Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002.

Area of Science:

• Synthetic biology
• Microbial biotechnology

Background:

• Marine cyanobacteria are key for sustainable chemical production.
• Conventional genetic engineering relies on antibiotic markers, limiting complex strain development.
• Existing markerless methods for PicoSynechococcus sp. PCC 7002 lack versatility.

Purpose of the Study:

• To develop a versatile, host-strain-independent markerless genetic manipulation method for PicoSynechococcus sp. PCC 7002.
• To enable efficient multigenic engineering of cyanobacteria for industrial applications.

Main Methods:

• A novel counter-selection system using a mutated phenylalanyl-tRNA synthetase (pheS) gene was developed.
• Specific amino acid substitutions (T261A and A303G) in pheS were identified for high susceptibility to p-chlorophenylalanine.
• The method was validated through markerless gene knockout (nblA) and gene knockin (lldD and lldP).

Main Results:

• A highly effective markerless transformation system for PicoSynechococcus sp. PCC 7002 was established.
• The mutated pheS system enabled precise gene editing without requiring host strain gene disruption.
• Successful markerless knockin and knockout of multiple genes were demonstrated, including a double knockin.

Conclusions:

• The developed mutated pheS-based counter-selection strategy is a powerful and repeatable tool for markerless genetic engineering in cyanobacteria.
• This method significantly advances the potential for complex, multigenic strain engineering for enhanced photosynthetic chemical production.
• The approach offers a versatile alternative to antibiotic markers, overcoming limitations in developing engineered cyanobacterial strains.