{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,8]],"date-time":"2026-06-08T21:28:59Z","timestamp":1780954139374,"version":"3.54.1"},"reference-count":82,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T00:00:00Z","timestamp":1773014400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Major Science and Technology Projects for Public Welfare of Henan Province in China","award":["201300110300"],"award-info":[{"award-number":["201300110300"]}]},{"name":"Zhongyuan Scholars of Henan Province in China","award":["192101510004"],"award-info":[{"award-number":["192101510004"]}]},{"name":"Zhongyuan Scholar Workstation Funded Project","award":["ZYGZZ2020015"],"award-info":[{"award-number":["ZYGZZ2020015"]}]},{"name":"Zhongyuan Scholar Workstation Funded Project","award":["214400510015"],"award-info":[{"award-number":["214400510015"]}]},{"name":"Central Government Guides the Local Science and Technology Development Special Fund","award":["Z20221341069"],"award-info":[{"award-number":["Z20221341069"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Cell-free protein synthesis has become a powerful tool for producing functional proteins, circumventing many limitations of live-cell systems. Platforms based on wheat germ extract are favored for their high efficiency in translating and folding complex eukaryotic proteins. To overcome the energy limitation common in such systems, we engineered an Escherichia coli strain to function as a self-renewing ATP source. This strain co-expresses a three-enzyme cascade\u2014adenosine kinase, adenylate kinase, and acetate kinase\u2014that efficiently converts adenosine and acetyl phosphate into ATP. Using the lysate from this biocatalyst to energize an optimized wheat germ extract, we established a high-performance cell-free synthesis platform. This integrated system supported the robust production of multiple recombinant proteins. As a key demonstration, we synthesized human vascular endothelial growth factor 165, which exhibited full biological activity. The cell-free-produced VEGF165 significantly stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs). It also potently induced angiogenic responses, including the formation of extensive, interconnected capillary-like networks by HUVECs in vitro and accelerated cell migration in scratch-wound assays. Our work establishes a scalable and efficient platform for on-demand production of bioactive eukaryotic proteins, highlighting its considerable potential for advancing regenerative medicine and related therapeutic applications.<\/jats:p>","DOI":"10.3390\/cimb48030290","type":"journal-article","created":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T10:16:12Z","timestamp":1773051372000},"page":"290","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Synthesis and Biological Activity Characterization of Vascular Endothelial Growth Factor Using an Optimized Wheat Germ Cell-Free System"],"prefix":"10.3390","volume":"48","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3373-5404","authenticated-orcid":false,"given":"Ming","family":"Liu","sequence":"first","affiliation":[{"name":"College of Grain and Food Science, Henan University of Technology, Zhengzhou 450001, China"},{"name":"Food Laboratory of Zhongyuan, Henan University of Technology, Zhengzhou 450001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ran","family":"Xiao","sequence":"additional","affiliation":[{"name":"College of Agriculture, Henan University, Kaifeng 475004, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuiyang","family":"Kong","sequence":"additional","affiliation":[{"name":"College of Agriculture, Henan University, Kaifeng 475004, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aimei","family":"Liao","sequence":"additional","affiliation":[{"name":"College of Grain and Food Science, Henan University of Technology, Zhengzhou 450001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1149-8081","authenticated-orcid":false,"given":"Long","family":"Pan","sequence":"additional","affiliation":[{"name":"College of Grain and Food Science, Henan University of Technology, Zhengzhou 450001, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jihong","family":"Huang","sequence":"additional","affiliation":[{"name":"Food Laboratory of Zhongyuan, Henan University of Technology, Zhengzhou 450001, China"},{"name":"College of Agriculture, Henan University, Kaifeng 475004, China"},{"name":"School of Food and Pharmacy, Xuchang University, Xuchang 461000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"251","DOI":"10.1111\/jipb.13055","article-title":"The plant cell wall: Biosynthesis, construction, and functions","volume":"63","author":"Zhang","year":"2021","journal-title":"J. Integr. Plant Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e70078","DOI":"10.1111\/1541-4337.70078","article-title":"A comprehensive review of recombinant technology in the food industry: Exploring expression systems, application, and future challenges","volume":"24","author":"Liu","year":"2025","journal-title":"Compr. Rev. Food Sci. Food Saf."