High-Temperature Catalytic Platform Powered by Thermophilic Microorganisms and Thermozymes

Abstract

Thermophilic microorganisms, capable of thriving under high temperatures, are emerging as key platforms for next-generation industrial biotechnology (NGIB), driving innovations in lignin biorefining, bioplastics synthesis, biodiesel production, and environmental remediation. Enzymes derived from thermophilic microorganisms, thermozymes, exhibit remarkable stability and efficiency under extreme conditions, making them highly suitable for diverse industrial applications. This review highlights recent advances in leveraging thermophilic microorganisms and thermozymes for high-temperature catalysis, focusing on their economic and environmental benefits. It also emphasizes progress in high-throughput screening and artificial intelligence (AI), which have revolutionized the bioprospecting, engineering, and application potential of thermozymes. Challenges and potential solutions for industrial implementation of high-temperature catalytic platforms are also discussed, highlighting their transformative impact on sustainable biotechnology.

Keywords

Thermophiles / Thermozymes / NGIB / Synthetic biology / Machine learning / High-throughput screening

Acknowledgments

Our experiments related to this review were carried out in Biological & Medical Engineering Core Facilities of Beijing Institute of Technology.

Author Contributions

J.L.: Investigation, Writing—original draft, Visualization, Writing—review & editing. L.S.: Conceptualization, Writing—original draft, Writing—Review & Editing, Project administration. Y.-X.H.: Conceptualization, Writing—Review & Editing, Project administration.

Ethics Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Funding

This work was supported by Hebei Natural Science Foundation (C2023105022), Science and Technology Program of Tangshan (23130228E), the Beijing Institute of Technology Research Fund Program for Young Scholars, and the BIT Research and Innovation Promoting Project (Grant No. 2024YCXY048).

Declaration of Competing Interest

The authors declare no competing financial interest.

References

[1] Kircher M. Bioeconomy—Present status and future needs of industrial value chains. New Biotechnol. 2021, 60, 96-104.

[2] Zhang Y-HP, Sun J, Ma Y. Biomanufacturing: history and perspective. J. Ind. Microbiol. Biotechnol. 2017, 44, 773-784.

[3] Scown CD, Keasling JD.Sustainable manufacturing with synthetic biology. Nat. Biotechnol. 2022, 40, 304-307.

[4] Chen G-Q, Jiang X-R. Next generation industrial biotechnology based on extremophilic bacteria. Curr. Opin. Biotechnol. 2018, 50, 94-100.

[5] Yu L-P, Wu F-Q, Chen G-Q. Next-Generation Industrial Biotechnology-Transforming the Current Industrial Biotechnology into Competitive Processes. Biotechnol. J. 2019, 14, 1800437.

[6] Xiao Z, Wang X, Huang Y, Huo F, Zhu X, Xi L, et al. Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain. Biotechnol. Biofuels 2012, 5, 88.

[7] Karim AA, Martínez-Cartas ML, Cuevas-Aranda M. Fermentation of Sugar by Thermotolerant Hansenula polymorpha Yeast for Ethanol Production. Fermentation 2024, 10, 260. doi:10.3390/fermentation10050260.

[8] Zeldes BM, Keller MW, Loder AJ, Straub CT, Adams MWW, Kelly RM. Extremely thermophilic microorganisms as metabolic engineering platforms for production of fuels and industrial chemicals. Front. Microbiol. 2015, 6, 1209.

[9] Ye J-W, Lin Y-N, Yi X-Q, Yu Z-X, Liu X, Chen G-Q. Synthetic biology of extremophiles: A new wave of biomanufacturing. Trends Biotechnol. 2023, 41, 342-357.

[10] Jing X, Evangelista Falcon W, Baudry J, Serpersu EH. Thermophilic Enzyme or Mesophilic Enzyme with Enhanced Thermostability: Can We Draw a Line? J. Phys. Chem. B 2017, 121, 7086-7094.

[11] Atalah J, Cáceres-Moreno P, Espina G, Blamey JM. Thermophiles and the applications of their enzymes as new biocatalysts. Bioresour. Technol. 2019, 280, 478-488.

[12] Dumorné K, Córdova DC, Astorga-Eló M, Renganathan P. Extremozymes: A Potential Source for Industrial Applications. J. Microbiol. Biotechnol. 2017, 27, 649-659.

[13] Lee JA, Kim HU, Na J-G, Ko Y-S, Cho JS, Lee SY. Factors affecting the competitiveness of bacterial fermentation. Trends Biotechnol. 2023, 41, 798-816.

[14] Lieph R, Veloso FA, Holmes DS. Trends Microbiol. 2006, 14, 423-426.

[15] Leng H, Wang Y, Zhao W, Sievert SM, Xiao X. Identification of a deep-branching thermophilic clade sheds light on early bacterial evolution. Nat. Commun. 2023, 14, 4354.

[16] Arbab S, Ullah H, Khan MIU, Khattak MNK, Zhang J, Li K, et al. Diversity and distribution of thermophilic microorganisms and their applications in biotechnology. J. Basic. Microbiol. 2022, 62, 95-108.

[17] Narsing Rao MP, Luo Z-H, Dong Z-Y, Li Q, Liu B-B, Guo S-X, et al. Metagenomic analysis further extends the role of Chloroflexi in fundamental biogeochemical cycles. Environ. Res. 2022, 209, 112888.

[18] Lin L, Xu J. Dissecting and engineering metabolic and regulatory networks of thermophilic bacteria for biofuel production. Biotechnol. Adv. 2013, 31, 827-837.

[19] Zhu D, Adebisi WA, Ahmad F, Sethupathy S, Danso B, Sun J. Recent Development of Extremophilic Bacteria and Their Application in Biorefinery. Front. Bioeng. Biotechnol. 2020, 8, 843.

[20] Zhai Y, Li X, Bai Y, Jin Z, Svensson B. Maltogenic α-amylase hydrolysis of wheat starch granules: Mechanism and relation to starch retrogradation. Food Hydrocoll. 2022, 124, 107256.

[21] Febriani, Aura N, Kemala P, Saidi N, Iqbalsyah TM. Novel thermostable lipase produced by a thermo-halophilic bacterium that catalyses hydrolytic and transesterification reactions. Heliyon 2020, 6, e04520.

[22] Abro AA, Qureshi AS, Naqvi M, Khushk I, Jatt AN, Ali CH, et al. Lipase Production from alkalophilic-thermophilic-Ionic liquid tolerant Bacillus cereus using agricultural residues for its applications in biodiesel and detergents. Ind. Crops Prod. 2024, 220, 119208.

[23] Mohammadi S, Pol A, van Alen TA, Jetten MSM, Op den Camp HJM. Methylacidiphilum fumariolicum SolV, a thermoacidophilic ‘Knallgas’ methanotroph with both an oxygen-sensitive and -insensitive hydrogenase. ISME J. 2017, 11, 945-958.

[24] Pawar SS, van Niel EWJ. Thermophilic biohydrogen production: how far are we? Appl. Microbiol. Biotechnol. 2013, 97, 7999-8009.

[25] Morra S, Valetti F, Gilardi G. [FeFe]-hydrogenases as biocatalysts in bio-hydrogen production. Rend. Lincei 2017, 28, 183-194.

[26] Wang S, Lei H, Ji Z. Exploring Oxidoreductases from Extremophiles for Biosynthesis in a Non-Aqueous System. Int. J. Mol. Sci. 2023, 24, 6396. doi:10.3390/ijms24076396.

[27] Espina G, Atalah J, Blamey JM. Extremophilic Oxidoreductases for the Industry: Five Successful Examples with Promising Projections. Front. Bioeng. Biotechnol. 2021, 9, 710035.

[28] Artzi L, Bayer EA, Moraïs S. Cellulosomes: Bacterial nanomachines for dismantling plant polysaccharides. Nat. Rev. Microbiol. 2017, 15, 83-95.

