Glucose-to-Starch Conversion by a Synthetic Enzymatic Cascade

Yiwen Shen; Ziqi Liang; Lanxue Li; Wei Zhang; Yaru Wang; Jian Tian; Bin Yao; Huoqing Huang; Huiying Luo; Tao Tu

doi:10.1016/j.eng.2026.04.021

Engineering ›› :202604021 DOI: 10.1016/j.eng.2026.04.021

research-article

Glucose-to-Starch Conversion by a Synthetic Enzymatic Cascade

Author information +

History +

PDF (4433KB)

Abstract

Starch is a vital global commodity for food and industry, yet its agricultural production is unsustainable, relying on inefficient photosynthesis, arable land, and stable climates. Meanwhile, the annual production of ~180 million tons of cellulose primarily from crop residues is largely wasted through incineration or landfilling. While enzymatic saccharification efficiently depolymerizes cellulose into glucose, its conversion to starch is fundamentally limited by the insufficient catalytic efficiency, poor substrate selectivity of key enzymes, and the reversibility of the core reactions they catalyze. Here, we optimized a cell-free three-enzyme cascade, designated the carbon-economical starch synthesis (CESS) pathway, to address this limitation. This in vitro cascade integrates three core enzymes: polyphosphate glucokinase (PPGK) for adenosine triphosphate (ATP)-free glucose activation, phosphoglucomutase (PGM), and α-glucan phosphorylase (αGP). We engineered the rate-limiting αGP fromThermotoga petrophila to generate the M7 variant (TpαGPM7), achieving a 4.54-fold higher catalytic efficiency, and optimized cofactors, identifying 20 mmol∙L⁻¹ Mn²⁺ to critically shift PGM’s preference toward starch synthesis. The optimized pathway converts 5.4 g∙L⁻¹ glucose (equivalent to 30 mmol∙L⁻¹) to 3.50 g∙L⁻¹ starch at 60 °C with a record-breaking 71.93% conversion yield and a space-time yield of 2.33 g∙L⁻¹∙h⁻¹, while minimizing carbon loss < 30%. The product was characterized as amylose. This photosynthesis-independent framework advances a sustainable route to valorize cellulosic waste into food-grade starch, offering a resilient strategy for food security and a circular bioeconomy.

Keywords

Artificially synthesized starch / Glucose / α-Glucan phosphorylase / Phosphoglucomutase

Cite this article

Download citation ▾

Yiwen Shen, Ziqi Liang, Lanxue Li, Wei Zhang, Yaru Wang, Jian Tian, Bin Yao, Huoqing Huang, Huiying Luo, Tao Tu. Glucose-to-Starch Conversion by a Synthetic Enzymatic Cascade. Engineering 202604021 DOI:10.1016/j.eng.2026.04.021

登录浏览全文

4963

注册一个新账户忘记密码

References

Publishing order | Descend order by publishing year | Descend order by cited within

[1]	Guo K, Liang W, Wang S, Guo D, Liu F, Persson S, et al. Strategies for starch customization: agricultural modification. Carbohydr Polym 2023; 321:121336.

[2]	Rodríguez—Rivera C, González—Martín C, Fernández—Millán E, Álvarez C, Escrivá F, Alguacil LF . Perinatal undernourishment provokes long—lasting alterations of clusterin and fumarate hydratase expression in the rat nucleus accumbens. Nutr Neurosci 2022; 25(9):1796-800.

[3]	Xu X, Sharma P, Shu S, Lin TS, Ciais P, Tubiello FN, et al. Global greenhouse gas emissions from animal—based foods are twice those of plant—based foods. Nat Food 2021; 2(9):724—32.

[4]	Willett W, Rockström J, Loken B, Springmann M, Lang T, Vermeulen S, et al. Food in the Anthropocene: the EAT—Lancet Commission on healthy diets from sustainable food systems. Lancet 2019; 393(10170):447-92.

[5]	Sharma A, Balda S, Capalash N, Sharma P . Engineering multifunctional enzymes for agro—biomass utilization. Bioresour Technol 2022; 347:126706.

[6]	You C, Chen H, Myung S, Sathitsuksanoh N, Ma H, Zhang XZ, et al. Enzymatic transformation of nonfood biomass to starch. Proc Natl Acad Sci USA 2013; 110(18):7182—7.

[7]	Xu X, Zhang W, You C, Fan C, Ji W, Park JT, et al. Biosynthesis of artificial starch and microbial protein from agricultural residue. Sci Bull 2023; 68(2):214-23.

[8]	Niu K, Ni Z, Yuan S, Wang Z, Fang X . One—pot synthesis of γ—cyclodextrin of high purity from non—food cellulose via an in vitro ATP—free synthetic enzymatic biosystem. Carbohydr Polym 2025; 347:122735.

[9]	Liu D, Wang Z, Yuan S, Ni Z, Niu K, Wu Z, et al. One—pot coproduction of high—value—added γ—cyclodextrin and myo—inositol from nonfood cellulose using an in vitro ATP—free enzymatic biosystem. ACS Sustain Chem Eng 2025; 13(24):9132-7.