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1080\/07388551.2022.2090314","article-title":"Research progress on the application of cell-free synthesis systems for enzymatic processes","volume":"43","author":"Liu","year":"2023","journal-title":"Crit. Rev. Biotechnol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Chipman, D.M., Woolley, A.C., Chau, D.N., Lance, W.A., Talley, J.P., Green, T.P., Robbins, B.C., and Bundy, B.C. (2025). Cell-Free Protein Synthesis Reactor Formats: A Brief History and Analysis. SynBio, 3.","DOI":"10.3390\/synbio3030010"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"117015","DOI":"10.1016\/j.trac.2023.117015","article-title":"Cell-free protein synthesis system for bioanalysis: Advances in methods and applications","volume":"161","author":"Gu","year":"2023","journal-title":"TrAC Trends Anal. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1016\/j.copbio.2020.12.012","article-title":"Toward sustainable, cell-free biomanufacturing","volume":"69","author":"Rasor","year":"2021","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1007\/s12257-024-00016-6","article-title":"Microfluidics-driven high-throughput phenotyping and screening in synthetic biology: From single cells to cell-free systems","volume":"29","author":"Kim","year":"2024","journal-title":"Biotechnol. Bioprocess Eng."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Rolf, J., Rosenthal, K., and L\u00fctz, S. (2019). Application of Cell-Free Protein Synthesis for Faster Biocatalyst Development. Catalysts, 9.","DOI":"10.3390\/catal9020190"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2762","DOI":"10.1016\/j.febslet.2014.05.061","article-title":"Wheat germ systems for cell-free protein expression","volume":"588","author":"Harbers","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"ysz027","DOI":"10.1093\/synbio\/ysz027","article-title":"Enhancing control of cell-free metabolism through pH modulation","volume":"5","author":"Karim","year":"2020","journal-title":"Synth. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Brookwell, A., Oza, J.P., and Caschera, F. (2021). Biotechnology Applications of Cell-Free Expression Systems. Life, 11.","DOI":"10.3390\/life11121367"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1408","DOI":"10.1021\/acssynbio.1c00641","article-title":"A Temperature-Controlled Cell-Free Expression System by Dynamic Repressor","volume":"11","author":"Yang","year":"2022","journal-title":"ACS Synth. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2519","DOI":"10.1038\/s41467-019-10409-4","article-title":"Importance of potassium ions for ribosome structure and function revealed by long-wavelength X-ray diffraction","volume":"10","author":"Rozov","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"7815","DOI":"10.1021\/bi00149a011","article-title":"Roles of magnesium in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: Kinetic evidence for a second open complex requiring magnesium","volume":"31","author":"Suh","year":"1992","journal-title":"Biochemistry"},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Fehlau, M., Westarp, S., Neubauer, P., and Kurreck, A. (2025). Advances in the Enzymatic Synthesis of Nucleoside-5\u2032-Triphosphates and Their Analogs. Catalysts, 15.","DOI":"10.3390\/catal15030270"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"25210","DOI":"10.1021\/acs.jafc.4c05520","article-title":"Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides","volume":"72","author":"Mao","year":"2024","journal-title":"J. Agric. Food. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Yan, G., Li, X., Yang, J., Li, Z., Hou, J., Rao, B., Hu, Y., Ma, L., and Wang, Y. (2021). Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins. Biomolecules, 11.","DOI":"10.3390\/biom11111706"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9077926","DOI":"10.1155\/2024\/9077926","article-title":"Role of Angiogenesis and Its Biomarkers in Development of Targeted Tumor Therapies","volume":"2024","author":"Pathak","year":"2024","journal-title":"Stem Cells Int."