[29] Guo Z-W, Ni Z-F, Zong M-H, Lou W-Y. Modular Metabolic Engineering of Bacillus licheniformis for Efficient 2,3-Butanediol Production by Consolidated Bioprocessing of Jerusalem Artichoke Tubers. ACS Sustain. Chem. Eng. 2022, 10, 9624-9634.

[30] Saini N, Pal K, Sujata, Deepak B, Mona S. Thermophilic algae: A new prospect towards environmental sustainability. J. Clean. Prod. 2021, 324, 129277.

[31] Wang J, Yin Y. Progress in microbiology for fermentative hydrogen production from organic wastes. Crit. Rev. Environ. Sci. Technol. 2019, 49, 825-865.

[32] Woon JM, Khoo KS, Al-Zahrani AA, Alanazi MM, Lim JW, Cheng CK, et al. Epitomizing biohydrogen production from microbes: Critical challenges vs opportunities. Environ. Res. 2023, 227, 115780.

[33] Taleb MA, Gomaa SK, Wahba MI, Zaki RA, El-Fiky AF, El-Refai HA, et al. Bioscouring of wool fibres using immobilized thermophilic lipase. Int. J. Biol. Macromol. 2022, 194, 800-810.

[34] Peng Y, Ma L, Xu P, Tao F. High-Performance Production of N-Acetyl-d-Neuraminic Acid with Whole Cells of Fast-Growing Vibrio natriegens via a Thermal Strategy. J. Agric. Food Chem. 2023, 71, 20198-20209.

[35] Li T, Chen X-B, Chen J-C, Wu Q, Chen G-Q. Open and continuous fermentation: Products, conditions and bioprocess economy. Biotechnol. J. 2014, 9, 1503-1511.

[36] Han X, Liu J, Wu Y, Yang Y, Tao F, Xu P. Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis. iScience 2023, 26, 106397.

[37] Ge Y, Li K, Li L, Gao C, Zhang L, Ma C, et al. Contracted but effective: production of enantiopure 2,3-butanediol by thermophilic and GRAS Bacillus licheniformis. Green Chem. 2016, 18, 4693-4703.

[38] Lü C, Ge Y, Cao M, Guo X, Liu P, Gao C, et al. Metabolic Engineering of Bacillus licheniformis for Production of Acetoin. Front. Bioeng. Biotechnol. 2020, 8, 125.

[39] Li L, Li K, Wang K, Chen C, Gao C, Ma C, et al. Efficient production of 2,3-butanediol from corn stover hydrolysate by using a thermophilic Bacillus licheniformis strain. Bioresour. Technol. 2014, 170, 256-261.

[40] Ohair J, Jin Q, Yu D, Wu J, Wang H, Zhou S, et al. Non-sterile fermentation of food waste using thermophilic and alkaliphilic Bacillus licheniformis YNP5-TSU for 2,3-butanediol production. Waste Manag. 2021, 120, 248-256.

[41] Liu J, Han X, Tao F, Xu P. Metabolic engineering of Geobacillus thermoglucosidasius for polymer-grade lactic acid production at high temperature. Bioresour. Technol. 2024, 393, 130164.

[42] Yang Z, Sun Q, Tan G, Zhang Q, Wang Z, Li C, et al. Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production. Microb. Biotechnol. 2021, 14, 363-373.

[43] Bu C-Y, Yan Y-X, Zou L-H, Zheng Z-J, Ouyang J. One-pot biosynthesis of furfuryl alcohol and lactic acid via a glucose coupled biphasic system using single Bacillus coagulans NL01. Bioresour. Technol. 2020, 313, 123705.

[44] Sun W, Jiang B, Zhao D, Pu Z, Bao Y. Integration of metabolic pathway manipulation and promoter engineering for the fine-tuned biosynthesis of malic acid in Bacillus coagulans. Biotechnol. Bioeng. 2021, 118, 2597-2608.

[45] Tian X, Liu X, Zhang Y, Chen Y, Hang H, Chu J, et al. Metabolic engineering coupled with adaptive evolution strategies for the efficient production of high-quality L-lactic acid by Lactobacillus paracasei. Bioresour. Technol. 2021, 323, 124549.

[46] Li Y, Hu J, Qu C, Chen L, Guo X, Fu H, et al. Engineered Thermoanaerobacterium aotearoense with nfnAB knockout for improved hydrogen production from lignocellulose hydrolysates. Biotechnol. Biofuels 2019, 12, 214.

[47] Yang X, Lai Z, Lai C, Zhu M, Li S, Wang J, et al. Efficient production of l-lactic acid by an engineered Thermoanaerobacterium aotearoensewith broad substrate specificity. Biotechnol. Biofuels 2013, 6, 124.

[48] Dai K, Qu C, Li X, Lan Y, Fu H, Wang J. Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity. Bioresour. Technol. 2024, 402, 130784.

[49] Dai K, Qu C, Feng J, Lan Y, Fu H, Wang J. Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT 27 for biofuels production from sucrose and molasses. Biotechnol. Biofuels Bioprod. 2023, 16, 155.

[50] Zhu M, Lu Y, Wang J, Li S, Wang X. Carbon Catabolite Repression and the Related Genes of ccpA, ptsH and hprK in Thermoanaerobacterium aotearoense. PLoS ONE 2015, 10, e0142121.

[51] Qu C, Zhang Y, Dai K, Fu H, Wang J. Metabolic engineering of Thermoanaerobacterium aotearoense SCUT27 for glucose and cellobiose co-utilization by identification and overexpression of the endogenous cellobiose operon. Biochem. Eng. J. 2021, 167, 107922.

[52] Akinosho H, Yee K, Close D, Ragauskas A. The emergence of Clostridium thermocellum as a high utility candidate for consolidated bioprocessing applications. Front. Chem. 2014, 2, 66.

[53] Lin PP, Mi L, Morioka AH, Yoshino KM, Konishi S, Xu SC, et al. Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum. Metab. Eng. 2015, 31, 44-52.

[54] Tian L, Conway PM, Cervenka ND, Cui J, Maloney M, Olson DG, et al. Metabolic engineering of Clostridium thermocellum for n-butanol production from cellulose. Biotechnol. Biofuels 2019, 12, 186.

[55] Seo H, Singh P, Wyman CE, Cai CM, Trinh CT. Rewiring metabolism of Clostridium thermocellum for consolidated bioprocessing of lignocellulosic biomass poplar to produce short-chain esters. Bioresour. Technol. 2023, 384, 129263.

[56] Xiong W, Reyes LH, Michener WE, Maness P-C, Chou KJ. Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously. Biotechnol. Bioeng. 2018, 115, 1755-1763.

[57] Singh B. Myceliophthora thermophila syn. Sporotrichum thermophile: A thermophilic mould of biotechnological potential. Crit. Rev. Biotechnol. 2016, 36, 59-69.

[58] Liu J, Chen M, Gu S, Fan R, Zhao Z, Sun W, et al. Independent metabolism of oligosaccharides is the keystone of synchronous utilization of cellulose and hemicellulose in Myceliophthora. PNAS Nexus 2024, 3, pgae053.

[59] Li J, Zhang Y, Li J, Sun T, Tian C. Metabolic engineering of the cellulolytic thermophilic fungus Myceliophthora thermophila to produce ethanol from cellobiose. Biotechnol. Biofuels 2020, 13, 23.

[60] Zhang Y, Sun T, Wu T, Li J, Hu D, Liu D, et al. Consolidated bioprocessing for bioethanol production by metabolically engineered cellulolytic fungus Myceliophthora thermophila. Metab. Eng. 2023, 78, 192-199.

[61] Gu S, Li J, Chen B, Sun T, Liu Q, Xiao D, et al. Metabolic engineering of the thermophilic filamentous fungus Myceliophthora thermophila to produce fumaric acid. Biotechnol. Biofuels 2018, 11, 323.