[10]	Cai T, Sun H, Qiao J, Zhu L, Zhang F, Zhang J, et al. Cell—free chemoenzymatic starch synthesis from carbon dioxide. Science 2021; 373(6562):1523-7.

[11]	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):277-81.

[12]	Sun S, Wei X, Zhou X, You C . Construction of an artificial in vitro synthetic enzymatic platform for upgrading low—cost starch to value—added disaccharides. J Agric Food Chem 2021; 69(1):302-14.

[13]	Eddy SR . Accelerated profile hmm searches. PLOS Comput Biol 2011; 7(10):e1002195.

[14]	Yu H, Deng H, He J, Keasling JD, Luo X . UniKP: a unified framework for the prediction of enzyme kinetic parameters. Nat Commun 2023; 14(1):8211.

[15]	Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 2024; 630(8016):493-500.

[16]	Adams PD, Grosse—Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, Moriarty NW, et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr D Biol Crystallogr 2002; 58(11):1948-54.

[17]	Emsley P, Cowtan K . Coot: model—building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; 60(Pt 12):2126-32.

[18]	Regni C, Naught L, Tipton PA, Beamer LJ . Structural basis of diverse substrate recognition by the enzyme PMM/PGM from P. aeruginosa . Structure 2004; 12(1):55-63.

[19]	Trott O, Olson AJ . AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010; 31(2):455-61.

[20]	Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C . Ff14sb: improving the accuracy of protein side chain and backbone parameters from ff99sb. J Chem Theory Comput 2015; 11(8):3696-713.

[21]	Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA . Development and testing of a general amber force field. J Comput Chem 2004; 25(9):1157—74.

[22]	Bayly CI, Cieplak P, Cornell W, Kollman PA . A well—behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J Phys Chem 1993; 97(40):10269-80.

[23]	Case DA, Aktulga HM, Belfon K, Cerutti DS, Cisneros GA, Cruzeiro VWD, et al. AmberTools. J Chem Inf Model 2023; 63(20):6183-91.

[24]	Li P, Merz Jr KM . MCPB.py: a python based metal center parameter builder. J Chem Inf Model 2016; 56(4):599-604.

[25]	Søndergaard CR, Olsson MHM, Rostkowski M, Jensen JH . Improved treatment of ligands and coupling effects in empirical calculation and rationalization of pK_a values. J Chem Theory Comput 2011;7(7):2284-95.

[26]	Kräutler V, van Gunsteren WF, Hünenberger PH . A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations. J Comput Chem 2001; 22(5):501-8.

[27]	Grant BJ, Skjaerven L, Yao XQ . The Bio3D packages for structural bioinformatics. Protein Sci 2021;30(1):20-30.

[28]	Zhou W, Huang R, Zhu Z, Zhang YPJ . Coevolution of both thermostability and activity of polyphosphate glucokinase from Thermobifida fusca YX. Appl Environ Microbiol 2018; 84(16):e01224—18.

[29]	Rashid N, Kanai T, Atomi H, Imanaka T . Among multiple phosphomannomutase gene orthologues, only one gene encodes a protein with phosphoglucomutase and phosphomannomutase activities in Thermococcus kodakaraensis . J Bacteriol 2004; 186(18):6070—6.

[30]	Bibel M, Brettl C, Gosslar U, Kriegshäuser G, Liebl W . Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima . FEMS Microbiol Lett 1998; 158(1):9-15.

[31]	Ewert J, Glück C, Strasdeit H, Fischer L, Stressler T . Influence of the metal ion on the enzyme activity and kinetics of PepA from Lactobacillus delbrueckii . Enzyme Microb Technol 2018; 110:69-78.

[32]	Nishitani Y, Maruyama D, Nonaka T, Kita A, Fukami TA, Mio T, et al. Crystal structures of N—acetylglucosamine—phosphate mutase, a member of the α—D—phosphohexomutase superfamily, and its substrate and product complexes. J Biol Chem 2006; 281(28):19740—7.

[33]	Lee Y, Villar MT, Artigues A, Beamer LJ . Promotion of enzyme flexibility by dephosphorylation and coupling to the catalytic mechanism of a phosphohexomutase. J Biol Chem 2014; 289(8):4674-82.

[34]	Figueroa CM, Asencion Diez MD, Ballicora MA, Iglesias AA . Structure, function, and evolution of plant ADP—glucose pyrophosphorylase. Plant Mol Biol 2022; 108(4—5):307-23.

[35]	Ubiparip Z, Beerens K, Franceus J, Vercauteren R, Desmet T . Thermostable alpha—glucan phosphorylases: characteristics and industrial applications. Appl Microbiol Biotechnol 2018; 102(19):8187-202.

[36]	Daniel RM, Peterson ME, Danson MJ, Price NC, Kelly SM, Monk CR, et al. The molecular basis of the effect of temperature on enzyme activity. Biochem J 2010; 425(2):353-60.

