{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T02:33:24Z","timestamp":1793414004471,"version":"4.5.0"},"reference-count":44,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2024,8,14]],"date-time":"2024-08-14T00:00:00Z","timestamp":1723593600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Biological Breeding-National Science and Technology Major Project","award":["2022ZD04021"],"award-info":[{"award-number":["2022ZD04021"]}]},{"name":"Biological Breeding-National Science and Technology Major Project","award":["2024YFHZ0347"],"award-info":[{"award-number":["2024YFHZ0347"]}]},{"name":"Sichuan Province Key R&amp;D Program, Hong Kong, Macao and Taiwan Science and Technology Innovation Cooperation Project","award":["2022ZD04021"],"award-info":[{"award-number":["2022ZD04021"]}]},{"name":"Sichuan Province Key R&amp;D Program, Hong Kong, Macao and Taiwan Science and Technology Innovation Cooperation Project","award":["2024YFHZ0347"],"award-info":[{"award-number":["2024YFHZ0347"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>HxTx-Hv1h, a neurotoxic peptide derived from spider venom, has been developed for use in commercial biopesticide formulations. Cell Penetrating Peptides (CPPs) are short peptides that facilitate the translocation of various biomolecules across cellular membranes. Here, we evaluated the aphidicidal efficacy of a conjugated peptide, HxTx-Hv1h\/CPP-1838, created by fusing HxTx-Hv1h with CPP-1838. Additionally, we aimed to establish a robust recombinant expression system for HxTx-Hv1h\/CPP-1838. We successfully achieved the secretory production of HxTx-Hv1h, its fusion with Galanthus nivalis agglutinin (GNA) forming HxTx-Hv1h\/GNA and HxTx-Hv1h\/CPP-1838 in yeast. Purified HxTx-Hv1h exhibited contact toxicity against Megoura crassicauda, with a 48 h median lethal concentration (LC50) of 860.5 \u03bcg\/mL. Fusion with GNA or CPP-1838 significantly enhanced its aphidicidal potency, reducing the LC50 to 683.5 \u03bcg\/mL and 465.2 \u03bcg\/mL, respectively. The aphidicidal efficacy was further improved with the addition of surfactant, decreasing the LC50 of HxTx-Hv1h\/CPP-1838 to 66.7 \u03bcg\/mL\u2014over four times lower compared to HxTx-Hv1h alone. Furthermore, we engineered HxTx-Hv1h\/CPP-1838 multi-copy expression vectors utilizing the BglBrick assembly method and achieved high-level recombinant production in laboratory-scale fermentation. This study is the first to document a CPP fusion strategy that enhances the transdermal aphidicidal activity of a natural toxin like HxTx-Hv1h and opens up the possibility of exploring the recombinant production of HxTx-Hv1h\/CPP-1838 for potential applications.<\/jats:p>","DOI":"10.3390\/toxins16080358","type":"journal-article","created":{"date-parts":[[2024,8,14]],"date-time":"2024-08-14T09:20:51Z","timestamp":1723627251000},"page":"358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Cell Penetrating Peptide Enhances the Aphidicidal Activity of Spider Venom-Derived Neurotoxin"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9122-9796","authenticated-orcid":false,"given":"Wenxian","family":"Wu","sequence":"first","affiliation":[{"name":"Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu Agricultural Science and Technology Center, Chengdu 610000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3674-7811","authenticated-orcid":false,"given":"Abid","family":"Ali","sequence":"additional","affiliation":[{"name":"Department of Entomology, University of Agriculture Faisalabad, Faisalabad 38040, Pakistan"},{"name":"College of Life Science, Shenyang Normal University, Shenyang 110034, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinbo","family":"Shen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 310000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maozhi","family":"Ren","sequence":"additional","affiliation":[{"name":"Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu Agricultural Science and Technology Center, Chengdu 610000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Cai","sequence":"additional","affiliation":[{"name":"College of Life Science, Sichuan Agricultural University, Ya\u2019an 625000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5055-746X","authenticated-orcid":false,"given":"Limei","family":"He","sequence":"additional","affiliation":[{"name":"Institute of Urban Agriculture, Chinese Academy of Agricultural Sciences, Chengdu Agricultural Science and Technology Center, Chengdu 610000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,8,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.tibtech.2013.08.001","article-title":"Delivery of intrahemocoelic peptides for insect pest management","volume":"32","author":"Bonning","year":"2014","journal-title":"Trends Biotechnol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1126\/science.aat3466","article-title":"Increase in crop losses to insect pests in a warming climate","volume":"361","author":"Deutsch","year":"2018","journal-title":"Science"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"20201265","DOI":"10.1098\/rspb.2020.1265","article-title":"Do novel insecticides pose a threat to beneficial insects?","volume":"287","author":"Siviter","year":"2020","journal-title":"Proc. R. Soc. B"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Gupta, S., and Gupta, K. (2020). Bioaccumulation of pesticides and its impact on biological systems. Pesticides in Crop Production: Physiological and Biochemical Action, Wiley.","DOI":"10.1002\/9781119432241.ch4"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1016\/j.pestbp.2014.11.014","article-title":"IRAC: Mode of action classification and insecticide resistance management","volume":"121","author":"Sparks","year":"2015","journal-title":"Pestic. Biochem. Physiol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"730","DOI":"10.1126\/science.341.6147.730","article-title":"Infographic: Pesticide planet","volume":"341","author":"Stokstad","year":"2013","journal-title":"Science"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1111\/j.1467-7652.2011.00595.x","article-title":"Bacillus thuringiensis: A century of research, development and commercial applications","volume":"9","author":"Sanahuja","year":"2011","journal-title":"Plant Biotechnol. J."