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

Nannochloropsis is an industrially relevant marine microalga with exceptional potential as a chassis for sunlight-driven CO₂ 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, CO₂-based production of value-added biochemicals.