Engineering biomimetic ensembles for energy biomanufacturing: from local chemical constraints to functional performance

Abstract

The integration of renewable reducing power with biological C1 assimilation has established energy biomanufacturing as an important direction for low-carbon chemical synthesis under mild, and aqueous conditions. Yet pathway-level carbon, electron and cofactor demands are rarely matched at the spatial and temporal scales experienced by individual enzymes. The resulting local constraints remain obscured by bulk measurements. This review examines these constraints through four overlapping diagnostic lenses: transport, intermediate lifetime, resource allocation, and reaction selectivity. Natural protein shells, membrane-bounded spaces, multienzyme assemblies, and phase-separated domains provide mechanistic precedents for controlling molecular exchange, intermediate retention, cofactor distribution, and reactant concentration. Recent engineering studies have translated these principles into protein microcompartments, encapsulins, scaffolded enzyme assemblies, and biomolecular condensates, while related spatial-control principles have been extended to cell–material reaction environments. Comparison across these systems connects structural assembly with measurable changes in permeability, enzyme stoichiometry, local redox state, pathway flux, and sustained conversion. It also reveals coupled design constraints, because changes that improve retention or local enrichment may alter molecular exchange, cellular resource demand, or responsiveness to fluctuating inputs. Integrating quantitative characterization with kinetic, transport, and metabolic models provides a basis for developing and testing design rules for spatial organization in energy biomanufacturing.

Graphical abstract

Keywords

biomimetic ensembles / energy biomanufacturing / metabolic compartmentalization / compartmentalized biocatalysis / redox and electron transfer

References

[1] Claassens N J , Sousa D Z , Dos Santos V A , de Vos W M , van der Oost J . Harnessing the power of microbial autotrophy. Nature Reviews Microbiology, 2016, 14(11): 692–706

[2] Kornienko N , Zhang J Z , Sakimoto K K , Yang P D , Reisner E . Interfacing nature’s catalytic machinery with synthetic materials for semi-artificial photosynthesis. Nature Nanotechnology, 2018, 13(10): 890–899

[3] Puiggene O , Favoino G , Federici F , Partipilo M , Orsi E , Alván-Vargas M V G , Hernández-Sancho J M , Dekker N K , Ørsted E C , Bozkurt E U . et al. Seven critical challenges in synthetic one-carbon assimilation and their potential solutions. FEMS Microbiology Reviews, 2025, 49: fuaf011

[4] Claassens N J , Cotton C A R , Kopljar D , Bar-Even A . Making quantitative sense of electromicrobial production. Nature Catalysis, 2019, 2(5): 437–447

[5] Volk M J , Tran V G , Tan S I , Mishra S , Fatma Z , Boob A , Li H , Xue P , Martin T A , Zhao H. . Metabolic engineering: methodologies and applications.. Chemical Reviews, 2023, 123(9): 5521–5570

[6] Chowdhury C , Sinha S , Chun S , Yeates T O , Bobik T A . Diverse bacterial microcompartment organelles. Microbiology and Molecular Biology Reviews, 2014, 78(3): 438–468

[7] Kerfeld C A , Aussignargues C , Zarzycki J , Cai F , Sutter M . Bacterial microcompartments. Nature Reviews Microbiology, 2018, 16(5): 277–290

[8] Castellana M , Wilson M Z , Xu Y F , Joshi P , Cristea I M , Rabinowitz J D , Gitai Z , Wingreen N S. . Enzyme clustering accelerates processing of intermediates through metabolic channeling.. Nature Biotechnology, 2014, 32(10): 1011–1018

[9] Dueber J E , Wu G C , Malmirchegini G R , Moon T S , Petzold C J , Ullal A V , Prather K L J , Keasling J D . Synthetic protein scaffolds provide modular control over metabolic flux. Nature Biotechnology, 2009, 27: 753–759

[10] Wan L , Zhu Y , Ke J , Zhang W , Mu W . Compartmentalization of pathway sequential enzymes into synthetic protein compartments for metabolic flux optimization in Escherichia coli. Metabolic Engineering, 2024, 85: 167–179

[11] Bar-Even A , Noor E , Lewis N E , Milo R . Design and analysis of synthetic carbon fixation pathways. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(19): 8889–8894

[12] Stoll I K , Boukis N , Sauer J . Syngas fermentation to alcohols: reactor technology and application perspective. Chemie Ingenieur Technik, 2020, 92(1): 125–136

[13] Jakobson C M , Tullman-Ercek D , Slininger M F , Mangan N M . A systems-level model reveals that 1,2-propanediol utilization microcompartments enhance pathway flux through intermediate sequestration. Plos Computational Biology, 2017, 13(5): e1005525

