A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Area of Science:

• Synthetic Biology
• Biotechnology
• Microbial Engineering

Background:

• Synechococcus sp. PCC 11901 is a fast-growing marine cyanobacterium with high biomass accumulation potential.
• Limited genetic tools hinder the engineering of PCC 11901 for biotechnological applications.

Purpose of the Study:

• To develop and characterize a comprehensive suite of genetic tools for the engineering of Synechococcus sp. PCC 11901.
• To establish PCC 11901 as a robust model strain for synthetic biology and green biotechnology.

Main Methods:

• Characterization of neutral sites for stable genomic integration.
• Testing of constitutive and inducible promoters, including a novel 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system.
• Development of a DAPG-inducible CRISPR interference (CRISPRi) system and CRISPR-Cas12a for markerless genome editing.

Main Results:

• Identified neutral sites that do not impact growth at high cell densities.
• Demonstrated tight regulation and a 228-fold dynamic range for the PhlF repressor system in cyanobacteria.
• Achieved high efficiencies for CRISPRi-based repression and markerless genome editing with Cas12a (31-81% for single insertion, 25% for double insertion).

Conclusions:

• The developed genetic tools significantly enhance the engineering potential of Synechococcus sp. PCC 11901.
• PCC 11901 shows high responsiveness to CRISPRi and efficient genome editing, supporting its adoption as a model strain.
• These advancements lay the foundation for utilizing PCC 11901 in synthetic biology and green biotechnology applications.