{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,19]],"date-time":"2026-09-19T04:03:35Z","timestamp":1789790615197,"version":"4.0.1"},"reference-count":80,"publisher":"National Academy of Sciences","issue":"45","license":[{"start":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T00:00:00Z","timestamp":1762473600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100020595","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["NSTC113-2321-B-005-011"],"award-info":[{"award-number":["NSTC113-2321-B-005-011"]}],"id":[{"id":"10.13039\/100020595","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100020595","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["MOST 110-2221-E-039-002-MY3"],"award-info":[{"award-number":["MOST 110-2221-E-039-002-MY3"]}],"id":[{"id":"10.13039\/100020595","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,11,11]]},"abstract":"<jats:p>\n                    Cellulosic biomass represents a promising feedstock for biofuel and biochemical production. However, its recalcitrant structure strongly hinders enzymatic degradation. Cellulosomes are large multienzyme complexes, highly efficient at degrading cellulose. A cellulase in a cellulosome has a dockerin domain that binds to a cohesin module on the CipA (cellulosome integrating protein A). In a native cellulosome all cohesins are identical, so that the cellulase types and their positions in a CipA cannot be controlled. Here, we constructed the largest designer CipA known to date. Using innovative techniques, we synthesized a designer CipA gene that encodes nine distinct cohesins and two cellulose-binding modules, which we named\n                    <jats:italic toggle=\"yes\">DCipA2B9C<\/jats:italic>\n                    . Then, we fused nine distinct fungal cellulases separately with nine distinct dockerins for their precise positioning on DCipA2B9C to achieve enzyme proximity-effect. We constructed three yeast hosts to compare their performances. First, an enzyme host (EH) secretes nine dockerin-fused cellulases, including endoglucanases (EgIII-a, EgIII-m, and EgIII-c), exoglucanases (CBHII-j and EXG2-r), \u03b2-glucosidases (BGS-f and BGS-l), and cellulase boosters, including a LPMO-t and CDH-b. Second, the scaffoldin host (SH) expresses DCipA2B9C. Third, the cellulosome-9 host expresses DCipA2B9C and nine dockerin-fused cellulases. Native-PAGE and ELISA confirmed specific interactions between dockerins and cohesins. Additionally, native-PAGE, SDS-PAGE, and LC-MS verified the successful assembly of the multienzyme complex. Our performance evaluation showed that coculturing of EH and SH outperformed the cellulosome-9 host. It degraded microcrystalline cellulose efficiently to produce 14.29 g\/L bioethanol, which surpassed all previously constructed yeast cellulosomes by fourfold or more. In summary, our study provides an effective approach to biomass degradation.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2517490122","type":"journal-article","created":{"date-parts":[[2025,11,7]],"date-time":"2025-11-07T16:28:36Z","timestamp":1762532916000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":7,"title":["Creating a large designer cellulosome in yeast to boost ethanol production"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6862-3509","authenticated-orcid":false,"given":"Zeba","family":"Khatoon","sequence":"first","affiliation":[{"name":"Biodiversity Research Center, Academia Sinica","place":["Taipei, Taiwan"]},{"name":"Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica","place":["Taipei, Taiwan"]},{"name":"Graduate Institute of Biotechnology, National Chung Hsing 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Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jui-Jen","family":"Chang","sequence":"additional","affiliation":[{"name":"Department of Medical Research, China Medical University Hospital, China Medical University","place":["Taichung, Taiwan"]},{"name":"Graduate Institute of Integrated Medicine, China Medical University","place":["Taichung, Taiwan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wen-Hsiung","family":"Li","sequence":"additional","affiliation":[{"name":"Biodiversity Research Center, Academia Sinica","place":["Taipei, Taiwan"]},{"name":"Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica","place":["Taipei, Taiwan"]},{"name":"Department of Applied Mathematics, National Chung Hsing University","place":["Taichung, Taiwan"]},{"name":"Department of Ecology and Evolution, University of 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