[14] Mangan N , Brenner M . Systems analysis of the CO₂ concentrating mechanism in cyanobacteria. Elife, 2014, 3: e02043

[15] Sarkar D , Maffeo C , Sutter M , Aksimentiev A , Kerfeld C A , Vermaas J V . Atomic view of photosynthetic metabolite permeability pathways and confinement in synthetic carboxysome shells. Proceedings of the National Academy of Sciences of the United States of America, 2024, 121(45): e2402277121

[16] Lim S , Clark D S . Phase-separated biomolecular condensates for biocatalysis. Trends in Biotechnology, 2024, 42(4): 496–509

[17] Qian Z G , Huang S C , Xia X X . Synthetic protein condensates for cellular and metabolic engineering. Nature Chemical Biology, 2022, 18(12): 1330–1340

[18] Ceroni F , Algar R , Stan G B , Ellis T . Quantifying cellular capacity identifies gene expression designs with reduced burden. Nature Methods, 2015, 12(5): 415–8

[19] Rugbjerg P , Myling-Petersen N , Porse A , Sarup-Lytzen K , Sommer M O A . Diverse genetic error modes constrain large-scale bio-based production. Nature Communications, 2018, 9: 787

[20] Rugbjerg P , Sarup-Lytzen K , Nagy M , Sommer M O A . Synthetic addiction extends the productive life time of engineered Escherichia coli populations. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(10): 2347–52

[21] Cotton C A R , Claassens N J , Benito-Vaquerizo S , Bar-Even A . Renewable methanol and formate as microbial feedstocks. Current Opinion in Biotechnology, 2020, 62: 168–80

[22] Moon J , Poehlein A , Daniel R , Müller V . Redirecting electron flow in acetobacterium woodii enables growth on CO and improves growth on formate. Nature Communications, 2024, 15: 5424

[23] Yishai O , Lindner S N , de la Cruz J G , Tenenboim H , Bar-Even A . The formate bio-economy. Current Opinion in Chemical Biology, 2016, 35: 1–9

[24] Buckel W , Thauer R K . Flavin-based electron bifurcation, a new mechanism of biological energy coupling. Chemical Reviews, 2018, 118(7): 3862–86

[25] Schuchmann K , Müller V . Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nature Reviews Microbiology, 2014, 12(12): 809–21

[26] Dong H , Gu Y Y , Liu X Y , Li H Y , Liu C Q , Sun D Z , Qiu B , Li P , Xu H , Wu H . et al. Role of vht hydrogenase in electron uptake by methanosarcina barkeri from a cathode. Cell Reports Physical Science, 2025, 6(7): e106772

[27] Jahn M , Crang N , Gynnå A H , Kabova D , Frielingsdorf S , Lenz O , Charpentier E , Hudson E P . The energy metabolism of cupriavidus necator in different trophic conditions. Applied and Environmental Microbiology, 2024, 90(10): e0074824

[28] Rieth A J , Nocera D G . Hybrid inorganic-biological systems: Faradaic and quantum efficiency, necessary but not sufficient. Joule, 2020, 4(10): 2051–5

[29] Cowan A E , Hillers M , Rainaldi V , Collas F , Choudhary H , Zakaria B S , Bieberach G G , Carruthers D N , Grabovac M , Gin J W . et al. Fast growth and high-titer bioproduction from renewable formate via metal-dependent formate dehydrogenase in Escherichia coli. Nature Communications, 2025, 16: 5908

[30] Ning X , Liu L M , Lin R C , O'Shea R , Deng C , Xuan X X , Xia R , Wall D M , Murphy J D . Microbe-electrode interactions on biocathodes are facilitated through tip-enhanced electric fields during CO₂-fed microbial electrosynthesis. Cell Reports Physical Science, 2024, 5(11): 102262

[31] Du J , Liang H B , Zou Y B , Li B , Li X Y , Lin L . Boosting microbial CO₂ electroreduction by the biocompatible and electroactive bimetallic fe-mn oxide cathode for acetate production. ACS Sustainable Chemistry & Engineering, 2024, 12(42): 15659–70

[32] Zhang X Y , Song W T , Liu Y , Wang D D , Mao X W , Hu W P , Liu B . Dual atoms anchoring at microbe-semiconductor interfaces boost charge transfer for efficient biohydrogen production. Advanced Energy Materials, 2026, 16(15): e06772

[33] Sheng Y K , Guo F , Guo B C , Wang N , Sun Y Y , Liu H , Feng X , Han Q , Yu Y , Li C . Light-driven CO₂ reduction with a surface-displayed enzyme cascade-C₃N₄ hybrid. ACS Synthetic Biology, 2023, 12(9): 2715–24

