{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T05:17:19Z","timestamp":1788585439297,"version":"build-2803163510"},"reference-count":26,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004837","name":"Spanish Ministry of Science and Innovation","doi-asserted-by":"publisher","award":["PID2021-122914OB-I00"],"award-info":[{"award-number":["PID2021-122914OB-I00"]}],"id":[{"id":"10.13039\/501100004837","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003359","name":"Generalitat Valenciana","doi-asserted-by":"publisher","award":["GVPROMETEO2020-010"],"award-info":[{"award-number":["GVPROMETEO2020-010"]}],"id":[{"id":"10.13039\/501100003359","id-type":"DOI","asserted-by":"publisher"}]},{"award":["GVPROMETEO2020-010"],"award-info":[{"award-number":["GVPROMETEO2020-010"]}],"id":[{"id":"https:\/\/ror.org\/0097mvx21","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000780","name":"European Union","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/019w4f821","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Scientific and Technological Research Council of T\u00fcrkiye"},{"id":[{"id":"https:\/\/ror.org\/04w9kkr77","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>Vip3 proteins secreted by the entomopathogenic bacterium Bacillus thuringiensis (Bt) have an important role in biological control against economically important lepidopteran pests. The elucidation of Vip3 protein structures has helped to address the roles of domains and amino acid positions involved in toxicity, especially in the N-terminal domains I and II, thereby supporting their more efficient utilization. In this study, we evaluated the impact of combinations of critical amino acid substitutions, selected from previous studies, in domains IV and V of the Vip3Aa90 protein on its insecticidal activity against three lepidopteran pests. The double mutant S543N\/I544L, triple mutants S543N\/I544L\/E627A and S543N\/I544L\/S686R, and quadruple mutant S543N\/I544L\/E627A\/S686R were constructed in Escherichia coli by site-directed mutagenesis. Among these, only the Vip3Aa mutant proteins S543N\/I544L\/E627A and S543N\/I544L\/E627A\/S686R could be expressed and purified for bioassays. Both mutant proteins had similar toxicity against Spodoptera littoralis, showing higher insecticidal activity than the wild-type (WT) Vip3Aa90 at the LC90 level. However, at the LC50 level, only a slight improvement in toxicity was observed for the quadruple mutant. In the case of S. exigua, no significant difference in toxicity was observed for either of the two mutant proteins with respect to the WT at either LC level. Interestingly, for G. molesta, though the toxicity of the triple mutant did not differ significantly compared to that of the WT protein, that of the quadruple mutant showed a marked decrease in toxicity of over 10-fold. This study revealed that combining selected amino acid substitutions in domains IV and V can enhance Vip3Aa90 toxicity against some lepidopteran species but can be either neutral or even deleterious in others.<\/jats:p>","DOI":"10.3390\/biology15171508","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T09:20:43Z","timestamp":1788427243000},"page":"1508","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Amino Acid Substitutions in the C-Terminal Domains of Vip3Aa Enhance Insecticidal Toxicity"],"prefix":"10.3390","volume":"15","author":[{"given":"Burcu","family":"\u015eahin","sequence":"first","affiliation":[{"name":"Department of Biology, Faculty of Science, Mu\u011fla S\u0131tk\u0131 Ko\u00e7man University, 48000 Mu\u011fla, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3263-5786","authenticated-orcid":false,"given":"Patricia","family":"Hern\u00e1ndez-Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Department of Genetics, Institute of Biotechnology and Biomedicine (BIOTECMED), Universitat de Val\u00e8ncia, 46100 Burjassot, Spain"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5535-0612","authenticated-orcid":false,"given":"Juan","family":"Ferr\u00e9","sequence":"additional","affiliation":[{"name":"Department of Genetics, Institute of Biotechnology and Biomedicine (BIOTECMED), Universitat de Val\u00e8ncia, 46100 Burjassot, Spain"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kumar, P., Kamle, M., Borah, R., Mahato, D.K., and Sharma, B. (2021). Bacillus thuringiensis as microbial biopesticide: Uses and application for sustainable agriculture. Egypt. J. Biol. Pest Control, 31.","DOI":"10.1186\/s41938-021-00440-3"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3296","DOI":"10.3390\/toxins6123296","article-title":"Bacillus thuringiensis toxins: An overview of their biocidal activity","volume":"6","author":"Palma","year":"2014","journal-title":"Toxins"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1146\/annurev-ento-052620-073348","article-title":"Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis","volume":"66","author":"Heckel","year":"2021","journal-title":"Annu. Rev. Entomol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1128\/MMBR.00060-15","article-title":"Bacterial vegetative insecticidal proteins (Vip) from entomopathogenic bacteria","volume":"80","author":"Chakroun","year":"2016","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1612","DOI":"10.1002\/ps.5804","article-title":"Bacillus thuringiensis vegetative insecticidal protein family Vip3A and mode of action against pest Lepidoptera","volume":"76","author":"Chakrabarty","year":"2020","journal-title":"Pest Manag. Sci."