[62] Li J, Lin L, Sun T, Xu J, Ji J, Liu Q, et al. Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila. Metab. Eng. 2020, 61, 416-426.

[63] Li J, Tseng J, Tang D, Yong Y, Sun L, Huo Y-X. Upcycling C gas-derived resources in future food system. Resour. Conserv. Recycl. 2024, 210, 107827.

[64] Müller JEN, Heggeset TMB, Wendisch VF, Vorholt JA, Brautaset T. Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity production from methanol. Appl. Microbiol. Biotechnol. 2015, 99, 535-551.

[65] Brautaset T, Jakobsen ØM, Degnes KF, Netzer R, Nærdal I, Krog A, et al. Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for l-lysine production from methanol at 50 °C. Appl. Microbiol. Biotechnol. 2010, 87, 951-964.

[66] Klein VJ, Brito LF, Perez-Garcia F, Brautaset T, Irla M. Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol. Microb. Biotechnol. 2023, 16, 1011-1026.

[67] Holwerda EK, Olson DG, Ruppertsberger NM, Stevenson DM, Murphy SJL, Maloney MI, et al. Metabolic and evolutionary responses of Clostridium thermocellum to genetic interventions aimed at improving ethanol production. Biotechnol. Biofuels 2020, 13, 40.

[68] Mazzoli R, Olson DG, Lynd LR. Construction of lactic acid overproducing Clostridium thermocellum through enhancement of lactate dehydrogenase expression. Enzym. Microb. Technol. 2020, 141, 109645.

[69] Zhang H, Han L, Dong H. An insight to pretreatment, enzyme adsorption and enzymatic hydrolysis of lignocellulosic biomass: Experimental and modeling studies. Renew. Sustain. Energy Rev. 2021, 140, 110758.

[70] Wang M, Han J, Dunn JB, Cai H, Elgowainy A. Well-to-wheels energy use and greenhouse gas emissions of ethanol from corn, sugarcane and cellulosic biomass for US use. Environ. Res. Lett. 2012, 7, 045905.

[71] Hamann PRV, Noronha EF. Xylan-breakdown apparatus of Clostridium thermocellum. Cellulose 2022, 29, 7535-7553.

[72] Argyros DA, Tripathi Shital A, Barrett Trisha F, Rogers Stephen R, Feinberg Lawrence F, Olson Daniel G, et al. High Ethanol Titers from Cellulose by Using Metabolically Engineered Thermophilic, Anaerobic Microbes. Appl. Environ. Microbiol. 2011, 77, 8288-8294.

[73] Jiang Y, Jiang W, Xin F, Zhang W, Jiang M. Thermophiles: Potential chassis for lignocellulosic biorefinery. Trends Biotechnol. 2022, 40, 643-646.

[74] Kiyoshi K, Furukawa M, Seyama T, Kadokura T, Nakazato A, Nakayama S. Butanol production from alkali-pretreated rice straw by co-culture of Clostridium thermocellum and Clostridium saccharoperbutylacetonicum. Bioresour. Technol. 2015, 186, 325-328.

[75] Lu J, Lv Y, Jiang Y, Wu M, Xu B, Zhang W, et al. Consolidated Bioprocessing of Hemicellulose-Enriched Lignocellulose to Succinic Acid through a Microbial Cocultivation System. ACS Sustain. Chem. Eng. 2020, 8, 9035-9045.

[76] Pason P, Tachaapaikoon C, Panichnumsin P, Ketbot P, Waeonukul R, Kosugi A, et al. One-step biohydrogen production from cassava pulp using novel enrichment of anaerobic thermophilic bacteria community. Biocatal. Agric. Biotechnol. 2020, 27, 101658.

[77] de Vrije T, Bakker RR, Budde MAW, Lai MH, Mars AE, Claassen PAM. Efficient hydrogen production from the lignocellulosic energy crop Miscanthus by the extreme thermophilic bacteria Caldicellulosiruptor saccharolyticus and Thermotoga neapolitana. Biotechnol. Biofuels 2009, 2, 12.

[78] Jiang Y, Lu J, Lv Y, Wu R, Dong W, Zhou J, et al. Efficient hydrogen production from lignocellulosic feedstocks by a newly isolated thermophlic Thermoanaerobacterium sp. strain F6. Int. J. Hydrogen Energy 2019, 44, 14380-14386.

[79] Rodge SP, Shende KS, Patil NP. Polyhydroxyalkanoate biosynthesis and optimisation of thermophilic Geobacillus stearothermophilus strain K4E3_SPR_NPP. Extremophiles 2023, 27, 13.

[80] James-Pearson LF, Dudley KJ, Te’o VSJ, Patel BKC. A hot topic: Thermophilic plastic biodegradation. Trends Biotechnol. 2023, 41, 1117-1126.

[81] An J, Ha B, Lee SK. Production of polyhydroxyalkanoates by the thermophile Cupriavidus cauae PHS1. Bioresour. Technol. 2023, 371, 128627.

[82] Pernicova I, Novackova I, Sedlacek P, Kourilova X, Kalina M, Kovalcik A, et al. Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers-1. Isolation and Characterization of the Bacterium. Polymers 2020, 12, 1235.

[83] Svetlitchnyi VA, Svetlichnaya TP, Falkenhan DA, Swinnen S, Knopp D, Läufer A. Direct conversion of cellulose to l-lactic acid by a novel thermophilic Caldicellulosiruptor strain. Biotechnol. Biofuels Bioprod. 2022, 15, 44.

[84] Aulitto M, Alfano A, Maresca E, Avolio R, Errico ME, Gentile G, et al. Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid. Appl. Microbiol. Biotechnol. 2024, 108, 155.

[85] Aulitto M, Fusco S, Bartolucci S, Franzén CJ, Contursi P. Bacillus coagulans MA-13: A promising thermophilic and cellulolytic strain for the production of lactic acid from lignocellulosic hydrolysate. Biotechnol. Biofuels 2017, 10, 210.

[86] Ma Y, Gao Z, Wang Q, Liu Y. Biodiesels from microbial oils: Opportunity and challenges. Bioresour. Technol. 2018, 263, 631-641.

[87] Chisti Y.Biodiesel from microalgae. Biotechnol. Adv. 2007, 25, 294-306.

[88] Gilmour DJ. Chapter One—Microalgae for biofuel production. In Advances in Applied Microbiology; Gadd GM, Sariaslani S,Eds.; Academic Press: San Diego, CA, USA, 2019; p. 1-30.

[89] D’Alessandro EB, Soares AT, Pereira J, Antoniosi Filho NR. Viability of biodiesel production from a thermophilic microalga in conventional and alternative culture media. Rev. Bras. de Bot. 2018, 41, 319-327.

[90] Zili F, Mezhoud N, Trabelsi L, Chreif I, Ben Ouada H. Fatty acid composition of the thermophilic Gloeocapsa gelatinosa under different combinations of temperature, light intensity, and NaNO₃ concentration. J. Appl. Phycol. 2015, 27, 97-107.

[91] Li T, Zheng Y, Yu L, Chen S. High productivity cultivation of a heat-resistant microalga Chlorella sorokiniana for biofuel production. Bioresour. Technol. 2013, 131, 60-67.

[92] Atanasova N, Stoitsova S, Paunova-Krasteva T, Kambourova M.Plastic Degradation by Extremophilic Bacteria. Int. J. Mol. Sci. 2021, 22, 5610.

[93] Atanasova N, Paunova-Krasteva T, Stoitsova S, Radchenkova N, Boyadzhieva I, Petrov K, et al. Degradation of Poly(ε-caprolactone) by a Thermophilic Community and Brevibacillus thermoruber Strain 7 Isolated from Bulgarian Hot Spring. Biomolecules 2021, 11, 1488.

