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Chemically synthesised Glucagon\u2010like peptide\u20101 receptor agonists (GLP\u20101RAs) containing artificial amino acids reduce haemoglobin A1c and obesity but are not yet affordable and require invasive injections. High dosage requirement and gastrointestinal complications are among the current limitations of oral GLP\u20101RAs. Therefore, we expressed codon optimised Exenatide and Lixisenatide fused with Cholera\u2010toxin B\u2010subunit (CTB) in lettuce chloroplasts to facilitate their oral delivery, increase affordability, and patient compliance. Site\u2010specific integration of transgene expression cassettes into the chloroplast genome and removal of the selectable marker gene from marker\u2010free lettuce transplastomic lines were confirmed using three sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. CTB\u2010Exenatide and CTB\u2010Lixisenatide expression levels were 1.94 and 3.64\u2009mg\/g plant powder in T0 generation and increased ~31 and ~48%, respectively in marker\u2010removed T1 lines. Maternal inheritance of transgenes was confirmed by lack of segregation when seedlings were germinated in the selection medium before removal of the antibiotic resistance gene (\n                    <jats:italic>aadA<\/jats:italic>\n                    ). Monosialotetrahexosylganglioside (GM1) ELISA confirmed pentameric assembly efficiency of both CTB\u2010fusion proteins similar to commercial CTB standards. GLP\u20101 receptor binding confirmed functionality of CTB\u2010Exenatide\/CTB\u2010Lixisenatide with statistical significance (***\n                    <jats:italic>p<\/jats:italic>\n                    \u2009&lt;\u20090.001 by\n                    <jats:italic>t<\/jats:italic>\n                    \u2010test) and post\u2010translational amidation in chloroplasts. Expression of functional CTB\u2010Exenatide and CTB\u2010Lixisenatide in an edible marker\u2010free system for the first time and much lower dosage requirement for functionality than recently developed synthetic GLP\u20101RAs paves the way for clinical studies to advance oral delivery of these affordable biologics.\n                  <\/jats:p>","DOI":"10.1111\/pbi.70554","type":"journal-article","created":{"date-parts":[[2026,1,24]],"date-time":"2026-01-24T11:15:07Z","timestamp":1769253307000},"page":"3158-3171","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Engineering Marker\u2010Free Lettuce Chloroplast Genome to Express Functional Glucagon\u2010Like Peptide\u20101 Receptor Agonists Exenatide and Lixisenatide"],"prefix":"10.1111","volume":"24","author":[{"given":"Rahul","family":"Singh","sequence":"first","affiliation":[{"name":"Department of Basic and Translational Sciences, School of Dental Medicine University of Pennsylvania  Philadelphia Pennsylvania USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4485-1176","authenticated-orcid":false,"given":"Henry","family":"Daniell","sequence":"additional","affiliation":[{"name":"Department of Basic and Translational Sciences, School of Dental Medicine University of Pennsylvania  Philadelphia Pennsylvania 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