One-Step Multi-fragment Assembly and Targeted Genomic Integration in Corynebacterium glutamicum.

Area of Science:

• Synthetic Biology
• Metabolic Engineering
• Microbial Biotechnology

Background:

• Corynebacterium glutamicum is a crucial industrial microorganism for producing valuable chemicals like amino acids.
• Efficiently integrating large DNA fragments into C. glutamicum is essential for metabolic engineering but currently challenging.
• Existing methods for large DNA integration are often inefficient or labor-intensive.

Purpose of the Study:

• To optimize a RecET variant-assisted homologous recombination system for efficient large DNA fragment integration in C. glutamicum.
• To develop a novel strategy, One-Step Multi-Fragment Assembly Integration (OMAI), to further enhance the size limit of DNA integration.
• To establish a rapid and efficient method for genetic engineering of C. glutamicum.

Main Methods:

• Optimization of a RecET variant-assisted homologous recombination system.
• Development of the One-Step Multi-Fragment Assembly Integration (OMAI) strategy using cotransformation of overlapping PCR fragments.
• In vivo assembly and integration of multiple DNA fragments.

Main Results:

• Achieved single-step integration of DNA fragments larger than 11.3 kb.
• The OMAI strategy enabled integration of heterologous sequences approximately 50% longer than the conventional single-fragment limit.
• Each OMAI editing cycle was completed within 3 days, demonstrating a rapid method for large-fragment insertion.

Conclusions:

• The optimized RecET system and OMAI strategy significantly improve the efficiency and speed of large DNA fragment integration in C. glutamicum.
• OMAI represents a breakthrough for engineering C. glutamicum, enabling the introduction of larger genetic elements for enhanced bioproduction.
• This rapid method is expected to accelerate metabolic engineering efforts in C. glutamicum for industrial applications.