[94] Chua T-K, Tseng M, Yang M-K. Degradation of Poly(ε-caprolactone) by thermophilic Streptomyces thermoviolaceus subsp. thermoviolaceus 76T-2. AMB Express 2013, 3, 8.

[95] Yan F, Wei R, Cui Q, Bornscheuer UT, Liu Y-J. Thermophilic whole-cell degradation of polyethylene terephthalate using engineered Clostridium thermocellum. Microb. Biotechnol. 2021, 14, 374-385.

[96] Tomita K, Hayashi N, Ikeda N, Kikuchi Y. Isolation of a thermophilic bacterium degrading some nylons. Polym. Degrad. Stab. 2003, 81, 511-514.

[97] Wollmann F, Dietze S, Ackermann J-U, Bley T, Walther T, Steingroewer J, et al. Microalgae wastewater treatment: Biological and technological approaches. Eng. Life Sci. 2019, 19, 860-871.

[98] Al-rasheedi S, Tawabini B, Nazal M, Khalil A. Application of Thermophilic Bacteria for the Treatment of Municipal Wastewaters. Arab. J. Sci. Eng. 2022, 47, 6881-6888.

[99] Selvaratnam T, Pegallapati AK, Montelya F, Rodriguez G, Nirmalakhandan N, Van Voorhies W, et al. Evaluation of a thermo-tolerant acidophilic alga, Galdieria sulphuraria, for nutrient removal from urban wastewaters. Bioresour. Technol. 2014, 156, 395-399.

[100] Sloth JK, Jensen HC, Pleissner D, Eriksen NT. Growth and phycocyanin synthesis in the heterotrophic microalga Galdieria sulphuraria on substrates made of food waste from restaurants and bakeries. Bioresour. Technol. 2017, 238, 296-305.

[101] Henkanatte-Gedera SM, Selvaratnam T, Karbakhshravari M, Myint M, Nirmalakhandan N, Van Voorhies W, et al. Removal of dissolved organic carbon and nutrients from urban wastewaters by Galdieria sulphuraria: Laboratory to field scale demonstration. Algal Res. 2017, 24, 450-456.

[102] Delanka-Pedige HMK, Munasinghe-Arachchige SP, Cornelius J, Henkanatte-Gedera SM, Tchinda D, Zhang Y, et al. Pathogen reduction in an algal-based wastewater treatment system employing Galdieria sulphuraria. Algal Res. 2019, 39, 101423.

[103] Mohanty SS, Mohanty K. Valorization of Chlorella thermophila biomass cultivated in dairy wastewater for biopesticide production against bacterial rice blight: a circular biorefinery approach. BMC Plant Biol. 2023, 23, 644.

[104] Padri M, Boontian N, Teaumroong N, Piromyou P, Piasai C. Co-culture of microalga Chlorella sorokiniana with syntrophic Streptomyces thermocarboxydus in cassava wastewater for wastewater treatment and biodiesel production. Bioresour. Technol. 2022, 347, 126732.

[105] Pradhan D, Sukla LB, Sawyer M, Rahman PKSM. Recent bioreduction of hexavalent chromium in wastewater treatment: A review. J. Ind. Eng. Chem. 2017, 55, 1-20.

[106] Kulkarni S, Ballal A, Apte SK. Bioprecipitation of uranium from alkaline waste solutions using recombinant Deinococcus radiodurans. J. Hazard. Mater. 2013, 262, 853-861.

[107] Donati ER, Castro C, Urbieta MS.Thermophilic microorganisms in biomining. World J. Microbiol. Biotechnol. 2016, 32, 179.

[108] Xia M-C, Wang Y-P, Peng T-J, Shen L, Yu R-L, Liu Y-D, et al. Recycling of metals from pretreated waste printed circuit boards effectively in stirred tank reactor by a moderately thermophilic culture. J. Biosci. Bioeng. 2017, 123, 714-721.

[109] Gan M, Jie S, Li M, Zhu J, Liu X. Bioleaching of multiple metals from contaminated sediment by moderate thermophiles. Mar. Pollut. Bull. 2015, 97, 47-55.

[110] Brock TD.Life at High Temperatures. Science 1985, 230, 132-138.

[111] Scoma A, Garrido-Amador P, Nielsen Søren D, Røy H, Kjeldsen Kasper U. The Polyextremophilic Bacterium Clostridium paradoxum Attains Piezophilic Traits by Modulating Its Energy Metabolism and Cell Membrane Composition. Appl. Environ. Microbiol. 2019, 85, e00802-19.

[112] Imanaka T. Molecular bases of thermophily in hyperthermophiles. Proc. Jpn.Acad. Ser. B 2011, 87, 587-602.

[113] Ishino Y, Narumi I. DNA repair in hyperthermophilic and hyperradioresistant microorganisms. Curr. Opin. Microbiol. 2015, 25, 103-112.

[114] Jolly SM, Gainetdinov I, Jouravleva K, Zhang H, Strittmatter L, Bailey SM, et al. Thermus thermophilus Argonaute Functions in the Completion of DNA Replication. Cell 2020, 182, 1545-1559.e18.

[115] Shu W-S, Huang L-N.Microbial diversity in extreme environments. Nat. Rev. Microbiol. 2022, 20, 219-235.

[116] Takemata N. How Do Thermophiles Organize Their Genomes? Microbes Environ. 2024, 39, ME23087.

[117] Dubrez L, Causse S, Borges Bonan N, Dumétier B, Garrido C.Heat-shock proteins: chaperoning DNA repair. Oncogene 2020, 39, 516-529.

[118] Dong Z, Chen X, Cai K, Shen P, Tian K, Jin P, et al. Overexpression of the Bacillus licheniformis GroES enhances thermotolerance of Bacillus subtilis WB600. Biotechnol. Biotechnol. Equip. 2018, 32, 1527-1532.

[119] Hofman-Bang J, Lange M, Conway de Macario E, Macario AJL, Ahring BK. The genes coding for the hsp70(dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1. Gene 1999, 238, 387-395.

[120] Jin J, Li Y, Zhou Z, Zhang H, Guo J, Wan F. Heat Shock Factor Is Involved in Regulating the Transcriptional Expression of Two Potential Hsps (AhHsp70 and AhsHsp21) and Its Role in Heat Shock Response of Agasicles hygrophila. Front. Physiol. 2020, 11, 562204.

[121] Roncarati D, Scarlato V. Regulation of heat-shock genes in bacteria: from signal sensing to gene expression output. FEMS Microbiol. Rev. 2017, 41, 549-574.

[122] Zeldes BM, Straub CT, Otten JK, Adams MWW, Kelly RM. A synthetic enzymatic pathway for extremely thermophilic acetone production based on the unexpectedly thermostable acetoacetate decarboxylase from Clostridium acetobutylicum. Biotechnol. Bioeng. 2018, 115, 2951-2961.

[123] Hendrikse NM, Charpentier G, Nordling E, Syrén P-O. Ancestral diterpene cyclases show increased thermostability and substrate acceptance. FEBS J. 2018, 285, 4660-4673.

[124] Vieille C, Zeikus Gregory J. Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol. Mol. Biol. Rev. 2001, 65, 1-43.

[125] Turner P, Mamo G, Karlsson EN. Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb. Cell Fact. 2007, 6, 9.

[126] Borhani S, Arab SS. Investigation of thermal stability characteristic in family A DNA polymerase—A theoretical study. Prog. Biophys. Mol. Biol. 2023, 182, 15-25.

[127] Pack SP, Kang TJ, Yoo YJ. Protein Thermostabilizing Factors: High Relative Occurrence of Amino Acids, Residual Properties, and Secondary Structure Type in Different Residual State. Appl. Biochem. Biotechnol. 2013, 171, 1212-1226.

[128] Modarres HP, Mofrad MR, Sanati-Nezhad A.Protein thermostability engineering. RSC Adv. 2016, 6, 115252-115270.