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Syed, T., Askari, M., Meng, Z., Li, Y., Abid, M.A., Wei, Y., Guo, S., Liang, C., and Zhang, R. (2020). Current insights on vegetative insecticidal proteins (Vip) as next generation pest killers. Toxins, 12.","DOI":"10.3390\/toxins12080522"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"103000","DOI":"10.1016\/j.jbc.2023.103000","article-title":"Functional characterization of Vip3Aa from Bacillus thuringiensis reveals the contributions of specific domains to its insecticidal activity","volume":"299","author":"Jiang","year":"2023","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Jiang, K., Zhang, Y., Chen, Z., Wu, D., Cai, J., and Gao, X. (2020). Structural and functional insights into the C-terminal fragment of insecticidal Vip3A toxin of Bacillus thuringiensis. Toxins, 12.","DOI":"10.1101\/2020.01.24.918433"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Gupta, M., Kumar, H., and Kaur, S. (2021). Vegetative insecticidal protein (Vip): A potential contender from Bacillus thuringiensis for efficient management of various detrimental agricultural pests. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.659736"},{"key":"ref_10","first-page":"93","article-title":"Chapter Three\u2014Vip3 insecticidal proteins: Structure and mode of action","volume":"Volume 65","year":"2023","journal-title":"Advances in Insect Physiology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3974","DOI":"10.1038\/s41467-020-17758-5","article-title":"Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis","volume":"11","author":"Huesa","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"583","DOI":"10.1007\/s00284-012-0201-9","article-title":"Ser-substituted mutations of Cys residues in Bacillus thuringiensis Vip3Aa7 exert a negative effect on its insecticidal activity","volume":"65","author":"Dong","year":"2012","journal-title":"Curr. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"7539","DOI":"10.1038\/s41598-018-25346-3","article-title":"Critical amino acids for the insecticidal activity of Vip3Af from Bacillus thuringiensis: Inference on structural aspects","volume":"8","author":"Banyuls","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Quan, Y., and Ferr\u00e9, J. (2019). Structural domains of the Bacillus thuringiensis Vip3Af protein unraveled by tryptic digestion of alanine mutants. Toxins, 11.","DOI":"10.20944\/preprints201905.0203.v1"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"107439","DOI":"10.1016\/j.jip.2020.107439","article-title":"Effect of substitutions of key residues on the stability and the insecticidal activity of Vip3Af from Bacillus thuringiensis","volume":"186","author":"Banyuls","year":"2021","journal-title":"J. Invertebr. Pathol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2324","DOI":"10.1002\/ps.6858","article-title":"Vip3Aa domain IV and V mutants confer higher insecticidal activity against Spodoptera frugiperda and Helicoverpa armigera","volume":"78","author":"Yang","year":"2022","journal-title":"Pest Manag. Sci."},{"key":"ref_17","unstructured":"Sambrook, J., and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press. [3rd ed.]."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"\u015eahin, B., Gomis-Cebolla, J., G\u00fcnes, H., and Ferr\u00e9, J. (2018). Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0206813"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1093\/aesa\/69.2.365","article-title":"Techniques for rearing laboratory colonies of tobacco hornworms and pinkboll worms","volume":"69","author":"Bell","year":"1976","journal-title":"Ann. Entomol. Soc. Am."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"16204","DOI":"10.1038\/s41598-018-34214-z","article-title":"Efficacy and resistance management potential of a modified Vip3C protein for control of Spodoptera frugiperda in maize","volume":"8","author":"Kahn","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1002\/ps.8402","article-title":"A Vip3Af mutant confers high resistance to broad lepidopteran insect pests","volume":"81","author":"Sun","year":"2025","journal-title":"Pest Manag. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1363","DOI":"10.1080\/09583157.2017.1399309","article-title":"Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests","volume":"27","author":"Chi","year":"2017","journal-title":"Biocontrol Sci. Technol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1448","DOI":"10.1007\/s12010-018-2880-6","article-title":"In silico structure-based identification and validation of key residues of Vip3Aa involving in lepidopteran brush border receptor binding","volume":"187","author":"Chi","year":"2019","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"123407","DOI":"10.1016\/j.ijbiomac.2023.123407","article-title":"Recombinant protein expression: Challenges in production and folding related matters","volume":"233","author":"Beygmoradi","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"108079","DOI":"10.1016\/j.biotechadv.2022.108079","article-title":"Folding of heterologous proteins in bacterial cell factories: Cellular mechanisms and engineering strategies","volume":"63","author":"Rong","year":"2023","journal-title":"Biotechnol. Adv."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1002\/0471140864.ps0524s61","article-title":"Strategies to optimize protein expression in E. Coli","volume":"61","author":"Francis","year":"2010","journal-title":"Curr. Protoc. Protein Sci."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/17\/1508\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T04:50:31Z","timestamp":1788583831000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/17\/1508"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":26,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["biology15171508"],"URL":"https:\/\/doi.org\/10.3390\/biology15171508","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}