{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T12:27:42Z","timestamp":1785414462655,"version":"3.56.0"},"reference-count":28,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,4,23]],"date-time":"2026-04-23T00:00:00Z","timestamp":1776902400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32472293"],"award-info":[{"award-number":["32472293"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32472293"],"award-info":[{"award-number":["32472293"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Hebei Province Program for Full-time Introduction of National High-level Innovative Talents","award":["2025HBQZYCXY005"],"award-info":[{"award-number":["2025HBQZYCXY005"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid detection method, but its application is often limited by the suboptimal performance of wild-type Bacillus stearothermophilus (Bst) DNA polymerase. This study employed a combined deep learning and semi-rational design strategy to engineer Bst DNA polymerase. High-throughput screening identified the A0A150MFP3 sequence and the L105M mutation, which increased enzymatic activity by 32.92%. Fusion with the CL7 protein generated a CL7-Bst mutant with enhanced thermal stability and tolerance to common inhibitors, including 7% (v\/v) ethanol, 0.18\u2030 (w\/v) SDS, 80 mmol\/L NaCl, and 0.8 mmol\/L EDTA. Systematic optimization of the LAMP reaction system determined the optimal pH (9.0), enzyme concentration (0.20 U\/\u03bcL), and temperature (64 \u00b0C). When applied to Escherichia coli O157:H7 detection, the CL7-Bst mutant achieved Tt values of 15.13 and 12.78 for crude and purified DNA, respectively, with a limit of detection of 1 \u00d7 103 CFU\/mL. In summary, integrating deep learning with semi-rational design and fusion protein engineering yielded a high-performance DNA polymerase that facilitates rapid, sensitive, and field-deployable LAMP-based pathogen detection.<\/jats:p>","DOI":"10.3390\/microorganisms14050954","type":"journal-article","created":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T17:26:18Z","timestamp":1777051578000},"page":"954","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Deep Learning-Guided Engineering of Bst DNA Polymerase Improves LAMP-Based Detection of Foodborne Pathogens"],"prefix":"10.3390","volume":"14","author":[{"given":"Haoting","family":"Chen","sequence":"first","affiliation":[{"name":"College of Life Sciences, South China Agricultural University, Guangzhou 510642, China"},{"name":"Guangzhou Double Helix Gene Technology Co., Ltd., Guangzhou 510700, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2238-6533","authenticated-orcid":false,"given":"Jingfeng","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Food Science, Instrumental Analysis & Research Center, South China Agricultural University, Guangzhou 510642, China"},{"name":"Guangzhou Double Helix Gene Technology Co., Ltd., Guangzhou 510700, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoli","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Food Science, Instrumental Analysis & Research Center, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Guangzhou Double Helix Gene Technology Co., Ltd., Guangzhou 510700, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanlei","family":"Chang","sequence":"additional","affiliation":[{"name":"Guangzhou Double Helix Gene Technology Co., Ltd., Guangzhou 510700, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Shi","sequence":"additional","affiliation":[{"name":"College of Life Sciences, Hebei Agricultural University, Baoding 071000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lichao","family":"Zhao","sequence":"additional","affiliation":[{"name":"College of Food Science, Instrumental Analysis & Research Center, South China Agricultural University, Guangzhou 510642, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e63","DOI":"10.1093\/nar\/28.12.e63","article-title":"Loop-mediated isothermal amplification of DNA","volume":"28","author":"Notomi","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2201174","DOI":"10.1002\/adem.202201174","article-title":"Point-of-care diagnostic platforms for loop-mediated isothermal amplification","volume":"25","author":"Atceken","year":"2023","journal-title":"Adv. Eng. Mater."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4519","DOI":"10.1016\/j.csbj.2023.09.008","article-title":"Bst polymerase\u2014A humble relative of Taq polymerase","volume":"21","author":"Oscorbin","year":"2023","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"768","DOI":"10.1093\/bbb\/zbae059","article-title":"PEG modification increases thermostability and inhibitor resistance of Bst DNA polymerase","volume":"88","author":"Yang","year":"2024","journal-title":"Biosci. Biotechnol. Biochem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"5391","DOI":"10.1021\/acscatal.4c07614","article-title":"FADS-Based Directed Evolution of a Robust Bst DNA Polymerase Adapting High-Temperature Loop-Mediated Isothermal Amplification (HT-LAMP)","volume":"15","author":"Li","year":"2025","journal-title":"ACS Catal."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"133243","DOI":"10.1016\/j.ijbiomac.2024.133243","article-title":"Double domain fusion improves the reverse transcriptase activity and inhibitor tolerance of Bst DNA polymerase","volume":"274","author":"Xiang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Grigorakis, K., Ferousi, C., and Topakas, E. (2025). Protein engineering for industrial biocatalysis: Principles, approaches, and lessons from engineered PETases. Catalysts, 15.","DOI":"10.3390\/catal15020147"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1007\/s11157-024-09688-1","article-title":"Metabolic and enzymatic engineering strategies for polyethylene terephthalate degradation and valorization","volume":"23","author":"Satta","year":"2024","journal-title":"Rev. Environ. Sci. Bio\/Technol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"4845","DOI":"10.1007\/s00253-022-12067-x","article-title":"Thermostability engineering of industrial enzymes through structure modification","volume":"106","author":"Nezhad","year":"2022","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"108096","DOI":"10.1016\/j.bej.2021.108096","article-title":"Protein engineering design from directed evolution to de novo synthesis","volume":"174","author":"Xiong","year":"2021","journal-title":"Biochem. Eng. J."