[34] Fan J P , Wang H T , Liao G D , Song C H , Zou J . Potassium-doped g-C₃N₄/nitrogen-doped g-C₃N₄ step-scheme homojunction for enhanced H₂ evolution photocatalysis. Science China-Technological Sciences, 2025, 68(6): 1620206

[35] Liu J , Xu B C , Mao M Q , Yang L , Shang C Q , Miao Y P , Peng X , Huang Z . Bimetallic electronic coupling engineering in CO-Fe-O heterostructure enabling industrial-grade alkaline hydrogen evolution at 2 A∙cm⁻². Science China-Technological Sciences, 2025, 68(9): 1920208

[36] Sampson E M , Bobik T A . Microcompartments for b12-dependent 1,2-propanediol degradation provide protection from DNA and cellular damage by a reactive metabolic intermediate. Journal of Bacteriology, 2008, 190(8): 2966–71

[37] Zhu C , Chen Y , Sun W , Li J , Liu H , Peng J , Bai Y , Gonzalez R , Tan Z. . Repair of DNA and protein damages caused by formaldehyde improves methanol assimilation.. Fundamental Research, 2025,

[38] Liu H Y , Chen Y , Li J , Zhu C , Peng J H , Gonzalez R , Bai Y , Tan Z . Scavenging intracellular reactive oxygen species to boost methanol assimilation. Chemical Engineering Journal, 2025, 516: 164002

[39] Penrod J T , Roth J R . Conserving a volatile metabolite: a role for carboxysome-like organelles in salmonella enterica. Journal of Bacteriology, 2006, 188(8): 2865–74

[40] Chen H Z , Xia A , Huang Y , Ji J Y , Zhang J M , Zhu X Q , Zhu X , Liao Q . Engineering a semi-artificial photosynthetic biofilm for robust and high-efficiency CO₂-to-methane conversion. Chemical Science, 2026, 17(12): 5857–68

[41] Liew F E , Nogle R , Abdalla T , Rasor B J , Canter C , Jensen R O , Wang L , Strutz J , Chirania P , De Tissera S . et al. Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale. Nature Biotechnology, 2022, 40: 335–44

[42] Boto S T , Gerges K , Bardl B , Rosenbaum M A . Evaluation of yeast alcohol acetyltransferases for ethyl acetate production in clostridium ljungdahlii. Engineering in Life Sciences, 2025, 25: e202400076

[43] Mahamkali V , Valgepea K , Lemgruber R D P , Plan M , Tappel R , Köpke M , Simpson S D , Nielsen L K , Marcellin E . Redox controls metabolic robustness in the gas-fermenting acetogen clostridium autoethanogenum. Proceedings of the National Academy of Sciences of the United States of America, 2020, 117(23): 13168–13175

[44] Grimalt-Alemany A , Etler C , Asimakopoulos K , Skiadas I , Gavala H N . Orp control for boosting ethanol productivity in gas fermentation systems and dynamics of redox cofactor NADH/NAD⁺ plus under oxidative stress. Journal of CO₂ Utilization, 2021, 50: 101589

[45] Oh S , Jeong J , Park B , Kang B , Kim J Y , Park S , Lee D H , Jung S , Lee M , Lee W . et al. Acetogenesis to ethanologenesis: facilitating nadh oxidation via reductive acetate uptake. Trends in Biotechnology, 2025, 43(3): 696–714

[46] Mangan N M , Flamholz A , Hood R D , Milo R , Savage D F . pH determines the energetic efficiency of the cyanobacterial CO₂ concentrating mechanism. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(36): E5354–E5362

[47] Flamholz A I , Dugan E , Blikstad C , Gleizer S , Ben-Nissan R , Amram S , Antonovsky N , Ravishankar S , Noor E , Bar-Even A . et al. Functional reconstitution of a bacterial CO₂ concentrating mechanism in Escherichia coli. Elife, 2020, 9: e59882

[48] Szyszka T N , Wijaya D S , Siddiquee R , Loustau A , Rhodes T , Paul N , Whitney S M , Lau Y H . Reprogramming encapsulins into modular carbon-fixing nanocompartments. Nature Communications, 2025, 16: 9493

[49] Page C C , Moser C C , Chen X X , Dutton P L . Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature, 1999, 402(6757): 47–52

[50] Edwards M J , White G F , Butt J N , Richardson D J , Clarke T A . The crystal structure of a biological insulated transmembrane molecular wire. Cell, 2020, 181(3): 665–673.e10

[51] Wang F B , Gu Y Q , O'Brien J P , Yi S M , Yalcin S E , Srikanth V , Shen C , Vu D , Ing N L , Hochbaum A I . et al. Structure of microbial nanowires reveals stacked hemes that transport electrons over micrometers. Cell, 2019, 177(2): 361–369

