Ultra-rapid and high-titer biomanufacturing of trehalose 6-phosphate by an in vitro synthetic biology platform

Keywords

Chemical intervention / Food security / In vitro BioTransformation / In vitro synthetic biology / Multi-enzyme molecular machine / Trehalose-6-phosphate

References

[1] Billerbeck S, Härle J, Panke S. The good of two worlds: increasing complexity in cell-free systems. Curr Opin Biotechnol, 2013, 24(6): 1037-1043

[2] Bowie JU, Sherkhanov S, Korman TP, Valliere MA, Opgenorth PH, Liu H. Synthetic biochemistry: The bio-inspired bell-free approach to commodity chemical production. Trends Biotechnol, 2020, 38(7): 766-778

[3] Chen H, Zhang Y-HPJ. Enzymatic regeneration and conservation of ATP: challenges and opportunities. Crit Rev Biotechnol, 2021, 41(1): 16-33

[4] Chen A, Peng X, Shen T, Zheng L, Wu D, Wang S. Discovery, design, and engineering of enzymes based on molecular retrobiosynthesis. mLife, 2025, 4(2): 107-125

[5] Deng X, Song C-C, Gu W-J, Wang Y-J, Feng L, Zhou X-J, Zhou M-Q, Yuan W-C, Chen Y-Z. Regioselective and enantioselective propargylic hydroxylations catalyzed by P450tol monooxygenases. Bioresour Bioprocess, 2024, 11(1): 64

[6] Doi J, Yokoigawa K, Isobe Y, Kawai H. Trehalose 6-phosphate production with energy coupling fermentation by yeast cells. Biosci Biotechnol Biochem, 1998, 62(4): 735-739

[7] Fan L, Shi T, Chen X, Li Y, Han P, Jensen P, Zhang Y-HPJ. Biosynthesis of a healthy natural sugar D-tagatose: advances and opportunities. Crit Rev Biotechnol, 2025, 23: 1-16

[8] Feng Y, Wang N, Cao Q, Li S, Xu Y, Che X, Zhao J, Yang C, Xue S. Structural insights into trehalose-6-phosphate phosphorylase and its role in trehalose 6-phosphate biosynthesis via a multienzyme cascade. J Agri Food Chem, 2025, 73(30): 19065-19075

[9] Fichtner F, Lunn JE. The role of trehalose 6-phosphate (Tre6P) in plant metabolism and development. Annu Rev Plant Biol, 2021, 72(1): 737-760

[10] Gao Y, Saburi W, Taguchi Y, Mori H. Biochemical characteristics of maltose phosphorylase MalE from Bacillus sp. AHU2001 and chemoenzymatic synthesis of oligosaccharides by the enzyme. Biosci Biotechnol Biochem, 2019, 83(11): 2097-2109

[11] Griffiths CA, Sagar R, Geng Y, Primavesi LF, Patel MK, Passarelli MK, Gilmore IS, Steven RT, Bunch J, Paul MJ, Davis BG. Chemical intervention in plant sugar signalling increases yield and resilience. Nature, 2016, 540(7634): 574-578

[12] Griffiths CA, Xue X, Miret JA, Salvagiotti F, Acevedo-Siaca LG, Gimeno J, Reynolds MP, Hassall KL, Halsey K, Puranik S, Oszvald M, Kurup S, Davis BG, Paul MJ. Membrane-permeable trehalose 6-phosphate precursor spray increases wheat yields in field trials. Nat Biotechnol, 2025, 44(2): 316-325

[13] Han P, Wang X, Li Y, Wu H, Shi T, Shi J. Synthesis of a healthy sweetener D-tagatose from starch catalyzed by semiartificial cell factories. J Agri Food Chem, 2023, 71(8): 3813-3820

[14] Han P, You C, Li Y, Shi T, Wu H, Zhang Y-HPJ. High-titer production of myo-inositol by a co-immobilized four-enzyme cocktail in biomimetic mineralized microcapsules. Chem Eng J, 2023, 461: 141946

[15] Honda K, Maya S, Omasa T, Hirota R, Kuroda A, Ohtake H. Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes. J Biotechnol, 2010, 148(4): 204-207

[16] Honda K, Hara N, Cheng M, Nakamura A, Mandai K, Okano K, Ohtake H. In vitro metabolic engineering for the salvage synthesis of NAD. Metab Eng, 2016, 35: 114-120

[17] Iwamoto S, Motomura K, Shinoda Y, Urata M, Kato J, Takiguchi N, Ohtake H, Hirota R, Kuroda A. Use of an Escherichia coli recombinant producing thermostable polyphosphate kinase as an ATP regenerator to produce fructose 1,6-diphosphate. Appl Environ Microbiol, 2007, 73(17): 5676-5678

[18] Kim J-E, Zhang Y-HPJ. Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade. Biotechnol Bioeng, 2016, 113: 275-282

[19] Kwon SJ, Mora-Pale M, Lee M-Y, Dordick JS. Expanding nature’s small molecule diversity via in vitro biosynthetic pathway engineering. Curr Opin Chem Biol, 2012, 16(1–2): 186-195

