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

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Molecular Biology

Background:

• Efficient multi-gene expression is crucial for metabolic engineering and synthetic biology.
• Current methods for optimizing gene expression are often low-throughput and labor-intensive.
• Standardized genetic elements and high-throughput platforms are needed to overcome these limitations.

Purpose of the Study:

• To develop a high-throughput platform for engineering and optimizing multi-gene expression in Escherichia coli.
• To create standardized genetic parts (promoters, 5' UTRs) and libraries for modular assembly.
• To demonstrate the platform's utility in balancing multi-gene pathways for metabolite production.

Main Methods:

• Engineered standardized promoters and 5' UTRs with fluorescent reporters (e.g., eGFP, mCherry, TagBFP) for expression quantification.
• Assembled single-, dual-, and tri-gene constructs using Golden Gate assembly.
• Applied the platform to lycopene biosynthesis by replacing fluorescent genes with crtE, crtI, and crtB via Gibson assembly.
• Validated construct uniformity using quantitative PCR (qPCR).

Main Results:

• Developed a modular, dual-plasmid platform for high-throughput multi-gene expression analysis.
• Generated libraries of single-, dual-, and tri-gene constructs.
• Successfully applied the platform to optimize lycopene biosynthesis in E. coli BL21(DE3) strains.
• Demonstrated the platform's ability to balance multi-gene pathways and enable variable metabolite production.

Conclusions:

• The developed modular platform enables rapid exploration of multi-gene expression landscapes.
• This scalable tool facilitates metabolic engineering and the co-expression of multiple enzymes.
• The platform accelerates the optimization of complex genetic pathways in engineered microorganisms.