[129] Seychell BC, Beck T.Molecular basis for protein-protein interactions. Beilstein J. Org. Chem. 2021, 17, 1-10.

[130] Mundlapati VR, Sahoo DK, Bhaumik S, Jena S, Chandrakar A, Biswal HS.Noncovalent Carbon-Bonding Interactions in Proteins. Angew. Chem. Int. Ed. 2018, 57, 16496-16500.

[131] Liu Z, Lemmonds S, Huang J, Tyagi M, Hong L, Jain N. Entropic contribution to enhanced thermal stability in the thermostable P 450 CYP119. Proc. Natl. Acad. Sci. USA 2018, 115, E10049-E10058.

[132] Camilloni C, Bonetti D, Morrone A, Giri R, Dobson CM, Brunori M, et al. Towards a structural biology of the hydrophobic effect in protein folding. Sci. Rep. 2016, 6, 28285.

[133] Li J, Hou C, Ma X, Guo S, Zhang H, Shi L, et al. Entropy-Enthalpy Compensations Fold Proteins in Precise Ways. Int. J. Mol. Sci. 2021, 22, 9653.

[134] Matsumura M, Signor G, Matthews BW. Substantial increase of protein stability by multiple disulphide bonds. Nature 1989, 342, 291-293.

[135] Pu M, Xu Z, Peng Y, Hou Y, Liu D, Wang Y, et al. Protein crystal quality oriented disulfide bond engineering. Protein Cell 2018, 9, 659-663.

[136] Karshikoff A, Nilsson L, Ladenstein R. Rigidity versus flexibility: The dilemma of understanding protein thermal stability. FEBS J. 2015, 282, 3899-3917.

[137] Yu H, Yan Y, Zhang C, Dalby PA. Two strategies to engineer flexible loops for improved enzyme thermostability. Sci. Rep. 2017, 7, 41212.

[138] Xu Z, Cen Y-K, Zou S-P, Xue Y-P, Zheng Y-G. Recent advances in the improvement of enzyme thermostability by structure modification. Crit. Rev. Biotechnol. 2020, 40, 83-98.

[139] Yang W, Yang Y, Zhang L, Xu H, Guo X, Yang X, et al. Improved thermostability of an acidic xylanase from Aspergillus sulphureus by combined disulphide bridge introduction and proline residue substitution. Sci. Rep. 2017, 7, 1587.

[140] Zou J, Song B, Simmerling C, Raleigh D. Experimental and Computational Analysis of Protein Stabilization by Gly-to-d-Ala Substitution: A Convolution of Native State and Unfolded State Effects. J. Am. Chem. Soc. 2016, 138, 15682-15689.

[141] Charoenwongpaiboon T, Sommanat N, Wangpaiboon K, Puangpathanachai M, Pongsawasdi P, Pichyangkura R. Improving the thermostability and modulating the inulin profile of inulosucrase through rational glycine-to-proline substitution. RSC Adv. 2024, 14, 2346-2353.

[142] Jiang F, Bian J, Liu H, Li S, Bai X, Zheng L, et al. Creatinase: Using Increased Entropy to Improve the Activity and Thermostability. J. Phys. Chem. B 2023, 127, 2671-2682.

[143] Ferreira PHB, Freitas FC, McCully ME, Slade GG, de Oliveira RJ. The Role of Electrostatics and Folding Kinetics on the Thermostability of Homologous Cold Shock Proteins. J. Chem. Inf. Model. 2020, 60, 546-561.

[144] Khan S, Farooq U, Kurnikova M. Exploring Protein Stability by Comparative Molecular Dynamics Simulations of Homologous Hyperthermophilic, Mesophilic, and Psychrophilic Proteins. J. Chem. Inf. Model. 2016, 56, 2129-2139.

[145] Gómez-Calvo A, Gallardo ME, Ladero M. Lipozyme^® TL IM Biocatalyst for Castor Oil FAME and Triacetin Production by Interesterification: Activity, Stability, and Kinetics. Catalysts 2022, 12, 1673.

[146] Janusz G, Pawlik A, Świderska-Burek U, Polak J, Sulej J, Jarosz-Wilkołazka A, et al. Laccase Properties, Physiological Functions, and Evolution. Int. J. Mol. Sci. 2020, 21, 966.

[147] Ayubi MM, Werner A, Steudler S, Haase S, Lange R, Walther T, et al. Enhanced xylan conversion to xylitol in a bio- and chemocatalytic one-pot process. Catal. Today 2021, 367, 137-144.

[148] Rabbani G, Ahmad E, Ahmad A, Khan RH. Structural features, temperature adaptation and industrial applications of microbial lipases from psychrophilic, mesophilic and thermophilic origins. Int. J. Biol. Macromol. 2023, 225, 822-839.

[149] Cai J, Xie Y, Song B, Wang Y, Zhang Z, Feng Y. Fervidobacterium changbaicum Lip1: identification, cloning, and characterization of the thermophilic lipase as a new member of bacterial lipase family V. Appl. Microbiol. Biotechnol. 2011, 89, 1463-1473.

[150] Yang W, Vethanayagam RR, Dong Y, Cai Q, Hu BH. Activation of the antigen presentation function of mononuclear phagocyte populations associated with the basilar membrane of the cochlea after acoustic overstimulation. Neuroscience 2015, 303, 1-15.

[151] Salameh MA, Wiegel J. Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl. Environ. Microbiol. 2007, 73, 7725-7731.

[152] Mathew GM, Madhavan A, Arun KB, Sindhu R, Binod P, Singhania RR, et al.Thermophilic Chitinases: Structural, Functional and Engineering Attributes for Industrial Applications. Appl. Biochem. Biotechnol. 2021, 193, 142-164.

[153] Toharisman A, Suhartono MT, Spindler-Barth M, Hwang J-K, Pyun Y-R. Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2. World J. Microbiol. Biotechnol. 2005, 21, 733-738.

[154] Juturu V, Wu JC. Microbial cellulases: Engineering, production and applications. Renew. Sustain. Energy Rev. 2014, 33, 188-203.

[155] Shad M, Rehman HM, Akhtar MW, Sajjad M. Structural and functional insights of starch processing α-amylase from hyperthermophilic archaeon Pyrococcus abyssi. Carbohydr. Res. 2024, 539, 109122.

[156] Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 1976, 127, 1550-1557.

[157] Kwon K-M, Kang SG, Sokolova TG, Cho SS, Kim YJ, Kim C-H, et al. Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR. Enzyme Microb. Technol. 2016, 86, 117-126.

[158] Yamashita M, Xu J, Morokuma D, Hirata K, Hino M, Mon H, et al. Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System. Mol. Biotechnol. 2017, 59, 221-233.

[159] Chambers CR, Patrick WM. Archaeal Nucleic Acid Ligases and Their Potential in Biotechnology. Archaea 2015, 2015, 170571.

[160] Pezeshgi Modarres H, Dorokhov BD, Popov VO, Ravin NV, Skryabin KG, Dal Peraro M. Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp. 1519. Biochemistry 2015, 54, 3076-3085.

[161] Seo MS, Kim YJ, Choi JJ, Lee MS, Kim JH, Lee J-H, et al. Cloning and expression of a DNA ligase from the hyperthermophilic archaeon Staphylothermus marinus and properties of the enzyme. J. Biotechnol. 2007, 128, 519-530.

[162] Berezina OV, Herlet J, Rykov SV, Kornberger P, Zavyalov A, Kozlov D, et al. Thermostable multifunctional GH 74 xyloglucanase from Myceliophthora thermophila: High-level expression in Pichia pastoris and characterization of the recombinant protein. Appl. Microbiol. Biotechnol. 2017, 101, 5653-5666.

[163] Kengen SWM, Luesink EJ, Stams AJM, Zehnder AJB. Purification and characterization of an extremely thermostable β-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur. J. Biochem. 1993, 213, 305-312.