},{"key":"ref_8","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_9","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1093\/bioinformatics\/btx661","article-title":"ArachnoServer 3.0: An online resource for automated discovery, analysis and annotation of spider toxins","volume":"34","author":"Pineda","year":"2018","journal-title":"Bioinformatics"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3665","DOI":"10.1007\/s00018-013-1315-3","article-title":"The insecticidal potential of venom peptides","volume":"70","author":"Smith","year":"2013","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2437","DOI":"10.1002\/ps.5452","article-title":"Tying pest insects in knots: The deployment of spider-venom-derived knottins as bioinsecticides","volume":"75","author":"King","year":"2019","journal-title":"Pest Manag. Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1002\/ps.7198","article-title":"Enhancing the oral and topical insecticidal efficacy of a commercialized spider venom peptide biopesticide via fusion to the carrier snowdrop lectin (Galanthus nivalis agglutinin)","volume":"79","author":"Sukiran","year":"2023","journal-title":"Pest Manag. Sci."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"107616","DOI":"10.1016\/j.toxicon.2024.107616","article-title":"Spider venom neurotoxin based bioinsecticides: A novel bioactive for the control of the Asian citrus psyllid Diaphorina citri (Hemiptera)","volume":"239","author":"Miranda","year":"2024","journal-title":"Toxicon"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Nakasu, E.Y., Williamson, S.M., Edwards, M.G., Fitches, E.C., Gatehouse, J.A., Wright, G.A., and Gatehouse, A.M. (2014). Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees. Proc. R. Soc. B Biol. Sci., 281.","DOI":"10.1098\/rspb.2014.0619"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1093\/jee\/tox280","article-title":"Effects of pesticides on the survival of rove beetle (Coleoptera: Staphylinidae) and insidious flower bug (Hemiptera: Anthocoridae) adults","volume":"111","author":"Cloyd","year":"2018","journal-title":"J. Econ. Entomol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1007\/s10340-019-01143-3","article-title":"Demonstrating the potential of a novel spider venom-based biopesticide for target-specific control of the small hive beetle, a serious pest of the European honeybee","volume":"93","author":"Powell","year":"2020","journal-title":"J. Pest Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"601","DOI":"10.1016\/j.toxicon.2004.02.010","article-title":"Australian funnel-web spiders: Master insecticide chemists","volume":"43","author":"Tedford","year":"2004","journal-title":"Toxicon"},{"key":"ref_18","unstructured":"Sollod, B.L. (2006). From Venoms to Insecticides: Exploring the Structure, Function, and Evolution of Peptide Toxins Found in the Venom of Australian Funnel-Web Spiders, University of Connecticut."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"44133","DOI":"10.1074\/jbc.M404006200","article-title":"Scanning mutagenesis of \u03c9-atracotoxin-Hv1a reveals a spatially restricted epitope that confers selective activity against insect calcium channels","volume":"279","author":"Tedford","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1016\/j.bcp.2007.05.017","article-title":"The \u03c9-atracotoxins: Selective blockers of insect M-LVA and HVA calcium channels","volume":"74","author":"Chong","year":"2007","journal-title":"Biochem. Pharmacol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1002\/1873-3468.13435","article-title":"Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor","volume":"593","author":"Chambers","year":"2019","journal-title":"FEBS Lett."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Fitches, E.C., Pyati, P., King, G.F., and Gatehouse, J.A. (2012). Fusion to snowdrop lectin magnifies the oral activity of insecticidal \u03c9-hexatoxin-Hv1a peptide by enabling its delivery to the central nervous system. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0039389"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.ibmb.2014.01.007","article-title":"A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders","volume":"47","author":"Yang","year":"2014","journal-title":"Insect Biochem. Mol. Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/nbt.2753","article-title":"Toxin delivery by the coat protein of an aphid-vectored plant virus provides plant resistance to aphids","volume":"32","author":"Bonning","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1279","DOI":"10.1007\/s10340-018-1016-7","article-title":"Assessment of a commercial spider venom peptide against spotted-wing Drosophila and interaction with adjuvants","volume":"91","author":"Fanning","year":"2018","journal-title":"J. Pest Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"406","DOI":"10.1016\/j.tips.2017.01.003","article-title":"Cell-penetrating peptides: From basic research to clinics","volume":"38","author":"Guidotti","year":"2017","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"16010","DOI":"10.1074\/jbc.272.25.16010","article-title":"A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus","volume":"272","author":"Vives","year":"1997","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"D1098","DOI":"10.1093\/nar\/gkv1266","article-title":"CPPsite 2.0: A repository of experimentally validated cell-penetrating peptides","volume":"44","author":"Agrawal","year":"2016","journal-title":"Nucleic Acids Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1093","DOI":"10.1038\/sj.bjp.0706279","article-title":"Characterisation of cell-penetrating peptide-mediated peptide delivery","volume":"145","author":"Jones","year":"2005","journal-title":"Br. J. Pharmacol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"928","DOI":"10.1080\/10717544.2018.1458921","article-title":"Cell penetrating peptides: A comparative transport analysis for 474 sequence motifs","volume":"25","author":"Ramaker","year":"2018","journal-title":"Drug Deliv."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Darif, N., Vogelsang, K., Vorgia, E., Schneider, D., Deligianni, E., Geibel, S., Vontas, J., and Denecke, S. (2023). Cell penetrating peptides are versatile tools for enhancing multimodal uptake into cells from pest insects. Pestic. Biochem. Physiol., 190.","DOI":"10.1016\/j.pestbp.2022.105317"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"De Brabander, P., Uitterhaegen, E., Delmulle, T., De Winter, K., and Soetaert, W. (2023). Challenges and progress towards industrial recombinant protein production in yeasts: A review. Biotechnol. Adv., 64.","DOI":"10.1016\/j.biotechadv.2023.108121"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"999","DOI":"10.1016\/j.ijbiomac.2017.11.008","article-title":"Combined effect of gene dosage and process optimization strategies on high-level production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris fed-batch culture","volume":"108","author":"Dagar","year":"2018","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1237","DOI":"10.1007\/s10295-014-1466-8","article-title":"Optimising expression of the recombinant fusion protein biopesticide \u03c9-hexatoxin-Hv1a\/GNA in Pichia pastoris: Sequence modifications and a simple method for the generation of multi-copy strains","volume":"41","author":"Pyati","year":"2014","journal-title":"J. Ind. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1111\/j.1365-2672.2009.04279.x","article-title":"Efficient generation of multi-copy strains for optimizing secretory expression of porcine insulin precursor in yeast Pichia pastoris","volume":"107","author":"Zhu","year":"2009","journal-title":"J. Appl. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1016\/j.addr.2012.09.039","article-title":"Fusion protein linkers: Property, design and functionality","volume":"65","author":"Chen","year":"2013","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"475","DOI":"10.1146\/annurev-ento-120811-153650","article-title":"Spider-venom peptides: Structure, pharmacology, and potential for control of insect pests","volume":"58","author":"King","year":"2013","journal-title":"Annu. Rev. Entomol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1080\/17425247.2019.1676720","article-title":"Cell penetrating peptides: The potent multi-cargo intracellular carriers","volume":"16","author":"Kardani","year":"2019","journal-title":"Expert Opin. Drug Deliv."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1016\/B978-0-12-800197-4.00008-7","article-title":"Methods for deployment of spider venom peptides as bioinsecticides","volume":"Volume 47","author":"Herzig","year":"2014","journal-title":"Advances in Insect Physiology"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1260","DOI":"10.1111\/j.1567-1364.2009.00561.x","article-title":"Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus","volume":"9","author":"Marx","year":"2009","journal-title":"Fems Yeast Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1016\/j.tibtech.2005.02.008","article-title":"Seamless cloning and gene fusion","volume":"23","author":"Lu","year":"2005","journal-title":"Trends Biotechnol."},{"key":"ref_42","unstructured":"Wolf, K., Breunig, K.D., and Barth, G. (2012). Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols, Springer Science & Business Media."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"169","DOI":"10.1016\/S0076-6879(09)63013-5","article-title":"Expression in the yeast Pichia pastoris","volume":"463","author":"Cregg","year":"2009","journal-title":"Methods Enzymol."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Nord\u00e9n, K., Agemark, M., Danielson, J.\u00c5., Alexandersson, E., Kjellbom, P., and Johanson, U. (2011). Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris. BMC Biotechnol., 11.","DOI":"10.1186\/1472-6750-11-47"}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/8\/358\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T15:36:38Z","timestamp":1760110598000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/16\/8\/358"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,8,14]]},"references-count":44,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2024,8]]}},"alternative-id":["toxins16080358"],"URL":"https:\/\/doi.org\/10.3390\/toxins16080358","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,8,14]]}}}