{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T06:27:37Z","timestamp":1787120857786,"version":"build-2736575974"},"reference-count":91,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      Laccases are versatile oxidoreductases found in bacteria, fungi, and plants., which have a wide range of industrial applications, including the removal of pharmaceutical micropollutants from wastewater. Bacterial laccases are particularly attractive because of their high catalytic activity and stability over broad temperature and pH ranges. One example is the copper efflux oxidase (CueO) from\n                      <jats:italic>Escherichia coli<\/jats:italic>\n                      , which has been fused to the adhesion-promoting peptide Macaque Histatin (MacHis) because immobilized enzymes have proven especially effective for solid-phase wastewater treatment. However, the practical application of this enzyme is constrained by the relatively low production yields of 25\u201350 mg\/L when expressed in recombinant\n                      <jats:italic>E. coli<\/jats:italic>\n                      or\n                      <jats:italic>Pichia pastoris<\/jats:italic>\n                      .\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods and results<\/jats:title>\n                    <jats:p>Here, we used transient expression in Nicotiana benthamiana to demonstrate that the engineered CueO-MacHis can be produced at substantially higher levels, reaching 250\u2013320 (286 \u00b1 35) mg\/kg biomass in the prokaryotic-like environment of chloroplasts in 6-week-old plants, which are typically used for production purposes. Subsequent systematic optimization of the transient expression process based on statistical experimental designs increased the accumulation of recombinant CueO-MacHis approximately threefold to 846 \u00b1 57 mg\/kg biomass. Furthermore, we established a simplified and cost-effective purification strategy that leverages the enzyme\u2019s temperature and pH stability to clarify extracts before anion exchange chromatography, achieving 76% recovery and 92% purity. Moreover, the plant-derived CueO-MacHis had a specific activity of 7.22 \u00b1 1.55 U\/mg, which is in the same range as that reported for other CueO enzymes.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>Our approach paves the way for the large-scale production of CueO-MacHis as a technical enzyme for industrial applications.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fpls.2026.1907380","type":"journal-article","created":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T05:46:41Z","timestamp":1787118401000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Plant chloroplasts enable high-yield and cost-effective production of the engineered bacterial enzyme CueO-MacHis as a technical protein via transient expression in Nicotiana benthamiana"],"prefix":"10.3389","volume":"17","author":[{"given":"Henrik","family":"Nausch","sequence":"first","affiliation":[{"name":"Department Model-based Product and Bioprocess Engineering, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)","place":["Aachen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catherine Rose","family":"Bernau","sequence":"additional","affiliation":[{"name":"Department Model-based Product and Bioprocess Engineering, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)","place":["Aachen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dirk","family":"Scheffler","sequence":"additional","affiliation":[{"name":"Department Model-based Product and Bioprocess Engineering, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)","place":["Aachen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"J\u00f6rg Ingmar","family":"Schuphan","sequence":"additional","affiliation":[{"name":"Department Model-based Product and Bioprocess Engineering, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)","place":["Aachen, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"B1","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1016\/B978-0-443-22072-2.00003-6","article-title":"Chapter 6 - Role of microbial enzymes in wastewater processing and treatment","volume-title":"Enzyme Biotechnology for Environmental Sustainability: Progress in Biochemistry and Biotechnology","author":"Ahmad","year":"2024"},{"key":"B2","doi-asserted-by":"publisher","first-page":"200","DOI":"10.1186\/s12934-019-1248-0","article-title":"Laccases: structure, function, and potential application in water bioremediation","volume":"18","author":"Arregui","year":"2019","journal-title":"Microb. 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