[164] Flores-Fernández CN, Cárdenas-Fernández M, Lye GJ, Ward JM. Synergistic action of thermophilic pectinases for pectin bioconversion into D-galacturonic acid. Enzyme Microb. Technol. 2022, 160, 110071.

[165] Gu X, Lu H, Chen W, Meng X. Characterization of a Novel Thermophilic Mannanase and Synergistic Hydrolysis of Galactomannan Combined with Swollenin. Catalysts 2021, 11, 254.

[166] Brunecky R, Donohoe BS, Yarbrough JM, Mittal A, Scott BR, Ding H, et al. The Multi Domain Caldicellulosiruptor bescii CelA Cellulase Excels at the Hydrolysis of Crystalline Cellulose. Sci. Rep. 2017, 7, 9622.

[167] Abol-Fotouh D, AlHagar OEA, Hassan MA. Optimization, purification, and biochemical characterization of thermoalkaliphilic lipase from a novel Geobacillus stearothermophilus FMR12 for detergent formulations. Int. J. Biol. Macromol. 2021, 181, 125-135.

[168] Buzzo Bárbara B, Lima Natália Sarmanho M, Pereira Pâmela Aparecida M, Gomes-Pepe Elisângela S, Sartini Camila Cesario F, Lemos Eliana Gertrudes de M. Lignin degradation by a novel thermophilic and alkaline yellow laccase from Chitinophaga sp. Microbiol. Spectr. 2024, 12, e04013-23.

[169] Wayllace NM, Hedín N, Busi MV, Gomez-Casati DF. Identification, molecular and biochemical characterization of a novel thermoactive and thermostable glucoamylase from Thermoanaerobacter ethanolicus. Biotechnol. Lett. 2022, 44, 1201-1216.

[170] Xu Y, Xuan X, Gao R, Xie G. Increased Expression Levels of Thermophilic Serine Protease TTHA 0724 through Signal Peptide Screening in Bacillus subtilis and Applications of the Enzyme. Int. J. Mol. Sci. 2023, 24, 15950.

[171] Curci N, Strazzulli A, De Lise F, Iacono R, Maurelli L, Dal Piaz F, et al. Identification of a novel esterase from the thermophilic bacterium Geobacillus thermodenitrificans NG80-2. Extremophiles 2019, 23, 407-419.

[172] Yang W, He Y, Xu L, Zhang H, Yan Y. A new extracellular thermo-solvent-stable lipase from Burkholderia ubonensis SL-4: Identification, characterization and application for biodiesel production. J. Mol. Catal. B Enzym. 2016, 126, 76-89.

[173] Xu Y, OuYang B, Deng L, Liao M, Tang T, Lan D, et al. Biochemical characterization of a novel hyperthermophilic chitinase from a deep-sea Thermotogae bacterium. Process Biochem. 2024, 143, 60-72.

[174] Li W, Shi H, Ding H, Wang L, Zhang Y, Li X, et al. Characterization of two novel thermostable esterases from Thermoanaerobacterium thermosaccharolyticum. Protein Expression Purif. 2018, 152, 64-70.

[175] Sharma N, Leung IKH. Novel Thermophilic Bacterial Laccase for the Degradation of Aromatic Organic Pollutants. Front. Chem. 2021, 9, 711345.

[176] Lv C, Gu T, Ma R, Yao W, Huang Y, Gu J, et al. Biochemical characterization of a GH 19 chitinase from Streptomyces alfalfae and its applications in crystalline chitin conversion and biocontrol. Int. J. Biol. Macromol. 2021, 167, 193-201.

[177] Saghatelyan A, Panosyan H, Trchounian A, Birkeland N-K. Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1. MicrobiologyOpen 2021, 10, e1149.

[178] Madavi TB, Chauhan S, Keshri A, Alavilli H, Choi K-Y, Pamidimarri SDVN. Whole-cell biocatalysis: Advancements toward the biosynthesis of fuels. Biofuels Bioprod. Biorefining 2022, 16, 859-876.

[179] Ninh Pham H, Honda K, Yokohigashi Y, Okano K, Omasa T, Ohtake H. Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst. Appl. Environ. Microbiol. 2013, 79, 1996-2001.

[180] Schwarz FM, Müller V. Whole-cell biocatalysis for hydrogen storage and syngas conversion to formate using a thermophilic acetogen. Biotechnol. Biofuels 2020, 13, 32.

[181] Adachi D, Koh F, Hama S, Ogino C, Kondo A. A robust whole-cell biocatalyst that introduces a thermo- and solvent-tolerant lipase into Aspergillus oryzae cells: Characterization and application to enzymatic biodiesel production. Enzyme Microb. Technol. 2013, 52, 331-335.

[182] Li Q, Li G, Ma F, Zhang Z, Zheng B, Feng Y. Highly efficient ring-opening polymerization of ɛ-caprolactone catalyzed by a recombinant Escherichia coli whole-cell biocatalyst. Process Biochem. 2011, 46, 477-481.

[183] Wei B, Kang Y, Zhao Y, Xu H, Liang H. Rational Design of Spontaneous Self-Cyclization Enzymes In Vivo and In Vitro with Improved Thermal Tolerance and Activity. ACS Catal. 2024, 14, 5469-5480.

[184] Straub CT, Zeldes BM, Schut GJ, Adams MWW, Kelly RM. Extremely thermophilic energy metabolisms: biotechnological prospects. Curr. Opin. Biotechnol. 2017, 45, 104-112.

[185] Cea PA, Pérez M, Herrera SM, Muñoz SM, Fuentes-Ugarte N, Coche-Miranda J, et al. Deciphering Structural Traits for Thermal and Kinetic Stability across Protein Family Evolution through Ancestral Sequence Reconstruction. Mol. Biol. Evol. 2024, 41, msae127.

[186] Wu Y, Liu J, Han X, Meng X, Li M, Wang J, et al. Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine. iScience 2023, 26, 105774.

[187] Li X, Gui P, Yang R, Lu Z, Wang X, Luo C, et al. Identification and characterization of a novel thermostable transaminase (TATP) from Thermorudis peleae. Biocatal. Biotransform 2024, 42, 378-387.

[188] Dong Y-Q, Shen J-D, Pan L, Huang J-H, Liu Z-Q, Zheng Y-G. Mining and Characterization of Thermophilic Glucose Isomerase Based on Virtual Probe Technology. Appl. Biochem. Biotechnol. 2023, 195, 4399-4413.

[189] Chen J, Ni D, Zhu Y, Xu W, Moussa TAA, Zhang W, et al. Discovery of a Thermostable Tagatose 4-Epimerase Powered by Structure- and Sequence-Based Protein Clustering. J. Agric. Food Chem. 2024, 72, 18585-18593.

[190] Qi H, Wang T, Li H, Li C, Guan L, Liu W, et al. Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering to Improve Enzyme Activity. J. Agric. Food Chem. 2023, 71, 18431-18442.

[191] Olalla L-L, Maria EC, Maria IGS. New Extremophilic Lipases and Esterases from Metagenomics. Curr. Protein Peptide Sci. 2014, 15, 445-455.

[192] Ariaeenejad S, Gharechahi J, Foroozandeh Shahraki M, Fallah Atanaki F, Han J-L, Ding X-Z, et al. Precision enzyme discovery through targeted mining of metagenomic data. Nat. Prod. Bioprospect. 2024, 14, 7.

[193] DeCastro M-E, Rodríguez-Belmonte E, González-Siso M-I. Metagenomics of Thermophiles with a Focus on Discovery of Novel Thermozymes. Front. Microbiol. 2016, 7, 1521.

[194] Shahraki MF, Atanaki FF, Ariaeenejad S, Ghaffari MR, Norouzi-Beirami MH, Maleki M, et al. A computational learning paradigm to targeted discovery of biocatalysts from metagenomic data: A case study of lipase identification. Biotechnol. Bioeng. 2022, 119, 1115-1128.

