Exploiting Polyploidy for Markerless and Plasmid-Free Genome Engineering in Cyanobacteria

Area of Science:

• Microbiology
• Synthetic Biology
• Genetics

Background:

• Cyanobacteria are vital microorganisms with complex polyploid genomes.
• Traditional genome engineering in cyanobacteria often relies on plasmids and can be inefficient.
• Developing efficient, plasmid-free genome engineering tools is crucial for cyanobacterial biotechnology.

Purpose of the Study:

• To develop a universal, plasmid-free genome engineering approach for cyanobacteria.
• To leverage the natural polyploidy of cyanobacterial chromosomes for counterselection.
• To create a method for efficient construction of stable, unmarked cyanobacterial mutants.

Main Methods:

• Genes for DNA modifying enzymes are integrated into essential chromosomal genes via allelic exchange under antibiotic selection.
• The polyploid nature of chromosomes maintains the engineered locus as a merodiploid under selection.
• Removal of antibiotic selection eliminates the DNA modifying enzyme gene from the population.

Main Results:

• Demonstrated proof of concept using CRE-lox and DRE-rox recombination systems in *Synechococcus* sp. PCC 7002 and *Synechocystis* sp. PCC 6803.
• Successfully constructed a markerless, high-CO2-requiring mutant (Δndh3 Δndh4 ΔbicA ΔsbtA) in *Synechococcus* sp. PCC 7002, showcasing method reusability.
• Achieved efficient and stable genome modification without the need for shuttle vectors or specific counterselection systems.

Conclusions:

• The developed method offers a generalizable and efficient strategy for plasmid-free genome engineering in cyanobacteria.
• This approach simplifies the creation of stable, unmarked mutants, advancing cyanobacterial genetic manipulation.
• The technique bypasses the need for developing additional shuttle vectors or counterselection systems, streamlining genetic engineering workflows.