Abstract

Zeaxanthin is an oxygenated carotenoid with established physiological functions in ocular health and antioxidant protection. Currently, industrial zeaxanthin production relies primarily on plant extraction and chemical synthesis, which is costly and poses a significant environmental burden. Microbial biosynthesis offers a sustainable and eco-friendly alternative, where the gene dosage of key enzymes critically influences biosynthetic performance. However, optimizing copy number remains challenging for pathways requiring tandem insertion of introduced genes into limited genomic loci. Accordingly, this study developed a genetic system rNTS, which targets the r ibosomal n on- t ranscribed s pacer region for one-step, high-copy integration of multiple genes and direct assessment of gene dosage effects. F luorescence I ntensity R atio s (FIRs) served as indicators for the direct and quantitative estimation of gene copy number and the selection of optimal dosage. An individual rNTSi ( integration ) vector integrated up to 40 single-gene copies, while parallel rNTSi vectors enabled multi-gene strains totaling 20 copies with independent visual selection. Following marker removal with rNTSr ( removal ) vector via the Cre/LoxP system, selection markers were efficiently reused for iterative integrations to achieve 32 copies. Applying rNTS to zeaxanthin biosynthesis rapidly identified the optimal dosage of the rate-limiting enzyme CrtZ, yielding titers of 1.10 g/L under glucose fermentation and 1.19 g/L under methanol-induced fermentation. Owing to its high efficiency, multi-gene copy number readability, and iterative integration capacity, the rNTS strategy offers an additional solution for quantitative gene-dosage assessment and microbial biosynthetic pathway optimization. Graphical abstract The rNTS system enables one-step, multi-copy integration anditerative assembly of synthetic genes, allowing rapid optimization ofgene dosage in microbial cell factories. This facil