[195] Sonnendecker C, Oeser J, Richter PK, Hille P, Zhao Z, Fischer C, et al. Low Carbon Footprint Recycling of Post-Consumer PET Plastic with a Metagenomic Polyester Hydrolase. ChemSusChem 2022, 15, e202101062.

[196] Sun B, Zhao X, Xu B, Su E, Kovalevsky A, Shen Q, et al. Discovering and Designing a Chimeric Hyperthermophilic Chitinase for Crystalline Chitin Degradation. ACS Sustain. Chem. Eng. 2023, 11, 4690-4698.

[197] Li G, Rabe KS, Nielsen J, Engqvist MKM. Machine Learning Applied to Predicting Microorganism Growth Temperatures and Enzyme Catalytic Optima. ACS Synth. Biol. 2019, 8, 1411-1420.

[198] Gado JE, Beckham GT, Payne CM. Improving Enzyme Optimum Temperature Prediction with Resampling Strategies and Ensemble Learning. J. Chem. Inf. Model. 2020, 60, 4098-4107.

[199] Zhang Y, Guan F, Xu G, Liu X, Zhang Y, Sun J, et al. A novel thermophilic chitinase directly mined from the marine metagenome using the deep learning tool Preoptem. Bioresour. Bioprocess. 2022, 9, 54.

[200] Sun J, Zhu T, Cui Y, Wu B. Structure-based self-supervised learning enables ultrafast prediction of stability changes upon mutation at the protein universe scale. bioRxiv 2023. doi:10.1101/2023.08.09.552725.

[201] Delgado J, Radusky LG, Cianferoni D, Serrano L. FoldX 5.0: Working with RNA, small molecules and a new graphical interface. Bioinformatics 2019, 35, 4168-4169.

[202] Leaver-Fay A, Tyka M, Lewis SM, Lange OF, Thompson J, Jacak R, et al. Chapter nineteen—Rosetta3:An Object-Oriented Software Suite for the Simulation and Design of Macromolecules. In Methods Enzymol; Johnson ML, Brand L,Eds.; Academic Press: San Diego, CA, USA, 2011; pp. 545-574.

[203] Musil M, Jezik A, Horackova J, Borko S, Kabourek P, Damborsky J, et al. FireProt 2.0: Web-based platform for the fully automated design of thermostable proteins. Brief. Bioinform. 2024, 25, bbad425.

[204] Jung F, Frey K, Zimmer D, Mühlhaus T. DeepSTABp: A Deep Learning Approach for the Prediction of Thermal Protein Stability. Int. J. Mol. Sci. 2023, 24, 7444.

[205] Wang S, Tang H, Zhao Y, Zuo L. BayeStab: Predicting effects of mutations on protein stability with uncertainty quantification. Protein Sci. 2022, 31, e4467.

[206] Xu Y, Liu D, Gong H. Improving the prediction of protein stability changes upon mutations by geometric learning and a pre-training strategy. Nat. Comput. Sci. 2024, 4, 840-850.

[207] Sumbalova L, Stourac J, Martinek T, Bednar D, Damborsky J. HotSpot Wizard 3.0: Web server for automated design of mutations and smart libraries based on sequence input information. Nucleic Acids Res. 2018, 46, W356-W362.

[208] Nikam R, Kulandaisamy A, Harini K, Sharma D, Gromiha MM. ProThermDB: thermodynamic database for proteins and mutants revisited after 15 years. Nucleic Acids Res. 2021, 49, D420-D424.

[209] Jeske L, Placzek S, Schomburg I, Chang A, Schomburg D. BRENDA in 2019: A European ELIXIR core data resource. Nucleic Acids Res. 2019, 47, D542-D549.

[210] Xavier JS, Nguyen T-B, Karmarkar M, Portelli S, Rezende PM, Velloso JPL, et al. ThermoMutDB: A thermodynamic database for missense mutations. Nucleic Acids Res. 2021, 49, D475-D479.

[211] Wang Y, Xue P, Cao M, Yu T, Lane ST, Zhao H. Directed Evolution: Methodologies and Applications. Chem. Rev. 2021, 121, 12384-12444.

[212] Tobin MB, Gustafsson C, Huisman GW. Directed evolution: the ‘rational’ basis for ‘irrational’ design. Curr. Opin. Struct. Biol. 2000, 10, 421-427.

[213] Chowdhury R, Maranas CD. From directed evolution to computational enzyme engineering—A review. AICHE J. 2020, 66, e16847.

[214] Li C, Zhang W, Wei C, Gao X, Mao S, Lu F, et al. Continuous Spectrophotometric Assay for High-Throughput Screening of Predominant d-Allulose 3-Epimerases. J. Agric. Food Chem. 2021, 69, 11637-11645.

[215] Shi L, Liu P, Tan Z, Zhao W, Gao J, Gu Q, et al. Complete Depolymerization of PET Wastes by an Evolved PET Hydrolase from Directed Evolution. Angew. Chem. Int. Ed. 2023, 62, e202218390.

[216] Zhou W, Huang R, Zhu Z, Zhang Yi-Heng PJ. Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX. Appl. Environ. Microbiol. 2018, 84, e01224-18.

[217] Xiong Z, Tian X, Wang G, Song X, Xia Y, Zhang H, et al. Development of a high-throughput screening method for exopolysaccharide-producing Streptococcus thermophilus based on Congo red. Food Res. Int. 2022, 162, 112094.

[218] Li M, Chen Z, Huo Y-X. Application Evaluation and Performance-Directed Improvement of the Native and Engineered Biosensors. ACS Sens. 2024, 9, 5002-5024.

[219] Mannan AA, Liu D, Zhang F, Oyarzún DA. Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors. ACS Synth. Biol. 2017, 6, 1851-1859.

[220] Reyes-De-Corcuera JI, Olstad HE, García-Torres R. Stability and Stabilization of Enzyme Biosensors: The Key to Successful Application and Commercialization. Annu. Rev. Food Sci. Technol. 2018, 9, 293-322.

[221] Li Z, Hu Y, Yu C, Fei K, Shen L, Liu Y, et al. Semi-rational engineering of D-allulose 3-epimerase for simultaneously improving the catalytic activity and thermostability based on D-allulose biosensor. Biotechnol. J. 2024, 19, 2400280.

[222] Li C, Gao X, Li H, Wang T, Lu F, Qin H-M. Growth-Coupled Evolutionary Pressure Improving Epimerases for D-Allulose Biosynthesis Using a Biosensor-Assisted In Vivo Selection Platform. Adv. Sci. 2024, 11, 2306478.

[223] Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 2005, 23, 102-107.

[224] Mo H-M, Xu Y, Yu X-W. Improved Soluble Expression and Catalytic Activity of a Thermostable Esterase Using a High-Throughput Screening System Based on a Split-GFP Assembly. J. Agric. Food Chem. 2018, 66, 12756-12764.

[225] 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, 493-500.

[226] Jumper J, Hassabis D. Protein structure predictions to atomic accuracy with AlphaFold. Nat. Methods 2022, 19, 11-12.

[227] Peccati F, Alunno-Rufini S, Jiménez-Osés G. Accurate Prediction of Enzyme Thermostabilization with Rosetta Using AlphaFold Ensembles. J. Chem. Inf. Model. 2023, 63, 898-909.

[228] Wang Z-K, Feng D-T, Su C, Li H, Rao Z-M, Rao Y-J, et al. Designing ASSMD Strategy for Exploring and Engineering Extreme Thermophilic Ancestral Nitrilase for Nitriles Biocatalysis. ACS Catal. 2024, 14, 13825-13838.

[229] Graff DE, Shakhnovich EI, Coley CW. Accelerating high-throughput virtual screening through molecular pool-based active learning. Chem. Sci. 2021, 12, 7866-7881.