[52] Yalcin S E , O'Brien J P , Gu Y Q , Reiss K , Yi S M , Jain R , Srikanth V , Dahl P J , Huynh W , Vu D . et al. Electric field stimulates production of highly conductive microbial OmcZ nanowires. Nature Chemical Biology, 2020, 16(10): 1136–42

[53] Portela P C , Shipps C C , Shen C , Srikanth V , Salgueiro C A , Malvankar N S . Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE. Nature Communications, 2024, 15: 2434

[54] Kotloski N J , Gralnick J A . Flavin electron shuttles dominate extracellular electron transfer by shewanella oneidensis. Mbio, 2013, 4(1): e00553–12

[55] Okamoto A , Hashimoto K , Nealson K H , Nakamura R . Rate enhancement of bacterial extracellular electron transport involves bound flavin semiquinones. Proceedings of the National Academy of Sciences of the United States of America, 2013, 110(19): 7856–7861

[56] Okamoto A , Kalathil S , Deng X , Hashimoto K , Nakamura R , Nealson K H . Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH. Scientific Reports, 2014, 4: 5628

[57] Raza S , Sarkar D , Chan L J G , Mae J , Sutter M , Petzold C J , Kerfeld C A , Ralston C Y , Gupta S , Vermaas J V . Comparative pore structure and dynamics for bacterial microcompartment shell protein assemblies in sheets or shells. ACS Omega, 2024, 9(33): 35503–36614

[58] Sweetlove L J , Fernie A R . The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nature Communications, 2018, 9: 2136

[59] An S , Kumar R , Sheets E D , Benkovic S J . Reversible compartmentalization of de novo purine biosynthetic complexes in living cells. Science, 2008, 320(5872): 103–106

[60] Pareek V , Tian H , Winograd N , Benkovic S J . Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells. Science, 2020, 368(6488): 283–90

[61] Kumar A , Kremp F , Roth J , Freibert S A , Müller V , Schuller J M . Molecular architecture and electron transfer pathway of the stn family transhydrogenase. Nature Communications, 2023, 14: 5484

[62] Oltrogge L M , Chaijarasphong T , Chen A W , Bolin E R , Marqusee S , Savage D F . Multivalent interactions between CsoS2 and rubisco mediate α-carboxysome formation. Nature Structural & Molecular Biology, 2020, 27: 281–287

[63] Sun Y Q , Sheng Y W , Ni T , Ge X W , Sarsby J , Brownridge P J , Li K , Hardenbrook N , Dykes G F , Rockliffe N . et al. Rubisco packaging and stoichiometric composition of the native β-carboxysome in synechococcus elongatus pcc7942. Plant Physiology, 2025, 197: kiae665

[64] Kilgore H R , Mikhael P G , Overholt K J , Boija A , Hannett N M , Van Dongen C , Lee T I , Chang Y T , Barzilay R , Young R A . Distinct chemical environments in biomolecular condensates. Nature Chemical Biology, 2024, 20: 291–301

[65] Thody S A , Clements H D , Baniasadi H , Lyon A S , Sigman M S , Rosen M K . Small-molecule properties define partitioning into biomolecular condensates. Nature Chemistry, 2024, 16: 1794–802

[66] Wang H , Yan X , Aigner H , Bracher A , Nguyen N D , Hee W Y , Long B M , Price G D , Hartl F U , Hayer-Hartl M . Rubisco condensate formation by CcmM in β-carboxysome biogenesis. Nature, 2019, 566(7742): 131–5

[67] Lee D , Walls M T , Siu K H , Dai Y F , Xu K , Brangwynne C P , Chilkoti A , Avalos J L , You L . Principles of metabolic pathway control by biomolecular condensates in cells. Nature Chemical Engineering, 2025, 2(3): 198–208

[68] Sutter M , Greber B , Aussignargues C , Kerfeld C A . Assembly principles and structure of a 6.5-MDa bacterial microcompartment shell. Science, 2017, 356(6344): 1293–1297

[69] Liu S R , Cai L F , Wang L Y , Yi X F , Peng Y J , He N , Wu X , Wang Y P . Polydopamine coating on individual cells for enhanced extracellular electron transfer. Chemical Communications, 2019, 55(71): 10535–10538

[70] Lin S , Wang T L , Tao Z Y , Li Z H , Li S S , Liu X M , Liu J , Huang X . Shewanella oneidensis-based artificial conductive micro-niche for hydrogen augmentation. Chemical Engineering Journal, 2024, 488: 150850