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"E5138","DOI":"10.1073\/pnas.1704872114","article-title":"Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins","volume":"114","author":"Vassylyeva","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Chow, L.T., and Vassylyev, D.G. (2022). Application of a Novel CL7\/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins. Affinity Chromatography: Methods and Protocols, Springer.","DOI":"10.1007\/978-1-0716-2176-9_5"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Li, Z., Wang, Y., Wang, X., Niu, S., Su, Z., Wang, F., Ni, J., Gong, Y., and Rao, B. (2024). A fusion of Taq DNA polymerase with the CL7 protein from Escherichia coli remarkably improves DNA amplification. Molecules, 29.","DOI":"10.3390\/molecules29051145"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"113503","DOI":"10.1016\/j.bios.2021.113503","article-title":"A high-specificity flap probe-based isothermal nucleic acid amplification method based on recombinant FEN1-Bst DNA polymerase","volume":"192","author":"Ye","year":"2021","journal-title":"Biosens. Bioelectron."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1021\/acs.biochem.1c00451","article-title":"Improved Bst DNA polymerase variants derived via a machine learning approach","volume":"62","author":"Paik","year":"2021","journal-title":"Biochemistry"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1038\/s41929-022-00798-z","article-title":"Deep learning-based k cat prediction enables improved enzyme-constrained model reconstruction","volume":"5","author":"Li","year":"2022","journal-title":"Nat. Catal."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"4607","DOI":"10.1021\/acs.biochem.8b00200","article-title":"Evolution of a thermophilic strand-displacing polymerase using high-temperature isothermal compartmentalized self-replication","volume":"57","author":"Milligan","year":"2018","journal-title":"Biochemistry"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e109","DOI":"10.1093\/nar\/gks316","article-title":"Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes","volume":"40","author":"Zweitzig","year":"2012","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1197","DOI":"10.1093\/nar\/gkh271","article-title":"A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro","volume":"32","author":"Wang","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"17592","DOI":"10.1073\/pnas.1906569116","article-title":"Energy metabolism controls phenotypes by protein efficiency and allocation","volume":"116","author":"Chen","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"16754","DOI":"10.1073\/pnas.1310377110","article-title":"Molecular crowding limits translation and cell growth","volume":"110","author":"Klumpp","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","unstructured":"Zhang, B.L. (2017). Study on Improving the Activity of Large-Fragment Thermostable DNA Polymerase from Bacillus stearothermophilus by Site-Directed Mutagenesis. [Doctoral Dissertation, South China University of Technology]."},{"key":"ref_23","unstructured":"Zhang, J.G. (2022). Modification of Bst DNA Polymerase and Its Application in the Detection of African Swine Fever Virus. [Doctoral Dissertation, South China University of Technology]."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"620","DOI":"10.2353\/jmoldx.2010.090190","article-title":"Immiscible phase nucleic acid purification eliminates PCR inhibitors with a single pass of paramagnetic particles through a hydrophobic liquid","volume":"12","author":"Sur","year":"2010","journal-title":"J. Mol. Diagn."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2872","DOI":"10.3748\/wjg.14.2872","article-title":"A simple and rapid method for extracting bacterial DNA from intestinal microflora for ERIC-PCR detection","volume":"14","author":"Yang","year":"2008","journal-title":"World J. Gastroenterol. WJG"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"57","DOI":"10.20450\/mjcce.2010.173","article-title":"Influence of salts and pcr inhibitors on the amplification capacity of three thermostable DNA polymerases","volume":"29","author":"Davalieva","year":"2010","journal-title":"Maced. J. Chem. Chem. Eng."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3432","DOI":"10.1038\/s41598-018-21041-5","article-title":"Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis","volume":"8","author":"Chang","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Yang, N., Zhang, H., Han, X., Liu, Z., and Lu, Y. (2024). Advancements and applications of loop-mediated isothermal amplification technology: A comprehensive overview. Front. Microbiol., 15.","DOI":"10.3389\/fmicb.2024.1406632"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/5\/954\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,25]],"date-time":"2026-05-25T15:18:42Z","timestamp":1779722322000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/5\/954"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,23]]},"references-count":28,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["microorganisms14050954"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14050954","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,23]]}}}