angew. Chem. Int. Ed., 2023, 62, e202308562.
[18]	W. Kang, X. Ma, D. Kakarla, H. Zhang, Y. Fang, B. Chen, K. Zhu, D. Zheng, Z. Wu, B. Li, C. Xue, Angew. Chem. Int. Ed., 2022, 61, e202214001.
[19]	Y. Wang, M. Liu, Q. Wei, W. Wu, Y. He, J. Gao, R. Zhou, L. Jiang, J. Qu, J. Xia, Angew. Chem. Int. Ed., 2022, 61, e202203909.
[20]	Y. Azuma, T. G. W. Edwardson, D. Hilvert, Chem. Soc. Rev., 2018, 47, 3543-3557.
[21]	Z. Chen, T. Wu, S. Yu, M. Li, X. Fan, Y. X. Huo, Trends Biotechnol., 2024, 42, 43-60.
[22]	J. E. Dueber, G. C. Wu, G. R. Malmirchegini, T. S. Moon, C. J. Petzold, A. V. Ullal, K. L. Prather, J. D. Keasling, Nat. Biotechnol., 2009, 27, 753-759.
[23]	D. Zheng, Y. Zheng, J. Tan, Z. Zhang, H. Huang, Y. Chen, Nat. Commun., 2024, 15, 5510.
[24]	X. Gong, R. Li, J. Wang, J. Wei, K. Ma, X. Liu, F. Wang, Angew. Chem. Int. Ed., 2020, 59, 21648-21655.
[25]	A. Tanaka, Y. Fukuoka, Y. Morimoto, T. Honjo, D. Koda, M. Goto, T. Maruyama, J. Am. Chem. Soc., 2015, 137, 770-775.
[26]	L. Yang, J. Wei, W. Feng, Colloids Surf. B, 2023, 231, 113541.
[27]	P. Wu, Y. Zhao, X. Zhang, Y. Fan, S. Zhang, W. Zhang, F. Huo, JACS Au, 2023, 3, 2413-2435.
[28]	W. Hu, L. Wang, M. Wang, T. Zhong, Q. Wang, L. Zhang, F. Chen, K. Li, Z. Miao, D. Yang, H. Yang, Nat. Commun., 2021, 12, 1440.
[29]	Z. Luo, L. Qiao, H. Chen, Z. Mao, S. Wu, B. Ma, T. Xie, A. Wang, X. Pei, R. A. Sheldon, Angew. Chem. Int. Ed., 2024, 63, e202403539.
[30]	W. Zhang, D. Chen, J. Chen, W. Xu, Q. Chen, H. Wu, C. Guang, W. Mu, Crit. Rev. Food Sci. Nutr., 2023, 63, 5661-5679.
[31]	C. Li, X. Gao, H. Qi, W. Zhang, L. Li, C. Wei, M. Wei, X. Sun, S. Wang, L. Wang, Y. Ji, S. Mao, Z. Zhu, M. Tanokura, F. Lu, H.M. Qin, Angew. Chem. Int. Ed., 2023, 62, e202216721.
[32]	X. Gao, M. Wu, W. Zhang, C. Li, R.T. Guo, Y. Dai, W. Liu, S. Mao, F. Lu, H.M. Qin, J. Agric. Food Chem., 2021, 69, 11616-11625.
[33]	X. Gao, S. Fang, X. Ma, T. Wang, C. Li, F. Lu, H.-M. Qin, Chem. Eng. J., 2024, 484, 149453.
[34]	C. Li, W. Zhang, C. Wei, X. Gao, S. Mao, F. Lu, H. M. Qin, J. Agric. Food Chem., 2021, 69, 11637-11645.
[35]	B. Hess, C. Kutzner, D. van der Spoel, E. Lindahl, J. Chem. Theory Comput., 2008, 4, 435-447.
[36]	A. Mecklenfeld, G. Raabe, ADMET DMPK, 2020, 8, 274-296.
[37]	L. Chen, A. Cruz, D. R. Roe, A.C. Simmonett, L. Wickstrom, N. Deng, T. Kurtzman, J. Chem. Theory Comput., 2021, 17, 2714-2724.
[38]	R. Meli, P. C. Biggin, J. Cheminform., 2020, 12, 49.
[39]	G. Chen, L. Tong, S. Huang, S. Huang, F. Zhu, G. Ouyang, Nat. Commun., 2022, 13, 4816.
[40]	H. Qi, T. Wang, H. Li, C. Li, L. Guan, W. Liu, J. Wang, F. Lu, S. Mao, H. M. Qin, J. Agric. Food Chem., 2023, 71, 18431-18442.
[41]	I. Wheeldon, S. D. Minteer, S. Banta, S. C. Barton, P. Atanassov, M. Sigman, Nat. Chem., 2016, 8, 299-309.
[42]	J. A. Gable, T. L. Poulos, A. H. Follmer, J. Am. Chem. Soc., 2023, 145, 4254-4265.
[43]	N. Cao, R. Guo, P. Song, S. Wang, G. Liu, J. Shi, L. Wang, M. Li, X. Zuo, X. Yang, C. Fan, M. Li, Y. Zhang, Nano Lett., 2024, 24, 4682-4690.
[44]	P. Gullapalli, G. Takata, W. Poonperm, D. Rao, K. Morimoto, K. Akimitsu, S. Tajima, K. Izumori, Biosci. Biotechnol. Biochem., 2007, 71, 3048-3054.
[45]	W. Poonperm, G. Takata, Y. Ando, V. Sahachaisaree, P. Lumyong, S. Lumyong, K. Izumori, J. Biosci. Bioeng., 2007, 103, 282-285.
[46]	L. Wang, K. Chen, P. Zheng, X. Huo, F. Liao, L. Zhu, M. Hu, Y. Tao, Enzyme Microb. Technol., 2023, 163, 110172.