[71] Liu Q J , Xu W L , Ding Q R , Zhang Y , Zhang J Q , Zhang B C , Yu H , Li C , Dai L , Zhong C . et al. Engineering shewanella oneidensis-carbon felt biohybrid electrode decorated with bacterial cellulose aerogel-electropolymerized anthraquinone to boost energy and chemicals production. Advanced Science, 2024, 11(39): 2407599

[72] Mouhib M , Reggente M , Li L , Schuergers N , Boghossian A A . Extracellular electron transfer pathways to enhance the electroactivity of modified Escherichia coli. Joule, 2023, 7(9): 2092–2106

[73] Mouhib M , Reggente M , Wang H X , Roullier C , Boghossian A A . Flavin biosynthesis enhances extracellular electron transfer in bioengineered Escherichia coli. Advanced Science, 2026, 13(2): 2412230

[74] Luo B F , Wang Y Z , Li D , Shen H Q , Xu L X , Fang Z , Xia Z , Ren J , Shi W , Yong Y C . A periplasmic photosensitized biohybrid system for solar hydrogen production. Advanced Energy Materials, 2021, 11(19): 2100256

[75] Zhu X Y , Xu Z J , Tang H T , Nie L H , Nie R , Wang R F , Liu X , Huang X . Photosynthesis-mediated intracellular biomineralization of gold nanoparticles inside cells towards hydrogen boosting under green light. Angewandte Chemie-International Edition, 2023, 62(33): e202308437

[76] Shao S N , Wu J J , Guo Y M , He Y , Luo N C , Li Z J , Lee S Y , Tan T , Lv Y . Spatial organization of biohybrids governing electron utilization in microbial electrosynthesis. Journal of Energy Chemistry, 2026, 122: 33–45

[77] Tao Z Y , Lin S , Wang S L , Li H , Li S S , Huang X , Liu J . Enhanced methanol to hydrogen transformation via formate self-augment metabolism in a genetic engineered hybrid biosystem. Fuel, 2025, 386: 134271

[78] Cobb S J , Dharani A M , Oliveira A R , Pereira I A C , Reisner E . Carboxysome-inspired electrocatalysis using enzymes for the reduction of CO₂ at low concentrations. Angewandte Chemie-International Edition, 2023, 62(26): e202218782

[79] Lawrence A D , Frank S , Newnham S , Lee M J , Brown I R , Xue W F , Rowe M L , Mulvihill D P , Prentice M B , Howard M J . et al. Solution structure of a bacterial microcompartment targeting peptide and its application in the construction of an ethanol bioreactor. ACS Synthetic Biology, 2014, 3(7): 454–465

[80] Lee M F S , Jakobson C M , Tullman-Ercek D . Evidence for improved encapsulated pathway behavior in a bacterial microcompartment through shell protein engineering. ACS Synthetic Biology, 2017, 6(10): 1880–1891

[81] Mills C E , Waltmann C , Archer A G , Kennedy N W , Abrahamson C H , Jackson A D , Roth E W , Shirman S , Jewett M C , Mangan N M . et al. Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology. Nature Communications, 2022, 13: 3746

[82] Ferlez B , Sutter M , Kerfeld C A . A designed bacterial microcompartment shell with tunable composition and precision cargo loading. Metabolic Engineering, 2019, 54: 286–291

[83] Hagen A , Sutter M , Sloan N , Kerfeld C A . Programmed loading and rapid purification of engineered bacterial microcompartment shells. Nature Communications, 2018, 9: 2881

[84] Tefft N M , Wang Y L , Jussupow A , Feig M , Teravest M A . Controlled enzyme cargo loading in engineered bacterial microcompartment shells. Biochemistry, 2025, 64(6): 1285–1292

[85] Young E J , Kirst H , Dwyer M E , Vermaas J V , Kerfeld C A . Quantitative measurement of molecular permeability to a synthetic bacterial microcompartment shell system. ACS Synthetic Biology, 2025, 14(5): 1405–1413

[86] Giessen T W , Silver P A . Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nature Microbiology, 2017, 2: 17029

[87] Kwon S , Andreas M P , Giessen T W . Structure and heterogeneity of a highly cargo-loaded encapsulin shell. Journal of Structural Biology, 2023, 215(4): 108022

[88] Ji M Q , Yao B H , Zhou J Y , Wang Y Z , Ding Q . Engineering a silk protein-mediated customizable compartment for modular metabolic synthesis. ACS Synthetic Biology, 2024, 13(12): 4180–4190

[89] Ke J T , Wan L , Chen M Q , Lv Y Z , Liu Y L , Zhu Y Y , Zhang W , Mu W . Design of a diblock-based membraneless organelle system for metabolic process control. Chemical Engineering Journal, 2025, 506: 160239

[90] Wan L , Ke J T , Zhu Y Y , Zhang W L , Mu W M . Intracellular construction of organelle-like compartments facilitates metabolic flux in Escherichia coli. Journal of Agricultural and Food Chemistry, 2024, 72(37): 20582–20591