[37]	Liao M, Dong R, Li L, Liu X, Wang Y, Bai Y, et al. High production of maltooligosaccharides in the starch liquefaction process: a study on the hyperthermophilic mechanism of α—amylase. J Agric Food Chem 2023; 71(16):6480—9.

[38]	Liao H, Myung S, Zhang YHP . One—step purification and immobilization of thermophilic polyphosphate glucokinase fromThermobifida fusca YX: glucose—6—phosphate generation without ATP. Appl Microbiol Biotechnol 2012; 93(3):1109-17.

[39]	Dai Y, Zhang T, Jiang B, Mu W, Chen J, Hassanin HA . Dictyoglomus turgidum DSM 6724 α—glucan phosphorylase: characterization and its application in multi—enzyme cascade reaction for D—tagatose production. Appl Biochem Biotechnol 2021; 193(11):3719-31.

[40]	Fan L, Su H, Sun S, Zhang M, Jensen PR, Sheng X, et al. Protein engineering of an inositol—1—phosphate synthase based on the active—site redesign facilitates the biomanufacturing of myo—inositol from starch via in vitro synthetic enzymatic biosystem. J Agric Food Chem 2025; 73(30):18853-63.

[41]	Kang L, Li H, Lin K, Hu S, Liu S, Qiao Y, et al. Regioselectivity switching in CYP107Pdh—catalyzed VD3 hydroxylation: a structure—guided approach to improve calcidiol production. ACS Catal 2025; 15(5):4160-71.

[42]	Meng D, Wei X, Bai X, Zhou W, You C . Artificial in vitro synthetic enzymatic biosystem for the one—pot sustainable biomanufacturing of glucosamine from starch and inorganic ammonia. ACS Catal 2020; 10(23):13809—19.

[43]	Zhang P, Shao Z, Li L, Liu S, Yao J, Duan D . Molecular characterisation and biochemical properties of phosphomannomutase/phosphoglucomutase (PMM/PGM) in the brown seaweedSaccharina japonica . J Appl Phycol 2018; 30(4):2687-96.

[44]	Kourtoglou E, Anasontzis GE, Mamma D, Topakas E, Hatzinikolaou DG, Christakopoulos P . Constitutive expression, purification and characterization of a phosphoglucomutase from Fusarium oxysporum . Enzyme Microb Technol 2011; 48(3):217-24.

[45]	Wang J, Li Y, Lu M, Guo Q, Chen Y, Li Y, et al. Complete biotransformation of cellulose to starch in vitro. Natl Sci Rev 2026; 13(1):nwaf503.

[46]	Chiou WC, Lai WH, Cai YL, Du ML, Lai HM, Chen JC, et al. Gut microbiota—directed intervention with high—amylose maize ameliorates metabolic dysfunction in diet—induced obese mice. Food Funct 2022; 13(18):9481-95.

[47]	Sciarini LS, Rolland—Sabaté A, Guilois S, Decaen P, Leroy E, Le Bail P . Understanding the destructuring of starch in water—ionic liquid mixtures. Green Chem 2015; 17(1):291—9.

[48]	Correa Pinto B, Nunes NGDS, Favacho Ribeiro A, Viegas BM, Souza JADS, Del Nero J, et al. Arrowroot starch/ZnO nanoparticle nanocomposite films: thermal, morphological, and tensile properties. ACS Omega 2025; 10(27):28920—31.

[49]	Zhao F, Ren W, Zhang A, Jiang N, Liu W, Wang F . Effects of different amylose to amylopectin ratios on rumen fermentation and development in fattening lambs. Asian—Australas J Anim Sci 2018; 31(10):1611—8.

[50]	Feng Y, Yao M, Wang Y, Ding M, Zha J, Xiao W, et al. Advances in engineering UDP—sugar supply for recombinant biosynthesis of glycosides in microbes. Biotechnol Adv 2020; 41:107538.

[51]	Zhang S, Ni D, Zhu Y, Xu W, Zhang W, Mu W . A comprehensive review on the properties, production, and applications of functional glucobioses. Crit Rev Food Sci Nutr 2024; 64(33):13149—62.

[52]	Wu CH, Rismondo J, Morgan RML, Shen Y, Loessner MJ, Larrouy—Maumus G, et al. Bacillus subtilis YngB contributes to wall teichoic acid glucosylation and glycolipid formation during anaerobic growth. J Biol Chem 2021; 296:100384.

[53]	Ronchera—Oms CL, Jiménez NV, Peidro J . Stability of parenteral nutrition admixtures containing organic phosphates. Clin Nutr 1995; 14(6):373-80.

[54]	Fujinaka H, Nakamura J, Kobayashi H, Takizawa M, Murase D, Tokimitsu I, et al. Glucose 1—phosphate increases active transport of calcium in intestine. Arch Biochem Biophys 2007; 460(2):152-60.