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Here, we describe two yeast-based strategies for the direct recombinant production of this cyclic, disulfide-rich peptide. In the first approach, using Pichia pastoris, a fusion protein comprising the conotoxin precursor and a full-length asparaginyl ligase connected by a poly-His linker was secreted into the culture medium. Cyclization of the secreted \u03b1-conotoxin was initiated by a two-step pH adjustment protocol for generating the correctly folded conotoxin. In the second approach, Saccharomyces cerevisiae was engineered to display the activated ligase on its cell surface while secreting Vc1.1 precursors extracellularly. Cyclized peptides were detected in the culture supernatant without further processing. These two strategies provide efficient routes for the direct recombinant production of disulfide-rich and backbone-cyclized peptides.<\/jats:p>","DOI":"10.1021\/acschembio.6c00509","type":"journal-article","created":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T19:02:11Z","timestamp":1786647731000},"page":"2394-2403","source":"Crossref","is-referenced-by-count":0,"title":["Direct Heterologous Expression of Cyclic Conotoxin [G22N]Vc1.1 in Yeast"],"prefix":"10.1021","volume":"21","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0397-8341","authenticated-orcid":true,"given":"Jing","family":"Xie","sequence":"first","affiliation":[{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9409-5287","authenticated-orcid":true,"given":"Kuok","family":"Yap","sequence":"additional","affiliation":[{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]},{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7973-7632","authenticated-orcid":true,"given":"Conan K.","family":"Wang","sequence":"additional","affiliation":[{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Durek","sequence":"additional","affiliation":[{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0007-6796","authenticated-orcid":true,"given":"David J.","family":"Craik","sequence":"additional","affiliation":[{"name":"The University of Queensland , , , ,","place":["Brisbane, QLD, Australia, 4072"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,13]]},"reference":[{"issue":"22","key":"2026101511062451900_cit1","doi-asserted-by":"publisher","first-page":"6904","DOI":"10.1021\/bi034043e","article-title":"A novel \u03b1-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo","volume":"42","author":"Sandall","year":"2003","journal-title":"Biochemistry"},{"issue":"2","key":"2026101511062451900_cit2","doi-asserted-by":"publisher","first-page":"149","DOI":"10.1016\/j.brainres.2005.08.009","article-title":"Alpha-conotoxin Vc1 1 alleviates neuropathic pain and accelerates functional recovery of injured neurones","volume":"1059","author":"Satkunanathan","year":"2005","journal-title":"Brain Res."},{"issue":"37","key":"2026101511062451900_cit3","doi-asserted-by":"publisher","first-page":"6545","DOI":"10.1002\/anie.201000620","article-title":"The engineering of an orally active conotoxin for the treatment of neuropathic pain","volume":"49","author":"Clark","year":"2010","journal-title":"Angew. 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