[91] Yu W W , Jin K , Wang D D , Wang N K , Li Y Y , Liu Y F , Li J , Du G , Lv X , Chen J . et al. De novo engineering of programmable and multi-functional biomolecular condensates for controlled biosynthesis. Nature Communications, 2024, 15: 7989

[92] Pan F , Zu H , Zhu Y J , Qian Z G , Xia X X . Spatiotemporal organization of functional cargoes by light-switchable condensation in Escherichia coli cells. JACs Au, 2024, 4(4): 1480–1488

[93] Su D Y , Qi J R , Liu X M , Wang L , Zhang H , Xie H , Huang X . Enzyme-modulated anaerobic encapsulation of chlorella cells allows switching from O₂ to H₂ production. Angewandte Chemie-International Edition, 2019, 58(12): 3992–3995

[94] Wang S L , Xu Z J , Lin S , Liu X M , Wang L , Huang X . Polymer-chlorella cells conjugating with aggregation-induced functionality switch towards hydrogen evolution. Science China-Technological Sciences, 2020, 63(8): 1416–1425

[95] Li S S , Xu Z J , Lin S , Li L X , Huang Y , Qiao X , Huang X . Temperature modulated sustainable on/off photosynthesis switching of microalgae towards hydrogen evolution. Chemical Science, 2024, 15(16): 6141–6150

[96] Li S S , Li L X , Tao Z Y , Lin S , Huang X . Efficient and sustained photosynthetic hydrogen production by algae under high light intensity. Trends in Biotechnology, 2025, 43(8): 2073–2086

[97] Tao Z Y , Chen D Q , Li B Y , Li L X , Lin S , Li S S , Huang X . Core-shell bio-gel microreactor for efficient hydrogen production through multi-bacterial synergistic catalysis. Chemical Engineering Journal, 2025, 514: 163308

[98] Li B Y , Xu Z J , Wang R F , Nie R , Tao Z Y , Huang X . Mineralizing biofilm towards sustainable conversion of plastic wastes to hydrogen. Angewandte Chemie-International Edition, 2025, 64(4): e202416577

[99] Battle A L L , Barrett A W , Fricker M D , Sweetlove L J . Localising enzymes to biomolecular condensates increases their accumulation and benefits engineered metabolic pathway performance in nicotiana benthamiana. Plant Biotechnology Journal, 2026, 24: 171–186

[100] Ding Q , Su X Y , Yao B H , Zhao Y N , Wu J Y , Zhou J Y , Ji M , Wang Y . Biomolecular condensate-based artificial organelle for driving compartmentalized flux control. ACS Synthetic Biology, 2025, 14(6): 2044–2052

[101] Razaghi-Moghadam Z , Babadi F S , Nikoloski Z . Harnessing the optimization of enzyme catalytic rates in engineering of metabolic phenotypes. Plos Computational Biology, 2024, 20(11): e1012576

[102] Yadav N S , Raza S , Wang Y L , Landa J F , Hegg E L , Hausinger R P , Vermaas J V . Quantifying selective metabolite transport for the bacterial microcompartment from Haliangium ochraceum with molecular dynamics simulations. Nanoscale, 2026, 18(11): 6092–6104

[103] Mu X Y , Evans T D , Zhang F Z . ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction. Nature Communications, 2024, 15: 5299

[104] Scherschel M , Niemeier J O , Jacobs L , Hoffmann M D A , Diederich A , Bell C , Höhne P , Raetz S , Kroll J B , Steinbeck J . et al. A family of NADPH/NADP⁺ biosensors reveals in vivo dynamics of central redox metabolism across eukaryotes. Nature Communications, 2024, 15: 10704

[105] Sutter M , Utschig L M , Niklas J , Paul S , Kahan D N , Gupta S , Poluektov O G , Ferlez B H , Tefft N M , TerAvest M A . et al. Electrochemical cofactor recycling of bacterial microcompartments. Proceedings of the National Academy of Sciences of the United States of America, 2024, 121(49): e2414220121

[106] Savigny J , Shabestary K , Portela M , Klemm C , Sum Y , Hapeta P , Storch M , Rowlands C , Ledesma-Amaro R . Single cell profiling framework reveals metabolic subpopulations as drivers of bioproduction heterogeneity. Nature Communications, 2026, 17: 645

[107] Lee M , Moon H C , Jeong H , Kim D W , Park H Y , Shin Y . Optogenetic control of mRNA condensation reveals an intimate link between condensate material properties and functions. Nature Communications, 2024, 15: 3216