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Chen, X., Gao, C.-Y., Chu, X.-Y., Zheng, C.-Y., Luan, Y.-Y., He, X., Yang, K., and Zhang, D.-L. (2022). VEGF-Loaded Heparinised Gelatine-Hydroxyapatite-Tricalcium Phosphate Scaffold Accelerates Bone Regeneration via Enhancing Osteogenesis-Angiogenesis Coupling. Front. Bioeng. Biotechnol., 10.","DOI":"10.3389\/fbioe.2022.915181"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Fu, J., Gao, J., Liang, Z., and Yang, D. (2021). PDI-Regulated Disulfide Bond Formation in Protein Folding and Biomolecular Assembly. Molecules, 26.","DOI":"10.3390\/molecules26010171"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Yamaoka, Y., Jeremiah, S.S., Funabashi, R., Miyakawa, K., Morita, T., Mihana, Y., Kato, H., and Ryo, A. (2022). Characterization and Utilization of Disulfide-Bonded SARS-CoV-2 Receptor Binding Domain of Spike Protein Synthesized by Wheat Germ Cell-Free Production System. Viruses, 14.","DOI":"10.3390\/v14071461"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"4160","DOI":"10.1111\/j.1742-4658.2006.05434.x","article-title":"Wheat germ cell-free platform for eukaryotic protein production","volume":"273","author":"Vinarov","year":"2006","journal-title":"FEBS J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e00122","DOI":"10.1042\/BSR20140050","article-title":"Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins","volume":"34","author":"Gad","year":"2014","journal-title":"Biosci. Rep."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"6445","DOI":"10.1021\/acssuschemeng.3c00410","article-title":"Efficient Bioproduction of 3-Hydroxypropionic Acid from Methanol by a Synthetic Yeast Cell Factory","volume":"11","author":"Wu","year":"2023","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1608","DOI":"10.1021\/acssynbio.0c00173","article-title":"Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer","volume":"9","author":"Takahashi","year":"2020","journal-title":"ACS Synth. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"12030","DOI":"10.1038\/s41598-017-12364-w","article-title":"Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates","volume":"7","author":"Stech","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1038\/nprot.2009.207","article-title":"Practical cell-free protein synthesis system using purified wheat embryos","volume":"5","author":"Takai","year":"2010","journal-title":"Nat. Protoc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"4780","DOI":"10.1046\/j.1432-1033.2003.03880.x","article-title":"Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ","volume":"270","author":"Kawasaki","year":"2003","journal-title":"Eur. J. Biochem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"e00420-22","DOI":"10.1128\/jb.00420-22","article-title":"Initiator AUGs Are Discriminated from Elongator AUGs Predominantly through mRNA Accessibility in C. crescentus","volume":"205","author":"Ghosh","year":"2023","journal-title":"J. Bacteriol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"10602","DOI":"10.1093\/nar\/gkaa786","article-title":"Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor","volume":"48","author":"Ding","year":"2020","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"548","DOI":"10.1016\/j.bbrc.2015.10.120","article-title":"Internal ribosome entry site (IRES) from Encephalomyocarditis virus (EMCV) as a tool for shuttle expression plasmids","volume":"468","year":"2015","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"510","DOI":"10.1007\/s00284-025-04498-2","article-title":"Coexpression of the Ectoine Biosynthetic Gene Cluster from Nocardiopsis dassonvillei NCIM 5124 in Escherichia coli by Using a Novel Strategy","volume":"82","author":"Kadam","year":"2025","journal-title":"Curr. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"108977","DOI":"10.1016\/j.isci.2024.108977","article-title":"HetMM: A Michaelis-Menten model for non-homogeneous enzyme mixtures","volume":"27","author":"Douglas","year":"2024","journal-title":"iScience"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"133978","DOI":"10.1016\/j.ijbiomac.2024.133978","article-title":"Efficient and easible biocatalysts: Strategies for enzyme improvement. A review","volume":"276","author":"Albayati","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11590","DOI":"10.1039\/D4CS00404C","article-title":"Nanomaterial-based regulation of redox metabolism for enhancing cancer therapy","volume":"53","author":"Jia","year":"2024","journal-title":"Chem. Soc. Rev."