Reusable combinatorial libraries with high diversity for efficient multi-gene expression optimization in Escherichia coli.

Efficient multi-gene expression in Escherichia coli is critical for advancing metabolic engineering and synthetic biology. However, existing strategies for combinatorial optimization remain labor-intensive and low-throughput. In addressing this challenge, a high-throughput platform was developed, encompassing the engineering of standardized genetic elements (promoters and 5' UTRs) with fluorescent reporters (e.g. eGFP, mCherry, TagBFP) to quantify expression variability. Libraries of single-, dual-, and tri-gene (dual-plasmid) constructs were assembled via Golden Gate, validated by IPTG induction, and applied to lycopene biosynthesis by replacing fluorescent genes with crtE, crtI, and crtB using Gibson assembly. The optimized tri-gene library was used to generate E. coli BL21(DE3) strains exhibiting variable levels of lycopene production, thereby demonstrating the platform's capacity to balance multi-gene pathways. Subsequent quantitative analysis by qPCR confirmed the uniformity of promoter-UTR combinations across the plasmid library. This modular platform, featuring reusable libraries and a dual-plasmid system, enables rapid exploration of multi-gene expression landscapes, offering a scalable tool for metabolic engineering and multi-enzyme co-expression.