[108] Jin K , Yu W W , Liu Y F , Li J H , Du G C , Chen J , Liu L , Lv X . Light-induced programmable solid-liquid phase transition of biomolecular condensates for improved biosynthesis. Trends in Biotechnology, 2025, 43(6): 1403–1424

[109] Jerez-Longres C , Weber W . Metabolite-responsive control of transcription by phase separation-based synthetic organelles. ACS Synthetic Biology, 2025, 14(3): 711–718

[110] Zhang R , Yang W F , Zhang R X , Godar A , Rijal S , Youssef A , Nielsen D R , Zheng W , Tian X J . Phase separation to buffer growth-mediated dilution in synthetic circuits. Cell, 2025, 188(26): 7413–7427

[111] Beentjes M , Ortega-Arbulú A S , Meiners C , Kremling A , Pflüger-Grau K . Going beyond size: exploring the metabolic burden in pseudomonas putida during heterologous protein production. Synthetic Biology, 2026, 11(1): ysag004

[112] Mannan A A , Darlington A P S , Tanaka R J , Bates D G . Design principles for engineering bacteria to maximise chemical production from batch cultures. Nature Communications, 2025, 16: 279

[113] Mao J W , Zhang H Y , Chen Y , Wei L , Liu J , Nielsen J , Chen Y , Xu N . Relieving metabolic burden to improve robustness and bioproduction by industrial microorganisms. Biotechnology Advances, 2024, 74: 108401

[114] Snoeck S , Guidi C , De Mey M . ‘Metabolic burden’ explained: stress symptoms and its related responses induced by (over)expression of (heterologous) proteins in Escherichia coli. Microbial Cell Factories, 2024, 23: 96

[115] Zuo X B , Jussupow A , Ponomarenko N S , Grant T D , Tefft N M , Yadav N S , Range K L , Ralston C Y , TerAvest M A , Sutter M . et al. Structure characterization of bacterial microcompartment shells via X-ray scattering and coordinate modeling: evidence for adventitious capture of cytoplasmic proteins. ACS Applied Biology Materials, 2025, 8(3): 2090–2103

[116] Hoang Y , Azaldegui C A , Dow R E , Ghalmi M , Biteen J S , Vecchiarelli A G . An experimental framework to assess biomolecular condensates in bacteria. Nature Communications, 2024, 15: 3222

[117] Branon T C , Bosch J A , Sanchez A D , Udeshi N D , Svinkina T , Carr S A , Feldman J L , Perrimon N , Ting A Y . Efficient proximity labeling in living cells and organisms with turboid. Nature Biotechnology, 2018, 36(9): 880–887

[118] Rhee H W , Zou P , Udeshi N D , Martell J D , Mootha V K , Carr S A , Ting A Y . Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science, 2013, 339(6125): 1328–1331

[119] Roux K J , Kim D I , Raida M , Burke B . A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. Journal of Cell Biology, 2012, 196(6): 801–810

[120] Christoforou A , Mulvey C M , Breckels L M , Geladaki A , Hurrell T , Hayward P C , Naake T , Gatto L , Viner R , Arias A M . A draft map of the mouse pluripotent stem cell spatial proteome. Nature Communications, 2016, 7: 9992

[121] Bayne L , Ulijn R V , Halling P J . Effect of pore size on the performance of immobilised enzymes. Chemical Society Reviews, 2013, 42(23): 9000–9010

[122] Diamanti E , Santiago-Arcos J , Grajales-Hernández D , Czarnievicz N , Comino N , Llarena I , Di Silvio D , Cortajarena A L , López-Gallego F . Intraparticle kinetics unveil crowding and enzyme distribution effects on the performance of cofactor-dependent heterogeneous biocatalysts. ACS Catalysis, 2021, 11(24): 15051–15067

[123] Liese A , Hilterhaus L . Evaluation of immobilized enzymes for industrial applications. Chemical Society Reviews, 2013, 42(15): 6236–6249

[124] Stoffel F , Papp M , Gil-Garcia M , Küffner A M , Benitez-Mateos A I , Jacquat R P B , Galvanetto N , Faltova L , Arosio P . Enhancement of enzymatic activity by biomolecular condensates through ph buffering. Nature Communications, 2025, 16: 6368

[125] Testa A , Dindo M , Rebane A A , Nasouri B , Style R W , Golestanian R , Dufresne E R , Laurino P . Sustained enzymatic activity and flow in crowded protein droplets. Nature Communications, 2021, 12: 6293

[126] Sprague B L , McNally J G . Frap analysis of binding: proper and fitting. Trends in Cell Biology, 2005, 15(2): 84–91

[127] Kelley F M , Ani A , Pinlac E G , Linders B , Favetta B , Barai M , Ma Y , Singh A , Dignon G L , Gu Y . Controlled and orthogonal partitioning of large particles into biomolecular condensates. Nature Communications, 2025, 16: 3521