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"e00092-22","DOI":"10.1128\/mbio.00092-22","article-title":"A Central Role for Magnesium Homeostasis during Adaptation to Osmotic Stress","volume":"13","author":"Pi","year":"2022","journal-title":"mBio"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1152\/physrev.00009.2022","article-title":"Determinants, maintenance, and function of organellar pH","volume":"103","author":"Freeman","year":"2022","journal-title":"Physiol. Rev."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"100512","DOI":"10.1016\/j.crfs.2023.100512","article-title":"Extraction, bioactive function and application of wheat germ protein\/peptides: A review","volume":"6","author":"Zhang","year":"2023","journal-title":"Curr. Res. Food Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"102228","DOI":"10.1016\/j.molmet.2025.102228","article-title":"Three-dimensional network of creatine metabolism: From intracellular energy shuttle to systemic metabolic regulatory switch","volume":"100","author":"Su","year":"2025","journal-title":"Mol. Metab."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"2374","DOI":"10.1021\/acs.biochem.1c00012","article-title":"Functional Roles of Chelated Magnesium Ions in RNA Folding and Function","volume":"60","author":"Yamagami","year":"2021","journal-title":"Biochemistry"},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Wiseman, R.W., Brown, C.M., Beck, T.W., Brault, J.J., Reinoso, T.R., Shi, Y., and Chase, P.B. (2023). Creatine Kinase Equilibration and \u0394GATP over an Extended Range of Physiological Conditions: Implications for Cellular Energetics, Signaling, and Muscle Performance. Int. J. Mol. Sci., 24.","DOI":"10.20944\/preprints202308.1711.v1"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Xu, B., Liu, L., and Song, G. (2022). Functions and Regulation of Translation Elongation Factors. Front. Mol. Biosci., 8.","DOI":"10.3389\/fmolb.2021.816398"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Yang, L., and Liu, X. (2025). The inhibitory effect of tyrosine kinase inhibitors on angiogenesis in zebrafish and interrelationship with the activation of the PI3K\/AKT pathway. BMC Pharmacol. Toxicol., 26.","DOI":"10.1186\/s40360-025-00987-z"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"H1403","DOI":"10.1152\/ajpheart.00002.2021","article-title":"Enhanced endothelial barrier function by monoclonal antibody activation of vascular endothelial cadherin","volume":"320","author":"Park","year":"2021","journal-title":"Am. J. Physiol.-Heart Circ. Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"2054","DOI":"10.1021\/acsabm.3c01227","article-title":"Unraveling Endothelial Cell Migration: Insights into Fundamental Forces, Inflammation, Biomaterial Applications, and Tissue Regeneration Strategies","volume":"7","author":"Jerka","year":"2024","journal-title":"ACS Appl. Bio Mater."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"102441","DOI":"10.1016\/j.ceb.2024.102441","article-title":"Endothelial cell mechanics and dynamics in angiogenesis","volume":"91","author":"Huveneers","year":"2024","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1007\/s10495-022-01764-7","article-title":"CACNA1C-AS2 inhibits cell proliferation and suppresses cell migration and invasion via targeting FBXO45 and PI3K\/AKT\/mTOR pathways in glioma","volume":"27","author":"Cao","year":"2022","journal-title":"Apoptosis"},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Omotajo, D., Tate, T., Cho, H., and Choudhary, M. (2015). Distribution and diversity of ribosome binding sites in prokaryotic genomes. BMC Genom., 16.","DOI":"10.1186\/s12864-015-1808-6"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1007\/s12257-021-0354-0","article-title":"Ribosome-Binding Sequences (RBS) Engineering of Key Genes in Escherichia coli for High Production of Fatty Alcohols","volume":"27","author":"Chen","year":"2022","journal-title":"Biotechnol. Bioprocess Eng."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1021\/acssynbio.4c00551","article-title":"Fine-Tuning Genetic Circuits via Host Context and RBS Modulation","volume":"14","author":"Chan","year":"2025","journal-title":"ACS Synth. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1038\/nature14219","article-title":"Initiation of translation in bacteria by a structured eukaryotic IRES RNA","volume":"519","author":"Colussi","year":"2015","journal-title":"Nature"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"9355","DOI":"10.1093\/nar\/gkac697","article-title":"The prokaryotic activity of the IGR IRESs is mediated by ribosomal protein S1","volume":"50","author":"Roberts","year":"2022","journal-title":"Nucleic Acids Res."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"110","DOI":"10.1016\/j.bbrc.2022.12.022","article-title":"IRES-mediated translation in bacteria","volume":"641","author":"Takallou","year":"2023","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"He, J.J., Liu, X.X., Li, Y., Wang, Z., Shi, H.L., Kan, Y.C., Yao, L.G., and Tang, C.D. (2023). High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiae and its purification and immobilization by one-step method. Front. Bioeng. Biotechnol., 11.","DOI":"10.3389\/fbioe.2023.1134152"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1002\/yea.1039","article-title":"Expression in Escherichia coli of a recombinant adenosine kinase from Saccharomyces cerevisiae: Purification, kinetics and substrate analyses","volume":"20","author":"Barrado","year":"2003","journal-title":"Yeast"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1016\/j.pep.2014.11.011","article-title":"Recombinant expression, purification and preliminary characterization of the mRNA export factor MEX67 of Saccharomyces cerevisiae","volume":"107","author":"Chhetri","year":"2015","journal-title":"Protein Expr. Purif."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1016\/j.jbiosc.2016.06.005","article-title":"Effect of NADH kinase on poly-3-hydroxybutyrate production by recombinant Escherichia coli","volume":"122","author":"Hong","year":"2016","journal-title":"J. Biosci. Bioeng."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.pep.2019.02.012","article-title":"Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli","volume":"159","author":"Lopes","year":"2019","journal-title":"Protein Expr. Purif."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Sandomenico, A., Sivaccumar, J.P., and Ruvo, M. (2020). Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21176324"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1186\/s12934-021-01569-4","article-title":"Whole cell-based catalyst for enzymatic production of the osmolyte 2-O-\u03b1-glucosylglycerol","volume":"20","author":"Schwaiger","year":"2021","journal-title":"Microb. Cell Fact."},{"key":"ref_61","unstructured":"Lacapere, J.-J. (2017). Wheat Germ Cell-Free Overexpression for the Production of Membrane Proteins. Membrane Protein Structure and Function Characterization: Methods and Protocols, Springer."},{"key":"ref_62","doi-asserted-by":"crossref","unstructured":"Nagy, S.K., K\u00e1llai, B.M., Andr\u00e1s, J., and M\u00e9sz\u00e1ros, T. (2020). A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression. BMC Biotech., 20.","DOI":"10.1186\/s12896-020-00610-5"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"MSB198875","DOI":"10.15252\/msb.20198875","article-title":"Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems","volume":"15","author":"Yim","year":"2019","journal-title":"Mol. Syst. Biol."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/1477-5956-8-32","article-title":"Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts","volume":"8","author":"Henderson","year":"2010","journal-title":"Proteome Sci."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"109361","DOI":"10.1016\/j.bej.2024.109361","article-title":"Synthesis of myrcene using a hybrid cell-free reaction system","volume":"208","author":"Zhang","year":"2024","journal-title":"Biochem. Eng. J."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"2121","DOI":"10.1002\/bit.28711","article-title":"Efficient production of l-glutathione by whole-cell catalysis with ATP regeneration from adenosine","volume":"121","author":"Zuo","year":"2024","journal-title":"Biotechnol. Bioeng."},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"11157","DOI":"10.1021\/acs.jafc.5c00892","article-title":"Promoter Engineering and Two-Phase Whole-Cell Catalysis Improve the Biosynthesis of Naringenin in E. coli","volume":"73","author":"Zhang","year":"2025","journal-title":"J. Agric. Food. Chem."