Multiplex iterative plasmid engineering for combinatorial optimization of metabolic pathways and diversification of...

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Molecular Biology

Background:

• Engineering complex biological systems requires combinatorial optimization for desired functionality.
• Current methods for sequence diversification can be inefficient or lack customization.

Purpose of the Study:

• To present Multiplex Iterative Plasmid Engineering (MIPE), a novel method for efficient combinatorial diversification of plasmid sequences.
• To demonstrate MIPE's utility in optimizing biological pathways and protein-coding sequences.

Main Methods:

• MIPE utilizes single-stranded DNA (ssDNA) mediated lambda Red recombineering for targeted mutations.
• It allows simultaneous targeting of multiple sites, generating libraries up to 10^7 sequences.
• Restriction digestion mediated co-selection (RD CoS) enhances recombineering efficiency and simplifies selection.

Main Results:

• MIPE was applied to optimize the riboflavin biosynthetic pathway, yielding a clone with 2.67-fold improved production in under a week.
• The method successfully diversified 23 codons in a 750 bp protein-coding sequence simultaneously.
• High-efficiency library generation (up to 10^7 sequences) was achieved in a single reaction.

Conclusions:

• MIPE offers a highly efficient and customizable approach for combinatorial plasmid diversification.
• This method is broadly applicable for optimizing diverse biological systems in synthetic biology and metabolic engineering.
• MIPE significantly accelerates the engineering cycle for complex biological systems.