[128] Bennett B D , Kimball E H , Gao M , Osterhout R , Van Dien S J , Rabinowitz J D . Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nature Chemical Biology, 2009, 5(8): 593–599

[129] Hackett S R , Zanotelli V R T , Xu W X , Goya J , Park J O , Perlman D H , Gibney P A , Botstein D , Storey J D , Rabinowitz J D . Systems-level analysis of mechanisms regulating yeast metabolic flux. Science, 2016, 354(6311): aaf2786

[130] Park J O , Rubin S A , Xu Y F , Amador-Noguez D , Fan J , Shlomi T , Rabinowitz J D. . Metabolite concentrations, fluxes, and free energies imply efficient enzyme usage.. Nature Chemical Biology, 2016, 12(7): 482–489

[131] Antoniewicz M R . Methods and advances in metabolic flux analysis: a mini-review. Journal of Industrial Microbiology & Biotechnology, 2015, 42(3): 317–325

[132] Sauer U . Metabolic networks in motion: 13C-based flux analysis. Molecular Systems Biology, 2006, 2: 62

[133] Zamboni N , Fendt S M , Rühl M , Sauer U . 13C-based metabolic flux analysis. Nature Protocols, 2009, 4(6): 878–892

[134] Yuan J , Bennett B D , Rabinowitz J D . Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nature Protocols, 2008, 3(8): 1328–1340

[135] Dai X F , Shen L . Advances and trends in omics technology development. Frontiers in Medicine, 2022, 9: 911861

[136] Zenobi R . Single-cell metabolomics: analytical and biological perspectives. Science, 2013, 342(6163): 1243259

[137] Orth J D , Thiele I , Palsson B O. . What is flux balance analysis?. Nature Biotechnology, 2010, 28(3): 245–248

[138] Chen Y , Gustafsson J , Rangel A T , Anton M , Domenzain I , Kittikunapong C , Li F , Yuan L , Nielsen J , Kerkhoven E J . Reconstruction, simulation, and analysis of enzyme-constrained metabolic models using gecko toolbox 3.0. Nature Protocols, 2024, 19: 629–667

[139] Henry C S , Broadbelt L J , Hatzimanikatis V . Thermodynamics-based metabolic flux analysis. Biophysical Journal, 2007, 92(5): 1792–1805

[140] Mahadevan R , Edwards J S , Doyle F J III . Dynamic flux balance analysis of diauxic growth in Escherichia coli. Biophysical Journal, 2002, 83(3): 1331–1340

[141] Zhang Y J , Pyo A G , Kliegman R , Jiang Y Y , Brangwynne C P , Stone H A , Wingreen N S . The exchange dynamics of biomolecular condensates. Elife, 2024, 12: RP91680

[142] Gutenkunst R N , Waterfall J J , Casey F P , Brown K S , Myers C R , Sethna J P . Universally sloppy parameter sensitivities in systems biology models. Plos Computational Biology, 2007, 3(10): 1871–1878

[143] Cook M , Anastasakis S , Heydarabadipour A , Shin J , Burbano D A , Carothers J M , Sauro H M . A standardized workflow for kinetic metabolic model curation and dissemination. Plos Computational Biology, 2026, 22(4): e1014227

[144] Puiman L , Benalcázar E A , Picioreanu C , Noorman H J , Haringa C . High-resolution computation predicts that low dissolved co concentrations and Co gradients promote ethanol production at industrial-scale gas fermentation. Biochemical Engineering Journal, 2024, 207: 109330

[145] Park C , Ha G S , Kim M , Bae S , Jeong Y , Sim M G , Logan B E , Baek G . A data-driven approach to evaluate performance trends across different scales of bioelectrochemical systems. Chemical Engineering Journal, 2025, 519: 164918

[146] Soriano-Jerez Y , Gallardo-Rodríguez J J , López-Rosales L , García-Camacho F , Bressy C , Molina-Grima E , Cerón-García M C . Preventing biofouling in microalgal photobioreactors. Bioresource Technology, 2024, 407: 131125

[147] Guerrero-Sodric O , Baeza J A , Guisasola A . Enhancing bioelectrochemical hydrogen production from industrial wastewater using ni-foam cathodes in a microbial electrolysis cell pilot plant. Water Research, 2024, 256: 121616

[148] Jiang J Y , Du L , Si B C , Kawale H D , Wang Z X , Summers S , Lopez-Ruiz J A , Li S , Zhang Y , Ren Z J . Pilot microbial electrolysis cell closes the hydrogen loop for hydrothermal wet waste conversion to jet fuel. Water Research, 2025, 268: 122644

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