[230] Foroozandeh Shahraki M, Ariaeenejad S, Fallah Atanaki F, Zolfaghari B, Koshiba T, Kavousi K, et al. MCIC: Automated Identification of Cellulases from Metagenomic Data and Characterization Based on Temperature and pH Dependence. Front. Microbiol. 2020, 11, 567863.

[231] Xi Y, Ye L, Yu H. Enhanced thermal and alkaline stability of L-lysine decarboxylase CadA by combining directed evolution and computation-guided virtual screening. Bioresour. Bioprocess. 2022, 9, 24.

[232] Li Y, Fu Y, Chen X, Fan S, Cao Z, Xu F. A Dual-Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase. Angew. Chem. Int. Ed. 2024, 136, e202410881.

[233] Xu S-Y, Chu R-L, Liu H-T, Weng C-Y, Wang Y-J, Zheng Y-G. Computer-directed rational design enhanced the thermostability of carbonyl reductase LsCR for the synthesis of ticagrelor precursor. Biotechnol. Bioeng. 2024, 121, 1532-1542.

[234] Xian-Fang W, Peng G, Yi-Feng L, Hong-Fei L, Fan L. Predicting Thermophilic Proteins by Machine Learning. Curr. Bioinform. 2020, 15, 493-502.

[235] Greener JG, Kandathil SM, Moffat L, Jones DT. A guide to machine learning for biologists. Nat. Rev. Mol. Cell Biol. 2022, 23, 40-55.

[236] Yang KK, Wu Z, Arnold FH. Machine-learning-guided directed evolution for protein engineering. Nat. Methods 2019, 16, 687-694.

[237] Kouba P, Kohout P, Haddadi F, Bushuiev A, Samusevich R, Sedlar J, et al.Machine Learning-Guided Protein Engineering. ACS Catal. 2023, 13, 13863-13895.

[238] Zhang Z, Li Z, Yang M, Zhao F, Han S. Machine learning-guided multi-site combinatorial mutagenesis enhances the thermostability of pectin lyase. Int. J. Biol. Macromol. 2024, 277, 134530.

[239] Yoshida K, Kawai S, Fujitani M, Koikeda S, Kato R, Ema T. Enhancement of protein thermostability by three consecutive mutations using loop-walking method and machine learning. Sci. Rep. 2021, 11, 11883.

[240] Khakzad H, Igashov I, Schneuing A, Goverde C, Bronstein M, Correia B. A new age in protein design empowered by deep learning. Cell Syst. 2023, 14, 925-939.

[241] Shroff R, Cole AW, Diaz DJ, Morrow BR, Donnell I, Annapareddy A, et al. Discovery of Novel Gain-of-Function Mutations Guided by Structure-Based Deep Learning. ACS Synth. Biol. 2020, 9, 2927-2935.

[242] Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 2022, 604, 662-667.

[243] Fang X, Huang J, Zhang R, Wang F, Zhang Q, Li G, et al. Convolution Neural Network-Based Prediction of Protein Thermostability. J. Chem. Inf. Model. 2019, 59, 4833-4843.

[244] Liao M, Feng S, Liu X, Xu G, Li S, Bai Y, et al. “Short Board” Novel Insights into Enzymatic Thermostability: The Theory and Zero-Shot Hamiltonian Model. Adv. Sci. 2024, 11, 2402441.

[245] Ouyang J, Cai C, Chen H, Jiang T, Zheng Z. Efficient non-sterilized fermentation of biomass-derived xylose to lactic acid by a thermotolerant Bacillus coagulans NL01. Appl. Biochem. Biotechnol. 2012, 168, 2387-2397.

[246] Georgieva T, Mikkelsen M, Ahring B. High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1. Cent. Eur. J. Biol. 2007, 2, 364-377.

[247] Wang Q, Guo S, Ali M, Song X, Tang Z, Zhang Z, et al. Thermally enhanced bioremediation: A review of the fundamentals and applications in soil and groundwater remediation. J. Hazard. Mater. 2022, 433, 128749.

[248] He Y, He Y, Abdullah Al-Dhabi N, Gao P, Huang H, Yan B, et al. Effects of exogenous thermophilic bacteria and ripening agent on greenhouse gas emissions, enzyme activity and microbial community during straw composting. Bioresour. Technol. 2024, 407, 131114.

[249] Camacho CI, Estévez S, Conde JJ, Feijoo G, Moreira MT. Dark fermentation as an environmentally sustainable WIN-WIN solution for bioenergy production. J. Clean. Prod. 2022, 374, 134026.

[250] Obruča S, Dvořák P, Sedláček P, Koller M, Sedlář K, Pernicová I, et al. Polyhydroxyalkanoates synthesis by halophiles and thermophiles: Towards sustainable production of microbial bioplastics. Biotechnol. Adv. 2022, 58, 107906.

[251] Wittner N, Vasilakou K, Broos W, Vlaeminck SE, Nimmegeers P, Cornet I. Investigating the Technical and Economic Potential of Solid-State Fungal Pretreatment at Nonsterile Conditions for Sugar Production from Poplar Wood. Ind. Eng. Chem. Res. 2023, 62, 18292-18302.

[252] Wu P, Guo Y, Golly MK, Ma H, He R, Luo S, et al. Feasibility study on direct fermentation of soybean meal by Bacillus stearothermophilus under non-sterile conditions. J. Sci. Food Agric. 2019, 99, 3291-3298.

[253] Ma C, Wang A, Qin J, Li L, Ai X, Jiang T, et al. Enhanced 2,3-butanediol production by Klebsiella pneumoniae SDM. Appl. Microbiol. Biotechnol. 2009, 82, 49-57.

[254] Dhatt S, Nandi M, Chaudhury P. Substrate inhibition versus product feedback inhibition: In the perspective of single molecule enzyme kinetics. Int. J. Chem. Kinet. 2021, 53, 775-786.

[255] Le Y, Sun J. Chapter One—CRISPR/Cas genome editing systems in thermophiles:Current status, associated challenges, and future perspectives. In Advances in Applied Microbiology; Gadd GM, Sariaslani S,Eds.; Academic Press: San Diego, CA, USA, 2022; p. 1-30.

[256] Houghton KM, Carere CR, Stott MB, McDonald IR.Thermophilic methanotrophs: in hot pursuit. FEMS Microbiol. Ecol. 2019, 95, fiz125.

[257] Sugden S, Lazic M, Sauvageau D, Stein Lisa Y. Transcriptomic and Metabolomic Responses to Carbon and Nitrogen Sources in Methylomicrobium album BG8. Appl. Environ. Microbiol. 2021, 87, e00385-21.

[258] Johnson SR, Fu X, Viknander S, Goldin C, Monaco S, Zelezniak A, et al. Computational scoring and experimental evaluation of enzymes generated by neural networks. Nat. Biotechnol. 2024. doi: 10.1038/s41587-024-02214-2.

[259] Zhou J, Huang M. Navigating the landscape of enzyme design: from molecular simulations to machine learning. Chem. Soc. Rev. 2024, 53, 8202-8239.

[260] Gong X, Zhang J, Gan Q, Teng Y, Hou J, Lyu Y, et al. Advancing microbial production through artificial intelligence-aided biology. Biotechnol. Adv. 2024, 74, 108399.

[261] Li L, Liu X, Bai Y, Yao B, Luo H, Tu T. High-Throughput Screening Techniques for the Selection of Thermostable Enzymes. J. Agric. Food Chem. 2024, 72, 3833-3845.

[262] Stucki A, Vallapurackal J, Ward TR, Dittrich PS. Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey. Angew. Chem. Int. Ed. 2021, 60, 24368-24387.

[263] Rolf J, Rosenthal K, Lütz S. Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development. Catalysts 2019, 9, 190.

[264] Salas-Bruggink DIJ, Martín JS-S, Leiva G, Blamey JM. Extremozymes: Challenges and opportunities on the road to novel enzymes production. Process Biochem. 2024, 143, 323-336.