[20] Li Z, Wei X, Tong X, Zhao J, Liu X, Wang H, Tang L, Shu Y, Li G, Wang Y, Ying J, Jiao G, Hu H, Hu P, Zhang J. The OsNAC23-Tre6P-SnRK1a feed-forward loop regulates sugar homeostasis and grain yield in rice. Mol Plant, 2022, 15(4): 706-722

[21] Li X, Yu H, Liu S, Ma B, Wu X, Zheng X, Xu Y. Discovery, characterization and mechanism of a Microbacterium esterase for key d-biotin chiral intermediate synthesis. Bioresour Bioprocess, 2024, 11(1): 59

[22] Li Z, Zhong Y, Qing Z, Li Z. A circuitous route for in vitro multi-enzyme cascade production of cytidine triphosphate to overcome the thermodynamic bottleneck. Bioresour Bioprocess, 2024, 11(1): 6

[23] Liu Y, Li B, Chen T, Tian S, Zhang Z. The synthesis, degradation and biological function of trehalose- 6-phosphate. Stress Biol, 2025, 5(1): 38

[24] Nuccio ML, Wu J, Mowers R, Zhou H-P, Meghji M, Primavesi LF, Paul MJ, Chen X, Gao Y, Haque E, Basu SS, Lagrimini LM. Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in well-watered and drought conditions. Nat Biotechnol, 2015, 33(8): 862-869

[25] Opgenorth PH, Korman TP, Iancu L, Bowie JU. A molecular rheostat maintains ATP levels to drive a synthetic biochemistry system. Nat Chem Biol, 2017, 13(9): 938-942

[26] Saheki S, Takeda A, Shimazu T. Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity. Anal Biochem, 1985, 148(2): 277-281

[27] Shen K, Zhu C-N, Xu J-H, Zheng G-W, Chen Q. Development of an in vitro multi-enzyme system for efficient one-pot biosynthesis of sorbitol from fructose-6-phosphate. Bioresour Bioprocess, 2025, 12(1): 104

[28] Sperl JM, Sieber V. Multienzyme cascade reactions-status and recent advances. ACS Catal, 2018, 8(3): 2385-2396

[29] Taguchi Y, Saburi W, Imai R, Mori H. Efficient one-pot enzymatic synthesis of trehalose 6-phosphate using GH65 α-glucoside phosphorylases. Carbohydr Res, 2020, 488: 107902

[30] Tasnádi G, Staśko M, Ditrich K, Hall M, Faber K. Preparative-scale enzymatic synthesis of rac-glycerol-1-phosphate from crude glycerol using acid phosphatases and phosphate. Chemsuschem, 2020, 13(7): 1759-1763

[31] Wang W, Liu M, You C, Li Z, Zhang Y-H. ATP-free biosynthesis of a high-energy phosphate metabolite fructose 1,6-diphosphate by in vitro metabolic engineering. Metab Eng, 2017, 42: 168-174

[32] Wang J, Li Y, Lu M, Guo Q, Chen Y, Li Y, Jing Y, Zhai Z, Shi T, Zhang Y, Jiang B. Complete biotransformation of cellulose to starch in vitro. Natl Sci Rev, 2025, 13(1): nwaf503

[33] Wen L, Huang K, Liu Y, Wang PG. Facile enzymatic synthesis of phosphorylated ketopentoses. ACS Catal, 2016, 6(3): 1649-1654

[34] Wohlgemuth R, Liese A, Streit W. Biocatalytic phosphorylations of metabolites: Past, present, and future. Trends Biotechnol, 2017, 35(5): 452-465

[35] Wu H, Chen Q, Zhang W, Mu W. Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges. Crit Rev Food Sci Nutr, 2023, 63(14): 2057-2073

[36] Yang R, Sun Y, Jin C (2023) Manufacturing trehalose-6-phosphate by synthetic biology. Hebei University

[37] You C, Shi T, Li Y, Han P, Zhou X, Zhang, Y-H P, J (2017) An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch. Biotechnol Bioeng 114(8):1855–1864. https://doi.org/10.1002/bit.26314

[38] Zhang Y-HPJ, Zhu Z, You C, Zhang L, Liu K. In vitro BioTransformation (ivBT): definitions, opportunities, and challenges. Synth Biol Eng, 2023, 1(2): 10013

[39] Zhang Y-HPJ, Chen X, Han P. PE and PX values in biomanufacturing: definitions and applications. Syn Biol J, 2025, 6(4): 715-727

[40] Zhou W, Huang R, Zhu Z, Zhang Y-HP. Coevolution of both thermostability and activity of polyphosphate glucokinase from YX. Appl Environ Microbiol, 2018, 84(16): e01224-e01218

Funding

Key Technologies Research and Development Program(2022YFC3401700)

Hubei Technological Innovation Special Fund(22HHSWSS00015)

RIGHTS & PERMISSIONS

The Author(s)