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Despite substantial progress in pathway design and prototyping, most efforts stay at the proof-of-concept stage, with frequent failures observed even under in vitro conditions. This review identifies seven major barriers constraining the deployment of synthetic C1 metabolism in microorganisms and proposes targeted strategies for overcoming these issues. A primary limitation is the low catalytic activity of carbon-fixing enzymes, particularly carboxylases, which restricts the overall pathway performance. In parallel, challenges in expressing multiple heterologous genes\u2014especially those encoding metal-dependent or oxygen-sensitive enzymes\u2014further hinder pathway functionality. At the systems level, synthetic C1 pathways often exhibit poor flux distribution, limited integration with the host metabolism, accumulation of toxic intermediates, and disruptions in redox and energy balance. These factors collectively reduce biomass formation and compromise product yields in biotechnological setups. Overcoming these interconnected challenges is essential for moving synthetic C1 assimilation beyond conceptual stages and enabling its application in scalable, efficient bioprocesses towards a circular bioeconomy.<\/jats:p>","DOI":"10.1093\/femsre\/fuaf011","type":"journal-article","created":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T02:30:45Z","timestamp":1743647445000},"source":"Crossref","is-referenced-by-count":32,"title":["Seven critical challenges in synthetic one-carbon assimilation and their potential solutions"],"prefix":"10.1093","volume":"49","author":[{"given":"\u00d2scar","family":"Puiggen\u00e9","sequence":"first","affiliation":[{"name":"The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby ,","place":["Denmark"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giusi","family":"Favoino","sequence":"additional","affiliation":[{"name":"The Novo Nordisk Foundation Center for Biosustainability, 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