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"15859","DOI":"10.1021\/acssuschemeng.5c04717","article-title":"Engineering the Distal Loci of SAM Synthase for High-Yield Synthesis of SAM Using Whole-Cell Catalysis","volume":"13","author":"Huang","year":"2025","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_69","doi-asserted-by":"crossref","first-page":"9807","DOI":"10.1021\/acs.jafc.5c02080","article-title":"Enhanced Production of Leucine\u2013Leucine Dipeptide by Whole-Cell Catalysis","volume":"73","author":"Zhu","year":"2025","journal-title":"J. Agric. Food. Chem."},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"125157","DOI":"10.1016\/j.biortech.2021.125157","article-title":"A cost-practical cell-recycling process for xylonic acid bioproduction from acidic lignocellulosic hydrolysate with whole-cell catalysis of Gluconobacter oxydans","volume":"333","author":"Han","year":"2021","journal-title":"Bioresour. Technol."},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"e70145","DOI":"10.1111\/1751-7915.70145","article-title":"Reconstitution of Methionine Cycle With ATP Regeneration for Whole-Cell Catalysis of Creatine Production in Engineered Escherichia coli","volume":"18","author":"Sheng","year":"2025","journal-title":"Microb. Biotechnol."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"110379","DOI":"10.1016\/j.enzmictec.2023.110379","article-title":"Efficient production of \u03b3-aminobutyric acid using engineered Escherichia coli whole-cell catalyst","volume":"174","author":"Chang","year":"2024","journal-title":"Enzym. Microb. Technol."},{"key":"ref_73","first-page":"174","article-title":"Use of endogenous acetate kinase for ATP regeneration system of whole-cell L-theanine production catalyzed by \u03b3-glutamylmethylamide synthetase","volume":"30","author":"Lee","year":"2025","journal-title":"Biotechnol. Bioprocess Eng."},{"key":"ref_74","doi-asserted-by":"crossref","unstructured":"Gao, S., Ma, D., Wang, Y., Zhang, A., Wang, X., and Chen, K. (2023). Whole-cell catalyze L-dopa to dopamine via co-expression of transport protein AroP in Escherichia coli. BMC Biotech., 23.","DOI":"10.1186\/s12896-023-00794-6"},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1186\/s12934-017-0763-0","article-title":"Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts","volume":"16","author":"White","year":"2017","journal-title":"Microb. Cell Fact."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"346","DOI":"10.1007\/s12011-023-03768-z","article-title":"Antibacterial Activity of Boron Compounds Against Biofilm-Forming Pathogens","volume":"202","author":"Celebi","year":"2024","journal-title":"Biol. Trace Elem. Res."},{"key":"ref_77","doi-asserted-by":"crossref","first-page":"231","DOI":"10.1016\/j.jiec.2018.06.033","article-title":"Synthesis of (R,R)-2,3-butanediol from starch in a hybrid cell-free reaction system","volume":"67","author":"Yi","year":"2018","journal-title":"J. Ind. Eng. Chem."},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"MSB200857","DOI":"10.1038\/msb.2008.57","article-title":"An integrated cell-free metabolic platform for protein production and synthetic biology","volume":"4","author":"Jewett","year":"2008","journal-title":"Mol. Syst. Biol."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"211","DOI":"10.3390\/polym2030211","article-title":"Borate Minerals and RNA Stability","volume":"2","author":"Cossetti","year":"2010","journal-title":"Polymers"},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1007\/s11084-013-9350-5","article-title":"Selective Stabilization of Ribose by Borate","volume":"43","author":"Furukawa","year":"2013","journal-title":"Orig. Life Evol. Biosph."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"246","DOI":"10.1016\/j.chroma.2006.02.066","article-title":"Boric acid inhibits adenosine diphosphate-ribosyl cyclase non-competitively","volume":"1115","author":"Kim","year":"2006","journal-title":"J. Chromatogr. A"},{"key":"ref_82","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.1111\/tpj.15248","article-title":"Global analysis of boron-induced ribosome stalling reveals its effects on translation termination and unique regulation by AUG-stops in Arabidopsis shoots","volume":"106","author":"Sotta","year":"2021","journal-title":"Plant J."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/3\/290\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T10:59:05Z","timestamp":1773053945000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/3\/290"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,9]]},"references-count":82,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["cimb48030290"],"URL":"https:\/\/doi.org\/10.3390\/cimb48030290","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,9]]}}}