A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis

Abstract Nannochloropsis is an industrially relevant marine microalga with exceptional potential as a chassis for sunlight-driven CO 2 valorization. However, its broad application in synthetic biology has been constrained by the lack of a standardized and modular genetic toolbox. Here, we report the development of a comprehensive Modular Cloning (MoClo) toolkit for Nannochloropsis , based on Golden Gate assembly and a standard syntax. The toolkit comprises 91 domesticated genetic parts spanning promoters, signal peptides, selectable markers, reporter genes, tags and terminators. A large subset of these parts, including several not previously evaluated in Nannochloropsis , was functionally validated, enabling convenient and reliable transformant selection, immunodetection, and subcellular localization. To demonstrate the utility of the toolkit for multi-gene pathway engineering, modularly assembled keto-carotenoid biosynthetic pathways were introduced into Nannochloropsis , leading to substantial accumulation of canthaxanthin (4.5 mg g −1 ) or astaxanthin (2.8 mg g −1 ). Collectively, this flexible and expandable MoClo toolkit establishes a standardized foundation for synthetic biology in Nannochloropsis , enables rapid design-build-test cycles for multi-gene constructs, and advances the use of industrial microalga for sustainable, CO 2 -based production of value-added biochemicals.